Rank Order Of Inhibitory Potency Research Articles

Ionic requirements for the specific binding of [3H]GBR 12783 to a site associated with the dopamine uptake carrier.

At 0 degrees C, when Na+ was the only cation present in the incubation medium, increasing the Na+ concentration from 3 to 10 mM enhanced the affinity of [3H]1-[2-(++di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperaz ine [( 3H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the Bmax. For higher Na+ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na+. In a 10 mM Na+ medium, the KD and the Bmax were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [3H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na+. A similar Na+ dependence was observed at 20 degrees C. Scatchard plots indicated that K+, Ca2+, Mg2+, and Tris+ acted like competitive inhibitors of the specific binding of [3H]GBR 12783. The inhibitory potency of various cations (K+, Ca2+, Mg2+, Tris+, Li+, and choline) was enhanced when the Na+ concentration was decreased from 130 to 10 mM. In a 10 mM Na+ medium, the rank order of inhibitory potency was Ca2+ (0.13 mM) greater than Mg2+ greater than Tris+ greater than K+ (15 mM). The requirement for Na+ was rather specific, because none of the other cations acted as a substitute for Na+. No anionic requirement was found: Cl-, Br-, and F- were equipotent. These results suggest that low Na+ concentrations are required for maximal binding; higher Na+ concentrations protect the specific binding site against the inhibitory effect of other cations.

Effect of chlorpromazine on sympathetic neuroeffector transmission in the rabbit isolated pulmonary artery and aorta.

1. The effects of chlorpromazine on sympathetic neuroeffector transmission have been studied in the rabbit isolated pulmonary artery and aorta. 2. Chlorpromazine (10(-8)-10(-5) M), prazosin (10(-9)-10(-7) M) and phentolamine (3 x 10(-8)-3 x 10(-5) M) decreased the contractions of pulmonary artery evoked by electrical field stimulation (150 pulses; 3 Hz). The rank order of inhibitory potency (ID50) was prazosin greater than chlorpromazine greater than phentolamine. 3. Rauwolscine (3 x 10(-9) M-4 x 10(-6) M) enhanced the neurogenic response by up to 201%. However, higher concentrations (6 x 10(-6)-3 x 10(-5) M) reduced the contractions evoked by transmural stimulation. 4. The inhibitory effect of prazosin (10(-6) M) was reversible, while that of chlorpromazine (10(-8) M) was not. 5. Chlorpromazine (10(-8)-10(-4) M), desmethylimipramine (3 x 10(-9)-10(-5) M), cocaine (10(-7)-3 x 10(-4) M) and phentolamine (10(-5)-3 x 10(-4) M) reduced the accumulation of [3H]-noradrenaline ([3H]-NA, 10(-8) M) by aorta. The rank order of inhibitory potency (ID50) was: desmethylimipramine greater than chlorpromazine greater than cocaine greater than phentolamine. Prazosin (10(-7)-10(-5) M) and rauwolscine (10(-8)-10(-4) M) did not reduce [3H]-NA accumulation. 6. Chlorpromazine (10(-8)-10(-6) M) and prazosin (3 x 10(-9)-10(-7) M) antagonized the contractions of aorta evoked by exogenous noradrenaline (10(-9)-3 x 10(-4) M) and phenylephrine (10(-9)-3 x 10(-3) M). The pA2 values for chlorpromazine on the alpha 1-adrenoceptors were 8.24 (noradrenaline) and 8.27 (phenylephrine). The corresponding values for prazosin were 8.64 and 8.57, respectively. 7. It is concluded that chlorpromazine and prazosin are potent inhibitors of postsynaptic alpha 1-adrenoceptors. Chlorpromazine and phentolamine, unlike prazosin and rauwolscine, are also inhibitors of Uptake.

Characterization of synaptosomal dopamine uptake in post-mortem brain regions of schizophrenics.

The drug sensitivity of synaptosomal high-affinity dopamine (DA) uptake was investigated in post-mortem brain regions of schizophrenics, in comparison to controls matched for age, sex, and post-mortem delay, and in model experiments in rats. DA uptake was inhibited by nomifensine in the investigated regions of rat brain in a concentration-dependent manner; the regional rank order of inhibitory potency was: nucleus (n) caudatus greater than n. accumbens greater than frontal cortex. Furthermore, it was shown that the inhibitory potency of nomifensine is unchanged after in situ storage of rat brain tissue for 48 h and after the cryopreservation method used. In post-mortem brain of human controls, nomifensine inhibited DA uptake with the same regional differences as in rats; however, the inhibitory potencies were three-fourfold weaker. In schizophrenia, on the other hand, synaptosomal DA uptake inhibition by nomifensine was significantly weaker than in the corresponding control brains for all regions studied. This suggests a decreased affinity of the DA uptake carrier to nomifensine, similar to DA shown in recent studies with schizophrenic patients. The possible relevance of investigating functional parameters for understanding patho-biochemical mechanisms in schizophrenia is discussed.

ChemInform Abstract: Comparative Antiaggregatory Activity in Human Platelets of a Benzopyranone aci‐Reductone, Clofibric Acid, and a 2,3‐Dihydrobenzofuran Analogue.

A synthetic method for the preparation of aci-reductone 6-chloro-3,4-dihydroxy-2H-1-benzopyran-2-one (3) from 5-chlorosalicylate is presented. In human platelets, the benzopyranone derivative 3, clofibric acid (1), and the 2,3-dihydrobenzofuran analogue 4 inhibited aggregation and serotonin secretory responses to adenosine diphosphate (ADP) with a rank order of potency 3 greater than or equal to 4 greater than 1. Only analogues 3 and 4 consistently blocked the aggregatory responses (greater than 50%) to arachidonic acid (AA) and U46619, a thromboxane A2 agonist. Further, the rank order of inhibitory potency against U46619-induced serotonin secretion was 4 greater than 3 greater than 1. Benzopyranone 3 is of interest since it was the most potent inhibitor of thrombin-induced [3H]AA release (3 much greater than 4 = 1) and more potent than 1 or 4 for the blockade of the ADP- or AA-mediated pathway of platelet aggregation.

The effect of benzodiazepines and β-carbolines on gabastimulated chloride influx by membrane vesicles from the rat cerebral cortex

Benzodiazepine agonists such as diazepam, flunitrazepam and clonazepam enhanced GABA (30 microM)-stimulated 36Cl- uptake in membrane vesicles from the rat cerebral cortex. The rank order of potencies was flunitrazepam greater than diazepam = clonazepam. beta-Carboline-3-carboxylate esters beta-CCM, beta-CCE and DMCM inhibited GABA-stimulated 36Cl- uptake. The rank order of inhibitory potencies was DMCM greater than beta-CCM greater than beta-CCE. The benzodiazepine antagonist Ro15-1788 antagonized the enhancement of flunitrazepam and the inhibition of DMCM on GABA-stimulated 36Cl- uptake in a competitive inhibitory manner. These results suggest that benzodiazepine receptors regulate GABA-stimulated 36Cl- uptake and there is a functional coupling between the GABA and benzodiazepine receptors, and chloride channels in membrane vesicles from the rat cerebral cortex.

Alpha-adrenoceptor-mediated actions of optical isomers and desoxy analogs of catecholimidazoline and norepinephrine in human platelets: In vitro

Adrenoceptor-mediated effects of the enantiomers of optically active imidazoline, 2-(3,4,α-trihydroxybenzyl imidazoline (catecholimidazoline; CI), and norepinephrine (NE), and the corresponding desoxy derivatives, 2-(3,4-dihydroxybenzyl)imidazoline (desoxy-CI) and dopamine, have been investigated in human platelets. Differences between responsiveness of platelets from donor to donor were observed in the presence of the isomers and the desoxy analogs of NE and CI. In certain platelet preparations, all compounds gave concentration-dependent stimulatory responses, whereas in other preparations, only R(−)-NE and R(−)-CI were inducers of platelet aggregation and serotonin release. The rank order of stimulatory potencies ( ec 50; μM) for CI and NE was R(−)-NE (1.3) > R(−)-CI (7.5) > S(+)-NE (19) = S (+)-CI (20) = dopamine (22) > desoxy-CI (>35). Unlike R(−)-CI, both S(+)-CI and desoxy-CI were either agonists or antagonists of human platelet function. In preparations unresponsive to the S(+)-isomers or desoxy analogs, the potencies (EC 50) for R(-)-NE and R(−)-CI were 1.7 and 7.7 μM respectively. The corresponding inactive CIs [ S(+)-CI and desoxy-CI] were inhibitors of both primary and secondary phases of aggregation and serotonin release responses to R(−)-CI and R(−)-NE, respectively. In contrast, the aggregation responses to ADP, arachidonic acid or U46619 were not blocked by S(+)-CI or desoxy CI. The rank order of inhibitory potencies for selected α-adrenoceptor agents against R(−)-NE was phentolamine > clonidine > desoxy-CI > S(+)-CI. Moreover, the relative inhibitory potencies of phentolamine and desoxy-CI against aggregation responses to R(−)-NE and R(−)-CI, respectively, were the same. These results suggest that (1) the enantiomers and desoxy derivatives of CI and NE mediate their effects in human platelets by an interaction with α-adrenoceptors; (2) catecholamines and imidazolines interact with the same α-adrenoceptors in human platelets; (3) the stereochemical requirements of both chemical classes for stimulatory activity in human platelets adhere to the Easson-Stedman hypothesis in this α 2-adrenoceptor system; and (4) desoxy-CI possessed the highest potency as an antagonist of α-adrenoceptors which suggests that the hydroxy group at the benzylic carbon atom of these imidazolines may not be required for maximal binding to adrenoceptors in platelets.

Cholinergic inhibition of follicle-stimulating hormone-induced progestin production by cultured rat granulosa cells.

The influence of cholinomimetics on follicle-stimulating hormone (FSH)-induced progestin production was studied in a primary culture of rat granulosa cells. Cells were cultured for 2 days with FSH and delta 4-androstenedione in the presence or absence of increasing concentrations of cholinergic agonists. Although ineffective as stimulators of steroidogenesis by themselves, the three nicotinic receptor-selective agonists lobeline, dimethylphenylpiperazinium iodide (DMPP), and phenyltrimethylammonium iodide (PTMA) inhibited FSH-induced progesterone and 20 alpha-hydroxypregn-4-en-3-one production in dose-dependent fashions. The rank order of inhibitory potencies was lobeline greater than DMPP greater than PTMA with IC50 values of 2 X 10(-6) M, 3 X 10(-5) M, and 3 X 10(-4) M, respectively. In contrast, the muscarinic receptor-selective agonists muscarine and bethanechol failed to inhibit steroid production. The inhibitory effect of lobeline on the time course of FSH-induced induced steroid production indicated an immediate inhibitory action; however, this inhibition was readily reversed upon removal of the drug. Further studies demonstrated that the FSH-stimulated increase in intracellular cAMP levels, as well as progesterone production induced by cholera toxin and forskolin (agents that stimulate cAMP production) and by dibutyryl cAMP (a cAMP analog), were also suppressed by lobeline. The present observations indicate that nicotinic, but not muscarinic, cholinergic agonists inhibit progesterone biosynthesis in cultured granulosa cells and suggest that endogenous acetylcholine may play a modulatory role in ovarian steroidogenesis.

Characterization of "high-affinity" [3H]ouabain binding in the rat central nervous system.

The characteristics of [3H]ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. [3H]Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific [3H]ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for [3H]ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H]ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H]ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the "high-affinity" [3H]ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of [3H]ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.

Antagonism of prostaglandin-mediated responses in platelets and vascular smooth muscle by 13-azaprostanoic acid analogs: Evidence for selective blockade of thromboxane A 2 responses

Studies were undertaken to examine the pharmacological properties and stereochemical requirements of a limited series of prostanoic acid analogs for inhibition of arachidonic acid (AA) and/ or endoperoxide (U46619)-mediated responses in human platelets and rat aorta. To assess the role of stereochemistry, a set of trans- and cis-isomers of 13-azaprostanoic acid (APA) and 11a-homo-13-azaprostanoic acid (HAPA) were prepared. Each prostanoic acid analog blocked AA- or U46619-induced aggregatory and secretory responses in platelets, and U46619-mediated contractions of rat aorta in a concentration-dependent manner (0.1 to 100 μM). The azaprostanoic acid analogs blocked responses to both inducers of platelet activation with ic 50 values ranging from 3.4 to 27.5μM. Trans-APA was about 2- to 3-fold more active as an antagonist of serotonin release induced by AA or U46619 than the remaining analogs. The rank order of inhibitory potency ( ic 50; μM) for these analogs against U46619-induced serotonin release in human platelets was trans-APA (3.4) > cis-APA (8.9) = cis-HAPA (8.7) = trans-HAPA (9.1). Concentrations of the prostanoic acid analogs required to block these responses to AA and U46619 were similar, and the highest concentration used (100 μM) did not modify AA-induced malondialdehyde production in human platelet preparations. In contrast, the isomers of APA and HAPA were equally active as antagonists of U46619-indueed contractions of rat vascular tissue, possessing K B values varying from 7.1 to 13.2 μM. Each azaprostanoic acid analog shifted the concentration-response curve of U46619 in rat aorta to the right, indicating a competitive-type inhibition. In addition, the azoprostanoic acid analog (U51605) was a more potent competitive antagonist of U46619 in this preparation and possessed an average p K B value of 6.18. In summary, the results show that (1) expansion of the five-membered ring of APA to the six-membered ring analogs (HAPA) led to a retention of potent inhibitory activity against U46619 in human platelets and rat vascular smooth muscle, (2) the antiaggregatory and antisecretory actions of the azaprostanoic acid analogs were mediated by a blockade of the responses to AA and U46619, and not by an inhibition of AA metabolism, (3) the blocking activity for the APA isomers was stereoselective ( trans > cis) whereas the isomers of HAPA were equally effective as inhibitors of platelet function; and (4) these azaprostanoic acid analogs act as selective endoperoxide (U46619)/thromboxane A 2 antagonists in these two tissues.

Characterization of the adenosine receptor responsible for the inhibition of histamine and SRS-A release from human lung fragments.

The inhibitory effects of a range of natural and synthetic derivatives of adenosine on the antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung has been studied. The nucleotides ATP, ADP and AMP appear to act by being converted to adenosine. The rank order of inhibitory potency of the synthetic analogues indicates that these compounds act at an extracellular A2/Ra purinoceptor. The xanthines, 1, 3-diethyl-8-phenylxanthine, 8-phenyltheophylline and theophylline antagonized the inhibitory action of N-ethyl-carboxamideadenosine competitively. Theobromine was inactive. This supports the view that the inhibitory receptor is of the A/R type. Hexobendine and dipyridamole, reported to antagonize the uptake of adenosine, failed to modify the response of human lung fragments to adenosine. The P site agonist 2',5' dideoxyadenosine inhibited the release of histamine and SRS-A. This effect was not prevented by the inhibitors of uptake, hexobendine and dipyridamole, nor was it antagonized by 8-phenyltheophylline.

Single cell activity in the noradrenergic A-5 region: Responses to drugs and peripheral manipulations of blood pressure

Single unit recordings were obtained from the region of the A-5 noradrenergic cell group in the ventrolateral tegmentum at the level of the exiting VIIth nerve. In this region a distinct cell population was identified which exhibited moderate spontaneous rates of activity, a regular firing pattern, wide action potentials, and a characteristic response to sensory stimulation consisting of a brief excitation followed by a more prolonged period of inhibition. Simultaneous visualization of recording sites and catecholamine cells by fluorescence histochemistry indicated a close association between the location of the recorded units and the A-5 noradrenergic cell group. Antidromic responses could be elicited by stimulation of the spinal cord and forebrain indicating that A-5 cells project both anteriorly and posteriorly. The spontaneous activity of A-5 cells was invariably suppressed by low doses of clonidine and l-amphetamine, while adjacent cells showed varying responses to these agents. The effects of clonidine and l-amphetamine could be reversed by low doses of the α 2-adrenoceptor antagonist, piperoxane. When applied by microiontophoresis, clonidine and norepinephrine were effective in inhibiting A-5 neurons. The rank order of inhibitory potencies for agonists applied microiontophoretically to A-5 neurons was characteristic of an α 2-receptor (i.e. clonidine > norepinephrine > phenylephrine). Thus, the physiological and pharmacological properties of the A-5 neurons closely matched those previously reported for noradrenergic neurons elsewhere. Sixty percent of A-5 neurons responded to peripherally induced changes in blood pressure with reciprocal changes in spontaneous firing rate. Increases in blood pressure led to decreases in spontaneous activity while decreases in pressure led to the opposite response. These observations are consistent with the proposed vasomotor role for the A-5 noradrenergic cell group.

Differential effects of various phosphodiesterase inhibitors, pyrimidine and purine compounds, and inorganic phosphates on cyclic CMP, cyclic AMP and cyclic GMP phosphodiesterases

The effects of various compounds on homogeneous cyclic CMP phosphodiesterase (cyclic CMP-PDE) from pig liver were compared with the effects on cyclic AMP phosphodiesterase (cyclic AMP-PDE) and cyclic GMP phosphodiesterase (cyclic GMP-PDE). Of the conventional inhibitors for AMP-PDE and cyclic GMP-PDE, only Sch 15280 was found to inhibit cyclic CMP-PDE. Nucleoside monophosphates, orthophosphate, and 2':3'-cyclic nucleotides were rather specific and were more effective in inhibiting cyclic CMP-PDE, compared to their effects on cyclic AMP-PDE and cyclic GMP-PDE. On the other hand, nucleoside di- and triphosphates and pyrophosphate (PP i) were less effective in inhibiting cyclic CMP-PDE and were without marked effect on cyclic AMP-PDE and cyclic GMP-PDE. Orthophosphate (P i) was more potent than CMP, CDP and CTP in inhibiting cyclic CMP-PDE, with a rank order of inhibitory potency of P i > CMP > CDP > CTP. Of the 3' :5'-cyclic nucleotides examined, cyclic UMP was more specific in inhibiting cyclic CMP-PDE compared to its effect on cyclic AMP-PDE and cyclic GMP-PDE. In all experiments similar results were obtained when either cyclic CMP or cyclic AMP was used as a substrate for this multifunctional cyclic CMP-PDE, supporting the contention that a single catalytic site on the enzyme is responsible for the hydrolysis of both cyclic CMP and cyclic AMP. The present studies further support our original suggestion that cyclic CMP-PDE is a unique enzyme that is distinguishable from the conventional enzymes for purine cyclic nucleotides.

Release of Slow-Reacting Substance from Anaphylactic Lung Tissue and Its Modification by β-sympathomimetics

Anaphylactic release of slow-reacting substance of anaphylaxis (SRS-A) from chopped guinea pig lung was studied. The antibodies mediating the anaphylactic reaction were classified as IgG antibodies by PCA technique. Isoprenaline was found to inhibit the release of SRS-A in the concentration range 10-8-10-6M, while the beta2-selective agonist terbutaline showed inhibition in the concentration range 10-6-10-5M. The beta1-selective beta-agonist tazolol was ineffective when tested in the concentration range 10-7-10-5M. The rank order of inhibitory potency of the compounds on SRS-A release agree with that for histamine release.

Inhibition of lymphocyte proliferation stimulated by lectins and allogeneic cells by normal plasma lipoproteins.

Lipoproteins, isolated by sequential flotation at densities 1.006, 1.019, 1.063, and 1.21, were examined for their ability to inhibit human lymphocytes stimulated by allogeneic cells and by lectins (phytohemagglutinin-P and concanavalin A). All the classes of normal plasma lipoproteins inhibited lymphoproliferation when peripheral blood lymphocytes were cultured in autologous, heterologous, or lipoprotein-deficient plasma (d greater than 1.21). The rank order of inhibitory potency was intermediate density lipoprotein (IDL) greater than very low density lipoproteins (VLDL) greater than low density lipoproteins (LDL) greater than high density lipoproteins (HDL), regardless of the mode of stimulation. The concentrations of IDL, VLDL, and LDL required for complete inhibition of stimulated lymphoproliferation were considerably below the levels of each of these lipoproteins normally found in human plasma. In addition, the concentration of HDL required for 50-90% inhibition was in the range of HDL levels normally found in human plasma. Moreover, at relatively higher concentrations, lipoproteins suppressed the incorporation of [3H]thymidine into DNA below the levels seen with reseting, unstimulated lymphocytes. The results suggest that circulating lymphocytes may normally be highly suppressed by the combined effects of all the endogenous lipoproteins and that the lipoproteins may play important roles in vivo in modulating lymphocyte functions and responses.