bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in endothelium‐dependent hyperpolarization and relaxation in mesenteric arteries from streptozotocin‐induced diabetic rats
Authors: Fukao Mitsuhiro , Hattori Yuichi , Kanno Morio , Sakuma Ichiro , Kitabatake Akira
Published: August 2006

The aim of this study was to determine whether endothelium‐dependent hyperpolarization and relaxation are altered during experimental diabetes mellitus. Membrane potentials were recorded in mesenteric arteries from rats with streptozotocin‐induced diabetes and age‐matched controls. The resting membrane potentials were not significantly different between control and diabetic mesenteric arteries (−55.3±0.5 vs −55.6±0.4 mV). However, endothelium‐dependent hyperpolarization produced by acetylcholine (ACh; 10−8–10−5 M) was significantly diminished in amplitude in diabetic arteries compared with that in controls (maximum −10.4±1.1 vs −17.2±0.8 mV). Furthermore, the hyperpolarizing responses of diabetic arteries were more transient.

ACh‐induced hyperpolarization observed in control and diabetic arteries remained unaltered even after treatment with 3×10−4 M NG‐nitro‐L‐arginine (L‐NOARG), 10−5 M indomethacin or 60 u ml−1 superoxide dismutase.

Endothelium‐dependent hyperpolarization with 10−6 M A23187, a calcium ionophore, was also decreased in diabetic arteries compared to controls (−8.3±1.4 vs −18.0±1.9 mV). However, endothelium‐independent hyperpolarizing responses to 10−6 M pinacidil, a potassium channel opener, were similar in control and diabetic arteries (−20.0±1.4 vs −19.2±1.1 mV).

The altered endothelium‐dependent hyperpolarizations in diabetic arteries were almost completely prevented by insulin therapy. Endothelium‐dependent relaxations by ACh in the presence of 10−4 M L‐NOARG and 10−5 M indomethacin in diabetic arteries were also reduced and more transient compared to controls.

These data indicate that endothelium‐dependent hyperpolarization is reduced by diabetes, and this would, in part, account for the impaired endothelium‐dependent relaxations in mesenteric arteries from diabetic rats.
Spatial heterogeneity of the effects of calcitonin gene‐related peptide (CGRP) on the microvasculature of ligaments in the rabbit knee joint
Authors: Ferrell W R , McDougall J J , Bray R C
Published: August 2006

Experiments were performed in anaesthetized rabbits to examine the effects of calcitonin gene‐related peptide (CGRP) and the CGRP antagonist CGRP8–37 on blood flow to the medial collateral ligament of the knee joint.

Topical application of CGRP (10−13 to 10−9 mol) to the exposed external surface of eight knee joints resulted in dose‐dependent dilatation of vessels in both the ligament and the joint capsule. The magnitude of this response varied significantly in different regions of the medial collateral ligament, with the 10−9 mol dose of CGRP giving the maximum response (101.5±25.3% increase) at the femoral insertion site of the medial collateral ligament and lowest (23.1±8.8%) at the tibial insertion site.

Topical application of CGRP8–37 (0.1, 1 and 10 nmol) produced dose‐dependent constriction of vessels in the ligament and the joint capsule in five knees, with a trend towards the greatest effect occurring at the femoral insertion site (45.8±8.1% reduction in blood flow). With the 10 nmol dose, the vasoconstrictor response at the femoral insertion site differed significantly (P<0.05) from the responses obtained at the tibial insertion and joint capsule sites.

Topical application of CGRP8–37 (0.1, 1 and 10 nmol) to four chronically denervated knees produced substantially smaller vasoconstrictor responses at all sites. At the femoral insertion site, where 10 nmol CGRP8–37 normally produces a 45.8±8.1% reduction in blood flow (n=8), ten days following denervation this response was reduced to 6.5±6.1%, this difference being significant (P=0.01).

Adrenaline was applied topically to augment blood vessel tone, in order to establish how effectively co‐administration of CGRP would offset this increase in tone. Adrenaline (10−10 mol) produced vasoconstriction at all sites (n=6). In the capsule this vasoconstriction was virtually abolished when CGRP (10−9 mol) was co‐administered with adrenaline but in the ligament vasodilatation occurred at all sites. This vasodilatation was significantly greater at the femoral insertion site compared to the tibial insertion and mid ligament sites (P<0.05 for both) and the capsule (P<0.01).

Topical application of substance P (10−10 or 10−9 mol) failed to elicit dilatation of ligament blood vessels.

These results suggest that endogenous CGRP may play an important role in regulating blood flow to different structures in and around the knee joint.
Dissociation of the effects of the antitumour ether lipid ET‐18‐OCH3 on cytosolic calcium and on apoptosis
Authors: Teresa Alonso Maria , Gajate Consuelo , Mollinedo Faustino , Modolell Manuel , Alvarez Javier , García‐Sancho Javier
Published: August 2006

We have compared the effects of 1‐O‐octadecyl‐2‐O‐methyl‐sn‐glycero‐3‐phosphocholine (ET‐18‐OCH3) on the cytosolic free calcium concentration ([Ca2+]i) and on apoptosis in several normal and leukaemia cells, including human polymorphonuclear neutrophils (PMNs), U937 cells, and undifferentiated as well as dimethylsulphoxide‐differentiated HL60 cells (uHL60 and dHL60, respectively).

ET‐18‐OCH3 produced apoptosis, as evidenced by DNA degradation into oligonucleosome‐size fragments, in U937 and uHL60 cells, but not in dHL60 cells or PMNs.

ET‐18‐OCH3 induced an increase in [Ca2+]i mediated through the platelet‐activating factor (PAF) receptor in U937, dHL60 cells and PMNs, as shown by cross‐desensitization experiments and by prevention of the [Ca2+]i changes by the PAF antagonist WEB‐2170. The EC50 values for the increase in [Ca2+]i induced by PAF and ET‐18‐OCH3 were 5×10−11 and 2.5×10−7 M, respectively. In uHL60 cells the effect of ET‐18‐OCH3 on [Ca2+]i was very small and was not affected by WEB‐2170.

PAF did not produce apoptosis in any of the cell types tested. WEB‐2170 did not prevent the apoptosis induced by ET‐18‐OCH3.

The uptake of [3H]‐ET‐18‐OCH3 was much larger in U937 and uHL60 cells than in dHL60 cells and PMNs.

Our results indicate that the apoptotic effect of ET‐18‐OCH3 is not related to the changes in [Ca2+]i, effected by interaction with plasma membrane PAF receptors, but to other actions which are associated with the uptake of this drug into the cells.
Capsazepine block of voltage‐activated calcium channels in adult rat dorsal root ganglion neurones in culture
Authors: Docherty R J , Yeats J C , Piper A S
Published: August 2006

We have found that capsazepine, a competitive antagonist at the vanilloid (capsaicin) receptor, blocks voltage‐activated calcium currents in sensory neurones.

The block of calcium current was slow to develop with a half time of about one minute at 100 μM and lasted for the duration of the experiment. The rate of block of calcium current was strongly concentration‐dependent.

The EC50 for the blocking effect at 0 mV was 7.7±1.4 μM after 6 min exposure to capsazepine. The EC50 at equilibrium was estimated to be 1.4±0.2 μM.

The block of calcium current showed some voltage‐dependence but there was no indication of any selectivity of action for a calcium channel subtype. The characteristics of the blocking action of capsazepine on the residual current of cells which were pretreated with either □Omega;‐conotoxin or nimodipine were similar to control.

The data suggest that capsazepine, in addition to its competitive antagonism of vanilloid receptors, has a non‐specific blocking action on voltage‐activated calcium channels which should be taken into account when interpreting the effects of this substance on intact preparations in vitro or in vivo.
Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation
Authors: Elimadi Aziz , Morin Didier , Albengres Edith , Chauvet‐Monges Anne‐Marie , Allain Valérie , Crevat Aimé , Tillement Jean‐Paul
Published: August 2006

The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV‐3P) on Ca2+‐induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria.

ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV‐3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC50=3.0±0.9 μM).

The effect of ZDV‐3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV‐3P (8 μM) totally inhibited the rate of swelling.

ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV‐3P on mitochondrial swelling. Indeed, the IC50 value of ZDV‐3P increased from 3.0 to 17.4, 93.6 and 66.5 μM, in the presence of 20 μM, ADP, ATP or atractyloside, respectively.

ZDV‐3P did not displace [3H]‐ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV‐3P and [3H]‐ATP do not share the same binding sites.

ZDV‐3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca2+‐dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV‐3P).
Cellular mechanisms underlying carbachol‐induced oscillations of calcium‐dependent membrane current in smooth muscle cells from mouse anococcygeus
Authors: Wayman Christopher P , McFadzean Ian , Gibson Alan , Tucker John F
Published: August 2006

At a holding potential of −40 mV, carbachol (50 μM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole‐cell configuration of the patch‐clamp technique. Previous work has identified the first, transient component as a calcium‐activated chloride current (ICl(Ca)) and the second sustained component as a store depletion‐operated non‐selective cation current (IDOC). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (Ioscil) superimposed on IDOC.

Carbachol‐induced Ioscil (amplitude 97±11 pA; frequency 0.26±0.02 Hz) was inhibited by the chloride channel blocker anthracene‐9‐carboxylic acid (A‐9‐C; 1 mM), and by inclusion of 1 mM EGTA in the patch‐pipette filling solution.

In calcium‐free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; Ioscil could be restored by re‐admission of calcium. The frequency, but not the amplitude, of Ioscil increased with increasing concentrations of extracellular calcium (0.5–10 mM).

Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml−1) in the patch‐pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), prevented the activation of Ioscil by carbachol. Caffeine (10 mM) activated both ICl(Ca) and IDOC and prevented the induction of Ioscil by carbachol. Caffeine and CPA also abolished Ioscil in the presence of carbachol, as did both a low (3 μM) and a high (30 μM) concentration of ryanodine.

Carbachol‐induced Ioscil was abolished by the general calcium entry blocker SKF 96365 (10 μM) and by Cd2+ (100 μM), but was unaffected by La3+ (400 μM). As found previously, IDOC was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of IDOC preceded the abolition of Ioscil by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 μM) produced a partial inhibition of the carbachol‐induced Ioscil frequency at holding potentials of −20 mV and −60 mV and, in addition, reduced IDOC at −60 mV by 18%.

It is concluded that carbachol‐induced inward current oscillations in mouse anococcygeus cells are due to a calcium‐activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via IDOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re‐filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.
Role of endothelium in regulation of smooth muscle membrane potential and tone in the rabbit middle cerebral artery
Authors: Yamakawa Nobutaka , Ohhashi Masuo , Waga Shiro , Itoh Takeo
Published: August 2006

The characteristic features of the endothelium‐mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium‐intact and ‐denuded strips taken from the rabbit middle cerebral artery (MCA).

In endothelium‐intact strips, histamine (His, 3–10 μM) and high K+ (20–80 mM) concentration‐dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 μM), 5‐hydroxytryptamine (10 μM) and 9,11‐epithio‐11, 12‐methano‐thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+‐induced contraction).

NG‐nitro‐L‐arginine (L‐NOARG, 100 μM), but not indomethacin (10 μM), greatly enhanced the phasic and the tonic contractions induced by His (1–10 μM) in endothelium‐intact, but not in endothelium‐denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His‐induced contractions. Acetylcholine (ACh, 0.3–3 μM) caused concentration‐dependent relaxation (maximum relaxation by 89.7±7.5%, n=4, P<0.05) when applied to endothelium‐intact strips precontracted with His. L‐NOARG had little effect on this ACh‐induced relaxation (n=4; P<0.05). Apamin (0.1 μM), but not glibenclamide (3 μM), abolished the relaxation induced by ACh (0.3–3 μM) in L‐NOARG‐treated strips (n=4, P<0.05).

In endothelium‐intact tissues, His (3 μM) depolarized the smooth muscle membrane potential (by 4.4±1.8 mV, n=12, P<0.05) whereas ACh (3 μM) caused membrane hyperpolarization (−20.9±3.0 mV, n=25, P<0.05). The ACh‐induced membrane hypepolarization persisted after application of L‐NOARG (−23.5±5.9 mV, n=8, P<0.05) or glibenclamide (−20.6±5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to −5.8±3.2 mV, n=3, P<0.05).

Sodium nitroprusside (0.1–10 μM) did not hyperpolarize the smooth muscle cell membrane potential (0.2±0.3 mV, n=4, P>0.05) but it greatly attenuated the His‐induced contraction in endothelium‐denuded strips (n=4, P<0.05).

These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist‐induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium‐derived hyperpolarizing factor (EDHF) in rabbit MCA.
Effects of extracellular pH on agonism and antagonism at a recombinant P2X2 receptor
Authors: King B F , Wildman S S , Ziganshina L E , Pintor J , Burnstock G
Published: August 2006

Under voltage‐clamp conditions, the activity of agonists and antagonists at a recombinant P2X2 receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pHe).

In normal Ringer (Mg2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pHe (8.0–5.5). ATP‐responses reached a maximum at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.05±0.05.

Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP‐responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.35±0.05.

ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important.

The potency order for agonists of P2X2 receptors was: ATP>2‐MeS‐ATPATPγS> ATPαS>>CTPBzATP, while other nucleotides were inactive. EC50 and nH values for full agonists were determined at pH 7.4 and re‐examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2‐MeSATP, ATPγS and ATPαS), without altering the potency order.

The potency order for antagonists at P2X2 receptors was: Reactive blue‐2>trinitrophenol‐ATPPalatine fast blackCoomassie brilliant bluePPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re‐examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC50 values: 10.4±2 μM, at pH 7.4; 78±5 nM, at pH 6.5; 30±6 nM, at pH 5.5).

In summary, a lowered pHe enhanced the activity of all agonists at P2X2 receptors but, with the exception of suramin, not antagonists. Since a lowered pHe is also known to enhance agonist activity at P2X receptors on sensory neurones containing P2X2 transcripts, the sensitization by metabolic acidosis of native P2X receptors containing P2X2 subunits may have a significant effect on purinergic cell‐to‐cell signalling.
Pharmacological properties of the Ca2+‐release mechanism sensitive to NAADP in the sea urchin egg
Authors: Genazzani A A , Mezna M , Dickey D M , Michelangeli F , Walseth T F , Galione A
Published: August 2006

The sea urchin egg homogenate is an ideal model to characterize Ca2+‐release mechanisms because of its reliability and high signal‐to‐noise‐ratio. Apart from the InsP3‐ and ryanodine‐sensitive Ca2+‐release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist.

The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+‐releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors.

NAADP‐induced Ca2+‐release was blocked by L‐type Ca2+‐channel blockers and by Bay K 8644, while InsP3‐ and cyclic ADPR‐induced Ca2+‐release were insensitive to these agents. L‐type Ca2+‐channel blockers did not displace [32P]‐NAADP binding, suggesting that their binding site was different. Moreover, stopped‐flow kinetic studies revealed that these agents blocked NAADP in a all‐or‐none fashion.

Similarly, a number of K+‐channel antagonists blocked NAADP‐induced Ca2+‐release selectively over InsP3‐ and cyclic ADPR‐induced Ca2+‐release. Radioligand studies showed that these agents were not competitive antagonists.

As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+‐release mechanisms.

The presence of K+ was not essential for NAADP‐induced Ca2+‐release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3‐sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP‐induced Ca2+‐release. While the alkaline condition permitted InsP3 and cyclic ADPR‐induced Ca2+‐release, the acidic condition completely hampered both Ca2+‐release mechanisms.

The present results characterize pharmacologically and biochemically the novel Ca2+‐release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.
Characterization of α2‐adrenoceptors mediating contraction of dog saphenous vein: identity with the human α2A subtype
Authors: MacLennan S. J. , Luong L. A. , Jasper J. R. , To Z. P. , Eglen R. M.
Published: August 2006

In the dog saphenous vein α1‐ and α2‐adrenoceptors mediate noradrenaline‐induced contractions in vitro. In order to study the α2‐adrenoceptor in isolation, α1‐adrenoceptors were inactivated by treatment of tissues with the alkylating agent phenoxybenzamine (3.0 μM for 30 min) in the presence of rauwolscine (1 μM) to protect α2‐adrenoceptors.

Noradrenaline‐induced contractions of tissues treated with phenoxybenzamine were antagonized competitively by the selective α2‐adrenoceptor antagonist rauwolscine, pKB=8.63±0.07 (means±s.e.mean; n=3), consistent with an interaction at α2‐adrenoceptors.

Noradrenaline was a full agonist at α2‐adrenoceptors in dog saphenous vein. By use of the method of partial receptor alkylation and analysis of concentration‐effect curve data by direct, operational model fitting methods, the affinity (pKA) and efficacy (τ) were 5.74±0.07 and 7.50±1.05, respectively (n=6). Nine other agonists which were examined each had affinities higher than noradrenaline, but with the exception of the imidazoline, A‐54741 (5,6‐dihydroxy‐1,2,3,4‐tetrahydro‐1‐naphthyl‐imidazoline) had relatively lower efficacies.

To compare the α2‐adrenoceptor in dog saphenous vein to the human recombinant subtypes, the affinities of twenty‐one compounds were estimated in functional studies in the dog saphenous vein and in radioligand binding studies for the human α2A, α2B and α2C receptor subtypes expressed in Chinese hamster lung (CHL) cells.

Of twenty‐one compounds examined in ligand binding studies, only nine had greater than ten fold selectivity for one human receptor subtype over either of the other two. These compounds were A‐54741, oxymetazoline, guanfacine, guanabenz, prazosin, spiroxatrine, tolazoline, WB 4101 and idazoxan. In dog saphenous vein, their affinities (pKA and pKB for agonists and antagonists respectively) were: A‐54741 (pKA=8.03±0.05), oxymetazoline (pKA=7.67±0.09), guanfacine (pKA=6.79±0.03); guanabenz (pKA=7.02±0.13); prazosin (pKB=5.19±0.08), spiroxatrine (pKB=6.59±0.04), tolazoline (pKB=6.21±0.07), WB 4101 (pKB=7.42±0.09) and idazoxan (pKB=7.11±0.08).

Comparisons of affinity estimates for these nine compounds at the receptor in dog saphenous vein and at the human recombinant subtypes suggest that the vascular receptor is most similar to the h α2A subtype; correlation coefficients (r) were 0.82 (h α2A), 0.24 (h α2B) and 0.04 (h α2C).

British Journal of Pharmacology (1997) 121, 1721–1729; doi:10.1038/sj.bjp.0701296
Contribution of phosphodiesterase isozymes to the regulation of the L‐type calcium current in human cardiac myocytes
Authors: Kajimoto Katsuya , Hagiwara Nobuhisa , Kasanuki Hiroshi , Hosoda Saichi
Published: August 2006

To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L‐type calcium current (ICa(L)) in the human myocardium, we investigated the effect of selective and non‐selective PDE inhibitors on ICa(L) in single human atrial cells by use of the whole‐cell patch‐clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison.

In human atrial cells, 100 μM pimobendan increased ICa(L) (evoked by depolarization to +10 mV from a holding potential of −40 mV) by 250.4±45.0% (n=15), with the concentration for half‐maximal stimulation (EC50) being 1.13 μM. ICa(L) was increased by 100 μM UD‐CG 212 by 174.5±30.2% (n=10) with an EC50 value of 1.78 μM in human atrial cells. These two agents inhibit PDE III selectively.

A selective PDE IV inhibitor, rolipram (1–100 μM), did not itself affect ICa(L) in human atrial cells. However, 100 μM rolipram significantly enhanced the effect of 100 μM UD‐CG 212 on ICa(L) (increase with UD‐CG 212 alone, 167.9±33.9, n=5; increase with the two agents together, 270.0±52.2%; n=5, P<0.05). Rolipram also enhanced isoprenaline (5 nM)‐stimulated ICa(L) by 52.9±9.3% (n=5) in human atrial cells.

In rabbit atrial cells, ICa(L) at +10 mV was increased by 22.1±9.0% by UD‐CG 212 (n=10) and by 67.4±12.0% (n=10) by pimobendan (each at 100 μM). These values were significantly lower than those obtained in human atrial cells (P<0.0001). Rolipram (1–100 μM) did not itself affect ICa(L) in rabbit atrial cells. However, ICa(L) was increased by 215.7±65.2% (n=10) by the combination of 100 μM UD‐CG 212 and 100 μM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 μM UD‐CG 212 alone.

These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating ICa(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under β‐adrenoceptor‐mediated stimulation.

British Journal of Pharmacology (1997) 121, 1549–1556; doi:10.1038/sj.bjp.0701297
Differential effects of lovastatin on mitogen induced calcium influx in human cultured vascular smooth muscle cells
Authors: Clunn G. F. , Lymn J. S. , Schachter M , Hughes A. D.
Published: August 2006

In this study the effect of lovastatin, an inhibitor of cholesterol and isoprenoid synthesis, on the rises in intracellular calcium concentration ([Ca2+]i) induced by platelet derived growth factor BB (PDGF‐BB), angiotensin II (AII), low density lipoproteins (LDL) and foetal calf serum (FCS) was examined in human cultured vascular smooth muscle cells (VSMC) from saphenous vein. Changes in [Ca2+]i were measured in cell suspensions by the Ca2+ sensitive probe, fura 2.

Incubation with lovastatin for 24–26 h markedly reduced the peak rise and sustained phase of [Ca2+]i elevation in response to PDGF‐BB but the responses to AII, LDL and FCS were unaffected. Further experiments showed that lovastatin pretreatment inhibited PDGF‐BB induced Ca2+ influx but not intracellular Ca2+ release. This inhibition could be overcome by co‐incubation with mevalonic acid.

Pretreatment of cells with the heterotrimeric G protein inhibitor pertussis toxin for up to 24 h completely abolished AII‐induced [Ca2+]i rises but the response to PDGF‐BB was unaffected.

The tyrosine kinase inhibitor genistein largely abolished PDGF‐BB‐induced [Ca2+]i elevation but had no significant effect on AII‐induced responses.

Pre‐incubation with lovastatin had no effect on the level of tyrosine phosphorylation of PDGF‐β receptors (as measured by Western blot) in response to the PDGF‐BB ligand.

PDGF‐BB elicits Ca2+ influx via a tyrosine kinase‐dependent mechanism distinct from the heterotrimeric G protein coupled pathway utilized by AII. Lovastatin most likely acts by inhibition of isoprenylation (via blockade of isoprenoid synthesis) of an intermediate molecule involved in PDGF‐BB‐induced Ca2+ influx.

British Journal of Pharmacology (1997) 121, 1789–1795; doi:10.1038/sj.bjp.0701299
Involvement of protein kinase C in the UTP‐mediated potentiation of cyclic AMP accumulation in mouse J774 macrophages
Authors: Lin W. W. , Chen B. C.
Published: August 2006

We have investigated the effects of nucleotide analogues on cyclic AMP formation in mouse J774 macrophages and the mechanisms involved.

UTP, in the concentration range 0.1–100 μM, induced concentration‐dependent potentiation of prostaglandin E1 (PGE1)‐induced cyclic AMP formation, but had no effect on basal cyclic AMP formation. UDP showed an equal potency, while 2‐methylthio ATP, α,β‐methylene ATP and β,γ‐methylene ATP gave either a slight increase or had no effect at concentrations up to 100 μM. ATP, although 100 fold less effective than UTP, also caused cyclic AMP potentiation, but had no effect on agonist‐stimulated or basal cyclic AMP levels.

The cyclic AMP potentiation effect of UTP correlated with increased [Ca2+]i and inositol phosphate (IP) formation over the same concentration range.

Ionomycin, which evokes an increase in [Ca2+]i without affecting IP formation, did not cause an increase in cyclic AMP content, indicating that UTP‐induced cyclic AMP regulation is not due to activation of Ca2+‐sensitive adenylyl cyclase isoforms.

Although reduced, UTP potentiation was seen in cells incubated in a Ca2+‐free and/or BAPTA‐containing medium. Under these conditions, the UTP‐increased IP accumulation was similarly reduced.

Exposure of cells to phorbol 12‐myristate 13‐acetate (PMA) also increased PGE1 stimulation of cyclic AMP levels, and the UTP‐induced potentiation of cyclic AMP formation was inhibited by either staurosporine or Ro 31‐8220. Pretreatment of cells with PMA for 4–24 h resulted in marked attenuation of UTP‐stimulated cyclic AMP potentiation.

Pretreatment with pertussis toxin (24 h, 100 ng ml−1) did not significantly affect UTP‐induced cyclic AMP potentiation and IP formation, although it increased the cyclic AMP response to PGE1.

Analysis of J774 cells by Western blotting with antibodies specific for different protein kinase C (PKC) isoforms shows the presence of the βI, βII, δ, ε, ζ, μ, λ and ζ isoforms. Moreover, UTP significantly increased the level of PKC βI, βII, δ, ε, μ, λ and ζ immunoreactivity in the membrane fraction and decreased the cytosolic reactivity of PKC βII, δ, ε and ζ.

Immunoblot studies also indicate the presence of type II adenylyl cyclase.

These results indicate that PKC is required for the potentiation of adenylyl cyclase activity by macrophage pyrimidinoceptors, which exhibit a higher specificity for UTP and UDP than for ATP.

British Journal of Pharmacology (1997) 121, 1749–1757; doi:10.1038/sj.bjp.0701300
Inhibition by cannabinoid receptor agonists of acetylcholine release from the guinea‐pig myenteric plexus
Authors: Coutts Angela A. , Pertwee Roger G.
Published: August 2006

The dose‐related inhibition of the twitch responses of the myenteric plexus‐longitudinal muscle preparation of the guinea‐pig small intestine by cannabinoid (CB) agonists, (+)‐WIN 55212 and CP 55940 during stimulation at 0.1 Hz with supramaximal voltage was confirmed. These agonists inhibited acetylcholine (ACh) release in the presence of physostigmine (7.7 μM) thus indicating a prejunctional site of action.

Inhibition of twitch responses and ACh release by CB agonists was reversed by the CB1‐selective cannabinoid receptor antagonist, SR141716A. Dose‐response curves to (+)‐WIN 55212 and CP 55940 were shifted to the right, with no reduction of maximal response, by pretreatment with SR141716A (31.6–1000 nM), but not its vehicle, Tween 80 (1 μM). However, at very high concentrations (25–400 μM), Tween 80 itself caused a dose‐related inhibition of the twitch response which was significantly reduced in the presence of SR141716A (1 μM). The opioid receptor antagonist, naloxone (1 μM) had no significant effect on the inhibition by CP 55940 of the twitch response.

(+)‐WIN 55212, CP 55940 and Tween 80 (50 μM) had no effect on responses to exogenous ACh, confirming that their actions were prejunctional. SR141716A (1 μM) did not increase the sensitivity of the longitudinal muscle to either ACh or histamine, but inhibited the responses to high doses of ACh.

The (−)‐enantiomer of WIN 55212, was approximately 300 times less active than the (+) enantiomer in inhibiting the twitch response, had no CB1 antagonist activity against the active isomer and did not inhibit the release of ACh in the presence of physostigmine.

The dissociation constant (KD) values for SR 141716A against the inhibitory effect of (+)‐WIN 55212 and CP 55940 on the twitch response were 12.07 nM (95% confidence intervals 8.55 and 20.83) and 6.44 nM (95% confidence intervals 4.70 and 10.24), respectively. In experiments in which the release of ACh was inhibited by (+)‐WIN 55212, the KD values were 9.21 nM and 10.53 nM at SR141716A concentrations of 31.6 nM and 100 nM, respectively. The KD values for the antagonism by naloxone of the inhibition of the twitch responses and the inhibition of ACh release by normorphine in this preparation were found to be 2.38±0.69 nM and 2.00±0.9 nM, respectively.

During maximal inhibition of ACh release by (+)‐WIN 55212, the addition of normorphine (400 nM) caused a further significant decrease in ACh output.

SR141716A alone produced a significant increase in ACh release in both the absence and presence of exogenous cannabinoid drugs, hence we conclude that it has a presynaptic site of action. We also conclude that SR141716A acts either by antagonizing the effect of an endogenous CB1 receptor agonist or by having an inverse agonist effect at these receptors.

British Journal of Pharmacology (1997) 121, 1557–1566; doi:10.1038/sj.bjp.0701301
Evidence that methyl arachidonyl fluorophosphonate is an irreversible cannabinoid receptor antagonist
Authors: Fernando Susanthi R. , Pertwee Roger G.
Published: August 2006

Methyl arachidonyl fluorophosphonate (MAFP) (1 μM) significantly attenuated the ability of WIN 55,212‐2, CP 55,940, (−)‐Δ9‐tetrahydrocannabinol (THC), nabilone and (R)‐(+)‐arachidonoyl‐1′‐hydroxy‐2′‐propylamide (methanandamide) to inhibit electrically‐evoked isometric contractions of the myenteric plexus‐longitudinal muscle preparation of guinea‐pig small intestine.

The sizes of the maximal responses to WIN 55,212‐2 and CP 55,940 decreased significantly in the presence of 1 μM MAFP.

MAFP (1 μM) essentially abolished the inhibitory effects on the twitch response of the highest concentration of methanandamide used (3.162 μM). The dextral shift it induced in the log concentration‐response curve of nabilone was non‐parallel. In contrast, the dextral shift in the log concentration‐response curve of THC produced by MAFP did not deviate significantly from parallelism and was relatively small with a mean value of 3.45 and 95% confidence limits of 1.19 and 13.08.

MAFP (1 μM) did not attenuate the effects of normorphine or clonidine on the twitch response of the myenteric plexus‐longitudinal muscle preparation or affect the contractile response of this preparation to acetylcholine.

When administered by itself at concentrations of 1 to 1000 nM, MAFP had no detectable effect on the twitch response of the myenteric plexus‐longitudinal muscle preparation.

These results support the hypothesis that MAFP is an irreversible cannabinoid CB1 receptor antagonist that possesses some degree of selectivity.

British Journal of Pharmacology (1997) 121, 1716–1720; doi:10.1038/sj.bjp.0701303
Investigation of the mechanisms underlying the hypophagic effects of the 5‐HT and noradrenaline reuptake inhibitor, sibutramine, in the rat
Authors: Jackson Helen C. , Bearham M. Clair , Hutchins Lisa J. , Mazurkiewicz Sarah E. , Needham Andrew M. , Heal David J.
Published: August 2006

Sibutramine is a novel 5‐hydroxytryptamine (5‐HT) and noradrenaline reuptake inhibitor (serotonin‐ noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine‐induced hypophagia.

Individually‐housed male Sprague‐Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.

Sibutramine (10 mg kg−1, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α1‐adrenoceptor antagonist, prazosin (0.3 and 1 mg kg−1, i.p.), and partially antagonized by the β1‐adrenoceptor antagonist, metoprolol (3 and 10 mg kg−1, i.p.) and the 5‐HT receptor antagonists, metergoline (non‐selective; 0.3 mg kg−1, i.p.); ritanserin (5‐HT2A/2C; 0.1 and 0.5 mg kg−1, i.p.) and SB200646 (5‐HT2B/2C; 20 and 40 mg kg−1, p.o.).

By contrast, the α2‐adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg−1, i.p.) and the β2‐adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg−1, i.p.) did not reduce the decrease in food intake induced by sibutramine.

These results demonstrate that β1‐adrenoceptors, 5‐HT2A/2C‐receptors and particularly α1‐adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine‐induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5‐HT, with the subsequent activation of a variety of noradrenaline and 5‐HT receptor systems.

British Journal of Pharmacology (1997) 121, 1613–1618; doi:10.1038/sj.bjp.0701311
Study of the in vivo and in vitro cardiovascular effects of a hydralazine‐like vasodilator agent (HPS‐10) in normotensive rats
Authors: Orallo Francisco
Published: August 2006

In this work, the cardiovascular effects of HPS‐10, a new vasodilator agent, were studied in rats.

In conscious normotensive rats, oral administration of HPS‐10 (4–9 mg kg−1) produced a dose‐related and long‐lasting fall in systolic arterial blood pressure (ED30 of 5.32 mg kg−1), accompanied by an increase in heart rate (ED30 of 8.43 mg kg−1). This tachycardia was totally inhibited by pretreatment with (±)‐propranolol (10 mg kg−1, p.o.).

In anaesthetized normotensive rats, HPS‐10 (0.3–0.6 mg kg−1, i.v.) produced a gradual, dose‐dependent and sustained decrease in systolic, diastolic and mean arterial pressure (MAP) (ED30 for MAP of 0.41 mg kg−1, i.v.), accompanied by a significant bradycardia at high doses (>0.4 mg kg−1; ED20 of 0.61 mg kg−1, i.v.). HPS‐10 (0.5 mg kg−1, i.v.) did not modify the positive chronotropic effects induced by intravenous administration of noradrenaline (NA; 5 μg kg−1), angiotensin II (AII; 0.2 μg kg−1) and nicotine (200 μg kg−1) but markedly inhibited the hypertensive response produced by these agents.

In rat isolated rubbed aorta, HPS‐10 (0.1–1 mM) non‐competitively and with almost equal effectiveness antagonized the contractions induced by NA, AII (in normal Krebs solution) and Ca2+ (in depolarizing Ca2+‐free high‐K+ 50 mM solution). In the experiments in Ca2+‐free medium, HPS‐10 (1 mM) considerably inhibited the contractions induced by NA, AII and caffeine in rat aorta.

Furthermore, in the studies with radioactive Ca2+, HPS‐10 (1 mM) did not modify the basal uptake of 45Ca2+ but strongly decreased the influx of 45Ca2+ induced by NA, AII and K+ in rat aortic rings.

In rat isolated atria, HPS‐10 (1 mM) produced a positive inotropic/negative chronotropic effect.

HPS‐10 (0.3 mM) significantly inhibited the sustained and transient Ba2+ inward current (IBa) recorded in whole‐cell clamped rat aortic myocytes.

These results indicate that the non‐selective vasorelaxant effects of HPS‐10 in rat aortic rings can be attributed to transmembrane Ca2+‐antagonist activity and an intracellular action on smooth muscle cells. The direct vasodilator action of HPS‐10 observed in rat isolated aorta may be responsible for the HPS‐10 hypotensive activity in anaesthetized normotensive rats.

British Journal of Pharmacology (1997) 121, 1627–1636; doi:10.1038/sj.bjp.0701314
Differential effect of L‐NAME and S‐methyl‐isothiourea on leukocyte emigration in carrageenin‐soaked sponge implants in rat
Authors: Iuvone Teresa , Van Osselaer Nancy , D'Acquisto Fulvio , Carnuccio Rosa , Herman Arnold G.
Published: August 2006

The role of nitric oxide (NO) in leukocyte (polymorphonuclear cells, monocytes and lymphocytes) emigration was studied in a model of carrageenin‐sponge implants in rats.

The subcutaneous implantation of 1% (w/v) of λ‐carrageenin‐soaked sponges elicited an inflammatory response that was characterized by a time‐related increase in leukocyte infiltration in the sponges and increased levels of nitrite in the exudate. Total leukocyte infiltration and nitrite production were maximal at 24 h and decreased after 48 and 96 h. The mononuclear cell influx was maximal at 48 h (21% of the total leukocytes). Therefore, this time point was used in the successive experiments.

Polymorphonuclear cell (PMN) and lymphocyte infiltration in the sponges significantly increased when rats were treated with the non‐specific NO‐synthase (NOS) inhibitor, NG‐nitro‐L‐arginine methylester (L‐NAME) (1 mg ml−1 in drinking water ad libitum). Monocyte emigration was not affected by L‐NAME treatment. The nitrite levels in the exudate of L‐NAME‐treated rats were significantly reduced. The concomitant ingestion of L‐arginine (30 mg ml−1) resulted in a reversion of the L‐NAME effect, while D‐arginine (30 mg ml−1) had no effect, indicating the involvement of the L‐arginine: NO pathway.

Administration of L‐NAME resulted also in an increased release of tumour necrosis factor‐α (TNF‐α) and prostacyclin (measured as the stable metabolite, 6‐keto‐PGF1α). L‐NAME had no effect on monocyte chemoattractant protein‐1 (MCP‐1) release in the exudate.

Since L‐NAME may have effects on the local blood flow, phenylephrine (0.034 mg ml−2 in drinking water) was used as it has an effect on the local blood flow similar to L‐NAME. Phenylephrine had no effect on either leukocyte emigration, or on nitrite, TNF‐α, prostacyclin or MCP‐1 accumulation in the exudate.

In contrast, the more selective iNOS inhibitor S‐methyl‐isothiourea (SMT) (10 μg ml−1 in drinking water) significantly reduced PMNs and lymphocyte influx in the sponge, having no effect on monocyte influx. Moreover, SMT decreased nitrite production in the exudate to a comparable extent as L‐NAME.

Administration of SMT significantly reduced MCP‐1 release in the exudate, without an effect on TNF‐α or prostacyclin production. Moreover SMT did not produce any changes in local blood flow.

Our results show that a different outcome of the inflammatory process can be obtained depending on the types of NOS inhibitor used.

British Journal of Pharmacology (1997) 121, 1637–1644; doi:10.1038/sj.bjp.0701317
Capsaicin and neurokinin A‐induced bronchoconstriction in the anaesthetised guinea‐pig: evidence for a direct action of menthol on isolated bronchial smooth muscle
Authors: Wright C. E. , Laude E. A. , Grattan T. J. , Morice A. H.
Published: August 2006

For many years menthol has been used in the treatment of respiratory disorders although, a bronchodilator effect of menthol has yet to be described. Using the bronchoconstrictors capsaicin (acting via stimulating the release of neuropeptides from sensory afferents) and neurokinin A (NKA) we have raised airways resistance in the guinea‐pig (GP) and studied the effect of menthol on both capsaicin and NKA‐induced bronchoconstriction in vivo. In vitro the effect of menthol on acetylcholine (ACh) and KCl precontracted GP bronchi was also studied.

GP (n=13) were anaesthetized (urethane 1.5 g kg−1, i.p.) and a bolus injection of capsaicin (7.5 μg ml−1, i.v.) or infusion of NKA (1 μg min−1, i.v.) was given either in the presence of air (0.81 min−1) or air impregnated with menthol vapour (7.5 μg l−1) freely breathed from a tracheal cannula via a T‐piece. Airways resistance (Raw) and ventilation were measured throughout. Bronchi of mean internal diameter (1029+73.6 μm; n=24) were removed from GP (n=16) and mounted in the Cambustion myograph. Bronchial rings were maximally precontracted with 80 mM KC1 or 2 mM ACh. Relaxation due to a cumulative dose of menthol (1–3000 μM) was measured.

Menthol produced a significant (P<0.05) 51.3% reversal of the capsaicin‐induced increase in Raw, and also inhibited the significant (P<0.05) reduction in minute ventilation (Ve) associated with the capsaicin‐induced increased in Raw. Menthol also caused a significant (P<0.05) 41% reversal of the NKA‐induced increase in Raw. The NKA‐induced decrease in Ve was again significantly (P<0.05) reversed with menthol inhalation. Menthol caused a significant (P<0.001) dose‐dependent relaxation of KCl and ACh precontracted bronchi.

We have shown that menthol attenuates both capsaicin and NKA‐induced bronchoconstriction in vivo and relaxes KCl and ACh preconstricted bronchi in vitro. Menthol inhibition of NKA and capsaicin‐induced bronchoconstriction could be, in part, explained by a direct action of menthol on bronchial smooth muscle.

British Journal of Pharmacology (1997) 121, 1645–1650; doi:10.1038/sj.bjp.0701319
The role of cyclic nucleotides in guinea‐pig bladder contractility
Authors: Longhurst Penelope A. , Briscoe Janice A K. , Rosenberg David J. , Leggett Robert E.
Published: August 2006

The effects of phosphodiesterase (PDE) inhibition and forskolin pretreatment on the contractile responses of guinea‐pig urinary bladder strips to electrical field stimulation, carbachol, ATP and KCl were studied.

Inhibition of cyclic AMP‐specific PDE4 isozymes by rolipram significantly reduced the contractile response of bladder strips to field stimulation. Rolipram also suppressed the contractile response to low concentrations of carbachol, but potentiated the response to high concentrations. The contractile response to ATP was significantly reduced by rolipram treatment, but that to KCl was unaltered.

Inhibition of cyclic GMP‐specific PDE5 isozymes by zaprinast had no effects on the contractile response of bladder strips to field stimulation, ATP or KCl. Zaprinast suppressed the contractile responses to 1 μM carbachol and potentiated the response to high concentrations.

Contractile responses to field stimulation and to carbachol after pretreatment with the adenylyl cyclase activator, forskolin, were qualitatively similar to those caused by rolipram treatment. β‐Adrenoceptor blockade with propranolol partially reversed the inhibitory effects of rolipram on the response to field stimulation.

Rolipram significantly reduced the contractile response of bladder strips from sensitized guinea‐pigs to ovalbumin challenge, but zaprinast was ineffective. PDE inhibition had similar effects on the responsiveness of control and of sensitized guinea‐pig bladder strips to field stimulation, carbachol, ATP and KCl.

The data suggest that the contractile response of guinea‐pig bladder strips can be modified by increases in cyclic AMP levels.

British Journal of Pharmacology (1997) 121, 1665–1672; doi:10.1038/sj.bjp.0701328
Characterization of [3H]‐prostaglandin E2 binding to prostaglandin EP4 receptors expressed with Semliki Forest virus
Authors: Marshall Fiona H. , Patel Kalpana , Lundstrom Kenneth , Camacho Janet , Foord Steven M. , Lee Melanie G.
Published: August 2006

The human prostaglandin EP4 receptor has been expressed by use of the Semliki Forest virus system.

In cell membranes [3H]‐prostaglandin E2 ([3H]‐PGE2) bound to a high affinity site with a Kd of 1.12±0.3 nM and a Bmax of 3.1±0.3 pmol mg−1 protein.

In competition studies the rank order of potency for prostaglandins was PGE2  = PGE1  ∼#62;  PGF2α =PGI2.

The binding of [3H]‐PGE2 to cell membranes was inhibited by approximately 60% by the addition of guanylnucleotides, suggesting that this proportion of the receptors was G‐protein coupled.

[3H]‐PGE2 binding was increased by greater than 200% by the addition of divalent cations, with little change in the IC50 of PGE2.

In saturation studies removal of divalent cations and addition of GTPγS resulted in a 65% reduction in the Bmax with no change in the Kd. These results are consistent with the ligand labelling two states of the receptor R*, a high affinity state and R*G, a high affinity G protein coupled state.

British Journal of Pharmacology (1997) 121, 1673–1678; doi:10.1038/sj.bjp.0701332