Gastrointestinal hormone receptors on isolated smooth muscle cells from gastric antrum of the rabbit

The regulation by gastrointestinal polypeptide hormones of contraction and relaxation of functionally isolated smooth muscle cells from gastric antrum of the rabbit has been investigated. Gastrin, cholecystokinin (CCK-8) and motilin induced a rapid contraction of isolated cells: significant response occurred within a 5-sec incubation with these peptides and maximal response (40% decrease in cell length) after 30 sec. A higher sensitivity of smooth muscle cells to gastrin and CCK-8 than to motilin stimulations was demonstrated (EC50 = 10 pM for both gastrin and CCK-8 and EC50 = 1 nM for motilin). The minimal gastrin fragment required to get full contraction was the C-terminal pentapeptide amide common to gastrin and CCK. Proglumide inhibited gastrin- or CCK-8- but not motilin-induced contractions with an IC50 of 50 microM. contraction induced by gastrin and motilin required normal levels of extracellular calcium, whereas that due to CCK-8 seemed to be independent of extracellular calcium. Vasoactive intestinal polypeptide (VIP) caused a relaxation of smooth muscle cells maximally contracted by carbachol or CCK-8 or gastrin (EC50 = 2.2 nM) with a parallel increase in intracellular cAMP content.     

5-HT4 receptor activation induces relaxation and associated cAMP generation in rat esophagus.

Changes in mechanical events and intracellular levels of cAMP induced by the activation of the 5-HT4 receptor were investigated in the rat esophagus tunica muscularis mucosae preparation. Serotonin (5-HT) and 5 methoxytryptamine (5-MOT; 5-HT4 agonist) caused concentration-related relaxation responses, while 5-carboxamidotryptamine (5-CT; 5-HT1 agonist), 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI; 5-HT2 agonist) and 2-methyl-serotonin (2-methyl-5-HT; 5-HT3 agonist) were less active. The prokinetic agents, cisapride and renzapride also induced concentration-dependent relaxation of rat esophagus which was intermediate to 5-HT and 5-MOT in potency. The relaxation was not due to activity at receptors other than the 5-HT4 since methysergide (5-HT1 and 5-HT2 antagonist) and granisetron (5-HT3 antagonist) did not block the relaxant response to 5-HT while ICS 205930 (5-HT4 antagonist) antagonized this response (pA2 = 6.45). Serotonin also caused concentration-related increases in tunica muscularis mucosae cAMP with the rank order of efficacy of 5-HT agonists in raising tissue cAMP levels reflecting their relaxant activities (5HT greater than or equal to 5-MOT greater than 5-CT greater than DOI = 2-methyl-5-HT = control). Enhancement of cAMP concentrations was also observed following renzapride treatment. This cAMP relaxation response was specific for 5-HT4 receptor activation as demonstrated by the lack of ICS 205930 inhibition of rat esophagus relaxation caused by isoproterenol, 16,16-dimethyl-prostaglandin E2 and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of endothelin-1 on guinea pig gallbladder smooth muscle in vitro.

The purpose of this study was to investigate the pharmacological activity of endothelin-1 (ET-1) on guinea pig gallbladder smooth muscle. Guinea pig gallbladder muscle strips were mounted in 10-ml siliconized organ baths containing Krebs' solution. After 1 hr of equilibration, ET-1 was added cumulatively. ET-1 induced slow-developing and long-duration contractile responses. The EC50 was approximately 10 nM. ET-1 was 5 times less potent than cholecystokinin (EC50, 2 nM), but 20 and 40 times more potent than carbachol (EC50, 200 nM) and histamine (EC50, 400 nM), respectively. The concentration-response curve to ET-1 was not affected by tetrodotoxin (0.1 microM) or by the muscarinic antagonist, atropine (10 microM). The neuronal N-type calcium channel blocker, omega-conotoxin (0.1 microM), had no significant effect on the ET-1 concentration-response curve. In contrast, the contractile effect to ET-1 was reduced markedly by removal of extracellular calcium or by the voltage-dependent calcium channel blockers nicardipine and diltiazem. Substitution of strontium (an inhibitor of intracellular calcium release) for Ca++ significantly reduced the response to ET-1. The cyclooxygenase inhibitor indomethacin had no significant effect on the contractile activity of ET-1. These finding suggest that ET-1 is a potent contractile stimulant of guinea pig gallbladder and that it acts directly on the smooth muscle. The activity depends on extracellular Ca++, suggesting involvement of Ca++ influx via the voltage-dependent Ca++ channel and on intracellular calcium.     

Treatment of ovarian cysts by laparoscopic surgery: a retrospective study of 40 cases

The ovarian cysts treatment has taken advantage of the improvement made on the operating laparoscopy, that gives progressively better results than those of the laparotomy. On the one hand, laparoscopic surgery reduces the traditional surgery constraints by giving an exact histologic diagnosis and a radical cyst treatment. On the other hand, it prevents against inherent recurrence related to a sample puncture, and en the same time diminish adhesion risks. The laparoscopy can treat all kind of histologic benign ovarian cysts. We report the first experience of the Maternité Universitaire des Orangers in treating ovarian cysts by laparoscopic surgery.     

Neurokinin A-induced guinea pig gallbladder contraction: Potential mechanism of action

The present study was performed to examine the mechanism of action of neurokinin A (NKA) on guinea pig gallbaldder smooth muscle. Muscle strips were prepared and mounted in 10 mL tissue bath containing Krebs' solution under 1 g tension. NKA induced a concentration-dependent increase in gallbladder muscle tension and reached a maximal response at 1 μM. The EC₅₀ value was approximately 30nM. Preincubation of the muscle strips with neurotoxins, tetrodotoxin (1 μM), or omega-conotoxin (0.1 μM) had no effect on the NKA contractile response. NKA-induced gallbladder contractions were insensitive to cyclooxygenase inhibitors (5 μM) piroxicam and indomethacin. In contrast, the calcium channel blockers verapamil and diltiazem (0.1–1 μM) significantly blocked the contractile response to NKA. The intracellular calcium chelator BAPTA/AM had no significant effect on NKA activity. The removal of extracellular calcium, however, completely abolished the contractile response of NKA. These data suggest that NKA has a direct contractile effect on guinea pig gallbladder smooth muscle, which is independent of prostaglandin release. The primary source of calcium involved in mediating the NKA contractile response is the extracellular pool, suggesting that NKA might act via activation of L-type voltage-operated calcium channels to mediate its action. © 1992 Wiley-Liss, Inc.     

Oxidant-evoked release of acetylcholine from enteric neurons of the rat colon.

In the presence of halides, granulocytes generate hypochlorous acid and, subsequently, chlorinated amines (chloramines). These lipophilic, potent reactive oxygen metabolites may contribute to the mucosal pathophysiology associated with inflammatory bowel disease. A common symptom of inflammatory bowel disease is mucosal secretion of fluid and electrolytes, leading to diarrhea. Because acetylcholine (Ach) can stimulate colonic fluid secretion, we determined the effect of monochloramine (NH2Cl) on Ach release by mucosal/submucosal nerves. Mucosa from the rat colon was separated from outer muscle layers and minced before incubation with [14C]choline to label stores of Ach in cholinergic neurons. Release of [14C]Ach was evoked with NH2Cl in the absence and presence of 5-aminosalicylic acid, glutathione, nordihydroguaiarectic acid or the cyclooxygenase inhibitor piroxicam. NH2Cl produced concentration-related increases in [14C] Ach release into the medium; greater than 100% over base line was observed at 0.5 mM. Glutathione inhibited the NH2Cl-evoked release in a concentration-dependent fashion. Release induced by 0.1 mM NH2Cl was abolished by 5-aminosalicylic acid and significantly inhibited by nordihydroguaiarectic acid. Piroxicam also prevented the effect of NH2Cl on release of [14C] Ach. None of these agents alone had any effect on base line [14C]Ach release. Tetrodotoxin (5 microM) did not significantly inhibit the NH2Cl-evoked transmitter release. We conclude that NH2Cl, at concentrations believed to exist in inflamed tissue, causes the release of Ach from mucosal/submucosal nerves primarily through nonspecific neural membrane injury. Endogenous prostaglandins, possibly liberated as a consequence of the injury, may be involved in the Ach release process.     

Differential effects of reactive oxygen metabolites on neurally stimulated and nonstimulated guinea pig ileum.

Large numbers of polymorphonuclear leukocytes which generate reactive oxygen metabolites are found in mucosa and submucosa of the intestinal wall of subjects suffering from inflammatory bowel disease. We have, therefore, examined the relative influences of hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and N-chloramines such as NH2Cl, on the neurally stimulated and nonstimulated guinea pig ileum. In separate experiments the oxidants were tested in the presence and absence of the cyclooxygenase inhibitor piroxicam and the antioxidant glutathione. All three oxidants, in concentrations produced by activated neutrophils, increased the muscle tone (concentration-dependent, peak at 0.3 mM for NH2Cl and H2O2 and 1 mM for HOCl). Tetrodotoxin (0.5 microM) inhibited the NH2Cl and H2O2 effects by 50% and 70%, respectively. Piroxicam (5 microM) partially blocked maximal contractions induced by all three oxidants. The contractile response to carbachol (10 microM) was blocked by 0.3 mM NH2Cl, but not by H2O2 and HOCl. In electrically stimulated ileum the oxidants produced a concentration-dependent biphasic response (transient enhancement of neurally mediated twitch contraction followed by marked inhibition). This response was not modified by piroxicam, hexamethonium, atropine and pyrilamine. The inhibition of twitch contraction was irreversible for NH2Cl and HOCl, in contrast to H2O2, which was reversed by repeated washing. Neither the contractile effect nor the effects on nerve stimulation-induced contraction were affected by preincubation of the tissue with glutathione, whereas prior combination of NH2Cl with glutathione prevented the effects of NH2Cl. Oxidant-induced contraction of guinea pig ileum appears to be via release of prostaglandins and one or more neurotransmitters. High concentrations of reactive oxygen metabolites may alter receptor function.(ABSTRACT TRUNCATED AT 250 WORDS)     

The discovery of a new structural class of cyclin-dependent kinase inhibitors, aminoimidazo[1,2-a]pyridines

The protein kinase family represents an enormous opportunity for drug development. However, the current limitation in structural diversity of kinase inhibitors has complicated efforts to identify effective treatments of diseases that involve protein kinase signaling pathways. We have identified a new structural class of protein serine/threonine kinase inhibitors comprising an aminoimidazo[1,2-a]pyridine nucleus. In this report, we describe the first successful use of this class of aza-heterocycles to generate potent inhibitors of cyclin-dependent kinases that compete with ATP for binding to a catalytic subunit of the protein. Co-crystal structures of CDK2 in complex with lead compounds reveal a unique mode of binding. Using this knowledge, a structure-based design approach directed this chemical scaffold toward generating potent and selective CDK2 inhibitors, which selectively inhibited the CDK2-dependent phosphorylation of Rb and induced caspase-3-dependent apoptosis in HCT 116 tumor cells. The discovery of this new class of ATP-site-directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis for a new medicinal chemistry tool to be used in the search for effective treatments of cancer and other diseases that involve protein kinase signaling pathways.