pKB Values Research Articles

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated urinary bladder.

1. We have studied several tachykinin NK2 receptor antagonists, bearing a monocyclic pseudopeptide (MEN 10,508, MEN 10,573, MEN 10,581, MEN 10,612, MEN 10,619 and MEN 10,677), or bicyclic peptide (MEN 10,627, MEN 10,692, MEN 10,771, MEN 10,882 and MEN 10,993) structure, on the human isolated urinary bladder detrusor muscle against neurokinin A as an agonist, and compared their affinities in this preparation with those for NK2 receptors expressed in the rabbit isolated pulmonary artery and hamster isolated trachea. 2. In the human bladder, all the antagonists tested produced a concentration-dependent and competitive antagonism of neurokinin A-mediated contractions: among the cyclic pseudopeptides MEN 10,677 (pKB = 8.0) was the most potent antagonist, while among the bicyclic analogues it was MEN 10,993 (pKB = 8.8). 3. In general, the bicyclic peptide antagonists tested were more potent than the monocyclic pseudopeptide compounds, either in the human urinary bladder or in the rabbit pulmonary artery or hamster trachea, showing a nanomolar affinity for the human NK2 receptor. 4. A highly significant correlation was found between the estimated pKB values of all the antagonists tested in the human urinary bladder and rabbit pulmonary artery (r2 = 0.94, n = 12, P < 0.01), whereas no linear correlation was found between pKB values measured in the human urinary bladder and hamster trachea (r2 = 0.52, n = 12, P > 0.05): these observations provide further pharmacological evidence for receptor homology between the human and rabbit NK2 receptor. 5. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN 10,627, as candidates for testing in pathological conditions, such as bladder hyperactivity, for which preclinical evidence indicates that a therapeutic effect could result from the block of the tachykinin NK2 receptor.

Naturally occurring clavines: antagonism/partial agonism at 5-HT2A receptors and antagonism at alpha 1-adrenoceptors in blood vessels.

The interaction of eight typical representatives of naturally occurring clavines (agroclavine, costaclavine, dihydrolysergol-l, elymoclavine, festuclavine, lysergene, lysergol, and pyroclavine) with 5-HT2A receptors and alpha 1-adrenoceptors was studied in rat tail artery and aorta, respectively. Clavines antagonized 5-HT-induced contractions with calculated pKB values (pKP values for partial agonists) of 4.84-7.81 and (R)-phenylephrine-induced contractions with calculated pKB values of 5.34-7.09. Specificity of clavines at 5-HT2A receptors relative to alpha 1-adrenoceptors was rather low. Low affinity for costaclavine at both 5-HT2A receptors (pKP = 4.84 +/- 0.06) and alpha 1-adrenoceptors (pKB = 5.34 +/- 0.05) indicates that the trans-junction of ring C and D of the ergoline pharmacophore is crucial for the binding of ergolines to these sites. Lysergol, lysergene, and costaclavine produced non-parallel displacements of the 5-HT concentration-response curve in the rat tail artery and caused small contractions by themselves. Lysergol contracted the rat tail artery with a pEC50 of 6.36 +/- 0.04 and an intrinsic activity of 0.18 +/- 0.03 with respect to 5-HT. Lysergol-induced contractile responses were surmountably antagonized by ketanserin (10 nM) with a pKB of 9.1 which is consistent with an interaction of lysergol with 5-HT2A receptors. The pKP for the lysergol-5-HT2A receptor complex calculated from concentration-response curves to lysergol was 6.88 +/- 0.07 and did not match the pKP of 7.66 +/- 0.02 calculated from antagonism by lysergol of the contractile response to 5-HT. This suggests that lysergol and 5-HT possibly bind in two slightly different orientations at the 5-HT2A receptor. It is concluded that partial agonism and pure antagonism at 5-HT2A receptors on the one side and antagonism at alpha 1-adrenoceptors on the other side may contribute to the noxious effects of naturally occurring clavines.

M1 and M3 muscarinic receptors in human pulmonary arteries.

1. Acetylcholine (ACh) and the M1 agonists (McN-A-343 or PD142505) relaxed human isolated pulmonary arteries which were pre-contracted with noradrenaline (10 microM). In preparations where the endothelium had been removed ACh induced a contractile response whereas the M1 agonists (McN-A-343 or PD142505) had no effect. 2. ACh- and McN-A-343-induced relaxations were abolished after treatment of endothelium-intact preparations with the drug combination NG-nitro-L-arginine (L-NOARG: 0.1 mM) and indomethacin (1.7 microM). 3. The affinity (pKB value) for pirenzepine was higher in human pulmonary arteries when tissues were relaxed with McN-A-343 as compared with ACh (pKB values, 7.71 +/- 0.30 (n = 4) and 6.68 +/- 0.15 (n = 8), respectively). In addition, the affinity for pFHHSiD against McN-A-343- and ACh-induced relaxations was 6.86 +/- 0.13 (n = 3) and 7.35 +/- 0.11 (n = 9) respectively. 4. The low affinities for methoctramine in human isolated pulmonary arteries with the endothelium either intact or removed, suggested the lack of involvement of M2 and M4 receptors in the Ach responses. 5. Phenoxybenzamine (3 microM: 30 min) abolished both ACh contraction and relaxation in human pulmonary artery. The ACh contraction was present when the phenoxybenzamine treatment was preceded by incubation with pFHHSiD (2 microM) but not with pirenzepine (1 microM). In addition, the ACh relaxation was present when preparations were treated with either pFHHSiD (2 microM) or pirenzepine (1 microM), before exposure to phenoxybenzamine. 6. These results in human isolated pulmonary arteries support the notion that only M3 receptors, on smooth muscle, mediate the ACh-induced contraction whereas M3 and M1 receptors are involved in the endothelium-dependent ACh-induced relaxation.

Pharmacological characterization of the 5-hydroxytryptamine receptor mediating relaxation in the rat isolated ileum.

1 The aim of the present study was to investigate a 5-HT4 receptor involvement in the mediation of a 5-HT-induced relaxation response in the rat isolated ileum in vitro. 2 Ileal segments were taken at regular intervals from the ileo-caecal junction to duodenum. 5-HT (1 microM) induced a relaxation or contraction response in segments taken from the terminal ileum: the relaxation decreased and finally disappeared as contractions dominated in the proximal tissues. The 5-HT-induced relaxations were enhanced in the terminal segments and the contractions attenuated in both terminal and proximal segments, in the presence of methysergide (1 microM) and atropine (0.1 microM). 3 In the presence of methysergide (1 microM) and atropine (0.1 microM), a cumulative addition of 5-HT (0.01-1 microM) induced a concentration-dependent relaxation in the terminal (1-20 cm from the ileo-ceacal junction) ileal segments which at higher concentrations of 5-HT (3-30 microM) reverted to contraction. 4 The rank order of potency of indole agonists in inducing a concentration-related relaxation response in tissues of the terminal ileum (pretreated with pargyline (100 microM) and in the presence of methysergide (1 or 100 microM) and atropine (0.1 microM) was 5-hydroxytryptamine (6.97 +/- 0.06), 5-methoxytryptamine (6.50 +/- 0.07), alpha-methyl-5-hydroxytryptamine (5.53 +/- 0.17), 5-carboxamidotryptamine (5.51 +/- 0.12) and 2-methyl-5-hydroxytryptamine (< 5), the pEC50 values (mean +/- s.e.mean) being shown in parentheses. 5 Pretreatment of tissues with pargyline (100 microM) selectively enhanced the potency of 5-methoxytryptamine by a factor of 19 but failed to modify the potency of the other indole agonists. 6 The 5-HT4 receptor antagonists, tropisetron, SDZ 205-557 and GR 113808 antagonized the relaxation response to 5-HT (in the presence of methysergide (1 or 10 microM) and atropine (0.1 microM)) with pKB values (95% CL) of 6.09 (5.94-6.24), 7.0 (6.9-7.09) and 8.95 (8.81-9.1) respectively. Apparent pKB values estimations for tropisetron (1 microM) and GR 113808 (10 nM) using the agonists 5-methoxytryptamine and 5-carboxamidotryptamine were 6.37 +/- 0.31, 5.91 +/- 0.38 and 8.83 +/- 0.11, 8.82 +/- 0.22 respectively. 7 Tropisetron (10 microM), SDZ 205-557 (3 microM) and GR 113808 (10-100 nM) caused an increase in basal tone of the rat terminal ileum when administered in the presence of methysergide and atropine. 8 The relaxation response to 5-HT in the rat terminal ileum was not antagonized by ritanserin (1 microM), ondansetron (1 microM) or N omega-nitro-L-arginine methyl ester (100 microM) and with only a twofold dextral shift of the concentration-response curve by tetrodotoxin (1 microM). 9 It is concluded that the relaxant response to 5-HT in the terminal region of the ileum is mediated directly at the smooth muscle; a ranked indole agonist potency and selective antagonism by 5-HT4 receptor antagonists tropisetron, SDZ 205-557 and GR 113808 indicate a 5-HT4 receptor involvement in the relaxation response.

Neural 5-HT4 receptors in the human isolated detrusor muscle: effects of indole, benzimidazolone and substituted benzamide agonists and antagonists.

1. In strips of human isolated detrusor muscle, the 5-hydroxytryptamine (5-HT) receptor (5-HT4) that mediates facilitation of neuromuscular cholinergic transmission was further characterized by using 5-HT and a series of ligands known for their 5-HT4 agonist (5-methoxytryptamine: 5-MeOT, cisapride, (R,S)-zacopride, BIMU 8) or antagonist (RS 23597, GR 125487, DAU 6285) properties. 2. In the presence of methysergide (1 microM) and ondansetron (3 microM) to isolate pharmacologically the 5-HT4 receptors, 5-HT (0.3 nM-1 microM), 5-MeOT (10 nM -30 microM), BIMU 8 (10 nM-3 microM), cisapride (0.1-10 microM) and (R,S)-zacopride (0.1-30 microM) potentiated cholinergic contractions to electrical field stimulation in a concentration-dependent manner. RS 23597 (10 nM-10 microM), a competitive 5-HT4 receptor antagonist in other systems, also showed agonist properties. The following rank order of potency as an agonist was obtained: 5-HT (pEC50 = 8.0) > RS 23597 (7.0) = BIMU 8 (6.9) > or = cisapride (6.6) > 5-MeOT (6.0) > or = (R,S)-zacopride (5.7). Relative to 5-HT (intrinsic activity = 1), 5-MeOT acted as a full agonist (1.03), while BIMU 8 (0.76), (R,S)-zacopride (0.61), RS 23597 (0.60) and cisapride (0.41) behaved as partial agonists. 3. The potentiation by 5-HT was competitively antagonized by the selective 5-HT4 receptor antagonist GR 125487 (0.3-3 nM) with a pA2 estimate of 9.75 (Schild slope of 1.09), and by DAU 6285 (1 microM; pK3 = 6.45). Additionally, GR 125487 (3 nM) antagonized the responses to 5-MeOT (pKB = 9.72) and reversed the potentiation induced by RS 23597. As an antagonist, RS 23597 (10, 30 and 100 nM) inhibited the response to 5-HT. In addition, 30 and 100 nM RS 23597 reduced the 5-HT response maximum by 30 and 50%, respectively. The pKB value calculated at 10 nM was 8.0. 4. Thus, in the human isolated detrusor muscle, the 5-HT4 receptors mediating facilitation of cholinergic neuromuscular transmission are activated by indoleamines (5-HT, 5-MeOT), substituted benzamide (cisapride, (R,S)-zacopride), benzoate (RS 23597) and benzimidazolone (BIMU 8) derivatives. The activities (in terms of both potency and efficacy) of most agonists, as well as the affinity estimates of the antagonists GR 125487 and DAU 6285, are comparable to those found in other peripheral tissues. Exceptions are RS 23597, which acted either as a partial agonist or as an antagonist of the response to 5-HT1 and 5-MeOT that showed an unusually low potency. The latter findings may be ascribed to differences in the efficiency of receptor coupling mechanisms and/or in the molecular structure (i.e. splice variants) of the 5-HT4 receptor.

Characterization of alpha-adrenoceptor subtype(s) mediating vasoconstriction in the perfused rabbit ovarian vascular bed.

1. alpha 1-Adrenoceptor agonists, noradrenaline, phenylephrine, methoxamine, oxymetazoline and SDZ NVI 085 but not alpha 2-adrenoceptor agonists, UK 14304, tizanidine or clonidine evoked dose-dependent vasoconstriction of the isolated perfused rabbit ovarian vascular bed. The rank order of agonist potency was noradenaline > oxymetazoline > phenylephrine > SDZ NVI 085 > methoxamine. 2. Prazosin (10(-8) M - 10(-5) M) displaced agonist dose-response curves to the right. The pA2/pKB values ranged between 7.27 and 7.66 against noradrenaline, phenylephrine, methoxamine and SDZ NVI 085 and were not significantly different from each other. Prazosin was however significantly less potent against oxymetazoline (pA2 6.38). Yohimbine (10(-6) M - 10(-5) M) was not very effective against any of the agonists. 3. WB 4101 (10(-8) M - 10(-5) M) displaced agonist dose-response curves to the right. The pA2/ pKB values ranged between 7.08 and 7.93 against noradrenaline, phenylephrine, methoxamine and SDZ NVI 085. WB 4101 was significantly less potent against oxymetazoline (pKB 6.85). 4. SZL-49 (5 x 10(-6) M) but not chloroethylclonidine (3 x 10(-5) M) significantly reduced vasoconstrictor responses to all the agonists. 5. Electrical field stimulation of the ovarian bed produced frequency-dependent vasoconstrictor effects which were abolished by 6-OHDA. The responses were also antagonized in a concentration-dependent by prazosin (10(-7) M - 10(-5) M) and WB 4101 (3 x 10(-8) M - 3 x 10(-7) M). Yohimbine reduced the response to electrical stimulation by 20% at 10(-5) M. The vasoconstrictor effect was also inhibited by SZL-49 but not by chloroethylclonidine. 6. These results would suggest that the vasoconstrictor responses of the ovarian vascular bed to adrenergic agonists and to electrical stimulation are mediated via the alpha 1A-adrenoceptor subtype.

Analysis of the effects of alpha 1-adrenoceptor antagonists on noradrenaline-mediated contraction of rat small mesenteric artery.

1. In this study, we examined the interaction between noradrenaline (NA) and phenylephrine (PE) with seven antagonists (prazosin, tamsulosin, phentolamine, WB-4101, 5-methylurapidil, spiperone and HV 723) in an attempt to characterize the alpha 1-adrenoceptor population of the rat isolated small mesenteric artery (SMA) preparation. 2. Six of the seven antagonists investigated produced concentration-dependent, parallel, rightward shift of the NA concentration-effect (E/[A]) curves. The exception was tamsulosin, which produced significant decrease of the upper asymptote. In the case of 5-methylurapidil and HV723, the Schild plot slope parameters were not significantly different from unity over the range of concentrations used. However, the Schild plot slopes obtained for the other antagonists were all significantly greater than unity, inconsistent with expectations for simple competitive antagonism. 3. HV723, prazosin and tamsulosin were also tested using PE as an agonist. All three antagonists produced concentration-dependent, parallel, rightward shifts of the PE curves and Schild analysis yielded slope parameters not significantly different from unity. The pKB estimates obtained for tamsulosin and prazosin were not significantly different from the pA2 values obtained when NA was used as agonist. In the case of HV723, the 95% confidence intervals for the pKB values yielded with NA and PE did not overlap (pKB = 8.80-9.13 and 8.15-8.77 for NA and PE, respectively). 4. In the absence of evidence to indicate that the steep Schild plots were due to failure to satisfy the basic criteria for quantitative analysis in a one-receptor system, we considered the possibility that the complexity was caused by an action of NA at inhibitory D1 receptors. The selective D1 receptor antagonists, SCH-23390 (10 nM), had no significant effect on the NA E/[A] control curve, but the apparent potency of 100 nM prazosin was reduced by approximately 3.5 fold. 5. This study indicates that the steep Schild plots obtained from the interaction between NA and alpha 1-adrenoceptor antagonists were due to the simultaneous activation of inhibitory D1 receptors by NA. Notwithstanding this complexity, our explanatory model of the system (see Appendix) suggests that the antagonist affinity values estimated in the absence of D1 receptor block were not significantly affected by this other action of NA. The low affinity estimate obtained for prazosin suggests that the pharmacologically-defined alpha IL-subtype operates in the SMA.

P-18-10 - Effect of substance abuse on the course of Bipolar Disorder

The 5-HT receptor mediating postjunctional relaxation of precontracted guinea-pig ileum has been characterized using several agonists and antagonists. Substance P precontracted tissues were potently relaxed by 5-HT (5-hydroxytryptamine, serotonin), 5-CT (5-carboxamidotryptamine) and several other indoles. The rank order of potency, with pEC50 values in parentheses, was 5-CT (7.6) > 5-methoxytryptamine (5.7) > 5-HT (5.5) > α-methyl-5-HT (4.7) > 2-methyl-5-HT (< 4.0) = tryptamine (< 4.0) = N,N-dimethyl-tryptamine (< 4.0) = N,N-dimethyl-5-HT (< 4.0) = dipropyl-5-CT (< 4.0) = sumatriptan (< 4.0). 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)-tetralin) acted as a potent (6.3), but partial, agonist with respect to 5-HT. The responses to 5-CT were antagonized by several compounds with the following rank order of affinity, with pKB values in parentheses: LSD (lysergic acid diethylamide; 8.1) = mesulergine (7.8) > methysergide (7.6) = spiperone (7.6) > clozapine (7.3) ⪢ (−)-pindolol (< 6.0) > ketanserin (< 6.0) = ondansetron (< 6.0) = GR 113808 ([1-(2-methane-sulphonamido-ethyl)-piperidin-4-yl]-methyl-indole-3-carboxylate maleate; < 6.0). The relaxant responses to 5-HT were also resistant to tetrodotoxin. These data are consistent with a functional 5-HT receptor, mediating relaxation of guinea-pig ileum, which exhibits an operational profile similar to that of the cloned guinea-pig 5-ht7 receptor. This study, therefore, provides evidence for a functional correlate of the 5-ht7 gene product.

In vitro pharmacological profile of YM-43611, a novel D2-like receptor antagonist with high affinity and selectivity for dopamine D3 and D4 receptors.

1. We investigated some neurochemical properties of a novel benzamide, YM-43611, [(S)-N-(1-benzyl-3-pyrrolidinyl)-5-chloro-4-cyclopropylcarbonylamino+ ++-2- methoxybenzamide] in comparison with putative D2-like receptor antagonists using both rat and human cloned dopamine D2-like receptors in vitro. 2. Receptor binding studies revealed that YM-43611 had appropriately potent affinities for both rat and human D2-like receptors, with moderate selectivity for D3 receptors and high selectivity for D4 receptors over D2 receptors (Ki values (nM) for rat receptors: D2, 165; D3, 35.5; D4, 1.85, and for human receptors: D2, 42.9; D3, 11.2; D4, 2.10). 3. YM-43611 displayed weak or negligible affinity for other neurotransmitter receptors, namely D1, D5, alpha(1), alpha(2), beta, 5-HT1A, 5-HT2A, 5-HT3, H1, M1 and M2 receptors. 4. Dopamine stimulated low-Km GTPase activity on membranes from Chinese hamster ovary (CHO) cells expressing the human D2-like receptor subtype. This response to dopamine of low-Km GTPase activity was inhibited by use of putative D2-like receptor antagonists. YM-43611 showed a moderate selectivity for D3 receptors (Ki = 45.5 nM) and a high selectivity for D4 receptors (Ki = 3.28 nM) over D2 receptors (Ki = 70.6 nM). 5. Dopamine inhibited forskolin-stimulated adenylate cyclase in intact CHO cells expressing the human D2-like receptor subtype. YM-43611 shifted the inhibition curve of dopamine on respective D2-like receptor subtype-mediated cyclic AMP formation to the right in a parallel fashion, showing a pA2 value of 7.42 (38.1 nM) for D2 receptors, a pKB value of 8.06 (8.68 nM) for D3 receptors, and a pA2 value of 8.42 (3.77 nM) for D4 receptors. 6. YM-43611 but not the other D2-like receptor antagonists exhibited good selectivity with respect to dual antagonism for D3 and D4 receptors in both receptor binding and functional assays. 7. These results indicate that YM-43611 is a novel D2-like receptor antagonist with high potency and selectivity for both D3 and D4 receptors. YM-43611 is therefore expected to be valuable in exploration of the physiological role of D3 and D4 receptors.

Atypical responses of rat ileum to pindolol, cyanopindolol and iodocyanopindolol

1. Pindolol, cyanopindolol (CYP) and iodocyanopindolol (IodoCYP) have been reported to act either as antagonists, agonists or partial agonists at the beta 3-adrenoceptor in different preparations. A comprehensive investigation has not yet been described with these compounds tested in one tissue from one species. This study was conducted to delineate the pharmacological effects of pindolol, CYP and IodoCYP and to provide data on their affinities at the predominant beta-adrenoceptor in rat ileum. 2. The beta-adrenoceptors present in rat ileum were characterized in the presence of CGP 20712A and ICI 118 551, atropine and corticosterone, with (-)-isoprenaline used as an agonist. The role of the beta 1 and beta 2-adrenoceptors was determined by the omission of either CGP 20712A, ICI 118 551, or both, from the buffers. Conversely, the effectiveness of the beta 1- and beta 2-adrenoceptor blockade was examined by use of the beta 1-adrenoceptor-selective agonist, RO 363 and the beta 2-adrenoceptor-selective agonist, salbutamol. 3. There was no evidence for the presence of functional beta 1-adrenoceptors, and no strong evidence that beta 2-adrenoceptor stimulation contributed to the relaxant effects of (-)-isoprenaline. (-)-Phenylephrine did not produce relaxation of the tissue and 5-hydroxytryptamine produced contraction. 4. The beta 3-adrenoceptor-selective agonist, BRL 37344 and (-)-isoprenaline were potent full agonists (pD2 8.35 +/- 0.04 and 7.76 +/- 0.14 respectively), whereas ICI D7114 was less potent (pseudo pD2 6.92 +/- 0.15). These results indicate that the predominant functional beta-adrenoceptors in rat ileum are beta 3-adrenoceptors. 5. Partial agonist effects were produced by CYP (pD2 5.28 +/- 0.26) and IodoCYP (pD2 7.0 +/- 0.26), but not pindolol. All three compounds antagonized the effects of (-)-isoprenaline with pKb values of 6.68 +/- 0.10, 7.59 +/- 0.07 and 7.59 +/- 0.11 for pindolol, CYP and IodoCYP respectively. Likewise, CYP and IodoCYP antagonized the effects of BRL 37344 with pKb values of 7.20 +/- 0.22 and 7.21 +/- 0.14 respectively. This study provides the first functional data on the effects of IodoCYP, the ligand with the highest known affinity for the beta 3-adrenoceptor, at the characterized rat ileum beta 3-adrenoceptor. 6. In conclusion, whereas pKb values suggest that CYP and IodoCYP have a similar affinity for the beta 3-adrenoceptor in rat ileum, the higher potency of IodoCYP suggests that it promotes a greater coupling efficiency, or that its partial agonist effects are produced through a site other than the beta 3-adrenoceptor. The similar pKb values for CYP and IodoCYP at the beta 3-adrenoceptor contrast with their order of known affinities at the beta 1- and beta 2-adrenoceptors, where IodoCYP is far more potent than CYP. This provides evidence of further differences in the characteristics of the beta 3-adrenoceptors compared to the beta 1- and beta 2-adrenoceptors. Finally, the utility of IodoCYP as a beta 3-adrenoceptor antagonist would appear to be limited because of the greater magnitude of partial agonist effects that it produces.

Biphasic antagonisms by beta-blockers against positive inotropic response through beta1-adrenoceptors in isolated canine right ventricular muscles: possible involvement of two beta 1-adrenoceptor subtypes.

In isolated canine right ventricular muscles, we investigated the differences in antagonisms by beta-blockers against the positive inotropic effects (PIEs) of isoproterenol, a nonselective agonist, and T-0509, a beta1-selective agonist. The selective beta1-blockers atenolol and bisoprolol antagonized the PIE of T-0509 monophasically in Schild analysis, showing pA2 values of 7.05 and 7.63, respectively. On the other hand, both blockers produced biphasic antagonism against the PIE of isoproterenol (ISO); therefore, two pKB values were obtained (7.75 and 4.25 and 7.82 and 5.76, respectively). Nadolol, a nonselective beta-blocker, also antagonized the PIE of T-0509 monophasically (pA2 value 7.58), but antagonized the PIE of ISO biphasically (pKB values 7.42 and 4.39). Because the different mode of antagonism by three beta-blockers between T-0509 and ISO could not be explained by the selectivities of beta-agonists and blockers for beta1- and beta2-adrenoceptors in the heart, two subtypes of beta1-adrenoceptors may exist together in canine ventricular muscles, and atenolol, bisoprolol, and nadolol may act as antagonists for the two subtypes with two different affinities.

A Functional Comparison of the Antagonists Bicuculline and Picrotoxin at Recombinant GABAA Receptors

Allosteric modulation of GABAA receptor function by a number of ligands has been shown to be dependent on the subunit composition of the receptor complex. In this respect, modulation of GABAA receptors by the antagonists bicuculline and picrotoxin was examined in Xenopus laevis oocytes expressing recombinant GABAA receptors composed of combinations of murine α1, β1, γ2S and γ2L subunits. Bicuculline and picrotoxin reduced GABA-activated responses mediated by GABAA receptors composed of α1β1, α1β1γ2S and α1β1γ2L subunits in a dose-dependent manner. GABA equilibrium concentration-response curves for each receptor construct were shifted to the right by increasing concentrations of bicuculline in a competitive manner, whereas picrotoxin induced a slight lateral shift as well as a depression of the maximum response consistent with a mixed/non-competitive inhibitory mechanism. GABA concentration-response curves in the absence and presence of bicuculline were subjected to Schild analysis, which revealed similar pKB values of approximately 5.9 for α1β1, α1β1γ2S and α1β1γ2L receptor constructs. Concentration inhibition curves were used to estimate IC50 values for picrotoxin which approximated to 0.4–0.6 μM. The apparent pKB values for bicuculline and the IC50s for picrotoxin were relatively unaffected by the GABAA receptor isoforms used in this study, and in particular, by the absence of the γ2 subunit in the α1β1 GABAA receptor complex. The similarity of the pKBs reported in this study to those previously reported using native neuronal preparations, which are likely to represent heterogeneous GABAA receptor populations, further indicates the lack of dependence on receptor subunit composition for the inhibitory action of bicuculline. Copyright © 1996 Elsevier Science Ltd.

Pharmacological Characteristics of BDTI, a New Isoquinoline-Derived &amp;beta;&lt;sub&gt;2&lt;/sub&gt;-Adrenoceptor Agonist, in Canine Trachea and Rat Heart

The tracheal relaxing effects and beta 2-selectivity of BDTI (1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline HBr) were investigated in canine trachea and rat heart by radioligand binding assay and pharmacological experiments in comparison with those of other beta-adrenoceptor agonists, salbutamol and isoprenaline. The potency of relaxing effect on carbachol-induced contraction in isolated canine trachea was in the order of isoprenaline (pD2 = 6.70 +/- 0.08) > BDTI (6.11 +/- 0.06) approximately salbutamol (6.14 +/- 0.08). ICI-118,551 (a selective beta 2-antagonist) and atenolol (a selective beta 1-antagonist) inhibited the relaxant action of BDTI with pKB values of 8.4 and 5.3, respectively, corresponding to high affinity for ICI-118,551 and low affinity for atenolol in antagonizing this response. The Kd values of radioligand ([3H]-CGP12177) were 453.3 +/- 30.8 and 563.4 +/- 96.7 pmol/l in cultured canine tracheal smooth muscle cells (TSMCs) and rat cardiomyocytes, respectively, and the Bmax values were 64.6 +/- 10.7 and 245.7 +/- 44.5 fmol/mg protein, respectively. BDTI, salbutamol and isoprenaline inhibited the binding of [3H]-CGP12177 in a concentration-dependent manner in cultured canine TSMCs (Ki 0.73 +/- 0.15, 0.75 +/- 0.21 and 0.24 +/- 0.05 mumol/l, respectively) and rat cardiomyocytes (Ki 2.76 +/- 0.36, 2.31 +/- 0.26 and 0.22 +/- 0.03 mumol/l, respectively). These results demonstrated that BDTI possessed moderate selectivity (3.8-fold) to beta 2-adrenoceptors as judged from the Ki (heart)/Ki (trachea) value (salbutamol 3.1-fold, isoprenaline 0.92-fold). BDTI and salbutamol also stimulated cAMP formation in a concentration-dependent manner in cultured canine TSMCs (EC50 0.5 +/- 0.2 and 0.4 +/- 0.1 mumol/l, respectively) and rat cardiomyocytes (EC50 6.2 +/- 0.5 and 5.7 +/- 0.6 mumol/l, respectively). The selectivity of BDTI and salbutamol for beta 2-adrenoceptors on the cAMP response were 12.4 and 14.3 times, respectively. It is concluded that BDTI is a beta 2-selective adrenoceptor agonist.

Synthesis and thromboxane A2 antagonistic activity of [[1-aryl(or benzyl)-1-(benzenesulfonamido)methyl]phenyl]alkanoic acid derivatives.

In order to find new antiasthmatic and antithrombotic agents, various [[1-aryl(or benzyl)-1-(benzenesulfonamido)methyl]phenyl]alkanoic acid derivatives were synthesized. Evaluation of these compounds for thromboxane A2 (TXA2) antagonistic activities indicated that 4-[4-[(4-chlorobenzenesulfonamido)phenylmethyl]phenyl]butyric acid (6h) ,4-[4-[1-(4-chlorobenzenesulfonamido)-2-phenylethyl]phenyl]butyric acid (6y) and many other compounds have potent inhibitory effects on U-46619-induced guinea-pig platelet aggregation. No significant difference in the inhibitory effect between (+)-6h and its antipode could be detected, although (+)-6h and its antipode could be detected, although (+)-6y was about 10 times more potent than (-)6y. The pKb values of 6h and 6y were estimated to be 8.9 and 10, respectively on U-46619-induced contraction of guinea-pig trachea as a pharmacological measure of TXA2 antagonistic activity. These compounds also showed potent inhibitory effects on U-46619-induced bronchoconstriction in guinea-pig after oral administration in vivo. They were also evaluated for other related pharmacological effects involving the arachidonic acid cascade. It was found that these compounds possess TXA2 synthase inhibitory activity together with TXA2 antagonistic activity, and 6h also possesses weak leukotriene D4 (LTD4) antagonistic activity. Structure-activity relationships for TXA2 antagonistic activity of these derivatives are discussed.

Design and synthesis of new linear and cyclic bradykinin antagonists.

We report here on the synthesis and pharmacological properties of a new series of small linear and cyclic peptides derived from the five C-terminal amino acid residues of second-generation bradykinin receptor antagonists. Variations of the two first residues of the pentapeptide (Thi-Ser-D-Tic-Oic-Arg) were shown to modulate the biological activities of the analogs on bradykinin-induced smooth muscle contractions in rabbit jugular vein (RJV), a tissue preparation specific of the B2 bradykinin receptor. Several analogs showed pA2 values around 7 on this tissue preparation, and one cyclic compound, c[-Gly-Thi-D-Tic-Oic-Arg-], 24, in which Thi-Ser was replaced by Gly-Thi, displayed a pA2 of 7.4 on RJV. On the basis of these results, three cyclic molecules and their linear counterparts (compounds 22-24 and 4-6, respectively) were tested on human umbilical vein, a tissue specific of the human B2 receptor. The pKB values obtained for these compounds on these tissue preparations were equivalent to those obtained for the decapeptide NPC 567 (4.8 < pA2 < 5.1). NMR and molecular modeling studies performed on compound 24 clearly demonstrated a type II' beta-turn structure. This analog may serve as a new lead for the design of nonpeptide ligands of the bradykinin B2 receptor subtype.

α1B-Adrenoceptor Subtype Mediating the Phenylephrine-Induced Contractile Response in Rabbit Corpus Cavernosum Penis

The α1-adrenoceptor subtype mediating contraction to phenylephrine in rabbit corpus cavernosum penis (CCP) was investigated using selective α1-adrenoceptor subtype antagonists. WB4101 ((2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane) hydrochloride), 5-methylurapidil and tamsulosin concentration-dependently produced a parallel rightward shift of the concentration-response curve to phenylephrine, yielding pKB values of 8.05, 7.59 and 9.21, respectively. The slopes of the Schild plots were not different from unity. These antagonists did not affect the maximum response to phenylephrine. Oxymetazoline (1 μM), which initially caused a small contraction, produced a parallel rightward shift of the concentration-response curve to phenylephrine with an apparent pKB value of 6.99. However, oxymetazoline seemed to act as a non-surmountable antagonist to the phenylephrine-induced contraction, reducing the maximum response by 71.1 %. Chloroethylclonidine (25 and 100 μM) produced a parallel rightward shift of the concentration-response curve to phenylephrine without altering the maximum response. These results show that the α1-adrenoceptor in rabbit CCP has a relatively low affinity for WB4101, 5-methylurapidil, tamsulosin and oxymetazoline and is sensitive to inactivation by chloroethylclonidine. It is suggested that the α1-adrenoceptor subtype mediating contraction to phenylephrine in rabbit CCP has the characteristics of the α1B-adrenoceptor subtype.

Electromechanical effects of acetylcholine on the atrial tissues of the cultured tilapia (Oreochromis nilotica � O. aureus)

The effects of acetylcholine (ACh) on the action potential and twitch force of atrial tissues isolated from 15 tilapia (Oreochromis nilotica × O. aureus) were studied by means of conventional microelectrode techniques. In isolated whole atrium or sinoatrial tissue, scattered pacemaker-like cells with spontaneous diastolic depolarization were found mainly near the sinoatrial junction but also occasionally throughout the atrial wall. However, most of the atrial cells recorded were myocardial fibers as judged by a stable diastolic potential and a markedly reduced action potential duration (APD) in response to low concentrations of ACh (0.1-1 μM). The shortening in APD in atrial myocardial fibers was correlated with a significant fall in twitch force in the atrial preparations. ACh at high concentrations (10-300 μM) decreased moderately the APD and the slope of diastolic depolarization of the pacemakers and prolonged the spontaneous cycle length but did not induce hyperpolarization. The negative chronotropic action of ACh was competitively inhibited by atropine, a muscarinic antagonist. The means (± SEM) negative logarithm of the dissociation constant (pKb or pA2 value) for atropine against the ACh action on muscarinic receptors were 9.10 (± 0.13) (n = 6), similar to those values obtained in mammalian atria. The present findings indicate that while the negative inotropic effects of ACh in tilapia atria are comparable to those observed in mammalian hearts, unique electrophysiological responses to ACh exist in different types of tilapia atrial cells.

Competitive and selective antagonistic action of NPC 17731 on bradykinin-induced relaxant and contractile responses of the guinea pig isolated ileum

1. 1. The aim of the present study was to evaluate, by means of functional studies, the effect of the newly developed bradykinin (BK) receptor antagonist NPC 17731, D-Arg 0, Arg 1-Pro 2-Hyp 3-Gly 4-Phe 5-Ser 6-[DHype (transpropyl) 7]-Oic 8-Arg 9, against BK-mediated biphasic response in the guinea pig ileum “in vitro” (circular and longitudinal layers). 2. 2. In the circular muscle, NPC 17731 (0.1–1000 nM) dose dependently and reversibly antagonized, with similar potency, both the contractile and relaxant responses caused by BK, as well as BK-induced contraction in longitudinal smooth muscle with IC 50 of 23, 29 and 37 nM, respectively. 3. 3. NPC 17731 (10–300 nM) also caused a concentration-dependent displacement to the right of BK-induced contraction and relaxant responses in the circular smooth muscle of guinea pig ileum, without changing BK maximal response. Schild plot analyses were linear (correlation close to 1), yielding pKb values of 8.89 ± 0.19 and 8.73 ± 0.18, respectively, and slopes not significantly different from unity, providing evidence that NPC 17731 acts as a pure B 2 competitive receptor antagonist against both BK responses. 4. 4. Similarly, NPC 17731 (3-100 nM) antagonized, in a graded and competitive manner, BK-induced contraction in longitudinal muscle from guinea pig ileum with a slope not different from unity, yielding a pKb value of 8.62 ± 0.13. 5. 5. These results indicate that the new B 2 BK receptor antagonist NPC 17731 antagonizes, with high affinity and with similar potency through simple competitive and selective mechanisms, BK receptor-mediating contraction or relaxant responses in circular and longitudinal smooth muscles from guinea pig ileum. In addition, these results also suggest that although BK-induced contraction or relaxation responses in guinea pig ileum are coupled to distinct second messengers, NPC 17731 interacts with a homogeneous population of B 2 receptors in both guinea pig ileum preparations.

Pharmacological evidence for a single bradykinin B2 receptor in the guinea‐pig

1. The present study addresses the possibility of the existence of different kinin B2 receptor subtypes in the guinea-pig by evaluating the affinity of peptide and nonpeptide receptor antagonists. For this purpose, jugular vein rings, ileum segments, lung parenchymal and trachea strips were set up in organ baths for isometric tension measurements. The experiments were conducted in the presence o indomethacin (3 microM), atropine (10 microM) and captopril (10 microM). 2. BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50 of 13.2 +/- 1.4 nM (n=27), 11.2 +/- 2.1 (n=26), 23.6 +/- 6.3 (n=26), and 33.0 +/- 6.5 (n=27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.34.11) inhibitor and MERGETPA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a carboxypeptidase inhibitor, had no effect on the BK-induced contractions of JV, GPI and LP. In the GPT, thiorpan potentiated the contractile response to BK and was thus added in the corresponding experiments. 3. The peptide B2 receptor antagonist, Hoe 140 and the nonpeptide compound, WIN 64338, behaved as noncompetitive antagonists against contractile responses to cumulative BK in the four tissues although Hoe 140 appeared as a competitive inhibitor in the GPT only. IN order to compare the inhibitory potency of these compounds between tissues, pKB values were determined. Mean values of pKB for Hoe 140 were 8.05 +/- 0.07, 8.43 +/- 0.11, 8.13 +/- 0.18, and 8.52 +/- 0. 25 in the JV, GPI, GPT and LP, respectively. WIN 64338 gave mean pKB values of 6.89 +/- 0.10, 7.57 +/- 0.12, 7.36 +/- 0.12 adn 7.51 +/- 0.28 in the JV, GPI, LP and GPT, respectively. 4. D-Arg [Hyp3, D-Phe7, Leu8]BK and D-Arg[Hyp3, D-Phe7]BK (NPC 567) inhibited in a competitive fashion the concentration-response curves to BK. Values of pA2for each compound were not significantly different in the four tissues and were between 5.81 and 6.31 for D-Arg [Hyp3, D-Phe7, Leu8]BK and between 5.55 and 5.65 for NPC 567.

Characterization of the thromboxane (TP‐) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat

1. The effects of the thromboxane A2 (TxA2)-mimetic, U-46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta. 2. [3H]-U-46619 bound to A10 cells of passages 18-20 (p18-20) with two classes of sites. The high affinity site showed a Bmax of 3.0 +/- 1.8 fmol mg-1 protein with a KD value 1.0 +/- 0.1 nM, while the low affinity site showed a Bmax of 43.0 +/- 6.0 fmol mg-1 protein and KD value of 129.0 +/- 7.9 nM. However, [3H]-U-46619 bound to A10 cells from passages 28-30 (p28-30) at a single class of site with a Bmax 111.0 +/- 9.0 fmol mg-1 protein and a KD value of 175.4 +/- 22.0 nM. 3. Cinnamophilin and SQ29548 inhibited specific [3H]-U-46619 binding to p18-20 A10 cells in a concentration-dependent manner with Ki values of 390.0 +/- 3.2 and 4.6 +/- 1.0 nM, respectively at a high affinity site, and 2.6 +/- 0.2 microM and 310.0 +/- 6.4 nM, respectively at the low affinity site. 4. U-46619 produced isometric contractions of rat aorta in a concentration-dependent manner with an EC50 7.0 +/- 1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA2 values 6.3 +/- 0.1 and 8.2 +/- 0.2, respectively. 5. U-46619 increased [3H]-thymidine incorporation into DNA of p18-20 and p28-30 A10 cells in aconcentration-dependent manner with EC50 values 362.7 +/- 27.0 and 302.5 +/- 20.1 nm, respectively. The U-46619-induced increase of [3H]-thymidine incorporation into DNA of p28 -30 AO0 cells was potentiatedby PDGF (1 ng ml-1) and FCS (1%) and was inhibited by cinnamophilin (10 microM) and SQ29548 (1 microM)with estimated pKB values 5.4 +/- 1.2 and 6.3 +/- 0.9, respectively.6. Cell cycle analysis revealed that U-46619-increased cell cycle progression was primarily due to a rapidtransition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, U-46619 also increasedprotein synthesis and cell numbers in VSMC. All these effects of U-46619 were inhibited bycinnamophilin and SQ29548.7. U-46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration inVSMC, effects which were blocked by cinnamophilin and SQ29548.8 These data indicate there are two U-46619 binding sites in AlO VSMC. The high affinity site is correlated to U-46619-induced vasoconstriction while the low affinity site is correlated to U-46619-mediated VSMC proliferation. These data also reveal that U-46619 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G2/M. Since cinnamophilin inhibits TPreceptor-mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.