bluuebunny/crossref_metadata_2025 huggingface dataset:
Radioligand and functional estimates of the interaction of the 1,4‐dihydropyridines, isradipine and lacidipine, with calcium channels in smooth muscle
Authors: Salomone Salvatore , Godfraind Théophile
Published: July 2012

The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4‐dihydropyridine calcium antagonist.

We studied the effect of lacidipine on contraction evoked by 100 mm KCl solution in rat aorta, treated by Nω‐nitro‐l‐arginine (0.1 mm), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)‐isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine‐evoked inhibition was a pseudo‐first order association kinetics, we estimated the dissociation rate constant (k−1 = 0.031 min−1), the association rate constant (k1 = 2.70 × 108 m−1 min−1) and the dissociation constant (KD = k−1/k1 = 115 pm) which was close to the IC50 value in steady‐state conditions (160 pm).

The inhibitory effects of lacidipine and (+)‐isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mm KCl‐solution. Washout with drug‐free 40 mm K+‐depolarizing solution reversed inhibition in the (+)‐isradipine‐treated preparations, but not in the lacidipine‐treated ones.

Radioligand binding studies were performed with [3H]‐lacidipine and [3H]‐isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for [3H]‐lacidipine: KD = 23 ± 2.6 pm, Bmax = 380 ± 21 fmol mg−1 protein in membranes from aorta; KD = 23 ± 3.1 pm, Bmax = 790 ± 60 fmol mg−1 protein in membranes from ileum; for [3H]‐isradipine: KD = 140 ± 46 pm, Bmax = 350 ± 64 fmol mg−1 protein in membrane from aorta; KD = 68 ± 14 pm, Bmax = 760 ± 75 fmol mg−1 protein in membranes from ileum). After isotopic dilution, [3H]‐lacidipine and [3H]‐isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (k−1) were 0.0257 min−1 and 0.0595 min−1, for [3H]‐lacidipine and [3H]‐isradipine, respectively.

The non specific binding of [3H]‐lacidipine and [3H]‐isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine (1 μm). After incubation with [3H]‐lacidipine 77.6 ± 1.9 pm for 2 h the concentration of drug in the tissue was 15.15 ± 1.18 fmol mg−1 w.wt. and still amounted to 7.24 ± 0.61 fmol mg−1 w.wt. after 3.5 h washout in drug‐free solution. After incubation with [3H]‐isradipine 47.2 ± 0.4 pm for 2 h it was 2.26 ± 0.07 fmol mg−1 w.wt. and was undetectable after 3.5 h washout in a drug‐free solution.

It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a non specific binding in a tissue compartment different from the plasma membrane.
Effects of cicletanine on whole‐cell currents of single smooth muscle cells from the guinea‐pig portal vein
Authors: Noack Thomas , Deitmer Petra
Published: July 2012

Smooth muscle cells of the guinea‐pig portal vein were dispersed by enzymatic treatment and recordings of membrane currents were made in the whole‐cell mode by the patch‐clamp technique. The effects of extracellular application of cicletanine‐hydrochloride on the whole‐cell currents of isolated smooth muscle cells from the guinea‐pig portal vein were studied in solutions containing a normal concentration of calcium (2.5 mm).

Cicletanine, 10 to 100 μm, reduced the voltage‐dependent inward calcium current with an IC50 of 250 μm. These effects of cicletanine were reversible.

The action of cicletanine on calcium currents can be interpreted as a decrease of the availability of calcium channels but not by an alteration of the time course or voltage‐dependency of inactivation. 3 The control calcium current was enhanced by application of Bay K 8644. On this enhanced inward current, cicletanine also exerted inhibitory effects which were not use‐dependent.

Cicletanine, 1 to 100 μm, did not enhance outward potassium currents.

It is concluded that at least one component of the vasorelaxant effects of cicletanine is produced by inhibition of calcium currents.
The effect of lifarizine (RS‐87476), a novel sodium and calcium channel modulator, on ischaemic dopamine depletion in the corpus striatum of the gerbil
Authors: Brown C.M. , Calder C. , Alps B.J. , Spedding M.
Published: July 2012

Unilateral ligation of the right common carotid artery in the anaesthetized gerbil for 3 h caused a 62.7% decrease in ipsilateral dopamine in the corpus striatum from 1.40 (± 0.13, n = 27) μg g−1 in the non‐ischaemic hemisphere to 0.47 (± 0.07, n = 27) μg g−1 in the ischaemic hemisphere (all results are expressed as mean ± s.e.mean). In sham‐operated animals there were no differences in the dopamine levels (1.31 ± 0.14 μg g−1, n = 11, left; 1.27 ± 0.13 μg g−1, n = 11 in the right hemisphere). Animals with intact communicating arteries in the circulus arteriosus were excluded.

Lifarizine (RS‐87476; 250, 500, but not 50, μg kg−1, i.p.) protected against this dopamine depletion showing only a 9.2% decrease at 250 μg kg−1, i.p. (P < 0.01) and no decrease at 500 μg kg−1, i.p. (P < 0.01).

Nicardipine (250 μg kg−1, p.o.) was effective when administered chronically once daily for 10 days (26.6% decrease, P < 0.05) but not when administered acutely at 50 μg kg−1, i.p.
Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors
Authors: Mitchell Jane A. , Kohlhaas Kathy L. , Sorrentino Raffaella , Warner Timothy D. , Murad Ferid , Vane John R.
Published: July 2012

Male Sprague‐Dawley or Wistar rats were injected with bacterial lipopolysacharride (LPS; 5 mg kg−1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h.

The organs were homogenized in a semi‐frozen state and NO synthase (NOS) activity measured in tissues from both LPS‐treated and saline‐treated groups by the ability of homogenates to convert [3H]‐l‐arginine to [3H]‐l‐citrulline in a NADPH‐dependent manner.

The NOS activity in all organs taken from control animals was found to be calcium‐dependent, with the highest activity being in the brain. After LPS‐treatment an induced calcium‐independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS‐induced NOS activity. No induction of calcium‐dependent NOS was detected.

Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h.

The constitutive NOS in brain and mesentery and the LPS‐induced activities in the spleen, lung, liver and mesentery were inhibited by NG‐monomethyl‐l‐arginine (l‐NMMA) or NG‐nitro‐l‐arginine methyl ester (l‐NAME) according to concentration. The IC50 for l‐NAME was 2.5 μm against the constitutive NOS from brain, and 20–25 μm against the inducible NOS. For l‐NMMA the IC50 was 20–25 μm against either NOS isoform.

The vascular responses to endothelin‐1 (ET‐1), the thromboxane A2‐mimetic 11α,9α‐epoxymethano prostaglandin F2α (U46619), phenylephrine (PE) or 5‐hydroxytryptamine (5‐HT) were measured in the simultaneously perfused arterial and venous mesenteric vascular beds from both control and LPS‐treated (6 h) rats. Vasoconstrictor responses to all agonists tested were unaffected by LPS treatment. In the presence of l‐NAME (100 μm) vasoconstrictor responses were potentiated in both the arterial and venous portion of the mesenteric beds from both control and LPS‐treated rats. The potentiation of responses to U46619 was significantly greater in beds from LPS‐treated rats.

Injection of LPS i.p. is associated with induction of NOS in all organs tested, except for the brain. In the mesentery this is not accompanied by a hyporesponsiveness to constrictor agents suggesting an increased sensitivity, particularly to U46619. This may explain the poor perfusion and tissue damage in the splanchnic circulation associated with sepsis.
Differential effects of cocaine and cocaethylene on intracellular Ca24+ and myocardial contraction in cardiac myocytes
Authors: Qiu Zhihua , Morgan James P.
Published: July 2012

Isolated cardiac myocytes of the ferret were used to investigate the influence of cocaine and cocaethylene on the intracellular Ca2+ transient indicated by the indo‐1 405/480 nm ratio signal, and peak cell shortening.

Both cocaine and cocaethylene produced significant decreases in peak intracellular Ca2+ and peak cell shortening in a dose‐dependent manner. Of interest, (1) the minimally effective dose of cocaethylene was ten fold lower (10−8 m versus 10−7 m) than that of cocaine; (2) the log EC50 of cocaethylene was −5.99 ± 0.13 (1.0 × 10−6 m), which was about ten fold lower than that of cocaine (−5.02 ± 0.11, 9.6 × 10−6 m); and (3) 1 × 10−4 m cocaethylene decreased the contraction amplitude by 71 ± 7%, while the same concentration of cocaine decreased the amplitude only by 55 ± 5%, indicating that cocaethylene is more potent than cocaine.

The negative inotropic effects of either cocaine or cocaethylene could be overcome by noradrenaline (∼ 5 μm) or calcium.

In contrast to cocaine, cocaethylene shifted the peak [Ca2+]i‐peak shortening relationship downward, indicating that cocaethylene decreased myofilament Ca2+‐responsiveness.

These data indicate that both cocaine and cocaethylene act directly on cardiac myocytes to produce a negative inotropic effect that is due to decreased Ca2+ availability. In contrast to cocaine, cocaethylene produces more potent inhibition by an additional action to decrease myofilament Ca2+‐responsiveness.
High‐affinity uptake of noradrenaline in postsynaptic neurones
Authors: Al‐Damluji S. , Krsmanovic L.Z. , Catt K.J.
Published: July 2012

Neurotransmitters released from nerve endings are inactivated by re‐uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of α1‐adrenoceptors in the hypothalamus, we observed a high‐affinity uptake process for noradrenaline in postsynaptic peptidergic neurones.

In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]‐prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10−10 to 10−7 m. However, at concentrations of unlabelled prazosin above 10−7 m, there was a paradoxical increase in apparent [3H]‐prazosin binding.

Methoxamine, an α1‐adrenoceptor ligand that is not subject to significant neuronal uptake, displaced [3H]‐prazosin but did not cause the paradoxical increase in the apparent binding of [3H]‐prazosin. Cooling the cells to 4°C reduced the total amount of prazosin associated with the cells; under these conditions, methoxamine almost completely inhibited [3H]‐prazosin binding to the cells.

In the presence of desipramine (DMI), unlabelled prazosin displaced [3H]‐prazosin as before, but no paradoxical increase in apparent binding was seen above 10−7 m.

The paradoxical increase of [3H]‐prazosin binding was not observed in membrane preparations of hypothalamic neurones. These findings indicated that the paradoxical increase in apparent [3H]‐prazosin binding was due to a cellular uptake process that becomes evident at high concentrations of the ligand.

DMI (10−5 m) had no effect on the specific binding of [3H]‐prazosin. The presence of α1‐adrenoceptors was confirmed by binding of [125I]‐HEAT, but [3H]‐idazoxan (an α2‐ ligand) did not bind to the cells.

The uptake of prazosin obeyed the Michaelis‐Menten model, with similar Km and Vmax values in both types of cultures.

Noradrenaline was taken up with high affinity by both types of cultures. (±)‐[3H]‐noradrenaline uptake was reduced by DMI and by excluding sodium from the medium, indicating that this process has some of the properties of uptake1. (±)‐[3H]‐noradrenaline uptake in the cell line was unaffected by testosterone.

The measured uptake of (−)‐noradrenaline in the cell line was considerably increased by blockade of catechol‐O‐methyl‐transferase and monoamine oxidase, suggesting that (−)‐noradrenaline is metabolized to lipophilic products that escape across the plasma membrane.

Studies in rats, in which the noradrenaline isomer 6‐hydroxydopamine was used, suggested that the postsynaptic uptake process is operative in hypothalamic CRH and vasopressin neurones in vivo.

The Km for (−)‐noradrenaline was within the range for the high affinity uptake1 process in noradrenergic neurones. Uptake takes place in concentrations at which noradrenaline activates α1‐adrenoceptors. Removal of noradrenaline from the vicinity of the receptors may prevent desensitization, thus maintaining the responsiveness of postsynaptic neurones to the actions of the neurotransmitter.
Effects of potassium channel openers and their antagonists on rat locus coeruleus neurones
Authors: Finta E.P. , Harms L. , Sevcik J. , Fischer H.‐D. , Illes P.
Published: July 2012

Intracellular recordings were obtained from a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Two openers of ATP‐sensitive potassium (KATP) channels, RO 31–6930 (10 μm) and cromakalim (100 μm) decreased the spontaneous discharge of action potentials without altering their amplitude or duration. Neither compound changed the resting membrane potential.

Of two KATP channel blockers, tolbutamide (300 μm) increased the firing rate, while glibenclamide (3 μm) only tended to do so. In addition, both compounds antagonized the effect of RO 31–6930 (10 μm). Neither glibenclamide (3 μm) nor tolbutamide (300 μm) altered the resting membrane potential.

Tetrodotoxin (0.5 μm) depressed the firing, but did not influence the inhibitory action of RO 31–6930 (10 μm). The excitatory amino acid antagonist, kynurenic acid (500 μm), did not change the spontaneous discharge of action potentials.

Small shifts (2–4 mV) of the membrane potential by hyper‐ or depolarizing current injections markedly decreased and increased the firing rate, respectively.

Noradrenaline (100 μm) hyperpolarized the cells and decreased their input resistance. This effect was not antagonized by glibenclamide (3 μm) or tolbutamide (300 μm). Ba2+ (2 mm), a blocker of both ATP‐sensitive and inwardly rectifying potassium channels, abolished the effects of RO 31–6930 (10 μm) and noradrenaline (100 μm).

These data suggest that KATP channels are present on the noradrenergic LC neurones, but are not coupled to α2‐adrenoceptors.
Nitric oxide and arachidonic acid modulation of calcium currents in postganglionic neurones of avian cultured ciliary ganglia
Authors: Khurana G. , Bennett M.R.
Published: July 2012

A study has been made of the modulation of high‐voltage activated transient and sustained calcium currents in cultured neurones of avian ciliary ganglia by nitric oxide (NO) and arachidonic acid.

Sodium nitroprusside (100 μm) reduced the transient calcium current (ICa) on average by 31% and the sustained ICa by 32% during a test depolarization to + 20 mV from a holding potential of −100 mV. This reduction was maintained for at least 30 min following a single application of sodium nitroprusside.

l‐Arginine (270 μm) reduced the transient ICa on average by 28% and the sustained ICa by 22% and these effects were prevented by the presence of the NO‐synthase competitive blocker NG‐nitro‐l‐arginine methylester (l‐NAME; 100 μm) in the bathing solution.

Arachidonic acid (50 μm) reduced the transient ICa on average by 28% and the sustained ICa by 33%. When added together, arachidonic acid (50 μm) and l‐arginine (270 μm) produced the same effects as arachidonic acid alone.

Blocking the conversion of arachidonic acid to prostaglandins by addition of indomethacin (20 μm) to the bathing solution did not prevent the depression of either the transient or the sustained calcium current during application of arachidonic acid (50 μm). The effects of arachidonic acid were also not occluded by l‐NAME (100 μm) when present in the bathing solution.

Inhibiting the biosynthesis of leukotrienes by applying L‐663,536 (MK‐886; 3 μm) to the bathing solution prevented the depression of both components of ICa during application of arachidonic acid (50 μm).

These results indicate that endogenous NO and arachidonic acid pathways are present in parasympathetic ciliary neurones, and that both act to depress high‐voltage, gated, calcium channel activity.
Induction of endothelium‐dependent relaxation in the rat aorta by IRL 1620, a novel and selective agonist at the endothelin ETB receptor
Authors: Karaki Hideaki , Sudjarwo Sri Agus , Hori Masatoshi , Takai Michihiro , Urade Yoshihiro , Okada Toshikazu
Published: July 2012

The effects of a novel and selective agonist at the endothelin ETB receptor, IRL 1620 (Suc‐[Glu9, Ala11,15] endothelin‐1 (8–21)), were examined in the isolated aorta of the rat.

IRL 1620 (1–300 nm) changed neither the resting tone nor the cytosolic Ca2+ level ([Ca2+]i) of the aorta without endothelium. In the presence of endothelium, however, IRL 1620 increased endothelial [Ca2+]i with little effect on the muscle tone. In the absence of external Ca2+, IRL 1620 still induced a transient increase in endothelial [Ca2+]i.

Noradrenaline (100 nm) increased both muscle [Ca2+]i and tension. IRL 1620 (1–300 nm) relaxed the muscle with an increase in endothelial [Ca2+]i only in the presence of endothelium. An inhibitor of nitric oxide synthase, 100 μm NG‐monomethyl‐l‐arginine, inhibited the relaxant effect of IRL 1620 but not the increase in endothelial [Ca2+]i.

In resting and noradrenaline‐stimulated aorta, the effects of IRL 1620 were inhibited by a selective antagonist of the ETB receptor, IRL 1038 (0.3–3 μm), although a selective antagonist of the ETA receptor, BQ‐123 (3 μm), was ineffective. Verapamil (10 μm) did not alter the effects of IRL 1620.

A muscarinic receptor agonist, carbachol (1 μm), also induced endothelium‐dependent relaxation with an increase in endothelial [Ca2+]i. However, the effects of carbachol were not inhibited by the ETB antagonist, IRL 1038 (3 μm).

These results suggest that IRL 1620 is a selective agonist at the ETB receptor which increases endothelial [Ca2+]i by releasing Ca2+ from storage sites and by opening non‐L type Ca2+ channels, activates nitric oxide synthase, releases nitric oxide, and relaxes vascular smooth muscle.
Roles of prostacyclin, EDRF and active oxygens in leukocyte‐dependent platelet adhesion to endothelial cells induced by platelet‐activating factor in vitro
Authors: Hirafuji Masahiko , Shinoda Hisashi
Published: July 2012

The mechanism of polymorphonuclear leukocyte (PMN)‐dependent platelet adhesion to cultured endothelial cells induced by platelet‐activating factor (PAF) was investigated to determine whether PMNs release or generate any factor(s) capable of inducing platelet adhesion, and the roles of prostacyclin and endothelium‐derived relaxing factor (EDRF).

Cell‐free supernatants, sonicates or rapid filtrates of PAF‐stimulated PMN suspensions did not induce platelet adhesion to endothelial cells, but the PMN sonicates induced platelet adhesion when endothelial cells were pretreated with both aspirin and NG‐nitro‐lm‐arginine (l‐NOARG). Its microphotograph showed that mainly platelet aggregates adhered to the endothelial cell surface.

Platelet adhesion induced by the PMN sonicates to aspirin‐ and l‐NOARG‐pretreated endothelial cells was dose‐dependently prevented by OP‐41483 (1–100 nm), and slightly by l‐arginine (1 mm). The inhibition of platelet adhesion by OP‐41483 and l‐arginine was potentiated by their combination.

WEB 2170 (3 μm), a PAF antagonist, inhibited platelet adhesion induced by the PMN sonicates. However, PAF alone did not induce significant platelet adhesion to aspirin‐ and l‐NOARG‐treated endothelial cells.

Platelet adhesion induced by the PMN sonicates was not suppressed by AA‐861 and indomethacin. However, both superoxide dismutase and catalase significantly inhibited platelet adhesion, and, in combination, their inhibitory effect was synergistically potentiated. Mannitol had no effect. It was also significantly inhibited by α1‐antitrypsin, whereas chymostatin and elastatinal had no effect.

PAF‐induced platelet adhesion to endothelial cells in the presence of intact PMNs was not suppressed by indomethacin and AA‐861, or by protease inhibitors. SOD alone, and in combination with catalase, caused a slight but significant inhibition, while catalase and mannitol by themselves had no effect.

PMN‐induced platelet adhesion was slightly inhibited by OP‐41483 (100 nm). l‐Arginine (1 mm) alone had no effect, but slightly potentiated the effect of OP‐41483. This platelet adhesion was not accompanied by suppression of prostacyclin synthesis.

The results with the PMN sonicates show that prostacyclin, EDRF and active oxygens are important modulators of intercellular interactions between platelets and endothelial cells. These results further suggest that the mechanism of intact PMN‐dependent platelet adhesion is primarily through platelet‐endothelial cell interactions in which leukocyte‐derived active oxygens play a role, but does not involve platelet‐platelet interactions inhibitable by prostacyclin.
Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia
Authors: Scholz W. , Albus U. , Lang H.J. , Linz W. , Martorana P.A. , Englert H.C. , Schölkens B.A.
Published: July 2012

The benzoylguanidine derivative Hoe 694 ((3‐methylsulphonyl‐4‐piperidino‐benzoyl) guanidine methanesulphonate) was characterized as an inhibitor of Na+/H+ exchange in rabbit erythrocytes, rat platelets and bovine endothelial cells. The potency of the compound was slightly lower or comparable to ethylisopropyl amiloride (EIPA).

To investigate a possible cardioprotective role of the Na+/H+ exchange inhibitor Hoe 694, rat isolated working hearts were subjected to ischaemia and reperfusion. In these experiments all untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 10−7 m Hoe 694 to the perfusate almost abolished reperfusion arrhythmias in the rat isolated working hearts.

Hoe 694 reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK), which are indicators of cellular damage during ischaemia, into the venous effluent of the hearts by 60% and 54%, respectively.

The tissue content of glycogen at the end of the experiments was increased by 60% and the high energy phosphates ATP and creatine phosphate were increased by 240% and 270% respectively in the treated hearts as compared to control hearts.

Antiischaemic effects of the Na+/H+ exchange inhibitor, Hoe 694, were investigated in a second experiment in anaesthetized rats undergoing coronary artery ligation. In these animals, pretreatment with Hoe 694 caused a dose‐dependent reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation down to doses of 0.1 mg kg−1, i.v. Blood pressure and heart rate remained unchanged.

We conclude that the new Na+/H+ exchange inhibitor, Hoe 694, shows cardioprotective and antiarrhythmic effects in ischaemia and reperfusion in rat isolated hearts and in anaesthetized rats. In view of the role which Na+/H+ exchange seems to play in the pathophysiology of cardiac ischaemia these effects could probably be attributed to Na+/H+ exchange inhibition.
Functional characterization of three adenosine receptor types
Authors: Gurden M.F. , Coates J. , Ellis F. , Evans B. , Foster M. , Hornby E. , Kennedy I. , Martin D.P. , Strong P. , Vardey C.J. , Wheeldon A.
Published: July 2012

The purpose of the present study was to classify adenosine receptors into A1 and A2 subtypes in a wide range of isolated tissues and cell types (rat adipocytes and atria, guinea‐pig ileum and atria (A1); guinea‐pig aorta, dog coronary artery and human platelets and neutrophils (A2)) using the R‐ and S‐diastereoisomers of N‐phenylisopropyladenosine (PIA), N‐cyclopentyladenosine (CPA), the novel compound, N‐[(1S,trans)‐2‐hydroxycyclopentyl]adenosine (GR79236), N‐[(2‐methylphenyl)methyl]adenosine (metrifudil), 2‐(phenylamino)adenosine (CV1808), and 2‐[[2‐[4‐(2‐carboxyethyl)phenyl]ethyl]amino]‐N‐ethylcarboxamidoadenosine (CGS21680); N‐ethylcarboxamidoadenosine (NECA) was used as a standard.

Results obtained in all tissue preparations previously reported to contain A1‐receptors could be described by a single rank order of agonist potency: CPA ≥ GR79236, R‐PIA ≥ NEC A > > S‐PIA ≥ metrifudil ≥ CV1808, CGS21680.

In contrast, two distinct rank orders of agonist potency were observed in preparations previously reported to contain A2‐receptors. In dog coronary artery, human neutrophils and platelets the rank order of potency was: CV1808, CGS21680 ≥ NECA > R‐PIA ≥ metrifudil ≥ CPA > GR79236, S‐PIA. However, in guinea‐pig aorta the rank order was: NECA > metrifudil > R‐PIA, CPA > CV1808, GR79236 ≥ S‐PIA, CGS21680.

The results of this study are consistent with the existence of three types of adenosine receptor: A1‐and two subtypes of A2‐receptor. The receptor present in dog coronary artery, human platelets and neutrophils, probably corresponds to the A2a subtype, whilst that present in the guinea‐pig aorta may be of the A2b subtype.
Effect of RP 67580, a non‐peptide neurokinin1 receptor antagonist, on facilitation of a nociceptive spinal flexion reflex in the rat
Authors: Laird J.M.A. , Hargreaves R.J. , Hill R.G.
Published: July 2012

In order to examine the contribution of neurokinin1 (NK1) receptors to facilitation of a spinal nociceptive reflex in the rat, we have investigated the effects of RP 67580 ((3aR,7aR)‐7,7‐diphenyl‐2(1‐imino‐2‐(2‐methoxyphenyl/ethyl)perhydroisoindole)), a non‐peptide neurokinin1 (NK1) receptor antagonist, selective for the rodent receptor sub‐type, on the activity of individual motorunits. These results were compared with the effects of RP 68651, the inactive 3aS, 7aS enantiomer of RP 67580, as a control for non‐specific activity.

Experiments were performed on 15 rats anaesthetized with a continuous i.v. infusion of alphaxalone/alphadalone and spinalized at T9–10. Single unit recordings of motorunit activity from biceps femoris/semitendinosus were made with a concentric needle electrode. In each experiment, a vehicle dose followed by 4 sequential rising doses of either RP 67580 or RP 68651 were given at 15 min intervals. High intensity electrical stimuli were applied to the hindlimb receptive field of the motorunit at a rate of 1 per 60 s throughout the experiment to establish a baseline. A conditioning stimulus (20 of these stimuli at 1 Hz) was delivered 5 min after each dose and the effect of the size of the baseline response examined.

The conditioning stimulus evoked a facilitation of the baseline at the start of all experiments (mean increase ± s.e.mean = 151 ± 20%). RP 67580 attenuated this facilitation, with an ID50 (± s.e.mean) of 2.5 ± 4.2 μg kg−1, i.v., whereas RP 68651 at doses of up to 3 mg kg−1, i.v. did not. There was no statistically significant effect of drug on the baseline reflex, nor on the response to the conditioning stimulus. Doses of 300 and 3000 μg kg−1 of both RP 67580 and RP 68651 evoked small depressor effects on systemic arterial blood pressure.

We conclude that the facilitation of a spinal flexor reflex by noxious conditioning stimuli in the rat is mediated by NK1 receptors whereas the baseline reflex is not. The results suggest that brain penetrant NK1 receptor antagonists may have central anti‐nociceptive effects.
Pharmacology of a cloned potassium channel from mouse brain (MK‐1) expressed in CHO cells: effects of blockers and an ‘nactivation peptide’
Authors: Robertson Brian , Owen David G.
Published: July 2012

Chinese hamster ovary cells (CHO), maintained in cell culture, were stably transfected with DNA for the MK‐1 voltage‐activated potassium channel, previously cloned from a mouse brain library.

Voltage‐activated currents were recorded by the whole‐cell patch clamp method. In CHO cells transfected with the vector only, there were no significant outward voltage activated currents. However, large outward voltage‐activated potassium currents were always observed in those cells which had been transfected with the vector containing the DNA encoding for MK‐1.

These potassium currents activated from −40 mV, and reversed at the potassium equilibrium potential. The half‐maximal conductance of MK‐1 was at −10 mV and had a slope factor of 11 mV when fitted with a Boltzmann function. There was only very slight (< 10%) inactivation of MK‐1 even at very large positive voltages.

MK‐1 was reversibly blocked by: 4‐aminopyridine (4‐AP, 0.1–4 mm), Toxin I (10–100 nm), mast cell degranulating peptide (1 μm), tetraethylammonium (TEA, 4–10mm), tedisamil (100 μm), quinine (100 μm) and ciclazindol (100 μm); all applied to the outside of the cell from a ‘U tube’ rapid perfusion system. 4‐AP may block closed as well as open MK‐1 potassium channels.

A synthetic 20 amino acid peptide derived from the N‐terminus sequence of the Shaker B potassium channel (the ‘inactivation peptide’) produced dramatic inactivation of MK‐1 when applied to the inside, but not the outside of the cell. Reducing peptide concentration or ‘degrading’ the peptide produced less inactivation.

The block of MK‐1 by the synthetic inactivation peptide was quite different in time dependence from block by internal TEA (0.4–4 mm), which probably blocks much more quickly but less potently than the peptide.
Differential effects of BQ‐123 against endothelin‐1 and endothelin‐3 on the rat vas deferens: evidence for an atypical endothelin receptor
Authors: Eglezos Anthony , Cucchi Paola , Patacchini Riccardo , Quartara Laura , Maggi Carlo Alberto , Mizrahi Jacques
Published: July 2012

Endothelin‐1 and endothelin‐3 enhanced concentration‐dependently the rat vas deferens twitch response to electrical stimulation, endothelin‐1 being three times more potent. Sarafotoxin S6c was at least 200 times less active than endothelin‐1.

The response to endothelin was antagonized in a competitive manner by the supposedly selective ETA receptor antagonist, BQ‐123 (pA2:7.0 ± 0.1). In contrast, the endothelin‐1 concentration‐response curve was only shifted two fold in the presence of 10 μm BQ‐123, while no effect was observed at 1 μm.

This evidence suggests the rat vas deferens contains an endothelin receptor not conforming to the ETA/ETB receptor subtype classification so far proposed.
Effects of rolipram and siguazodan on the human isolated bronchus and their interaction with isoprenaline and sodium nitroprusside
Authors: Qian Y. , Naline E. , Karlsson J.‐A. , Raeburn D. , Advenier C.
Published: July 2012

The effects of the selective inhibitors of cyclic AMP phosphodiesterase type IV (rolipram) and type III (siguazodan) and their interactions with isoprenaline and sodium nitroprusside have been studied in the human isolated bronchus.

On bronchi under resting tone rolipram was, in terms of potency (pD2 = 7.77 ± 0.14, n = 8), very similar to isoprenaline (pD2 = 7.31 ± 0.12, n = 12) and salbutamol (pD2 = 7.12 ± 0.17, n = 10) and approximately 10 fold more potent than siguazodan (pD2 = 6.80 ± 0.12, n = 6). In terms of efficacy (Emax, expressed as percentage of maximal effect induced by theophylline 3 mm), both rolipram and siguazodan were less efficient (Emax = 74 ± 6.7%, n = 8 and 66 ± 7.5%, n = 6, respectively) than isoprenaline (Emax = 98 ± 0.4%, n = 12) and salbutamol (Emax = 83 ± 2.4%, n = 10).

During precontraction induced by methacholine (3 × 10−7 m) or acetylcholine (10−3 m), concentration‐response curves to rolipram and siguazodan were shifted to the right and maximal effects reduced. Rolipram was more potent than siguazodan and, in terms of efficacy, it was less active.

Rolipram 10−8 and 10−7 m but not siguazodan potentiated the effects of isoprenaline as shown by the shift to the left of the concentration‐response curve to isoprenaline. Sodium nitroprusside‐induced relaxation was not modified by either drug.

These results show that rolipram is a potent relaxant of the human isolated bronchus, potentiating the effects of β‐adrenoceptor stimulation and suggest that, as previously demonstrated in other species (guinea‐pig, cow) (Tomkinson et al., 1993), there may be a connection between the β2‐adrenoceptor subtype, which predominate in human airway smooth muscle, and the cyclic AMP phosphodiesterase type IV.
Thiocyanate ions selectively antagonize AMPA‐evoked responses in Xenopus laevis oocytes microinjected with rat brain mRNA
Authors: Bowie Derek , Smart Trevor G.
Published: July 2012

Responses to kainate (KA), willardiine and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) were recorded from rat brain mRNA‐injected Xenopus laevis oocytes by use of a two‐electrode voltage clamp.

Thiocyanate (SCN−; 50 μm − 4 mm) ions reversibly and selectively inhibited the membrane current responses to AMPA in a non‐competitive manner without affecting KA or willardiine‐induced responses.

The inhibition of AMPA‐induced responses by SCN− was dependent on the SCN− concentration with an estimated IC50 of 1 mm. The antagonism was not dependent on the AMPA concentration.

The response to a high concentration of AMPA (100–200 μm) exhibited a peak inward current which declined to a steady‐state. SCN− inhibited the steady‐state current more than the peak response. The inhibition was unaffected by prior incubation with concanavalin‐A (Con‐A; 10 μm).

Responses to KA were antagonized by AMPA in a competitive manner, suggesting that both agonists may activate a common receptor‐channel complex. This interaction between two non‐NMDA agonists was not affected by the SCN−‐induced inhibition of the AMPA response.

AMPA‐induced responses recorded from large cultured cerebellar neurones by whole‐cell recording were also inhibited by SCN− in a non‐competitive manner. The AMPA‐induced peak current was less affected than the steady‐state response.

We conclude that SCN− can inhibit the response to AMPA in expressed non‐NMDA receptors in Xenopus oocytes and also in native receptors in cultured cerebellar neurones. One possible mechanism of action for SCN− inhibition of responses to AMPA may involve a Con‐A‐insensitive, non‐NMDA receptor‐mediated desensitization.
Inhibition by nitric oxide‐donors of human polymorphonuclear leucocyte functions
Authors: Moilanen E. , Vuorinen P. , Kankaanranta H. , Metsä‐Ketelä T. , Vapaatalo H.
Published: July 2012

The study was designed to test the hypothesis that nitric oxide (NO)‐releasing compounds increase guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) production in human polymorphonuclear leucocytes (PMNs) and concomitantly inhibit PMN functions, i.e. leukotriene B4 (LTB4) synthesis, degranulation, chemotaxis and superoxide anion (O2−) release. The effects of two new NO‐releasing compounds, GEA 3162 and GEA 5024 were compared to 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP).

GEA 3162 and GEA 5024 (1–100 μm) inhibited Ca ionophore A23187‐induced LTB4 and β‐glucuronidase release, chemotactic peptide FMLP‐induced chemotaxis and opsonized zymosan‐triggered chemiluminescence dose‐dependently in human PMNs. SIN‐1 and SNAP were weaker inhibitors.

Cellular cyclic GMP production was increased after exposure to NO‐donors concomitantly with the inhibition of PMN functions. No alterations in the levels of adenosine 3′:5′‐cylic monophosphate (cyclic AMP) were detected.

The results suggest that NO, possibly through increased cyclic GMP, inhibits the activation of human PMNs and may thus act as a local modulator in inflammatory processes.
3,4 Dichlorobenzamil‐sensitive, monovalent cation channel induced by palytoxin in cultured aortic myocytes
Authors: Van Renterghem Catherine , Frelin Christian
Published: July 2012

Smooth muscle cells were dispersed from rat aorta and then cultured. The action of palytoxin on rat aortic myocytes was analysed by measurement of 22Na+ uptake and single channel recording techniques.

Palytoxin induced an increase in 22Na+ uptake, with a concentration of 50 nm producing half‐maximal activation. The action of palytoxin was inhibited by amiloride derivatives and by ouabain. The concentrations of inhibitor producing half‐maximal inhibition were 10 μm for 3,4 dichlorobenzamil, 30 μm for benzamil, 100 μm for phenamil and 1 mm for ouabain.

In outside‐out patches, palytoxin induced single channel currents that reversed near 0 mV with NaCl or KCl in the extracellular solution, but were outward with N‐methyl‐d‐glucamine chloride or CaCl2 (110 mm), indicating that palytoxin induced a cation channel permeable to Na+ and K+ (PK/PNa = 1.2) but not to Ca2+ (PK/PCa > 30) or to N‐methyl‐d‐glucamine (NMDG) (PK/PNMDG > 11). The unit channel conductance was 11–14 pS.

A high (> 0.1 mm) extracellular concentration of Ca2+ was necessary to observe channel activation by palytoxin. A high (150 mm) extracellular concentration of K+ partially prevented and reversed channel activation by palytoxin.

The channel activity was fully blocked by 3,4 dichlorobenzamil (20 μm) and partially blocked by phenamil (50 μm). It was not reduced by ouabain (200 μm).
Enhancement by endothelin‐1 of microvascular permeability via the activation of ETA receptors
Authors: Filep János G. , Sirois Martin G. , Földes‐Filep Éva , Rousseau Anne , Plante Gérard E. , Fournier Alain , Yano Mitsuo , Sirois Pierre
Published: July 2012

The objective of the present experiments was to assess the involvement of endothelin‐A (ETA) receptors in mediating the effects of endothelin‐1 on microvascular permeability in conscious rats.

Bolus injection of endothelin‐1 (0.1 and 1 nmol kg−1, i.v.) resulted in a dose‐dependent prolonged pressor effect preceded by a transient depressor response. These changes were accompanied by a dose‐dependent loss of plasma volume. Endothelin‐1 (1 nmol kg−1) enhanced the vascular permeability of the upper and lower bronchi, kidney, stomach, duodenum and spleen (up to 270%) as measured by the extravasation of Evans blue dye.

Pretreatment of the animals with the selective ETA receptor antagonist, BQ‐123 (1 mg kg−1, i.v.) significantly blunted the pressor response to endothelin‐1 without affecting the depressor response. BQ‐123 inhibited by 87% the endothelin‐1 (1 nmol kg−1)‐induced plasma volume loss. BQ‐123 markedly attenuated protein extravasation elicited by endothelin‐1 in the upper and lower bronchi and kidney, whereas it completely inhibited the permeability effect of endothelin‐1 in the stomach and duodenum. BQ‐123 by itself had no significant effect on the parameters studied.

The endothelin‐1 analogue, [Trp(For)21]‐endothelin‐1, in which Trp21 is formylated, was as potent a pressor agent as endothelin‐1, but had no depressor action. Bolus injection of [Trp(For)21]‐endothelin‐l (0.1 and 1 nmol kg−1, i.v.) evoked similar plasma volume losses to those observed following administration of equimolar doses of endothelin‐1. Furthermore, 1 nmol kg−1 [Trp(For)21]‐endothelin‐l evoked increases in protein extravasation similar to endothelin‐1, 1 nmol kg−1.

The present findings suggest that endothelin‐1 enhances microvascular permeability, in part, via the activation of ETA receptors.
Selective inactivation of muscarinic M2 and M3 receptors in guinea‐pig ileum and atria in vitro
Authors: Eglen R.M. , Harris G.C.
Published: July 2012

The role of muscarinic M2 and M3 receptors in ileal smooth muscle has been evaluated by use of selective receptor alkylation. The alkylating agents, 4‐diphenylacetoxy‐N‐(2‐chloroethyl)‐piperidine (4‐DAMP mustard) was studied for effects against (+)‐cis‐dioxolane, at muscarinic M2 and M3 receptors in guinea‐pig atria or ileum, respectively. 4‐DAMP mustard (10 nm, 40 min exposure) did not discriminate between these muscarinic receptors. In ileum, 4‐DAMP mustard, at 100 nm, resulted in a large dextral shift (197 fold) and depression in maxima. In atria there was a smaller dextral shift (14 fold) but no depression in maxima.

The muscarinic antagonists, atropine (non‐selective), methoctramine (M2‐selective) and para‐fluoro‐hexahydro‐siladiphenidol (pFHHSiD; M3 selective) were studied in protection studies against alkylation by phenoxybenzamine. Washout studies following equilibration of the tissues with atropine (30 nm), methoctramine (0.3 μm) or pFHHSiD (3 μm), showed the compounds to be reversible. No temporal changes in sensitivity to (+)‐cis‐dioxolane were observed.

Exposure, for 20 min, of atria and ileum to phenoxybenzamine (3 and 10 μm respectively) caused dextral shifts and depressions in the maxima of the concentration‐response curve to (+)‐cis‐dioxolane. These effects were inhibited by prior equilibration with atropine (30 nm) and methoctramine (0.1 μm) in atria or atropine (30 nm) and pFHHSiD (3 μm) in ileum. Similar results in ileum were obtained when pilocarpine was used as the agonist.

These data were consistent with muscarinic M2 receptors mediating responses in atria and M3 receptors mediating responses in ileum. No evidence was provided for a direct role of muscarinic M2 receptors in ileal contraction.

It is concluded that receptor protection by reversible antagonists for muscarinic M2 or M3 receptors provides a means to isolate pharmacologically a single subtype in a tissue possessing heterogeneous populations. This technique may prove useful in defining the role of the respective subtypes in smooth muscle contraction.
Vasoreactivity and prostacyclin release in streptozotocin‐diabetic rats: effects of insulin or aldose reductase inhibition
Authors: Stevens E.J. , Willars G.B. , Lidbury P. , House F. , Tomlinson D.R.
Published: July 2012

Alterations in vasoreactivity and endothelial cell function could underlie some of the vascular abnormalities in diabetes. To examine aspects of these phenomena we studied the effects of 4‐ 6 weeks streptozotocin‐induced diabetes in the rat on basal and angiotensin II (AII)‐stimulated prostacyclin release from isolated lung, perfused at constant flow. In addition, pressure was monitored throughout the lung perfusion as an index of vasomotor tone.

The experiment also included lungs from groups of diabetic rats treated with either insulin or an aldose reductase inhibitor (imirestat), to determine whether these treatments influenced the development of any defects seen in untreated diabetes.

Despite some indication of a trend towards reduced prostacyclin release in lungs from diabetic rats, neither the basal nor AII‐stimulated release was significantly different from that seen in tissues from control animals. There were no significant differences between groups in the average basal perfusion pressure and in either the absolute pressure response to AII or the time of this peak.

The area under the perfusion pressure curve during AII infusion was greater in lungs from diabetic animals than in controls indicating a prolonged vasoconstrictor response. This increased pressor response may indicate increased sensitivity of diabetic tissue to AII or a reduced production of vasodilators in response to the vasoconstriction.

Whichever mechanism was responsible, this alteration was prevented by insulin treatment but not by aldose reductase inhibition, implicating mechanisms probably unrelated to exaggerated polyol pathway flux.
Tachykinin receptors mediating responses to sensory nerve stimulation and exogenous tachykinins and analogues in the rabbit isolated iris sphincter
Authors: Hall Judith M. , Mitchell Debra , Morton Ian K.M.
Published: July 2012

We have used selective tachykinin receptor agonists and antagonists to investigate the nature of the receptors mediating responses to endogenous and exogenous tachykinins in the rabbit iris sphincter preparation in vitro.

The NK1‐selective agonist, substance P methyl ester, induced contraction with a pD2 of 9.16 indicating the presence of NK1 receptors. In confirmation, the NK1‐selective antagonist, GR82334, competitively antagonized responses to substance P methyl ester with high affinity (pKB 7.46).

NK3 receptors also mediate contraction since NK3‐selective agonists exhibited high potency, e.g. the pD2 of [Me‐Phe7]‐neurokinin B was 9.67, and their responses were not inhibited by GR82334 (10 μm).

NK2 receptor activation does not seem to contribute to contraction since the NK2‐selective agonist [β‐Ala8]‐neurokinin A(4–10) had relatively low potency (pD2 6.43), and the NK2‐selective antagonists MEN10207 (1 μm) and L‐659,877 (10 μm) were inactive or had low affinity, respectively.

GR82334 (1 μm) significantly inhibited responses to electrical field‐stimulation of non‐adrenergic non‐cholinergic sensory nerves (3, 10 and 30 Hz), and caused a rightward shift of the log concentration‐response curve to bradykinin (lateral shift ca. 1000 fold). Higher concentrations of GR82334 (10 μm) significantly attenuated responses to capsaicin (1–60 μm) whilst completely abolishing responses to field‐stimulation (3, 10 and 30 Hz) and bradykinin (1 nm– 3 μm).

In conclusion, NK1 and NK3 receptor activation results in contraction of the rabbit iris sphincter. The contractile response following sensory nerve stimulation by bradykinin, capsaicin and electrical field stimulation results from NK1 receptor activation.
Calcium modulatory properties of 2,6‐dibutylbenzylamine (B25) in rat isolated vas deferens, cardiac and smooth muscle preparations
Authors: Pirisino R. , Banchelli G. , Ignesti G. , Mantelli L. , Matucci R. , Raimondi L. , Buffoni F.
Published: July 2012

In rat isolated vas deferens the new compound 2,6‐dibutylbenzylamine (B25) evoked a series of repeating rhythmic contractions. Concentration‐response curves constructed for this effect were bell‐shaped, indicating a biphasic effect for this compound. By contrast, B25 depressed heart contractility without any visible positive inotropic or chronotropic activity.

Experiments with tetrodotoxin, reserpine, capsaicin, α‐adrenoceptor blocking compounds and other agents permit us to exclude a release of neuromediators or a direct stimulation of post‐synaptic receptors to account for the rhythmic effect of B25 in the rat vas deferens.

In the same tissue, the increase in 45Ca2+ uptake, the voltage‐dependency as well as the dependence of the B25‐induced rhythmic activity upon the external calcium concentration indicate a direct activation of voltage‐sensitive calcium channels (VSCC).

Verapamil paradoxically stimulated the rhythmic effect of B25 in the rat vas deferens. La3+ was inactive while nifedipine was a weak inhibitor. By contrast Ni2+ and Mn2+ ions were good inhibitors (IC50 < 10−4m), suggesting that a possible opening of T‐type VSCC underlies the rhythmic effect of B25.

In radioligand binding studies competition experiments with [3H]‐nitrendipine indicated that only at high concentrations was B25 able to interact with dihydropyridine‐sensitive binding sites of heart and vas deferens smooth muscle.

B25 (3–30 μm) counteracted the inhibitory effects of ω‐conotoxin GVIA in field‐stimulated rat vas deferens.