bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasorelaxant mechanism of KRN2391 and nicorandil in porcine coronary arteries of different sizes
Authors: Miwa Atsushi , Kaneta Soichiro , Motoki Kazuhiro , Jinno Yasuhiro , Kasai Hideo , Okada Yuji , Fukushima Hideaki , Ogawa Nobuyuki
Published: July 2012

The relaxant mechanisms of action of KRN2391, a novel vasodilator, and nicorandil on epi‐myocardial coronary artery (2.5–3.0 mm outer diameter) and mid‐myocardial coronary artery (0.8–1.0 mm outer diameter) were investigated in porcine isolated coronary arteries. In addition, the vasorelaxant responses of KRN2391 and nicorandil were compared with those of nitroglycerin and cromakalim, a K+ channel opener, in epi‐ and mid‐myocardial coronary arteries.

Nitroglycerin showed a more potent relaxant effect on epi‐myocardial coronary arteries than on mid‐myocardial coronary arteries, whereas cromakalim produced greater relaxation responses in mid‐myocardial coronary arteries. There was no difference between epi‐ and mid‐myocardial coronary arteries in terms of the relaxant effect of KRN2391 and nicorandil.

Relaxation induced by KRN2391 in epi‐ and mid‐myocardial coronary arteries was inhibited by oxyhaemoglobin, a pharmacological antagonist of nitrovasodilators, and glibenclamide, a pharmacological antagonist of K+ channel opening drugs. However, the inhibitory effect of glibenclamide on KRN2391‐induced relaxation was greater in mid‐myocardial coronary artery than in epi‐myocardial coronary artery.

Relaxation induced by nicorandil was inhibited by oxyhaemoglobin alone in epi‐myocardial coronary arteries and by both oxyhaemoglobin and glibenclamide in mid‐myocardial coronary arteries.

In epi‐ and mid‐myocardial coronary arteries, relaxation induced by cromakalim was inhibited by glibenclamide but not by oxyhaemoglobin, whereas relaxation induced by nitroglycerin was inhibited by oxyhaemoglobin but not by glibenclamide.

These results suggest that KRN2391 and nicorandil exhibit a dual mechanism of action acting partly as a nitrate and partly as a K+ channel opener. The mechanism of action of these drugs depend on the segment of coronary artery studied. Furthermore, the dual mechanism of action of KRN2391 and nicorandil seems to contribute to the equipotent relaxant effect between epi‐ and mid‐myocardial coronary arteries.
Modification by charybdotoxin and apamin of spontaneous electrical and mechanical activity of the circular smooth muscle of the guinea‐pig stomach
Authors: Suzuki Keiko , Ito Kaoru M. , Minayoshi Yukie , Suzuki Hikaru , Asano Masahisa , Ito Katsuaki
Published: July 2012

The effects of charybdotoxin and apamin, putative blockers of Ca2+‐activated K+ channels, on spontaneous electrical and mechanical activity of circular smooth muscle of the guinea‐pig stomach antrum were examined in the presence of 1 μm tetrodotoxin and 1 μm atropine.

Both charybdotoxin (> 3 nm) and apamin (> 3 nm) dose‐dependently increased the amplitude of spontaneous contractions without altering their frequency. The maximum effect of charybdotoxin was much greater than that of apamin. Both toxins increased the amplitude of intracellular Ca2+ oscillations measured with fura‐2.

When the extracellular Ca2+ concentration was lowered to 1.5 mm or less, apamin did not significantly potentiate the contractions whereas charybdotoxin still potentiated them but with less potency.

Charybdotoxin (30 nm) increased the amplitude of spikes and slow waves, and slightly decreased the resting membrane potential. On the other hand, apamin (100 nm) preferentially increased the slow wave amplitude with no effect on the resting membrane potential.

These results suggest that both toxins affect the spontaneous contraction by modifying the electrical activity and that charybdotoxin‐sensitive K+ channels and apamin‐sensitive ones are differently involved in the spontaneous electrical activity.
Cellular mechanism of the positive inotropic effect of hydralazine in mammalian myocardium
Authors: Hurrell David G. , Perreault Cynthia L. , Miao Lin , Ransil Bernard J. , Morgan James P.
Published: July 2012

The purpose of this study was to elucidate the cellular mechanism of the positive inotropic effect of hydralazine, a vasodilator widely used for afterload reduction in patients with heart failure that has also been reported to have positive inotropic effects on the heart. After isolation, right ventricular papillary muscles from the ferret were maintained in bicarbonate‐buffered salt solution (30°C). A concentration‐response relationship was obtained for hydralazine (10−6 to 10−3 m). In order to mimic different levels of catecholamine release found in heart failure, we utilized two methods of stimulation: (a) threshold punctate pulses and (b) suprathreshold punctate stimulation with voltage approximately 10% above threshold.

In a first group of muscles (n = 16), a maximally effective concentration of hydralazine (10−3 m) increased peak isometric tension by 39 ± 9% (P < 0.05). Doses lower than 10−5 m had no significant effect. The bioluminescent Ca2+ indicator, aequorin, was loaded into a subset of these muscles (n = 7). A significant increase in peak light (i.e., intracellular Ca2+) developed, concurrently with an increase in peak tension (38 ± 5% to 66 ± 8%). This inotropic response was associated with a decrease in time to peak tension (ms), 221 ± 7 to 186 ± 5 (P < 0.05), and time to peak light, 65 ± 4 to 52 ± 2 (P < 0.05). These effects were markedly attenuated by pretreatment with autonomic blocking agents.

In a second group of muscles (n = 12), histamine was used to stimulate cyclic AMP production in the presence of propranolol. Hydralazine (3 × 10−4 m) led to a shift in the pD2 (i.e. the negative log of the concentration of histamine producing 50% of the maximal response) from 6.1 ± 0.1 to 5.9 ± 0.1 (P < 0.05), thus increasing the sensitivity of the muscles to histamine. Hydralazine also increased maximum tension from 160 ± 77% to 195 ± 57% (P < 0.05) above baseline. Thus, hydralazine altered the potency and efficacy of histamine despite the presence of β‐adrenoceptor blockade.

A third group of muscles were chemically skinned to examine the effects of hydralazine on myofilament Ca2+ responsiveness. Pretreatment of ferret papillary muscles with hydralazine (10−3 m) before skinning did not shift the force‐pCa curve after skinning (n = 16). However, hydralazine added to previously skinned fibres desensitized the myofilaments, as indicated by a rightward shift of the force‐pCa curve (n = 12). Maximum tension development was not changed.

The pharmacological effects of hydralazine are characteristic of inotropic drugs that act mainly via cyclic AMP; however, the increase in peak tension demonstrated with histamine in the presence of hydralazine also suggests an effect on cyclic AMP‐independent second messenger pathways. These data are consistent with reports that large doses of hydralazine may increase cellular levels of cyclic AMP, as well as other second messengers, by direct cardiac and indirect neuronal mechanisms.
The novel 5‐HT1A receptor antagonist, SDZ 216–525, decreases 5‐HT release in rat hippocampus in vivo
Authors: Sharp T. , McQuade R. , Fozard J.R. , Hoyer D.
Published: July 2012

Recent evidence suggests that the novel compound SDZ 216–525 is a selective and possibly silent 5‐HT1A receptor antagonist. Here we have examined the action of SDZ 216–525 on central 5‐HT1A autoreceptor function. The experiments involved measurement of drug effects on extracellular 5‐HT in the ventral hippocampus of the chloral hydrate anaesthetized rat by use of microdialysis.

Acute injection of SDZ 216–525 (0.1, 0.3, 1.0 and 3 mg kg−1, s.c.) caused a dose‐related decrease in 5‐HT output with an estimated ED50 of at least 0.3 mg kg−1. This ED50 value is 20–30 times greater than ED50 values previously obtained for 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) and NAN‐190. In comparison, SDZ 216–525 is reported to have slightly higher affinity for the 5‐HT1A site than 8‐OH‐DPAT and NAN‐190.

The inhibitory effect of SDZ 216–525 (1 mg kg−1, s.c.) on 5‐HT was blocked by the 5‐HT1/β‐adrenoceptor antagonist, (−)‐pindolol (8 mg kg−1, s.c.) but not by a combination of the β1 and β2‐selective adrenoceptor antagonists metoprolol and ICI 118,551 (4 mg kg−1, each).

Although in several experimental models SDZ 216–525 has high affinity, selectivity and lacks intrinsic activity at the 5‐HT1A receptor, our experiments show that the drug decreases extracellular 5‐HT in ventral hippocampus of the chloral hydrate anaesthetized rat via a pindolol‐sensitive mechanism. We conclude that either SDZ 216–525 promotes (with low potency in vivo) 5‐HT1A receptor/G‐protein interactions, or that the 5‐HT1A autoreceptor is a 5‐HT1A receptor subtype different from the postsynaptic 5‐HT1A receptor.
Inhibition of nitric oxide synthesis reveals non‐cholinergic excitatory neurotransmission in the canine proximal colon
Authors: Shuttleworth C. William , Sanders Kenton M. , Keef Kathleen D.
Published: July 2012

Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques.

Electrical field stimulation (EFS; 0.1–20 HZ) produced frequency‐dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These reponses were enhanced by NG‐monomethyl‐l‐arginine (l‐NMMA; 300 μm) and reduced by atropine (1 μm). The effects of l‐NMMA were reversed by l‐arginine (3 mm). All responses to EFS were abolished by tetrodotoxin (1 μm).

In the presence of atropine, phentolamine and propranolol (all at 1 μm; ‘non‐adrenergic, non‐cholingergic (NANC) conditions’), EFS evoked frequency‐dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post‐stimulus excitation.

Under NANC conditions and in the presence of l‐NG‐nitroarginine methyl ester (l‐NAME; 200 μm), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co‐incubation with l‐arginine (2 mm) and abolished by tetrodotoxin (1 μm).

In the presence of atropine (1 μm), EFS (5–20 Hz) caused frequency‐dependent inhibition of electrical slow waves. In the presence of l‐NAME (100 μm) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than

Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 μm).

Atropine‐resistant contractions to EFS were enhanced by indomethacin (10 μm) and reduced or abolished by the non‐selective NK1/NK2 tachykinin receptor antagonist d‐Pro2, d‐Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 μm).

Exogenous tachykinins mimicked non‐cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A > neurokinin B > substance P, suggesting a predominance of the NK2 sub‐type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.

These results suggest that: (i) in previous studies, non‐cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non‐cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as ‘rebound’ excitation; (iii) one or more tachykinins, possibly, acting via NK2 receptors, may mediate non‐cholinergic excitatory responses.
Protective effects of ranolazine in guinea‐pig hearts during low‐flow ischaemia and their association with increases in active pyruvate dehydrogenase
Authors: Clarke B. , Spedding M. , Patmore L. , McCormack J.G.
Published: July 2012

In isolated Langendorff‐perfused, electrically‐paced, hearts of guinea‐pigs, global low‐flow‐ischaemia (LFI; at 0.7 ml min−1) resulted in marked increases in the rates of release of lactate, lactate dehydrogenase (LDH) and creatine kinase (CK) over a 30 min period. At the end of the LFI period, tissue ATP content was significantly reduced from a control value of 11.8 ± 0.8 (5) to 5.6 ± 0.8 (5) μmol g−1 dry weight.

The presence of ranolazine [(±)‐N‐(2,6‐dimethyl‐phenyl)‐4[2‐hydroxy‐3‐(2‐methoxy‐phenoxyl)‐propyl]‐1‐piperazine acetamide dihydro‐chloride; RS‐43285‐193] at 10 μm, from 20 min prior to and during LFI, resulted in significant reductions in the release of lactate, LDH and CK during the ischaemic period and a significant preservation of tissue ATP (9.0 ± 1.1 (6) μmol g−1 dry wt.). Ranolazine did not prevent the reductions in creatine phosphate or glycogen observed in LFI, nor did it have any significant effects on any contractile parameters before or during the LFI period.

Neither ranolazine nor LFI affected the total amounts of tissue pyruvate dehydrogenase (PDH) activity; however, the significant reduction in the amount of active, non‐phosphorylated PDH caused by LFI (from 88.2 ± 5.5 to 44.2 ± 3.2% of total activity) was partially but significantly prevented by ranolazine (67.2 ± 6.8%). This effect of ranolazine on PDH may be part of the mechanism whereby the compound reduces lactate release and preserves tissue ATP during ischaemia.
Binding of angiotensin antagonists to rat liver and brain membranes measured ex vivo
Authors: Marshall Fiona H. , Clark Samantha A. , Michel Anton D. , Barnes Julie C.
Published: July 2012

The effects of the angiotensin antagonists GR117289, losartan and Sar1Ala8‐angiotensin II on the ex vivo binding of [125I]‐Sar1Ile8‐angiotensin II to rat liver and cortex/hippocampus (Cx/H) membranes have been investigated.

GR117289 (0.1–30 mg kg−1, s.c., 2 h pretreatment) caused a dose‐dependent reduction in [125I]‐Sar1Ile8‐angiotensin II binding to both liver and cortex/hippocampus membranes.

Administration of a submaximal dose of GR117289 (1 mg kg−1, s.c.) indicated that the peak inhibition of binding in the liver occurred within 0.5 h, whereas the peak inhibition of binding in the Cx/H occurred 2 h after drug treatment.

The effect of GR117289 was long lasting. Binding was still reduced in the Cx/H 48 h after drug treatment (10 mg kg−1, s.c.) but had returned to normal 72 h after drug treatment. In the liver binding was still reduced 72 h after treatment with the same dose.

Losartan (1 − 30 mg kg−1, s.c.) was equipotent with GR117289 in its ability to reduce liver binding, but was less effective at inhibiting binding to central receptors.

The non‐peptide antagonist Sar1Ala8‐angiotensin II (3 and 10 mg kg−1) reduced binding in the liver but not in the Cx/H membranes.

These results suggest that, unlike the peptide antagonist Sar1Ala8‐angiotensin II, the non‐peptide angiotensin antagonists, GR117289 and losartan, are able to cross the blood brain barrier and occupy central angiotensin II receptors.
Postjunctional inhibitory effect of the NK2 receptor antagonist, SR 48968, on sensory NANC bronchoconstriction in the guinea‐pig
Authors: Lou Ya‐Ping , Lee Lu‐Yuan , Satoh Hiroyuki , Lundberg Jan M.
Published: July 2012

The effects of a selective NK2 receptor antagonist, SR 48968, on non‐adrenergic non‐cholinergic (NANC) bronchoconstriction in the guinea‐pig were investigated in both in vitro and in vivo studies.

In isolated bronchus, the electrical field stimulation (EFS, 1 Hz for 1 min)‐induced NANC bronchoconstriction was inhibited by 83% after preincubation with SR 48968 (10−7 m) for 1 h. The selective NK1 receptor antagonist, CP 96,345 (10−6 m), together with SR 48968 completely abolished the remaining EFS‐evoked NANC bronchial contraction. ST 48968 (10−7 m) totally blocked the bronchial contraction caused by neurokinin A (NKA), but reduced only slightly the bronchoconstriction caused by high concentrations of substance P (SP) and did not influence the response to acetylcholine (ACh).

In the guinea‐pig isolated perfused lung, SR 48968 (5 × 10−7 m) perfusion for 30 min markedly reduced, by 95% and 68% respectively, the increase in lung resistance (RL) and the decrease in dynamic compliance (CDyn) evoked by vagal stimulation (1 Hz for 1 min). Capsaicin (10−8 m)‐evoked bronchoconstriction was also significantly inhibited by SR 48968 (5 × 10−7 m). However, the same concentration of SR 48968 did not affect the release of neuropeptide calcitonin gene‐related peptide (CGRP)‐like immunoreactivity (LI) evoked by either vagal stimulation or capsaicin in the isolated perfused lung, suggesting no prejunctional action. SR 48968 (5 × 10−7 m) caused a parallel shift of the concentration‐response curve to the right by a factor of 10 for the bronchoconstriction evoked by NKA (10−9 − 3 × 10−7 m) in the isolated lung, while it abolished the contraction induced by the selective NK2 receptor agonist, Nle10 NKA(4–10) (10−9 − 3 × 10−7 m).

In in vivo studies, ST 48968 (0.3 mg kg−1, i.v.) also greatly inhibited the increase in insufflation pressure evoked by either capsaicin (10 μg kg−1, i.v.) or NKA (1 μg kg−1, i.v.), without any measurable effect on the accompanying hypotensive responses.

The results suggest: (i) ST 48968 is a selective and potent NK2 postjunctional receptor antagonist both in vitro and in vivo in the guinea‐pig, and (ii) the NANC bronchoconstriction evoked by sensory nerve activation either by antidromic nerve stimulation or by capsaicin is mediated mainly via NK2 receptors and only to a minor extent via NK1 receptors.
Inhibition by sumatriptan of central trigeminal neurones only after blood‐brain barrier disruption
Authors: Kaube Holger , Hoskin Karen L. , Goadsby Peter J.
Published: July 2012

The 5‐hydroxytryptamine (5‐HT1)‐like agonist, sumatriptan, is highly efficient in the relief of migraine headache and its accompanying symptoms.

Experimental evidence has indicated that its site of action may be on the cranial vessels or on the trigeminal innervation of the cranium, or both, since sumatriptan does not pass the blood‐brain barrier easily under normal circumstances. It is, however, not clear whether the blood‐brain barrier is normal or abnormal during a migraine attack.

In this study, single unit activity and trigeminal somatosensory evoked potentials in central trigeminal neurones were monitored during electrical stimulation of the superior sagittal sinus.

Intravenous administration of sumatriptan (100 μg kg−1) did not alter trigeminal evoked activity unless the permeability of the blood‐brain barrier had been increased by infusion of an hyperosmolar mannitol solution. After blood‐brain barrier disruption, sumatriptan decreased the peak‐to‐peak amplitude of evoked potentials by 40 ± 6% and the probability of firing of single units by 30 ± 9%. Mannitol infusions alone in control animals caused no changes in evoked potentials or single unit activity.

The data suggest that in normal circumstances sumatriptan does not have sufficient access to trigeminal neurones to alter their function.
Hypercholesterolaemia severely impairs EDRF‐dependent collateral perfusion following acute arterial occlusion in rabbit isolated ear
Authors: Randall Michael D. , Smith Jerry A. , Griffith Tudor M.
Published: July 2012

We have used a rabbit isolated buffer‐perfused ear as a model of acute arterial occlusion to investigate the effects of dietary‐induced hypercholesterolaemia on EDRF‐dependent collateral perfusion. The effects of hypercholesterolaemia on endothelium‐dependent relaxations to acetylcholine were also investigated in the unligated isolated buffer‐perfused ear of the rabbit.

In rabbits receiving a high cholesterol diet (1%) for 4 weeks, blood cholesterol levels were significantly (P < 0.001) increased (26.0 ± 3.6 vs. 2.6 ± 0.6 mmol 1−1), EDRF‐dependent collateral perfusion was similar to that in age‐matched controls for the first 15 min after occlusion but then decreased and was significantly (P < 0.01) less than control during the maintenance phase of collateral perfusion.

Cholesterol feeding for 8 weeks (blood cholesterol = 56.2 ± 3.8 vs. 1.3 ± 0.1 mmol 1−1) was associated with almost complete impairment of collateral perfusion, an effect previously observed following inhibition of EDRF synthesis.

Endothelium‐dependent relaxations to acetylcholine in isolated perfused ears were impaired in the rabbits fed the diet for 8 weeks but not those fed for 4 weeks. In the 8 week group, the maximum relaxation of tone was 32.6 ± 11.6% and was significantly (P < 0.01) less than that in the controls (77.9 ± 5.7%).

We conclude that EDRF‐dependent collateral perfusion is severely impaired in hypercholesterolaemia and that the level of impairment is related to the duration of feeding.
Non‐adrenergic, non‐cholinergic neural activation in guinea‐pig bronchi: powerful and frequency‐dependent stabilizing effect on tone
Authors: Lindén A. , Ullman A. , Löfdahl C.‐G. , Skoogh B.‐E.
Published: July 2012

We examined non‐adrenergic, non‐cholinergic (NANC) stimulation for its stabilizing effect on bronchial smooth‐muscle tone with respect to its regulatory power and the effect of variations in neural impulse frequency.

The guinea‐pig isolated main bronchus (n = 4–12) was pretreated with indomethacin (10 μm) and incubated with atropine (1 μm) and guanethidine (10 μm). Electrical field stimulation (EFS: 1200 mA, 0.5 ms, 240 s) was applied at various levels of tone prior to EFS: first without tone, then at a moderate tone induced by histamine (0.3 μm) and, finally, at a high tone induced by histamine (6 μm). Three different stimulation frequencies (1, 3 or 10 Hz) were used in order to produce moderate to near‐maximum contractile and relaxant NANC neural responses. Both the contractile and the relaxant NANC responses were tetrodotoxin‐sensitive in the guinea‐pig isolated main bronchus (3 Hz).

Without tone prior to EFS, NANC activation (1, 3 or 10 Hz) induced a pronounced contractile response. At a moderate level of tone prior to EFS, NANC activation induced a less pronounced contractile response. At the highest level of tone prior to EFS, NANC activation induced a relaxant response. All these NANC responses adjusted the tone towards a similar level and this ‘stabilization level’ was 56(6)% at 1 Hz, 65(3)% at 3 Hz and 56(5)% at 10 Hz, expressed as a percentage of the maximum histamine‐induced (0.1 mm) tone in each airway preparation.

There was a difference of approximately 90% of maximum between the highest and the lowest tone level prior to NANC activation. This difference was reduced by the converging contractile and relaxant NANC responses and the magnitude of this ‘convergence effect’ was 40(8)% at 1 Hz, 72(4)% at 3 Hz and 90(2)% at 10 Hz.

These findings indicate that NANC neural activation stabilizes bronchial smooth‐muscle tone via a contraction when the tone is low prior to activation and via a relaxation when the tone is high prior to activation. The NANC stabilizing effect on tone appears to be powerful and its magnitude can be controlled by the neural impulse frequency. The level of tone towards which the NANC responses converge does not appear to be markedly altered by variations in the impulse frequency. Our findings are consistent with a regulatory role for NANC responses in the control of bronchial smooth‐muscle tone.
Block of voltage‐dependent sodium currents by the substance P receptor antagonist (±)‐CP‐96,345 in neurones cultured from rat cortex
Authors: Caeser Manfred , Seabrook Guy R. , Kemp John A.
Published: July 2012

Whole cell patch clamp recordings of voltage‐ and tetrodotoxin‐sensitive Na+ currents were made from cultured rat neocortical neurones (E18). The effects of the non‐peptide NK1 receptor antagonist, (±)‐CP‐96,345 on Na+ currents was examined, relative to the effect of the local anaesthetic lignocaine and tetrodotoxin.

Sodium currents were reversibly depressed by bath application of (±)‐CP‐96,345 with a half‐maximally effective concentration of 18 ± 2 μm at a stimulation frequency of 0.1 Hz. Likewise the concentrations required to half‐maximally inhibit sodium currents by tetrodotoxin and lignocaine were 10 ± 2 nm and 1.3 ± 0.2 mm respectively.

The depression of sodium currents by (±)‐CP‐96,345 (10 μm) was use‐dependent in that raising the stimulus frequency from 0.1 Hz to 10 Hz further decreased the magnitude of sodium currents from 60 ± 5% to 37 ± 5% of control values respectively. Similarly, the depression of sodium currents by lignocaine (500 μm) and tetrodotoxin (30 nm) was also accentuated by raising the stimulus frequency from 0.1 Hz to 10 Hz.

The effect of (±)‐CP‐96,345 was not associated with a change in either the activation or steady‐state inactivation characteristics of these currents, suggesting that its mechanism of action was via open channel blockade.

These data demonstrate that in addition to antagonizing NK1 receptors, (±)‐CP‐96,345 also acts as a channel blocker on sodium channels at micromolar concentrations, an effect which should be taken into consideration when examining the antinociceptive or anti‐inflammatory action of this compound.
Effects of rubidium on responses to potassium channel openers in rat isolated aorta
Authors: Greenwood I.A. , Weston A.H.
Published: July 2012

In a physiological salt solution (PSS) in which potassium (K) was replaced by rubidium (Rb), segments of rat aorta precontracted with 20 mm RbCl were fully relaxed by K‐channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were inhibited by glibenclamide.

Segments of rat aorta bathed in normal PSS and precontracted with 20 mm KCl were also relaxed by these K‐channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were glibenclamide‐sensitive. However, the absolute potencies of the K‐channel openers were approximately four times greater in normal PSS than in RbPSS.

In RbPSS, minoxidil sulphate relaxed segments of aorta precontracted with 20 mm RbCl by approximately 20% whereas in normal PSS it fully relaxed those contracted with 20 mm KCl.

In RbPSS, levcromakalim‐induced relaxation of aortic segments precontracted with 20 mm RbCl was initially well‐maintained but then faded by approximately 60% of the initial relaxation to a new, stable level. Subsequent exposure to RP 49356 or to higher concentrations of levcromakalim was without further relaxant effect. Similar changes were observed when RP 49356 was the initial relaxant and tissues were exposed to either RP 49356 or levcromakalim. In normal PSS, levcromakalim‐ or RP 49356‐induced relaxation of contractions produced by 20 mm KCl was well‐maintained.

In RbPSS, minoxidil sulphate‐induced relaxation of aortic segments precontracted with 20 mm RbCl was well‐maintained. Subsequent exposure to either levcromakalim or to RP 49356 produced further tissue relaxation.

In RbPSS, levcromakalim produced no detectable increase in either 86Rb‐ or 42K‐efflux from rat aortic strips. In normal PSS, a significant increase in the exchange of both isotopes was detected.

Levcromakalim hyperpolarized segments of rat aorta bathed both in normal PSS and after depolarization by the addition of 20 mm KCl. Exposure to RbPSS depolarized the tissue and under these conditions, levcromakalim had no effect on membrane potential.

In Rb‐ and normal PSS, levcromakalim produced a similar degree of inhibition of the refilling of the noradrenaline‐sensitive Ca store.

It is concluded that millimolar concentrations of Rb inhibit the plasmalemmal ATP‐sensitive K‐channels (KAtp) which are the target of the K‐channel openers. The relaxant actions of the K‐channel openers in both normal and Rb‐PSS and the inhibition of these effects by glibenclamide may reflect a functional interaction between these agents at ATP‐binding sites associated with both KATP and with intracellular structures including Ca stores.
A mechanism underlying dopamine D1 and D2 receptor‐mediated inhibition of dopaminergic neurones in the ventral tegmental area in vitro
Authors: Momiyama Toshihiko , Todo Naoyuki , Sasa Masashi
Published: July 2012

An intracellular recording study was performed to elucidate the mechanism underlying D1 and D2 receptor‐mediated inhibition of neuronal activities of dopaminergic neurones in the ventral tegmental area (VTA) using slice preparations of the rat brain.

VTA neurones were classified into type I and type II neurones according to the shape of the action potential, which correspond to dopaminergic and non‐dopaminergic neurones, respectively.

Addition of dopamine (10 μm) and quinpirole (1–100 μm) to the bath hyperpolarized the membrane of the type I neurones concomitantly with an increase in membrane conductance and an inhibition of action potentials which occurred spontaneously and were elicited by depolarizing pulses applied to the cell. However, quinpirole (10 μm) had no effect on the threshold for action potentials induced by a depolarizing pulse.

These quinpirole (10 μm)‐induced effects were antagonized by simultaneous application of domperidone (5 μm), a D2 receptor antagonist.

The amplitude of quinpirole (10 μm)‐induced hyperpolarization was decreased by increasing the potassium concentration in the perfusing fluid or simultaneous application of tetraethylammonium (10 μm).

SKF 38393 (10 or 100 μm), a D1 receptor agonist, had no effect on the resting membrane potential or action potential firing induced by a depolarizing pulse applied to the cell. However, when SKF 38393 (10 μm) was applied simultaneously with quinpirole (10 μm), the threshold for action potential generation was elevated by 5–6 mV, although there was no enhancement of hyperpolarization induced by quinpirole.

The elevation of the threshold for action potentials induced by SKF 38393 in the presence of quinpirole was antagonized by simultaneous application of SCH 23390 (5 μm), a D1 receptor antagonist.

Dopamine (10 μm), quinpirole (10 or 100 μm) and SKF 38393 (10 or 100 μm) had no effect on the resting membrane potential or spontaneously occurring action potentials in type II neurones.

These findings suggest that activation of dopamine D2 receptors of dopaminergic neurones in the VTA increases potassium conductance, thereby hyperpolarizing the membrane and eventually inhibiting neuronal activities. They also suggest that simultaneous activation of both D1 and D2 receptors enhances the D2 receptor‐mediated inhibitory effects by elevation of the threshold for action potential generation.
Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells
Authors: Motte S. , Pirotton S. , Boeynaems J.M.
Published: July 2012

The response of bovine aortic endothelial cells to adenosine 5′‐triphosphate (ATP) is mediated by both P2y and nucleotide/P2u receptors. In order to determine which form of the nucleotide is the true ligand of these receptors, we have investigated the effects of divalent cations on ATP‐, uridine 5′‐triphosphate (UTP)‐ and 2 methylthioadenosine 5′‐triphosphate (2MeSATP)‐induced inositol phosphate accumulation in these cells.

Omisson of Mg2+ from a calcium‐free incubation buffer caused a shift to the left of the ATP concentration‐action curve.

In the presence of EDTA (1 mm), the basal level of inositol trisphosphate (InsP3) was markedly increased and the absolute maximal response to ATP was decreased; however, the response to low concentrations of ATP was enhanced.

When the results were plotted in terms of calculated ATP4‐ concentrations, the concentration‐response curves obtained in the presence of 1.25 mm Mg2+ lay closer to the respective curves obtained when Mg2+ was omitted from the medium or when Mg2+ was omitted and EDTA (1 mm) was added. The curves became almost superimposable when the baseline value was subtracted.

A similar shift to the left of the concentrations‐action curves was also observed with both UTP and 2MeSATP.

Our data provide evidence that a form of ATP uncomplexed with divalent cation is the preferential agonist of both the nucleotide/P2u and the P2y receptors expressed on bovine aortic endothelial cells.
The effects of evening primrose oil on nerve function and capillarization in streptozotocin‐diabetic rats: modulation by the cyclo‐oxygenase inhibitor flurbiprofen
Authors: Cameron Norman E. , Cotter Mary A. , Dines Kevin C. , Robertson Sharon , Cox Dominic
Published: July 2012

The aims of this study were first, to examine whether deficits in nerve conduction in streptozotocin‐diabetic rats could be reversed by a 10% dietary supplement of evening primrose oil. Second, to determine the time‐course of reversal, and third, to assess whether the effects could be blocked by the cylco‐oxygenase inhibitor flurbiprofen (5 mg kg−1 day−1).

One‐month diabetes produced 20% and 15% deficits in sciatic motor and saphenous sensory conduction velocity respectively, which were maintained over 2 months diabetes.

The effect of 1‐month evening primrose oil treatment on abnormalities caused by an initial month of untreated diabetes was examined. Motor and sensory nerve conduction velocity were restored to the non‐diabetic level.

Resistance to hypoxic conduction failure was investigated for sciatic nerve trunk in vitro. The 80% conduction failure times were 29% and 55% prolonged by 1‐ and 2‐month diabetes respectively. Evening primrose oil did not reverse the increased hypoxic resistance following 1‐month untreated diabetes.

Sciatic nerve endoneurial capillary density was not significantly affected by diabetes, but was 16% increased in diabetic rats with reversal by evening primrose oil treatment for 1 month compared to 2‐month untreated diabetes.

Serial motor conduction velocity measurement after 3‐month untreated diabetes revealed complete normalization by evening primrose oil within 4 days. Cessation of treatment resulted in a rapid decline in conduction velocity over 24 h.

In a preventive study of 2‐month duration, 6 groups of rats were used. These comprised non‐diabetic controls, diabetic rats, and evening primrose oil‐treated diabetic rats, both with and without flurbiprofen treatment. Flurbiprofen had no significant effect in non‐diabetic rats, but produced an 11% worsening of motor conduction velocity and a 21% reduction of sciatic capillary density in diabetic rats. Evening primrose oil prevented the decreases in conduction velocity and increased hypoxic resistance with diabetes, and caused a 23% increase in capillary density. Flurbiprofen completely blocked the effect of evening primrose oil on conduction velocity, resistance to hypoxia, and capillarization.

Six main conclusions were reached. First, evening primrose oil rapidly reverses conduction deficits in diabetic rats. Second, the effects of treatment may be very short‐lived, suggesting a primary metabolic action. Third, evening primrose oil cannot reverse established changes in hypoxic resistance over 1‐month treatment. Fourth, long‐term treatment causes angiogenesis, suggesting a vascular action. Fifth, products of cyclo‐oxygenase‐mediated metabolism are necessary for maintaining vasa nervorum integrity in diabetic rats. Sixth, evening primrose oil probably acts by providing substrate for vasodilator prostanoid synthesis by vasa nervorum.
Acetylcholine‐induced endothelium‐independent relaxations in monkey isolated superior and inferior caval veins
Authors: Fukushima Sachiyo , Ohhashi Toshio
Published: July 2012

We examined the effects of acetylcholine (ACh), isoprenaline (Isop) and Ca‐ionophore, A23187 on monkey isolated superior (SCV) and inferior caval veins (ICV) with and without intact endothelium, which had been partially contracted by 2× 10−6–5 × 10−6 m prostaglandin F2α (PGF2α).

Low concentrations of ACh (10−10–10−9m) produced a dose‐dependent relaxation in the precontracted venous segments with endothelium. ACh at concentrations more than 10−7 m elicited a transient contraction followed by a relaxation in these segments.

An addition of 5 × 10−7 m A23187 induced about 60% of maximum relaxation produced by 10−5 m sodium nitroprusside (SNP) in each venous segment with endothelium.

Isop (10−10–10−5m) caused a dose‐related relaxation in the precontracted caval veins with intact endothelium.

Removal of endothelium caused no significant effect on the ACh‐induced dual responses but a significant inhibition of the A23187‐induced relaxation.

Pretreatment with atropine antagonized competitively the ACh‐induced relaxations in the endothelium‐intact and endothelium‐denuded caval veins. The Schild plot analysis showed that the pA2 values of the segments with and without endothelium were 9.72 ± 0.14 (n = 5) and 10.01 ± 0.23 (n = 6) in the ICV; and 9.95 ± 0.20 (n = 5) and 9.70 ±0.10 (n = 5) in the SCV, respectively.

Pretreatment with 5 × 10−5 m aspirin, 3 × 10−5 m NG‐nitro‐l‐arginine methylester, 1 mm tetraethylammonium, or 3 × 10−6 m glibenclamide caused no significant effect on the basal tone, ACh‐induced transient contraction, and ACh‐induced relaxation in the precontracted venous segments with and without endothelium.

Pretreatment with 10−5 m methylene blue produced a significant reduction of the ACh‐ and SNP‐induced relaxations in the precontracted venous segments with and without endothelium. The pretreatment with the same concentration of methylene blue, however, caused no significant effect on the Isop‐induced relaxation in venous segments with endothelium.

The results suggest that ACh acts directly on the venous smooth muscle cells via a high‐affinity muscarinic receptor subtype to accumulate cellular cyclic GMP producing endothelium‐independent relaxation in the monkey caval veins.
Metabolism of endothelin‐1 and big endothelin‐1 by recombinant neutral endopeptidase EC.3.4.24.11
Authors: Abassi Zaid A. , Golomb Eliahu , Bridenbaugh Robert , Keiser Harry R.
Published: July 2012

Inhibitors of neutral endopeptidase EC.3.4.24.11 (NEP) have been shown to attenuate the hypertensive effect of big‐endothelin‐1 (BET‐1) in rats. To determine whether NEP converts BET‐1 to endothelin‐1 (ET‐1), the effect of a recombinant NEP (rNEP) on BET‐1 and on ET‐1 was assessed in vitro.

Incubation of [125I]‐ET‐1 with 1 μg ml−1 of rNEP resulted in degradation of the peptide within minutes. Increase in the amount of rNEP to 10 μg ml−1 led to total cleavage of [125I]‐ET‐1 within seconds.

Phosphoramidon (10 μm) or SQ‐28,603 (100 μm) totally suppressed the degradation of [125I]‐ET‐1 by rNEP.

The degradation of [125I]‐BET‐1 by either 1 or 10 μg ml−1 of rNEP was much slower than that of [125I]‐ET‐1. Again, both phosphoramidon and SQ 28,603 protected the peptide from degradation.

Intact [125I]‐ET‐1 was not observed when [125I]‐BET‐1 was incubated with rNEP.

These data show that neutral endopeptidase EC.3.4.24.11 is not an endothelin converting enzyme.
The effects of α2‐adrenoceptor antagonists on the inhibition of 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI)‐induced head shakes by 5‐HT1A receptor agonists in the mouse
Authors: Dursun Serdar M. , Handley Sheila L.
Published: July 2012

8‐Hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT), gepirone, buspirone and ipsapirone dose‐dependently antagonized the head‐shakes induced by 1‐(2,5‐dimethoxy 4‐iodophenyl)‐2‐amino propane hydrochloride (DOI) (1.0 mg kg−1) in mice, when these agents were given i.p. 10 min beforehand.

para‐Chlorophenylalanine (pCPA) abolished the effect of 8‐OH‐DPAT (0.1 mg kg−1) and of buspirone (1.0 mg kg−1). (±)‐Pindolol (5.0 mg kg−1) also antagonized the effect of 8‐OH‐DPAT(0.1 mg kg−1).

The α2‐adrenoceptor antagonists, RX811059 (1.0 mg kg−1), idazoxan (0.5 mg kg−1), yohimbine (1.0 mg kg−1) and 1‐(2‐pyrimidinyl)‐piperazine (1‐PP) (2.0 mg kg−1) i.p. prevented the antagonistic effect of 8‐OH‐DPAT (0.1 mg kg−1) on DOI‐head‐shakes.

Orally‐administered buspirone, given 60 min beforehand, only reduced DOI‐head‐shakes at doses of 60 mg kg−1 and above. However, when buspirone (1.0 mg kg−1) was administered orally twice daily for 21 days, DOI‐head‐shakes were significantly reduced when tested 60 min after the first daily dose on days 5, 12 and 21 and 48 h after withdrawal.

A single oral dose of buspirone (1.0 mg kg−1) strongly antagonized DOI‐head‐shakes when given 24 h after the last of 4 daily doses of 1‐PP (2.0 mg kg−1, p.o.) but had no effect on DOI‐head‐shakes 24 h after the last of 4 daily doses of water (p.o.).

A single oral dose of 1‐PP (2.0 mg kg−1) abolished the inhibitory effect of i.p. buspirone (1.0 mg kg−1) on DOI‐head‐shakes in mice which had received water (p.o.) daily on the 4 previous days but not in mice which had received 1‐PP (2.0 mg kg−1, p.o.) on these days.

The ability of 5‐HT1A receptor agonists to antagonize DOI‐head‐shakes may be due to an effect at presynaptic 5‐HT receptors. It is suggested that 1‐PP, formed from buspirone, may act at α2‐adrenoceptors to prevent acutely administered oral buspirone from antagonizing DOI‐head shakes, but that tolerance occurs to this effect of 1‐PP, thus revealing the inhibitory effect of buspirone when the latter is given repeatedly.
Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation
Authors: Fozard J.R. , Milavec‐Krizman M.
Published: July 2012

A series of adenosine receptor agonists of varying degrees of selectivity induced concentration‐dependent contraction of the rat isolated spleen. With the exception of the response to the selective A2A receptor agonist, 2‐[p‐(2‐carboxyethyl)phenylethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680), responses to each ligand were blocked surmountably and to a broadly similar extent by 8‐p‐sulphophenyltheophylline (10−5 m).

There was a significant correlation between the pEC50 values obtained on the spleen and the binding affinities (pKD; measured with [3H]‐NECA) for the A1 receptor of pig striatum (r = 0.98, P < 0.001) but not the A2A receptor (r = 0.14, NS).

The antagonist potencies of 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) and 9‐chloro‐2‐furyl [1,2,3]triazolo[1,5‐C]quinazoline‐5‐amine (CGS 15943) were measured against the prototype selective A1 receptor agonist, R‐N6‐phenylisopropyladenosine (R‐PIA). The resulting pKB values of 8.67 and 7.70, respectively are consistent with the A1 receptor subtype mediating splenic contraction.

The response to R‐PIA was unaltered in the presence of a concentration (10−7 m) of CGS 21680 which is 6 fold its KD concentration at the A2A binding site in pig striatum but below the threshold for causing contraction per se; thus, A2A receptors inhibitory to contraction appear to be absent.

The response to R‐PIA was resistant to blockade by prazosin (10−7 m) and by nifedipine (10−6 m) but partially blocked by indomethacin (10−6 m).

The results show that the rat isolated spleen responds to adenosine receptor agonists with contraction. Both the relative potencies of agonists and the effects of antagonists indicate mediation by the A1 receptor subtype. α1‐Adrenoceptor activation is not involved in contraction but a role for products of cyclo‐oxygenase and calcium from a source not dependent on entry through L‐channels is implicated.
α2‐Adrenoceptor‐mediated modulation of the nitrergic innervation of the canine isolated ileocolonic junction
Authors: Boeckxstaens G.E. , De Man J.G. , Pelckmans P.A. , Herman A.G. , Van Maercke Y.M.
Published: July 2012

The effects of specific α‐adrenoceptor agonists and antagonists on electrically‐evoked non‐adrenergic non‐cholinergic (NANC) relaxations, previously demonstrated as nitrergic, were investigated in isolated circular muscle strips of the canine ileocolonic junction.

During a substance P‐induced contraction and in the presence of atropine and guanethidine, the specific α1‐adrenoceptor agonist, phenylephrine and antagonist, prazosin, as well as the specific α2‐adrenoceptor antagonist, yohimbine, had no effect on the NANC relaxations evoked by electrical field stimulation. In contrast, clonidine and the more specific α2‐adrenoceptor agonist, UK‐14,304, significantly reduced the electrically‐induced relaxations, preferentially those in response to low frequency stimulation. The inhibitory effect of UK‐14,304 on these relaxations was antagonized by yohimbine.

During a noradrenaline‐induced contraction, clonidine, but not UK‐14,304 significantly augmented the relaxations to electrical stimulation.

The adrenoceptor agonists and antagonists used had no effect on concentration‐response curves to NO or on the relaxation induced by nitroglycerin.

These results indicate that stimulation of prejunctional α2‐adrenoceptors inhibits the nitrergic NANC relaxations induced by field stimulation and thus suggest prejunctional regulation of nitric oxide release via α2‐adrenoceptors in the canine ileocolonic junction.
Signal transduction pathways involved in the acute potentiation of NMDA responses by 1S,3R‐ACPD in rat hippocampal slices
Authors: Harvey Jenni , Collingridge Graham L.
Published: July 2012

A grease‐gap recording technique has been used to investigate the mechanisms underlying the acute potentiation of N‐methyl‐d‐aspartate (NMDA) responses by aminocyclopentane‐1S,3R‐dicarboxylic acid (1S,3R‐ACPD) in area CA1 of rat hippocampal slices.

1S,3R‐ACPD (10 μm), but not 1R,3S‐ ACPD (10 μm), potentiated submaximal responses to NMDA (dose‐ratio of 0.81 ± 0.02 (mean ± s.e.mean); n = 55), but not to α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), in a readily reversible manner. Potentiation also occurred in slices treated with 0.2 μm tetrodotoxin, and in slices perfused with Mg2+‐free medium.

1S,3R‐ACPD‐induced potentiation was unaffected by the protein kinase inhibitors K‐252b (0.1 μm) and staurosporine (1 μm) and the intracellular Ca2+ store depletor, thapsigargin (10 μm). Coapplication of staurosporine and thapsigargin was also without effect.

1S,3R‐ACPD‐induced potentiation was unaffected by inhibitors of arachidonic acid formation, bromophenacyl bromide (50 μm) and RG80267 (100 μm). Arachidonic acid (10–50 μm) depressed reversibly NMDA‐induced responses. The potentiation was unaffected by 8‐bromo cyclic AMP (500 μm) or the PKA inhibitor Rp‐adenosine 3,5‐cyclic monophosphothioate triethylamine (Rp‐cAMPS; 50 μm).

1S,3R‐ACPD‐induced potentiation was abolished in slices perfused with Ca2+‐free medium. The potentiation was also blocked by phorbol‐12,13‐diacetate (1 μm), in a staurosporine‐sensitive manner.

It is concluded that the potentiation of NMDA responses by 1S,3R‐ACPD is not mediated by protein kinase A or C., by release of Ca2+ from intracellular stores or by arachidonic acid. It involves a Ca2+‐sensitive process and is negatively regulated by protein kinase C.
Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng‐Prusoíf equations
Authors: Lazareno S. , Birdsall N.J.M.
Published: July 2012

The estimation of antagonist affinity from functional experiments in which the effect of a fixed agonist concentration is reduced by a range of antagonist concentrations (‘functional inhibition curves’) has been considered from both a theoretical and experimental viewpoint.

Theoretical predictions are compared with results obtained from the stimulation of [35S]‐GTPγS binding by acetylcholine to membranes of Chinese hamster ovary (CHO) cells stably transfected with human m1–m4 muscarinic receptors, and inhibition of the stimulated binding by pirenzepine and AQ‐RA 741.

The usual procedure of applying the Cheng‐Prusoff correction is shown to be theoretically invalid, and predictions are made of the size and distribution of errors associated with this procedure.

A different procedure for estimating antagonist affinity, using the principles of dose‐ratio analysis and analogous to use of the Gaddum equation, is found to be accurate and theoretically valid.

A novel method of analysis allows accurate estimation of both antagonist affinity and Schild slope, by fitting the combined data from an antagonist inhibition curve and an agonist activation curve directly to a form of the Schild equation (derived by Waud) using non‐linear regression analysis.

It is shown that the conventional Schild analysis can be enhanced by treating part of the data as a family of inhibition curves and including in the Schild plot dose‐ratios estimated from the inhibition curves.