Neutralization of EP217609, a new dual-action FIIa/FXa anticoagulant, by its specific antidote avidin: a phase I study.

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.

First in man study of EP217609, a new long-acting, neutralisable parenteral antithrombotic with a dual mechanism of action.

UNLABELLED: EP217609 is a parenteral antithrombotic compound combining in one molecule an indirect anti-factor Xa inhibitor, a direct thrombin active site inhibitor and a biotin moiety. AIMS: The aim of the study is to investigate the safety, pharmacokinetics and pharmacodynamics of single ascending intravenous doses of EP217609. METHODS: In this randomised double-blind placebo-controlled study, healthy male subjects were administered intravenously single ascending doses (1, 3 or 10 mg) of EP217609 or placebo. Each treatment group consisted of 10 subjects of whom 8 received EP217609 and 2 received placebo. RESULTS AND CONCLUSIONS: All doses of EP217609 were well tolerated. A total of five treatment-emergent adverse events were reported, all considered unrelated, but no bleedings or other significant adverse events occurred during this study. In both plasma and urine, there was a strong correlation between EP217609 concentrations as measured by anti-factor IIa and Xa specific bioassays indicating that the two pharmacological activities of EP217609 did not dissociate in vivo. EP217609 pharmacokinetics were dose proportional and characterised by a low clearance, a small volume of distribution and a terminal half-life of 20.4 h. The long half-life was reflected in long-lasting, dose-dependent effects on activated and ecarin clotting time, thrombin and prothrombin time, activated partial thromboplastin time, thrombin generation time and anti-factor Xa activity. Pharmacokinetic/pharmacodynamic modelling indicated that the concentration of EP217609 producing 50 % of the pharmacodynamic effect was 3400 and 2210 ng/mL for activated clotting time and anti-factor Xa activity, respectively. These results warranted further clinical development of EP217609. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: • There is a limited number of neutralisable anticoagulants, particularly when rapid neutralisation is required. • Synthetic anti-Xa compounds have predictable pharmacokinetic profiles. However, problems with thrombin rebound remain because of the inability to inhibit clot-bound thrombin. WHAT THIS STUDY ADDS: • This manuscript provides a comprehensive investigation of the pharmacokinetics, pharmacodynamics and safety of EP217609, and the results were the basis of future clinical studies in both healthy subjects and patients. • The pharmacokinetic/pharmacodynamic modelling provided information for dose selection in such future studies.

In vitro and in vivo pharmacokinetic characteristics of clazosentan, an intravenous endothelin receptor antagonist, in humans.

OBJECTIVE: In this study, the distribution, metabolism and excretion of the endothelin receptor antagonist clazosentan were investigated. SUBJECTS AND METHODS: 4 healthy male subjects received an intravenous 3-h infusion at a rate of 0.2 mg/kg/h of 14C-labeled clazosentan and blood, urine and feces samples were collected for a period of 8 days. Experiments were performed to investigate the plasma protein binding, the binding to red blood cells and the inhibition potential of cytochrome P450 isoenzymes of clazosentan. RESULTS: Clazosentan was mainly excreted unchanged into feces whereas about 15% of the radioactive dose was recovered in urine. No metabolites representing more than 5% of total radioactivity were identified. No relevant inhibition of the human cytochrome P450 isoenzymes, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4, was observed in vitro at clazosentan concentrations largely exceeding those observed in clinical trials. In human blood, clazosentan was highly bound to plasma proteins and did hardly penetrate into red blood cells. CONCLUSION: The primary route of excretion of clazosentan was via the feces, mainly as unchanged drug.

Inhibitory and inductive effects of rifampin on the pharmacokinetics of bosentan in healthy subjects.

This study was conducted to investigate the effect of rifampin on the pharmacokinetics of bosentan. Healthy male subjects received bosentan 125 mg b.i.d. for 6.5 days in the presence or absence of rifampin 600 mg once a day. In vitro experiments were performed to investigate the effect of rifampin on the uptake of bosentan into Chinese hamster ovary cells expressing the human organic anion-transporting polypeptide (OATP)1B1, -1B3, and -2B1. Following the first concomitant administration, there was a fivefold increase in bosentan trough concentrations. At steady state, concomitant rifampin significantly decreased exposure to bosentan by 58%. Rifampin potently inhibited the uptake of bosentan into cells expressing human OATP1B1 and -1B3. Rifampin decreased the exposure to bosentan consistent with its known cytochrome P450 enzyme-inductive properties. The initial increase in bosentan concentrations can be explained by an inhibitory effect of rifampin on hepatic drug transporters.

Pharmacokinetic interaction between bosentan and the oral contraceptives norethisterone and ethinyl estradiol.

OBJECTIVE: Bosentan has been shown in vitro and in vivo to induce the cytochrome P450 enzymes CYP2C9 and CYP3A4. The present study was conducted to investigate the effect of bosentan on the pharmacokinetics of a combined oral contraceptive. SUBJECTS AND METHODS: In a randomized, 2-way crossover study, 20 healthy female subjects received Treatments A and B. Treatment A consisted of a single dose of OrthoNovum containing 1 mg norethisterone (norethindrone) and 35 microg ethinyl estradiol. Treatment B consisted of bosentan, 125 mg b.i.d. for 7 days plus concomitant norethisterone and ethinyl estradiol on Day 7. Plasma concentrations of norethisterone and ethinyl estradiol were measured on days of oral contraceptive administration. RESULTS: In the absence of bosentan, the pharmacokinetics of norethisterone and ethinyl estradiol were characterized by Cmax and AUC0-infinity values (95% CI) of 9.8 (8.1, 11.9) ng/ml and 72.9 (57.0, 93.1) ng x h/ml, and 53.0 (47.0, 59.9) pg/ml and 758 (655, 878) pg x h/ml, respectively. Concomitant bosentan did not affect the Cmax but significantly decreased the AUC of norethisterone and ethinyl estradiol by 13.7% (-23.5, -2.6) and 31.0% (-40.5,-20.2), respectively. The maximum decrease in AUC of norethisterone and ethinyl estradiol in an individual subject was 56% and 66%, respectively. CONCLUSIONS: Bosentan decreases the AUC of norethisterone and ethinyl estradiol in healthy female subjects. In patients treated with bosentan, reduced efficacy of hormonal contraceptives should be considered.

Possible involvement of brain opioid peptides in clonidine-induced hypotension in spontaneously hypertensive rats.

The effect of intracisternal pretreatment with opiate antagonists or antisera against various opioid peptides on the hypotension and bradycardia induced by cumulative intracisternal administration of clonidine (0.02-2.5 microgram) was studied in conscious Wistar-Kyoto rats and in spontaneously hypertensive rats. No effect of any pretreatment on basal values of blood pressure and heart rate was detected in either of these strains. In spontaneously hypertensive rats intracisternal pretreatment with naltrexone resulted in a dose-dependent inhibition of clonidine-induced hypotension and bradycardia. DL-naloxone also antagonized the hypotension but not influence the hypotensive and bradycardic response to clonidine. In Wistar-Kyoto rats naltrexone and the beta-endorphin antiserum B4 failed to affect the cardiovascular effects of clonidine. B4 and an antiserum against dynorphin(1-13) inhibited clonidine-induced hypotension in spontaneously hypertensive rats, whereas a [Met5]enkephalin antiserum had no effect. Use of antisera specifically recognizing the C-terminus of beta-, alpha-, and gamma-endorphin, respectively, revealed that only the beta-endorphin antiserum inhibited the fall in blood pressure in spontaneously hypertensive rats after cumulative administration of clonidine. None of the antisera used affected clonidine-induced bradycardia. These results indicate that activation of stereospecific opiate receptors in spontaneously hypertensive, but not in Wistar-Kyoto rats, plays a role in the central hypotensive effect of clonidine. beta-Endorphin(1-31) and dynorphin(1-13) might be the endogenous ligands for these receptors.

A toxin from the green mamba Dendroaspis angusticeps: amino acid sequence and selectivity for muscarinic m4 receptors.

Muscarinic toxin 3 (MT3) (65 amino acids, four disulphides, M(r) 7379) was isolated from the venom of the African snake Dendroaspis angusticeps (green mamba) and its amino acid sequence determined. Its ability to inhibit the binding of [3H]N-methylscopolamine ([3H]NMS) to Chinese hamster ovary cells stably expressing subtypes of muscarinic receptors was studied. MT3 displayed high affinity for the m4 receptor (pKi = 8.7 +/- 0.06), 40-fold lower affinity at ml receptors (pKi = 7.11 +/- 0.17) whereas no inhibition of [3H]NMS binding to m2, m3 and m5 receptors was observed at concentrations up to 1 microM. This makes MT3 the most selective m4 receptor ligand known to date.

Human muscarinic receptors expressed in A9L and CHO cells: activation by full and partial agonists.

1. A comparative study of receptor activation by ten full and partial muscarinic agonists was undertaken on the five subtypes of human muscarinic receptors expressed at similar receptor densities in Chinese hamster ovary (CHO-K1) cells. In addition, m1, m2 and m3 receptors were expressed in mouse fibroblast A9L cells in order to compare the influences of cell type on agonist activation of these receptors. 2. Receptor-effector coupling efficiencies were greater in CHO than A9L cells and agonists displayed greater potencies and similar or greater intrinsic activities at CHOm1 and CHOm3 than A9Lm1 and A9Lm3 receptors. Although m2 receptor density was 6 fold higher in A9L than CHO cells, carbachol elicited significantly greater inhibition of adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in CHOm2 cells. These data suggest that not only receptor density but receptor-effector coupling and/or coupling efficiencies play significant roles in agonist-induced responses. 3. In CHO cells, receptor-effector coupling efficiencies were m3 = m1 > m5. Although CHOm5 receptors were the least efficiently coupled, some partial agonists displayed higher intrinsic efficacies at m5 than m3 receptors suggesting that, in CHO cells, m5 and m3 receptors may activate different G proteins and/or effectors to stimulate inositol monophosphate (IP1) formation. 4. McN-A-343 was a functionally selective m4 agonist. It had little or no agonist activity at m3 receptors expressed in either A9L or CHO cells. The slopes of McN-A-343 concentration-response curves inCHOm2 cells were significantly lower than the slopes obtained with this compound in CHOm4 cells suggesting that the mode of activation by McN-A-343 differed between the two muscarinic receptors negatively coupled to adenylyl cyclase.5. Cloned receptors provide valuable tools for the study of agonist-receptor interaction and agonist receptor activation but caution should be applied in assuming that the results are valid for all cell types or for tissue-expressed receptors.

Alpha-methyldopa induces a naltrexone-insensitive antinociception and hypomotility in rats.

1. This study served to investigate whether endogenous opioid peptides play a role in the putative antinociceptive and the sedative actions of alpha-methyldopa. 2. In conscious normotensive rats, alpha-methyldopa induced hypotension, starting around 1 h and reaching a maximum 3-4 h after administration. Pretreatment with naltrexone resulted in an inhibition of alpha-methyldopa-induced hypotension. 3. alpha-Methyldopa dose-dependently increased hot plate latency which became evident after a 4 h lag period and reaching a maximum effect at 6 h. The antinociceptive effect of alpha-methyldopa was not affected by naltrexone. 4. In a small open field, alpha-methyldopa dose-dependently suppressed locomotion and sniffing behaviour. These effects of alpha-methyldopa were apparent 1 h after administration and were naltrexone-insensitive. 5. No changes in the level of beta-endorphin-like immunoreactivity in plasma and cerebrospinal fluid were observed after administration of alpha-methyldopa. 6. The results indicate that endogenous opioid peptides are involved in the hypotensive action of alpha-methyldopa but not in alpha-methyldopa-induced hypomotility and antinociception.

The receptors for endothelins and their analogues in SK-N-MC neuroblastoma cells.

The potency order of peptides to inhibit [125I]endothelin-1 binding and to stimulate phosphatidylinositol phosphate (PtdInsP) turnover in SK-N-MC cells was consistent with the presence of ETA-endothelin receptors. Divalent cations enhanced [125I]endothelin-1 binding by, in the case of Mn2+, increasing radioligand affinity. Mn2+ did not induce conformational changes in endothelin-1, and its effect was maintained in solubilized receptors. Hence, metal ions may directly interact with endothelin receptors. The effects of BQ-123 and [Ala1,3,11,15]endothelin-1 on PtdInsP turnover were investigated. Concentration-response curves of endothelins were modeled by a second-order equation that assumes pseudoirreversible ligand binding.

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.

Affinity of R 396, an NK-2 tachykinin receptor antagonist, for NK-2 receptors in preparations from different species.

We have assessed the affinity of R 396 (Ac. Leu-Asp-Gln-Trp-Phe-Gly NH2) in a number of NK-2 tachykinin receptor bearing-tissues from several species. The cyclic analog of R 396, (MEN 10354) was less potent and selective than the linear hexapeptide at NK-2 tachykinin receptors subtypes in the rabbit pulmonary artery and hamster trachea. The affinity of R 396, as measured by a smooth muscle contraction assay and a radioligand binding assay, was higher (about 10 fold) for NK-2 receptors expressed in hamster tissues (urinary bladder, stomach and trachea) than in rat tissues (urinary bladder, vas deferens, colon and stomach) and a further drop in affinity was observed in bovine tissues (urinary bladder and stomach) or rabbit bronchus. The results are discussed in relation to the proposed existence of NK-2 receptor subtypes and raise the question of the existence of species-related differences as compared to the existence of true receptor subtypes.

The nigrostriatal dopamine system: role in the development of hypertension in spontaneously hypertensive rats.

The effects of lesions in the pars compacta of the substantia nigra of 4-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were studied on (a) changes in blood pressure during a period of 6 weeks following lesioning, and (b) dopamine and metabolite concentrations in the caudate nucleus and (c) the in vitro release of [3H]dopamine from caudate nucleus slices 6 weeks after the lesioning. We report here that substantia nigra lesions caused a profound attenuation of the development of hypertension in SHR. No effect on heart rate was observed. Whereas the concentrations of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the caudate nucleus were not different, the amount of homovanillic acid (HVA) and the ratios DOPAC/dopamine and HVA/dopamine were lower in sham-treated SHR than in sham-treated WKY, indicating a lower turnover of dopamine in SHR. Six weeks after lesioning, the concentrations of dopamine, DOPAC and HVA were decreased in the caudate nucleus (46-66%) of both strains, whereas DOPAC/dopamine and HVA/dopamine ratios tended to be increased in both SHR and WKY. Using an in vitro superfusion method, it was found, that the electrically stimulated release of [3H]dopamine from caudate nucleus slices of sham-treated SHR was lower than from slices of sham-treated WKY at the age of 10 weeks. Six weeks after lesioning, the stimulus-evoked release of [3H]dopamine from the remaining terminals was significantly increased in caudate nucleus slices of SHR, whereas the difference did not reach statistical significance in WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of opiate receptors located in the nucleus tractus solitarii in the hypotension induced by alpha-methyldopa.

The local administration of the opiate receptor antagonist, naltrexone, into the nucleus tractus solitarii (NTS) inhibited the hypotension induced by systemically injected alpha-methyldopa in conscious rats. In addition, the local injection into the NTS of a beta-endorphin antiserum but not of antisera against [Met5]enkephalin and dynorphin A(1-13) prevented the alpha-methyldopa-induced hypotension. These results suggest a role of opiate receptors in the NTS, or in a closely located medullary site, in the centrally mediated hypotension induced by alpha-methyldopa.

Activation of glucocorticoid receptors and the effect of naloxone during hemorrhagic hypotension.

Evidence indicates that endogenous opioid peptides and glucocorticoids participate in the control of cardiovascular regulation during hemorrhagic shock. In the present study, we investigated a possible interaction between brain opioid peptides and adrenal corticosteroids regarding the control of arterial pressure during hemorrhage. The bleeding volumes required to lower arterial pressure to 80, 60 and 40 mmHg were studied in anesthetized sham-operated (SHAM) and adrenalectomized (ADX) rats. I.c.v. administration of 10 micrograms of naloxone resulted in a significant increase in the bleeding volume required to lower arterial pressure from 60 to 40 mmHg in SHAM animals, whereas no effect of naloxone was observed in ADX animals. Replacement therapy with a 100% corticosterone pellet (100 mg, s.c.), but not with a 12.5% corticosterone pellet (12.5 mg corticosterone and 87.5 mg cholesterol, s.c.), resulted in an effect of naloxone on the bleeding volume in ADX animals. The effect of replacement therapy could be inhibited by i.c.v. pretreatment with the synthetic glucocorticoid receptor antagonist, RU38486 (100 ng). These data suggest that (1) opioid mechanisms are involved in the regulation of blood pressure during hemorrhage, and (2) occupancy of glucocorticoid receptors is required for naloxone to exert its hemodynamic effect during hemorrhagic hypotension in ADX rats.

Demonstration of a neurokinin A receptor subtype in transfected fibroblasts.

In competitive radioligand binding assays, the NK2 receptor antagonists [Tyr5,D-Trp6,8,9,Arg10]NKA(4-10) (MEN 10207) and [Tyr5,D-Trp6,8,9,Arg10]NKA(3-10) (MEN 10208) had high and low affinity, respectively, in bovine stomach membranes and SKLKB82#3 cells, a murine fibroblast cell line transfected with a cDNA encoding for the bovine NK2 receptor. These antagonists also had different affinities when inhibiting neurokinin A-induced polyphosphoinositide hydrolysis in SKLKB82#3 murine fibroblasts. Thus, the de novo protein expressed by the SKLKB82#3 murine fibroblasts may represent a distinct NK2 receptor subtype.

MDL 100,458 and MDL 102,288: two potent and selective glycine receptor antagonists with different functional profiles.

Glycine receptor antagonists have been proposed to have multiple therapeutic applications, including the treatment of stroke, epilepsy, and anxiety. The present study compared the biochemical and behavioral profiles of two strychnine-insensitive glycine receptor antagonists, MDL 100,458 (3-(benzoylmethylamino)-6-chloro-1H-indole-2- carboxylic acid) and MDL 102,288 (5,7-dichloro-1,4-dihydro-4-[[[4- [(methoxycarbonyl)amino]phenyl]sulfonyl]imino]-2-quinolinecarboxylic acid monohydrate). Both compounds potently inhibited [3H]glycine binding to rat cortical/hippocampal membranes (Ki = 136, 167 nM, respectively) without showing significant activity in 18 other receptor binding assays. In an in vitro functional assay, both compounds completely antagonized N-methyl-D-aspartate (NMDA)-stimulated cGMP accumulation in rat cerebellar slices. However, in contrast to their equipotency in the glycine receptor assay, MDL 100,458 was approximately 6-fold more potent than MDL 102,288 in the cGMP assay (IC50 values = 1.25, 7.8 microM, respectively). Behavioral tests demonstrated that MDL 102,288 and MDL 100,458 exhibited strikingly different in vivo profiles. MDL 100,458 antagonized audiogenic seizures in DBA/2J mice (ED50 = 20.8 mg/kg i.p.), whereas MDL 102,288 was without effect in the dose range tested (ED50 > 300 mg/kg i.p.). Central nervous system penetration did not appear to account for this difference. For example, MDL 102,288 was not active following direct intracerebroventricular administration (ED50 > 16 micrograms; vs. 0.78 microgram for MDL 100,458). In a test of anxiolytic activity, MDL 102,288 reduced separation-induced ultrasonic vocalizations in rat pups (ED50 = 6.3 mg/kg i.p.) whereas MDL 100,458 was only weakly active (ED50 = 80.8 mg/kg i.p.). Furthermore, the anxiolytic effect of MDL 102,288 was selective in that it occurred at doses that did not produce motoric disruption as measured by an inclined-plane test (ED50 > 160 mg/kg; therapeutic index > 25.4). In contrast, the anxiolytic activity of MDL 100,458 was non-selective in that it occurred at doses that also produced motoric disruption (ED50 = 57.7 mg/kg; therapeutic index = 0.7). Thus, two glycine receptor antagonists which have similar in vitro binding profiles as selective ligands for the strychnine-insensitive glycine receptor, demonstrate different in vitro and in vivo functional profiles. The reason for these differences is not clear, though one possibility could be that the compounds may act on different NMDA receptor subtypes. These data support the possibility that different glycine receptor antagonists may have different therapeutic targets.

Pharmacological characterization of MDL 105,519, an NMDA receptor glycine site antagonist.

MDL 105,519, (E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid, is a potent and selective inhibitor of [3H]glycine binding to the NMDA receptor. MDL 105,519 inhibits NMDA (N-methyl-D-aspartate)-dependent responses including elevations of [3H]N-[1,(2-thienyl)cyclohexyl]-piperidine ([3H]TCP) binding in brain membranes, cyclic GMP accumulation in brain slices, and alterations in cytosolic CA2+ and NA(+)-CA2+ currents in cultured neurons. Inhibition was non-competitive with respect to NMDA and could be nullified with D-serine. Intravenously administered MDL 105,519 prevented harmaline-stimulated increases in cerebellar cyclic GMP content, providing biochemical evidence of NMDA receptor antagonism in vivo. This antagonism was associated with anticonvulsant activity in genetically based, chemically induced, and electrically mediated seizure models. Anxiolytic activity was observed in the rat separation-induced vocalization model, but muscle-relaxant activity was apparent at lower doses. Higher doses impair rotorod performance, but were without effect on mesolimbic dopamine turnover or prepulse inhibition of the startle reflex. This pattern of activities differentiates this compound from (5R,10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and indicates a lower psychotomimetic risk.

Modulation of agonist binding by guanine nucleotides in CHO cells expressing muscarinic m1-m5 receptors.

In membranes prepared from CHO-m2 cells, inhibition of [3H]-N-methylscopolamine ([3H]NMS) binding by several muscarinic agonists resulted in competition curves with Hill slopes significantly different from unity. Addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) led to an increase in the IC50 value of the agonists with significant steepening of the inhibition curves. The shift in potency induced by Gpp(NH)p differed among the agonists with a rank order of oxotremorine-M = carbachol > oxotremorine > McN-A-343 = pilocarpine. In CHO-m4 membranes, Gpp(NH)p was less efficacious than in CHO-m2 membranes whereas no effect of the guanine nucleotide was found in membranes prepared from CHO-m1, -m3, and -m5 cells. No major differences in the effect of Gpp(NH)p among agonists were found in CHO-m4 cells. Atropine binding was not affected by the guanine nucleotide. Together, these results indicate that coupling of G-proteins to muscarinic receptors linked to inhibition of cyclic adenosine monophosphate (cAMP) (m2 and m4) but not of those linked to phosphoinositol turnover (m1, m3 and m5) can be perturbed by Gpp(NH)p. The differential effects observed with Gpp(NH)p between agonist binding to m2 and m4 receptors appear to be receptor-specific and may reflect differences in the G proteins activated by these receptors in CHO cells.

Comparison of the effects of neuropeptide K and neuropeptide gamma with neurokinin A at NK2 receptors in the hamster urinary bladder.

Neuropeptide K (NPK) and neuropeptide gamma (NP gamma) are two endogenous N-terminally extended forms of neurokinin A (NKA). Here, we compared their effects with those of NKA on 125I-NKA binding, phosphatidylinositol (PI) turnover and smooth muscle contraction in the hamster urinary bladder. NPK, NP gamma and NKA were equipotent in competing 125I-NKA from NK2 receptors in crude hamster bladder membranes. All three peptides stimulated PI turnover by approximately 750% with similar potency. In a third series of experiments, these peptides had similar efficacy in inducing a dose-dependent contraction of bladder smooth muscle. The NK2 receptor selective antagonist L-659,877 (cyclo[Leu-Met-Gln-Trp-Phe-Gly]) inhibited the stimulation of PI turnover and bladder contractions induced by all three tachykinins. The present results show that NKA, NPK and NP gamma display a similar biological profile. The N-terminal extensions of NPK and NP gamma appear not to influence binding of these peptides to NK2 receptors, NK2 receptor mediated stimulation of PI turnover, or smooth muscle contraction in hamster urinary bladder.