bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for ETA and ETB receptors in rat skin and an investigation of their function in the cutaneous microvasculature
Authors: Lawrence E. , Siney L. , Wilsoncroft P. , Knock G.A. , Terenghi G. , Polak J.M. , Brain S.D.
Published: July 2012

The relative contribution of ETA and ETB receptors in the response of rat skin to endothelins was investigated by use of the selective ETB agonist IRL‐1620 and the selective ETA antagonist BQ‐123.

Binding data suggest the presence of ETA and ETB receptors as preincubation with [Ala3′11,18Nle7]‐endothelin‐1 reduced ET‐1 binding by approximately 40%.

Intradermal injection of endothelin‐1 (ET‐1, 1–10 pmol/site) and ET‐3 (3–100 pmol/site) induced a dose‐dependent decrease in local blood flow assessed by 133Xe clearance at test sites in rat skin.

The endothelin analogue [Ala3′11,18Nle7]‐ET‐l (30–1000 pmol/site) induced significant vasoconstriction (P<0.05) at the highest doses used and the selective ETB receptor agonist, IRL‐1620 [Suc‐[Glu9, Ala11,15] endothelin (8–21)], (0.01–100 pmol/site) acted in a potent manner to induce a significant (P<0.01) dose‐dependent decrease in 133Xe clearance.

Co‐injection with the selective ETA receptor antagonist, BQ‐123 (1 nmol/site), completely abolished the vasoconstriction to ET‐1 and partially to ET‐3, but had no effect on IRL‐1620‐induced vasoconstriction. In addition, IRL‐1620 responses were not altered at sites treated with submaximal doses of a nitric oxide synthase inhibitor or a prostaglandin synthase inhibitor.

ET‐1 and IRL‐1620 (100 fmol‐1 pmol/site) did not induce oedema formation as measured by [125I]‐albumin accumulation in the presence or absence of the vasodilator, calcitonin gene‐related peptide (CGRP). ET‐1 (1–3 pmol/site) inhibited substance P‐induced oedema formation and this effect, suggested to be secondary to a vasoconstrictor effect, was significantly reversed by BQ‐123 (1 nmol/site).

The findings in this study indicate that there are ETA and ETB receptors in rat skin and agents which activate either receptor act to mediate a decrease in cutaneous blood flow, but have no effect on increased microvascular permeability.
Role of nitric oxide in learning and memory and in monoamine metabolism in the rat brain
Authors: Yamada Kiyofumi , Noda Yukihiro , Nakayama Shinobu , Komori Yumiko , Sugihara Hisayoshi , Hasegawa Takaaki , Nabeshima Toshitaka
Published: July 2012

We investigated the effects of NG‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide (NO) synthase, on the performance of rats in a radial arm maze and in habituation tasks, and on monoamine metabolism in the brain.

Daily administration of L‐NAME (10–60 mg kg−1) resulted in a dose‐dependent impairment of performance during the acquisition of the radial arm maze task, while it failed to affect performance in those rats that had previously acquired the task.

The rate of decrease in locomotor activity in the habituation task in the L‐NAME‐treated rats was significantly less than that in control rats.

NG‐nitro‐D‐arginine methyl ester (D‐NAME, a less active inhibitor of NO synthase) showed no effects in the above behavioural tasks.

NO synthase activity was significantly decreased in both the L‐NAME and D‐NAME‐treated rats, with the magnitude of inhibition being greater in the L‐NAME‐treated animals.

The content of 5‐hydroxyindoleacetic acid (5‐HIAA) in the hippocampus and the 5‐HIAA/5‐hydroxytryptamine ratio in the hippocampus and cortex were significantly decreased in the L‐NAME (60 mg kg)‐treated rats compared with these values in the controls.

Striatal 3,4‐dihydroxyphenylacetic acid (DOPAC) content was significantly increased in the L‐NAME (60 mg kg_1)‐treated rats compared with the values in the controls, while the DOPAC/dopamine ratio was not changed.

These results suggest that: (i) NO may play an important role in performance during the acquisition, but not retention, of the radial arm maze task, and (ii) that endogenous NO may be involved in the regulation of monoamine metabolism.
Effects of WIN 64338, a nonpeptide bradykinin B2 receptor antagonist, on guinea‐pig trachea
Authors: Scherrer Didier , Daeffler Laurent , Trifilieff Alexandre , Gies Jean‐Pierre
Published: July 2012

We investigated the effect of the nonpeptide bradykinin receptor antagonist, [[4‐[[2‐[[bis(cyclohexylamino)methylene] amino]‐3‐(2‐naphthalenyl) 1‐oxopropyl]amino]‐phenyl]‐tributyl, chloride, monohydrochloride (WIN 64338), on [3H]‐bradykinin binding and on bradykinin‐induced contraction of the guinea‐pig trachea. This non peptide bradykinin receptor antagonist inhibited [3H]‐bradykinin binding with a nanomolar range of affinity, Ki = 50.9 ± 19 nM and inhibited bradykinin‐induced contraction in a noncompetitive manner with a KB value of 6.43 10−8±2.34 10−8 m.
Effects of U46619 on contractions to 5‐HT, sumatriptan and methysergide in canine coronary artery and saphenous vein in vitro
Authors: Kemp B.K. , Cocks T.M.
Published: July 2012

The aim of this study was to investigate the mechanism of enhanced reactivity to 5‐hydroxytryptamine (5‐HT) and sumatriptan previously observed in human isolated coronary arteries when active force was raised with the thromboxane A2‐mimetic, U46619.

Ring segments of dog isolated coronary artery and saphenous vein were suspended in organ baths and cumulative concentration‐contraction curves to 5‐HT, sumatriptan and methysergide were constructed in the absence and presence of low concentrations of U46619.

In both endothelium‐intact and endothelium‐denuded rings of coronary artery, precontraction with U46619 to low (<10% Fmax; the contraction to a maximum depolarizing 125 mM KCl Krebs solution; KPSS) levels of active force had no effect on either the maximum contraction or sensitivity (pEC50) to 5‐HT, sumatriptan and methysergide.

Ketanserin (1 μm) had no effect on contractions to sumatriptan and methysergide in endothelium‐denuded coronary artery rings, but reduced the maximum contraction to 5‐HT by z 90% to a value (5% Fmax) similar to that for sumatriptan and methysergide. Under these conditions, U46619 precontraction had no effect on either pEC50 or maximum for 5‐HT, sumatriptan or methysergide. 5 In rings of saphenous vein with endothelium and treated with ketanserin (1 μm), 5‐HT and sumatriptan caused equal maximum responses of 65% Fmax which were approximately double that of methysergide (32% Fmax). The maximum responses and sensitivity to 5‐HT, sumatriptan, methysergide and noradrenaline were unaffected by precontraction with U46619.

Pretreatment of the saphenous vein with sodium nitroprusside (SNP; 10 μm) caused a small sustained relaxation and significantly depressed the maximal contraction to 5‐HT without affecting sensitivity and abolished the contraction curve to sumatriptan and methysergide. When the relaxation response to SNP was reversed with U46619 (1–4 nM), the contraction curves to 5‐HT, sumatriptan and methysergide were similar to those obtained prior to relaxation with SNP. In contrast, the same treatment with SNP had little affect on the contraction curve to noradrenaline.

In conclusion, the pattern of U46619‐enhanced reactivity of 5‐HT, sumatriptan and methysergide in SNP‐treated dog saphenous vein, highlights the importance of functional antagonism when assessing reactivity to contractile agonists in isolated blood vessels.
Possible role of Na+‐K+‐ATPase in the regulation of human corpus cavernosum smooth muscle contractility by nitric oxide
Authors: Gupta Sandeep , Moreland Robert B. , Munarriz Ricardo , Daley Jennifer , Goldstein Irwin , de Tejada Iñigo Saenz
Published: July 2012

This study was designed to determine the role of sodium‐potassium adenosine triphosphatase (Na+‐K+‐ATPase) in the regulation of human corpus cavernosum smooth muscle contractility by nitric oxide (NO). In addition, we determined if the modulation of Na+‐K+‐ATPase activity by NO is dependent on the increases in intracellular cyclic GMP concentration.

The effect of NO donors, sodium‐nitroprusside (SNP) and S‐nitroso‐glutathione (S‐NO‐Glu), and a permeable cyclic GMP analogue, 8‐bromo‐cyclic GMP, on Na+‐K+‐ATPase activity (measured as ouabain‐sensitive 86Rb‐uptake) was studied in human cultured corpus cavernosum smooth muscle cells (HCCSMC). In addition, the effect of the cyclic GMP lowering agent, methylene blue, on NO‐induced increase in Na+‐K+‐ATPase activity was studied.

SNP (1 μm) caused time‐dependent increases in ouabain‐sensitive Rb‐uptake (33–72%) over 2–20 min in HCCSMC. The stimulation of ouabain‐sensitive Rb‐uptake by SNP was concentration‐dependent (30 and 102% with 0.1 and 1 μm SNP, respectively). Similarly, significant increases in ouabain‐sensitive Rb‐uptake were obtained with 1 and 10 μm S‐NO‐Glu. In contrast, incubation of HCCSMC with 8‐bromo‐cyclic GMP (100 μm) did not increase ouabain‐sensitive Rb‐uptake.

S‐NO‐Glu induced‐increase in intracellular cyclic GMP synthesis, but not the increase in ouabain‐sensitive Rb‐uptake, was completely inhibited by methylene blue in HCCSMC.

The Na+‐K+‐ATPase inhibitor, ouabain, caused a concentration‐dependent increase in tension (0.5 to 2 fold) in tissues contracted with 15 mM KCL. SNP and S‐NO‐Glu caused a concentration‐dependent relaxation (concentration required to cause half maximal relaxation (ED50) = 0.04 and 0.2 μm, respectively) of HCC strips contracted with 15 mM K+. Ouabain (0.1 to 10 μm) inhibited the response to SNP and S‐NO‐Glu by shifting the concentration‐response curves to the right and preventing full smooth muscle relaxation.

These results indicate that the activity of Na+‐K+‐ATPase modulates the contractility of HCC smooth muscle, and that NO stimulates Na+‐K+‐ATPase activity in HCCSMC independently of its ability to increase the intracellular cyclic GMP concentration. They also suggest that stimulation of Na+‐K+‐ATPase activity plays an important role in NO‐induced relaxation of HCC smooth muscle
Electrical activity in rat tail artery during asynchronous activation of postganglionic nerve terminals by ciguatoxin‐1
Authors: Brock James A. , McLachlan Elspeth M. , Jobling P. , Lewis R.J.
Published: July 2012

The effects of ciguatoxin‐1 (CTX‐1) on the membrane potential of smooth muscle cells have been examined in rat proximal tail arteries isolated in vitro.

CTX‐1 (≥ 10 pM) increased the frequency of spontaneous excitatory junction potentials (s.ej.ps). At 100–400 pM, there was also a marked and maintained depolarization (19.7 ± 1.4 mV, n=14, at 400 pM).

In 20–400 pM CTX‐1, perivascular stimuli evoked excitatory junction potentials (s.e.j.ps) which were prolonged in time course relative to control.

Although threshold and latency of the e.j.p. were not affected by CTX‐1 (≤400 pM), propagated impulses were blocked at ≥ 100 pM.

The spontaneous activity and the depolarization produced by CTX‐1 were reduced in the presence of Ca2+ (0.1 mM)/Mg2+ (25 mM), ω‐conotoxin (0.1 μm) or Cd2+ (50–100 μm)

All effects of CTX‐1 were abolished by tetrodotoxin (0.3 μm).

Raised Ca2+ (6 mM) reduced the depolarization and spontaneous activity produced by CTX‐1.

In 400 pM CTX‐1, the membrane repolarized (17 ± 3.2 mV, n=4) following the addition of phentolamine (1 μm). S.e.j.ps and e.j.ps were selectively abolished by suramin (1 mM), and the membrane repolarized by 1.3±1.6 mV (n=4).

We conclude that CTX‐1 releases noradrenaline and ATP by initiating asynchronous discharge of postganglionic perivascular axons. In 100–400 pM CTX‐1, the smooth muscle was depolarized to levels resembling those recorded in this artery during ongoing vasoconstrictor discharge in vivo.
Lack of involvement of bradykinin in the vascular sympathoinhibitory effects of angiotensin converting enzyme inhibitors in spontaneously hypertensive rats
Authors: Chauvin Bénédicte , Richer Christine , Giudicelli Jean‐François
Published: July 2012

The aim of this study was to investigate the contribution of endogenous bradykinin to the vascular sympathoinhibitory effects exerted by angiotensin I converting enzyme inhibitors (ACEIs) in the spontaneously hypertensive rat (SHR).

Adult SHRs were treated daily for 8 days with either perindopril (3 mg kg−1), or a selective angiotensin II AT1 receptor antagonist, losartan (10 mg kg−1) both given orally ‐ these two doses being equipotent in inhibiting angiotensin I (AI)‐induced vascular responses ‐ or distilled water (controls). After pithing, the animals were instrumented for determination of blood pressure, heart rate, cardiac output, regional (renal, mesenteric, hindlimb) blood flows (pulsed Doppler technique) and corresponding vascular resistances. Afterwards, half of the animals of each group were given the selective bradykinin B2 receptor antagonist, icatibant, used in a dose (10 μg kg−1, i.v.) that achieved B2 receptor blockade, the other half received saline (10 μl kg−1, i.v.). Haemodynamic responses to increasing frequencies of spinal cord stimulation were then measured.

Pressor and vasoconstrictor responses to AI were significantly and similarly reduced in both perindopril‐ and losartan‐treated groups. Perindopril and losartan both decreased to a similar extent the pressor and vasoconstrictor responses to electrical stimulation of the spinal cord.

In the dose used, icatibant did not affect any of the investigated haemodynamic parameters in any of the experimental groups. Furthermore, icatibant did not affect the stimulation frequency‐response curves in the control animals and did not modify the vascular sympathoinhibitory effects exerted by perindopril and by losartan.

Taken together, these results demonstrate that endogenous bradykinin does not, through B2 receptor activation, contribute to the vascular sympathoinhibitory effects of ACEIs in SHRs.
Mediation by CCKB receptors of the CCK‐evoked hyperaemia in rat gastric mucosa
Authors: Heinemann Akos , Jociĉ Milana , Holzer‐Petsche Ulrike , Pethö Gábor , Peskar Brigitta M. , Horwell David C. , Holzer Peter
Published: July 2012

Cholecystokinin octapeptide (CCK‐8) and gastrin‐17 augment gastric mucosal blood flow in the rat. The present study examined whether the gastric vasodilator effect of these peptides is mediated by CCKA or CCKB receptors.

Intravenous injection of CAM‐1481 (1 mg kg−1), a dipeptoid antagonist of CCKA receptors, or CAM‐1028, a dipeptoid CCKB receptor antagonist (1 mg kg−1), had no effect on basal gastric mucosal blood flow as determined by the clearance of hydrogen in urethane‐anaesthetized rats.

Intravenous infusion of CCK‐8 or gastrin‐17 (8–200 pmol min−1) increased gastric mucosal blood flow in a dose‐dependent fashion. The CCKB receptor antagonist, CAM‐1028, significantly attenuated the hyperaemic response to CCK‐8 and gastrin‐17 whereas the CCKA receptor antagonist, CAM‐1481, did not antagonize CCK‐8 but caused a slight attenuation of the vasodilator response to gastrin‐17.

The selectivity of the two antagonists was proved by the findings that CAM‐1028, but not CAM‐1481, inhibited gastric acid secretion evoked by CCK‐8 or gastrin‐17 (CCKB receptor assay) while CAM‐1481, but not CAM‐1028, inhibited the CCK‐8‐induced contraction of guinea‐pig isolated gall bladder strips (CCKA receptor assay).

These data show that the actions of CCK‐8 and gastrin‐17 to increase mucosal blood flow in the rat stomach are primarily mediated by CCKB receptors.
Effects of 5‐HT and 5‐HT1Areceptor agonists and antagonists on dorsal vagal preganglionic neurones in anaesthetized rats: an ionophoretic study
Authors: Wang Yun , Jones James F.X. , Ramage Andrew G. , Jordan David
Published: July 2012

Effects of ionophoretic administration of 5‐hydroxytryptamine (5‐HT) and selective 5‐HT1Areceptor agonists and antagonists on identified dorsal vagal preganglionic and dorsal raphe neurones were studied in pentobarbitone sodium or chloral hydrate‐anaesthetized rats, respectively.

Extracellular recordings were made from 176 preganglionic neurones in the dorsal vagal nucleus (DVN). Application of 5‐HT at low currents (<10nA) increased the activity of these neurones. However, at increased currents (10–60 nA), it had a predominantly depressant effect. Application of selective 5‐HT1Areceptor antagonists, (±)‐pindolol or WAY‐100635, attenuated the excitatory responses evoked by 5‐HT.

Ionophoresis of the 5‐HT1Areceptor agonist, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) (5–30 nA) increased the firing rate of 19 and decreased that of 67 of the 104 vagal neurones tested. Other 5‐HT1Areceptor agonists, flesinoxan and N, N‐di‐n‐propyl‐5‐carboxamidotryptamine (DP‐5‐CT) also had predominantly depressant effects.

(±)‐Pindolol attenuated excitations but not inhibitions evoked by 8‐OH‐DPAT. Surprisingly, WAY‐100635 and 8‐OH‐DPAT produced the same effect on these neurones and when applied together, WAY‐100635 failed to attenuate the 8‐OH‐DPAT responses.

Dorsal raphe neurones were identified by their low, regular firing rate and their subsequent histological localization. 8‐OH‐DPAT reversibly reduced the activity in all 7 neurones tested and this was antagonized by WAY‐100635 in all 3 neurones tested.

In conclusion, 5‐HT applied to vagal preganglionic neurones evokes excitatory and inhibitory responses. The excitatory, but not the inhibitory responses may be mediated, at least in part, by activation of 5‐HT1Areceptors.
Involvement of G‐protein αil subunits in activation of G‐protein gated inward rectifying K+ channels (GIRK1) by human NPY1 receptors
Authors: Brown Nicola A. , McAllister George , Weinberg David , Milligan Graeme , Seabrook Guy R.
Published: July 2012

This study investigated the type of G‐protein a subunit(s) that human neuropeptide Y (NPY)1 receptors preferentially utilize when activating G‐protein gated K+ currents. Two electrode voltage‐clamp recordings were made from Xenopus oocytes that had been injected with cDNAs encoding either human NPY1 or D2(short) dopamine receptors, and GIRK1 a cloned rat brain K+ channel. These receptors were also co‐injected with G‐protein αil, αi2, αi3 and αol subunits to determine which subunit(s) modulate the efficiency of signal transduction. In NPY1 receptor injected cells neuropeptide Y (100 nM) caused a 53+10 nA inward current (n=14; EC50=3 nM) and this effect was blocked by pertussis toxin (500 ng ml−1 24 h). Activation of GIRK1 currents by neuropeptide Y was selectively potentiated by αil subunit cDNA whereas coupling dopamine of D2 receptors to this channel was not.
Endothelium‐dependent relaxations in sheep pulmonary arteries and veins: resistance to block by NG‐nitro‐L‐arginine in pulmonary hypertension
Authors: Kemp B.K. , Smolich J.J. , Ritchie B.C. , Cocks T.M.
Published: July 2012

The effect of the nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG), on endothelium‐dependent relaxation to a receptor‐independent agent, ionomycin, was examined in isolated pulmonary arteries and veins from control, short‐term and chronic pulmonary hypertensive sheep. All vessel segments were contracted to optimal levels of active force with endothelin‐1 to record endothelium‐dependent relaxation.

Pulmonary hypertension was induced by continuous pulmonary artery air embolization for 1 day (short‐term) and 14 days (chronic) and was associated with a 2 and 3 fold increase in pulmonary vascular resistance respectively.

L‐NOARG (0.1 mM) reduced the maximum relaxation (Rmax) to ionomycin in large and medium‐sized pulmonary arteries from control sheep by approximately 70%. By contrast, L‐NOARG (0.1 mM) did not inhibit the Rmax to ionomycin in matched vessels from short‐term and chronic pulmonary hypertensive sheep.

Resistance of ionomycin‐induced relaxations to inhibition by L‐NOARG, was confined to the arterial vasculature in chronic pulmonary hypertensive animals, as relaxations to ionomycin in large and medium‐sized chronic pulmonary hypertensive veins were, like those in control veins, abolished by L‐NOARG. Both large and medium‐sized pulmonary veins from short‐term pulmonary hypertensive sheep, however, were resistant to block by L‐NOARG.

Neither sensitivity (pEC50) nor Rmax, to ionomycin in large, short‐term pulmonary hypertensive arteries was affected when the extracellular concentration of K+ was increased isotonically to 30 mM. Nifedipine (0.3 μm) was present throughout to prevent high K+‐induced smooth muscle contraction. In the presence of this high extracellular K+, however, L‐NOARG (0.1 mM) caused complete inhibition of the relaxation to ionomycin, whereas in normal extracellular K+ (4.7 mM), L‐NOARG only weakly inhibited ionomycin relaxations.

In conclusion, the onset of pulmonary hypertension in sheep following air embolization, is associated with the development of resistance of endothelium‐dependent relaxations to block by L‐NOARG. The mechanism of L‐NOARG resistance appears to be due to the up‐regulation of a K+ channel‐mediated backup vasodilator mechanism which can compensate for the loss of nitric oxide (NO)‐mediated relaxation. Although this mechanism remains functionally ‘silent’ in the presence of NO it is able to maintain adequate endothelium‐dependent vasodilatation during pulmonary hypertension if NO synthesis is compromised.
Lack of effect of TNF antibodies on the cardiovascular sequelae of lipopolysaccharide infusion in conscious rats
Authors: Waller John , Gardiner Sheila M. , Jose Joby , Bennett Terence
Published: July 2012

The aims of the present study were to determine the profile of tumour necrosis factor (TNF) release, and the effect of monoclonal antibodies to TNF, on the changes in regional haemodynamics and the responses to vasodilator and vasoconstrictor challenges, during a continuous 24 h low dose infusion of lipopolysaccharide (LPS) in conscious rats.

Male Long Evans rats were chronically instrumented for measurement of regional haemodynamics (renal, superior mesenteric and hindquarters) and were challenged with 3 min infusions of acetylcholine (22 nmol min−1), methoxamine (120 nmol min−1), salbutamol (0.83 nmol min−1) and bradykinin (14.4 nmol min−1). The rats were given either saline, or the TNF antibodies, TN3gl or TN3g2a, 1 h before the start of a continuous infusion of LPS (150 μg kg−1 h−1) and were subsequently re‐tested with the vasodilator and vasoconstrictor challenges 2, 6 and 24 h after the start of the LPS infusion.

Prior to infusion of LPS, TNF was not detectable in the plasma. During continuous infusion of LPS there was a transient elevation in plasma TNF levels, reaching a maximum (∼2300 pg ml−1) after approximately 1 h, and returning to undetectable levels after approximately 3 h of LPS infusion.

In the saline pretreated group, after 1–2 h of LPS infusion, there was a small hypotension and a marked renal vasodilatation; 6 h after the start of LPS infusion there was a minor elevation in MAP above control levels, renal vasodilatation was maintained and a hindquarters vasoconstriction occurred; after 24 h of LPS infusion, there was a hypotension and renal and hindquarters vasodilatation. There were significant reductions in the tachycardic and renal vasodilator responses and an enhanced depressor response to acetylcholine after 24 h of LPS infusion. LPS infusion also caused a generalized hyporesponsiveness to the cardiovascular effects of methoxamine and salbutamol. The major changes in response to bradykinin were reduced tachycardic and enhanced depressor responses throughout the LPS infusion, a biphasic decrease and increase in renal conductance and enhanced hindquarters vasodilatation at 24 h.

Pretreatment with either TN3gl or TN3g2a antibodies had no major effects on the changes in resting haemodynamics, or on the changes in response to methoxamine, salbutamol or bradykinin challenges during LPS infusion. However, the tachycardic responses to acetylcholine were generally preserved, and its hypotensive effect after 24 h of LPS infusion was not enhanced after TN3gl or TN3g2a pretreatment. Thus, despite substantial, but transient, elevation of plasma TNF levels during continuous LPS infusion, it appears that the majority of cardiovascular changes were dependent on factors other than plasma TNF.
Effect of endotoxin on the angiotensin II receptor in cultured vascular smooth muscle cells
Authors: Burnier Michel , Centeno Gabriel , Waeber Gérard , Centeno Catherine , Bürki Ernst
Published: July 2012

In some tissues, a decrease in the number of cell surface receptors and alterations of the receptor coupling have been proposed as possible mechanisms mediating the deleterious effects of bacterial endotoxin in septic shock.

The effects of bacterial lipopolysaccharide (Escherichia coli 0111‐B4; LPS) on vascular angiotensin II and vasopressin receptors have been examined in cultured aortic smooth muscle cells (SMC) of the rat by use of radioligand binding techniques.

In vascular SMC exposed to 1 μg ml−1 endotoxin for 24 h, a significant increase in angiotensin II binding was found. The change in [125I]‐angiotensin II binding corresponded to an increase in the number of receptors whereas the affinity of the receptors was not affected by LPS. In contrast, no change in [3H]‐vasopressin binding was observed.

The pharmacological characterization of angiotensin II binding sites in control and LPS‐exposed cells demonstrated that LPS induced an increase in the AT, subtype of the angiotensin II receptors. Receptor coupling as evaluated by measuring total inositol phosphates was not impaired by LPS.

The effect of LPS on the angiotensin II receptor was dose‐, time‐ and protein‐synthesis dependent and was associated with an increased expression of the receptor gene.

The ability of LPS to increase angiotensin II binding in cultured vascular SMC was independent of the endotoxin induction of NO‐synthase.

These results suggest that, besides inducing factors such as cytokines and NO‐synthase, endotoxin may enhance the expression of cell surface receptors. The surprising increase in angiotensin II binding in LPS exposed VSM cells may represent an attempt by the cells to compensate for the decreased vascular responsiveness. It may also result from a non‐specific LPS‐related induction of genes.
Effect of a 5‐HT1 receptor agonist, CP‐122,288, on oedema formation induced by stimulation of the rat saphenous nerve
Authors: Kajekar Radhika , Gupta Paul , Shepperson Nicholas B. , Brain Susan D.
Published: July 2012

Neurogenic oedema formation in the rat hind paw skin induced by electrical stimulation of the saphenous nerve and measured by extravasation of [I25I]‐albumin, was inhibited by the 5‐HT1B receptor agonist, CP‐93,129, and the novel, tryptamine analogue, CP‐122,288. Significant inhibition of up to 66% of control was observed with CP‐122,288 (2 × 1014‐2 × 10−7 mol kg−1) and CP‐93,129 (5 × 10−7‐5 × 10−6 mol kg−1), with the minimum effective dose for CP‐122,288 being about 107 fold less than that for CP‐93,129. Oedema formation induced by the intradermal administration of exogenous mediators (substance P and histamine) in rat dorsal skin was not inhibited by CP‐122,288 (2 × 10−10 mol kg−1). These results suggest that CP‐122,288 is a potent inhibitor of neurogenic inflammation in rat skin and that the effect may be due to a prejunctional inhibition of neuropeptide release.
JB‐9322, a new selective histamine H2‐receptor antagonist with potent gastric mucosal protective properties
Authors: Palacios Beatriz , Montero Ma Jose , Sevilla Ma Angeles , Roman Luis San
Published: July 2012

JB‐9322 is a selective histamine H2‐receptor antagonist with gastric antisecretory activity and mucosal protective properties.

The affinity of JB‐9322 for the guinea‐pig atria histamine H2‐receptor was approximately 2 times greater than that of ranitidine.

In vivo, the ID50 value for the inhibition of gastric acid secretion in pylorus‐ligated rats was 5.28 mg kg−1 intraperitoneally. JB‐9322 also dose‐dependently inhibited gastric juice volume and pepsin secretion. In gastric lumen‐perfused rats, intravenous injection of JB‐9322 dose‐dependently reduced histamine‐, pentagastrin‐ and carbachol‐stimulated gastric acid secretion.

JB‐9322 showed antiulcer activity against aspirin and indomethacin‐induced gastric lesions and was more potent than ranitidine.

JB‐9322 effectively inhibited macroscopic gastric haemorrhagic lesions induced by ethanol. Intraperitoneal injection was effective in preventing the lesions as well as oral treatment. The oral ID50 value for these lesions was 1.33 mg kg−1. By contrast, ranitidine (50 mg kg−1) failed to reduce these lesions. In addition, the protective effect of JB‐9322 was independent of prostaglandin synthesis.

These results indicate that JB‐9322 is a new antiulcer drug that exerts a potent cytoprotective effect in addition to its gastric antisecretory activity.
Electrophysiological actions of phenytoin on N‐methyl‐D‐aspartate receptor‐mediated responses in rat hippocampus in vitro
Authors: Laffling Alison J. , Scherr Patrick , McGivern Joseph G. , Patmore Leslie , Sheridan Robert D.
Published: July 2012

The effects of the anticonvulsant, phenytoin, have been examined on N‐methyl‐D‐aspartate (NMDA) receptor‐mediated population spikes in the CA1 region of the rat hippocampus in vitro.

The ‘conventional’ (AMPA receptor‐mediated) CA1 population spike, evoked by electrical stimulation of the Schaffer collateral/commissural pathway, was abolished by 5 min treatment with 5 × 10−6M 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), after which superfusion with a nominally Mg2+‐free Krebs solution (containing 5 × 10−6 M CNQX) led to the appearance of an epileptiform population spike which was fully developed by 30–40 min.

The epileptiform population spike was abolished by the non‐competitive NMDA antagonist, dizocil‐pine (1 × 10−6 M, 20–30 min) and inhibited by the competitive NMDA receptor antagonist, D‐CPP (IC50 for reducing the amplitude of the first spike in the train = 8.3 × 10−7 M), demonstrating that the response was mediated by activation of NMDA receptors and validating its use as an assay for antagonists acting at the NMDA receptor/channel complex.

Phenytoin (0.1, 0.3 and 1 × 10−4M applied cumulatively for 30 min at each concentration) failed to inhibit the NMDA receptor‐mediated epileptiform population response (n = 7 slices).

Phenytoin (3 × 10−6 M to 1 × 10−4 M) attenuated the effects of the sodium channel activator, veratridine (2 × 10−6M), on the CA1 population spike amplitude (recorded in normal Krebs solution), indicating that the previously observed lack of effect of phenytoin on the NMDA receptor‐mediated response was not due to impaired access of phenytoin to the biophase.

These data support the conclusion that antagonism of NMDA receptor‐mediated events is not a pharmacological property of phenytoin and that such an action is therefore unlikely to contribute to the anticonvulsant activity of this drug.
Modulation of epidermal growth factor effects on epithelial ion transport by intestinal trefoil factor
Authors: Chinery R. , Cox H.M.
Published: July 2012

The direct epithelial effects of epidermal growth factor (EGF) and its modulation by intestinal trefoil factor (ITF) have been studied in a human colonic adenocarcinoma cell line called Colony‐29 (Col‐29).

When grown in culture as confluent monolayers and voltage‐clamped in Ussing chambers, these epithelia responded with an increase in short circuit current (SCC) to basolateral as well as to apically applied EGF although the latter responses (at 10 nM) were only 25% of those observed following basolateral peptide.

Recombinant rat ITF (added to the basolateral surface) did not alter basal SCC levels, but it did enhance the electrogenic effects of basolateral EGF. The EC50 values for EGF‐induced ion transport were 0.25 nM in control, and 0.26 nM in ITF pretreated Col‐29 epithelia. A significant increase in the size of EGF responses (0.1 nM −10 nM) was observed in the presence of 10 nM ITF and the half‐maximal concentration for this modulatory effect of ITF was 7.6 nM.

The EGF‐induced increases in SCC were partially inhibited (50%) by piretanide pretreatment, indicating that C1− secretion is involved. EGF responses either in the presence or absence of ITF were also significantly reduced (84% and 66% respectively) by the cyclo‐oxygenase inhibitor, piroxicam, therefore implicating prostaglandins as mediators of EGF‐stimulated anion secretion.

We conclude that in confluent Col‐29 epithelia, basolateral EGF stimulates a predominantly prostaglandin‐dependent increase in C1− secretion that is enhanced by basolateral ITF, and that these two peptides may interact in normal and damaged mucosa to alter the local apical solute and fluid environment.
Inhibitory actions of GABA on rabbit urinary bladder muscle strips: mediation by potassium channels
Authors: Ferguson D.R. , Marchant J.S.
Published: July 2012

The actions of γ‐aminobutyric acid (GABA) upon rabbit urinary bladder muscle were investigated to determine whether they were mediated through potassium channels.

In vitro experiments were undertaken in which bladder muscle strips were caused to contract with carbachol. Addition of GABA or baclofen reduced the size of such evoked contractions in the case of GABA by 20.7 ± 3.2%, in the case of baclofen by 22.4 ± 2.2%.

Electrical stimulation of autonomic nerves in bladder wall strips also evoked contractions which were significantly smaller in potassium‐free Krebs solution. The size of contractions produced by carbachol on the other hand were unaffected by the absence of potassium in the Krebs solution.

The inhibitory actions of GABA and baclofen on carbachol‐induced contractions of bladder muscle were detected at much lower concentrations in potassium‐free compared with potassium containing solutions.

The inhibitory effects of baclofen were completely reversed by tetraethyl ammonium chloride between 1 and 5 mM, caesium chloride between 0.5 and 3 mM and barium chloride between 0.5 and 2.5 mM. The actions of baclofen were only partially reversed by 4‐amino‐pyridine between 1 and 5 mM.

It was concluded that the GABAB receptor‐mediated inhibitory actions on rabbit urinary bladder smooth muscle cells were produced by activation of potassium channels.
Effect of 5‐hydroxytryptamine on the membrane potential of endothelial and smooth muscle cells in the pig coronary artery
Authors: Frieden M. , Beny J.‐L.
Published: July 2012

Many endothelium‐dependent vasodilators hyperpolarize the endothelial cells in blood vessels. It is not known whether these hyperpolarizations are linked to nitric oxide synthesis or to an endothelium‐derived hyperpolarizing phenomenon, since most of the vasodilators release both factors. In this context, we first verified that the endothelium‐dependent relaxations induced by 5‐hydroxytryptamine (5‐HT) on pig coronary arteries are due only to the activation of the nitric oxide pathway. Then we studied the effects of 5‐HT on membrane potential of endothelial and smooth muscle cells.

In the absence of endothelium, 5‐HT caused a concentration‐dependent contraction of coronary artery strips. No change of the smooth muscle cell membrane potential was observed during contraction to 1 μM 5‐HT.

In the presence of 1 μM ketanserin to suppress the contractile effect of 5‐HT, 5‐HT induced concentration‐dependent relaxation of endothelium‐intact strips precontracted by 10 μM prostaglandin F2α (PGF2α). These relaxations were suppressed by 1 μM NG‐nitro‐L‐arginine, an inhibitor of nitric oxide synthesis, showing that they were produced predominantly by nitric oxide.

In the presence of 1 μM ketanserin, 1 μM 5‐HT did not change the smooth muscle cell membrane potential of strips precontracted by either 10 μM PGF2α or by 10 μM acetylcholine (ACh). In the same conditions, 1 UM 5‐HT caused a weak 2.6 ± 0.4 mV hyperpolarization, of the endothelial cells.

In conclusion, the fact that 5‐HT did not change the membrane potential of smooth muscle cells and only weakly hyperpolarized the endothelial cells during relaxations, suggests that in both cell types no electrical events accompany activation of the nitric oxide pathway. This is in contrast to the hyperpolarizations observed in endothelial and smooth muscle cells when the endothelium‐derived hyperpolarization factor (EDHF) pathway is activated.
Characterization and distribution of putative 5‐ht7 receptors in guinea‐pig brain
Authors: To Z.P. , Bonhaus D.W. , Eglen R.M. , Jakeman L.B.
Published: July 2012

In the presence of (−)−cyanopindolol (1.0 μM) and sumatriptan (1.0 μM), 0.5 nM [3H]‐carboxamidotryptamine ([3H]‐5‐CT) labelled a single population of receptors in guinea‐pig cerebral cortex membranes.

5‐HT‐displaceable binding was rapid, saturable and reversible. A high affinity binding site was characterized both by equilibrium saturation (Kd = 0.76 ± 0.28 nM; Bmax = 68.1 ± 26.7 fmol mg−1 protein) and kinetic (Kd = 0.18 ± 0.05 nM) analysis. The pharmacological profile of this site was similar to the profile obtained in transfected CHO‐K1 cells expressing guinea‐pig 5‐ht7 receptors.

Autoradiographic analysis revealed a discrete localization of binding sites in guinea‐pig brain, with the highest density of sites in the medial thalamic nuclei and related limbic and cortical regions. Moderate levels of binding were detected in sensory relay nuclei, substantia nigra, hypothalamus, central grey and dorsal raphe nuclei. This distribution corresponded to that observed using in situ hybridization with [35S]‐UTP labelled riboprobes complementary to mRNA encoding the guinea‐pig 5‐ht7 receptor.

In conclusion, under appropriate conditions, [3H]‐5‐CT labelled a single population of saturable binding sites that corresponded to an endogenous 5‐ht7 receptor in guinea‐pig brain. The distribution of 5‐ht7 receptors in thalamocortical and limbic brain regions suggests a role for these receptors in sensory and affective behaviours.
Time‐dependent fading of the activation of KATP channels, induced by aprikalim and nucleotides, in excised membrane patches from cardiac myocytes
Authors: Thuringer Dominique , Cavero Icilio , Coraboeuf Edouard
Published: July 2012

The effects of the potassium channel opener (KCO) aprikalim (RP 52891) on the nucleotide‐induced modulation of ATP‐sensitive K+ (KATP) channels in freshly dissociated ventricular myocytes of guinea‐pig heart, were studied by use of the inside‐out patch‐clamp technique. The internal surface of the excised membrane patch was initially bathed with a standard solution (Mg2+‐free with EDTA), then sequentially superfused with solutions containing nucleoside diphosphates (NDPs: 200 UM ADP and 50 UM GDP) and NDPs plus 1 mM MgCl2 (with EGTA; referred to as Mg‐NDP solution).

The normalized concentration‐response (channel closing) relationship to ATP was shifted to the right when the standard solution was replaced by the Mg‐NDP solution. Hence, the internal concentration of ATP ([ATP]i) inhibiting the channel activity by half (Ki) increased from 56 UM to 180 μM, with an apparently constant slope factor (s = 2.37). NDPs in the absence of Mg2+ did not decrease the sensitivity of the channels to ATP.

In standard solution, aprikalim (100 μM) activated KATP channels in the presence of a maximally inhibitory [ATP]i (500 μM). This effect was strongly enhanced when aprikalim was applied to patches exposed to Mg‐NDP solution, as demonstrated by the 9 fold increase in Kifor [ATP]i (from 180μM to 1.5 mM and s = 2.37).

The ability of aprikalim to overcome the channel closing effects of ATP in Mg‐NDP solution waned rapidly. Similarly, the NDP‐induced activation of ATP‐blocked channels was also time‐dependent. Both activation processes disappeared before the channel run‐down phenomenon appeared in ATP‐free conditions.

In conclusion, aprikalim is much more potent in opening KATP channels in membrane patches bathed in Mg‐NDP solution than in standard solution. However, under the former experimental conditions, the effect of aprikalim waned rapidly. It is proposed that the waning phenomenon results from changes in the intrinsic enzymatic activity of the KATP channel protein (possibly linked to the experimental conditions) which lead to the channel closure.
Differences in response to 5‐HT4 receptor agonists and antagonists of the 5‐HT4‐like receptor in human colon circular smooth muscle
Authors: Tarn T.S‐F. , Hillier K. , Bunce K.T. , Grossman C.
Published: July 2012

In isolated circular smooth muscle strips of human colon 5‐hydroxytryptamine (5‐HT) produced a concentration‐related inhibition of spontaneous motility.

The azabicycloalkyl benzimidazolones, BIMU 8 and BIMU 1, which have 5‐HT4 receptor stimulant properties, inhibited motility with EC50 values of 0.76 μM and 3.19 μM respectively and their Emax values were not significantly different from 5‐HT (EC50, 0.13 μM).

The 5‐HT4 receptor antagonist, DAU 6285 (1–10 μM), displaced the 5‐HT concentration‐response curve to the right in a parallel concentration‐dependent manner without depressing the maximum. The Schild plot was linear and the slope did not differ significantly from unity giving a pA2 value of 6.32.

The high affinity selective 5‐HT4 receptor antagonist, GR 113808, at a concentration of 3 nM displaced the 5‐HT concentration‐response curve in a parallel manner giving an apparent pKB estimate of 8.9 ±0.24. However, higher concentrations of 10–100 nM GR 113808 did not result in a further significant displacement of the 5‐HT concentration‐response curve and there was no suppression of Emax.

GR 113808 (10 nM) also caused a parallel displacement of the concentration‐response curve to the 5‐HT4 receptor agonist, 5‐methoxytryptamine (5‐MeOT) giving apparent pKB values ranging from 8.3–9.3.

GR 113808 (3–100 nM) failed to displace 5‐HT or 5‐MeOT concentration‐response curves in tissue strips from 3 patients out of a total of 10 patients studied in whom the response to 5‐HT and 5‐MeOT was normal.

The 5‐HT4 receptor antagonist, SDZ 205–557 (0.3–10 μM), had no significant effect on 5‐HT‐induced inhibition of spontaneous motility.

The present results are discussed in the light of variability of response to GR 113808 and SDZ 205–557 in other tissues.

Overall, our data indicate that human colon circular smooth muscle can be regarded as a site in which 5‐HT4‐like receptors are present but it is as yet unclear whether these results are also an indication of receptor variation.
A study on P2x purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens
Authors: Khakh Baljit Singh , Surprenant Annmarie , Humphrey Patrick P.A.
Published: July 2012

We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine‐5′‐triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro.

Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential − 70 mV) with fast flow concentration‐clamp applications of agonists in single isolated smooth muscle cells. ATP, 2‐methylthio adenosine‐5′‐triphosphate (2‐MeSATP) and α, β methylene adenosine‐5′‐triphosphate (αβ‐meATP) all evoked inward currents over a similar concentration range (0.3–10 μM), being approximately equipotent with similar concentrations for threshold effects (0.3 μM). ADP (10 μM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 μM).

α,β‐meATP was the most potent agonist in producing contractions of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 UM). However, ATP and 2‐MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration‐effect curves were shallow with smaller maximal responses than could be achieved with α,β‐meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was α,β‐meATP≫ATP = 2‐MeSATP.

Measurement of inorganic phosphate (iP), as a marker of purine nucleotide metabolism in the vas deferens whole tissue preparation, indicated that ATP and 2‐MeSATP were rapidly metabolized, whereas α,β‐meATP was stable for up to 2 h. Removal of divalent cations prevented breakdown of ATP and 2‐MeSATP, suggesting that metabolism involved a Ca2+/Mg2+‐dependent enzyme.

It appears that in isolated preparations of rat vas deferens, the low potency of ATP and 2‐MeSATP can be explained by rapid agonist breakdown by ectonucleotidases. However, this is not the case in the single cell studies where the use of rapid concentration‐clamp applications revealed the true potency of the agonists. Under such conditions the three agonists were all equal in potency indicating that the rank order of agonist potencies of α,β‐meATP≫ATP = 2‐MeSATP is not in fact characteristic of smooth muscle P2X‐purinoceptors as commonly believed.
Intrinsic activity of the non‐prostanoid thromboxane A2 receptor antagonist, daltroban (BM 13,505), in human platelets in vitro and in the rat vasculature in vivo
Authors: Bertolino Frédéric , Valentin Jean‐Pierre , Maffre Myriam , Grelac Françoise , Bessac Anne‐Marie , Maclouf Jacques , Delhon André , Levy‐Toledano Sylviane , Patoiseau Jean‐François , Colpaert Francis C. , John Gareth W.
Published: July 2012

We evaluated the effects of daltroban on (i) human platelet shape change and aggregation in vitro, and (ii) mean systemic and pulmonary arterial pressures (MAP and MPAP, respectively) as well as haematocrit, in anaesthetized, open‐chest Sprague‐Dawley rats, compared with those of a chemically distinct prostanoid thromboxane A2 (TxA2) receptor antagonist, SQ 29,548, and agonist, U‐46619.

In human platelets in vitro, daltroban (10 nM‐100 μM; n = 6 per group) concentration‐dependently induced shape change, attaining at 50 μM, a maximum amplitude of 0.83 ± 0.09 mV representing 46.4 ±4.8% of that evoked by U‐46619 (1.78 ± 0.20 mV at 0.2 μM; n = 9); and inhibited U‐46619‐induced platelet aggregation with an IC50 of 77 (41–161)nM. SQ 29,548 (10 nM‐100 μM; n = 6 per group) failed to evoke any platelet shape change, but potently inhibited U‐46619‐induced platelet aggregation with an IC50< 10nM.

In anaesthetized rats in vivo, daltroban (10–2500 μg kg−1, i.v. infused over 2 min; n = 4–8 per group) produced a bell‐shaped dose‐response curve for MPAP and haematocrit, and evoked maximal increases of 12.7 ± 2.1 mmHg and 5.8 ± 1.5% at 80 μg kg−1 (n = 6) and 630 μg kg−1 (n = 8), respectively (both P<0.05) with ED50S of 20 (16–29) and 217 (129–331) μg kg−1, respectively. By comparison, U‐46619 (0.16–20 fig kg−1, i.v.), induced dose‐dependent increases in MPAP and haematocrit (25.4 ± 1.0 mmHg and 16.1 ±2.9% at the highest dose; n = 12, both P<0.01), with ED50s of 1.8 (1.3‐2.5) and 3.9 (3.5‐5.4) fig kg−1, respectively. Daltroban dose‐dependently increased MAP with a maximum amplitude of 42.2 ±4.4 mmHg at a dose of 80 μkg−1 [ED50 = 94 (64–125) fig kg1], similar to that induced by U‐46619 (41.3 ±9.6 mmHg) at a dose of 0.63 fig kg−1 [ED50 = 0.22 (0.13‐0.24) fig kg−1]. SQ 29,548 (10–2500 μg kg−1, i.v.; n = 4 per group) failed to modify significantly any of these parameters.

Our results clearly demonstrate that daltroban, in a similar manner to the TxA2 analogue, U‐46619, but unlike the TxA2 receptor antagonist, SQ 29,548, exhibits significant intrinsic activity in human platelets in vitro and in the rat vasculature in vivo, possibly through TxA2 receptor activation.