bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐peptide antagonists, CP‐96,345 and RP 67580, distinguish species variants in tachykinin NK1 receptors
Authors: Barr A.J. , Watson S.P.
Published: July 2012

The potency of the non‐peptide antagonists CP‐96,345 and RP 67580 on NK1 receptor‐stimulated [3H]‐inositol phosphate accumulation in cell lines or tissue from three different species has been examined.

We have used: UC11 cells, derived from a human astrocytoma, and rat LRM55 glial cells, both of which express large numbers of functional NK1 receptors, and the well characterized guinea‐pig ileum which expresses both NK1 and NK3 receptors.

RP 67580 has an ∼25 fold lower affinity for NK1 receptors in human UC11 cells (Kd = 194 nm) than in rat LRM55 cells (Kd = 7.9 nm), in contrast CP‐96,345 has an ∼200 fold lower affinity in rat LRM55 cells (Kd = 210 nm) relative to human UC11 cells (Kd = 0.99 nm). The pharmacological profile of CP‐96,345 and RP 67580 in guinea‐pig ileum was similar to that observed in human UC11 cells.

In conclusion, we have demonstrated that previously reported species differences in binding affinities for the non‐peptide NK1 antagonists, CP‐96,345 and RP 67580, are also observed in inhibition of NK1 receptor stimulated hydrolysis of inositol phospholipids.
Actions and interactions of NG‐substituted analogues of l‐arginine on NANC neurotransmission in the bovine retractor penis and rat anococcygeus muscles
Authors: Martin William , Gillespie John S. , Gibson Ian F.
Published: July 2012

The effects and interactions of a series of NG‐substituted analogues of l‐arginine known to inhibit nitric oxide synthase were examined on non‐adrenergic, non‐cholinergic (NANC) neurotransmission in the bovine retractor penis (BRP) and rat anococcygeus muscles.

Treatment of BRP muscle strips with either NG‐nitro l‐arginine (l‐NOARG: 0.1–10 μm) or NG‐nitro l‐arginine meythl ester (l‐NAME; 0.1–100 μm) produced a concentration‐dependent blockade of NANC relaxation: blockade was complete at the highest concentration of each.

Pretreatment with l‐arginine (1–10 mm) had no effect on NANC relaxation by itself, but inhibited, in a concentration‐dependent manner, the subsequent ability of both l‐NOARG (0.1–300 μm) and l‐NAME (0.1–1 mm) to produce blockade. l‐Arginine (1–10 mm) reversed established submaximal blockade of NANC relaxation induced by l‐NOARG (1 μm) or l‐NAME (1 μm), but had little effect on maximal blockade induced by these agents.

In contrast to l‐NOARG and l‐NAME, NG‐monomethyl l‐arginine (l‐NMMA; 1 μm‐1 mm) had no effect by itself on NANC relaxation of the BRP. l‐NMMA (0.1–1 mm) did, however, like l‐arginine, inhibit, in a concentration‐dependent manner, the subsequent ability of both l‐NOARG (0.1–1 mm) and l‐NAME (0.1–3 mm) to produce blockade, but was more potent. As with l‐arginine, l‐NMMA (0.1–1 mm) reversed established submaximal blockade of NANC relaxation induced by l‐NOARG (1 μm) or l‐NAME (1 μm), but had little effect on maximal blockade induced by these agents.

In contrast to the effects on BRP, treatment of rat anococcygeus muscle with either l‐NOARG (0.1–10 μm) or l‐NMMA (1–100 μm) produced concentration‐dependent inhibition of NANC relaxation: the maximal inhibition induced by l‐NOARG and l‐NMMA was 100% and 40.1 ± 5.9% (n = 8), respectively. l‐Arginine (1–10 mm) reversed established submaximal inhibition of NANC relaxation induced by l‐NOARG (1 μm), had little effect on maximal blockade by this agent, and reversed maximal blockade induced by l‐NMMA (100 μm).

In the presence of partial blockade of NANC relaxation on rat anococcygeus by a maximal concentration of l‐NMMA (100 μm), subsequent blockade by l‐NOARG (0.1–100 μm) was inhibited. l‐NMMA (100 μm) produced a partial reversal of established submaximal blockade of NANC relaxation induced by l‐NOARG (1 μm), but had little effect on maximal blockade induced by this agent.

These findings suggest a complex series of interactions between l‐arginine and certain of its NG‐substituted analogues that are commonly used to inhibit nitric oxide synthase. The most striking new finding is that l‐NMMA does not block NANC relaxation in the BRP, but acts with greater potency than the endogenous substrate, l‐arginine, to inhibit the blockade induced by l‐NOARG or l‐NAME. Even on rat anococcygeus where l‐NMMA acts as a partial blocker of NANC relaxation, further blockade by l‐NOARG is inhibited.
A factor released by monocytes in the presence of dexamethasone stimulates neutrophil locomotion
Authors: Chettibi S. , Lawrence A.J. , Stevenson R.D.
Published: July 2012

Steroid‐treated monocyte supernatants cause a dramatic increase in the speed of locomotion of human neutrophils and a significant decrease in their adhesion to protein‐coated glass. In contrast, control monocyte supernatants have a smaller effect on the speed of locomotion, but cause a large increase in their adhesiveness.

This supernatant activity was produced equally well in the presence or absence of serum after 24 h culture at 37°C with 10−6 m dexamethasone.

The effect of the steroid‐treated monocyte supernatants on the speed of locomotion of human peripheral blood neutrophils was not altered by rabbit polyclonal antisera against lipocortins 1–6.

Rabbit anti‐interleukin‐8 antibody which blocked the effect of IL‐8 on the speed of locomotion of neutrophils did not antagonize the locomotion stimulating action of steroid‐treated monocyte supernatants.

The exocellular release of this factor(s) by human mononuclear leucocytes suggests that it may be an in vivo mediator of the anti‐inflammatory effect of glucocorticoids.
Pharmacological characteristics of the positive inotropic effect of angiotensin II in the rabbit ventricular myocardium
Authors: Ishihata Akira , Endoh Masao
Published: July 2012

In order to elucidate the mechanism underlying the positive inotropic effect (PIE) of angiotensin II (AII), we measured changes in phosphoinositide hydrolysis and contractile force induced by AII in the rabbit ventricular myocardium.

AII (1.0 nm−3 μm) produced a PIE in a concentration‐dependent manner in the presence of bupranolol (0.3 μm) and prazosin (0.1 μm), the maximal response being about 40% of that to isoprenaline and the EC50 30 nm.

The PIE of AII was associated with a concentration‐dependent increase in the total duration of contraction; the time to peak force and the relaxation time were prolonged.

AII (10 nm −30 μm) elicited an accumulation of [3H]‐inositol monophosphate in a concentration‐dependent manner in rabbit ventricular slices prelabelled with myo‐[3H]‐inositol.

The PIE and the accumulation of [3H]‐inositol monophosphate induced by AII were inhibited by a non‐selective AII receptor antagonist, saralasin (10 nm −1 μm) and by a selective AT1 receptor antagonist, losartan (10 nm −1 μm), but not a selective AT2 receptor antagonist, PD 123319 (1 μm).

A tumour‐promoting phorbol ester, phorbol 12,13‐dibutyrate (PDBu, 10–100 nm), inhibited the AII‐induced PIE and [3H]‐inositol monophosphate accumulation in a concentration‐dependent manner.

These results suggest that AII exerts a PIE through activation of AT1 receptors and subsequent acceleration of phosphoinositide hydrolysis. Activation of protein kinase C by PDBu may inhibit the AII‐induced stimulation of phosphoinositide hydrolysis and thereby the PIE of AII in the rabbit ventricular myocardium.
Regulation of neural responses in the canine pyloric sphincter by opioids
Authors: Bayguinov Orline , Sanders Kenton M.
Published: July 2012

Regulation of excitatory and inhibitory junction potentials (e.j.ps and i.j.ps) by opioid peptides was studied in isolated muscle strips from the pyloric sphincter of the dog.

Methionine enkephalin (MetEnk; 10−10 to 10−6 m) and [d‐Ala2, d‐Leu5] enkephalin (DADLE; 10−11 to 10−7 m), a δ‐specific opioid agonist, inhibited i.j.ps and e.j.ps recorded from cells in the myenteric and submucosal regions of the circular muscle layer. These compounds had no effect on resting potential or slow wave activity suggesting that the effects on junction potentials were not due to direct effects on smooth muscle cells.

MetEnk and DADLE caused similar effects on junction potentials in preparations in which the myenteric plexus was removed, suggesting that opioids inhibit pre‐junctional effects on nerve fibres within the muscularis externa.

Inhibition of junction potentials by MetEnk and DADLE was blocked by approximately the same extent by naloxone (10−6 m) and ICI 174,864 (10−6 m), a δ‐specific antagonist.

MetEnk and DADLE blocked a portion of the i.j.p. that was sensitive to arginine analogues; after treatment with Nω‐nitro‐l‐arginine methyl ester (l‐NAME, 10−4 m), MetEnk and DADLE had no further effect on i.j.ps. These data suggest that opioids regulate nitric oxide‐dependent neurotransmission.

Naloxone (10−6 m) alone had no effect on i.j.ps elicited by short trains of electrical field stimuli.

I.j.p. amplitude was reduced after a period of conditioning stimulation (2 min, 30 Hz, 30 V). Naloxone blocked the post‐stimulation inhibition. Repetitive stimulation at high frequencies (30 Hz) resulted in sustained hyperpolarization. Naloxone increased the amplitude of the hyperpolarization responses elicited by high frequency stimulation.

These results show that e.j.ps and i.j.ps in the canine pylorus are inhibited by opioids. A portion of the inhibitory effects appears to be mediated via δ receptors.

Although pyloric muscles are richly innervated by nerves containing opioid peptides, brief trains of stimuli do not appear to release concentrations of opioids that are effective in regulating junction potentials. Higher frequency stimulation (or longer durations of stimulation) appear to be necessary to release concentrations of opioids that are effective in modulating the amplitude of junction potentials.
Inhibition of nerve stimulation‐induced vasodilatation in corpora cavernosa of the pithed rat by blockade of nitric oxide synthase
Authors: Finberg John P.M. , Levy Shulamit , Vardi Yoram
Published: July 2012

The effect of inhibition of nitric oxide synthase by NG‐nitro‐l‐arginine methyl ester (l‐NAME) on nerve stimulation‐induced vasodilatation in corpora cavernosa was studied in the pithed rat. Corporal vasodilatation was estimated by the increase in ratio (corpora cavernosal pressure/systemic blood pressure; CP/BP) following electrical stimulation of the sacral part of the spinal cord.

l‐NAME (2, 5, 10 and 25 mg kg−1) caused an increase in BP and a dose‐dependent inhibition of the rise in the CP/BP ratio following stimulation.

The inhibitory effect of l‐NAME (25 mg kg−1) on the corporal response to spinal cord stimulation, as well as the pressor response, was partially prevented by prior administration of l‐ but not d‐arginine (400 mg kg−1, i.v.).

l‐NAME (20 mg kg−1, i.v.) did not inhibit the rise in corporal pressure resulting from direct intracavernosal administration of papaverine (400 μg over 2 min). However, this response was inhibited by 5‐hydroxytryptamine (20 μg kg−1, i.v.).

The results are indicative of a role of nitric oxide (NO) in the corporal vasodilator response to erectile stimulation.
Modulation of cholinergic and substance P‐like neurotransmission by nitric oxide in the guinea‐pig ileum
Authors: Wiklund Claes U. , Olgart Caroline , Wiklund N. Peter , Gustafsson Lars E.
Published: July 2012

The role of endogenous nitric oxide (NO) as a modulator of enteric neurotransmission was investigated in longitudinal muscle myenteric plexus (LMMP) preparations of guinea‐pig isolated ileum.

In tissues previously incubated with [3H]‐choline, exogenous NO inhibited electrically‐evoked [3H]‐choline overflow as well as responses to exogenous agonists, indicating that NO has the potential of neuromodulation both pre‐ and postjunctionally.

A series of NO synthase inhibitors enhanced contractile responses to nerve stimulation indicating inhibitory neuromodulation by endogenous NO.

The potency order of the NO synthase inhibitors and their consistent effects after dexamethasone, on responses to nerve stimulation, indicate action on a constitutive NO synthase.

Responses enhanced by NO synthase inhibitors were inhibited by the substance P receptor antagonist, spantide, suggesting a neuromodulatory influence on substance P‐like neurotransmission by the endogenous NO.

NO synthase inhibition did not modify contractile responses to application of acetylcholine or substance P, or [3H]‐choline overflow, indicating that endogenous NO mainly has a prejunctional inhibitory action on substance P‐like neurotransmission. Nor did it modify responses to direct electrical muscle stimulation in the presence of tetrodotoxin. This suggests a prejunctional enhancing effect by NO synthesis inhibition.

Evidence for endogenous NO modulation of acetylcholine release was obtained when NO synthase inhibition modified atropine‐sensitive, nerve‐mediated contractile responses. However, [3H]‐choline overflow was unaltered by NO synthase inhibition.

NO synthase inhibition did not modify responses to inhibitory neurotransmission.

The findings suggest that endogenous NO inhibits substance P‐like motor neurotransmission, probably via prejunctional mechanisms. Cholinergic transmission may also be reduced by endogenous NO, acting prejunctionally.
The effect of cyclic AMP elevating agents on bradykinin‐ and carbachol‐induced signal transduction in canine cultured tracheal smooth muscle cells
Authors: Yang Chuen Mao , Hsia Hui‐Chuan , Luo Shue‐Fen , Hsieh Jen‐Tsung , Ong Richard
Published: July 2012

The effects of cholera toxin (CTX), forskolin and dibutyryl cyclic AMP on bradykinin (BK)‐ and carbachol‐induced accumulation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The BK‐induced responses were mediated via a G protein which was not inhibited by CTX or pertussis toxin treatment.

BK‐stimulated IPs accumulation and Ca2+ mobilization were potentiated by CTX (10 μg ml−1) pretreatment which was time‐dependent. Maximal increase of these responses occurred after 24 h treatment with CTX. The concentration‐effect relationship of BK‐induced responses were shifted to the left and BK was substantially more effective in CTX‐treated cells than in the control cells. This enhancing effect of CTX did not occur with carbachol.

Short‐term (< 4 h) treatment with forskolin (10 μm) or dibutyryl cyclic AMP (1 mm) failed to accentuate BK‐induced responses, but long‐term (> 4 h) treatment of TSMCs with these agents mimicked the enhancing effect of CTX, suggesting that CTX‐induced enhancement of BK responsiveness might be due to a rise in cyclic AMP.

Prolonged treatment of TSMCs with these agents was accompanied by an increase in cell surface [3H]‐BK binding sites, which was inhibited by concurrent incubation with cycloheximide, an inhibitor of protein biosynthesis. Cycloheximide also abolished the potentiating actions of CTX, forskolin, and dibutyryl cyclic AMP on BK‐induced IPs formation and Ca2+ mobilization.

The locus of this enhancement was further investigated by examining the effects of CTX, forskolin and dibutyryl cyclic AMP on AlF4‐induced IPs accumulation in canine TSMCs. AlF4−‐induced IPs accumulation was not affected by CTX, forskolin, or dibutyryl cyclic AMP treatment, supporting the contention that this stimulatory effect is located at the BK receptor level.

These results demonstrate that the augmentation of BK‐induced IPs accumulation and Ca2+ mobilization produced by CTX, forskolin and dibutyryl cyclic AMP involves a cyclic AMP‐dependent mechanism which is induced by a sustained increase in the level of intracellular cyclic AMP. CTX and forskolin may promote an increase of the synthesis of BK receptors, and thereby enhance BK‐induced responses.
Effects of bosentan (Ro 47–0203), an ETA‐, ETB‐receptor antagonist, on regional haemodynamic responses to endothelins in conscious rats
Authors: Gardiner S.M. , Kemp P.A. , March J.E. , Bennett T.
Published: July 2012

Regional haemodynamic responses to endothelin (ET)‐1, −2 and −3 and big ET‐1 (all at 500 pmol kg−1) were assessed in the same conscious Long Evans rats (n = 8) in the absence or presence of the mixed ETA‐, ETB‐receptor antagonist, Ro 47–0203 (bosentan; 30 mg kg−1).

Bosentan blocked the initial depressor, tachycardic and hindquarters hyperaemic vasodilator effects of ET‐1, −2 and −3, and substantially curtailed the primary renal and secondary hindquarters vasoconstrictor responses. Bosentan did not inhibit the initial mesenteric vasoconstrictor action of ET‐1, but reduced the duration of the later mesenteric vasoconstriction. In contrast, bosentan delayed the rate of onset, and reduced the duration, of the mesenteric vasoconstrictor actions of ET‐2 and ET‐3. The most likely explanation of this finding is that ET‐1, but not ET‐2 or ET‐3, triggered a covert mesenteric vasodilator mechanism which was antagonized by bosentan.

Bosentan blocked all the effects of big ET‐1, and, in a separate group of rats (n = 7), blocked all the haemodynamic effects of a lower dose of ET‐1 (50 pmol kg−1), with the exception of a slight mesenteric vasoconstriction.

The most straightforward explanation of the results is that the major haemodynamic effects of ET‐1, −2 and −3, and all the effects of big ET‐1, are mediated through ETA‐ and/or ETB‐receptors that are effectively antagonized by bosentan.
Enhancement of arterial relaxation by long‐term atenolol treatment in spontaneously hypertensive rats
Authors: Kähönen Mika , Mäkynen Heikki , Arvola Pertti , Pörsti Ilkka
Published: July 2012

The effects of long‐term atenolol (25 mg kg−1 day−1) therapy on arterial function were studied in spontaneously hypertensive rats (SHR) and Wistar‐Kyoto (WKY) rats. The 14‐week treatment attenuated the increase in blood pressure by approximately 30 mmHg in SHR, but did not affect blood pressure in WKY rats.

Responses of mesenteric arterial rings in vitro were examined at the end of the study. The relaxation to acetylcholine was similar in WKY rats and atenolol‐treated SHR and more pronounced than in untreated SHR, whereas the relaxation to the nitric oxide donor 3‐morpholinosydnonimine (SIN‐1) was comparable in all study groups. Moreover, after maximal relaxations to acetylcholine, marked recontractions developed in untreated SHR but not in the other groups. Vasorelaxation to isoprenaline was also attenuated in SHR and was moderately improved by the atenolol therapy.

Arterial relaxation induced by return of potassium to the organ bath upon precontractions elicited by potassium‐free solution were used to evaluate vascular smooth muscle Na+,K+‐ATPase. The rate of potassium relaxation was fastest in WKY rats and was also faster in atenolol‐treated than in untreated SHR.

The ability of vascular smooth muscle to sequester calcium was evaluated by eliciting responses to caffeine or noradrenaline after loading periods in different organ bath calcium concentrations. The subsequent contractions were lower in untreated SHR than in WKY rats, and augmented in SHR by the atenolol treatment.

Smooth muscle contractions to noradrenaline were comparable in SHR and WKY rats, while atenolol treatment slightly increased the maximal response to this agonist in SHR. Responses to potassium chloride were not affected by atenolol and contractions following cumulative re‐addition of calcium to the organ bath after precontraction with potassium chloride and noradrenaline in calcium‐free solution were comparable in all study groups.

In conclusion, the moderate antihypertensive effect of atenolol in SHR was accompanied by enhancement of β‐adrenoceptor‐mediated and normalization of endothelium‐dependent arterial relaxation. Furthermore, ability to sequester calcium into cellular stores, and function of Na+,K+‐ATPase were augmented in vascular smooth muscle. Therefore, the present results suggest that the long‐term blood pressure‐lowering action of atenolol in this type of genetic hypertension is accompanied by improved arterial relaxation and normalization of endothelial function.
Mediation by 5‐HT1D receptors of 5‐hydroxytryptamine‐induced contractions of rabbit middle and posterior cerebral arteries
Authors: Deckert Valérie , Pruneau Didier , Elghozi Jean‐Luc
Published: July 2012

5‐Hydroxytryptamine (5‐HT) receptor‐mediated contraction of endothelium denuded rabbit middle (MCA) and posterior (PCA) cerebral arteries was characterized by use of selective agonists and antagonists for different 5‐HT receptor subtypes.

5‐HT and various 5‐HT receptor agonists contracted the arteries with the following rank order of potency in MCA: 5‐carboxamidotryptamine (5‐CT) > 5‐HT > 5‐methoxytryptamine (5‐MeOT) > sumatriptan > α‐methyl‐5‐HT (α‐Me‐5‐HT) >> 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT) and in PCA: 5‐CT > 5‐HT >sumatriptan > 5‐MeOT > α‐Me‐5‐HT >> 8‐OH‐DPAT. With few exceptions, the maximal contractile responses of these agonists were similar to that induced by 5‐HT.

The selective antagonists of 5‐HT2A/2C (ketanserin), 5‐HT4 (SDZ 205–557) and 5‐HT1A/1B (S‐(−)‐propranolol) sites were devoid of inhibitory effect on 5‐HT‐mediated contraction in both MCA and PCA, thus excluding activation of the corresponding receptors.

In both arteries, the contraction‐response curve to 5‐HT was unaffected by the 5‐HT3 receptor antagonist, ICS 205–930 (0.01 and 0.1 μm) whilst a small (3 and 6 fold displacement) was seen with MDL 72222 (0.1 and 1 μm).

The mixed 5‐HT1‐like/5‐HT2A receptor antagonist, methiothepin (0.001–0.1 μm), was a potent antagonist of 5‐HT‐induced contractions in both arteries, giving pA2 values of 9.4 ± 0.7 and 9.6 ± 0.8 in MCA and PCA, respectively.

Rauwolscine (0.1–10 μm) and yohimbine (0.3, 3 μm) inhibited contractions to 5‐HT in a competitive manner, pA2 values of 7.1 ± 0.6 and 6.7 ± 0.6 were determined for rauwolscine in MCA and PCA, respectively. An apparent pA2 value of 6.9 ± 0.2 was calculated for yohimbine (3 μm) in both MCA and PCA.

In conclusion, these results suggest that the contractile response to 5‐HT in rabbit isolated MCA and PCA is predominantly mediated by the 5‐HT1D receptor subtype, although a small contribution by 5‐HT3 receptors cannot be excluded.
Potentiation by adenosine of ATP‐evoked dopamine release via a pertussis toxin‐sensitive mechanism in rat phaeochromocytoma PC12 cells
Authors: Koizumi Schuichi , Watano Tomokazu , Nakazawa Ken , Inoue Kazuhide
Published: July 2012

The effects of adenosine on adenosine 5′‐triphosphate (ATP)‐evoked dopamine release from rat phaeochromocytoma PC12 cells was investigated to determine whether adenosine exerts a regulatory effect on the ATP‐evoked response. Adenosine potentiated ATP (30 μm)‐evoked dopamine release in a concentration‐dependent manner over a concentration‐range of 1 to 100 μm. Adenosine (100 μm) shifted the concentration‐dependence of the ATP‐evoked response to the left without affecting the maximal response.

Aminophylline, a non‐selective adenosine receptor antagonist, and CP66713, a selective antagonist at the A2 subclass of adenosine receptors, abolished the adenosine‐induced potentiation. Furthermore, 8‐cyclopentyltheophylline, a selective antagonist at the adenosine A1 receptor partially inhibited the adenosine‐evoked potentiation. CGS22492, a selective A2 receptor agonist, potentiated ATP‐evoked dopamine release whereas N6‐cyclohexyladenosine (CHA), a selective A1 receptor agonist, had no effect.

Pertussis toxin (PTX), a bacterial exotoxin which catalyzes the ADP‐ribosylation of guanosine 5′‐triphosphate (GTP)‐binding proteins (G‐proteins), inhibited the adenosine‐induced potentiation of dopamine release. Dibutyryl cyclic AMP (db cyclic AMP), an analogue of cyclic AMP, had no effect on the release on the ATP‐evoked response.

Adenosine potentiated the ATP‐evoked rise in intracellular Ca2+ concentration ([Ca]i) in PC12 cells. This potentiation was also observed with CGS 22492 but not with CHA. PTX completely inhibited the adenosine‐induced potentiation of the rise in [Ca]i.

On the basis of these findings, we suggest that the adenosine‐induced potentiation of ATP‐evoked dopamine release was due to an increase in [Ca]i in the cells. Although the potentiation is most likely mediated by a subclass of A2 receptors, the subclass may be different from those previously reported since the potentiation was sensitive to PTX and was not reproduced by db cyclic AMP.
Reduced endothelium‐dependent relaxation at enhanced NO release in hearts of hypercholesterolaemic rabbits
Authors: Woditsch Isabelle , Schrör Karsten
Published: July 2012

Langendorff hearts, perfused at constant volume, were prepared from rabbits fed a cholesterol‐enriched diet for 4 months. Coronary perfusion pressure and nitric oxide (NO) release (oxyhaemoglobin technique) into the coronary effluent were measured continuously. Prostacyclin (PGI2) in the effluents was determined by radioimmunoassay (6‐oxo‐PGF1α).

Basal NO release was not different between control and hypercholesterolaemic rabbits. However, the coronary vasculature of hypercholesterolaemic rabbits showed a considerably (> 50%) reduced endothelium‐dependent relaxation in response to short‐term (3 min) infusion of bradykinin (50 nm) and substance P (50 nm) (P<0.05, n = 8–9). Under these conditions, NO release into the vessel lumen was increased, by 26%, in hypercholesterolaemic hearts (P<0.05, n = 8–9). NG‐nitro‐l‐arginine (l‐NOARG, 30 μm) significantly attenuated both bradykinin‐induced NO formation and vessel relaxation in control hearts but only NO release in hypercholesterolaemia. l‐Arginine (200 μm) restored the response to that before l‐NOARG but did not improve the reduced endothelium‐dependent relaxation in cholesterol‐fed rabbits.

Superoxide dismutase (10 u ml−1) significantly improved vessel relaxation without changing the hypercholesterolaemia‐related coronary dysfunction. Vasodilatation in response to exogenous NO donors (linsidomine) was diminished in hypercholesterolaemia as compared to controls.

Basal PGI2 release was unchanged in hypercholesterolaemic hearts. There was a tendency in these hearts for greater PGI2 formation after stimulation by substance P and bradykinin (P ≥ 0.05). The coronary relaxation to iloprost was unchanged.

The data demonstrate impaired endothelium‐dependent relaxation of coronary arterial resistance vessels in hypercholesterolaemia. This diminished vascular response was not due to reduced NO generation but probably a reduced action of released NO, either by accelerated degradation and/or disturbed signal transduction pathways to vascular smooth muscle cells. There was no significant change in PGI2 related pathways of vasomotor control in hypercholesterolaemia.
A nucleotide receptor that mobilizes Ca2+in the mouse submandibular salivary cell line ST885
Authors: Gibb C.A. , Singh S. , Cook D.I. , Poronnik P. , Conigrave A.D.
Published: July 2012

We have identified a Ca2+‐mobilizing receptor that responds to extracellular ATP (0.1–1000 μm) in a salivary epithelial cell line (ST885). The activated receptor also stimulates Ca2+entry from the extracellular fluid.

The receptor shows an agonist selectivity profile consistent with the behaviour of the nucleotide receptor class. In particular, the pyrimidine nucleotide, UTP, was equipotent with ATP.

Analysis of concentration‐response data for the ATP and UTP‐activated receptor using the Hill equation yielded EC50 values (concentrations of agonist inducing a half‐maximal response) for ATP of 4.2 μm and for UTP of 4.6 μm.

ATP and UTP induced cross‐desensitization and were not additive when administered simultaneously at maximal concentration. These findings are consistent with the hypothesis that both nucleotides act upon a common receptor.

The ATP analogue, 2‐methylthio ATP, mobilized Ca2+ions with higher potency (EC50 = 0.23 μm) but lower efficacy than either ATP or UTP. However, 2‐methylthio ATP was not a partial agonist of the ATP/UTP receptor. At maximal concentration (30 μm), it failed to antagonize the effects of near maximal concentrations of ATP or UTP. We conclude that 2‐methylthio ATP acts upon a distinct receptor.
Bradykinin binding sites in healthy and carcinomatous human lung
Authors: Trifilieff Alexandre , Lach Estelle , Dumont Pascal , Gies Jean‐Pierre
Published: July 2012

Direct ligand binding techniques have been used to compare bradykinin receptors in squamous‐ or adeno‐carcinoma and healthy lung membranes removed from patients during operations.

The binding of [3H]‐bradykinin to healthy lung membrane is time‐dependent and saturable with a KD value of 1.08 ± 0.18 nm and a Bmax value of 46.1 ± 3.1 fmol mg−1protein (n = 10). In squamous‐carcinoma tissue (n = 8) the same amount of receptors are present, Bmax = 52.2 ± 3.3 fmol mg−1protein (P = 0.22) but the KD value is significantly higher 2.57 ± 0.40 nm (P = 0.004). Similar measurements were obtained for adeno‐carcinoma tissue (n = 3), KD = 2.80 ± 0.29 mm (P = 0.001) and Bmax = 49.8 ± 2.1 fmol mg−1protein (P = 0.56).

In both healthy and squamous‐carcinoma preparations, bradykinin analogues displace [3H]‐bradykinin binding with the following relative order of potency: Hoe 140 > bradykinin > kallidin > d‐Arg0[Hyp3,d‐Phe7]bradykinin >>>des‐Arg9‐bradykinin. Of the analogues used, bradykinin and d‐Arg0[Hyp3,d‐Phe7]bradykinin appear to be able to differentiate the bradykinin receptors present in both preparations.

It is concluded that bradykinin receptors present in healthy and carcinomatous human lung are of the B2 type.
Characterization of the 5‐HT receptor potentiating neuromuscular cholinergic transmission in strips of human isolated detrusor muscle
Authors: Tonini M. , Messori E. , Franceschetti G.P. , Rizzi C.A. , Castoldi A.F. , Coccini T. , Candura S.M.
Published: July 2012

In human isolated detrusor strips, submaximal contractile responses evoked by electrical stimulation were resistant to hexamethonium (30 μm) and abolished by tetrodotoxin (0.6 μm) and hyoscine (1 μm), indicating the activation of postganglionic cholinergic nerves. In methysergide (1 μm) and ondansetron (3 μm) pretreated tissues, 5‐hydroxytryptamine (5‐HT) (0.3 nm − 1 μm) caused a concentration‐dependent increase in the amplitude of contractions (pEC50 = 8.1), which was antagonized by the selective 5‐HT4 receptor antagonist GR 113808 (3, 10 and 30 nm) in a competitive manner. Schild analysis yielded a pA2 estimate of 8.9, a value comparable to that reported for GR 113808 in other animal and human peripheral tissues (8.8–9.7). Our findings indicate that neuromuscular cholinergic transmission in human isolated detrusor muscle is facilitated by neural 5‐HT receptors belonging to the 5‐HT4 subtype. The human urinary bladder can thus be regarded as an additional site in which 5‐HT4 receptors are distributed.
Photon pharmacology of an iron‐sulphur cluster nitrosyl compound acting on smooth muscle
Authors: Matthews E.K. , Seaton E.D. , Forsyth M.J. , Humphrey P.P.A.
Published: July 2012

The mechanisms of action on smooth muscle of the iron‐sulphur cluster nitrosyl compound, heptanitrosyl‐tri‐μ3‐thioxotetraferrate (1‐), (RBS), a photosensitive nitric oxide donor, have been investigated in the guinea‐pig taenia caeci (coli) in vitro.

After exposure to RBS (50 μm) for 30 min, and subsequent washout, a sustained contraction was recorded in the absence of light to either the agonist carbachol (50 μm) or a depolarizing concentration of KC1 (23.5 mm). Photon irradiation (>400 nm) caused a prompt relaxation of precontracted RBS‐treated muscle, the magnitude of which depended upon the intensity (1.1 × 103 to 1.1 × 105 lux), duration (30 s to 20 min) and wavelength (400 to 800 nm), of the incident illumination.

Repeated periods of illumination at 1.1 × 104 lux produced a reversible relaxation of both carbachol and KC1‐evoked tone in muscle pretreated with RBS (50 μm). These photorelaxations were reproducible at 10 min intervals for several hours with a maximal relaxation amounting to 80 to 90% that of the tone produced by carbachol (50 μm).

The nitric oxide synthase inhibitor, NG‐nitro‐l‐arginine (60 μm), caused no inhibition of the photon‐induced relaxation of RBS‐treated muscle. In contrast, N‐methylhydroxylamine (2 mm), l‐cysteine (10 mm), dl‐dithiothreitol (2 mm), methylene blue (30 μm), and haemoglobin (20 μm), all reversibly but significantly inhibited (P < 0.001) the photorelaxation response. However, neither the aminothiol N‐acetyl‐l‐cysteine (10 mm) nor the tripeptide glutathione (10 mm) blocked the RBS‐induced photorelaxation.

The photolytic cleavage of RBS depended on the intensity and duration of illumination; it was accompanied by a corresponding decrease in absorbance and by the liberation of NO as measured by the Griess diazo reaction with sulphanilic acid. l‐Cysteine (10 mm) prevented the decrease in absorbance and the photolytic liberation of NO.

It is concluded that (i) sequestered or bound RBS, when photon‐activated, liberates NO by a process which can be controlled by the wavelength, intensity and duration of the incident light, (ii) the photon‐released NO rapidly relaxes the smooth muscle cells of the taenia coli primarily via cyclic GMP‐dependent pathways which can be blocked by use of appropriate inhibitors, and (iii) the RBS‐induced photorelaxation effect does not involve the activation of NO synthase. RBS is therefore a valuable photosensitive NO donor for establishing the functional and pharmacological significance of NO.
Anti‐ischaemic efficacy of a nitric oxide synthase inhibitor and a N‐methyl‐d‐aspartate receptor antagonist in models of transient and permanent focal cerebral ischaemia
Authors: Dawson D.A. , Graham D.I. , McCulloch J. , Macrae I.M.
Published: July 2012

We have recently developed a new model of transient focal ischaemia in the rat utilising topical application of endothelin‐1 to the left middle cerebral artery (MCA). In order to validate this approach the present study assessed the neuroprotective efficacy of the NMDA receptor antagonist dizocilpine (MK‐801) in the endothelin‐1 model. The anti‐ischaemic efficacy of the nitric oxide (NO) synthase inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME) was subsequently evaluated, and contrasted with its efficacy against permanent focal ischaemia, to determine the utility of the endothelin‐1 model for identification of novel pharmacoprotective agents.

MK‐801 (0.12 mg kg−1 bolus, 108 μg kg−1 h−1 infusion i.v., either 1 or 2.5 h pre‐transient MCA occlusion (MCAO)) induced hypotension that persisted for approximately 1.5 h so that mean arterial blood pressure (MABP) at the time of MCAO was significantly lower in the 1 h group compared with control (MABP: 86 ± 11, 68 ± 6 and 84 ± 4 mmHg (mean ± s.d.) for saline, 1h MK‐801 and 2.5 h MK‐801 groups respectively). The 2.5 h pretreatment schedule resulted in significant reduction (71%) in the volume of hemispheric damage (assessed 4 h post onset of ischaemia) while the 1 h pretreatment schedule did not (volumes of hemispheric damage: 59 ± 38, 51 ± 51 and 17 ± 28 mm3 for saline, 1 h and 2.5 h MK‐801 groups).

Thus the considerable neuroprotective effect of MK‐801 in the endothelin‐1 model of transient focal cerebral ischaemia was highly sensitive to drug‐induced hypotension. This result is in contrast to previous studies of permanent MCAO where MK‐801‐induced hypotension did not compromise its neuroprotective action.

l‐NAME (3 mg kg−1, i.v. 30 min pre‐MCAO) moderately, but significantly, reduced (16%) the volume of ischaemic damage 4 h post‐permanent MCA occlusion, whereas the 29% reduction in volume of damage achieved in the model of transient focal ischaemia did not attain significance due to the greater variability associated with this model. l‐NAME did not significantly alter MABP in either model.

The modest neuroprotection achieved with NO synthase inhibition suggests NO is of relatively minor importance as a mediator of neurotoxicity following permanent focal cerebral ischaemia. In addition the comparable efficacy of l‐NAME against transient focal ischaemia suggests the presence of reperfusion does not enhance the contribution of NO to neuronal injury in the acute (4 h) phase following a focal ischaemic insult.
In vivo pharmacological characterization of the non‐peptide endothelin receptor antagonist SB 209670
Authors: Douglas Stephen A. , Edwards Richard M. , Elliott John D. , Ohlstein Eliot H.
Published: July 2012

The aim of the present study was to assess the ability of SB 209670, a high affinity non‐peptide endothelin receptor antagonist (0.4 and 18 nm Kis at human cloned ETA and ETB receptors, respectively), to inhibit the haemodynamic actions of endothelin‐1 in vivo.

Systemic administration of (±)‐SB 209670, given either as a bolus i.v. injection or as a continuous i.v. infusion, did not alter basal haemodynamic parameters in the anaesthetized rat.

Infusion of (±)‐SB 209670 (10 μg kg−1 min−1) selectively inhibited the depressor and carotid vasodilator response to exogenous endothelin‐1: 100 μg kg−1 min−1 was required to inhibit significantly the biphasic haemodynamic actions of endothelin‐1. The haemodynamic actions of angiotensin II and calcitonin gene‐related peptide were unaltered by 100 μg kg−1 min−1 (±)‐SB 209670.

Bolus i.v. administration of (±)‐SB 209670 (1 mg kg−1) selectively inhibited the depressor and carotid vasodilator actions of endothelin‐1: 10 mg kg−1 (±)‐SB 209670 was required to inhibit the secondary vasoconstrictor actions of endothelin‐1.

(±)‐SB 209670 (10 mg kg−1) was also effective at antagonizing the pressor actions of endothelin‐1 in the conscious rat for up to 3 h after intraduodenal administration thereby demonstrating that the antagonist was bioavailable upon enteric administration. This dose of (±)‐SB 209670 did not alter basal haemodynamic parameters in the conscious rat.

Thus, (±)‐SB 209670 is an effective endothelin receptor antagonist in vivo. Using the doses defined in this study, SB 209670 may, therefore, serve as a useful tool for understanding the role of endogenous endothelin‐1 in the control of cardiovascular function under both physiological and pathophysiological conditions.
Pharmacological evidence that nitric oxide may be a retrograde messenger in the enteric nervous system
Authors: Yuan S.Y. , Bornstein J.C. , Furness J.B.
Published: July 2012

The effects of inhibition of nitric oxide synthase on neuro‐neuronal and neuromuscular transmission during motility reflexes in the small intestine of the guinea‐pig were examined.

Isolated segments of intestine were secured in a three chambered organ bath so that different parts of the reflex pathways could be independently exposed to drug‐containing solutions. Reflexes were evoked by distension or compression of the mucosa in two adjacent chambers and reflex responses were recorded from the circular muscle with intracellular microelectrodes in the third chamber. Thus, the actions of drugs at connections between sensory neurones and interneurones, between interneurones and other interneurones and at motor neurones could be distinguished.

NG‐monomethyl‐l‐arginine (l‐NMMA; 100 μm), an inhibitor of nitric oxide synthase, did not affect the ascending excitatory reflex when added to either the central stimulation chamber or the recording chamber.

In contrast, l‐NMMA (100 μm) enhanced the descending inhibitory reflex when added to the chamber in which stimuli were applied. This effect was prevented by prior exposure to l‐arginine (100 μm), which had no effect by itself. Conduction of reflexes between the stimulus chamber and the recording chamber was unaffected by the presence of l‐NMMA in an intervening chamber.

l‐NMMA (100 μm) added to the recording chamber depressed the descending inhibitory reflex, an effect that was prevented by previous exposure to l‐arginine.

The nitric oxide donor, sodium nitroprusside (100 μm), added to the stimulus chamber, depressed both ascending excitatory and descending inhibitory reflexes. When added to the middle chamber, sodium nitroprusside had no effect on conduction of reflexes through this chamber.

It is deduced that nitric oxide, released from the cell bodies of descending interneurones, suppresses transmission from synaptic connections made with them by enteric sensory neurones.
Desensitization of the nicotine‐induced mesolimbic dopamine responses during constant infusion with nicotine
Authors: Benwell Maureen E.M. , Balfour David J.K. , Birrell Claire E.
Published: July 2012

The effects of constant nicotine infusions (0.25, 1.0 and 4.0 mg kg−1 day−1) on extracellular dopamine levels in the nucleus accumbens (NAc) and on locomotor activity have been compared with the changes evoked by repeated daily injections (0.4 mg kg−1 day−1 for 5 days) of the drug.

The extracellular dopamine concentration in the NAc was significantly increased (P<0.05) following a challenge dose of nicotine (0.4 mg kg−1, s.c.) in animals which had been pretreated with daily injections of the drug. This effect was accompanied by an enhanced locomotor response to nicotine.

The stimulant effects of nicotine on mesolimbic dopamine secretion and on locomotor activity were significantly inhibited (P<0.01) by the prior administration of mecamylamine (2.0 mg kg−1, s.c.) but not by hexamethonium (2.0 mg kg−1, s.c).

The constant infusion of nicotine at a rate of 1 and 4 but not 0.25 mg kg−1 day−1 abolished the sensitized dopamine response in the NAc to an injection of nicotine in animals pretreated with the drug. The locomotor responses to nicotine in the nicotine‐pretreated rats were significantly attenuated by the infusion of nicotine at all 3 doses, although the nicotine induced locomotor activity, in the rats infused with 0.25 mg kg−1 day−1 was also significantly (P<0.05) higher than that observed in the rats treated acutely with nicotine.

Significantly (P<0.01) enhanced mesolimbic dopamine responses, to a challenge injection of nicotine (0.4 mg kg−1, s.c), were observed 2 and 7 days after termination of the infusion of nicotine (4 mg kg−1 day−1 for 14 days); locomotor responses were enhanced (P<0.01) 1, 2 and 7 days after termination of the infusion.

The results suggest that sensitized mesolimbic dopamine responses to nicotine occur as a result of stimulation of centrally located nicotinic receptors but that these receptors may be desensitized during periods of chronic exposure to nicotine at doses which may be relevant to smoking.
Mediation of noradrenaline‐induced contractions of rat aorta by the (α1B‐adrenoceptor subtype
Authors: Testa R. , Guarneri L. , Poggesi E. , Simonazzi I. , Taddei C. , Leonardi A.
Published: July 2012

The subtypes of α1‐adrenoceptor mediating contractions to exogenous noradrenaline (NA) in rat aorta have been examined in both biochemical and functional studies.

Incubation of rat aortic membranes with the irreversible α1B‐adrenoceptor antagonist, chloroethylclonidine (CEC: 10 μm) did not change the KD of [3H]‐prazosin binding in comparison to untreated membranes, but reduced by 88% the total number of binding sites (Bmax).

Contractions of rat aortic strips to NA after CEC (50 μm for 30 min) incubation followed by repetitive washing, showed a marked shift in the potency of NA and a partial reduction in the maximum response. The residual contractions to NA after CEC incubation were not affected by prazosin (10 nm).

The competitive antagonists prazosin, terazosin, (R)‐YM‐12617, phentolamine, 5‐methylurapidil and spiperone inhibited contractions to NA with estimated pA2 values of 9.85, 8.54, 9.34, 7.71, 7.64 and 8.41, respectively.

The affinity of the same antagonists for the α1A‐ and α1B‐adrenoceptors was evaluated by utilizing membranes from rat hippocampus pretreated with CEC, and rat liver, respectively. 5‐Methylurapidil and phentolamine were confirmed as selective for the α1A‐adrenoceptors, whereas spiperone was α1B‐selective.

A significant correlation was found between the pA2 values of the α1‐adrenoceptor antagonists tested and their affinity for the α1B‐adrenoceptor subtype, but not for the α1A‐subtype.

In conclusion, these findings indicate that in rat aorta most of the contraction is mediated by α1B‐adrenoceptors, and that the potency (pA2) of an antagonist in this tissue should be related to its antagonistic effect on this subtype of the α1‐adrenoceptor population.
Effects of the selective I1 imidazoline receptor agonist, moxonidine, on gastric secretion and gastric mucosal injury in rats
Authors: Glavin Gary B. , Smyth Donald D.
Published: July 2012

Previous reports of the effects of α2‐adrenoceptor stimulation on gastric secretion are inconsistent because it was not clear whether the compounds were activating α2‐adrenoceptors and/or newly described imidazoline receptors. In the present experiments, the effects of moxonidine, an I1‐imidazoline receptor agonist and antihypertensive agent, on gastric secretion and on experimental gastric mucosal injury were examined.

Moxonidine (0.01, 0.1 and 1.0 mg kg−1, i.p.) potently inhibited basal (non‐stimulated) gastric acid secretion in conscious rats with an ED50 of 0.04 mg kg−1. Two hours following administration of the highest dose of moxonidine (1.0 mg kg−1), gastric acid output was completely suppressed. Moxonidine also significantly increased intragastric pH, at the two highest doses.

The α2‐adrenoceptor agonist, clonidine (0.01, 0.1 and 1.0 mg kg−1, i.p.) decreased basal acid secretion at the lowest dose (37%) and at the highest dose (46%), while the intermediate dose did not affect gastric acid output.

In an ethanol‐induced model of gastric mucosal injury, moxonidine decreased the length of lesions at the lowest and highest doses (0.01 and 1.0 mg kg−1) as well as the number of the lesions, at the highest dose (1.0 mg kg−1).

In pylorus‐ligated rats, moxonidine significantly decreased acid secretion (all doses), total secretory volume (1.0 mg kg−1) as well as pepsin output (1.0 mg kg−1).

In comparison to clonidine, moxonidine appears to be a more potent anti‐secretory and gastric‐protective compound. These data indicate a potential role for imidazoline receptor agonists in the management of gastroduodenal diseases associated with hypertension. The relative contribution of the central and peripheral effects of moxonidine to these gastrointestinal actions remains to be determined.
Pertussis toxin‐sensitive muscarinic relaxation in the rat iris dilator muscle
Authors: Yamahara Natsuki S. , Tanaka Masako , Imaizumi Yuji , Watanabe Minora
Published: July 2012

The effects of pertussis toxin (PTX) on contraction and/or relaxation induced by agonists or transmural nerve stimulation (TNS) were examined in the rat iris dilator and sphincter muscles.

TNS in the presence of phentolamine induced an atropine‐sensitive biphasic response: initial contraction followed by relaxation in dilator muscles. Exogenously applied acetylcholine (ACh) elicited a large relaxation at low doses (3 μm or less) and a concentration at high doses.

Only the ACh‐induced relaxation was affected by injection of PTX (10 ng) into the anterior eye chamber. Relaxation was decreased 12 h after injection and had completely disappeared after 24 h. Relaxation recovered in part 3 weeks and almost completely 8 weeks after PTX treatment. A gradual decrease in muscarinic relaxation in a dilator muscle was also observed in vitro after addition of PTX to the bathing solution.

The pA2 values of muscarinic blockers, pirenzepine, AF‐DX 116, 4‐DAMP, and himbacine for competitive antagonism to ACh‐induced contraction were 7.14, 6.53, 9.03, and 6.80, respectively, in PTX‐pretreated dilator muscles. These values are comparable to those obtained in parasympathectomized dilator muscles and may indicate involvement of M3 or M3‐like receptors in muscle contraction.

Pretreatment with PTX did not significantly affect contraction induced by noradrenaline or 5‐hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles.

In conclusion, among several agonist‐induced responses in the rat iris dilator and sphincter muscles, only muscarinic relaxation in dilator muscle occurs via activation of PTX‐sensitive GTP binding proteins.