European Journal of Pharmacology (v.388, #1)

Dopamine D2 receptor ribozyme inhibits quinpirole-induced stereotypy in rats by Peter Salmi; Brian S Sproat; Janos Ludwig; Ruth Hale; Nicola Avery; Johanna Kela; Claes Wahlestedt (R1-R2).
The injection of a dopamine D2 receptor hammerhead ribozyme (20 μg) once daily for 5 days into the nucleus accumbens of rats resulted in an inhibition of stereotyped sniffing and locomotor activation produced by the selective dopamine D2 receptor agonist, quinpirole (0.4 mg kg−1 s.c.). The results suggest that ribozymes may be useful in the study of in vivo gene function in the brain.
Keywords: Ribozyme; Dopamine D2 receptor; Stereotypy;

Influence of age on nitric oxide modulatory action on Na+, K+-ATPase activity through cyclic GMP pathway in proximal rat trachea by Cristoforo Scavone; Isaias Glezer; Carolina Demarchi Munhoz; Cristiane de Sena Bernardes; Regina Pekelmann Markus (1-7).
Age-related changes in the modulatory action of nitric oxide (NO) on cyclic GMP levels and Na+,K+-ATPase activity in the proximal rat trachea were investigated using sodium nitroprusside, 8-bromo-cyclic GMP and okadaic acid. At 24 months, both control activities of Na+,K+-ATPase and Mg2+-ATPase were decreased when compared to the segments from 4- and 12-month-old animals. However, cyclic GMP levels were similar among the three ages. Sodium nitroprusside (100 μM) induced stimulation of Na+,K+-ATPase activity in segments from both 4- and 12-month-old animals, but not 24-month-old animals. The effect was specific for Na+,K+-ATPase since Mg2+-ATPase activity was unaffected. Sodium nitroprusside induced an increase in nitrates/nitrites and cyclic GMP levels in proximal segments at 4, 12 and 24 months. The 8-bromo-cyclic GMP (100 μM) induced a similar specific stimulation of Na+,K+-ATPase activity in segments from 4- and 12- but not 24-month-old animals. Okadaic acid (1 μM), a phosphatase-1 inhibitor, increased proximal Na+,K+-ATPase but not Mg2+-ATPase activity in tissues from 4-, 12- and 24-month-old animals. Our results suggest that aging affects cyclic GMP pathway in proximal rat trachea by an action at the level of the cyclic GMP-dependent protein kinase.
Keywords: Nitric oxide (NO); cGMP-dependent protein kinase; Na+,K+-ATPase; Aging; Trachea;

Affinity of cholecystokinin receptor antagonists for the gastrin-binding protein by Kristy A Rorison; Zhiyu Yang; Graham S Baldwin (9-15).
A 78 kDa gastrin-binding protein is a likely target for the anti-proliferative effects of the cholecystokinin (CCK) receptor antagonists d,l-4-benzamido-N,N-dipropylglutaramic acid (proglumide) and N-4-chlorobenzoyl-l-tryptophan (benzotript) on colorectal carcinoma cell lines [Baldwin, G.S., 1994. Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein. Proc. Natl. Acad. Sci. USA 91, 7593–7597.]. Definition of the physiological role of the gastrin-binding protein has been hampered by the very low affinity of benzotript for the gastrin-binding protein. Benzotript analogues were therefore tested for their ability to inhibit the binding of iodinated gastrin to the gastrin-binding protein. The affinity of the most potent analogue (the d-isomer of benzotript, CR 665) was similar to the value reported previously for the l-isomer. In order to isolate more potent binding inhibitors, several selective CCK receptor antagonists were also tested as inhibitors of the binding of gastrin to the gastrin-binding protein. The affinity of the most potent binding inhibitor PD 149164 (benzenebutanoic acid, 4-fluoro-!b/-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo-[,7]dec-2-yloxy)carbonyl]amino]propyl]amino]-, [R-(R*,S*)]-) was approximately 10-fold higher than the l-isomer of benzotript. PD 149164 may serve as the lead compound for the future development of more potent and selective gastrin-binding protein inhibitors.
Keywords: Gastrin; CCK receptor antagonist; Gastrin-binding protein;

A novel effect of rebamipide: generation of [Ca2+]i oscillations through activation of CCK1 receptors in rat pancreatic acinar cells by Seok Jun Moon; Wooin Ahn; Min Goo Lee; Hyeyoung Kim; Syng-Ill Lee; Jeong Taeg Seo; R.Maynard Case; Kyung Hwan Kim (17-20).
The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca2+ concentration ([Ca2+]i) through the activation of cholecystokinin subtype 1 (CCK1) receptors. At concentrations higher than 5 μM, rebamipide induced [Ca2+]i oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca2+]i oscillations decreased with increasing concentration. The [Ca2+]i oscillations evoked by rebamipide were inhibited by the CCK1 receptor antagonist L-364,718 but not by atropine or the CCK2 receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK1 receptor agonist.
Keywords: Rebamipide; CCK1 receptor; [Ca2+]i oscillation; Pancreatic acinar cell;

Angiotensin II type 1 receptor-mediated increase in cytosolic Ca2+ and proliferation in mesothelial cells by Masayoshi Kuwahara; Takako Miyaji; Hirokazu Tsubone; Maki Kuwahara (21-27).
We investigated the Ca2+ signaling pathways of the response to angiotensin II in pleural mesothelial cells and the role of these Ca2+ signaling pathways in mesothelial cell proliferation. Rat pleural mesothelial cells were maintained in vitro, and the Ca2+ movement to angiotensin II was evaluated using the fluorescent Ca2+ indicator fura 2. Furthermore, proliferation of mesothelial cells was assessed using a spectrophotometric 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl-2H-tetrasodium bromide (MTT) assay. Angiotensin II (1 pM–100 μM) induced in mesothelial cells a biphasic elevation of intracellular Ca2+ concentration ([Ca2+]i) that consisted of a transient initial component, followed by a sustained component. Neither removal of extracellular Ca2+ nor inhibition of Ca2+ influx by 1 μM nifedipine affected the angiotensin II-induced initial transient elevation of [Ca2+]i in mesothelial cells. Nifedipine did not block angiotensin II-induced sustained elevation of [Ca2+]i. Angiotensin II (1 pM–100 μM) had a proliferative effect on mesothelial cells in a dose-dependent manner. Angiotensin II type 1 (AT1) receptor antagonist ([Sar1, Ile8]angiotensin II) inhibited both angiotensin II-induced elevation of [Ca2+]i and proliferation of mesothelial cells. Pertussis toxin did not affect angiotensin II-induced responses. These results suggest that angiotensin II-induced responses to mesothelial cells are extremely dependent on the angiotensin AT1 receptor coupled with pertussis toxin-insensitive G protein.
Keywords: Angiotensin II; Angiotensin II type 1 receptor; Ca2+; Mesothelial cell; Pleura;

ABT-627, an endothelin ETA receptor-selective antagonist, attenuates tactile allodynia in a diabetic rat model of neuropathic pain by Michael F Jarvis; Jerry L Wessale; Chang Z Zhu; James J Lynch; Brian D Dayton; Samuel V Calzadilla; Robert J Padley; Terry J Opgenorth; Elizabeth A Kowaluk (29-35).
Tactile allodynia, the enhanced perception of pain in response to normally non-painful stimulation, represents a common complication of diabetic neuropathy. The activation of endothelin ETA receptors has been implicated in diabetes-induced reductions in peripheral neurovascularization and concomitant endoneurial hypoxia. Endothelin receptor activation has also been shown to alter the peripheral and central processing of nociceptive information. The present study was conducted to evaluate the antinociceptive effects of the novel endothelin ETA receptor-selective antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), in the streptozotocin-induced diabetic rat model of neuropathic pain. Rats were injected with 75 mg/kg streptozotocin (i.p.), and drug effects were assessed 8–12 weeks following streptozotocin treatment to allow for stabilization of blood glucose levels (≥240 mg/dl) and tactile allodynia thresholds (≤8.0 g). Systemic (i.p.) administration of ABT-627 (1 and 10 mg/kg) was found to produce a dose-dependent increase in tactile allodynia thresholds. A significant antinociceptive effect (40–50% increase in tactile allodynia thresholds, P<0.05) was observed at the dose of 10 mg/kg, i.p., within 0.5–2-h post-dosing. The antinociceptive effects of ABT-627 (10 mg kg−1 day−1, p.o.) were maintained following chronic administration of the antagonist in drinking water for 7 days. In comparison, morphine administered acutely at a dose of 8 mg/kg, i.p., produced a significant 90% increase in streptozotocin-induced tactile allodynia thresholds. The endothelin ETB receptor-selective antagonist, 2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (A-192621; 20 mg/kg, i.p.), did not significantly alter tactile allodynia thresholds in streptozotocin-treated rats. Although combined i.p. administration of ABT-627 and A-192621 produced a significant, acute increase in tactile allodynia thresholds, this effect was significantly less than that produced by ABT-627 alone. These results indicate that the selective blockade of endothelin ETA receptors results in an attenuation of tactile allodynia in the streptozotocin-treated rat.
Keywords: Allodynia; Endothelin; Streptozotocin; Diabetic neuropathy;

Blockade of NMDA receptors in the nucleus accumbens elicits spontaneous tail-flicks in rats by Mark J. Millan; Valérie Audinot; Prisca Honoré; Karin Bervoets; Sylvie Veiga; Mauricette Brocco (37-47).
The open channel blocker at N-methyl-d-aspartate (NMDA) receptors, dizocilpine, stereospecifically elicited spontaneous tail-flicks in rats — a reaction similar to those elicited by other drugs (tenocyclidine, phencyclidine and ketamine) acting as open channel blockers. Their relative potencies were strongly correlated with affinities at NMDA binding sites and labeled by [3H]dizocilpine in the frontal cortex (r=0.94) and, as determined previously [Millan, M.J., Seguin, L., 1994. Chemically-diverse ligands at the glycine B site coupled to N-methyl-d-aspartate (NMDA) receptors selectively block the late phase of formalin-induced pain in mice, Neurosci. Lett., 178 (1994) 139–143], potency for eliciting antinociception (0.93). The competitive antagonists at the NMDA receptor recognition site, (±)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), 4-phosphonomethyl-2-piperidine carboxylic acid (CGS19755), d,l-(E)-2-amino-4-methylphosphono-3-pentanoic acid (CGP37849) and (3E)-1-ethyl ester-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP39551), likewise dose-dependently evoked spontaneous tail-flick. In contrast, antagonists/weak partial agonists at the coupled, glycine B site, 7-chloro-4-hydroxy-3-(3-phenoxy) phenyl-2(H)-quinolinone (L701,324), (+)-1-hydroxy-3-aminopyrrolidine-2-one ((+)-HA966), (3R,4R)-3-amino-1-hydroxy-4-methyl-2-pyrrolidinone (L687,414), 6,7-dichloro-1, 4-dihydro-5-nitro, 2,3 quinoxalinedione (ACEA1021) and 2-carboxy-4,6-dichloro (1H)-indole-3-propanoic acid (MDL29,951), were inactive. NMDA abolished induction of spontaneous tail-flick by CPP and CGS19755, but not by dizocilpine. Upon bilateral injection into the nucleus accumbens, dizocilpine immediately and dose-dependently elicited spontaneous tail-flick, but it was ineffective in the ventrotegmental area and striatum. Similarly, injection of CPP into the nucleus accumbens elicited spontaneous tail-flick. Neither dizocilpine nor CPP elicited spontaneous tail-flick upon administration onto lumbar spinal cord. In conclusion, a pharmacologically specific spontaneous tail-flick-response is elicited by both open channel blockers and recognition site antagonists, but not glycine B site antagonists, at NMDA receptors. Their actions, mediated in the nucleus accumbens, may be differentiated by their respective resistance and sensitivity to NMDA.
Keywords: NMDA receptors; Glutamate; Dizocilpine; Nucleus accumbens; Tail-flick;

Changes in hypothalamic paraventricular nucleus catecholaminergic activity after acute and chronic morphine administration by Gregorio Fuertes; M.Victoria Milanés; Manuel Rodrı́guez-Gago; M.Teresa Marı́n; M.Luisa Laorden (49-56).
The participation of hypothalamic noradrenaline in the expression of neuroendocrine signs of morphine withdrawal has been proposed. The present study in rats examined: (1) the relationships between corticosterone secretion and the possible modifications in noradrenaline and dopamine content and turnover in the hypothalamic paraventricular nucleus after acute and chronic morphine administration; (2) the changes in cyclic adenosine monophosphate (cAMP) levels in the paraventricular nucleus after the same treatments. The results showed that acute morphine injection in control rats increased corticosterone release, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) production, and noradrenaline turnover. Dopamine turnover in the paraventricular nucleus was decreased and the cAMP levels remained unchanged. In chronic morphine-treated rats, there was no elevation in noradrenaline turnover or in corticosterone secretion, indicating that tolerance developed to the acute effects of the opioid. Correspondingly, no alterations in dopamine turnover were observed when chronic morphine-treated rats were compared with control rats acutely injected with morphine. cAMP levels in the paraventricular nucleus were unchanged during the tolerant state. The results raise the possibility that noradrenergic afferents play a significant role in the alterations of paraventricular nucleus function and pituitary–adrenal axis activity in response to acute and chronic morphine and suggest that these modifications are not mediated through adenylate cyclase activation. The present data provide further support for the idea of adaptive changes in noradrenergic neurons projecting to the paraventricular nucleus during chronic morphine exposure.
Keywords: Noradrenaline; Dopamine; Hypothalamus–pituitary–adrenocortical axis; Tolerance; Morphine; Paraventricular nucleus;

Many antipsychotics have marked antagonist effects at 5-hydroxytryptamine (5-HT2C) receptors in vitro, which, however, have been difficult to show in behavioral assays. Here, we used two assays — hypolocomotion and hypophagia induced by the 5-HT2C receptor agonist 1-(3-chlorophenyl)piperazine (mCPP) — to try to characterize the 5-HT2C receptor antagonist properties of antipsychotics in vivo. Clozapine, olanzapine, pipamperone, and trans-5-chloro-2-methyl-2,3,3a,12b-tetrahydro-1H-dibenz-[2,3:6,7]oxepino[4,5-C] pyrrolidino maleate (ORG 5222), modestly, but significantly, attenuated mCPP (10 mg/kg)-induced hypolocomotion. In contrast, risperidone and loxapine were inactive. The putative antipsychotic ORG 5222 significantly attenuated mCPP (5 mg/kg)-induced hypophagia, whereas the other antipsychotics were inactive. Selective antagonists at dopamine D2-like receptors, α1-adrenoceptors, α2-adrenoceptors, or muscarinic receptors were not able to antagonize the effects of mCPP in either assay. The results suggest that mCPP-induced hypolocomotion can be used to characterize the 5-HT2C receptor antagonist properties of antipsychotics, whereas mCPP-induced hypophagia appeared to be sensitive only to compounds highly selective for 5-HT2C receptors. Together, these assays may help to characterize functional, in vivo, 5-HT2C receptor antagonist properties of antipsychotics.
Keywords: Antipsychotic; Neuroleptic; 5-HT (5-hydroxytryptamine); 5-HT2C receptor; mCPP-(3-chlorophenyl)piperazine; Behavior; Hypolocomotion; Hypophagia; Locomotor activity; Food intake;

Psychostimulant sensitization: differential changes in accumbal shell and core dopamine by Cristina Cadoni; Marcello Solinas; Gaetano Di Chiara (69-76).
The nucleus accumbens has been subdivided into a shell and a core compartment on the basis of histochemical and connectional differences. Recently, we reported that behavioral sensitization to morphine is associated with an increased dopamine transmission in the caudate-putamen and in the nucleus accumbens core as well as a decreased response in the nucleus accumbens shell following acute morphine challenge. We have now performed a similar study in rats sensitized to amphetamine and to cocaine. Behavioral sensitization was induced by daily administration of a single dose of 1 mg/kg s.c. of amphetamine for 10 days or of 10 mg/kg i.p. of cocaine twice a day for 14 days. Microdialysis was performed 10–14 days after the last injection of amphetamine and 7–10 days after the last injection of cocaine. Both schedules resulted in robust behavioral sensitization in response to challenge with 0.25 and 0.5 mg/kg of amphetamine and to 5 and 10 mg/kg of cocaine, respectively. Subjects pre-exposed to amphetamine showed a sensitization of dopamine transmission in the nucleus accumbens core but not in the nucleus accumbens shell. Subjects pre-exposed to cocaine showed sensitization of dopamine transmission in the core only to the lower dose of cocaine. In the shell no change was observed after the lower dose of cocaine while a significant reduction of the dopamine response was observed after the higher dose. These results suggest that behavioral sensitization might result from reciprocal changes in the response of nucleus accumbens dopamine in the shell and in the core to drug challenge.
Keywords: Psychostimulants sensitization; Cocaine; Amphetamine; Nucleus accumbens shell; Nucleus accumbens core;

The nuclei of the hypothalamus have been shown to be involved in central cardiovascular homeostasis. Recent studies suggest that glutamate-containing neurons have an important role in the regulation of central cardiovascular function. We report first on the effects of intracerebrally injected NMDA and non-NMDA receptor ligands on blood pressure and heart rate in conscious Sprague–Dawley rats. In the second part, we describe the effect of blockade of NMDA or kainate receptors in the paraventricular nucleus on glutamate receptor agonist-induced blood pressure responses. Intracerebroventricular injections of l-glutamic acid, NMDA and kainic acid produced increases in mean arterial pressure. Kainic acid produced significant decreases in heart rate. Microinjection of dl-2-amino-5-phosphonopentanoic acid (APV; 25 and 50 nmol), a competitive NMDA receptor antagonist, into the paraventricular nucleus blunted the increases in the mean arterial pressure evoked by intracerebroventricular injections of NMDA (1 nmol), whereas microinjection of dinitroquinoxaline (DNQX; 20, 40 and 80 pmol), which acts as an antagonist at kainate receptors, failed to antagonize the cardiovascular effects of intracerebroventricular kainic acid (10 pmol). Microinjections of NMDA (100 pmol) into the paraventricular nucleus produced pressor responses, but kainic acid (5 and 10 pmol) failed to affect either mean arterial pressure or heart rate. These results suggest participation of the glutamergic system in cardiovascular regulation via NMDA receptors located within the paraventricular nucleus of the hypothalamus in rats.
Keywords: Paraventricular nucleus; Excitatory amino acid; Cardiovascular regulation; Central; Microinjection; (Rat);

Intravenous administration of 0.3 mg/kg of quinpirole to conscious rabbits that had been pretreated with domperidone caused a marked increase in blood pressure and renal sympathetic nerve activity with a peak at 5–10 min after injection (25% and 3-fold increase, respectively). Spectral analysis of the blood pressure–renal sympathetic nerve activity relationship in the 0.2–0.4 Hz domain showed that baroreflex gain was markedly increased at 5–10 min (4-fold) and at 20–25 min after injection (3.7-fold). These results show that administration of the dopamine D2/D3 receptor agonist quinpirole causes profound and long-lasting changes in the central integration of the sympathetic baroreceptor–vasomotor reflex.
Keywords: Quinpirole; Baroreflex; Renal sympathetic nerve activity; Rabbit;

Rank-order inhibition by ω-conotoxins in human and animal autonomic nerve preparations by Gareth J Sanger; Elizabeth S Ellis; Mark H Harries; Nick S Tilford; Kay A Wardle; Christopher D Benham (89-95).
The inhibitory effects of the ω-conotoxins GVIA, MVIIA and MVIIC on electrically-evoked, tetrodotoxin (10−7 M)-sensitive, autonomic nerve activity were studied using human, rat or guinea-pig vas deferens and intestinal tissues. In each preparation from each species, nM concentrations of ω-conotoxins GVIA and MVIIA prevented the neuronally-mediated contractions, whereas ω-conotoxin MVIIC was either markedly less potent (IC50's 1.4 or 2.9 log units more than for ω-conotoxin GVIA in guinea-pig ileum and rat vas deferens, respectively) or was without significant activity (human vas deferens, human Taenia coli) when tested at similar concentrations. In contrast the differences in potency between ω-conotoxins GVIA and MVIIC were considerably less when assayed directly on Ca2+ channel currents evoked from rat superior cervical ganglion neurons in culture (approximately 0.1 log unit difference) and from a stable cell line expressing rat α1B, α2δ, β1b Ca2+ channel subunits (approximately 0.9 log unit). These different rank-orders of inhibitory activity of the conotoxins support the suggestion that there are pharmacologically distinct N-type Ca2+ channels in the peripheral nervous system, and that this tissue-dependent difference is seen in man.
Keywords: Ca2+ channel; ω-Conotoxin GVIA; ω-Conotoxin MVIIA; ω-Conotoxin MVIIC; Vas deferens; Intestine; Superior cervical ganglion; (Human); (Rat); (Guinea-pig);

Differences in mediator of nonadrenergic, noncholinergic relaxation of the distal colon between Wistar–ST and Sprague–Dawley strains of rats by Yutaka Okishio; Satomi Niioka; Tadayoshi Takeuchi; Hideaki Nishio; Fumiaki Hata; Koichi Takatsuji (97-105).
Participation of nitric oxide and vasoactive intestinal peptide (VIP) in electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle and in balloon distension-induced descending NANC relaxation of circular muscle were studied in the distal colon of Wistar–ST and Sprague–Dawley rats. The extent of the nitric oxide-mediated component was approximately 50% in longitudinal and circular muscle of Sprague–Dawley rats, whereas this component was absent in both muscles of Wistar–ST rats. The extent of the VIP-mediated component was approximately 40% in longitudinal muscle of Wistar–ST rats and circular muscle of Sprague–Dawley rats, whereas this component was absent in circular muscle of Wistar–ST rats and longitudinal muscle of Sprague–Dawley rats. In circular muscle of Sprague–Dawley rats, in which participation of both nitric oxide and VIP in the relaxation was suggested, inhibition of descending relaxation by N G-nitro-l-arginine (l-NOARG) together with VIP-(10–28) was similar to that by either of the antagonists, and exogenous VIP-induced relaxation was not affected by l-NOARG, but exogenous nitric oxide-induced relaxation was partly inhibited by VIP-(10–28). These results suggest a linkage of the pathways mediated by nitric oxide and VIP. In the immunohistochemical studies, nitric oxide synthase or VIP immunoreactive neurons were seen in the ganglia, primary internodal strands of the myenteric plexus and in the circular muscle layer. However, the overall appearance of immunoreactive cell bodies in the myenteric plexus and the numbers of immunoreactive fibers in the circular muscle layer appeared to be similar in Wistar–ST and Sprague–Dawley rats. These results suggest that mediators of NANC relaxation in the distal colon are different in different strains of rats, i.e., Wistar–ST and Sprague–Dawley, although no such difference was seen in immunohistochemical studies.
Keywords: NANC (nonadrenergic, noncholinergic) relaxation; Strain difference; Colon; Nitric oxide (NO); VIP (vasoactive intestinal peptide);

Shortening velocity of skeletal muscle from humans with malignant hyperthermia susceptibility: effects of halothane by Toussaint S Etchrivi; Ghislain Haudecoeur; Isabelle Stix; Hugo Reyford; Benoı̂t Tavernier; Renée M Krivosic-Horber; Pascal J Adnet (107-113).
The aim of this investigation was to assess the effect of halothane on the velocity of shortening and lengthening of muscle from normal subjects and from patients with malignant hyperthermia susceptibility. Strips were mounted horizontally at optimal length in normal Krebs–Ringer's solution and mechanical parameters were obtained before and after exposure to 3 vol.% halothane. The maximun shortening velocity at zero load (V max) was determined by using Hill's characteristic equation. The contraction and relaxation indices were measured under isotonic and isometric conditions: maximum shortening and lengthening velocities (maxV c and maxV r, respectively); isometric peak twitch tension; peak of the positive (+dP/dt max) and negative (−dP/dt max) twitch tension derivative; ratio R1=maxV c/maxV r and ratio R2=(+dP/dt max)/(−dP/dt max). In normal muscle, halothane markedly increased V max, maxV c and peak twitch tension by 30±10%, 30±5% and 40±15%, respectively. The maxV r values increased concomitantly with the maxV c values, such that no change in the ratio R1 was observed. Both +dP/dt max and −dP/dt max increased such that the ratio R2 did not vary. In malignant hyperthermia susceptibility muscle, halothane induced a significant decrease in V max (−30±10%) and maxV r (−45±15%) without changing maxV c. The decrease in maxV r was greater than that of maxV c, such that the ratio R1 increased significantly. Peak twitch tension and +dP/dt max remained unchanged whereas −dP/dt max decreased significantly; the ratio R2 increased by 40±10%. These results suggest that halothane alters the contractile properties of malignant hyperthermia susceptibility muscle.
Keywords: Malignant hyperthermia; Shortening velocity; Skeletal muscle;

Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder by Ahmet Akici; Atila Karaalp; Ece İskender; Arthur Christopoulos; Esam E El-Fakahany; Şule Oktay (115-123).
Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca2+ sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M1–M5 subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca2+-free or 5 mM Sr2+-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8±8.0% and 33.2±6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus–response characteristics of carbachol-mediated contraction appeared significantly altered in Ca2+-free PSS relative to normal or Sr2+-substituted PSS. The antagonist, methoctramine (1×10−7–3×10−5 M), exerted only a slight inhibition of carbachol (10−5 M)-induced contractions in 5 mM Sr2+-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10−5 M carbachol in the absence of extracellular Ca2+. Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca2+-free (pIC50: 6.85±0.11 for atropine and 5.75±0.32 for tripitramine) and Sr2+-substituted media (pIC50: 6.88±0.25 for atropine and 5.70±0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca2+-free PSS (pIC50: 5.66±0.23) than in Sr2+-substituted PSS (pIC50: 5.33±0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca2+ influx and intracellular Ca2+ release. These two subtypes may represent the muscarinic M3 and M4 receptors, although the presence of the muscarinic M2 receptor subtype is also suggested from the binding data.
Keywords: Muscarinic receptor; (Guinea pig); Gallbladder; Receptor heterogeneity; Radioligand binding; Kinetics; Ca2+; Ca2+; Sr2+;