J. Adolfo García-Sáinz

Role of phosphatidylinositol turnover in alpha1 and of adenylate cyclase inhibition in alpha2 effects of catecholamines

Abstract Ligand binding and pharmacological studies have indicated that alpha-adrenergic receptors can be divided into alpha1 and alpha2. We suggest that alpha1 receptors mediate those metabolic effects of alpha catecholamines which involve phosphatidylinositol turnover and the release of bound intracellular Ca2+ as well as the entry of extracellular Ca2+. In contrast, alpha effects of catecholamines are due to non-specific inhibition of adenylate cyclase through a mechanism independent of Ca2+. A similar classification for the effects of both histamine and serotonin suggests that they have separate type 1 or alpha receptors for Ca2+ dynamics which are different from type 2 or beta receptors which regulate adenylate cyclase. There is a significant correlation between hormone effects on phosphatidylinositol turnover and elevation of intracellular Ca2+. The available data suggest that the turnover of membrane-bound phosphatidylinositol is involved in Ca2+ gating in rat hepatocytes, rat and hamster adipocytes and blowfly salivary glands. In hamster adipocytes adenylate cyclase activity is also inhibited by alpha2 catecholamines through a Ca2+ independent mechanism.

α1-Adrenoceptors : function and phosphorylation

Abstract This review focuses on α 1 -adrenoceptor phosphorylation and function. Most of what is currently known is based on studies on the hamster α 1B -adrenoceptor. It is known that agonist stimulation leads to homologous desensitization of these receptors and current evidence indicates that such decrease in receptor activity is associated with receptor phosphorylation. Such receptor phosphorylation seems to involve G protein-receptor kinases and the receptor phosphorylation sites have been located in the carboxyl tail (Ser 404 , Ser 408 , and Ser 410 ). There is also evidence showing that in addition to desensitization, receptor phosphorylation is associated with internalization and roles of β-arrestins have been observed. Direct activation of protein kinase C leads to receptor desensitization/internalization associated with phosphorylation; the protein-kinase-C-catalyzed receptor phosphorylation sites have been also located in the carboxyl tail (Ser 394 and Ser 400 ). Activation of G q -coupled receptors, such as the endothelin ET A receptor induces α 1B -adrenoceptor phosphorylation and desensitization. Such effect involves protein kinase C and a yet unidentified tyrosine kinase. Activation of G i -coupled receptors, such as the lysophosphatidic acid receptor, also induces α 1B -adrenoceptor phosphorylation and desensitization. These effects involve protein kinase C and phosphatidyl inositol 3-kinase. Interestingly, activation of epidermal growth factor receptors also induces α 1B -adrenoceptor phosphorylation and desensitization involving protein kinase C and phosphatidyl inositol 3-kinase. A pivotal role of these kinases in heterologous desensitization is evidenced.

Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes

Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [(3)H]dihydroalprenolol binding (beta adrenergic) B(max) 280 fmol/mg protein, K(D) 0.38 nM; [(3)H]para-aminoclonidine binding (alpha-2 adrenergic) B(max) 166 fmol/mg protein, K(D) 0.49 nM; [(3)H]WB 4101 binding (alpha-1 adrenergic) B(max) 303 fmol/mg protein, K(D) 0.86 nM. In adipocytes from subcutaneous adipose tissue, [(3)H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 muM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 muM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [(32)P]phosphate and epinephrine (10 muM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in (32)P incorporation into phosphatidylinositol. Prazosin (0.1 muM) blocked this action whereas yohimbine (0.1 muM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.

α1-Adrenoceptors: Subtypes, Signaling, and Roles in Health and Disease

Alpha 1-adrenoceptors mediate some of the main actions of the natural catecholamines, adrenaline, and noradrenaline. They participate in many essential physiological processes, such as sympathetic neurotransmission, modulation of hepatic metabolism, control of vascular tone, cardiac contraction, and the regulation of smooth muscle activity in the genitourinary system. It is now clear that alpha 1-adrenoceptors mediate, in addition to immediate effects, longer term actions of catecholamines such as cell growth and proliferation. In fact, adrenoceptor genes can be considered as protooncogenes. Over the past years, considerable progress has been achieved in the molecular characterization of different alpha 1-adrenoceptor subtypes. Three main subtypes have been characterized pharmacologically and in molecular terms. Splice variants, truncated isoforms, and polymorphisms have also been detected. Similarly, it is now clear that these receptors are coupled to several classes of G proteins that, therefore, are capable of modulating different signaling pathways. In the present article, some of these aspects are reviewed, together with the distribution of the subtypes in different tissues and some of the known roles of these receptors in health and disease.

Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca++-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha 1-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha 1-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha 1-adrenergic actions seems to occur at an early step of the alpha 1-adrenergic action. TPA (10(-11) -10(-6)M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha 1-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca++ ionophore A-23187.

G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions

G protein-coupled receptors are dynamically regulated. Such regulation is frequently associated with covalent posttranslational modifications, such as phosphorylation, and with regulatory elements. G protein-coupled receptor kinases and casein kinase 1alpha play key roles in agonist-dependent receptor phosphorylations. Cross-talk between different receptors frequently involves second messenger-activated proteins, such as protein kinase C and protein kinase A. There is some evidence indicating that such kinases may not only turn off receptors but also switch their coupling to different G proteins. Receptor tyrosine kinases may phosphorylate and regulate G protein-coupled receptors and recent evidence indicates that other kinases, such as Akt/protein kinase B and phosphoinositide 3-kinase, may participate in such regulations as integrators of signalling. Recent approaches have shed new light on G protein-coupled receptor interactions that provide novel mechanisms of action and regulation. G protein-coupled receptor activities go beyond G proteins and receptors can be partners of exquisitely assembled signalling complexes through molecular bridges composed of multidomain proteins. The possibilities of interaction increase enormously through the diversity of structural and functional domains present in complex proteins, many of them just known as predicted sequences.

Role of alpha1 adrenoceptors in the turnover of phosphatidylinositol and of alpha2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

Abstract The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin⪢phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha1 adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ⪢prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha2 adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha1 and alpha2 adrenoceptors but about 90% of the binding sites were alpha2. These data support the hypothesis that alpha2 effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha1 adrenoceptor activation.

Activation of Endothelin ETA Receptors Induces Phosphorylation of α1b-Adrenoreceptors in Rat-1 Fibroblasts

The effect of endothelin-1 on the phosphorylation of α1b-adrenoreceptors, transfected into rat-1 fibroblasts, was studied. Basal α1b-adrenoreceptor phosphorylation was markedly increased by endothelin-1, norepinephrine, and phorbol esters. The effect of endothelin-1 was dose dependent (EC50 ≈ 1 nm), reached its maximum 5 min after stimulation, and was inhibited by BQ-123, an antagonist selective for ETA receptors. Endothelin-1-induced α1b-adrenoreceptor phosphorylation was attenuated by staurosporine or genistein and essentially abolished when both inhibitors were used together. The effect of norepinephrine was not modified by either staurosporine or genistein alone, and it was only partially inhibited when both were used together. These data suggest the participation of protein kinase C and tyrosine kinase(s) in endothelin-1-induced receptor phosphorylation. However, phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not of phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by endothelin, could act in a step previous to receptor phosphorylation. The effect of endothelin-1 on α1b-adrenoreceptor phosphorylation was not mediated through pertussis toxin-sensitive G proteins. Calcium mobilization induced by norepinephrine was diminished by endothelin-1. Norepinephrine and endothelin-1 increased [35S]GTPγS binding to control membranes. The effect of norepinephrine was abolished in membranes obtained from cells pretreated with endothelin-1. Interestingly, genistein plus staurosporine inhibited this effect of the endothelial peptide. Endothelin-1 did not induce α1b-adrenoreceptor internalization. Our data indicate that activation of ETA receptors by endothelin-1 induces α1b-adrenoreceptor phosphorylation and alters G protein coupling.

Species heterogeneity of hepatic α1-adrenoceptors : α1A-, α1B- and α1C-subtypes

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.

Modulation of basal intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing α1d-adrenoceptors

In rat‐1 fibroblasts stably expressing α1d‐adrenoceptors BMY 7378, phentolamine, chloroethylclonidine and 5‐methyl urapidil decreased basal [Ca2+]i. WB 4101 induced a very small effect on this parameter but when added before the other antagonists it blocked their effect. All these agents inhibited the action of norepinephrine. Phorbol myristate acetate also blocked the effect of norepinephrine and decreased basal [Ca2+]i. Staurosporine inhibited these effects of the phorbol ester. Our results suggest that: (1) α1d‐adrenoceptors exhibit spontaneous ligand‐independent activity, (2) BMY 7378, phentolamine, chloroethylclonidine and 5‐methyl urapidil act as inverse agonists and (3) protein kinase C activation blocks spontaneous and agonist‐stimulated α1d‐adrenoceptor activity.