Interaction of subtype-selective antagonists with alpha 1-adrenergic receptor-mediated second messenger responses in rat brain.

The selective antagonists (+)-niguldipine and 5-methylurapidil (5-MU) were used to more clearly identify the alpha 1-adrenergic receptor subtypes involved in second messenger responses in slice and culture preparations of rat brain. The alpha 1-adrenergic receptor activating [3H]inositol phosphate (InsP) formation in neocortical and hippocampal slices appeared to have mixed characteristics. Although the low potency of (+)-niguldipine indicated involvement of the alpha 1B subtype, 5-MU had an alpha 1A-like potency at this subtype. (+)-Niguldipine did not inhibit the alpha 1 receptor-mediated potentiation of the cAMP response to either isoproterenol or adenosine in cortical slices, even at high concentrations. 5-MU inhibited both cAMP responses, although this inhibition appeared non-competitive. Thus, these receptors are clearly different from those mediating InsP formation. In primary glial cultures, (+)-niguldipine also had a low potency in blocking norepinephrine-stimulated [3H]InsP formation, consistent with involvement of the alpha 1B subtype. However, both 5-MU and WB 4101 had high potencies in blocking this response, suggesting involvement of the alpha 1A subtype. Inactivation of the alpha 1B subtype by pretreatment of cultures with chloroethylclonidine did not increase the potencies of any of these antagonists. The inhibition by 5-MU and WB 4101 was competitive in both control and chloroethylclonidine-pretreated cultures, whereas the inhibition by (+)-niguldipine was primarily noncompetitive. The use of these more selective antagonists shows that the current alpha 1A/alpha 1B subclassification scheme is inadequate to identify the receptors mediating these responses. None of the responses were blocked by (+)-niguldipine with the high potency expected at the alpha 1A subtype, although all InsP responses were blocked by 5-MU with a relatively high (alpha 1A-like) potency. In addition, very low affinity and noncompetitive effects of (+)-niguldipine were observed. These data raise the possibility of additional subtypes of alpha 1-adrenergic receptors or as yet unidentified functional interactions between known subtypes.     

Interaction between antidepressants and alpha 1-adrenergic receptor antagonists on the binding to alpha 1-acid glycoprotein

alpha 1-Receptor antagonists and antidepressant agents are basic (cationic) drugs that are known to bind to alpha 1-acid glycoprotein (AAG). Since these drugs are frequently co-administered and since they bind to the same protein, this investigation was designed to evaluate the "in vitro" ability of antidepressants, alpha 1-receptor antagonists, and propranolol to displace [3H]imipramine and [3H]prazosin from the AAG binding site(s). Equilibrium dialysis was employed. Of the drugs studied, the following order of potency in displacing [3H]prazosin was found: trazodone greater than prazosin greater than doxazosin greater than propranolol greater than doxepin = amoxapine = trimazosin = amitriptyline greater than imipramine greater than nortriptyline = desipramine = nomifensine greater than bupropion = maprotiline. [3H]lmipramine binding from AAG was displaced with the following potency order: prazosin greater than imipramine greater than propranolol greater than doxazosin greater than nortriptyline greater than desipramine greater than trimazosin. Tricyclic antidepressants produced similar degrees of displacement of both [3H]imipramine and [3H]prazosin from AAG; whereas, alpha 1-receptor antagonists were more effective displacers of [3H]prazosin than of [3H]imipramine. Furthermore, the demethylated metabolites of imipramine and amitriptyline were less potent displacers than their parent compounds. These results suggest that more than a single binding site may be available for binding to AAG and that hydrophobic bonding is important in the binding of drugs to AAG.     

Phenoxybenzamine is more potent in inactivating alpha 1- than alpha 2-adrenergic receptor binding sites

The effects of phenoxybenzamine on alpha 1- and alpha 2-adrenergic receptor binding sites were examined directly using radioligand binding assays of the antagonist drugs [125I]BE 2254 and [3H]rauwolscine, respectively, in homogenates of rat cerebral cortex. Treatment with phenoxybenzamine caused an irreversible, dose-dependent decrease in the density of both alpha 1- and alpha 2-adrenergic receptors. Phenoxybenzamine was approximately 250-fold more potent at decreasing alpha 1-adrenergic receptor density than it was at decreasing alpha 2-adrenergic receptor density.     

Solubilization of rat liver alpha 1-adrenergic receptors. Agonist specific alteration in receptor binding affinity

An improved method for the solubilization of the alpha 1-adrenergic receptors in rat liver, utilizing digitonin, glycerol and sonication, is described. The yield of solubilized receptors was approximately 20%. The soluble receptors showed characteristics similar to the membrane-bound alpha 1 receptors. However, upon solubilization, the affinity for the agonists (-)norepinephrine and (-)epinephrine increased 35- to 66-fold when compared to the affinity in the membranes. The affinity for antagonists remained unchanged. A number of synthetic partial agonists showed a less marked (5- to 10-fold) increase in affinity upon solubilization. These data are consistent with the notion that these receptors might be capable of existing in two distinct conformational states with the high affinity state for agonists being favored by solubilization.     

Neuroleptic drug interactions with norepinephrine alpha receptor binding sites in rat brain.

Alpha adrenergic receptor sites in mammalian brain tissue can be labeled by the binding of [³H]WB-4101 (2-([2′,6′-dimethoxy] phenoxyethylamino) methyl benzodioxan), a potent α-adrenergic antagonist. Numerous neuroleptic drugs of phenothiazine, butyrophenone and thioxanthene classes are potent in competing for [³H]WB-4101 binding, with affinities resembling those of classic α-antagonists such as phentolamine and phenoxybenzamine. The potencies of neuroleptics in competing for WB-4101 binding sites correlate closely with their potencies in antagonizing norepinephrine and epinephrine induced lethality in rats, confirming that affinity for WB-4101 binding sites predicts α-receptor antagonism in vivo. The relative affinities of neuroleptics for WB-4101 binding sites and for dopamine receptors as labeled by [³H]haloperidol provides an index of the relative propensities of these drugs for eliciting autonomic side effects such as orthostatic hypotension and sedation.     

Expression of three alpha 2-adrenergic receptor subtypes in rat tissues: implications for alpha 2 receptor classification

Based on biochemical and ligand binding studies in various tissues and species, evidence for several alpha 2-adrenergic receptor subtypes has accumulated. The current alpha 2-adrenergic receptor classification (alpha 2A, alpha 2B, alpha 2C) is based exclusively on pharmacological criteria. The molecular cloning of three distinct genes for human alpha 2-adrenergic receptors has confirmed the existence of multiple alpha 2-adrenergic receptor subtypes. According to their localization on different human chromosomes, the receptor genes were termed alpha 2-C10, alpha 2-C4, and alpha 2-C2. The relationship, however, between the pharmacologically characterized alpha 2-adrenergic receptors and the isolated genes has yet to be clarified. Using Northern blot hybridization, we analyzed the expression of the three cloned alpha 2-adrenergic receptor genes in 13 rat tissues, as well as in cell lines previously described as model systems for the pharmacologically defined alpha 2-adrenergic receptor subtypes. The alpha 2-C10 receptor corresponds to the alpha 2A subtype and is expressed in rat brainstem, cerebral cortex, hippocampus, pituitary gland, cerebellum, kidney, aorta, skeletal muscle, spleen, and lung. Messenger RNA coding for the alpha 2-C4 receptor was detected only in brain regions, not in peripheral tissues, whereas the alpha 2-C2 message was found only in liver and kidney. Hybridization experiments with RNA derived from tissues and cells from which the pharmacological alpha 2-receptor classification has been developed lead to the conclusion that the alpha 2B subtype represents two distinct receptor molecules, the alpha 2-C4 and a subtype previously undetected by classical ligand binding approaches. Furthermore, our results suggest that the alpha 2C subtype characterized in opossum kidney cells is an interspecies variation of alpha 2-C4 rather than a separate subtype. Finally, the cloned alpha 2-C2 receptor was found to be "alpha 2B-like" and not covered by the current pharmacological classification.     

Adrenomedullin receptor binding sites in rat brain and peripheral tissues

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.     

Relationship between alpha 2-adrenergic receptor binding sites and the functional receptors inhibiting norepinephrine release in rat cerebral cortex

The properties of alpha 2-adrenergic receptor binding sites and the receptors inhibiting [3H]norepinephrine (3H-NE) release from slices of cerebral cortex were compared. [3H]RX 781094, an alpha 2-adrenergic receptor antagonist radioligand, labeled a single class of binding sites in membranes at 37 degrees with the pharmacological specificity expected of alpha 2-adrenergic receptors. 5'-Guanylimidodiphosphate (Gpp(NH)p) and NaCl caused small increases in the potencies of antagonists at the 3H-RX binding sites but decreased the potencies of agonists 4-22-fold. Antagonists blocked the inhibition of potassium-evoked 3H-NE release caused by exogenous NE with potencies similar to those in competing for 3H-RX binding sites. Partial receptor inactivation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine whether there was a receptor reserve. EEDQ dose-dependently decreased both the density of 3H-RX binding sites and the maximal inhibition of 3H-NE release by different agonists. For most agonists, KA values calculated after the receptor inactivation did not differ significantly from EC50 values; however, for epinephrine a small receptor reserve was apparent. The proportion of 3H-RX binding sites lost was similar to the proportion of functional receptors lost after EEDQ treatment. The functional KA values for agonists correlated most closely with KD values for the low affinity binding state observed in the presence of Gpp(NH)p and NaCl. However, both epinephrine and NE still showed two-site binding curves under these conditions, and it was the high affinity subpopulation of sites observed in the presence of Gpp(NH)p and NaCl which resembled most closely the functional KA values. These data suggest that 3H-RX labels binding sites with properties similar to the alpha 2-adrenergic receptors inhibiting 3H-NE release in cerebral cortex. There is little or no receptor reserve for this effect, and there appears to be a binding state for the natural catecholamine agonists which has an affinity lower than that for mediating this functional response.     

The hepatic alpha 1-adrenergic receptor

Since the relatively recent advent of radioligand binding techniques, it has been possible to directly identify and characterize hepatic adrenergic receptors as well as study their physiological regulation. While it is now clear that alpha 1-adrenergic receptors constitute the major population of hepatic adrenergic receptors and are primarily responsible for the actions of catecholamines in liver, relatively little is known about the molecular mechanisms underlying alpha 1-responses. Recent results suggest that guanine nucleotides may be implicated in the transmission of the hormonal signal from the hepatic alpha 1-receptor to its effectors in a manner analogous to that described for adenylate cyclase-linked receptors. The lack of an easily measurable proximal membrane response for the alpha 1-receptor has been a severe handicap in our understanding of the mechanism of transmission of the hormonal signal. It is likely that until such a response is defined, alpha 1-adrenergic research will continue to lag behind research on the beta-adrenergic receptor.     

Antagonism of apomorphine-enhanced startle by alpha 1-adrenergic antagonists

The present study investigated the possible involvement of central noradrenergic neurons in mediating the excitatory effect of the dopamine agonist apomorphine on the acoustic startle response in rats. Experiment 1 assessed the effects of intraperitoneal (i.p.) administration of adrenergic antagonists on apomorphine-enhanced startle. The excitation of startle produced by apomorphine (1.0-3.0 mg/kg i.p.) was blocked by the alpha 1-adrenergic antagonists prazosin (0.03-1.0 mg/kg) and WB-4101 (1.0 mg/kg). Prazosin was very potent in this regard, having an ED50 of 0.03 mg/kg. Blockade of beta-adrenergic receptors with propranolol (20 mg/kg) or blockade of peripheral alpha-adrenergic receptors with phentolamine (10 mg/kg) failed to alter the effect of apomorphine. Prazosin did not block the enhancement of startle produced by other drugs (5-methoxy-N,N-dimethyltryptamine, strychnine), nor did it alter the entry of apomorphine into the brain. The alpha 1-adrenergic antagonists piperoxane (0.03 mg/kg), yohimbine (0.03 mg/kg) or RX781094 (0.07 mg/kg) markedly potentiated apomorphine excitation. These data indicated that specific blockade of central alpha 1-adrenergic receptors prevents apomorphine-enhanced startle. In contrast to the effects of alpha 1-adrenergic antagonists, Experiment 2 found that other drugs that produce an acute (clonidine, 0.040 mg/kg) or chronic (intraventricular 6-hydroxydopamine, 2 X 200 micrograms; DSP4, 50 mg/kg i.p.) disruption of noradrenergic transmission failed to affect apomorphine excitation. Thus, the ability of alpha 1-adrenergic antagonists to block apomorphine's excitation of startle cannot be explained by a simple dopamine-norepinephrine interaction. Alternative hypothesis are discussed.     

Sustained diacylglycerol formation in norepinephrine-stimulated rat heart is associated with alpha 1-adrenergic receptor

We measured the amount of 1,2-diacylglycerol (DG) in rat hearts using thin-layer chromatography and a flame ionization detection technique, since 1,2-DG is thought to play a central role in the metabolism of phosphoinositide hydrolysis. In response to exogenous norepinephrine, a significant increase in 1,2-DG content in the myocardium was observed over 10-60 min, achieving an 80% increase over unstimulated controls at 60 min. On the other hand, the cholesterol content and six species of phospholipids were not significantly affected. In order to elucidate the mechanism of 1,2-DG accumulation elicited by norepinephrine, alpha- and beta-adrenergic antagonists were given. Pretreatment with phentolamine and prazosin, but not propranolol, inhibited the norepinephrine-induced 1,2-DG accumulation. Yohimbine appeared to have a partially inhibitory effect. These results suggest that myocardial norepinephrine-related 1,2-DG formation, which is likely to activate protein kinase C, is associated with alpha-adrenergic receptors, especially alpha 1-adrenergic receptors.     

Interactions of calmodulin antagonists with calcium antagonists binding sites.

Calmodulin antagonists have calcium entry blocking properties. In order to quantitatively investigate the interactions of these drugs with calcium channels, their effect on [3H]nitrendipine and [3H]d-cis-diltiazem binding to rat cerebral cortex membrane preparations was compared to their inhibitory effect on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The potency of most antagonists to inhibit [3H]nitrendipine binding was correlated with their calmodulin inhibitory potency. However, bepridil (K0.5 = 280 nM), chlorpromazine (K0.5 = 3 microM), triflupromazine (K0.5 = 1.5 microM), imipramine (K0.5 = 3 microM) and propranolol (K0.5 = 14 microM) were much more active on [3H]d-cis-diltiazem binding than on either [3H]nitrendipine binding or calmodulin, suggesting that these compounds bind to higher affinity sites on the calcium antagonist target protein. Moreover, the potencies of these compounds to compete with [3H]d-cis-diltiazem and to inhibit calcium-induced contractions in depolarized smooth muscle were correlated (R = 0.76, p less than 0.02). These data suggest that low concentrations of these hydrophobic drugs which have calcium and calmodulin antagonistic properties inhibit smooth muscle contraction through calcium entry blockade, not calmodulin antagonism.     

Adenosine receptor subtype-selective antagonists in inflammation and hyperalgesia

In this study, we examined the effects of systemic and local administration of the subtype-selective adenosine receptor antagonists PSB-36, PSB-1115, MSX-3, and PSB-10 on inflammation and inflammatory hyperalgesia. Pharmacological blockade of adenosine receptor subtypes after systemic application of antagonists generally led to a decreased edema formation after formalin injection and, with the exception of A₃ receptor antagonism, also after the carrageenan injection. The selective A_2B receptor antagonist PSB-1115 showed a biphasic, dose-dependent effect in the carrageenan test, increasing edema formation at lower doses and reducing it at a high dose. A₁ and A_2B antagonists diminished pain-related behaviors in the first phase of the formalin test, while the second, inflammatory phase was attenuated by A_2B and A₃ antagonists. The A_2B antagonist was particularly potent in reducing inflammatory pain dose-dependently reaching the maximum effect at a low dose of 3 mg/kg. Inflammatory hyperalgesia was totally eliminated by the A_2A antagonist MSX-3 at a dose of 10 mg/kg. In contrast to the A₁ antagonist, the selective antagonists of A_2A, A_2B, and A₃ receptors were also active upon local administration. Our results demonstrate that the blockade of adenosine receptor subtypes can decrease the magnitude of inflammatory responses. Selective A_2A antagonists may be useful for the treatment of inflammatory hyperalgesia, while A_2B antagonists have potential as analgesic drugs for the treatment of inflammatory pain.     

Imidazoline-guanidinium and alpha 2-adrenergic binding sites in basolateral membranes from human kidney.

In the present study, we used [3H]idazoxan and [3H]rauwolscine to characterize the imidazoline-guanidinium receptive site (IGRS) and alpha 2-adrenoceptors in the human renal proximal tubule, respectively. In purified basolateral membranes, 11-fold enriched in Na(+)-K+ ATPase. [3H]idazoxan and [3H]rauwolscine binding was twofold higher than in homogenates ([3H]idazoxan: 87 +/- 19 vs. 45 +/- 23.3 fmol/mg protein, P less than 0.05; [3H]rauwolscine: 56.4 +/- 21.4 vs. 25.2 +/- 7.3 fmol/mg protein, P less than 0.01). In competition studies performed at saturating concentration of [3H]idazoxan (15 NM), specific binding was competed for by epinephrine and rauwolscine only by 10-15% but was completely inhibited by imidazoline and guanidinium compounds. Thus, in human renal proximal tubule. [3H]idazoxan mainly binds to an IGRS. The highest density of alpha 2-adrenoceptors in basolateral membranes and of IGRS in partially purified membrane preparations, suggests that these two binding sites have a different subcellular localization. When compared to the rabbit renal IGRS, the human [3H]idazoxan binding site displays different affinities for guanabenz, rilmenidine, clonidine, amiloride and its derivatives that persist after membrane solubilization. In contrast, the human and rabbit renal IGRS share similar regulatory properties such as the sensitivity to K+ and the insensitivity to Na+, divalent cations and 5'-guanylylimidodiphosphate (Gpp(NH)p). In conclusion, we demonstrated that, in the human renal proximal tubule, alpha 2-adrenoceptors are mainly located in basolateral membranes while IGRS appear to be associated with another cell compartment. As indicated by their common interaction with imidazoline and guanidinium derivatives and by similar regulatory properties, human and rabbit IGRS belong to the same family of membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of neuronal nitric oxide synthase with alpha1-adrenergic receptor subtypes in transfected HEK-293 cells

Background

The effects of lindane, a gamma-isomer of hexachlorocyclohexane, were studied on transmembrane potentials and currents of frog atrial heart muscle using intracellular microelectrodes and the whole cell voltage-clamp technique.

Results

Lindane (0.34 microM to 6.8 microM) dose-dependently shortened the action potential duration (APD). Under voltage-clamp conditions, lindane (1.7 microM) increased the amplitude of the outward current (I_out) which developed in Ringer solution containing TTX (0.6 microM), Cd²⁺ (1 mM) and TEA (10 mM). The lindane-increased I_out was not sensitive to Sr²⁺ (5 mM). It was blocked by subsequent addition of quinidine (0.5 mM) or E-4031 (1 microM). E-4031 lengthened the APD; it prevented or blocked the lindane-induced APD shortening.

Conclusions

In conclusion, our data revealed that lindane increased the quinidine and E-4031-sensitive rapid delayed outward K⁺ current which contributed to the AP repolarization in frog atrial muscle.     

Identification of subtypes of [3H]prazosin-labelled alpha 1 receptor binding sites in rat brain

Detailed antagonist competition curves for [3H]prazosin-labelled binding sites in rat cerebral cortex membranes reveal anomalous binding characteristics. Dihydroergocryptine and indoramine compete in a steep, monophasic manner while WB4101 and phentolamine exhibit shallow competition curves. Computer-assisted analysis of binding data indicate that both WB4101 and phentolamine discriminate identical subpopulations of [3H]prazosin binding sites, which each comprise approximately 50% of specific [3H]prazosin binding. These data suggest the presence of subtypes of [3H]prazosin-labelled alpha 1 adrenergic receptors in rat cerebral cortex.     

Recent progress in α1-adrenergic receptor research

Alpha1-Adrenergic receptors (AR) play an important role in the regulation of physiological responses mediated by norepinephrine and epinephrine, particularly in the cardiovascular system. The three cloned alpha1-AR subtypes (alpha1A, alpha1B, and alpha1D) are G protein-coupled receptors that signal through the Gq/11 signaling pathway, each showing distinct pharmacological properties and tissue distributions. However, due to the lack of highly subtype-selective drugs, the functional roles of individual subtypes are still not clear. Development of new subtype-specific drugs will greatly facilitate the identification of the functions of each subtype. Conopeptide rho-TIA has been found to be a new alpha1B-AR selective antagonist with different modes of inhibition at alpha1-AR subtypes. In addition, recent studies using genetically engineered mice have shed some light on alpha1-AR functions in vivo, especially in the cardiovascular system and brain. Several proteins have been shown to interact directly with particular alpha1-AR, and may be important in regulating receptor function. Receptor heterodimerization has been shown to be important for cell surface expression, signaling and internalization. These new observations are likely to help elucidate the functional roles of individual alpha1-AR subtypes.     

Bolus intravenous injection of phosphorothioate oligonucleotides causes hypotension by acting as alpha(1)-adrenergic receptor antagonists.

Bolus intravenous injections of phosphorothioate oligonucleotides (PS-ODN) into primates cause profound hypotension, which has been attributed to complement activation, the biochemical pathway leading to acute inflammatory response. Because the hypotension was not accompanied by peripheral or pulmonary edema and epinephrine was not effective, but administration of 200 ml Ringer's lactate was effective, we examined the possibility that the 15-base PS-ODN interferes with sympathetic tone. We administered doses ranging from 3.3 to 10 mg/kg of a 15-base PS-ODN as a 30-60 s iv bolus into the right atrium of conscious Macaca mulatta. Blood pressure fell to 27 mm Hg following a 5.0 mg/kg dose, but no hypotension was observed after a 3.3 mg/kg dose; 10 mg/kg was lethal. Adrenergic receptor binding was evaluated in radioligand binding assays using rat cerebral cortex membranes with radiolabeled prazosin. The 15-base PS-ODN competes with prazosin for the alpha(1)-adrenergic receptor with an IC50 of 14 microM, which favors binding over serum albumin (K(d) = 37 to 48 microM). Admixing serum albumin with 5.0 mg/kg 15-base PS-ODN prior to injection prevented hypotension, suggesting that unbound PS-ODN interferes with sympathetic tone before binding to plasma proteins. Interactions of the 15-base PS-ODN with the alpha(1)-adrenergic receptor in vivo were confirmed by a decreased response to phenylephrine. Reducing the length from 15 to 9 or 5 bases abolished alpha(1)-adrenergic receptor binding in vitro and bolus infusion of 5.0 mg/kg of 9-base PS-ODN no longer produced hypotension. In conclusion, the 15-base PS-ODN shows cooperative binding to the alpha(1)-adrenergic receptor, which produces cardiovascular effects that are oligomer length, dose, and formulation dependent.     

Expression of renal alpha 1-adrenergic receptor subtypes in established hypertension.

Radioligand binding studies were undertaken in renal membranes of normotensive and hypertensive rats in order to test the hypothesis that there are alterations in renal alpha 1-adrenergic subtypes of genetic hypertensive animals. The highly selective competitive compound, (+)-niguldipine, was used to distinguish high-affinity (alpha 1a) from low-affinity (alpha 1b) sites, after initial studies demonstrated that this compound had greater selectivity than 5-methylurapidil in distinguishing alpha 1a and alpha 1b sites in rat renal membranes. In contrast to the significant difference in the blood pressure of the spontaneously hypertensive rats (delta BP = 74 mm Hg), there was no difference in the renal alpha 1-adrenergic receptor density. Membranes from the whole kidneys of spontaneously hypertensive rats (SHRs) possessed 31% alpha 1a and 69% alpha 1b sites with -log(Ki) values of 10.0 +/- 0.3 and 7.1 +/- 0.1, respectively, for (+)-niguldipine. However, these values were not different from those obtained from renal membranes of the normotensive Wistar-Kyoto (WKY) rats. These results indicate that in spite of the elevated blood pressure during the established phase of hypertension, the number, the affinity, and the ratio of the alpha 1a and alpha 1b appear not to be responsible for the manifestation of hypertension during this phase.     

Interactions of calmodulin antagonists with calcium antagonists binding sites

Calmodulin antagonists have calcium entry blocking properties. In order to quantitatively investigate the interactions of these drugs with calcium channels, their effect on [³H]nitrendipine and [³H]d-cis-diltiazem binding to rat cerebral cortex membrane preparations was compared to their inhibitory effect on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The potency of most antagonists to inhibit [³H]nitrendipine binding was correlated with their calmodulin inhibitory potency. However, bepridil (K_0.5 = 280 nM), chlorpromazine (K_0.5 = 3 μM), triflupromazine (K_0.5 = 1.5 μM), imipramine (K_0.5 = 3 μM) and propranolol (K_0.5 = 14 μM) were much more active on [³H]d-cis-diltiazem binding than on either [³H]nitrendipine binding or calmodulin, suggesting that these compounds bind to higher affinity sites on the calcium antagonist target proitein. Moreover, the potencies of these compounds to compete with [³H]d-cis-diltiazem and to inhibit calcium-induced contractions in depolarized smooth muscle were correlated (R = 0.76, p < 0.02). These data suggest that love concentrations of these hydrophobic drugs which have calcium and calmodulin antagonistic properties inhibit smooth muscle contraction through calcium entry blockade, not calmodulin antagonism.