bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of neuroeffector transmission in human vas deferens by sildenafil
Authors: Medina Pascual , Segarra Gloria , Torondel Belén , Chuan Pascual , Domenech Cristina , Vila José M , Lluch Salvador
Published: January 2005

Sildenafil (0.1–30 μM), a cyclic GMP phosphodiesterase 5 (PDE 5) inhibitor, induced inhibition of electrically evoked contractions of ring segments of human vas deferens from 34 vasectomies. Zaprinast (0.1–100 μM), another PDE 5 inhibitor, and the nitric oxide (NO) donor sodium nitroprusside (SNP) (0.1–100 μM) had no effect on neurogenic contractions. The inhibition induced by sildenafil was not modified by the inhibitor of guanylate cyclase 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxaline‐1‐one (ODQ) (1–30 μM) but it was abolished by the K+ channel blockers tetraethylammonium (TEA, 1 mM), iberiotoxin (0.1 μM) and charybdotoxin (0.1 μM). Sildenafil, zaprinast and SNP did not affect the contractions induced by noradrenaline. SNP (10 μM) caused elevation of cyclic GMP levels that was potentiated by sildenafil (10 μM) and zaprinast (100 μM). ODQ (10 μM) inhibited the increase in cyclic GMP. Sildenafil inhibits adrenergic neurotransmission in human vas deferens. The inhibition is not related to accumulation of cyclic GMP but is probably due to activation of prejunctional large‐conductance Ca2+‐activated K+ channels.

British Journal of Pharmacology (2000) 131, 871–874; doi:10.1038/sj.bjp.0703657
Pharmacological examination of contractile responses of the guinea‐pig isolated ileum produced by μ‐opioid receptor antagonists in the presence of, and following exposure to, morphine
Authors: Mundey M K , Ali A , Mason R , Wilson V G
Published: January 2005

We have assessed the potential of several μ‐opioid receptor antagonists to elicit a response in the guinea‐pig isolated ileum in the presence of, and following overnight exposure to, morphine.

Naloxone, D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP), (−)‐5,9α‐diethyl‐2‐(3‐furyl‐methyl)‐2′‐hydroxy‐6,7‐benzomorphan (MR2266), but not D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2 (CTAP), produced a transient inhibition of electrically‐evoked contractions of the guinea‐pig ileum. The effect of 1 μM CTOP, but not that to MR2266, was inhibited by 1 μM somatostatin.

Naloxone (0.3 μM), CTOP (3 μM), CTAP (3 μM) and MR2266 (0.3 μM) antagonized the inhibitory effect of morphine on electrically‐evoked contractions of the guinea‐pig to a similar degree and, following 60 min exposure to morphine, produced non‐sustained contractions. The response to 3 μM CTOP was significantly smaller than that to 3 μM CTAP. None of the antagonists produced a response in the absence of morphine.

Following overnight exposure of the ileum to 0.3 μM morphine (4°C), and repeated washing to remove the agonist, all four antagonists elicited non‐sustained contractions. However, the responses to 3 μM CTOP and 0.3 μM MR2266 were significantly smaller than those elicited by 0.3 μM naloxone and 3 μM CTAP. Somatostatin (1 μM) significantly reduced naloxone‐induced contractions, but not those to CTAP.

While all four μ‐opioid antagonists elicited contractions in the presence of, and following prolonged exposure to, morphine, differences between them were noted which may be a consequence of non‐opioid actions.

British Journal of Pharmacology (2000) 131, 893–902; doi:10.1038/sj.bjp.0703659
Levamisole induced apoptosis in cultured vascular endothelial cells
Authors: Artwohl Michaela , Hölzenbein Thomas , Wagner Ludwig , Freudenthaler Angelika , Waldhäusl Werner , Baumgartner‐Parzer Sabina M
Published: January 2005

To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5‐Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro‐ and macrovascular endothelial cells (ECs) and fibroblasts.

Cells exposed (24 h) to Levamisole (range: 0.5–2 mmol l−1) alone or in combination with antioxidants (10 mmol l−1 glutathione or 5 mmol l−1 N‐Acetylcysteine or 0.1 mmol l−1 Tocopherol) were evaluated for apoptosis (3H‐thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation (3H‐thymidine incorporation).

Levamisole dose‐dependently increased apoptosis in ECs to 230% (HUVECs‐human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%).

Levamisole increased in ECs integrin‐dependent matrix adhesion, inhibited proliferation (−70%), reduced expression of survival factors such as clusterin (−30%), endothelin‐1 (−43%), bcl‐2 (−34%), endothelial NO‐synthase (−32%) and pRb (Retinoblastoma protein: −89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%).

LMS (2 mmol l−1)‐induced apoptosis was inhibited by glutathione (−50%) and N‐Acetylcysteine (−36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes.

The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity.

British Journal of Pharmacology (2000) 131, 1577–1583; doi:10.1038/sj.bjp.0703660
(8‐Naphthalen‐1‐ylmethyl‐4‐oxo‐1‐phenyl‐1,3,8‐triaza‐spiro[4.5]dec‐3‐yl)‐acetic acid methyl ester (NNC 63‐0532) is a novel potent nociceptin receptor agonist
Authors: Thomsen Christian , Hohlweg Rolf
Published: January 2005

Spiroxatrine was identified as a moderately potent (Ki=118 nM) but non‐selective agonist at the human nociceptin/orphanin FQ receptor, ORL1. This compound was subject to chemical modification and one of the resulting compounds, (8‐naphthalen‐1‐ylmethyl‐4‐oxo‐1‐phenyl‐1,3,8‐triaza‐spiro[4.5]dec‐3‐yl)‐acetic acid methyl ester (NNC 63‐0532) was shown to have high affinity for ORL1 (Ki=7.3 nM).

NNC 63‐0532 showed only moderate affinity for the following receptors (Ki values in parentheses): μ‐opioid (140 nM), κ‐opioid (405 nM), dopamine D2S (209 nM), dopamine D3 (133 nM) and dopamine D4.4 (107 nM) out of 75 different receptors, ion‐channels and transporters.

In functional assays, NNC 63‐0532 was shown to be an agonist at ORL1 (EC50=305 nM), a much weaker agonist at the μ‐opioid receptor (EC50>10 μM) and an antagonist or weak partial agonist at dopamine D2S (IC50=2830 nM).

Thus, NNC 63‐0532 is a novel non‐peptide agonist with ∼12 fold selectivity for ORL1 and may be useful for exploring the physiological roles of this receptor owing to its brain‐penetrating properties.

British Journal of Pharmacology (2000) 131, 903–908; doi:10.1038/sj.bjp.0703661
Pharmacological profile of SL 59.1227, a novel inhibitor of the sodium/hydrogen exchanger
Authors: Lorrain Janine , Briand Véronique , Favennec Evelyne , Duval Nicole , Grosset Alain , Janiak Philip , Hoornaert Christian , Cremer Gérard , Latham Christopher , O'Connor Stephen E
Published: January 2005

The NHE1 isoform of the Na+/H+ exchanger plays an important role in the regulation of intracellular pH and in cardiac cell injury caused by ischaemia and reperfusion. SL 59.1227 is a novel imidazolypiperidine Na+/H+ antiport inhibitor which is structurally unrelated to previously described acylguanidine inhibitors such as cariporide.

Recovery of pHi following an intracellular acid load was measured in CCL39‐derived PS120 variant cells, selectively expressing either NHE1 or NHE2 isoforms of the Na+/H+ exchanger. pHi recovery was potently and selectively slowed by SL 59.1227 in NHE1‐expressing cells (IC50 3.3±1.3 nM) versus NHE2‐expressing cells (2.3±1.0 μM). The respective IC50 values for cariporide were 103±28 nM (NHE1) and 73±46 μM (NHE2).

In anaesthetized rats following left coronary artery occlusion (7 min) and reperfusion (10 min) SL 59.1227 (10–100 μg kg−1 min−1 i.v.) inhibited ischaemia‐mediated ventricular tachycardia (71–100%) and reperfusion‐induced ventricular fibrillation (75–87%) and prevented mortality. Bolus i.v. administration of SL 59.1227 (1 mg kg−1) produced anti‐arrhythmic effects when administered either before or during ischaemia.

Cardiac infarct size was determined in anaesthetized rabbits following left coronary artery occlusion (30 min) and reperfusion (120 min). Infarct size measured as a percentage of the area at risk was 36.2±3.4% (control group) versus 15.3±3.9% (SL 59.1227 0.6 mg kg−1 i.v.).

SL 59.1227 is the first example of a potent and NHE1‐selective non‐acylguanidine Na+/H+ exchanger inhibitor. It possesses marked cardioprotective properties.

British Journal of Pharmacology (2000) 131, 1188–1194; doi:10.1038/sj.bjp.0703671
Lack of involvement of endothelin‐1 in angiotensin II‐induced contraction of the isolated rat tail artery
Authors: Jiang Yanfen , Triggle Christopher R
Published: January 2005

The contribution of endothelin‐1 (ET‐1) to angiotensin II (Ang II)‐mediated contraction of the isolated rat tail artery was assessed with measurements of tension, and cytosolic calcium ([Ca2+]i). The distribution of the AT1 receptor was studied with RT–PCR and immunohistochemistry.

Ang II induced an endothelium‐independent contraction (pEC50 7.95±0.06 and Emax: 0.46 g±0.05 with endothelium vs 7.81±0.02 and 0.41 g±0.07 without endothelium; P>0.05). Ang II (0.003–0.3 μM)‐induced a non‐sustained contraction of endothelium‐intact preparations which was not antagonized by BQ‐123 (1 μM), but was inhibited by losartan (10 nM). In addition, the maximal contraction induced by ET‐1 (0.1 μM) could be further increased by the addition of 0.1 μM Ang II.

Ang II (0.001–0.3 μM) elevated [Ca2+]i in single vascular smooth muscle cells (VSMCs) in a dose‐dependent manner (pEC50 9.12±0.26) and the Ang II‐induced increases in [Ca2+]i were not affected by a Ca2+‐free solution, but were abolished by pretreatment with caffeine (5 mM). Ang II did not increase [Ca2+]i in endothelial cells. ET‐1 (0.1 μM) increased [Ca2+]i in single VSMCs in a normal Ca2+ containing physiological saline solution (PSS), but not in a Ca2+‐free solution.

Ang II‐induced contraction was insensitive to inhibition by nifedipine (0.1 μM), an antagonist of L‐type voltage‐gated Ca2+ channels, and SK&F96365 (10 μM), which blocks non‐selective cation channels, whereas that to ET‐1 was inhibited by SK&F69365.

RT–PCR data indicate the expression of AT1A and AT1B on both VSMCs and endothelial cells, but immunohistochemical evidence illustrates that the AT1 is located primarily on VSMCs.

These results indicate that endothelium‐derived ET‐1 is not involved in the Ang II‐mediated vasoconstriction of the rat tail artery and that Ang II‐ and ET‐1‐mediated VSM contractions utilize distinct pathways.

British Journal of Pharmacology (2000) 131, 1055–1064; doi:10.1038/sj.bjp.0703674
Modulation of histamine H2 receptor signalling by G‐protein‐coupled receptor kinase 2 and 3
Authors: Rodriguez‐Pena Maria Sol , Timmerman Henk , Leurs Rob
Published: January 2005

To evaluate the role of G‐protein‐coupled receptor kinases (GRK) in the desensitization of the histamine H2 receptor, the H2 receptor was transiently cotransfected with GRK2, 3, 5 or 6 in COS‐7 cells and the cyclic AMP levels in response to histamine were studied.

Coexpression of the H2 receptor with GRK2 and 3 significantly decreased both the basal cyclic AMP levels and the cyclic AMP response to 100 μM histamine. Moreover, preincubation with 100 μM histamine desensitized the H2 receptor response to 53±8%. Coexpression of GRK2 and 3 increased the H2 receptor desensitization to 27±4% and 24±4% respectively. No effect on either cyclic AMP response or desensitization was found when GRK5, GRK6 or dominant negative mutants of GRK2 or 3 (GRK2K220R and GRK3K220R) were coexpressed.

To study the role of the C‐terminal tail in the GRK‐mediated desensitization of the H2 receptor, three truncations of C‐tail were constructed: H2T295, H2T307 and H2T341. H2T307 and 341 H2T341 expressed and responded normally to 100 μM histamine. The interaction of the H2 receptor with GRK2 and 3 was also not altered upon truncation of the C‐terminal tail.

These findings strongly suggest a role of GRK2 and 3 in the desensitization of the H2 receptor. Furthermore, the finding that C‐terminal truncations of the H2 receptor did not abolish the effect of GRK2 and 3 suggests that the C‐terminus is not involved in the GRK mediated desensitization of the histamine H2 receptor.

British Journal of Pharmacology (2000) 131, 1707–1715; doi:10.1038/sj.bjp.0703676
Possible involvement of endothelium‐derived hyperpolarizing factor (EDHF) in the depressor responses to platelet activating factor (PAF) in rats
Authors: Tanaka Yoshio , Hayakawa Sachiko , Imai Toshiyasu , Akutsu Aya , Hirano Haruko , Tanaka Hikaru , Nakahara Tsutomu , Ishii Kunio , Shigenobu Koki
Published: January 2005

In anaesthetized rats, platelet activating factor (PAF; 1 μg kg−1) decreased mean arterial blood pressure by around 60 mmHg (n=18). This depressor response was completely blocked by the PAF antagonist, CV‐6209 (1 mg kg−1), indicating the role of PAF‐specific receptor in the response.

NG‐nitro‐L‐arginine methyl ester (L‐NAME; 50 mg kg−1), an NO synthase inhibitor, profoundly elevated systemic blood pressure (n=19), indicating an important role of NO in the basal blood pressure regulation. The depressor response to PAF (1 μg kg−1) normalized against that to sodium nitroprusside (SNP) (10 μg kg−1) was not substantially different between rats treated without and with L‐NAME (n=4). In contrast, the depressor effect of acetylcholine (0.03–1.0 μg kg−1) normalized against that of SNP (10 μg kg−1) was significantly attenuated by L‐NAME (n=5).

Charybdotoxin (0.4 mg kg−1) plus apamin (0.2 mg kg−1) significantly attenuated the depressor response to PAF (1 μg kg−1) (n=5) without affecting the blood pressure change due to SNP (1 mg kg−1) (n=3). Charybdotoxin (0.4 mg kg−1) (n=4) or apamin (0.2 mg kg−1) (n=4) alone did not affect the PAF‐induced depressor response.

These findings suggest that EDHF may make a significant contribution to the depressor response to PAF in rats. Although NO plays the determinant role in the basal blood pressure regulation, its contribution to PAF‐produced depressor response seems to be less as compared with that to the depressor response to acetylcholine.

British Journal of Pharmacology (2000) 131, 1113–1120; doi:10.1038/sj.bjp.0703681
The human GABAB1b and GABAB2 heterodimeric recombinant receptor shows low sensitivity to phaclofen and saclofen
Authors: Wood M D , Murkitt K L , Rice S Q , Testa T , Punia P K , Stammers M , Jenkins O , Elshourbagy N A , Shabon U , Taylor S J , Gager T L , Minton J , Hirst W D , Price G W , Pangalos M
Published: January 2005

The aim of this study was to characterize the pharmacological profile of the GABAB1/GABAB2 heterodimeric receptor expressed in Chinese hamster ovary (CHO) cells. We have compared receptor binding affinity and functional activity for a series of agonists and antagonists.

The chimeric G‐protein, Gqi5, was used to couple receptor activation to increases in intracellular calcium for functional studies on the Fluorimetric Imaging Plate Reader (FLIPR), using a stable GABAB1/GABAB2/Gqi5 CHO cell line. [3H]‐CGP‐54626 was used in radioligand binding studies in membranes prepared from the same cell line.

The pharmacological profile of the recombinant GABAB1/B2 receptor was consistent with that of native GABAB receptors in that it was activated by GABA and baclofen and inhibited by CGP‐54626A and SCH 50911.

Unlike native receptors, the GABAB1/GABAB2/Gqi5 response was not inhibited by high microMolar concentration of phaclofen, saclofen or CGP 35348.

This raises the possibility that the GABAB1/GABAB2/Gqi5 recombinant receptor may represent the previously described GABAB receptor subtype which is relatively resistant to inhibition by phaclofen.

British Journal of Pharmacology (2000) 131, 1050–1054; doi:10.1038/sj.bjp.0703682
Hyperpolarization‐induced dilatation of submucosal arterioles in the guinea‐pig ileum
Authors: Imaeda Kenro , Yamamoto Yoshimichi , Fukuta Hiroyasu , Koshita Makoto , Suzuki Hikaru
Published: January 2005

The effects of inhibition of acetylcholine (ACh)‐induced hyperpolarization on dilatation of submucosal arterioles were investigated in the guinea‐pig ileum.

In smooth muscles of the arterioles depolarized by Ba2+ (0.5 mM) to about −40 mV, ACh (3 μM) repolarized the membrane to about −65 mV (hyperpolarization), irrespective of the absence or presence of L‐Nω‐nitroarginine (L‐NOARG, 0.1 mM) and diclofenac (1 μM), and increased the diameter (dilatation).

Combined application of charybdotoxin (CTX, 50 nM) and apamin (0.1 μM), inhibitors of some types of K+‐channels, abolished the ACh‐induced hyperpolarization and dilatation.

18β‐Glycerrhetinic acid (18β‐GA, 30 μM), a known inhibitor of gap junctions, depolarized the membrane to about −36 mV, either in the absence or in the presence of Ba2+, with no associated contraction of the arterioles. In the presence of 18β‐GA, ACh‐induced hyperpolarization was abolished, however the dilatation was inhibited only partially, with associated inhibition of constriction produced by Ba2+ and NA.

18β‐GA inhibited the dilatation produced by sodium nitroprusside, an NO donor.

The ACh‐induced hyperpolarization and dilatation were abolished in the presence of 2‐aminoethoxydiphenyl borate (30 μM), an inhibitory modulator of inositol trisphosphate receptor‐mediated Ca2+ release from intracellular stores.

It is concluded that in submucosal arterioles, hyperpolarizations produced by ACh have causal relationship to the arteriolar dilatation. 18β‐GA did not induce parallel relationship between hyperpolarization and dilatation produced by ACh. 18β‐GA may have unidentified inhibitory effects on agonist‐mediated actions, in addition to the inhibition of gap junctions.

British Journal of Pharmacology (2000) 131, 1121–1128; doi:10.1038/sj.bjp.0703689
Differential vasoconstrictor activity of human urotensin‐II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey
Authors: Douglas Stephen A , Sulpizio Anthony C , Piercy Valerie , Sarau Henry M , Ames Robert S , Aiyar Nambi V , Ohlstein Eliot H , Willette Robert N
Published: January 2005

Urotensin‐II (U‐II) and its G‐protein‐coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys).

The vasoconstrictor activity of human U‐II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: −log[EC50]s 9.09±0.19 and 8.84±0.21, Rmaxs 143±21 and 67±26% 60 mM KCl, respectively (compared, for example, to −log[EC50] 7.90±0.11 and Rmax 142±12% 60 mM KCl for endothelin‐1 [ET‐1] in thoracic aortae). Responses were, however, absent in mice aortae (−log[EC50] <6.50). These findings were further contrasted by the observation that U‐II was a ‘coronary‐selective’ spasmogen in the dog (−log[EC50] 9.46±0.11, Rmax 109±23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (−log[EC50]s range from 8.96±0.15 to 9.92±0.13, Rmaxs from 43±16 to 527±135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET‐1 consistently).

Thus, human U‐II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U‐II influences the physiological regulation of mammalian cardiovascular function.

British Journal of Pharmacology (2000) 131, 1262–1274; doi:10.1038/sj.bjp.0703690
Effect of endothelin antagonists, including the novel ETA receptor antagonist LBL 031, on endothelin‐1 and lipopolysaccharide‐induced microvascular leakage in rat airways
Authors: Hele Dave J , Birrell Mark A , Webber Stephen E , Foster Martyn L , Belvisi Maria G
Published: January 2005

The effect of the novel ETA receptor antagonist LBL 031 and other selective and mixed endothelin receptor antagonists on endothelin‐1 (ET‐1)‐induced and lipopolysaccharide (LPS)‐induced microvascular leakage was assessed in rat airways

Intravenously administered ET‐1 (1 nmole kg−1) or LPS (30 mg kg−1) caused a significant increase in microvascular leakage in rat airways when compared to vehicle treated animals.

Pre‐treatment with the selective ETA receptor antagonists, LBL 031 or PD 156707, or the mixed ETA/B receptor antagonist, bosentan (each at 30 mg kg−1), reduced ET‐1‐induced leakage to baseline levels. ET‐1‐induced leakage was not reduced by pre‐treatment with the ETB selective antagonist BQ 788 (3 mg kg−1).

Pre‐treatment with the selective ETA receptor antagonist, LBL 031 (0.1 mg kg−1) or PD 156707 (10 mg kg−1), or the mixed ETA/B receptor antagonist, bosentan (30 mg kg−1), reduced LPS‐induced leakage by 54, 48 and 59% respectively. LPS‐induced leakage was not affected by pre‐treatment with the ETB selective antagonist BQ 788 (3 mg kg−1).

The data suggests that ET‐1‐induced microvascular leakage in the rat airway is ETA receptor mediated and that part of the increase induced by LPS may be due to the actions of ET‐1. Therefore, a potent ETA receptor selective antagonist, such as LBL 031, may provide a suitable treatment for inflammatory diseases of the airways, especially those involving LPS and having an exudative phase, such as the septic shock‐induced adult respiratory distress syndrome.

British Journal of Pharmacology (2000) 131, 1129–1134; doi:10.1038/sj.bjp.0703691
Effects of chronic and acute aminoguanidine treatment on tail artery vasomotion in ageing rats
Authors: Tabernero Antonia , Nadaud Sophie , Corman Bruno , Atkinson Jeffrey , Capdeville‐Atkinson Christine
Published: January 2005

This study was designed to evaluate the effects of aminoguanidine, a selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), on the reactivity and intracellular calcium ([Ca2+]i) mobilization induced by noradrenaline in the perfused tail artery from aged WAG/Rij rats. Global mean internal diameter was 350±15 microns and wall thickness 161±3 microns. The influence of the endothelium on these responses was also analysed. The intracellular dye fura‐2 for [Ca2+]i measurements was used.

Noradrenaline‐induced vasoconstriction decreased progressively from 3 to 20 and 30 months. Removal of the endothelium attenuated vasoconstriction in 20 and 30 month‐old rats (P<0.05) but not in young rats.

Chronic administration of aminoguanidine (50 mg kg−1 day−1, p.o.) to WAG/Rij rats from 20 to 30 months enhanced (P<0.01) the [Ca2+]i‐sensitivity of noradrenaline‐induced vasoconstriction.

Aminoguanidine (300 μM) in vitro significantly shifted the concentration‐vasoconstriction curve to noradrenaline to the left (P<0.01) in denuded vessels from both 20 and 30 month‐old rats. The acute inhibitory effect of aminoguanidine was also observed after chronic aminoguanidine treatment. Aminoguanidine failed to modify vasoconstriction in the presence of the endothelium.

Acute aminoguanidine (300 μM) treatment did not modify vasoconstriction induced by noradrenaline in young rats.

Quantification of iNOS mRNA expression in tail arteries from 3 and 20 month‐old WAG/Rij rats showed that expression was enhanced (×2.1, P<0.01) with age.

These results suggest that an inflammatory process develops in the media of the rat tail artery with age and that the subsequent increase in non‐endothelial iNOS activity attenuates noradrenaline‐induced vasoconstriction.

British Journal of Pharmacology (2000) 131, 1227–1235; doi:10.1038/sj.bjp.0703696
Chamber‐specific alterations of noradrenaline uptake (uptake1) in right ventricles of monocrotaline‐treated rats
Authors: Leineweber Kirsten , Seyfarth Torsten , Brodde Otto‐Erich
Published: January 2005

In rats a single injection of the alkaloid monocrotaline (60 mg MCT kg−1 body weight, i.p.) caused right ventricular hypertrophy and heart failure. The aim of this study was to find out whether, in these MCT‐treated rats, the cardiac neuronal noradrenaline uptake (uptake1) might undergo chamber‐specific alterations.

For this purpose we assessed in right and left ventricular slices, uptake1 activity (by [3H]‐noradrenaline accumulation), and in right and left ventricular membranes, uptake1 carrier protein density (by [3H]‐nisoxetine binding).

Uptake1‐inhibitors blocked [3H]‐noradrenaline accumulation in ventricular slices and [3H]‐nisoxetine binding in ventricular membranes with the order of potency: desipramine>nisoxetine>>cocaineGBR 12909, indicating that with both approaches noradrenaline uptake1 was determined.

In right ventricular slices of MCT‐treated rats uptake1 activity was significantly lower than in control rats (84.7±8.2 vs 145.1±6.2 pmol noradrenaline mg−1 tissue 15 min−1; P<0.05). This was accompanied by a significant decrease in the density of [3H]‐nisoxetine binding sites (73.7±14.4 vs 125.9±9.1 fmol mg−1 protein; P<0.05).

In left ventricular slices of MCT‐treated rats uptake1 activity was not significantly altered (131.2±10.5 vs 116.1±5.2 pmol noradrenaline mg−1 tissue 15 min−1); similarly, also the density of [3H]‐nisoxetine binding sites was unchanged (108±9.7 vs 123±7.7 fmol mg−1 protein).

We conclude that in MCT‐treated rats with right ventricular hypertrophy and heart failure uptake1 activity is chamber‐specifically reduced possibly due to a decrease in carrier protein density.

British Journal of Pharmacology (2000) 131, 1438–1444; doi:10.1038/sj.bjp.0703698
S‐Nitrosothiols cause prolonged, nitric oxide‐mediated relaxation in human saphenous vein and internal mammary artery: therapeutic potential in bypass surgery
Authors: Sogo Naoki , Campanella Ciro , Webb David J , Megson Ian L
Published: January 2005

Reduced endothelial nitric oxide (NO) production in conduit vessels for coronary artery bypass grafting (CABG) has been implicated in post‐operative complications, including spasm.

The brief effects of existing NO donors limits their applicability to improving patency of graft vessels. RIG200 is a novel S‐nitrosothiol that might have advantages over conventional drugs because it has sustained effects in areas of endothelial damage.

Here we tested the hypothesis that RIG200 and S‐nitrosoglutathione (GSNO) have prolonged, NO‐mediated effects in human saphenous vein (SV) and internal mammary artery (IMA), compared with glyceryl trinitrate (GTN) and sodium nitroprusside (SNP).

84 SV and 80 IMA rings from 64 patients undergoing CABG were studied in vitro. Rings were precontracted with phenylephrine (EC80 concentration) and the functional integrity of the endothelium tested with acetylcholine (10 μM).

Relaxation of precontracted SV and IMA rings to GTN and SNP (0.01–10 μM) generally recovered fully on washout. In contrast, responses to RIG200 and GSNO were sustained during washout (30 min). Sustained relaxation was reversed by the NO scavenger, ferrohaemoglobin (10 μM) but not by the NO synthase inhibitor, Nω‐nitro‐L‐arginine methyl ester (100 and 250 μM in SV and IMA respectively).

Pretreatment (30 min) of SV with both S‐nitrosothiols (10 μM) inhibited phenylephrine‐induced contraction for >180 min, compared with <90 min for GTN. In IMA, contractility was suppressed to 49±4% (GSNO) and 26±4% (RIG200) of baseline after 240 min washout.

Pretreatment of bypass conduits with S‐nitrosothiols might improve their patency in the early post‐operative period.

British Journal of Pharmacology (2000) 131, 1236–1244; doi:10.1038/sj.bjp.0703700
Pertussis toxin‐sensitive Gi‐proteins and intracellular calcium sensitivity of vasoconstriction in the intact rat tail artery
Authors: Spitzbarth‐Régrigny E , Petitcolin M ‐A , Bueb J ‐L , Tschirhart E J , Atkinson J , Capdeville‐Atkinson C
Published: January 2005

We studied the involvement of pertussis toxin (PTX)‐sensitive G‐proteins in the sensitivity of arterial constriction to intracellular calcium ([Ca2+]i) mobilization.

Vasoconstriction was measured in vitro in perfused, de‐endothelialized rat tail arteries loaed with the calcium‐sensitive dye, fura‐2 and treated or not with PTX (30–1000 ng ml−1). Arteries were stimulated with noradrenaline (NA, 0.1–100 μM) or KCl (15–120 mM).

KCl elicited a smaller vasoconstrictor response (Emax=94±8 mmHg) than NA (Emax=198±9 mmHg) although [Ca2+]i mobilization was similar (Emax=123±8 and 135±7 nM for KCl and NA, respectively). PTX (1000 ng ml−1) had no effect on [Ca2+]i mobilization but lowered NA‐ (but not KCl‐) induced vasoconstriction (Emax=118±7 mmHg).

Gi/o‐proteins were revealed by immunoblotting with anti‐Giα and anti‐Goα antibodies in membranes prepared from de‐endothelialized tail arteries. [α32P]‐ADP‐ribosylation of G‐proteins by PTX (1000 ng ml−1) was demonstrated in the intact rat tail artery (pixels in the absence of PTX: 3150, presence: 25053).

In conclusion, we suggest that smooth muscle cells possess a PTX‐sensitive Gi‐protein‐mediated intracellular pathway which amplifies [Ca2+]i sensitivity of contraction in the presence of agonists such as NA.

British Journal of Pharmacology (2000) 131, 1337–1344; doi:10.1038/sj.bjp.0703703
Effects of castration on contraction and α1‐adrenoceptor expression in rat prostate
Authors: Homma Yukio , Hamada Kozo , Nakayama Yasuhisa , Tsujimoto Gozoh , Kawabe Kazuki
Published: January 2005

The prostate function is regulated by androgens and α‐adrenergic activity. Clinically, antiandrogens and/or α1‐adrenergic antagonists are commonly used to treat symptomatic prostatic hypertrophy. To elucidate the effects of androgen deprivation on prostate contractility via α1‐adrenoceptor, the characteristics and expression of α1‐adrenoceptors were examined in castrated rats.

Isolated prostate strips from intact and castrated rats were subjected to a phenylephrine stimulated contraction. Prazosin (10 nM), [3H]‐prazosin and phenoxybenzamine (3–300 nM) were used for inhibition assay, receptor characterization and partial alkylation of α‐adrenoceptor, respectively. The mRNA content of three subtypes of α‐adrenoceptors was determined by reverse transcription combined with polymerase chain reaction (RT–PCR).

Contractile response to phenylephrine increased in castrated rats, which could be explained by a relative increase of the stromal component. A lowered contraction potency was also noted in castrated rats. Receptor binding assay indicated minimal changes in the affinity or density of α1‐adrenoceptor. Escalating alkylation of the α1‐adrenoceptor population resulted in a rightward shift in the contraction‐response curves before depressing maximal contractile force, and the suppression was detected at lower doses in castrated rats. RT–PCR study confirmed the expression of three types of α1‐adrenoceptor, α1a, α1b and α1d‐adrenoceptors, in intact rat prostate, and revealed that α1a‐adrenoceptor, but not α1b or α1d‐adrenoceptors, was down‐regulated in castrates.

The results show that androgen deprivation suppressed α1‐adrenergic contractility of rat prostate strips, and the suppression was associated with down‐regulation of receptor reserve for the α1a‐adreneroceptor population expressed in intact rat prostate.

British Journal of Pharmacology (2000) 131, 1454–1460; doi:10.1038/sj.bjp.0703706
Mechanical stretch reveals different components of endothelial‐mediated vascular tone in rat aortic strips
Authors: Franchi‐Micheli Sergio , Failli Paola , Mazzetti Luca , Bani Daniele , Ciuffi Mario , Zilletti Lucilla
Published: January 2005

Since the role of mechanical stretches in vascular tone regulation is poorly understood, we studied how stretch can influence endothelial tone.

Isometric contractions of isolated rat aortic helical strips were recorded. The resting tension was set at 0.7 g, 1.2 g or 2.5 g. Endothelium‐preserved strips were precontracted with either phenylephrine or prostaglandin F2α (PGF2α).

In control conditions, acetylcholine (ACh) dose‐dependently relaxed phenylephrine‐precontracted strips independently of resting tension.

At 0.7 g resting tension, nitric oxide synthase (NOS) inhibitors did not reduce ACh‐induced relaxation, while either a guanylyl cyclase inhibitor or a NO trapping agent prevented it. At 1.2 g and 2.5 g resting tensions, NOS inhibitors shifted the ACh dose‐response curve to the right.

After preincubation with indomethacin (5 μM) or ibuprofen (10 and 100 μM), at 0.7 g and 1.2 g resting tensions, ACh induced an endothelium‐dependent, dose‐dependent contraction. ACh (10−6 M) increased the contraction up to two times greater the phenylephrine‐induced one. Lipoxygenase inhibitors prevented it. At high stretch, the ACh vasorelaxant effect was marginally influenced by cyclooxygenase (COX) inhibition. Similar results were obtained when aortic strips were precontracted with PGF2α.

Our data indicate that when resting tension is low, ACh mobilizes a stored NO pool that, synergistically with COX‐derived metabolites, can relax precontracted strips. COX inhibition up‐regulates the lipoxygenase metabolic pathway, accounting for the ACh contractile effect. At an intermediate resting tension, NO production is present, but COX inhibition reveals a lipoxygenase‐dependent, ACh‐induced contraction. At high resting tension, NO synthesis predominates and COX metabolites influence ACh‐induced relaxation marginally.

British Journal of Pharmacology (2000) 131, 1355–1362; doi:10.1038/sj.bjp.0703707
Tachykinin‐induced contraction of the guinea‐pig isolated oesophageal mucosa is mediated by NK2 receptors
Authors: Kerr Karen P , Thai Binh , Coupar Ian M
Published: January 2005

The tachykinin receptor present in the guinea‐pig oesophageal mucosa that mediates contractile responses of the muscularis mucosae has been characterized, using functional in vitro experiments.

The NK1 receptor‐selective agonist, [Sar9(O2)Met11]SP and the NK3 receptor‐selective agonists, [MePhe7]‐NKB and senktide, produced no response at submicromolar concentrations. The NK2 receptor‐selective agonists, [Nle10]‐NKA(4–10), and GR 64,349 produced concentration‐dependent contractile effects with pD2 values of 8.20±0.16 and 8.30±0.15, respectively.

The concentration‐response curve to the non‐selective agonist, NKA (pD2=8.13±0.04) was shifted significantly rightwards only by the NK2 receptor‐selective antagonist, GR 159,897 and was unaffected by the NK1 receptor‐selective antagonist, SR 140,333 and the NK3 receptor‐selective antagonist, SB 222,200.

The NK2 receptor‐selective antagonist, GR 159,897, exhibited an apparent competitive antagonism against the NK2 receptor‐selective agonist, GR 64,349 (apparent pKB value=9.29±0.16) and against the non‐selective agonist, NKA (apparent pKB value=8.71±0.19).

The NK2 receptor‐selective antagonist, SR 48,968 exhibited a non‐competitive antagonism against the NK2 receptor‐selective agonist, [Nle10]‐NKA(4–10). The pKB value was 10.84±0.19.

It is concluded that the guinea‐pig isolated oesophageal mucosa is a useful preparation for studying the effects of NK2 receptor‐selective agonists and antagonists as the contractile responses to various tachykinins are mediated solely by NK2 receptors.

British Journal of Pharmacology (2000) 131, 1461–1467; doi:10.1038/sj.bjp.0703708
Relevance of the C‐terminal Arg‐Phe sequence in γ2‐melanocyte‐stimulating hormone (γ2‐MSH) for inducing cardiovascular effects in conscious rats
Authors: Nijsen M J M A , De Ruiter G J W , Kasbergen C M , Hoogerhout P , De Wildt D J
Published: January 2005

The cardiovascular effects by γ2‐melanocyte‐stimulating hormone (γ2‐MSH) are probably not due to any of the well‐known melanocortin subtype receptors. We hypothesize that the receptor for Phe‐Met‐Arg‐Phe‐amide (FMRFa) or Phe‐Leu‐Phe‐Gln‐Pro‐Gln‐Arg‐Phe‐amide (neuropeptide FF; NPFFa), other Arg‐Phe containing peptides, is the candidate receptor. Therefore, we studied various Arg‐Phe containing peptides to compare their haemodynamic profile with that of γ2‐MSH(6–12), the most potent fragment of γ2‐MSH.

Mean arterial pressure (MAP) and heart rate (HR) changes were measured in conscious rats after intravenous administration of γ2‐MSH related peptides.

Phe‐Arg‐Trp‐Asp‐Arg‐Phe‐Gly (γ2‐MSH(6–12)), FMRFa, NPFFa, Met‐enkephalin‐Arg‐Phe‐amide (MERFa), Arg‐Phe‐amide (RFa), acetyl‐Phe‐norLeu‐Arg‐Phe‐amide (acFnLRFa) and desamino‐Tyr‐Phe‐norLeu‐Arg‐Phe‐amide (daYFnLRFa) caused a dose‐dependent increase in MAP and HR. γ2‐MSH(6–12) showed the most potent cardiovascular effects (ED50=12 nmol kg−1 for ΔMAP; 7 nmol kg−1 for ΔHR), as compared to the other Arg‐Phe containing peptides (ED50=177–292 nmol kg−1 for ΔMAP; 130–260 nmol kg−1 for ΔHR).

Peptides, which lack the C‐terminal Arg‐Phe sequence (Lys‐Tyr‐Val‐Met‐Gly‐His‐Phe‐Arg‐Trp‐Asp‐Arg‐Pro‐Gly (γ2‐pro11‐MSH), desamino‐Tyr‐Phe‐norLeu‐Arg‐[L‐1,2,3,4 tetrahydroisoquinoline‐3‐carboxylic acid]‐amide (daYFnLR[TIC]a) and Met‐enkephalin (ME)), were devoid of cardiovascular actions.

The results indicate that the baroreceptor reflex‐mediated reduction of tonic sympathetic activity due to pressor effects is inhibited by γ2‐MSH(6–12) and that its cardiovascular effects are dependent on the presence of a C‐terminal Arg‐Phe sequence.

It is suggested that the FMRFa/NPFFa receptor is the likely candidate receptor, involved in these cardiovascular effects.

British Journal of Pharmacology (2000) 131, 1468–1474; doi:10.1038/sj.bjp.0703709
Inhibitory effects of JTV‐519, a novel cardioprotective drug, on potassium currents and experimental atrial fibrillation in guinea‐pig hearts
Authors: Nakaya Haruaki , Furusawa Yoshie , Ogura Takehiko , Tamagawa Masaji , Uemura Hiroko
Published: January 2005

We investigated the effects of JTV‐519 (4‐[3‐(4‐benzylpiperidin‐1‐yl)propionyl]‐7‐methoxy‐2,3,4,5‐tetrahydro‐1,4‐benzothiazepine monohydrochloride), a novel cardioprotective drug, on the repolarizing K+ currents in guinea‐pig atrial cells by use of patch‐clamp techniques. We also evaluated the effects of JTV‐519 on experimental atrial fibrillation (AF) in isolated guinea‐pig hearts.

In atrial cells stimulated at 0.2 Hz, JTV‐519 in concentrations of 0.3 and 1 μM slightly prolonged the action potential duration (APD). The drug also reversed the action potential shortening induced by the muscarinic agonist carbachol in a concentration‐dependent manner.

The muscarinic acetylcholine receptor‐operated K+ current (IK.ACh) was activated by the extracellular application of carbachol (1 μM), adenosine (10 μM) or by the intracellular loading of GTPγS (100 μM). JTV‐519 inhibited the carbachol‐, adenosine‐ and GTPγS‐induced IK.ACh with the IC50 values of 0.12, 2.29 and 2.42 μM, respectively, suggesting that the drug may inhibit IK.ACh mainly by blocking the muscarinic receptors.

JTV‐519 (1 μM) inhibited the delayed rectifier K+ current (IK). Electrophysiological analyses indicated that the drug preferentially inhibits IKr (rapidly activating component) but not IKs (slowly activating component).

In isolated hearts, perfusion of carbachol (1 μM) shortened monophasic action potential (MAP) and effective refractory period (ERP), and lowered atrial fibrillation threshold (AFT). Addition of JTV‐519 (1 μM) inhibited the induction of AF by prolonging MAP and ERP.

We conclude that JTV‐519 can exert antiarrhythmic effects against AF by inhibiting repolarizing K+ currents. The drug may be useful for the treatment of AF in patients with ischaemic heart disease.

British Journal of Pharmacology (2000) 131, 1363–1372; doi:10.1038/sj.bjp.0703713
Rapid activation of basolateral potassium transport in human colon by oestradiol
Authors: McNamara Brian , Winter Desmond C , Cuffe John , Taylor Colm , O'Sullivan Gerald C , Harvey Brian J
Published: January 2005

We investigated the effect of oestradiol on basolateral potassium channels in human colonic epithelium.

Ion transport was quantified using short circuit current (Isc) measurements of samples mounted in Ussing chambers. Serosal K transport was studied using nystatin permeabilization of the apical membrane. Intracellular pH changes were quantified using spectroflouresence techniques.

Experiments were performed with either 10 nM or 1 μM Ca2+ in the apical bathing solution. With 10 nM Ca2+ in the apical bathing solution addition of oestradiol (1 nM) to the basolateral bath produced a rapid increase in current (ΔIK=11.2±1.2 μA.cm−2, n=6). This response was prevented by treatment of the serosal membrane with tolbutamide (1 μM). With 1 μM Ca2+ in the apical bathing solution addition of oestradiol produced a rapid fall in current (ΔIK=−12.8±1.4μA.cm−2), this response was prevented by treatment of the basolateral membrane with tetra‐pentyl‐ammonium (TPeA). These responses were rapid and occurred independently of protein synthesis.

Inhibition of basolateral Na+/H+ exchange with either amiloride or a low sodium bathing solution prevented this response. These responses were prevented by inhibition of protein kinase C (PKC) with bis‐indolyl‐maleimide.

Oestradiol (1 nM) produced a rapid intracellular alkanization (mean increase=0.11 pH units; n=6; P<0.01).

These results suggest that oestradiol rapidly modulates serosal K transport in human colon. These effects depend upon intact Na+/H+ exchange and protein kinase C. We propose a non‐classical, possibly membrane linked, mechanism for oestradiol action in human colonic epithelium.

British Journal of Pharmacology (2000) 131, 1373–1378; doi:10.1038/sj.bjp.0703714
Essential role of L‐arginine uptake and protein tyrosine kinase activity for NO‐dependent vasorelaxation induced by stretch, isometric tension and cyclic AMP in rat pulmonary arteries
Authors: Hucks David , Khan Nayeem M , Ward Jeremy P T
Published: January 2005

The NO‐dependent component of cyclic AMP‐induced vasorelaxation in rat pulmonary arteries is critically dependent on extracellular L‐arginine but independent of endothelial cell intracellular [Ca2+]. We examined whether L‐arginine uptake was also essential for NO production induced by passive stretch or isometric tension, processes also reported to be Ca2+‐independent.

The passive length‐tension curve was depressed by physiological concentrations of L‐arginine (400 μM; P<0.05). Inhibition of the y+ transporter with 10 mM L‐lysine, NO synthase with L‐NAME (100 μM), or protein tyrosine kinase with erbstatin A (30 μM) caused identical upward shifts (P<0.001), alone or in combination. Tyrphostin 23 was similar to erbstatin A, whilst the inactive analogue tyrphostin A1 and genistein were without effect.

L‐arginine (400 μM) shifted the PGF2α concentration‐response curve under isometric conditions to the right (P<0.05), whereas L‐NAME or L‐lysine caused a leftward shift (P<0.001). Tyrphostin 23 (30 μM) more than reversed the L‐arginine‐induced suppression of PGF2α‐induced tension; subsequent addition of L‐NAME had no effect. The L‐lysine‐sensitive component of CPT cyclic AMP‐induced vasorelaxation was abolished by erbstatin A.

ACh‐induced vasorelaxation was ∼80% inhibited by L‐NAME, but was not affected by L‐lysine or 400 μM L‐arginine. Erbstatin A reduced the vasorelaxation by only ∼25%.

We conclude that activation of NO production by stretch, isometric tension, or cyclic AMP in rat pulmonary arteries is critically dependent on the presence and uptake of physiological concentrations of extracellular L‐arginine, and protein tyrosine kinase activity. This directly contrasts with ACh‐induced vasorelaxation, which was independent of extracellular L‐arginine, and relatively unaffected by tyrosine kinase inhibition.

British Journal of Pharmacology (2000) 131, 1475–1481; doi:10.1038/sj.bjp.0703718
P2X receptor expression in mouse urinary bladder and the requirement of P2X1 receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle
Authors: Vial C , Evans R J
Published: January 2005

We have used subtype selective P2X receptor antibodies to determine the expression of P2X1–7 receptor subunits in the mouse urinary bladder. In addition we have compared P2X receptor mediated responses in normal and P2X1 receptor deficient mice to determine the contribution of the P2X1 receptor to the mouse bladder smooth muscle P2X receptor phenotype.

P2X1 receptor immunoreactivity was restricted to smooth muscle of the bladder and arteries and was predominantly associated with the extracellular membrane. Diffuse P2X2 and P2X4 receptor immunoreactivity not associated with the extracellular membrane was detected in the smooth muscle and epithelial layers. Immunoreactivity for the P2X7 receptor was associated with the innermost epithelial layers and some diffuse staining was seen in the smooth muscle layer. P2X3, P2X5 and P2X6 receptor immunoreactivity was not detected.

P2X receptor mediated inward currents and contractions were abolished in bladder smooth muscle from P2X1 receptor deficient mice. In normal bladder nerve stimulation evoked contractions with P2X and muscarinic acetylcholine (mACh) receptor mediated components. In bladder from the P2X1 receptor deficient mouse the contraction was mediated solely by mACh receptors. Contractions to carbachol were unaffected in P2X1 receptor deficient mice demonstrating that there had been no compensatory effect on mACh receptors.

These results indicate that homomeric P2X1 receptors underlie the bladder smooth muscle P2X receptor phenotype and suggest that mouse bladder from P2X1 receptor deficient and normal animals may be models of human bladder function in normal and diseased states.

British Journal of Pharmacology (2000) 131, 1489–1495; doi:10.1038/sj.bjp.0703720
Block of the lymphocyte K+ channel mKv1.3 by the phenylalkylamine verapamil: Kinetic aspects of block and disruption of accumulation of block by a single point mutation
Authors: Röbe Raphael J , Grissmer Stephan
Published: January 2005

Phenylalkylamines (PAA) usually known for their action on L‐type Ca2+ channels potently block the C‐type inactivating lymphocyte Kv1.3 channel resulting in inhibition of activation of T lymphocytes. In order to design PAAs blocking Kv1.3 specifically over L‐type Ca2+ channels, we investigated the state‐dependent manner of mKv1.3 block by the PAA verapamil.

Verapamil seems to have access to the open state (OB) and, once bound to the channel, the channel‐verapamil complex is absorbed into a slowly recovering state. This state was proposed to be the inactivated blocked state (IB). Here we present a quantitative description of the transition into this state and provide evidence for the IB state through experiments with an inactivation lacking mutant channel. Since the inactivated state cannot be reached in this case the IB state cannot be reached either.

We show that the transition OB→IB is accelerated by verapamil most likely through a mechanism involving the reduction of [K+] at an inactivation modulating low affinity binding site for K+ at the outer vestibule.

Measurements of the voltage‐dependence of the off‐rate constants for verapamil suggest that verapamil can reach the channel in its neutral form and might get partially protonated while bound. Thus only those verapamil molecules that are protonated can more easily dissociate at hyperpolarizing voltages.

Since open block kinetics were shown to be similar for wild type mKv1.3 and the H404T mutant mKv1.3 channel, and since the block of the H404T mutant channels by verapamil could be described exactly by a simple three‐state open block model, the mutant channel could serve as a screening channel to determine open block affinities of new PAA derivatives in high through‐put experiments.

British Journal of Pharmacology (2000) 131, 1275–1284; doi:10.1038/sj.bjp.0703723