bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) activates capsaicin‐sensitive primary afferent nerves in guinea‐pig atria and urinary bladder
Authors: Giuliani Sandro , Santicioli Paolo , Tramontana Manuela , Geppetti Pierangelo , Maggi Carlo Alberto
Published: July 2012

We have investigated the ability of the N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) a synthetic analogue of a chemotactic peptide derived from a variety of bacteria, to activate capsaicin‐sensitive primary afferents in the guinea‐pig atria and urinary bladder.

In the isolated, electrically‐driven left atria from reserpine‐pretreated guinea‐pigs (atropine in the bath), FMLP (3 nm–1 μm) produced a biphasic positive inotropic response. The late component of this response was selectively abolished by in vitro capsaicin pretreatment while both the early and late responses were abolished by indomethacin.

The inotropic response to FMLP in the guinea‐pig atria was unaffected by ruthenium red. The late but not the early response was strongly inhibited or abolished by tetrodotoxin (TTX), ω‐conotoxin (CTX) or by the C‐terminal fragment (8–37) of human ω‐calcitonin gene‐related peptide (hCGRP). hCGRP‐(8–37) acts as competitive antagonist at CGRP receptors.

In the guinea‐pig isolated bladder, FMLP (10 nm–10 μm) produced a concentration‐dependent contraction which was unchanged by previous in vitro capsaicin, TTX or CTX pretreatment. The response to low concentrations of FMLP was suppressed by indomethacin, irrespective of the capsaicin pretreatment.

FMLP (10 μm) produced a significant increase in the outflow of CGRP‐like immunoreactivity (CGRP‐LI) from superfused guinea‐pig atria or urinary bladder. CGRP‐LI outflow induced by FMLP was blocked by indomethacin or in vitro capsaicin pretreatment.

These findings indicate that FMLP activates the ‘efferent’ function of capsaicin‐sensitive primary afferents via prostanoid generation. This action could provide a neurogenic contribution to the overall inflammatory response produced by bacteria‐derived peptides in inflamed tissues. In addition the present data indicate that endogenous prostanoids generated during exposure to FMLP produce peptide secretion from sensory nerves via a TTX‐ and CTX‐sensitive but ruthenium red‐resistant mechanism.
Morphine, but not sodium cromoglycate, modulates the release of substance P from capsaicin‐sensitive neurones in the rat trachea in vitro
Authors: Ray N.J. , Jones A.J. , Keen P.
Published: July 2012

Opioids have been shown to inhibit substance P (SP) release from primary afferent neurones (PAN). In addition, opioid receptors have been identified on PAN of the vagus nerves. Sodium cromoglycate (SCG) decreases the excitability of C‐fibres in the lung of the dog in vivo. We have utilised a multi‐superfusion system to investigate the effect of opioids and SCG on the release of SP from the rat trachea in vitro.

Pretreatment of newborn rats with capsaicin (50 mg kg−1 s.c. at day 1 and 2 of life) resulted in a 93.2 ± 6.3% reduction in tracheal substance P‐like immunoreactivity (SP‐LI) content when determined by radioimmunoassay in the adult.

Exposure to isotonically elevated potassium concentrations (37–90 mm), capsaicin (100 nm–10 μm), and bradykinin (BK; 10 nm–1 μm) but not des‐Arg9‐BK (1 μm) stimulated SP‐LI release by a calcium‐dependent mechanism.

SCG (1 μm and 100 μm) did not affect spontaneous, potassium (60 mm)‐ or BK (1 μm)‐stimulated SP‐LI release.

Morphine (0.1–100 μm) caused dose‐related inhibition of potassium (60 mm)‐stimulated SP‐LI release with the greatest inhibition of 60.4 ± 13.7% at 100 μm. The effect of morphine was not mimicked by the κ‐opioid receptor agonist, U50,488H (10 μm) or the ω‐opioid receptor agonist, Tyr‐(d‐Pen)‐Gly‐Phe‐(d‐Pen) (DPDPE).

The effect of morphine was totally abolished by prior and concomitant exposure to naloxone (100 nm) which had no effect on control release values.

We conclude that opioid receptors, predominantly of the μ‐opioid receptor subtype, inhibit SP‐LI release from PAN in the rat trachea and suggest that centrally inactive μ‐opioid receptor agonists may have therapeutic potential in the treatment of asthma.
Investigation of the 5‐hydroxytryptamine receptor mechanism mediating the short‐circuit current response in rat colon
Authors: Bunce K.T. , Elswood C.J. , Ball M.T.
Published: July 2012

5‐Hydroxytryptamine (5‐HT) stimulated an increase in short‐circuit current (SCC) in rat isolated colonic mucosa with an EC50 value of approximately 4 μm. The purpose of the present study was to investigate the 5‐HT receptor mechanism(s) involved in this response.

The relatively selective 5‐HT receptor agonists 5‐carboxamidotryptamine (5‐CT) and α‐methyl‐5‐HT stimulated SCC and were 6 to 8 times less potent than 5‐HT. 2‐Methyl‐5‐HT was inactive both as an agonist and an antagonist.

The following compounds produced no significant inhibition of the SCC response to 5‐HT: ketanserin (1 μm), methysergide (1 μm), methiothepin (0.3 μm), GR38032F (0.3 μm), tetrodotoxin (0.3 μm) and sulpiride (1 μm).

Both metoclopramide (3 and 10 μm) and cisapride (0.1 and 1 μm) inhibited the SCC responses to 5‐HT in a concentration‐related manner, and the higher doses similarly inhibited the responses to 5‐CT. With both agonists the inhibitory effects of metoclopramide and cisapride were insurmountable. However, these inhibitory actions appeared to be selective since neither metoclopramide nor cisapride affected the basal SCC or the SCC response to prostaglandin E2.

The SCC responses to 5‐HT and 5‐methoxytryptamine were selectively inhibited by ICS205‐930 at 3 μm, and respective pKB values of 6.0 and 6.6 were calculated.

It is concluded that 5‐HT stimulates an SCC response in rat colon via a receptor mechanism that cannot be clearly identified as 5‐HT1‐like, 5‐HT2 or 5‐HT3. This receptor is selectively antagonized by ICS205‐930 and by the benzamides, metoclopramide and cisapride. The 5‐HT receptor in rat colon therefore exhibits some of the properties associated with the so‐called 5‐HT4 receptor.
Run‐down of neuromuscular transmission during repetitive nerve activity by nicotinic antagonists is not due to desensitization of the postsynaptic receptor
Authors: Hong S.J. , Chang C.C.
Published: July 2012

Whether the function of the postsynaptic acetylcholine receptor is use‐dependently affected by repetitive nerve stimulation in the presence of competitive antagonists was studied in the mouse phrenic nerve‐hemidiaphragm preparation.

For electrophysiological experiments, the preparation was immobilized by synthetic μ‐conotoxin, which preferentially blocks muscular Na‐channels causing neither depolarization of the membrane potential, inhibition of quantal transmitter release, nor depression of nicotinic receptor function.

High concentrations of cobratoxin depressed indirect twitches and endplate potentials (e.p.ps) without inducing waning of contractilities or run‐down of trains of e.p.ps evoked at 10–100 Hz. However, waning and run‐down were accelerated after washout of the toxin despite diminished postsynaptic receptor blockade. Once the run‐down of e.p.ps was produced by washout or low concentrations of cobratoxin, further depression of e.p.p. amplitude with high concentrations of cobratoxin did not attenuate the e.p.p. rundown.

The degrees of waning of tetanus and trains of e.p.ps produced by a very high concentration of tubocurarine (20 μm) were also less than that caused at a 100 fold lower concentration, albeit the amplitudes of twitches and the first e.p.p. were depressed more rapidly and markedly.

Tubocurarine, like cobratoxin, depressed the amplitude of miniature endplate potentials (m.e.p.ps) more than e.p.ps.

In contrast to the steepened run‐down of successive e.p.ps in the presence of low concentrations of either nicotinic antagonists, the amplitude of m.e.p.ps observed during repetitive stimulation was uniform and was not different from that before stimulation.

The results suggest that the e.p.p. run‐down and tetanic fade induced by nicotinic antagonists are due to a slow kinetic blockade of presynaptic receptors and confirm that the e.p.p. run‐down is not produced by a use‐dependent failure of postsynaptic nicotinic receptors. The roles of the presynaptic nicotinic receptor in positive or negative feedback modulations of transmitter release are discussed.
l‐Arginine induces relaxation of rat aorta possibly through non‐endothelial nitric oxide formation
Authors: Moritoki Hideki , Ueda Haruyasu , Yamamoto Takaaki , Hisayama Tetsuhiro , Takeuchi Shougo
Published: July 2012

The relaxation of rings of rat thoracic aorta induced by l‐arginine and its derivatives was investigated.

l‐Arginine (0.3–100 μm), but not d‐arginine, induced relaxation of the arteries, which was detectable after 2 h and maximal after 4–6 h on its repeated application; it was endothelium‐independent.

l‐Arginine methyl ester, Nα‐benzoyl l‐arginine and l‐homo‐arginine had essentially similar effects to those of l‐arginine.

NG‐nitro l‐arginine methyl ester (l‐NAME, 3 μm), NG‐nitro l‐arginine (l‐NNA, 1 μm) and NG‐monomethyl l‐arginine (l‐NMMA, 10 μm), inhibitors of nitric oxide (NO) formation from l‐arginine, inhibited or reversed the l‐arginine‐induced relaxation, irrespective of the presence or absence of the endothelium. In contrast, NG‐nitro d‐arginine was without effect.

Haemoglobin (Hb, 10 nm) and methylene blue (MB, 0.3 μm) inhibited or reversed the l‐arginine‐induced relaxation.

l‐Arginine (1–100 μm), but not d‐arginine, increased guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels in the tissues that relaxed in response to l‐arginine. This effect of l‐arginine was suppressed by Hb (3 μm), MB (1 μm) and l‐NAME (100 μm). Removal of the endothelium did not significantly alter the l‐arginine‐induced cyclic GMP production.

These results suggest that l‐arginine itself caused a slowly developing relaxation of rat aorta, possibly via formation of NO by an endothelium‐independent mechanism.
Antagonism by reactive blue 2 but not by brilliant blue G of extracellular ATP‐evoked responses in PC12 phaeochromocytoma cells
Authors: Inoue Kazuhide , Nakazawa Ken , Ohara‐Imaizumi Mica , Obama Tomoko , Fujimori Kannosuke , Takanaka Akira
Published: July 2012

The effects of reactive blue 2 and brilliant blue G, which have been shown to block extracellular ATP‐evoked responses, were investigated to discover whether these compounds act as P2‐purinoceptor antagonists in PC12 phaeochromocytoma cells.

Reactive blue 2 (10 to 100 μm) suppressed the ATP‐stimulated dopamine secretion from PC12 cells in a dose‐dependent manner. The concentration‐response curve for ATP was shifted to the right and the maximal response was decreased by reactive blue (30 and 100 μm). Brilliant blue G (up to 100 μm) did not significantly affect the secretion.

Reactive blue 2 (10 to 100 μm) suppressed the ATP‐activated inward current recorded from the voltage‐clamped cells in a concentration‐dependent manner. Brilliant blue G (up to 100 μm) did not affect the current.

The results suggest that reactive blue 2 but not brilliant blue G is a P2‐purinoceptor antagonist in PC12 cells. The purinoceptors in these cells may be the same type as those involved in ATP‐evoked smooth muscle relaxation, judging from the antagonism by reactive blue 2.
A method for studying the pharmacodynamic profile of neuromuscular blocking agents on vocal cord movements in anaesthetized cats
Authors: Vandenbrom Ron H.G. , Houwertjes Martin C. , Agoston Sandor
Published: July 2012

A new in vivo experimental method is described whereby the neuromuscular blocking effects of muscle relaxants can be investigated on the intrinsic laryngeal muscles of anaesthetized cats. The peripheral tibialis anterior muscle preparation is employed in the same animal to compare the blocking effect on both preparations.

The intrinsic laryngeal muscles react with different sensitivities to the neuromuscular blocking agents when compared to the tibialis anterior muscle.

The neuromuscular response in both muscle preparations is similar with steroidal agents but appeared to be different after suxamethonium or isoquinoline analogues.

It is concluded that this preparation may become a useful tool for studying new muscle relaxants developed to facilitate rapid intubation conditions.
A patch‐clamp study of K+‐channel activity in bovine isolated tracheal smooth muscle cells
Authors: Green K.A. , Foster R.W. , Small R.C.
Published: July 2012

Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K+‐channels in these cells were studied by the patch‐clamp recording technique.

Recordings were made from inside‐out plasmalemmal patches when [K+] was symmetrically high (140 mm) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 μm. Large unitary currents of both Ca2+‐dependent and ‐independent types were observed. Measured between +20 and +40 mV, the slope conductances of the channels carrying these currents were 249 ± 18 pS and 268 ± 14 pS respectively.

Lowering [K+] on the cytosolic side of the patches from 140 to 6 mm, shifted the reversal potentials of the two types of unitary current from approximately zero to ≫ +40 mV, suggesting that both currents were carried by K+‐channels.

The Ca2+‐dependent and ‐independent K+‐channels detected in inside‐out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1–10 mm; Cs+, 10 mm; Ba2+, 1–10 mm; quinidine, 100 μm) applied to the cytosolic surface of the patches.

Recordings were made from outside‐out plasmalemmal patches when [K+] was symmetrically high (140 mm) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 μm. Ca2+‐dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 ± 5 pS.

Activity of the Ca2+‐dependent K+‐channel detected in outside‐out patches could be inhibited by application of TEA (1 mm), Cs+ (10 mm), Ba2+ (10 mm) or quinidine (100 μm) to the external surface of the patch. 4‐Aminopyridine (4‐AP; 1 mm) was ineffective as an inhibitor.

The activity of the Ca2+‐dependent K+‐channel recorded from outside‐out patches was reversibly inhibited by charybdotoxin (100 nm).

When whole‐cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2+] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nm–1 μm) applied to the external surface of the cell.

We conclude that bovine trachealis cells are richly endowed with charybdotoxin‐sensitive, large conductance, Ca2+‐dependent K+‐channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2+‐independent K+‐channels remains unclear.
The release of a non‐prostanoid inhibitory factor from rabbit bronchus detected by co‐axial bioassay
Authors: Spina D. , Page C.P.
Published: July 2012

Methacholine relaxed phenylephrine‐contracted aorta of the rat with the endothelium intact. This effect was inhibited by haemoglobin, methylene blue, gossypol, phenidone and l‐NG‐nitroarginine methyl ester (l‐NAME). Rat aorta denuded of endothelium failed to relax in response to methacholine, histamine and the peptidoleukotrienes C4, D4 and E4.

Methacholine and histamine but not leukotrienes C4, D4 and E4 relaxed phenylephrine‐contracted rat aorta without endothelium when surrounded by rabbit epithelium‐intact bronchus. The muscarinic antagonist atropine antagonized the methacholine‐induced relaxation.

Removal of the epithelium either mechanically or chemically, abolished methacholine‐induced relaxation of rat aorta in the co‐axial bioassay. These data indicate that the epithelium is responsible for the observed relaxant effect to methacholine and histamine.

The cyclo‐oxygenase inhibitor, indomethacin, the phospholipase A2 inhibitor, mepacrine and the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), failed to inhibit methacholine‐induced relaxation of rat aorta in the co‐axial bioassay. This indicates that the epithelium‐derived inhibitory factor (EpDIF) is not a product of the cyclo‐oxygenase or lipoxygenase pathway or a product derived from activation of phospholipase A2.

Haemoglobin, methylene blue, phenidone, gossypol and l‐NAME failed to inhibit the relaxation of rat aorta in the co‐axial bioassay. These results demonstrate that EpDIF detected in the co‐axial bioassay is not endothelium‐derived relaxing factor (EDRF) or nitric oxide. Similarly, catalase was without effect.

EpDIF is unlikely to be a peptide since papain and α‐chymotrypsin failed to alter the methacholine‐induced relaxation of rat aorta in the co‐axial bioassay. Furthermore, thiorphan, captopril and aprotinin were also without effect, suggesting that EpDIF is not a substrate for airway peptidases.

The results presented in this paper demonstrate the release of a vasoactive epithelium‐derived inhibitory factor (EpDIF) from rabbit intrapulmonary bronchi by use of a co‐axial bioassay preparation.
The mechanism of the sympathoinhibitory action of urapidil: role of 5‐HT1A receptors
Authors: Ramage Andrew G.
Published: July 2012

An investigation was carried out to determine if the sympathoinhibition caused by urapidil is due to activation of 5‐HT1A receptors by investigating whether it could be reversed by the non‐selective 5‐HT1A receptor antagonist spiperone. To control for the possibility of functional antagonism by spiperone, the ability of spiperone to reverse the sympathoinhibition caused by clonidine was also investigated. These experiments were carried out in anaesthetized prazosin‐pretreated cats to prevent the α1‐adrenoceptor antagonist action of urapidil and spiperone from masking any effects observed.

Cats were anaesthetized with α‐chloralose and simultaneous recordings were made of whole cardiac, splanchnic and renal nerve activities, blood pressure, heart rate and femoral arterial flow (from which conductance was derived). All animals were initially pretreated with prazosin (1 mg kg−1, i.v.) given in divided doses (0.75 followed 10 min later by 0.25 mg kg−1), then either urapidil (0.75 mg kg−1, i.v.) or clonidine (10 μg kg−1, i.v. in two divided doses) followed by 3 separate injections of spiperone (1 mg kg−1, i.v.). In another set of experiments urapidil was given followed by injections of the appropriate vehicle for spiperone, while in another set urapidil was replaced with an injection of the appropriate vehicle followed by injections of spiperone. In the experiments with clonidine, the α2‐adrenoceptor antagonist Wy 26392 (0.3 mg kg−1) was given after the last injection of spiperone.

The prazosin pretreatment caused a fall in blood pressure associated with femoral vasodilatation, a small bradycardia and little change in cardiac, splanchnic or renal nerve activities. Urapidil or clonidine injection after prazosin caused sympathoinhibition associated with an additional bradycardia. However, only urapidil caused an additional fall in blood pressure. Spiperone injections reversed the sympathoinhibition caused by urapidil but not that caused by clonidine. The sympathoinhibition caused by clonidine was reversed by the α2‐adrenoceptor antagonist Wy 26392.

These results show that the sympathoinhibition caused by urapidil in prazosin‐pretreated cats can be reversed by spiperone. The reversal of this sympathoinhibition is not due to functional antagonism. It is concluded that urapidil can cause sympathoinhibition by activation of 5‐HT1A receptors.
Effects of ATP‐sensitive K+ channel blockers on the action potential shortening in hypoxic and ischaemic myocardium
Authors: Nakaya Haruaki , Takeda Youji , Tohse Noritsugu , Kanno Morio
Published: July 2012

In order to determine whether activation of adenosine triphosphate (ATP)‐sensitive K+ channels exclusively explains the hypoxia‐ and ischaemia‐induced action potential shortening, effects of tolbutamide and glibenclamide on changes in action potential duration (APD) during hypoxia, metabolic blockade or experimental ischaemia were examined in guinea‐pig and canine isolated myocardium by standard microelectrode techniques.

With use of patch clamp techniques, activity of ATP‐sensitive K+ channels was recorded from open cell‐attached patches of guinea‐pig isolated ventricular myocytes. The probability of opening of the K+ channels was decreased by 2 mm tolbutamide and 20 μm glibenclamide to almost the same extent, whereas it was increased by 100 μm pinacidil.

In guinea‐pig papillary muscles a marked shortening of the action potential produced by 100 μm pinacidil was completely antagonized by 2 mm tolbutamide or 20 μm glibenclamide.

In guinea‐pig papillary muscles exposed to hypoxic, glucose‐free solution or dinitrophenol (10 μm)‐containing, glucose‐free solution, APD declined gradually and twitch tension decreased. Pretreatment with glibenclamide partially but significantly inhibited the action potential shortening, whereas tolbutamide failed to improve it during hypoxia or metabolic blockade.

When in canine isolated myocardium, experimental ischaemia was produced by the cessation of coronary perfusion, APD was gradually shortened. The action potential shortening was partially but not completely inhibited by pretreatment with 20 μm glibenclamide.

These results suggest that changes in membrane current(s) other than the outward current through ATP‐sensitive K+ channels also contribute to the action potential shortening in hypoxic or ischaemic myocardium.
Evidence that an l‐arginine/nitric oxide dependent elevation of tissue cyclic GMP content is involved in depression of vascular reactivity by endotoxin
Authors: Fleming Ingrid , Julou‐Schaeffer Géraldine , Gray Gillian A. , Parratt James R. , Stoclet Jean‐Claude
Published: July 2012

The aim of this investigation was to study the relationship between contractile responsiveness, activation of the l‐arginine pathway and tissue levels of guanosine 3′:5′‐cyclic monophosphate (cylic GMP) in aortic rings removed from rats 4 h after intraperitoneal administration of bacterial endotoxin (E.coli. lipopolysaccharide, LPS, 20 mg kg−1).

LPS‐treatment resulted in a reduction of the sensitivity and maximal contractile response to noradrenaline (NA).

Depression of the maximal contractile response was restored to control by 6‐anilo‐5,8‐quinolinedione (LY 83583, 10 μm), which prevents activation of soluble guanylate cyclase.

Cyclic GMP levels in tissue from LPS‐treated rats were 2 fold greater than cyclic GMP levels detected in tissue from control (saline‐treated) rats. The LPS‐induced increase in cyclic GMP content was observed both in the presence and absence of functional endothelium.

Addition of l‐arginine (1 mm) to maximally contracted aortic rings produced significant relaxation of rings from LPS‐treated rats but not rings from control animals. In the LPS‐treated group, addition of l‐arginine was also associated with a significant increase in cyclic GMP content. l‐Arginine had no effect on the cyclic GMP content of control rings. d‐Arginine (1 mm) was without effect.

In rings from LPS‐treated rats, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 300 μm) an inhibitor of nitric oxide (NO) production, increased the contractile response to NA and prevented the LPS‐induced increase in cyclic GMP content. In control rings, l‐NAME increased the NA sensitivity only when the endothelium remained intact and reduced the cyclic GMP content of these rings to that of control endothelium‐denuded rings.

These results demonstrate that LPS‐induced hyporeactivity to NA occurs secondarily to activation of the l‐arginine pathway and subsequent activation of soluble guanylate cyclase in vascular tissue. In addition they suggest that LPS induces the production of an NO‐like relaxing factor in non‐endothelial cells.
Characterization of bombesin receptors in peripheral contractile organs
Authors: Rouissi N. , Rhaleb N.‐E. , Nantel F. , Dion S. , Drapeau G. , Regoli D.
Published: July 2012

Guinea‐pig and rat urinary bladders, rat stomach and the guinea‐pig gall bladder, four isolated organs that show high sensitivity to bombesin, were used to characterize bombesin receptors in peripheral organs.

The order of potency of agonists was determined with several naturally occurring peptides of the bombesin series, namely bombesin (BBS), litorin (Lit), neuromedin B (NMB), the gastrin‐releasing peptide (GRP 18–27), neuromedin C (NMC) and with some bombesin fragments. It was found that bombesin, neuromedin C., litorin and two bombesin fragments, BBS (6–14) and AcBBS (6–14) had similar activities in the four preparations, while neuromedin B and [Phe6]‐neuromedin C were more active on the rat urinary bladder than on the other tissues.

The order of potency of agonists determined in the rat urinary bladder was as follows: BBS = NMB > Lit > NMC > [Phe6]NMC = GRP and it was found to be different from that observed in the other preparations: BBS > GRP = Lit ≥ NMC ≫ NMB > [Phe6]NMC, suggesting the existence of two different bombesin receptors, BBS1 and BBS2.

This interpretation was convalidated by the finding that bombesin antagonists, namely Ac.GRP(20–26)OCH3 and Ac.GRP(20–26)OC2H5 reduced or blocked the effects of bombesin‐related peptides on BBS2 receptor systems while being completely inactive on the rat urinary bladder (BBS1 system).
Modulation by the epithelium of the extent of bronchial narrowing produced by substances perfused through the lumen
Authors: Sparrow M. P. , Mitchell H.W.
Published: July 2012

Airway narrowing was determined in vitro as a measure of bronchial reactivity. A bronchial segment from pig lung was perfused with a Krebs solution and the change in flow rate to drugs and small ions perfused intraluminally was compared with that obtained by application to the serosal surface.

The sensitivity (EC50) to acetylchloline was 30 times greater on the serosal surface than on the luminal surface. Concentrations of histamine and carbachol which had threshold responses on flow rate when perfused intraluminally virtually stopped flow on the serosal surface. Potassium depolarizing solutions (containing either KCl or K2SO4) and vanadate (VO−3) had little or no effect intraluminally but completely stopped flow through the bronchial segment when applied to the serosal surface, i.e. they closed off the airway.

After removal of the epithelium the sensitivity to drugs and K+ perfused intraluminally was increased to equal that on the serosal surface.

No evidence for suppression of smooth muscle contraction by a putative epithelium‐derived inhibitory factor (EpDIF) could be obtained: no inhibition of smooth muscle contractility was seen when the agents listed above were perfused intraluminally and their perfusion continued while they were applied to outside.

It was concluded that the epithelium plays a crucial role as an impermeant barrier in modulating the responsiveness of the airways smooth muscle.
Effects of pretreatment with caffeine or ryanodine on the myocardial response to simulated ischaemia
Authors: Northover B.J.
Published: July 2012

The cytoplasmic calcium concentration ([Ca]c) of rat isolated atrial myocardium was assessed with the dye indo‐1. Dye‐loaded atria were superfused with physiological salt solution and excited with radiation at 360 nm, while epifluorescence emissions were collected simultaneously at 400 nm and 500 nm. The ratio of these emissions was used as a measure of [Ca]c.

Dye‐loaded atria showed a phasic rise and fall in [Ca]c with each applied electrical pacing stimulus. The amplitude of these oscillations was reduced by the presence of caffeine (10−3–10−2 m) or of ryanodine (10−8–10−6 m) in a concentration‐dependent manner.

Atria superfused with a solution the composition of which resembled that found extracellularly in regions of myocardial ischaemia rapidly lost systolic increments in [Ca]c, while end‐diastolic [Ca]c values gradually rose.

Pretreatment with caffeine (10−2 m) or ryanodine (10−7 m) protected atria against the rise in end‐diastolic [Ca]c that occurred when the tissue was exposed to conditions of simulated ischaemia.
Effects of intravenous μ and κ opioid receptor agonists on sensory responses of convergent neurones in the dorsal horn of spinalized rats
Authors: Dong Xiao‐wei , Parsons Chris G. , Headley P. Max
Published: July 2012

Electrophysiological experiments have been performed to assess the effects of intravenously administered μ and κ opioid agonists on the responses to noxious thermal and mechanical and non‐noxious tactile stimuli of single convergent neurones in laminae III‐VI of the dorsal horn of spinalized rats anaesthetized with α‐chloralose.

The μ receptor agonists tested were fentanyl (1–16 μg kg−1) and morphine (0.5–16 mg kg−1) and the κ‐receptor agonists U‐50,488 (1–16 mg kg−1) and tifluadom (0.1–1.6 mg kg−1). Multiple drug tests were made on each cell so that compounds could be compared under closely comparable conditions.

In one protocol, thermal and mechanical nociceptive responses of matched amplitudes were elicited alternately. Both μ and κ agonists dose‐dependently reduce the neuronal responses. Thermal nociceptive responses were as sensitive to the κ agents as were the mechanical nociceptive responses; the μ agonists similarly reduced both types of response in parallel.

In another protocol, nociceptive and non‐nociceptive responses were elicited alternately to permit the degree of selective antinociception to be assessed. The μ agonists were scarcely selective, fentanyl reducing nociceptive only slightly (but significantly at 4–16 μg kg−1) more than non‐nociceptive responses. The κ‐opioid agonist U50,488 reduced tactile responses somewhat more than nociceptive responses.

The spontaneous discharge of these cells with ongoing activity was reduced to a significantly greater degree than the evoked responses; this is likely to have contributed to the non‐selectivity of the reduction of the evoked responses.

The results are discussed with respect firstly to previous reports that κ opioids are ineffective in tests of thermal nociception, and secondly to the likely spinal mechanisms by which opioid receptor agonists mediate antinociception.
Haemodynamic effects of human α‐calcitonin gene‐related peptide following administration of endothelin‐1 or NG‐nitro‐l‐arginine methyl ester in conscious rats
Authors: Gardiner S.M. , Compton A.M. , Kemp P.A. , Bennett T. , Foulkes R. , Hughes B.
Published: July 2012

We investigated the peripheral haemodynamic effects of human α‐calcitonin gene‐related peptide (CGRP) following administration of endothelin‐1 or NG‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of nitric oxide production, in conscious, chronically‐instrumented, Long Evans rats.

Infusion of endothelin‐1 (3 nmol kg−1 h−1) caused hypertension, bradycardia and renal, mesenteric and hindquarters vasoconstrictions. Co‐infusion of human α‐CGRP (1.5 nmol kg−1 h−1) reduced the hypertension and abolished the hindquarters vasoconstriction caused by endothelin‐1 but the renal and mesenteric vasoconstrictor actions of endothelin‐1 were not affected.

Infusion of human α‐CGRP (15 nmol kg−1 h−1) in the presence of endothelin‐1 caused hypotension and hyperaemic vasodilatation in the hindquarters; the mesenteric vasoconstrictor effects of endothelin‐1 were diminished, but there was only a transient reversal of the renal vasoconstrictor effects of endothelin‐1.

Pretreatment with the non‐peptide angiotensin II receptor antagonist, DuP 753 (10 mg kg−1), caused slight hypotension associated with renal, mesenteric and hindquarters vasodilatations, but DuP 753 did not affect responses to endothelin‐1 infusion. However, under these conditions co‐infusion of human α‐CGRP (15 nmol kg−1 h−1) caused a sustained reversal of the renal vasoconstrictor effects of endothelin‐1.

These results indicate that the failure of human α‐CGRP to cause sustained reversal of the renal vasoconstrictor effects of endothelin‐1 in the absence of DuP 753 was due to activation of the renin‐angiotensin system (possibly as a consequence of the hypotension).

In the second experiment, l‐NAME (10 mg kg−1) caused renal, mesenteric and hindquarters vasoconstrictions similar to those seen in the presence of endothelin‐1. However, the renal vasoconstrictor effects of l‐NAME were reversed completely by human α‐CGRP (15 nmol kg−1 h−1), even though the latter caused hypotension comparable to that seen in the presence of endothelin‐1. These results are consistent with a lack of functional activation of the renin‐angiotensin system by human α‐CGRP in the presence of l‐NAME.

The vasoconstrictor effects of l‐NAME on the hindquarters were completely reversed by infusion of human α‐CGRP, but hindquarters flow and vascular conductance did not rise above baseline levels. Hence these results indicate the hindquarters hyperaemic vasodilator effects of human α‐CGRP seen in the presence of endothelin‐1 were contributed to by nitric oxide‐mediated mechanisms.
Block of ATP‐regulated potassium channels by phentolamine and other α‐adrenoceptor antagonists
Authors: Dunne M.J.
Published: July 2012

The patch clamp technique has been used to characterize the effects of phentolamine, an unselective blocker of α1 and α2‐adrenoceptors, on the electrical activity of isolated RINm5F insulin‐secreting cells and the gating of ATP‐regulated potassium (K+ATP) channels.

Current‐clamp experiments carried out by use of both conventional whole‐cell recordings and nystatin‐perforated cells, have demonstrated that phentolamine (5–20 μm) in the complete absence of α‐adrenoceptor agonists, caused a sharp depolarization of the cell membrane from approximately −66 mV to −42 mV. This depolarization was associated with the generation of calcium action potential‐like spikes. In the continued presence of phentolamine, diazoxide (100 μm) reversed these effects by causing a hyperpolarization of the cell, thereby preventing Ca2+ spikes.

Unitary current events from K+ATP channels were recorded from both outside‐out membrane patches and saponin permeabilized or open‐cells. When added to either the inside or the outside of the plasma membrane, phentolamine (0.1–100 μm) blocked openings from these channels. The effects of phentolamine were rapid, sustained and fully reversible. Phentolamine was apparently a more effective blocker of channels from the inside than the outside of the membrane.

The K1 value, corresponding to 50% inhibition of channels was estimated to be approximately 0.7 μm when phentolamine was added to the inside of the membrane and the Hill coefficient approximately 1.

Yohimbine (1–10 μm) and the chemically 2‐substituted imidazoline α‐adrenoceptor antagonists, antazoline (25 μm) and tolazoline (25 μm) were also found to block K+ATP channels in isolated patches of membrane.

In conclusion the present study demonstrates that phentolamine and other imidazoline adrenoceptor antagonists have effects upon ATP‐sensitive K+ channels that are not associated with stimulation of the adrenoceptor.
Kinin receptors of the central nervous system of spontaneously hypertensive rats related to the pressor response to bradykinin
Authors: Martins D.T.O. , Fior D.R. , Nakaie C.R. , Lindsey C.J.
Published: July 2012

Kinin analogues bradykinin (BK), T‐kinin, Met‐Lys‐BK, Lys‐Lys‐BK, Des‐Arg9‐BK with agonist activity and d‐Arg0‐Hyp3‐Thi5,8‐d‐Phe7‐BK (DAHTDBK) and Arg9‐Leu8‐BK with antagonist activity were injected into the posterior portion of the fourth cerebral ventricle of unanaesthetized rats implanted with permanent cannulae and arterial pressure was measured directly from the abdominal aorta.

The spontaneously hypertensive rats (SHR) were more sensitive than normotensive Wistar rats (NWR) to the pressor effect of BK and other kinin analogues. The SHR did not differ in sensitivity of the pressor response to centrally administered angiotensin II or endothelin‐1.

Experiments with selective kinin agonists and antagonists revealed that in the SHR, as in the NWR, the receptors which mediated the central pressor response are of the BK2 subtype.

Measurements of the pressor activity of kinins with different degrees of susceptibility to degradation, as well as experiments with kininase inhibitors, enalaprilat and CPP‐Ala‐Ala‐Phe‐pAB, suggest that the kininase activity in the central nervous system of SHR is reduced in comparison to that of NWR.

The SHR also showed increased sensitivity to BK and Lys‐Lys‐BK, compared with the NWR, when the kinins were injected following the administration of a mixture of the kininase inhibitors, suggesting that mechanisms other than kininase activity may play a role in the increased sensitivity of the SHR to the central pressor action of kinins.

An in vivo characterization of the kinin receptors which mediate the central pressor response showed that the interaction with DAHTDBK was reversible and of competitive nature. The pA2 in vivo estimated for the kinin receptors of the SHR was 0.7 logarithm units larger than that obtained in the NWR.

The kinin receptors which mediate the central BK pressor effect in the SHR are of the BK2 subtype and are similar to receptors in the NWR. The increased sensitivity to kinins in the SHR may be explained, at least in part, by their decreased kininase activity. At present it is impossible to conclude whether the difference observed in the pA2 represents an increased affinity of the kinin receptors or can be attributed to differences amongst strains in the enzymatic degradation of the antagonist.
Mechanisms controlling caffeine‐induced relaxation of coronary artery of the pig
Authors: van der Bent Vincent , Bény Jean‐Louis
Published: July 2012

We studied the effects of caffeine on coronary artery smooth muscle of the pig by measuring changes in isometric tension, cytosolic free Ca2+ concentration ([Ca2+]i) and transmembrane potential.

In the absence of tone, caffeine induced a concentration‐dependent transient contraction of coronary artery strips, followed by sustained relaxation. Simultaneously with the relaxation, caffeine, 25 mm, hyperpolarized the smooth muscle cells by 7.7 ± 0.9 mV.

Caffeine caused a concentration‐dependent relaxation of strips precontracted with 10−5 m acetylcholine (ACh). A supramaximal relaxing concentration of 25 mm caffeine produced an additional transient increase in [Ca2+]i on the Ca2+ plateau of ACh tonic contraction, which was followed by a decrease in [Ca2+]i to a level slightly below the basal concentration. This relaxation was accompanied by a hyperpolarization of 7.3 ± 0.9 mV.

KCl 120 mm (high K+) contracted the strips with a concomitant depolarization of 38.6 ± 1.6 mV and sustained increase in [Ca2+]i. Caffeine caused a concentration‐dependent relaxation of high K+‐induced contraction. Caffeine, 25 mm, decreased the Ca2+ plateau to a level that remained above the basal concentration of Ca2+ but did not change the membrane potential.

When strips were placed in a Ca2+‐free medium with EGTA 2 mm and, in addition, ACh was applied successively three times, both intracellular and extracellular mobilizable Ca2+ pools were depleted. In these conditions, phorbol 12,13 dibutyrate (PDBu) 10−7 m and prostaglandin F2α (PGF2α) 10−5 m contracted the strips. Caffeine (25 mm) inhibited these contractions with no change in [Ca2+]i.

Forskolin, 3 × 10−7 m, inhibited ACh induced‐contraction but did not affect those induced by PDBu.

In conclusion, these results show that caffeine has multiple cellular effects. During caffeine‐induced relaxation, [Ca2+]i, adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) content and membrane potential are modified. The findings suggest, however, that these effects are secondary, and that caffeine acts mainly by another unknown mechanism, possibly involving a direct inhibition of the contractile apparatus.
Characterization of a novel, hydrophilic dihydropyridine, NKY‐722, as a Ca2+ antagonist in bovine cultured adrenal chromaffin cells
Authors: Ohue Tetsuya , Lee Ken , Koshimura Kunio , Miwa Soichi
Published: July 2012

To characterize NKY‐722, a novel hydrophilic dihydropyridine derivative, as a Ca2+ antagonist, we examined its effects on 45Ca2+ influx, intracellular free Ca2+ concentrations ([Ca2+]i), and release of noradrenaline and adrenaline in bovine cultured adrenal chromaffin cells.

NKY‐722 had little effect on basal 45Ca2+ influx into the resting cells, but inhibited high K+ (35.9 mm)‐evoked 45Ca2+ influx in a concentration‐dependent manner with an IC50 value of 5.2 nm.

NKY‐722 inhibited high K+‐evoked increases in [Ca2+]i in a concentration‐dependent manner without effect on the resting [Ca2+]i.

NKY‐722 had little effect on basal release of noradrenaline and adrenaline but inhibited high K+‐evoked release of noradrenaline and adrenaline in a concentration‐dependent manner with IC50 values of 5.0 nm and 4.8 nm, respectively.

Nicardipine, a prototype of NKY‐722, also inhibited high K+‐evoked 45Ca2+ influx and release of noradrenaline and adrenaline in a concentration‐dependent manner: the IC50 value for high K+‐evoked 45Ca2+ influx was 51 nm, and the values for high K+‐evoked release of noradrenaline and adrenaline were 52 nm and 50 nm, respectively.

These results show that NKY‐722 is a hydrophilic Ca2+ antagonist ten times more potent than nicardipine.
Thrombin‐activated platelets promote leukotriene B4 synthesis in polymorphonuclear leucocytes stimulated by physiological agonists
Authors: Palmantier Rémi , Borgeat Pierre
Published: July 2012

The addition of 2 × 108 human platelets to 8 × 106 polymorphonuclear leucocytes (PMNL) incubated in presence of 2.5 u ml−1 thrombin and 0.1 μm N‐formyl‐Met‐Leu‐Phe (FMLP) (or C5a or PAF) led to enhancement of leukotriene B4 (LTB4) synthesis by the PMNL (measured by h.p.l.c. as 20‐hydroxy‐ and 20‐carboxy‐LTB4) from 4 ± 1 pmol (in absence of platelets) to 26 ± 4 pmol (mean ± s.e.mean, n = 9). Platelets and thrombin were both essential for the enhancement of LTB4 synthesis.

Platelets also caused enhancement of LTB4 synthesis from (30 ± 12 to 134 ± 25 pmol, n = 6) when PMNL pretreated with granulocyte‐macrophage colony‐stimulating factor were used in similar experiments.

Enhancement of LTB4 synthesis was also observed (from 5 ± 1.5 to 26.5 ± 5 pmol, n = 9) when the supernatants of thrombin‐activated platelet suspensions were added to FMLP‐stimulated PMNL.

Supernatants of platelet suspensions activated by thrombin in presence of cyclo‐oxygenase and 12‐lipoxygenase inhibitors led to greater enhancement (from 5 ± 3 to 153.5 ± 27.5 pmol, n = 3) of LTB4 synthesis by FMLP‐stimulated PMNL, suggesting that arachidonic acid itself, rather than its metabolites was responsible for the effects of platelets.

Addition of arachidonic acid to FMLP‐stimulated PMNL at a concentration comparable to that measured in thrombin‐activated platelet supernatants (0.2 ± 0.025 μm, n = 6) mimicked the effect of platelets or platelet supernatants on LTB4 synthesis in FMLP‐activated PMNL.

The present data indicate that under conditions of cell activation by physiological agonists, platelets can significantly increase the formation of the proinflammatory compound LTB4 in PMNL by providing arachidonic acid. These data lend support to the concept that platelet‐PMNL interactions could modulate the inflammatory process.
Inotropic and electrophysiological effects of BDF 9148, a congener of DPI 201–106, in guinea‐pig atria and papillary muscles
Authors: Brasch Helmut , Iven Heiko
Published: July 2012

BDF 9148 is a newly synthesized congener of DPI 201–106 in which the piperazidinyl moiety has been replaced by an azetidine‐3‐oxy‐moiety. The inotropic effect of both drugs was studied as well as their influence on the action potential, by use of standard microelectrode techniques.

BDF 9148 increased the contractile force of guinea‐pig atria and papillary muscles. The EC50 was 1.32 × 10−7mol l−1 and 0.7 × 10−6 mol l−1 respectively. DPI 201–106 was effective in a similar concentration‐range, the EC50 being 2.6 × 10−7mol l−1 for atria and 2.8 × 10−7 mol l−1 for papillary muscles.

Both drugs caused a concentration‐dependent prolongation of the relaxation time of the isometric contraction curve. With 10−6 mol l−1 BDF 9148, the mean increase was 39.1 ± 4.4 ms in atria and 39.4 ± 7.5 ms in papillary muscles while 10−6 mol l−1 DPI 201–106 caused increases of 56.7 ± 2.5 ms and 79.3 ± 11.7 ms, respectively. The effect of BDF 9148 was not prevented by propranolol, but was reversed by 3 × 10−6 mol l−1 tetrodotoxin. Pretreatment of atria with 3 × 10−8 mol l−1 BDF shifted the concentration‐response curve of ouabain to the left and reduced the EC50 of the glycoside from 3.21 × 10−7 mol l−1 to 2 × 10−7 mol l−1.

BDF 9148 and DPI 201–106 concentration‐dependently increased the action potential duration of papillary muscles and their functional refractory period. The drugs did not modify the resting potential, the action potential amplitude or the maximum depolarization velocity.

All effects of BDF 9148 persisted after washout. The lipophilic drug accumulated in the tissues and the tissue drug concentration was little reduced by washout of BDF 9148 from the organ bath.

In contrast to DPI 201–106, which had a prominent negative chronotropic effect in right atria, BDF 9148 caused only a slight reduction of the beating frequency with the largest (3 × 10−5 mol l−1) concentration.

The results are consistent with BDF 9148 being a sodium channel activator with much weaker influence on the beating frequency than the parent compound DPI 201–106.
Desensitization and functional antagonism by β‐adrenoceptor and muscarinic receptor agonists, respectively: a comparison with receptor alkylation for calculation of apparent agonist affinity
Authors: Eglen R.M. , Montgomery W.W. , Whiting R.L.
Published: July 2012

Apparent affinity constants (KD) for prenalterol, an agonist of low intrinsic efficacy at β1‐adrenoceptors in rat left atria, have been determined by use of receptor desensitization and functional antagonism induced by isoprenaline and carbachol, respectively. The values obtained have been compared to those values estimated with the irreversible β‐adrenoceptor antagonist, bromoacetylalprenololmenthane (BAAM).

The −log KD values for prenalterol estimated by desensitization or irreversible antagonism ranged from 6.8–7.1 and 6.2–7.1, respectively.

Carbachol produced functional antagonism of the response to prenalterol even though it was removed before addition of prenalterol. This effect was mediated by M2‐muscarinic receptors. Pretreatment of animals with pertussis toxin did not affect the functional antagonism elicited by carbachol. The apparent KD value obtained after pre‐exposure to carbachol (6.8) was similar to those estimated by use of either alkylation with BAAM or desensitization with isoprenaline (see above).

It is concluded that acute desensitization or functional antagonism of responses to agonists of low intrinsic efficacy provides a means to estimate apparent KD constants. This approach could be useful to characterize receptors for which an irreversible antagonist may not be available.