Kenneth P. Minneman

α1-Adrenoceptor subtypes

Abstract α1-Adrenoceptors are one of three subfamilies of receptors (α1, α2, β) mediating responses to adrenaline and noradrenaline. Three α1-adrenoceptor subtypes are known (α1A, α1B, α1D) which are all members of the G protein coupled receptor family, and splice variants have been reported in the C-terminus of the α1A. They are expressed in many tissues, particularly smooth muscle where they mediate contraction. Certain subtype-selective agonists and antagonists are now available, and α1A-adrenoceptor selective antagonists are used to treat benign prostatic hypertrophy. All subtypes activate phospholipase C through the Gq/11 family of G proteins, release stored Ca2+, and activate protein kinase C, although with significant differences in coupling efficiency (α1A>α1B>α1D). Other second messenger pathways are also activated by these receptors, including Ca2+ influx, arachidonic acid release, and phospholipase D. α1-Adrenoceptors also activate mitogen-activated protein kinase pathways in many cells, and some of these responses are independent of Ca2+ and protein kinase C but involve small G proteins and tyrosine kinases. Direct interactions of α1-adrenoceptors with proteins other than G proteins have not yet been reported, however there is a consensus binding motif for the immediate early gene Homer in the C-terminal tail of the α1D subtype. Current research is focused on discovering new subtype-selective drugs, identifying non-traditional signaling pathways activated by these receptors, clarifying how multiple signals are integrated, and identifying proteins interacting directly with the receptors to influence their functions.

Subtypes of α1adrenoceptors in rat blood vessels

Abstract Two α-adrenoceptor subtypes ( α 1A and α 1B ) have been distinguished by competitive antagonists and the alkylating agent chloroethylclonidine. The chloroethylclonidine-insensitive subtype ( α 1A ) has been suggestpd to selectively activate Ca 2+ influx through dihydropyridine-sensitive channels in smooth muscle. We compared the effects of chloroethylclonidine and nifedipine on contractile responses to norepinephrine (NE) in rat blood vessels. Pretreatment with chloroethylclonidine caused a large shift to the right and decrease in maximum for NE-induced contractions of rat aorta, however nifedipine had no effect in this tissue. Chloroethylclonidine had no effect on NE-induced contractions of rat renal arteries, which were ahnost completely abolished by nifedipine. Both chloroethylclonidine and nifedipine partially attenuated NE-induced contractions in rat mesenteric artery and portal vein. Addition of nifedipine abolished residual contractions remaining after chloroethylclonidine pretreatment in all four types of vessels. The pA 2 for the competitive antagonist WB 4101 was highest in renal artery and lowest in aorta. These results suggest that the sensitivity of rat blood vessels to chloroethylclonidine is inversely related to their sensitivity to nifedipine, and support the existence of two α-adrenoceptor subtypes in rat blood vessels. Rat aorta appears to contain primarily α 1B receptors, renal artery primarily α 1A and mesenteric artery and portal vein mixtures α 1A of and α 1b .

Differential Coupling of α1-, α2-, and β-Adrenergic Receptors to Mitogen-activated Protein Kinase Pathways and Differentiation in Transfected PC12 Cells

Three adrenergic receptor families that selectively activate three different G proteins (α1/Gq/11, α2/Gi, and β/Gs) were used to study mitogen-activated protein kinase (MAPK) activation and differentiation in PC12 cells. PC12 cells were stably transfected with α1A-, α2A-, or β1-adrenergic receptors (ARs) in an inducible expression vector, and subclones were characterized. Norepinephrine stimulated inositol phosphate formation in α1A-transfected cells, inhibited cyclic adenosine 3′5′-monophosphate (cAMP) formation in α2A-transfected cells, and stimulated cAMP formation in β1-transfected cells. Nerve growth factor activated extracellular signal-regulated kinases (ERKs) in all cell lines; however, norepinephrine activated ERKs only in α1A- and β1-transfected cells but not in α2A-transfected cells. Norepinephrine also activated c-Jun NH2-terminal kinase and p38 MAPK in α1A-transfected cells but not in β1- or α2A-transfected cells. Norepinephrine caused differentiation of PC12 cells expressing α1A-ARs but not those expressing β1- or α2A-ARs. However, norepinephrine acted synergistically with nerve growth factor in promoting differentiation of cells expressing β1-ARs. Whereas ERKs are activated by Gi- but not Gs-linked receptors in many fibroblastic cell lines, we observed the opposite in PC12 cells. The results show that activation of the different G protein signaling pathways has different effects on MAPKs and differentiation in PC12 cells, with Gq signaling pathways activating all three major MAPK pathways.

β1- andβ2-adrenoceptor binding and functional response in right and left atria of rat heart

SummaryThe properties ofβ1- andβ2-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses toβ-adrenoceptor agonists were examined. [125I](-)-pindolol (125IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek1s for association in right and left atria were 6.5×109 l/mol-min and 2.3×109 l/mol-min respectively, while thek−1s for dissociation were 0.20 min−1 and 0.17 min−1. The kinetically determinedKDs were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumKDs determined from Scatchard analysis of saturation isotherms of specific125IPIN binding. Inhibition of125IPIN binding byβ-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition byβ1- andβ2-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement byβ1-selective (practolol, atenolol and metoprolol) andβ2-selective (ICI 118,551) antagonists gave estimates of the proportion ofβ1- andβ2-adrenoceptors present in rat atria. Right atria contained 67±4.2%β2-adrenoceptors and 33±4.2%β2-adrenoceptor, while left atria contained 67±2.8%β1- and 33±2.8%β2-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused byβ-adrenoceptor agonists were also measured. pA2 values for non-subtype selectiveβ-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withKD values determined for specific125IPIN binding. pA2 values forβ1- andβ2-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pKD values of these drugs in binding toβ1-adrenoceptors, but not with the pKD values in binding toβ2-adrenoceptors. Dose-response curves for stimulation of both rate and force by theβ2-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade ofβ1-adrenoceptors with metoprolol than by selective blockade ofβ2-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activatingβ1-adrenoceptors. These results suggest thatβ1- andβ2-adrenoceptors coexist in both left and right atria of rat heart in approximately a 2∶1 ratio, however onlyβ1-adrenoceptors mediate the chronotropic and inotropic effects ofβ-adrenoceptor agonists.

Receptor-linked cyclic amp systems in rat neostriatum: Differential localization revealed by kainic acid injection

Various receptor-linked cyclic AMP systems were measured in rat neostriatum 2--14 days after selective destruction of neuronal cell bodies and dendrites by micro-injection of 3 microgram of kainic acid. Basal adenylate cyclase activity was reduced by up to 56% in the injected side and the sensitivity to dopamine was abolished. Up to 84% of cyclic nucleotide phosphodiesterase activity, hydrolyzing either cyclic AMP or cyclic GMP, was destroyed by kainic acid injection. Specific binding of [3H]etorphine and [3H]spiroperidol was reduced by up to 62% in the injected side, while non-specific binding was unchanged. All of these changes were time-dependent, and were greatest 7--14 days after kainic acid treatment. On the other hand, intrastriatal kainic acid injection caused no change in the steady-state concentration of cyclic AMP in striatal slices, or in the in vivo cyclic AMP content in the striatum of rats killed by microwave irradiation. Receptor-mediated increases in cyclic AMP accumulation in striatal slices were either unchanged or markedly potentiated by kainic acid treatment. The maximum response to adenosine was unchanged, while the response to isoprenaline was increased up to 3.7-fold, the response to dopamine increased up to 6.7-fold, and the response to PGE1 increased up to 30-fold. The effect of dopamine in kainic acid-treated striatal slices was no longer blocked by fluphenazine, but was blocked by propranolol, suggesting an interaction of dopamine with a beta-adrenoceptor in kainic acid-treated slices. The results suggest differential cellular localizations of the various receptor-linked cyclic AMP systems in rat neostriatum. Some dopamine and opiate receptors, as well as most of the phosphodiesterase activity, are associated with local neuronal elements, while beta-adrenoceptor, adenosine and PGE1 alterations in cyclic AMP are not. The potentiation of the beta-adrenoceptor and PGE1 responses suggests that they may occur in glial cells. In addition, the pool of adenylate cyclase destroyed by kainic acid appears to make little contribution to normal levels of cyclic AMP in the tissue.

Heterodimers of α1B and α1D-adrenergic receptors form a single functional entity

Heterologous expression of α1D-adrenergic receptors (α1D-ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with α1B-ARs promotes surface localization of α1D-ARs. Here, we report that the α1B-/α1D-AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of α1B-ARs and epitope-tagged α1D-ARs in both human embryonic kidney 293 and DDT1MF-2 cells, α1D-AR binding sites were not detectable with the α1D-AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro[4,5]decane-7,9-dione (BMY 7378), despite the ability to detect α1D-AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the α1B-AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in α1B/α1D-AR-expressing cells, revealing that α1D-ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in α1B/α1D-AR-coexpressing cells. Stable coexpression of an uncoupled mutant α1B-AR (Δ12) with α1D-ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that α1B/α1D-AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why α1D-ARs are often pharmacologically undetectable in native tissues when they are coexpressed with α1B-ARs.

Characterization of α1-adrenoceptors which increase cyclic AMP accumulation in rat cerebral cortex

Abstract The pharmacological properties of the α-adrenoceptors which increase cyclic AMP accumulation were studied in slices of rat cerebral cortex after inactivation of β-adrenoceptors with bromoacetylalprenololmenthane. Norepinephrine increased basal cyclic AMP accumulation 2-fold, and potentiated the effect of adenosine 5-fold. The K i and EC 50 values for antagonists and agonists for both the basal and potentiated responses were generally similar to those for α 1 -adrenoceptor-stimulated inositol phosphate accumulation in the same preparation. However, significant differences in the potencies of all agonists, and the antagonists phentolamine and BE2254 were observed between the basal and potentiated cyclic AMP responses. The differences in agonist potencies did not appear to be due to the existence of a receptor reserve. Norepinephrine, epinephrine, α-methylnorepinephrine and 6-fluoronorepinephrine were full agonists, while methoxamine and phenylephrine were partial agonists in both systems. The results suggest that norepinephrine increases cyclic AMP accumulation in rat cerebral cortex through α 1 -adrenoceptors similar to those increasing phosphatidylinositol metabolism in the same tissue.

Differential Activation of Mitogen-Activated Protein Kinase Pathways in PC12 Cells by Closely Related α1-Adrenergic Receptor Subtypes

Abstract: Coupling of the three known α1‐adrenergic receptor (α1‐AR) subtypes to mitogen‐activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing α1A‐, α1B‐, and α1D‐ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca2+ level in these cells in a manner dependent on receptor density. However, α1A‐ARs activated these second messenger responses more effectively than α1B‐ARs, whereas α1D‐ARs were least effective. NE stimulated activation of extracellular signal‐regulated kinases (ERKs) in cells expressing all three α1‐AR subtypes, although α1A‐ and α1B‐ARs caused larger ERK activation than did α1D‐ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in α1A‐ and α1B‐ but not α1D‐transfected cells and activated c‐Jun NH2‐terminal kinase (JNK) only in α1A‐transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70‐kDa protein only in α1A‐transfected PC12 cells. NE caused neutrite outgrowth only in α1A‐expressing PC12 cells, but not in α1B‐ or α1D‐transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that α1A‐ARs activate all three MAPK pathways, α1B‐ARs activate ERKs and p38 but not JNKs, and α1D‐ARs only activate ERKs. Only the α1A‐AR‐expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed.

Transcriptional responses to growth factor and G protein-coupled receptors in PC12 cells : Comparison of α1-Adrenergic receptor subtypes

Abstract: Transcriptional responses to growth factor and G protein‐coupled receptors were compared in PC12 cells using retroviral luciferase reporters. In cells stably expressing α1A‐adrenergic receptors, norepinephrine activated all five reporters [AP1 (activator protein‐1), SRE (serum response element), CRE (cyclic AMP response element), NFκB) (nuclear factor‐κB), and NFAT (nuclear factor of activated T cells)], whereas nerve growth factor (NGF) and epidermal growth factor activated only AP1 and SRE. Activation of P2Y2 receptors by UTP did not activate any reporters. Protein kinase C inhibition blocked NFκB activation by norepinephrine, but potentiated CRE. Mitogen‐activated protein kinase kinase inhibition blocked AP1 activation by norepinephrine, but also potentiated CRE. p38 mitogen‐activated protein kinase inhibition reduced most norepinephrine responses, but not NGF responses. inhibition of Src eliminated SRE responses to norepinephrine and NGF, and reduced all responses except CRE. Phosphatidylinositol 3‐kinase inhibitors markedly potentiated CRE activation by norepinephrine, with only small effects on the other responses. Comparison of the three human subtypes showed that the α1A activated all five reporters, the α1B showed smaller effects, and the α1D was ineffective. Cell differentiation caused by norepinephrine, but not NGF, was reduced by all inhibitors studied. These experiments suggest that α1A‐adrenergic receptors activate a wider array of transcriptional responses than do growth factors in PC12 cells. These responses are not linearly related to second messenger production, and different subtypes show different patterns of activation.

Recent progress in α 1 -adrenergic receptor research

Abstractα1-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 α1-AR subtypes (α1A, α1B, and α1D) 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 p-TIA has been found to be a new α1B-AR selective antagonist with different modes of inhibition at α1-AR subtypes. In addition, recent studies using genetically engineered mice have shed some light on α1-AR functions in vivo, especially in the cardiovascular system and brain. Several proteins have been shown to interact directly with particular α1-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 α1-AR subtypes.