Reproduction and development in Drosophila are dependent upon catecholamines.

Drosophila melanogaster, maintained on culture media containing alpha-methyl-p-tyrosine (alpha-MT) at millimolar levels for 7 days, fail to produce viable progeny. Lesser concentrations delay development. This effect of alpha-MT, an inhibitor of tyrosine hydroxylase (TH), is partially reversible by co-administration of L-dihydroxyphenylalanine, the product of TH. Potent inhibitors of other steps in the pathway for catecholamine biosynthesis are inactive except for a partial effect with dopamine B-hydroxylase inhibition. the effect of alpha-MT is due to a combination of ovulation suppression coupled with decreases in embryonic and larval viability. Effects similar to those of alpha-MT are found with millimolar levels of reserpine, prazosin and to a lesser extent, rauwolscine. No significant effects are found with propranolol, chlorpromazine, sulpiride and SK&F 83566. Mutant alleles of the gene coding for TH are known to be lethal at the embryonic stage when homozygous in both Drosophila and mice. Taken together, these results indicate that in addition to their established roles in the nervous system catecholamines function in animal development via an action mediated through alpha-adrenoceptors.

Inversion of (R)- to (S)-ketoprofen in eight animal species.

The (R)-enantiomer of the NSAID ketoprofen was administered orally at 20 mg/kg to a series of 8 animal species. In all species, a highly significant degree of inversion occurred after 1 h which varied from 27% (gerbil) to 73% (dog) and persisted or increased in plasma samples obtained 3 h after drug administration. Although the (R)-enantiomer was inactive as an inhibitor of cyclooxygenase, the analgesic effects of that isomer was almost the same as the (S)-isomer in animal analgesic assays, following oral administration of the drugs to mice and rats. Taken together, the present results suggest that (R)-ketoprofen administered alone functioned primarily as a prodrug for (S)-ketoprofen under the experimental conditions of this study.

Comparative effects of angiotensin II and angiotensin III in rabbit adrenal and aortic tissues.

The addition of angiotensin II (AII) and angiotensin III (AIII) to isolated tissue baths produced the same maximal contractile response of rabbit aortic strips. AIII was about 10 times less potent, the slope of its concentration-response curve was less steep and its rate of onset slower than that of AII. The responses of both AII and AIII were inhibited with equal potency by the surmountable AII antagonist Phe4, Tyr8-AII and its unsurmountable analog Sar1, Leu8-AII but the kinetic patterns of inhibition by both were less well defined with the agonist AIII than with AII. The addition of AIII to tissues which had exhibited a maximal response to AII did not increase the level of contraction, in contrast to the case when norepinephrine was added to tissues contracted by AII. Both AII and AIII displaced [125I]AII binding from rabbit adrenal membranes; AIII was 6 times less potent than AII but displayed competitive kinetics as an inhibitor of [125I]AII binding. In further studies two binding sites for [125I]AII were identified in adrenal membranes, having KD values of 2.0 +/- 0.2 and 19.6 +/- 2.3 nM, respectively. Each site was inhibited by both AII and AIII and the ratio of the apparent Ki values for the two hormones was not significantly different. The Hill coefficient for the high affinity site was, however, lower for AIII than AII. We interpret our data to suggest that AII and AIII act on the same receptors. AIII apparently binds less efficiently than does AII in both rabbit adrenal membranes and rabbit aortic strips.

RG 12915: a potent 5-hydroxytryptamine-3 antagonist that is an orally effective inhibitor of cytotoxic drug-induced emesis in the ferret and dog.

RG 12915 [4-[N-(1-azabicyclo[2.2.2.]octan-3-(S)-yl)]2-chloro-cis 5a-(S)-9a-(S)-5a,6,7,8,9,9a-hexahydrobenzofurancarboxamide hydrochloride] is a potent and effective agent against cisplatin-induced emesis in the ferret after i.v. or p.o. administration. This agent (p.o.) is also highly protective against cisplatin-induced emesis in the dog, as well as cyclophosphamide/doxorubicin-induced emesis in the ferret. When administered either p.o. or i.v., RG 12915 has a lower ED50 value (0.004 mg/kg) than GR 38032F, BRL 43694 and metoclopramide for attenuating cisplatin-induced emetic episodes in the ferret. It also has a long duration of action against cisplatin-induced emesis in the ferret. In contrast to metoclopramide, RG 12915 lacks significant antidopaminergic activity both in vitro [( 3H]spiroperidol displacement), as well as in vivo (apomorphine-induced emesis). In radioligand binding assays, RG 12915 is a potent and selective displacer of binding of 5-hydroxytryptamine (5-HT)3 binding sites (IC50 value = 0.16 nM), whereas failing to displace binding of ligands for the alpha-1, alpha-2 and beta adrenergic, 5-HT1 or 5-HT2 or cholinergic-muscarinic sites with IC50 values less than 1 microM. At a p.o. dose (1 mg/kg) in which RG 12915 is highly protective against cisplatin-induced emesis in the dog, RG 12915 has no significant gastroprokinetic activity in the same species. In summary, RG 12915 is a potent and p.o. effective agent against cytotoxic drug-induced emesis in animal models. The antiemetic potency of RG 12915 against cisplatin is unrelated to antidopaminergic or gastroprokinetic activity, but may be related to its affinity for 5-HT3 binding sites.

Antiinflammatory effects of various drugs on acetic acid induced colitis in the rat.

The efficacy of various drugs used to treat ulcerative colitis, (sulfasalazine, 5-aminosalicylate, hydrocortisone) was investigated in a model of acetic acid-induced colitis in the rat. Subsequently, we tested the ability of antioxidant/5-lipoxygenase inhibitors (gossypol and nordihydroguiaretic acid [NDGA]) and a cyclooxygenase inhibitor (indomethacin) to attenuate the macroscopic colonic damage and/or neutrophil influx (myeloperoxidase activity [MPO]) associated with this model of colitis. Oral pretreatment with either sulfasalazine, gossypol, or NDGA significantly decreased colonic MPO activity induced by acetic acid. Intrarectal administration of such drugs resulted in an even larger reduction of the colonic inflammation, with gossypol being the most potent compound. Oral or intrarectal administration of corticosteroids (dexamethasone, hydrocortisone) also attenuated the parameters of acetic acid induced colitis. In contrast, pretreatment with indomethacin was ineffective, or when administered daily after colitis induction, indomethacin actually increased colonic neutrophil influx significantly. Our data suggest that both the route of drug administration and dosing regimen employed affect the antiinflammatory potency and/or efficacy of compounds on colitis induced by acetic acid in the rat. Drugs which were effective against this colitis may act by scavenging of oxygen derived free radicals.

2,3-Diphosphoglycerate phosphatase/synthase: a potential target for elevating the diphosphoglycerate level in human red blood cells.

To exploit the well documented effect of 2,3-diphosphoglyceric acid (2,3-DPG) in enhancing oxygen delivery by human erythrocytes, we have investigated whether the DPG synthase/phosphatase enzyme system can be targeted to increase DPG levels in the cell. The hydrolytic activity (phosphatase) of the DPG metabolizing enzyme complex exhibits a marked dependence on a physiological effector, 2-phosphoglycolate. Little phosphatase activity is detected in the absence of this activator irrespective of the concentrations of the substrate. The phosphoglycolate-dependent phosphatase activity is competitively inhibited by a glycolytic intermediate, 3-phosphoglyceric acid (3-PGA). The 3-PGA inhibition persists when the 2,3-DPG concentration is raised to saturation level. In contrast, 3-PGA enhances the DPG synthase activity in a dose-dependent manner. In intact red cells, one-half of the cellular DPG content is depleted after 6 hr at 37 degrees C in glucose-free medium. The rate of 2,3-DPG degradation is accelerated when the cellular level of phosphoglycolate is increased by incubation with exogenous glycolate. Together, these results indicate that 2,3-DPG content in erythrocytes can be directly regulated through modulation of phosphatase/synthase activities. In support of this notion, a pyruvate kinase inhibitor, L-alanine, increases by 2-fold the cellular 3-PGA level. This is accompanied by a significant increase (30%) in 2,3-DPG content in human red blood cells. It is postulated that the DPG-promoting action of 3-PGA is mediated through simultaneous phosphatase inhibition and synthase activation. Furthermore, as a result of increased DPG accumulation, the oxygen-hemoglobin dissociation curve in L-alanine-treated cells is rightward shifted by 2.5 torr.

An LTC4 binding site in gastric mucosa is shared with glutathione.

Recent results from our laboratory and others have suggested a possible physiological functional role(s) for leukotrienes in gastric mucosa. In the present study 3H-LTC4 binds to washed rabbit gastric mucosal membranes at 4 degrees C with a Kd of 5 nM and a Bmax of 31.3 pmol/mg protein. Leukotrienes D4, E4, B4, oxidized glutathione (GSSG), cysteine, and mercaptoethanol were unable to displace 3H-LTC4 at 1 microM concentrations, while GSH inhibited binding with a Ki of 47 nM. Differential centrifugation of the membrane preparation to remove mitochondria resulted in Ki values for LTC4 and GSH of 14 and 23 nM, respectively. The similar binding affinities and competitive receptor binding kinetics for GSH and LTC4, the low affinity for other leukotrienes, and a Ki of 7 microM for hematin, a substrate for glutathione S-transferase, suggest that 3H-LTC4 binds to a GSH site which does not discriminate between LTC4 and GSH. Membranes fractionated to remove mitochondria were assayed for glutathione peroxidase, gamma-glutamyltranspeptidase, and glutathione S-transferase as possible binding sites for LTC4. We were unable to detect enzyme activity for any of the three enzymes. The binding of LTC4 in gastric mucosa differs from other tissues with respect to the high affinity for GSH, and thus becomes an appropriate tissue in which to investigate the relationships between LTC4 and GSH.

Studies defining minimal receptor domains for angiotensin II.

The purpose of this study was to define in a systematic experimental manner the minimal amino acid domain(s) present in the angiotensin II molecule that are required for binding to, as well as activation of, its receptor at physiological concentrations. Although removal of the C-terminal phenylalanine residue markedly reduced affinity for the rabbit adrenal cortical receptor, sequential additions of amino acids beginning with phenylalanine did not result in a molecule with significant receptor affinity until the hexapeptide stage was reached. Similar receptor affinities were obtained with the other two possible 6 amino acid fragments in the molecule. None of the possible pentapeptide fragments were active, as was also the case with representative 4, 3 and 2 amino acid sequences. Of the three hexapeptides, only the one containing phenylalanine as the C-terminal amino acid displayed agonist activity on the rabbit aortic strip. The other two behaved as competitive antagonists. These results indicate that 6 amino acids constitute the minimal receptor binding domain present in the angiotensin II molecule and that phenylalanine is crucial at the C-terminus for activating the receptor.

Tetrahydrothiadiazoloisoquinolines: synthesis and inhibition of phenylethanolamine-N-methyltransferase.

A series of 7,8-fused heterocyclic tetrahydroisoquinolines were synthesized and tested as inhibitors of rabbit adrenal phenylethanolamine-N-methyltransferase (PNMT). 6,7,8,9-Tetrahydro[1,2,3]thiadiazolo[5,4-h]isoquinoline 5 (SK&F 86607) was found to be a potent inhibitor of PNMT with an IC50 similar to that of 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (1, SK&F 64139). The isomeric tetrahydro[1,2,3]thiadiazolo[4,5-h]- and tetrahydro[1,2,5]thiadiazolo[3,4-h]isoquinolines, 13 and 20, were also potent PNMT inhibitors. However, substitution of Cl at position 5 (17) resulted in loss of potency similar to the loss observed in the 5-chloro analogue of 1. The 1,2,5 isomer 20 showed only a small drop in activity at 10(-6) M. All of the thiadiazoles were more potent than the 7,8-benzo-fused analogue 36. Fusion of other five-membered heterocyclic ring systems at the 7,8-position, e.g. triazole 22 and imidazoles 24 and 26, resulted in a decrease of PNMT inhibition. The alpha-adrenoreceptor affinities of 1 and 5 were also compared.

Acute effects of ranitidine, famotidine and omeprazole on plasma gastrin in the rat.

In the rat, treatment with gastric inhibitory drugs may result in hypergastrinemia, an effect thought to be in response to increased gastric pH caused by inhibition of acid secretion. This study compared 24-hr profiles of plasma gastrin levels associated with three different compounds at equivalent, highly effective antisecretory doses. Ranitidine, famotidine and omeprazole at 60, 20 and 40 mg/kg p.o., respectively, inhibited basal acid secretion of chronic gastric fistula rats by greater than 95% and raised intraluminal pH to above 7.0 for 5 hr. The peak plasma gastrin levels associated with each agent were observed 5 hr after dosing. Ranitidine, famotidine and omeprazole induced statistically significant and distinct peak hypergastrinemic responses of 312 +/- 20, 483 +/- 28 and 616 +/- 27 pg/ml, respectively. After 8 hr ranitidine and famotidine associated gastrin values returned to control levels, whereas those of omeprazole remained substantially above control values until the 12th hr. Differences in peak gastrin levels between compounds disappeared at increased dose levels of 500 mg/kg for ranitidine, 200 or 2000 mg/kg for famotidine and 140 mg/kg for omeprazole. Unlike high dose famotidine, omeprazole (140 mg/kg) maintained peak plasma gastrin levels at 8, 12, and 16 hr after dosing. These studies demonstrate clearly hypergastrinemic responses to single dose administration of ranitidine, famotidine and omeprazole. The differences observed in peak plasma gastrin levels at equivalent antisecretory doses of these agents suggests the presence of luminal acid independent components that effect gastrin release. Moreover, these studies indicate that, in the rat, the most unique aspect of omeprazole-associated hypergastrinemia is the magnitude of its prolonged response.

Studies on inhibition of angiotensin II receptors in rabbit adrenal and aorta.

Angiotensin II (AII) labeled with 125I binds to rabbit adrenal cortical membranes over a concentration range from 0.5 to 20 nM at an apparent single site with a KD of 5 nM. This binding was inhibited in a surmountable fashion with respect to AII by the peptide analogs sarcosine1 (Sar1),Leu8AII and Phe4, Tyr8 AII when added to the incubation media concomitant with AII addition. With a 30-min preincubation, however, the former inhibitor displayed nonsurmountable kinetics whereas the profile of the latter was unaffected. In rabbit aortic strips with the same preincubation time, the Sar1Leu8AII analog was a nonsurmountable antagonist of the contractile effect of AII whereas the inhibition produced by Phe4,Tyr8AII was surmountable by increasing agonist (AII) concentrations. The inhibitory effect of the former was maintained after repeated washing of the tissue whereas that of the latter was readily reversible. Addition of Phe4,Tyr8AII to the bath 5 min before preincubation protected the tissue from the prolonged AII inhibition by Sar1,Leu8AII. These findings indicate different kinetic modes of AII inhibition by these two antagonists. Phe4,Tyr8AII behaves as a reversible, competitive inhibitor of AII binding, whereas Sar1,Leu8AII combines with the AII receptor in a slowly dissociable manner and is therefore not readily displaced by AII.

Effect of metoclopramide, bethanechol and the cholecystokinin receptor antagonist, L-364,718, on gastric emptying in the rat.

The prokinetic effects of metoclopramide, bethanechol and L-364,718 on a semisolid meal and solid pellet gastric emptying were evaluated and compared. Each compound increased the rate of meal emptying as measured 90 min post-dose. L-364,718, a non-peptide CCK antagonist, was the most potent of these three agents with statistically significant activity observed at 0.03, 0.1 and 0.3 mg/kg p.o. Only metoclopramide significantly enhanced pellet emptying in a dose-dependent manner (3-30 mg/kg p.o.). The effects of each test agent and the potential physiologic role of cholecystokinin in regulating gastric emptying are discussed.

Lidamidine inhibits intrinsic contractile patterns of the rat proximal colon.

Lidamidine is a clinically effective antidiarrheal agent that inhibits intestinal secretion, reduces intestinal transit, and inhibits smooth muscle contraction. Yet, its specific effects upon colonic motility have not been thoroughly examined. The purpose of our studies was to examine lidamidine's inhibitory effects upon colonic contractile patterns in the rat and identify the responsible receptor mechanism. Fasted male rats were anesthetised and equipped with an intraluminal cannula positioned at the proximal end of a 10 cm fluid-filled segment of the ascending colon (basal pressure, 10 cm H2O). Intraluminal pressure was monitored by a transducer attached to a closed fluid-filled system. All drugs were administered intravenously by slow infusion. A regular pattern of distinct contractile complexes was observed over a 70 min period. These contractions increased intraluminal pressure to 39 +/- 1.2 cm H2O (mean +/- S.E.), occurred at a frequency of 0.3 per min and lasted from 1 to 2.5 min. Inhibition of these contractile patterns was observed with either atropine (0.1 mg/kg) or lidamidine (3.0 mg/kg). A 20 min pretreatment with idazoxan (3.0 mg/kg) antagonized lidamidine's but not atropine's effect. Trimazosin (1 mg/kg) or propranolol (0.3 mg/kg) pretreatment did not antagonize the lidamidine-generated inhibition. These results indicate that lidamidine inhibits an intrinsically generated, cholinergically controlled pattern of colonic contractions primarily by an alpha 2-receptor-mediated mechanism.

In vivo pharmacology of L-364,718, a new potent nonpeptide peripheral cholecystokinin antagonist.

The in vivo pharmacological activity of L-364,718, a new, potent peripheral cholecystokinin (CCK) antagonist, was characterized in several species using assay systems that measure various well known actions of CCK upon the gastrointestinal system. Administered p.o., L-364,178 was highly potent in antagonizing cholecystokinin octapeptide (CCK-8)-induced inhibition of gastric emptying in mice (ED50 = 38 micrograms/kg), rats (ED50 = 140 micrograms/kg) and dogs (ED50 = 91 micrograms/kg) as well as CCK-8-induced reduction in food consumption in rats (ED50 = 321 micrograms/kg). Administered i.v., L-364,718 effectively antagonized the contractile effects of CCK on the colon in rabbits (ED50 = 34 micrograms/kg) and the gallbladder in cats (ED50 = 210 micrograms/kg). Secretion of pancreatic protein and amylase elicited by CCK in cats was also antagonized by L-364,718 (ED50 less than 1.0 mg/kg i.v.). The CCK antagonism produced by L-364,718 in all species persisted for at least 2 to 5 hr. In the absence of exogenously administered CCK-8, L-364,718 per se had no effect in any of the assay systems studied, indicating a lack of CCK-like agonist properties. Specificity for CCK was demonstrated by the inability of L-364,718 (1.0-5.0 mg/kg) to antagonize either amino acid- or atropine-induced inhibition of gastric emptying in rats and dogs, respectively. L-364,718 also did not antagonize motilin-induced gallbladder contractions or secretin-induced pancreatic secretion in cats.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of endogenous cholecystokinin in biliary, pancreatic and gastric function: studies with L-364,718, a specific cholecystokinin receptor antagonist.

A new, highly potent antagonist of gut cholecystokinin (CCK) receptors has been examined for effects upon postprandial biliary and exocrine pancreatic secretion in conscious dogs with chronic duodenal pouches. This drug (L-364,718) markedly inhibited the postprandial increases in biliary volume, bile acid and bilirubin secretion. However, even at high p.o. doses relative to its ability to antagonize the effects of exogenous CCK, no effects were observed upon the pancreatic secretion of either fluid volume or amylase and lipase under similar conditions. In additional studies, pretreatment with L-364,718 did not significantly reverse the inhibitory effects of a fatty acid salt (sodium oleate) upon gastric emptying in gastric fistula dogs. Moreover, pretreatment with L-364,718 had no significant effects upon postprandial acid and pepsin secretion in lesser curvature (vagally innervated) pouch dogs or did it affect basal (interdigestive) gastric secretion in rats. These results suggest that endogenous CCK plays a critical physiological role in regulating postprandial biliary outflow, but not pancreatic enzyme secretion or the gastric emptying of at least liquid fatty substances. The latter two findings stand in contrast with classical views regarding the physiological function(s) of this hormone.

Effects of atropine upon various components mediating postprandial gastric acid secretion in dogs.

Dose-response curves in chronic gastric fistula dogs were first obtained to chemical stimulants of the three accepted physiological excitatory components regulating postprandial gastric acid secretion. These were: 2-deoxy-D-glucose, a central vagal stimulant; gastrin, a hormone; and histamine, a paracrine factor. Using equiactive doses of each, a dose of atropine just maximal for suppressing all of the anatomical phases of food-induced acid secretion in vagally innervated pouch dogs was found to inhibit substantially the responses to all three of the above stimulants. The above results argue in favor of an interdependent model among the above factors for regulating postprandial gastric acid secretion in the dog.

Evidence for differing leukotriene receptors in gastric mucosa.

Exogenously administered LTD4 was found to increase pepsin secretion from and decrease the transgastric electrical potential difference across the stomachs of anesthetized cats. The former response was completely blocked by prior administration of a new and selective LTD4 antagonist, L-649,923, while the potential difference response was unaffected. This result, coupled with previous evidence of a difference in leukotriene potency order for the two responses, suggests the presence of functionally different leukotriene receptor subtypes in the gastric mucosa.

Leukotriene effects upon the transgastric potential difference and pepsin secretion.

The effects of leukotrienes on gastric mucosal function in vivo, and on acid and pepsin secretion in vitro were investigated. In cats, treatment with leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) caused significant decreases in the transgastric electrical potential difference (P.D.) and significant increases in pepsin secretion, which returned toward control levels over 30-90 min. No detectable changes were observed in either acid concentration or gastric secretion volume over the entire 210 min experimental period. Leukotriene B4 (LTB4) had no effect upon any of these parameters. Treatment with LTD4 in isolated rabbit gastric glands resulted in significant increases in pepsin secretion, with no changes observed in aminopyrine accumulation (acid secretion). These results indicate that exogeneous LTC4, LTD4 and LTE4 can affect certain gastric mucosal functions.

Effects of H2-receptor antagonists upon physiological acid secretory states in animals.

The results reported in this paper indicate that representative H2-receptor antagonists are capable of maximally inhibiting gastric acid secretion in animals under the two general circumstances in which it occurs physiologically. Interdigestive or basal secretion was examined in chronic gastric fistula rats and food-stimulated secretion in vagally innervated, lesser curvature pouch dogs. The H2 antagonists studied and omeprazole, an inhibitor of the proton pump H+, K+-adenosine triphosphatase, also decreased pepsin secretion in rats, although not to the same maximal degree as acid secretion. Gastric emptying was increased by each H2 antagonist but only at high acid inhibitory doses. Omeprazole, in contrast, did not alter gastric emptying at a similar antisecretory dosage level. In dogs, a representative H2-receptor antagonist markedly inhibited food-stimulated acid secretion. These data suggest that the predominant effect of omeprazole and H2-receptor antagonists upon gastric function is to inhibit acid secretion and that H2-receptor antagonists may be capable of maximally inhibiting endogenous acid secretion in humans, as does omeprazole, if given under proper conditions.