Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries Capsaicin , a pungent constituent from red chilli peppers , activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 -LRB- TRPV1 -RRB- to release neuropeptides like calcitonin gene-related peptide -LRB- CGRP -RRB- and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature . In this study , we examined the mechanism of capsaicin-induced relaxations , with special emphasis on the role of CGRP , using various pharmacological tools . Segments of human and porcine proximal and distal coronary arteries , as well as cranial arteries , were mounted in organ baths . Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant -LRB- BIBN4096BS , 1 μM -RRB- , the neurokinin NK1 receptor antagonist L-733060 -LRB- 0.5 μM -RRB- , the voltage-sensitive calcium channel blocker ruthenium red -LRB- 100 μM -RRB- , the TRPV1 receptor antagonist capsazepine -LRB- 5 μM -RRB- , the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl -LRB- l-NAME ; 100 μM -RRB- , the gap junction blocker 18α-glycyrrhetinic acid -LRB- 10 μM -RRB- , as well as the RhoA kinase inhibitor Y-27632 -LRB- 1 μM -RRB- . Further , we also used the K + channel inhibitors 4-aminopyridine -LRB- 1 mM -RRB- , charybdotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- and iberiotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- . The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments . In distal coronary artery segments , we also measured levels of cyclic adenosine monophosphate -LRB- cAMP -RRB- after exposure to capsaicin , and in human segments , we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin . Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries , but none of the above-mentioned inhibitors did affect these relaxations . There was no increase in the cAMP levels after exposure to capsaicin , unlike after -LRB- exogenously administered -RRB- α-CGRP . Interestingly , there were significant increases in CGRP levels after exposure to vehicle -LRB- ethanol -RRB- as well as capsaicin , although this did not induce relaxant responses . In conclusion , the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP , NK1 , NO , vanilloid receptors , voltage-sensitive calcium channels , K + channels or cAMP-mediated mechanisms . Therefore , these relaxant responses to capsaicin are likely to be attributed to a non-specific , CGRP-independent mechanism . Introduction Capsaicin , a pungent constituent of red pepper , is known to activate sensory C-fibres via transient receptor potential vanilloid receptors type 1 -LRB- TRPV1 ; Caterina et al. 1997 ; Gunthorpe et al. 2002 ; Szallasi 2006 -RRB- , which are nonselective cation channels . Activation of these channels increases influx of mono - and divalent cations , which leads to an increase in the intracellular Ca2 + concentrations . Consequently , an array of neuropeptides like calcitonin gene-related peptide -LRB- CGRP -RRB- , substance P and neurokinin A are released -LRB- Franco-Cereceda et al. 1988 ; Mitchell et al. 1995 -RRB- . These neuropeptides play a role in the regulation of normal vascular smooth muscle tone and are also implicated in several pathological conditions like ischemic preconditioning -LRB- Chai et al. 2006 -RRB- , preeclampsia -LRB- Dong et al. 2005 -RRB- and migraine -LRB- Arulmani et al. 2004b -RRB- . In the pathophysiology of migraine , vasodilatation of cranial blood vessels , especially extracerebral intracranial blood vessels , which are richly innervated by nerves containing a number of peptides such as CGRP , seems pivotal -LRB- Arulmani et al. 2006 -RRB- . Vasodilatation of these intracranial arteries leads to activation of nociceptors , which stimulate the pain centres in the brain -LRB- Goadsby et al. 2002 -RRB- . Involvement of CGRP in the pathophysiology of migraine is further strengthened by the observation that olcegepant -LRB- BIBN4096BS , Doods et al. 2000 -RRB- , a CGRP receptor antagonist , is effective in the acute treatment of migraine attacks -LRB- Olesen et al. 2004 -RRB- . Capsaicin has been widely used in various in vivo models of migraine to induce cranial vasodilatation , which is attributed to endogenous release of neuropeptides , especially CGRP -LRB- Akerman et al. 2003 ; Arulmani et al. 2004a ; Gupta et al. 2006a -RRB- . The involvement of CGRP is further substantiated in all these in vivo studies by the fact that the capsaicin-induced vascular responses are all amenable to blockade with CGRP receptor antagonists . Repeated capsaicin challenges are reported to deplete a number of neurotransmitters , including CGRP in various animal models -LRB- Tang et al. 1997 ; Tang et al. 1999 ; Zhou et al. 2002 -RRB- . In addition to the in vivo models , also in vitro vascular models involving meningeal -LRB- Gupta et al. 2006b -RRB- and coronary -LRB- Edvinsson et al. 2002 ; Gupta et al. 2006c -RRB- arteries have been used in migraine research , but vasorelaxation in these models was induced by exogenously administered CGRP . Several major vascular beds , including the meningeal and coronary -LRB- Franco-Cereceda 1988 -RRB- , are richly innervated with capsaicin-sensitive sensory fibres containing CGRP -LRB- Edvinsson et al. 1987 -RRB- . Meningeal blood vessels have been shown to be involved in the plasma protein extravasation in rat dura mater -LRB- Delepine and Aubineau 1997 ; Seabrook et al. 1996 -RRB- , a model of neurogenic inflammation , one of the putative mechanisms in migraine pathophysiology . In this model , capsaicin pre-treatment has been shown to block plasma protein extravasation , again by depleting neuropeptides -LRB- Delepine and Aubineau 1997 -RRB- . Therefore , it is of interest to investigate the release of endogenous CGRP in various blood vessels and capsaicin seems most appropriate for such studies . Interestingly , very few studies have investigated capsaicin-induced relaxations in human isolated blood vessels -LRB- Franco-Cereceda 1991a ; Franco-Cereceda et al. 1987 -RRB- . Based on CGRP-like immunoreactivity , it was claimed that capsaicin-induced relaxations are mediated by CGRP -LRB- Franco-Cereceda 1991b -RRB- , but no CGRP receptor antagonists were used to unequivocally demonstrate the role of CGRP in these responses . Therefore , we were interested in studying capsaicin-mediated relaxations in human and porcine isolated arteries as a model to study endogenous release of CGRP in view of its relevance in the pathophysiology of migraine . Remarkably , some recent studies indicate that capsaicin-induced relaxations are mediated by non-CGRP-mediated mechanisms in guinea pig ileum , rabbit coronary arteries and equine tracheal smooth muscle preparations -LRB- Fujimoto et al. 2006 ; Yeon et al. 2001 ; Zhu et al. 1997 -RRB- , which is contrary to the observation that capsaicin-induced relaxations are mediated principally by CGRP . In these studies , relaxant responses to capsaicin were mainly attributed to different Ca2 + - activated K + channels . Therefore , in the present study , we tried to characterise capsaicin-induced relaxant response in human and porcine vessels , with special emphasis on the role of CGRP , and also investigated other mechanisms , using different pharmacological tools . Materials and methods Tissue preparation The study protocol was approved by the ethical committee of Erasmus MC . Human coronary artery was obtained from ` heart-beating ' organ donors -LRB- 13 men , 19 women ; 13 -- 65 years -RRB- who died due to non-cardiac disorders . The hearts were provided by the Heart Valve Bank , Rotterdam , The Netherlands , after donor mediation by Bio Implant Services Foundation/Eurotransplant Foundation -LRB- Leiden , The Netherlands -RRB- . The meningeal arteries were obtained from patients -LRB- four men , six women ; 41 -- 75 years -RRB- undergoing craniotomy at the neurosurgical unit -LRB- n = 6 -RRB- or from the Department of Pathology -LRB- postmortem , n = 4 -RRB- within 24 h of death , at Erasmus MC , Rotterdam . During the surgical procedure or autopsy , the dura mater together with a small piece of the meningeal artery was cut and placed in a plastic container filled with ice-cold -LRB- 0 -- 4 °C -RRB- , physiological salt solution . The artery segment was immediately transported to the laboratory and placed in cold oxygenated Krebs bicarbonate solution of the following composition -LRB- mM -RRB- : NaCl 119 , KCl 4.7 , CaCl2 1.25 , MgSO4 1.2 , KH2PO4 1.2 , NaHCO3 25 and glucose 11.1 ; pH 7.4 . Excess tissue was removed . The meningeal artery -LRB- internal diameter of 350 -- 800 μm -RRB- was used the same day or stored overnight in cold oxygenated modified Krebs solution and used the following day . Porcine hearts and heads -LRB- pigs of either sex ; 6 -- 12 months of age -RRB- were collected from a local slaughterhouse . Porcine basilar -LRB- internal diameter of 150 -- 200 μm -RRB- as well as meningeal -LRB- internal diameter of 100 -- 250 μm -RRB- arteries were dissected out from the skull and were placed in a cold oxygenated Krebs solution as described earlier for human meningeal arteries . In the laboratory , both human and porcine proximal -LRB- internal diameter , 2 -- 3.5 mm -RRB- and distal -LRB- internal diameter , 200 -- 600 μm -RRB- coronary arteries were dissected out of the right ventricle ; distal coronary arteries were dissected with the aid of a microscope and stored in bicarbonate solution of following composition -LRB- mM -RRB- : NaCl 118 , KCl 4.7 , CaCl2 2.5 , MgSO4 1.2 , KH2PO4 1.2 , NaHCO3 25 and glucose 11.1 ; pH 7.4 . Functional studies Both human and porcine proximal coronary artery segments -LRB- 3 -- 4 mm length -RRB- were suspended with the help of stainless steel hooks in 15-ml organ baths with a pretension of 15 mN -LRB- optimal tension shown in earlier experiments -RRB- . Distal coronary and cranial artery segments were cut into ring segments of 1 -- 2 mm length and were mounted in Mulvany myographs between two parallel titanium wires with a tension normalised to 90 % of l100 -LRB- distance when transmural pressure equals 100 mmHg -RRB- , thus achieving optimal conditions for active force development -LRB- Nyborg et al. 1987 -RRB- . Vessels were allowed to equilibrate for 30 min in Krebs solution at 37 °C , a similar equilibration period was repeated after each physical or pharmacological challenge . In case of human arteries , the cyclo-oxygenase inhibitor indomethacin -LRB- 0.1 μM -RRB- was added to prevent prostaglandin synthesis during the whole experimental protocol . Two successive challenges to KCl -LRB- 30 mM -RRB- were performed to test the reproducibility of responses . Endothelial integrity was assessed by observing relaxation to substance P -LRB- 10 nM -RRB- after precontraction with U46619 -LRB- 9,11-dideoxy-11α , 9α-epoxymethano-prostaglandin F2α ; 10 nM -- 1 μM -RRB- , followed by washout of the agonists . KCl -LRB- 18 -- 30 mM , except where higher concentrations are indicated -RRB- or U46619 -LRB- 10 nM -- 1 μM -RRB- , in case of the K + channel blockers , was used to obtain a stable contraction plateau of around 60 -- 75 % of the maximal contraction reached with KCl -LRB- 100 mM -RRB- . Subsequently , capsaicin was added in a cumulative manner in log steps . The different antagonists were used to antagonise capsaicin responses , and the concentration of each of these antagonists was decided on the basis of highest concentration in the literature unless mentioned otherwise . Concentration response curves were constructed in the absence or presence of the CGRP receptor antagonists olcegepant -LRB- 1 μM ; Gupta et al. 2006b , c -RRB- and CGRP8 -- 37 -LRB- 10 μM ; Gupta et al. 2006b , c -RRB- , the neurokinin NK1 receptor antagonist L-733060 -LRB- 0.5 μM ; Seabrook et al. 1996 -RRB- , the voltage-sensitive calcium channel blocker ruthenium red -LRB- 100 μM , 45 -- 90 mM KCl was required to get a similar precontraction as in the corresponding control segments ; Franco-Cereceda and Rudehill 1989 -RRB- , the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl -LRB- l-NAME ; 100 μM ; Batenburg et al. 2004a , b -RRB- , as well as the vanilloid receptor antagonist capsazepine -LRB- 5 μM ; Fujimoto et al. 2006 ; Gazzieri et al. 2006 -RRB- . At still higher concentrations of capsazepine -LRB- > 5 μM -RRB- and L-733006 -LRB- > 0.5 μM -RRB- , even higher concentrations of preconstricting agents could not produce a workable precontraction . We also investigated the role of different K + channels by using various inhibitors , 4-aminopyridine -LRB- 1 mM ; Fujimoto et al. 2006 ; Yeon et al. 2001 -RRB- , a voltage-dependent K + channel -LRB- Kv -RRB- blocker and charybdotoxin -LRB- 0.5 μM -RRB- , a blocker of Ca2 + - dependent K + channels for large conductance -LRB- BKCa -RRB- and intermediate conductance -LRB- IKCa -RRB- , in combination with apamin -LRB- 0.1 μM -RRB- , a blocker of small-conductance Ca2 + - dependent K + channels -LRB- SKCa ; Batenburg et al. 2004b ; Fujimoto et al. 2006 ; Zhu et al. 1997 -RRB- . The effect of the RhoA kinase inhibitor , Y-27632 -LRB- 1 μM ; Fujimoto et al. 2006 -RRB- was investigated both in the absence or presence of 4-aminopyridine -LRB- 1 mM -RRB- . We also investigated the effect of the gap junction inhibitor 18-α-glycyrrhetinic acid -LRB- 10 μM -RRB- on capsaicin-induced relaxations . Where indicated , the endothelium was removed with a human hair , and removal was confirmed by observing a relaxation of less than 10 % of precontraction of U46619 after addition of substance P. Further , in porcine distal coronary artery , we also studied the effect of repeated administration of capsaicin -LRB- 50 μM , four times -RRB- to verify the reproducibility of the responses in view of possible depletion of endogenous peptide pools or other agents . Measurements of cAMP Human proximal and distal , as well as porcine distal , coronary artery segments were incubated in a medium containing isobutylmethylxanthine -LRB- 0.5 mM -RRB- for 30 min in the absence or presence of olcegepant -LRB- 1 μM -RRB- . The arterial segments were exposed to KCl -LRB- 30 mM -RRB- , challenged with ethanol -LRB- vehicle of capsaicin , final concentration in the baths 0.56 % -RRB- , capsaicin -LRB- 10 μM -RRB- or h-αCGRP -LRB- 100 nM , serving as a positive control -RRB- for 5 min and then snap frozen . The samples were stored at − 80 °C until cyclic adenosine monophosphate -LRB- cAMP -RRB- assay . To determine cAMP , tissues were homogenised in 0.5 ml 0.1 M HCl using a stainless steel ultraturrax -LRB- Polytron , Staufen , Germany -RRB- . Homogenates were centrifuged at 3,300 × g , and cAMP was measured in 300 μl supernatant using the ELISA kit according to the instructions of the manufacturer -LRB- R&D Systems Europe , Abingdom , UK -RRB- . Measurements of CGRP in organ bath fluid Human distal coronary artery segments were subjected to a similar protocol as during the functional studies , while bath fluids were collected after construction of the concentration response curve . The bath fluids were collected from the segments treated with vehicle and capsaicin -LRB- 100 μM -RRB- , and Krebs solution was used as a control . Bath fluids were stored in tubes containing aprotinin -LRB- 0.6 TIU/ml -RRB- and stored at − 80 °C . A competitive radioimmunoassay -LRB- Peninsula Lab INC. , San Carlos , CA , USA -RRB- was used according to the instructions of the manufacturer to measure the CGRP concentrations in the bath fluid . Data presentation and statistical analysis The relaxant responses elicited by the agonists are expressed as percentage relaxation of the tone induced by 30 mM KCl or U46619 -LRB- in case of the K + channel blockers -RRB- . All data are presented as means ± SEM , and n represents the number of blood vessel segments , all obtained from different subjects . The effect of all potential inhibitors of the relaxations to capsaicin was investigated in a paired parallel setup ; that is , relaxations in segments with inhibitors were always compared to relaxations obtained in control segments from the same subject . The concentration response curves for all agonists were analysed using nonlinear regression analysis , and the potency of agonists was expressed as pEC50 using Graph Pad Prism 3.01 -LRB- Graph Pad Software , San Diego , CA , USA -RRB- , setting the Emax of capsaicin in the presence of potential inhibitors to that in the respective control segment in case it was lower than the control Emax . The blocking potency of the antagonists was estimated by calculating median effective concentration -LRB- EC50 -RRB- ratios , and apparent pKB values were calculated for the antagonists at each given concentration , with the slope set to unity . Statistical differences between concentration response curves to capsaicin in the absence and presence of potential inhibitors were determined using Student 's paired t-test with α set to 1.00 -- 0.95 -LRB- 1/n -RRB- -LRB- Motulsky 2003 -RRB- to correct for multiple comparisons . For the measurements of CGRP in the organ bath fluids , we could not exclude a non-Gaussian distribution due to the large degree of variability in the data . Therefore , the levels of CGRP in bath fluids were analysed by the non-parametric Kruskal -- Wallis test , followed by Dunn 's post hoc multiple comparison test . Significance was assumed at P ≤ 0.05 . Compounds Human α-CGRP and α-CGRP8 -- 37 were obtained from Polypeptide , -LRB- Wolfenbüttel , Germany -RRB- , olcegepant -LRB- BIBN4096BS , 1-piperidinecarboxamide , N - -LSB- 2 - -LSB- -LSB- 5-amino-1 - -LSB- -LSB- 4 - -LRB- 4-pyridinyl -RRB- -1-piperazinyl -RSB- carbonyl -RSB- pentyl -RSB- amino -RSB- -1 - -LSB- -LRB- 3,5-dibromo-4-hydroxyphenyl -RRB- methyl -RSB- -2-oxoethyl -RSB- -4 - -LRB- 1,4-dihydro-2-oxo-3 -LRB- 2H -RRB- - quinazolinyl -RRB- - , -LSB- R - -LRB- R * , S * -RRB- -RSB- - -RRB- was a gift from Boehringer Ingelheim Pharma -LRB- Biberach/Riss , Germany -RRB- ; 4-aminopyridine was purchased from ICN Biomedicals -LRB- Aurora , OH , USA -RRB- ; L-733060 was purchased from Tocris -LRB- Bristol , UK -RRB- ; apamin , capsaicin , capsazepine , 3-isobutyl-1-methyl-xanthine , l-NAME , charybdotoxin , ruthenium red , substance P , U46619 and Y-27632 were purchased from Sigma-Aldrich -LRB- Zwijndrecht , The Netherlands -RRB- , and KCl was obtained from Merck -LRB- Darmstadt , Germany -RRB- . Capsaicin was dissolved in 70 % ethanol , and the dilution series was also prepared in ethanol 70 % . Capsazepine was dissolved in methanol ; olcegepant was dissolved in a small amount of 1 N HCl and then diluted with distilled water . The other compounds were dissolved in distilled water , and all compounds were stored in aliquots at − 80 °C . Results Functional studies Human arteries Substance P relaxed artery segments precontracted with U46619 -LRB- 10 nM -- 1 μM -RRB- ; responses were equi-efficacious in distal coronary -LRB- 80 ± 5 % of contraction to U46619 , n = 28 -RRB- and meningeal -LRB- 75 ± 8 % , n = 8 -RRB- artery and significantly less in proximal coronary artery -LRB- 27 ± 15 % , n = 4 -RRB- . Both in the meningeal and distal coronary arteries , capsaicin induced concentration-dependent relaxations . In human proximal coronary artery , relaxant responses were only observed at the highest concentration of 100 μM , and the maximum relaxant response -LRB- 34 ± 14 % of contraction to 30 mM KCl -RRB- was significantly less than that observed in the distal arteries -LRB- 94 ± 1 % of contraction to 18 -- 30 mM KCl -RRB- . In human meningeal arteries , there was no difference in capsaicin-induced responses between arteries obtained perioperatively or postmortem ; therefore , these data were pooled for further analysis . Capsaicin was equipotent and equi-efficacious in human distal coronary and human meningeal artery . In human distal coronary artery segments , the lower concentrations of capsaicin -LRB- 0.1 nM -- 1 μM -RRB- in some cases induced contractions , but in all cases , we uniformly only measured the relaxant responses . The relaxations to capsaicin in proximal and distal coronary as well as meningeal arterial segments were insensitive to blockade by the CGRP antagonist olcegepant -LRB- 1 μM ; Fig. 1 , Table 1 -RRB- . Fig. 1Effect of capsaicin or its vehicle in the absence or presence of various pharmacological agents or interventions in precontracted human and porcine distal coronary arteriesTable 1Effect of various antagonists/interventions on capsaicin-induced relaxations in human isolated artery segmentsAntagonist or other intervention -LRB- n -RRB- Emax -LRB- % -RRB- Δ EmaxpEC50Δ pEC50Human distal coronary artery -LRB- Control -RRB- -LRB- 32 -RRB- 94 ± 15.27 ± 0.12 Olcegepant -LRB- 1 μM -RRB- -LRB- 10 -RRB- 89 ± 48 ± 44.84 ± 0.090.33 ± 0.16 CGRP8 -- 37 -LRB- 10 μM -RRB- -LRB- 5 -RRB- 96 ± 33 ± 44.79 ± 0.060.16 ± 0.08 Capsazepine -LRB- 5 μM -RRB- -LRB- 13 -RRB- 91 ± 31 ± 25.10 ± 0.130.07 ± 0.16 Ruthenium red -LRB- 0.1 mM -RRB- -LRB- 9 -RRB- 92 ± 35 ± 35.01 ± 0.130.21 ± 0.22L-733060 -LRB- 5 μM -RRB- -LRB- 7 -RRB- 94 ± 22 ± 26.03 ± 0.78 − 0.54 ± 0.34 Denuded endothelium -LRB- 5 -RRB- 90 ± 69 ± 55.34 ± 0.48 − 0.21 ± 0.58l-NAME -LRB- 0.1 mM -RRB- -LRB- 7 -RRB- 94 ± 23 ± 25.23 ± 0.48 − 0.24 ± 0.4518-α-Glycyrrhetinic acid -LRB- 10 μM -RRB- -LRB- 3 -RRB- 96 ± 20 ± 25.08 ± 0.28 − 0.15 ± 0.38 Olcegepant -LRB- 1 μM -RRB- + L-733060 -LRB- 5 μM -RRB- -LRB- 3 -RRB- 90 ± 61 ± 36.04 ± 0.230.06 ± 0.21 -LRB- Control -RRB- -LRB- 10 -RRB- 97 ± 15.91 ± 0.324-Aminopyridine -LRB- 1 mM -RRB- -LRB- 6 -RRB- 95 ± 22 ± 35.64 ± 0.380.94 ± 0.46 Charybdotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- -LRB- 8 -RRB- 97 ± 10 ± 15.80 ± 0.370.23 ± 0.37 Iberiotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- -LRB- 5 -RRB- 96 ± 20 ± 25.94 ± 0.650.15 ± 0.38Y-276323 -LRB- 1 μM -RRB- -LRB- 3 -RRB- 99 ± 1 − 1 ± 25.39 ± 0.21 − 0.58 ± 0.14Y-276323 -LRB- 1 μM -RRB- + 4-Aminopyridine -LRB- 1 mM -RRB- -LRB- 3 -RRB- 100 ± 0 − 3 ± 45.12 ± 0.180.33 ± 0.28 Human proximal coronary artery -LRB- Control -RRB- -LRB- 4 -RRB- 34 ± 144.30 ± 0.14 Olcegepant -LRB- 1 μM -RRB- -LRB- 4 -RRB- 36 ± 164.40 ± 0.17 Human meningeal artery -LRB- Control -RRB- -LRB- 10 -RRB- 91 ± 5 5.04 ± 0.09 Olcegepant -LRB- 1 μM -RRB- -LRB- 10 -RRB- 96 ± 1 − 5 ± 45.03 ± 0.07 − 0.07 ± 0.08 Capsazepine -LRB- 5 μM -RRB- -LRB- 4 -RRB- 81 ± 98 ± 74.90 ± 0.310.11 ± 0.31 Ruthenium red -LRB- 0.1 mM -RRB- -LRB- 3 -RRB- 74 ± 155 ± 145.13 ± 0.420.02 ± 0.62L-733060 -LRB- 5 μM -RRB- -LRB- 4 -RRB- 79 ± 128 ± 154.80 ± 0.31 − 0.09 ± 0.31 Emax is the maximum relaxant response , expressed as percentage of the respective precontraction ; pEC50 is the − logEC50 , where EC50 is the concentration of agonist required to produce half the maximal response . The arteries were precontracted with KCl -LRB- 18 -- 30 mM -RRB- except where KCl -LRB- 45 -- 90 mM ; bold -RRB- or U46619 -LRB- ital -RRB- . As the relaxant responses to capsaicin were small in human proximal coronary arteries and the availability of human meningeal arteries was very limited , further experiments were only carried out in human distal coronary artery . In this preparation , the CGRP receptor antagonist CGRP8 -- 37 -LRB- 10 μM -RRB- , the TRPV1 receptor antagonist capsazepine -LRB- 5 μM -RRB- and the NK1 receptor antagonist L-733006 -LRB- 0.5 μM -RRB- also did not attenuate capsaicin-induced relaxations -LRB- Table 1 -RRB- . Similarly , there was no significant difference in relaxant responses in endothelium-intact or endothelium-denuded segments -LRB- Table 1 -RRB- . Also , in the absence or presence of the NO synthase inhibitor l-NAME -LRB- 100 μM -RRB- or in the presence of the gap junction blocker 18-α-glycyrrhetinic acid , capsaicin caused equipotent relaxations compared to the respective control segments -LRB- Table 1 -RRB- . Various K + channel blockers , namely , 4-aminopyridine -LRB- 1 mM -RRB- , charybdotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- and iberiotoxin -LRB- 0.1 μM -RRB- + apamin -LRB- 0.1 μM -RRB- were also unable to block the relaxant responses to capsaicin . The RhoA kinase inhibitor Y-27632 -LRB- 1 μM -RRB- alone or in combination with 4-aminopyridine was also unable to block the relaxant responses to capsaicin . Ruthenium red -LRB- 100 μM -RRB- , a nonselective blocker of Ca2 + transport through membrane channels , also did not significantly block these responses -LRB- Table 1 -RRB- . It is noteworthy that at higher concentrations , capsazepine and L-733060 significantly attenuated the responses to their respective preconstriction agents , and also in case of preincubation with Y-27632 and ruthenium red , 2 - to 20-fold higher concentrations of precontracting agents were required to induce 60 -- 75 % of the maximal contraction reached with KCl -LRB- 100 mM -RRB- . At still higher concentrations of capsazepine -LRB- > 5 μM -RRB- and L-733060 -LRB- > 0.5 μM -RRB- , even higher concentrations of preconstricting agents could not produce a workable precontraction . Additionally , the combination of olcegepant -LRB- 1 μM -RRB- + L-733060 -LRB- 0.5 μM -RRB- in human distal coronary artery or 4-aminopyridine -LRB- 1 mM -RRB- + Y-27632 -LRB- 1 μM -RRB- both in human and porcine distal coronary artery were also unable to block the responses to capsaicin . The vehicle of capsaicin -LRB- 0.56 % ethanol -RRB- did not induce any significant relaxations . It should be noted that the potency of capsaicin was higher on a precontraction with U46619 -LRB- 10 nM -- 1 μM ; pEC50 of capsaicin , 5.91 ± 0.32 -RRB- than on a precontraction with KCl -LRB- 18 -- 30 mM ; pEC50 of capsaicin , 5.27 ± 0.12 ; P = 0.029 -RRB- . However , irrespective of the precontracting agent used , the antagonists behaved in a similar fashion towards capsaicin . For example , the effects of 4-aminopyridine on capsaicin-induced relaxations were similar after precontraction with KCl -LRB- pEC50 , 5.01 ± 0.05 and 5.12 ± 0.41 ; n = 7 , in the absence and presence of 4-aminopyridine , respectively -RRB- and with U46619 -LRB- pEC50 , 6.58 ± 0.47 and 5.64 ± 0.38 ; n = 6 , in the absence and presence of 4-aminopyridine , respectively -RRB- . In human meningeal artery , capsazepine , L-733006 and also ruthenium red did not block the responses to capsaicin -LRB- Table 1 -RRB- . Porcine arteries In porcine arteries , substance P induced relaxations in precontracted arteries to a varying degree in different vessels . The relaxations in distal coronary -LRB- 64 ± 6 % of contraction to U46619 , 10 nM -- 1 μM , n = 26 -RRB- basilar -LRB- 44 ± 3 % , n = 3 -RRB- and meningeal -LRB- 57 ± 15 % , n = 3 -RRB- artery were similar , whereas that in proximal coronary -LRB- 8 ± 8 % , n = 4 -RRB- artery was significantly smaller . In porcine proximal and distal coronary arteries , capsaicin induced concentration-dependent relaxations . Unlike in human coronary arteries , the maximum response was not significantly different between porcine proximal and distal coronary arteries -LRB- Table 2 -RRB- . In both the arteries , olcegepant -LRB- 1 μM -RRB- did not block the responses to capsaicin . Further experiments were carried out in distal coronary arteries and , similar as in the human distal coronary artery , capsazepine -LRB- 1 μM -RRB- , ruthenium red -LRB- 0.1 mM -RRB- , L-73360 -LRB- 0.5 μM -RRB- , 4-aminopyridine -LRB- 1 mM -RRB- , l-NAME -LRB- 0.1 mM -RRB- , charybdotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- , iberiotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- , Y-27632 -LRB- 1 μM -RRB- , 18-α-glycyrrhetinic acid -LRB- 10 μM -RRB- and endothelium denudation did not affect capsaicin-induced relaxations -LRB- Fig. 1 , Table 2 -RRB- . Additionally , Y-27632 -LRB- 1 μM -RRB- , alone or combined with 4-aminopyridine -LRB- 1 mM -RRB- , was also unable to block the responses to capsaicin . Similar as in the other arteries , the vehicle did not induce a relaxation -LRB- Fig. 1 -RRB- . Interestingly , in contrast to what we observed in the human distal coronary artery , precontracting the arteries with either KCl or U46619 did not change the relaxant responses to capsaicin . Four consecutive challenges to capsaicin -LRB- 50 μM -RRB- did not significantly affect the magnitude of the responses -LRB- Fig. 2 -RRB- . Table 2Effect of various antagonists/interventions on capsaicin-induced relaxations in porcine isolated artery segmentsAntagonist or other intervention -LRB- n -RRB- Emax -LRB- % -RRB- Δ EmaxpEC50Δ pEC50Porcine distal coronary artery -LRB- Control -RRB- -LRB- 56 -RRB- 96 ± 15.27 ± 0.09 Olcegepant -LRB- 1 μM -RRB- -LRB- 10 -RRB- 92 ± 23 ± 25.26 ± 0.11 − 0.02 ± 0.11 CGRP8 -- 37 -LRB- 10 μM -RRB- -LRB- 5 -RRB- 97 ± 3 − 2 ± 54.78 ± 0.060.00 ± 0.03 Capsazepine -LRB- 5 μM -RRB- -LRB- 7 -RRB- 99 ± 1 − 3 ± 25.15 ± 0.260.26 ± 0.16 Ruthenium red -LRB- 0.1 mm -RRB- -LRB- 12 -RRB- 88 ± 37 ± 44.89 ± 0.090.47 ± 0.23L-733060 -LRB- 5 μM -RRB- -LRB- 7 -RRB- 88 ± 34 ± 44.89 ± 0.09 − 0.04 ± 0.15 Denuded endothelium -LRB- 9 -RRB- 91 ± 42 ± 25.62 ± 0.490.21 ± 0.16l-NAME -LRB- 0.1 mM -RRB- -LRB- 6 -RRB- 90 ± 88 ± 84.84 ± 0.150.28 ± 0.31 -LRB- Control -RRB- -LRB- 15 -RRB- 99 ± 05.08 ± 0.1518-α-Glycyrrhetinic acid -LRB- 10 μM -RRB- -LRB- 3 -RRB- 99 ± 10 ± 15.73 ± 0.75 − 0.59 ± 0.534-Aminopyridine -LRB- 1 mM -RRB- -LRB- 7 -RRB- 96 ± 23 ± 24.84 ± 0.15 − 0.46 ± 0.35 Charybdotoxin -LRB- 0.5 μM -RRB- + apamin -LRB- 0.1 μM -RRB- -LRB- 11 -RRB- 99 ± 10 ± 15.27 ± 0.15 − 0.04 ± 0.20Y-276323 -LRB- 1 μM -RRB- -LRB- 9 -RRB- 96 ± 23 ± 25.17 ± 0.190.04 ± 0.21Y-276323 -LRB- 1 μM -RRB- + 4-Aminopyridine -LRB- 1 mM -RRB- -LRB- 8 -RRB- 96 ± 34 ± 35.22 ± 0.200.00 ± 0.11 Porcine proximal coronary artery -LRB- Control -RRB- -LRB- 4 -RRB- 100 ± 05.33 ± 0.42 Olcegepant -LRB- 1 μM -RRB- -LRB- 4 -RRB- 90 ± 65.79 ± 0.16 Porcine basilar artery -LRB- Control -RRB- -LRB- 3 -RRB- 97 ± 14.70 ± 0.05 Olcegepant -LRB- 1 μM -RRB- -LRB- 3 -RRB- 100 ± 04.97 ± 0.24 Capsazepine -LRB- 5 μM -RRB- -LRB- 3 -RRB- 100 ± 04.80 ± 0.01 Porcine meningeal artery -LRB- Control -RRB- -LRB- 3 -RRB- 99 ± 14.82 ± 0.02 Olcegepant 1 μM -LRB- 3 -RRB- 99 ± 14.88 ± 0.04 Emax is the maximum relaxant response , expressed as percentage of the respective precontraction ; pEC50 is the − logEC50 , where EC50 is the concentration of agonist required to produce half the maximal response . The arteries were precontracted with KCl -LRB- 18 -- 30 mM -RRB- except where KCl -LRB- 45 -- 90 mM ; bold -RRB- or U46619 -LRB- ital -RRB- . Fig. 2Effect of four consecutive challenges to capsaicin -LRB- 50 μM -RRB- in porcine distal coronary arteries precontracted with KCl -LRB- 30 mM -RRB- There were no significant differences in efficacy or potency of capsaicin in porcine basilar -LRB- Emax , 97 ± 1 % ; pEC50 , 4.70 ± 0.05 ; n = 3 -RRB- and meningeal -LRB- Emax , 99 ± 1 % ; pEC50 , 4.82 ± 0.02 ; n = 3 -RRB- arteries as compared to porcine distal coronary arteries . In both these arteries , responses to capsaicin were not affected by olcegepant -LRB- 1 μM -RRB- . Measurements of cAMP Capsaicin -LRB- 10 μM -RRB- did not affect cAMP levels in comparison to its vehicle or the control , which was only exposed to 30 mM KCl . In contrast , αCGRP -LRB- 100 nM -RRB- , which was used as a positive control , significantly increased cAMP levels in both human and porcine distal coronary arteries , which was blocked after incubation with olcegepant -LRB- 1 μM ; Fig. 3 -RRB- . Fig. 3Changes in cAMP levels in human -LRB- n = 4 -- 7 -RRB- and porcine -LRB- n = 4 -- 9 -RRB- distal coronary artery segments after exposure to various pharmacological agents . * Significantly different -LRB- P < 0.05 -RRB- from KCl -LRB- 30 mM -RRB- - treated segments Measurements of CGRP levels in organ bath fluid Capsaicin and its vehicle induced a significant CGRP release from human distal coronary artery segments into the organ bath fluid -LRB- Fig. 4 -RRB- as compared to levels observed in Krebs solution . There was no significant difference between CGRP levels obtained after incubation with capsaicin or its vehicle . Fig. 4CGRP levels measured in bath fluids -LRB- Krebs buffer , control -RRB- after capsaicin or vehicle challenge in precontracted human distal coronary artery segments . * Significantly different -LRB- P < 0.05 -RRB- from Krebs buffer Discussion In the present study , we investigated the role of CGRP in capsaicin-induced relaxations in human and porcine isolated arteries . In all arteries investigated , there does not seem to be any relevant role of CGRP in the relaxant responses to capsaicin . Further , the effects of capsaicin appear to be mediated by non-specific mechanisms . In all arteries investigated , capsaicin induced concentration-dependent relaxations , although these had a limited potency and efficacy in human proximal coronary artery . The responses to capsaicin were resistant to blockade with olcegepant in all tissues studied , which suggests no involvement of CGRP receptors . The potency of capsaicin is in line with what has earlier been observed in human coronary arteries -LRB- Franco-Cereceda 1991a -RRB- and guinea pig ileum -LRB- Fujimoto et al. 2006 -RRB- . As expected in view of its resistance to olcegepant , CGRP8 -- 37 -LRB- 10 μM -RRB- also did not block capsaicin-induced relaxations in human distal coronary artery segments . This is contrary to the observations by Franco-Cereceda and Rudehill -LRB- 1989 -RRB- , where the authors claim the relaxations to capsaicin are mediated by CGRP on the basis of the observation that the sustained relaxations in the human coronary induced by capsaicin are similar to those induced by exogenous CGRP , unlike the transient relaxations to substance P , which are followed by rapid tachyphylaxis . In another publication , these authors claim involvement of CGRP in responses to capsaicin in the human isolated coronary artery on the basis of increased CGRP-like immunoreactivity after exposure of the artery segments to capsaicin -LRB- Franco-Cereceda 1991b -RRB- . It should be noted that in the above study , the authors show an increase in CGRP-like immunoreactivity in large arteries , where no functional studies were performed . Further , the classical prerequisite for demonstrating the involvement of a particular pharmacological agent , by using the corresponding antagonist to block functional responses , was lacking in this study . Admittedly , the same group did demonstrate that CGRP8 -- 37 inhibited the responses to capsaicin in porcine coronary arteries -LRB- Franco-Cereceda 1991b -RRB- , although we could not block these responses even at 10-μM concentration . As CGRP-induced responses are mediated by increases in cAMP -LRB- Gupta et al. 2006c -RRB- , we measured the levels of this second messenger after exposure to capsaicin and CGRP . We observed no increase in cAMP after addition of capsaicin , in contrast to the increased levels of cAMP after exposure of the vessel segments to -LRB- exogenous -RRB- α-CGRP . These increased levels were , as expected , blocked by the CGRP receptor antagonist olcegepant . Interestingly , after exposure to capsaicin , we observed CGRP release in the organ bath fluid , where the human distal coronary arteries were mounted in accordance with earlier observations -LRB- Franco-Cereceda 1991a -RRB- . However , a similar increase was observed after administration of vehicle , while the control was not studied in the study of Franco-Cereceda et al. -LRB- 1991a -RRB- . Although capsaicin is known to activate TRPV1 , there are reports that ethanol , also via activation of TRPV1 , induces the release of CGRP as well -LRB- Trevisani et al. 2002 , 2004 -RRB- . Moreover , as obvious from Fig. 1 , the vehicle did not induce any relaxations in the precontracted arteries , and hence , the released CGRP can not account for relaxations induced by capsaicin . Interestingly , the concentration of CGRP detected in the bath fluid of about 3 pM should have been about 10,000 times higher in the vessel segments -LRB- ∼ 0.5 mg tissue in 5-ml organ bath fluid -RRB- . Thus , the concentration of CGRP in the vessel segments should have been in the nanomolar range , which is equal to or even higher than the pEC50 in human distal coronary artery under similar experimental conditions -LRB- Gupta et al. 2006c -RRB- and should , thus , have induced a detectable relaxation . Therefore , it is most likely that the radioimmunoassay displayed cross-reactivity to another ligand , not CGRP . Admittedly , our observation that ruthenium red , even at a very high concentration -LRB- 100 μM -RRB- , did not block the responses to capsaicin , is in contrast with the observations described by Franco-Cereceda -LRB- 1991a -RRB- , where ruthenium red completely blocked the responses to capsaicin . Additional evidence for the fact that CGRP and TRPV1 are not involved in relaxant responses to capsaicin is provided by the fact that capsazepine , a competitive antagonist of TRPV1 -LRB- Caterina et al. 1997 -RRB- , did not block responses to capsaicin in our study . In view of reports of involvement of various K + channels in the relaxant responses to capsaicin in various smooth muscle preparations -LRB- Yeon et al. 2001 ; Zhu et al. 1997 -RRB- , we also investigated various K + channel inhibitors in similar or even higher concentrations , but none of these blocked the responses to capsaicin . 4-Aminopyridine , a blocker of delayed rectifier K + channels , which is reported to block the responses to capsaicin in rabbit coronary artery -LRB- Yeon et al. 2001 -RRB- and guinea pig ileum -LRB- Fujimoto et al. 2006 -RRB- , was ineffective in human as well as porcine distal coronary artery . Similarly , charybdotoxin , a BKCa and IKCa blocker , which blocked capsaicin responses in equine tracheal smooth muscle -LRB- Zhu et al. 1997 -RRB- , did not block these responses at similar concentrations and even in combination with apamin , a SKCa blocker . We also used the RhoA kinase inhibitor Y-27632 , but unlike in guinea pig ileum , -LRB- Fujimoto et al. 2006 -RRB- it also did not antagonise responses to capsaicin in the isolated preparations used in the present study . The capsaicin-mediated responses appear to be endothelium-independent , as denudation of the endothelium did not significantly change the capsaicin-induced responses . Further , the vehicle of capsaicin was without effect and , hence , can not account for the relaxant responses to capsaicin . Moreover , repeated administration of capsaicin in porcine coronary artery did not significantly decrease the responses , suggesting that stored neuropeptides are not responsible for the relaxations , as these would most likely be depleted after repeated challenges to capsaicin -LRB- Sams-Nielsen et al. 2001 -RRB- . Unlike in the present study , relaxations in precontracted guinea pig isolated pulmonary artery could not be repeated with capsaicin indicating depletion of neurotransmitters -LRB- Maggi et al. 1990 -RRB- . Although we did not include a positive control for the various K + blockers in the current study , in the same set up at our laboratory , relaxant responses to l-S-nitrosocysteine were blocked by the combination of charybdotoxin -LRB- 100 nM -RRB- and apamin -LRB- 100 nM -RRB- in porcine distal arteries -LRB- Batenburg et al. 2004b -RRB- . Further , the concentrations of the various inhibitors that we used in our study were equal to -LRB- Yeon et al. 2001 ; Zhu et al. 1997 -RRB- or even higher -LRB- Yeon et al. 2001 -RRB- than those employed by others . Taken together , our observations in human and porcine distal coronary artery suggest that capsaicin-induced responses are not mediated by CGRP , substance P or TRPV1 receptors and also do not involve various Ca2 + - activated K + channels . The relaxations to capsaicin are mediated by a cAMP independent pathway . The major component of capsaicin-induced relaxations , therefore , appears to be mediated by non-specific actions of capsaicin , rather than from the release of neuropeptides like CGRP .

O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O

capsaicin
cgrp
human coronary artery
human meningeal artery
porcine coronary artery


Why are mineralocorticoid receptor antagonists cardioprotective ? Two clinical trials , the Randomized ALdosterone Evaluation Study -LRB- RALES -RRB- and the EPlerenone HEart failure and SUrvival Study -LRB- EPHESUS -RRB- , have recently shown that mineralocorticoid receptor -LRB- MR -RRB- antagonists reduce mortality in patients with heart failure on top of ACE inhibition . This effect could not be attributed solely to blockade of the renal MR-mediated effects on blood pressure , and it has therefore been proposed that aldosterone , the endogenous MR agonist , also acts extrarenally , in particular in the heart . Indeed , MR are present in cardiac tissue , and possibly aldosterone synthesis occurs in the heart . This review critically addresses the following questions : -LRB- 1 -RRB- is aldosterone synthesized at cardiac tissue sites , -LRB- 2 -RRB- what agonist stimulates cardiac MR normally , and -LRB- 3 -RRB- what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure ? Conclusions are that most , if not all , of cardiac aldosterone originates in the circulation -LRB- i.e. , is of adrenal origin -RRB- , and that glucocorticoids , in addition to aldosterone , may serve as the endogenous agonist of cardiac MR. MR-mediated effects in the heart include effects on endothelial function , cardiac fibrosis and hypertrophy , oxidative stress , cardiac inotropy , coronary flow , and arrhythmias . Some of these effects occur via or in synergy with angiotensin II , and involve a non-MR-mediated mechanism . This raises the possibility that aldosterone synthase inhibitors might exert beneficial effects on top of MR blockade . Introduction The renin-angiotensin-aldosterone system -LRB- RAAS -RRB- has been viewed conventionally as a circulating system , involved in the regulation of salt , fluid homeostasis and blood pressure . Kidney-derived renin cleaves liver-derived angiotensinogen to form angiotensin -LRB- Ang -RRB- I in circulating blood -LRB- Fig. 1 -RRB- . Angiotensin-converting enzyme -LRB- ACE -RRB- , located at the luminal side of the endothelium , subsequently converts Ang I to Ang II . Ang II exerts its effects via stimulation of Ang II type 1 -LRB- AT1 -RRB- and type 2 -LRB- AT2 -RRB- receptors . Besides acting as a vasoconstrictor via AT1 receptors , Ang II also stimulates the formation of the sodium-retaining hormone aldosterone . Aldosterone mediates its cellular effects by binding to the mineralocorticoid receptor -LRB- MR -RRB- , a member of the steroid/thyroid/retinoid / orphan receptor family of transcription factors . Fig. 1The renin-angiotensin-aldosterone system . Angiotensin II activates angiotensin II type 1 and type 2 -LRB- AT1 and AT2 -RRB- receptors . AT1 receptor activation in the adrenal results in the synthesis and release of aldosterone , which subsequently exerts ` genomic ' -LRB- after hours , involving protein synthesis -RRB- or ` non-genomic ' -LRB- within minutes , not involving protein synthesis -RRB- effects through either mineralocorticoid receptors -LRB- MR -RRB- or other , as yet unidentified , receptors . MR may occur both intracellular and on the cell membrane . Aldosterone upregulates both AT1 and AT2 receptors , and AT1 receptor activation also results in MR stimulation in an aldosterone-independent manner This classic concept has been updated in the past two decades . It is now believed that some or all of the components of the RAAS are synthesized locally in tissues such as the heart and vessel wall -LRB- Danser 2003 ; Tom et al. 2003 -RRB- . For instance , in the heart , Ang II is synthesized locally by cardiac ACE following uptake of renin and angiotensinogen from the circulation -LRB- Danser et al. 1994 ; van Kats et al. 1998 -RRB- . This Ang II subsequently stimulates cardiac AT1 and AT2 receptors -LRB- Batenburg et al. 2004 ; van Kats et al. 2000 -RRB- . Local synthesis of Ang II at cardiac tissue sites is in agreement with the observation that the beneficial effects of ACE inhibitors in heart failure are , at least in part , independent of their effect on blood pressure -LRB- Yusuf et al. 2000 -RRB- . Traditionally , treatment of heart failure and hypertension has been performed mainly on the basis of the renin-angiotensin system -LRB- RAS -RRB- rather than the RAAS , with the assumption that aldosterone will be suppressed once Ang II formation is blocked . However , aldosterone formation does not stay suppressed during prolonged RAS blocking therapy . After 3 months of therapy , aldosterone levels start to rise again and they continue to do so due to `` Ang II reactivation '' or `` aldosterone escape '' -LRB- Farquharson and Struthers 2002 ; Jorde et al. 2002 -RRB- . Two clinical trials , the Randomized ALdosterone Evaluation Study -LRB- RALES -RRB- -LRB- Pitt et al. 1999 -RRB- and the EPlerenone HEart failure and SUrvival Study -LRB- EPHESUS -RRB- -LRB- Pitt et al. 2003 -RRB- , have convincingly indicated that MR antagonists reduce mortality in patients with heart failure and systolic left ventricular dysfunction post-myocardial infarction on top of ACE inhibition . In particular , a reduction in the rate of sudden death was observed . These results draw attention to the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease . The benefit of MR antagonists during RAS blockade is not yet fully understood . Their effects can not be attributed solely to blockade of the renal MR-mediated effects on blood pressure -LRB- Pitt et al. 1999 , 2003 -RRB- , and it is now generally assumed that aldosterone also acts extrarenally , in agreement with the concept of local RAAS . Indeed , MR have been demonstrated in the heart , both at the mRNA and protein level -LRB- Lombès et al. 1995 -RRB- . Importantly , the enzymes required for the synthesis of aldosterone appear to be expressed in the human heart as well -LRB- Young et al. 2001 -RRB- . Together with the fact that Ang II is capable of increasing the aldosterone levels in isolated rat hearts and blood vessels -LRB- Silvestre et al. 1998 ; Takeda et al. 1997 -RRB- , these data suggest that aldosterone , like Ang II , may be formed and act locally in the heart . This review addresses the following questions : -LRB- 1 -RRB- is aldosterone really synthesized at cardiac tissue sites , -LRB- 2 -RRB- what agonist stimulates cardiac MR , and -LRB- 3 -RRB- what effects are mediated by aldosterone/MR in the heart that could explain the beneficial effects of MR blockade in heart failure ? Aldosterone synthesis at cardiac tissue sites ? Aldosterone , a steroid that was originally discovered in 1953 , is secreted by the zona glomerulosa cells of the adrenal cortex . The kidney is the major target for adrenal aldosterone to increase sodium -LRB- and consequently water -RRB- reabsorption and potassium excretion . The production of aldosterone is regulated at two critical enzyme steps : -LRB- 1 -RRB- the formation of pregnenolone from cholesterol by the mitochondrial enzyme P450scc -LRB- side-chain cleavage -RRB- , and -LRB- 2 -RRB- the conversion of corticosterone to aldosterone by cytochrome P450 11β-hydroxylase 2 -LRB- CYP11B2 , ` aldosterone synthase ' -RRB- . Aldosterone synthesis in the adrenal cortex is regulated by Ang II , potassium and , more weakly , sodium and adrenocorticotrophic hormone . Extra-adrenal aldosterone synthesis has been proposed in heart -LRB- Silvestre et al. 1998 -RRB- , brain -LRB- Gomez-Sanchez et al. 1997 -RRB- and vessel wall -LRB- Takeda et al. 1995 , 1997 -RRB- . The rat heart expresses the steroidogenic acute regulatory -LRB- StAR -RRB- protein and aldosterone synthase , although at 100-fold lower levels than the adrenal -LRB- Casal et al. 2003 ; Silvestre et al. 1998 -RRB- . The StAR protein facilitates intramitochondrial cholesterol transfer , the rate-limiting step of steroidogenesis . Aldosterone synthase expression has also been proposed in the human heart -LRB- Tsybouleva et al. 2004 ; Young et al. 2001 -RRB- . In support of the functional importance of such expression , net release of aldosterone was observed across the human coronary vascular bed -LRB- Nakamura et al. 2004 -RRB- . However , other studies demonstrated the opposite -LRB- i.e. , cardiac aldosterone extraction -RRB- -LRB- Hayashi et al. 2003 -RRB- , whereas it was also noted that , in humans , cardiac aldosterone correlated closely with the cardiac levels of renin . Since the latter is exclusively of renal origin -LRB- i.e. , blood-derived -RRB- -LRB- Danser et al. 1994 , 1997 ; Saris et al. 2001 -RRB- , its correlation with aldosterone argues against independent aldosterone synthesis at cardiac tissue sites . Furthermore , recent careful studies in rats , paying great attention to the measurement of aldosterone in cardiac tissue , did not confirm the idea of local synthesis of aldosterone in the rat heart -LRB- Fiebeler et al. 2005 ; Gomez-Sanchez et al. 2004 -RRB- . In these studies , the cardiac aldosterone levels were much lower than previously reported by others -LRB- Silvestre et al. 1998 -RRB- . Furthermore , the cardiac levels correlated closely with the plasma levels of aldosterone , and they decreased to levels at or below the detection limit after adrenalectomy . Subsequent perfusion studies with aldosterone in the isolated Langendorff heart -LRB- Chai et al. 2006 -RRB- showed that the steroid rapidly accumulated in cardiac tissue , not only in extracellular -LRB- interstitial -RRB- fluid but also in a second , as yet unidentified , compartment . At steady state , the aldosterone tissue levels -LRB- expressed per g wet weight -RRB- were higher than its levels in coronary effluent -LRB- expressed per ml -RRB- . Washout from the second compartment occurred relatively rapid -LRB- half life < 10 min -RRB- , suggesting that it represented cell surface-bound rather than internalized aldosterone -LRB- Fig. 2 -RRB- . This pattern resembles that of cardiac renin , which also accumulates in extracellular fluid and binds to membrane receptors -LRB- Danser et al. 1994 ; de Lannoy et al. 1997 ; Saris et al. 2001 -RRB- . After prolonged washout , cardiac aldosterone became undetectable . Thus , on the one hand , the heart displays a large capacity to accumulate aldosterone . This explains why the levels of cardiac aldosterone in rats can be up to 10-fold higher than in serum -LRB- Fiebeler et al. 2005 -RRB- . On the other hand , cardiac aldosterone disappears rapidly during perfusion with aldosterone-free buffer . This provides an explanation for the ` release ' of aldosterone across the coronary vascular bed in humans and rats -LRB- Takeda et al. 2000 -RRB- . The majority of cardiac aldosterone , if not all , is however derived from the circulation , i.e. , is not synthesized locally , both under normal and pathological conditions -LRB- Chai et al. 2006 ; Fiebeler et al. 2005 ; Gomez-Sanchez et al. 2004 -RRB- . Fig. 2Washout of aldosterone from the isolated perfused rat Langendorff heart after its exposure to 10 nmol/l aldosterone for 30 min . Data are modified from Chai et al. -LRB- 2006 -RRB- Activation of cardiac mineralocorticoid receptors by aldosterone ? MRs occur both in Na + - transporting epithelia -LRB- e.g. , kidney , colon -RRB- and non-epithelial tissues such as brain -LRB- de Kloet et al. 2000 -RRB- , heart -LRB- cardiomyocytes -RRB- -LRB- Lombès et al. 1995 -RRB- , and blood vessels -LRB- endothelial and smooth muscle cells -RRB- -LRB- Lombès et al. 1992 ; Oberleithner 2005 ; Oberleithner et al. 2004 -RRB- . The presence of MR in the cardiovascular system has been confirmed both at the mRNA and protein level in animals as well as in humans -LRB- Lombès et al. 1992 -RRB- . MRs bind mineralocorticoids and glucocorticoids with equal affinity -LRB- Kd ≈ 0.5 -- 2 nM -RRB- -LRB- Arriza et al. 1987 ; Lombès et al. 1994 -RRB- . Yet , the circulating concentrations of glucocorticoids are several orders of magnitude higher than those of aldosterone -LRB- Fig. 3 -RRB- . Selectivity of aldosterone binding to MR is achieved by co-expression of 11β-hydroxysteroid dehydrogenase type 2 -LRB- 11βHSD2 -RRB- -LRB- Alzamora et al. 2000 -RRB- . This enzyme converts cortisol -LRB- the endogenous glucocorticoid in humans -RRB- and corticosterone -LRB- the endogenous glucocorticoid in rats -RRB- to their non-MR-binding metabolites cortisone and 11-dehydrocorticosterone -LRB- Fig. 3 -RRB- . In addition , the off-rate of aldosterone from the MR is five times lower than that of glucocorticoids -LRB- Lombès et al. 1994 -RRB- , and thus it is possible that MR also discriminate aldosterone from glucocorticoids , at least in part , independently of 11βHSD2 . Fig. 3Diagram illustrating activation of glucocorticoid and mineralocorticoid receptors -LRB- GR , MR -RRB- by cortisol -LRB- top -RRB- or aldosterone -LRB- bottom -RRB- . Selectivity of aldosterone binding to MR is achieved by co-expression of 11β-hydroxysteroid dehydrogenase type 2 -LRB- 11βHSD2 -RRB- . 11β-hydroxysteroid dehydrogenase type 1 -LRB- 11βHSD1 -RRB- , which acts as a reductase in vivo , reactivates cortisone to cortisol . The affinity constants refer to aldosterone-MR and cortisol-GR binding -LRB- Arriza et al. 1987 ; Hagendorf et al. 2005 ; Lombès et al. 1994 -RRB- . Note that , in the absence of 11βHSD2 , MR bind cortisol with an affinity equal to that for aldosterone In the kidney , 11βHSD2 expression is high enough to allow selective MR stimulation by aldosterone . In contrast , in the heart , the 11βHSD2 levels are almost negligible -LRB- Nagata et al. 2006 -RRB- , and it has therefore been proposed that cardiac MR are occupied by cortisol/corticosterone rather than aldosterone -LRB- Funder 2005b ; Gomez-Sanchez et al. 2004 ; Nagata et al. 2006 -RRB- . In fact , the endogenous glucocorticoid levels are high enough to keep all cardiac MR permanently occupied -LRB- Funder 2005b -RRB- . Such permanent occupation does not apply to glucocorticoid receptors , since they have a ≈ 30-fold lower affinity for cortisol/corticosterone than MR -LRB- Fig. 3 -RRB- . Glucocorticoids are assumed to act as antagonists of MR -LRB- i.e. , they exert no effect following binding to MR -RRB- in kidney -LRB- Good et al. 2002 -RRB- and heart -LRB- Qin et al. 2003 -RRB- , whereas in the vessel wall , during 11βHSD2 blockade with carbenoxolone , they act as agonists -LRB- Alzamora et al. 2000 -RRB- . The latter could relate to the observation that glucocorticoid-MR complexes become activated as a result of the generation of reactive oxygen species -LRB- ROS -RRB- . Under such circumstances , MR antagonists may exert effects by blocking the consequences of glucocorticoid-MR complex activation rather than aldosterone-MR activation -LRB- Funder 2005b ; Nagata et al. 2006 -RRB- . The increased expression in the failing rat heart of the enzyme 11β-hydroxysteroid dehydrogenase type 1 -LRB- 11βHSD1 -RRB- , which reactivates 11-dehydrocorticosterone to corticosterone , further supports this concept -LRB- Nagata et al. 2006 -RRB- . Genomic versus non-genomic effects The classical MR-mediated effects of aldosterone are referred to as ` genomic ' effects . These effects involve binding of aldosterone to intracellular MRs , and the translocation of the steroid-MR complex to the nucleus , where it acts as a transcriptional regulator , inducing protein synthesis -LRB- in particular synthesis of the epithelial Na + channel -RRB- after several hours . Genomic effects can be inhibited by agents that block either transcription -LRB- e.g. , actinomycin D -RRB- or translation -LRB- e.g. , cycloheximide -RRB- . In addition to its genomic effects , which occur after hours , aldosterone also exerts rapid effects -LRB- within minutes -RRB- in various tissues , e.g. , heart and vasculature . These effects are usually described as ` non-genomic ' , since they do not involve DNA-directed , RNA-mediated protein synthesis . For instance , aldosterone rapidly affects cardiac inotropy and facilitates both vasodilation and vasoconstriction -LRB- Barbato et al. 2002 ; Liu et al. 2003 ; Mazak et al. 2004 ; Michea et al. 2005 ; Mihailidou et al. 2004 ; Schmidt et al. 2003 -RRB- . In many -LRB- but not all -RRB- cases , these effects could not be blocked by the MR antagonist spironolactone , and therefore the existence of a novel -LRB- membrane-associated ? -RRB- aldosterone receptor has been proposed -LRB- Funder 2005a ; Wehling et al. 1995 -RRB- . However , despite numerous efforts in the past decade , no convincing data toward the characterization of a membrane receptor for aldosterone have been put forward -LRB- Funder 2005a ; Wehling 2005 -RRB- . Consequently , it is not unlikely that these effects are after all also being mediated via the classical -LRB- intracellular -RRB- MR -LRB- Funder 2005a -RRB- . If so , an explanation must be provided for the lack of effect of spironolactone toward the rapid aldosterone-induced actions in vitro . Possibly , via modification or dimerization , classical MR can attain an atypical pharmacology , thereby no longer allowing the binding of MR antagonists such as spironolactone . In addition , spironolactone exerts MR-independent effects of its own , i.e. , it blocks human Ether-a-Go-Go-Related gene K + channels -LRB- Caballero et al. 2003 -RRB- and inhibits calcium entry -LRB- Cargnelli et al. 2001 -RRB- . Thus , to solve this issue , future studies should make use of alternative MR antagonists such as eplerenone . Eplerenone displays increased selectivity for the MR over other steroid receptors , although its affinity for the MR in vitro is 10 - to 20-fold lower than that of spironolactone . The rapid , non-genomic actions involve activation of the phospholipase C-protein kinase C -LRB- PKC -RRB- - inositol 1,4,5-trisphosphate -LRB- IP3 -RRB- -1,2 diacylglycerol -LRB- DAG -RRB- pathway , which leads to an increase in intracellular Ca2 + and stimulation of the Na + / H + exchanger -LRB- Barbato et al. 2004b ; Funder 2005a ; Liu et al. 2003 ; Lösel et al. 2002 ; Mihailidou et al. 2004 ; Sato et al. 1997 -RRB- . The latter causes a rise in intracellular Na + , which subsequently activates Na + / K + - ATPase . Interestingly , however , when the increase in intracellular Na + is prevented , aldosterone decreases Na + / K + - ATPase activity in a PKC-dependent manner -LRB- Mihailidou et al. 2004 -RRB- . Thus , aldosterone may exert both positive and negative inotropic effects . Other second messenger pathways that have been linked to the rapid effects of aldosterone include mitogen-activated protein -LRB- MAP -RRB- kinases , ROS and the epidermal growth factor receptor -LRB- Jaffe and Mendelsohn 2005 ; Mazak et al. 2004 -RRB- . Effects of aldosterone in the cardiovascular system Substantial evidence has emerged showing that aldosterone induces adverse effects in the cardiovascular system . The co-expression of 11βHSD2 and MR in human heart and blood vessels -LRB- Glorioso et al. 2005 ; Jaffe and Mendelsohn 2005 ; Lombès et al. 1995 -RRB- , albeit at low levels , supports the concept that these organs possess the cellular machinery required for direct aldosterone action , irrespective of the source of aldosterone . Endothelial dysfunction Aldosterone increases the volume and stiffness of endothelial cells and induces gap formation , allowing irregular diffusion pathways for large particles -LRB- Oberleithner 2005 ; Oberleithner et al. 2004 -RRB- . This mechanism could contribute to endothelial dysfunction observed in hyperaldosteronism . The normalization of endothelial function by spironolactone in patients with heart failure supports this concept -LRB- Abiose et al. 2004 ; Macdonald et al. 2004 -RRB- . Oxidative stress , inflammation , fibrosis and atherosclerosis A growing number of studies support a specific role of the MR as a mediator of oxidative stress and subsequent inflammation , fibrosis and atherosclerosis . Elevations in circulating aldosterone are accompanied by a pro-inflammatory/fibrogenic vascular phenotype -LRB- Ahokas et al. 2005 ; Blasi et al. 2003 ; Sun et al. 2002 -RRB- , and since this phenomenon can be blocked by both spironolactone and anti-oxidants -LRB- Sun et al. 2002 -RRB- , it appears that aldosterone , via MR , induces oxidative stress . Indeed , aldosterone upregulates various subunits of NADPH oxidase and induces ROS generation in mononuclear and vascular smooth muscle cells -LRB- Ahokas et al. 2005 ; Calo et al. 2004 ; Mazak et al. 2004 ; Sun et al. 2002 -RRB- . In addition , aldosterone stimulates collagen synthesis in cardiac fibroblasts -LRB- Brilla et al. 1994 -RRB- . Consequently , aldosterone-induced cardiac fibrosis may be due to both direct effects in the heart -LRB- mediated via fibroblasts -RRB- and indirect peripheral effects -LRB- mediated via oxidative stress-activated mononuclear cells -RRB- -LRB- Ahokas et al. 2005 -RRB- . The MR antagonist eplerenone inhibited atherosclerosis both in monkeys -LRB- Takai et al. 2005 -RRB- and apolipoprotein-E deficient mice -LRB- Suzuki et al. 2006 -RRB- fed a high-cholesterol diet , most likely by attenuating oxidative stress and inflammation . Cardiac inotropy and coronary flow In the isolated perfused rat Langendorff heart , aldosterone , like Ang II , rapidly increased left ventricular pressure and decreased coronary flow -LRB- Chai et al. 2005a ; Moreau et al. 1996 -RRB- . Barbato et al. -LRB- 2002 -RRB- observed an increase in cardiac contractility in combination with an increase in coronary flow , but this may relate to the fact that the much larger positive inotropic effects in their study had favoured coronary vasodilation . Spironolactone and eplerenone did not block the inotropic and vasoconstrictor/dilator effects of aldosterone in the rat heart -LRB- Barbato et al. 2002 ; Chai et al. 2005a -RRB- . In fact , spironolactone exerted similar inotropic effects on top of aldosterone -LRB- Barbato et al. 2002 ; Chai et al. 2005a -RRB- , and thus it appears that the effects of aldosterone on inotropy and flow occur in a non-MR-mediated manner . In human myocardial trabeculae , aldosterone induced a negative inotropic response -LRB- Fig. 4 -RRB- , in contrast to the well-known positive inotropic effects of Ang II in this preparation -LRB- Chai et al. 2005b -RRB- . The PKC inhibitor chelerythrine chloride , but not spironolactone or eplerenone , blocked this negative inotropic effect , suggesting that it is mediated via a non-MR in a PKC-dependent manner . The aldosterone concentrations required to induce this effect were in the high nanomolar range , i.e. a range that occurred in failing hearts only -LRB- Chai et al. 2005b -RRB- . Hydrocortisone , but not 17β-estradiol , mimicked the effects of aldosterone , although at lower potency -LRB- Fig. 4 -RRB- . The contrast between the effects of aldosterone in the rat heart -LRB- positive inotropy -RRB- and the human heart -LRB- negative inotropy -RRB- may relate to species differences . However , there are alternative explanations . First , inotropic effects in isolated trabeculae do not necessarily parallel inotropic effects in intact hearts , since the latter also reflect responses on coronary flow -LRB- Barbato et al. 2002 -RRB- . Second , similar diametrically differing effects of aldosterone have been observed on flow , either because such effects involve different cells , or because different second messengers are activated depending on the experimental circumstances -LRB- Barbato et al. 2004b ; Chai et al. 2005a ; Mihailidou et al. 2004 ; Schmidt et al. 2003 -RRB- . Finally , the consequences of PKC-induced regulation of Na + - K + pump activity are tissue-specific , and range from stimulation to inhibition , as described above -LRB- Mihailidou et al. 2004 ; Therien and Blostein 2000 -RRB- . Fig. 4Inotropic effects of aldosterone , hydrocortisone and 17β-estradiol in human atrial trabeculae . Left panel original tracing from an experiment with aldosterone -LRB- numbers represent - log -LSB- aldosterone -RSB- in mol/L -RRB- . Right panel % change from baseline contractile force . Data have been obtained from Chai et al. -LRB- 2005b -RRB- In human coronary arteries , aldosterone exerted no constrictor or dilator effect by itself . However , prior exposure to 1 μmol/L aldosterone greatly enhanced the constrictor response to Ang II -LRB- Chai et al. 2005b -RRB- . At the second messenger level , this was reflected by an increase in the level of phosphorylated p42/p44 MAP kinase . Hydrocortisone and 17β-estradiol induced similar potentiating effects , but only in the case of aldosterone did these effects occur at the subnanomolar level , i.e. , in a physiological range . Future investigations should now address to what degree this potentiation concerns aldosterone-induced endothelial dysfunction -LRB- Oberleithner 2005 ; Oberleithner et al. 2004 -RRB- , and/or an interaction with Ang II at the level of smooth muscle cells , involving some or all of the mediators that have recently been coupled to aldosterone , e.g. , the PKC-IP3-DAG pathway , Na + / H + exchange , Na + / K + - ATPase , p38 MAP kinase , ROS and/or the epidermal growth factor receptor -LRB- Jaffe and Mendelsohn 2005 ; Liu et al. 2003 ; Mazak et al. 2004 -RRB- . Finally , the possibility of aldosterone-induced , endothelium-dependent , NO-mediated vasodilation , as proposed by several investigators -LRB- Liu et al. 2003 ; Schmidt et al. 2003 -RRB- , needs to be addressed . Arrhythmias MR blockade , in addition to standard therapy , reduced sudden death in RALES and EPHESUS -LRB- Pitt et al. 1999 , 2003 -RRB- . The mechanism responsible for this favorable effect may rely on both renal changes in electrolyte excretion and myocardial fibrosis inhibition . In addition , conditional MR overexpression in the mouse heart , in the absence of aldosteronemia , has been reported to result in severe ventricular arrhythmias -LRB- Ouvrard-Pascaud et al. 2005 -RRB- . Apparently , cardiac MR trigger arrhythmias directly , thus providing an additional mechanism through which MR antagonists reduce sudden death in patients . In support of this possibility , spironolactone improved electrophysiological parameters such as QT interval dispersion -LRB- Yee et al. 2001 -RRB- , and , in combination with the ACE inhibitor fosinopril , reduced the arrhythmic score post-myocardial infarction -LRB- Beck et al. 2001 -RRB- . Furthermore , both spironolactone and eplerenone improved the condition of the isolated perfused rat Langendorff heart following ischemia and reperfusion , as evidenced by a decrease in infarct size , a decrease in arrhythmia incidence , and an increase in left ventricular pressure recovery -LRB- Chai et al. 2005a , 2006 -RRB- -LRB- Fig. 5 -RRB- . Given the virtual lack of aldosterone in the isolated perfused rat heart , it is unlikely that these effects are due to blockade of endogenous aldosterone . In fact , concomitant exposure to 100 nmol/l aldosterone did not further deteriorate the condition of the heart during ischaemia and reperfusion -LRB- Chai et al. 2006 -RRB- . A more likely explanation of these findings is therefore that spironolactone and eplerenone had blocked MR activation by endogenous glucocorticoids . Given the 1,000-fold higher levels of corticosterone in the rat heart -LRB- Gomez-Sanchez et al. 2004 -RRB- , and assuming that the washout of glucocorticoids resembles that of aldosterone , it can be calculated that , at the time of ischaemia , sufficient glucocorticoid levels are indeed present to allow cardiac MR activation . Such activation might occur particularly under conditions which facilitate ROS generation , such as ischaemia and reperfusion -LRB- Funder 2005b ; Nagata et al. 2006 -RRB- . Interestingly in this regard , epidemiological observations have recently shown that high-dose corticosteroids increase the risk of developing atrial fibrillation -LRB- van der Hooft et al. 2006 -RRB- . The cardioprotective effect of MR antagonism in the Langendorff heart during ischemia and reperfusion can not be explained on the basis of the vasoconstrictor effect of aldosterone , as proposed by Fujita et al. -LRB- 2005 -RRB- , since neither spironolactone nor eplerenone are capable of blocking aldosterone-induced vasoconstriction in vitro -LRB- Chai et al. 2005a , 2006 -RRB- . Fig. 5Infarct size -LRB- left panel -RRB- , recovery of left ventricular pressure -LRB- LVP -RRB- -LRB- middle panel -RRB- , and incidence of arrhythmias -LRB- right panel -RRB- in rat hearts that were subjected to 45 min left anterior descending coronary artery occlusion , followed by 3 h of reperfusion , after either no pretreatment -LRB- C , control -RRB- or a 15-min exposure to 100 nmol/l aldosterone -LRB- A -RRB- , 1 μmol/l spironolactone -LRB- S -RRB- , 1 μmol/l eplerenone -LRB- E -RRB- or 100 nmol/l aldosterone + 1 μmol/L eplerenone -LRB- A + E -RRB- . Data are from Chai et al. -LRB- 2005a , 2006 -RRB- . * P < 0.05 versus control Cardiac hypertrophy Serum aldosterone levels associate with the variability of left ventricular mass -LRB- LVM -RRB- in both healthy controls and subjects with hypertension -LRB- Schunkert et al. 1997 -RRB- . The CYP11B2 C-344T polymorphism associates with circulating aldosterone levels , subjects with the T allele having higher aldosterone levels than those with the CC genotype -LRB- Barbato et al. 2004a ; Brand et al. 1998 ; Hautanena et al. 1998 ; Schunkert et al. 1999 ; Stella et al. 2004 -RRB- . Given the association between circulating aldosterone and LVM , it is not surprising that the T allele also associates with LVM , both in subjects with hypertension -LRB- Stella et al. 2004 -RRB- and in patients with hypertrophic cardiomyopathy -LRB- Chai et al. 2005c -RRB- . Interestingly , urinary 11β-HSD2 activity correlated directly with LVM in essential hypertension -LRB- Glorioso et al. 2005 -RRB- . This suggests that glucocorticoids also take part in the regulation of LVM . Furthermore , two independent investigations have shown that the CYP11B2 C-344T polymorphism is in strong linkage disequilibrium with polymorphisms of the nearby CYP11B1 gene -LRB- Ganapathipillai et al. 2005 ; Keavney et al. 2005 -RRB- . Since CYP11B1 -LRB- 11β-hydroxylase -RRB- is the enzyme catalyzing the final step in the biosynthesis of cortisol , the association with the C-344T polymorphism might not only relate to increased aldosterone levels , but also to reduced 11β-hydroxylase activity -LRB- Hilgers and Schmidt 2005 -RRB- . Future studies , involving aldosterone , 11-deoxycortisol and cortisol measurements in serum and/or urine -LRB- White and Rainey 2005 -RRB- , should address this possibility . Interaction with angiotensin II Ang II stimulates the synthesis and release of aldosterone in the adrenal , and thus MR blockade and/or aldosterone synthase inhibition will exert beneficial effects in Ang II-dependent models -LRB- Fiebeler et al. 2005 ; Virdis et al. 2002 -RRB- . Remarkably , however , aldosterone also appears to exert its effects , at least in part , via Ang II -LRB- or its receptors -RRB- , and both agonists , when applied together , act synergistically -LRB- Gonzalez et al. 2005 ; Mazak et al. 2004 ; Xiao et al. 2004 -RRB- -LRB- Fig. 1 -RRB- . For instance , in cultured rat aortic smooth muscle cells , nanomolar concentrations of aldosterone enhanced the effect of Ang II on DNA synthesis -LRB- Chai et al. 2005a -RRB- . Unexpectedly , higher -LRB- micromolar -RRB- aldosterone concentrations reduced DNA synthesis , both in smooth muscle cells and in cardiac myocytes . Such high aldosterone concentrations also reduced collagen synthesis in cardiac fibroblasts . These latter inhibitory effects most likely reflect glucocorticoid receptor activation by aldosterone -LRB- Arriza et al. 1987 ; Sato and Funder 1996 -RRB- . Furthermore , in human coronary artery smooth muscle cells , Ang II stimulated MR-mediated gene expression in an aldosterone-independent manner , suggesting direct MR activation by post-translational modifications such as phosphorylation -LRB- Jaffe and Mendelsohn 2005 -RRB- . Clinical perspective : why are MR antagonists cardioprotective ? The beneficial effects of MR antagonists in heart failure can not be explained on the basis of their renal and/or blood pressure-lowering effects . Most likely they relate , at least in part , to blockade of a wide range of MR-mediated effects in the heart , including endothelial dysfunction , a decrease in cardiac inotropy and coronary flow , and the induction of fibrosis , hypertrophy , oxidative stress and arrhythmias . Some of these effects occur via or in synergy with Ang II . Given the virtual absence of aldosterone production in the heart , and in view of the low cardiac levels of the cortisol-inactivating enzyme 11β-HSD2 , it is unlikely that , under all conditions , aldosterone is the endogenous agonist of cardiac MR. Thus , some of the MR-mediated effects in the heart may be due to MR activation by glucocorticoids , particularly when ROS levels are high . The aldosterone levels in the failing human heart , but not those in the healthy heart , are high enough to be of functional importance . Whether some of the above -LRB- acute , ` non-genomic ' -RRB- in vitro effects of aldosterone are mediated via a non-MR-mediated mechanism remains to be proven , in particular because no -LRB- membrane receptor -RRB- candidate has been identified so far that induces these effects . If true , however , aldosterone synthase inhibitors might be expected to exert beneficial effects on top of MR blockade .

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O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O

aldosterone
mineralocorticoid receptor
cortisol
inotropy
arrhythmia