Effects of quinidine and verapamil on human cardiovascular alpha1-adrenoceptors.

BACKGROUND: The antiarrhythmic drugs quinidine and verapamil are known to block alpha1-adrenoceptors (alpha1ARs). Alpha1ARs are a heterogeneous family of three subtypes (alpha1A, alpha1B, and alpha1D), and little is known about the effects of quinidine and verapamil on the different human alpha1AR subtypes. METHODS AND RESULTS: Reverse transcriptase-polymerase chain reaction showed that all alpha1AR subtypes are expressed in both human heart (atrium and ventricle) and the mesenteric artery. Pharmacological profiles of quinidine and verapamil actions on the alpha1AR subtypes were characterized with Chinese hamster ovary cells stably expressing cloned human alpha1AR subtypes. Radioligand binding studies showed that quinidine and verapamil had high affinities for all alpha1AR subtypes. Also, both drugs synergistically inhibited alpha1AR-mediated inositol 1,4,5-triphosphate production at the clinical effective concentration range (1 micromol/L quinidine and 0.1 micromol/L verapamil). CONCLUSIONS: The results show that all alpha1AR subtypes are expressed in the human cardiovascular system and that quinidine and verapamil may have a potent, synergistic inhibitory effect on the alpha1ARs. Clinically observed hypotension after quinidine plus verapamil can be explained by their synergistic inhibitory effects on human alpha1ARs.

NS-49, a novel alpha 1a-adrenoceptor-selective agonist characterization using recombinant human alpha 1-adrenoceptors.

alpha 1-Adrenoreceptors comprise a heterogeneous family and subtype-selective ligands are valuable in studying the functional role of each receptor subtype. Using the Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a, alpha 1b, and alpha 1d)1, we have compared a newly synthesized phenethylamine class agonist (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride (NS-49) with imidazoline class agonist oxymetazoline in their binding affinities and intrinsic activities in causing transient increases of cytosolic Ca2+ concentrations ([Ca2+¿i response). Radioligand binding studies with 2-[beta-(4-hydroxyl-3-[125I]iodophenyl)ethylamino-methyl]tetralone ([125I]HEAT) showed NS-49 and oxymetazoline had higher affinities at alpha 1a-than at alpha 1b- and alpha 1d-subtypes (-log Ki values at alpha 1a-, alpha 1b-and alpha 1d-subtype: 6.18, 5.13, and 5.38 for NS-49: 8.19, 6.50, and 6.44 for oxymetazoline, respectively). In functional studies, both oxymetazoline and NS-49 worked as a selective and partial agonist at alpha 1a-subtype: however, NS-49 is more efficacious than oxymetazoline. NS-49 is the phenethylamine class of alpha 1-adrenoceptor partial agonist relatively selective and efficacious for the human alpha 1a-adrenoceptor subtype, NS-49 would be potentially useful for studying the physiological role of alpha 1-adrenoceptor subtype.

Selectivity of the imidazoline alpha-adrenoceptor agonists (oxymetazoline and cirazoline) for human cloned alpha 1-adrenoceptor subtypes.

1. To investigate the structure-activity relationships of alpha-adrenoceptor agonists for the alpha 1-adrenoceptor subtypes, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a-, alpha 1b-, and alpha 1d-subtypes). 2. Radioligand binding studies with [125I]-HEAT showed that cirazoline and oxymetazoline had higher affinities at alpha 1a-subtype than at alpha 1b- and alpha 1d-subtypes, while noradrenaline had higher affinity at the alpha 1d-subtype than at alpha 1a- and alpha 1b-subtypes. 3. In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+]i response) in a concentration-dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the alpha 1a-subtype, while it acted as a partial agonist at alpha 1b- and alpha 1d-adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at alpha 1a-adrenoceptors, and has no intrinsic activity at the other subtypes. 4. Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline-induced [Ca2+]i response for alpha 1a- and alpha 1d-subtypes were found to be linear, whereas it was moderately hyperbolic for the alpha 1b-subtype, indicating the absence of receptor reserves in CHO cells expressing alpha 1a- and alpha 1d-subtypes while there exists a small receptor reserve for CHO cells expressing the alpha 1b-subtype. 5 In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of alphal-adrenoceptor.

KMD-3213, a novel, potent, alpha 1a-adrenoceptor-selective antagonist: characterization using recombinant human alpha 1-adrenoceptors and native tissues.

alpha 1-Adrenoceptors (ARs) comprise a heterogeneous family, and subtype-selective ligands are valuable for studying the functional role of each receptor subtype. We characterized a newly synthesized, alpha 1-AR antagonist, KMD-3213, by using Chinese hamster ovary cells stably expressing the three cloned human alpha 1-ARs (alpha 1a, alpha 1b, and alpha 1d), as well as native rat and human tissues. KMD-3213 potently inhibited 2-[2-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-alpha-tetralone binding to the cloned human alpha 1a-AR, with a Ki value of 0.036 nM, but had 583- and 56-fold lower potency at the alpha 1b- and alpha 1d-ARs, respectively. KMD-3213 inhibited norepinephrine-induced increases in intracellular Ca2+ concentrations in alpha 1a-AR-expressing Chinese hamster ovary cells with an IC50 of 0.32 nM but had a much weaker inhibitory effect on the alpha 1b- and alpha 1d-ARs. Using pharmacologically well characterized native rat tissues [submaxillary gland (alpha 1A-AR-expressing tissue), liver (alpha 1B-AR-expressing tissue), and heart (mixed alpha 1A- and alpha 1B-AR-expressing tissue)], binding studies showed that inhibition curves for KMD-3213 in submaxillary gland and liver best fit a one-site model (with Ki values of 0.15 and 16 nM, respectively), whereas KMD-3213 had high and low affinity sites in heart membranes. Chloroethylclonidine treatment of rat heart membranes completely eliminated the low affinity sites for KMD-3213. Furthermore, in human liver and prostate KMD-3213 could identify high and low affinity sites, the Ki values of which corresponded well to those for the cloned human alpha 1a- and alpha 1b-ARs, respectively. Moreover, the affinity of KMD-3213 was found to be approximately 10-fold higher at the cloned human alpha 1a-AR than at the cloned rat alpha 1a-AR. KMD-3213 is a potent and highly selective antagonist for the human alpha 1a-AR and would be useful for studying the physiological roles of human alpha 1-AR subtypes.

Use of recombinant alpha 1-adrenoceptors to characterize subtype selectivity of drugs for the treatment of prostatic hypertrophy.

Several alpha 1-adrenoceptor antagonists have recently been developed for the treatment of benign prostatic hypertrophy because of their less frequent systemic side-effects compared to conventional alpha 1-adrenoceptor blockers. One potential explanation for their good tolerability would be the selectivity for a certain subtype of alpha 1-adrenoceptor. Utilizing COS-7 cells expressing the rat alpha 1A, the hamster alpha 1B and the human alpha 1C-adrenoceptors, we investigated affinities of alfuzosin, doxazosin, terazosin, indoramin and (+)- and (-)-5-[2-[[2-(o-ethoxyphenoxy)ethyl] amino]propyl]-2-methoxybenzesulfonamide HCl (YM 617) compared to prazosin. Radioligand binding studies showed that the affinities of alpha 1-adrenoceptor subtypes for alfuzosin (Ki value; alpha 1A: 2.4 nM, alpha 1B:1.4 nM, alpha 1C:4.2 nM), doxazosin (Ki value; alpha 1A:2.7 nM, alpha 1B:3.2 nM, alpha 1C:7.5 nM), terazosin (Ki value; alpha 1A:2.5 nM, alpha 1B:2.7 nM, alpha 1C:7.1 nM), indoramin (Ki value; alpha 1A:69 nM, alpha 1B:21 nM, alpha 1C:13 nM) and prazosin (Ki value; alpha 1A:0.16 nM, alpha 1B:0.19 nM, alpha 1C:0.2 nM) were equipotent to the three receptor subtypes. Unlike these antagonists, both (+)- and (-)-YM617 had relatively lower affinity for alpha 1B receptors compared to the other subtypes (Ki value; for (+)-YM617, alpha 1A:22 nM, alpha 1B:96 nM, alpha 1C:4.3 nM; for (-)-YM617, alpha 1A:0.11 nM, alpha 1B:0.7 nM, alpha 1C:0.035 nM). The data suggest that alpha 1-adrenoceptor antagonists currently used for the treatment of the benign prostatic hyperplasia do not show substantial subtype selectivity.

[Quantitative parameters of drug-receptor interactions and receptor reserves. I. Models I-3-R and I-3-S]. / Uvahy o kvantitativních parametrech farmakon-receptorové interakce a o receptorové rezerve. I: Modely I-3-R a I-3-S.

On a simple model of a reaction-chain from the pharmacon-receptor complex (RA) formation the formation of a ternal complex with substrate (S) and its activation to a product the authors investigate the meaning of the term "affinity" and "internal activity" and the behaviour of the model with substrate and velocity limits at various sites. If K1 = dissociation constant of the complex RA, K2 = dissociation constant of the complex RAS, then at S1 much greater than R1 (i.e. without receptor reserve) the obtained value ED50 described usually as KA = K1K2: (K2 + [St]) and if K1 is constant it is variable in relation to the substrate concentration. In this way continuous differences of apparent affinity of the same substance to the same receptor under different biological conditions are possible. At R1 much greater than St (i.e. with receptor reserve) apparent KA = K1K2: (K2 + [Rt]) and thus K1 can be assessed by blocking receptors with an irreversible antagonist. The authors describe the calculation and very simple graphical method for assessment of basic parameters using an irreversible antagonist. After linear plotting of ED50 (= KA) on the chi-axis and maximal effects on the gamma-axis and after extrapolation of the connecting line of the two points (results without antagonist and results with antagonist) the intersecting point with the chi-axis gives value K1. This makes it possible, among others, to assess also the "intrinsic efficacy" of the substance, the ratio of receptors eliminated by the antagonist, prediction of the behaviour of typical curves during gradual elimination of receptors by a blocker and to assess possibly also the half-life of binding of the irreversible blocker with the receptor.

[2 different populations of adrenergic alpha receptors in the splenic capsule in rats]. / Dve rozdílné populace adrenergních alpha-receptoru v capsula linenalis krysy.

Using the contraction of the smooth muscle of rat spleen capsula, studies of the character of the alpha-adrenergic receptors involved in this reaction where performed in vitro. By computer analysis, the flat dose-response curve of noradrenaline shows the presence of two different subpopulations of alpha-receptors, each being responsible for approximately 50% of the maximal overall contraction. The two subpopulations differ roughly one hundred times by their apparent affinity for noradrenaline (pD2 cca 8 and pD2 cca 6). Solely the high-affinity component is competitively antagonized by the alpha 1-blocker prazosin, needs necessarily extracellular Ca2+ for evoking the contraction and is desensitized by previous contact with a high concentration of noradrenaline. Neither of the two components is influenced in its action by calcium-channel blockers nifedipine and verapamil. The needed exogenous calcium thus enters the cell by different routes than by those affected by the mentioned blockers.

[Closer characterization of alpha-receptor subpopulations in the smooth muscle of the splenic capsule in rats]. / Pokus o blizsí charakteristiku alpha-receptorových supopulací v hladkém svalu krysí capsula lienalis.

Two different subpopulations of adrenergic alpha-receptors -- "high-affinity" (pD2 less than 8) and "low-affinity" (pD2 less than 6) ones -- are involved in alpha-adrenergic contractions of the rat spleen muscle evoked by noradrenaline in vitro. Only the "high-affinity" subpopulation is agonistically influenced by the alpha 1-mimetic phenylephrine (pD2 cca 6, pKA cca 4.8), antagonistically influenced by prazosin (pA2 cca 9); it has a substantial receptor reserve for noradrenaline and a somewhat lower reserve for phenylephrine, it can be desensitized by these mimetics and it requires the presence of exogenous Ca2+ in the medium for evoking the contraction. -- Only the "low-affinity" subpopulation is agonistically influenced by the alpha 2-mimetic UK-14304. Both subpopulations are activated by noradrenaline and competitively blocked by yohimbine. The effect of neither subpopulation is influenced by nifedipine. According to the new classification (11) the "high-affinity" receptors can be described as alpha 1A, the "low-affinity" ones as alpha 2A. Contrarily to current data, the peculiar feature of this second subpopulation is a low affinity for yohimbine (pA2 less than 7) and a certain extent of blockability by phenoxybenzamine.