Roles of alpha 1- and beta-adrenergic receptors in adrenergic responsiveness of liver cells formed after partial hepatectomy

The adrenergic receptor involved in the action of epinephrine changed dramatically during the process of active proliferation which follows partial hepatectomy. In control or sham-operated animals, the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by epinephrine was mediated through alpha 1-adrenergic receptors. In contrast, in hepatocytes obtained from animals partially hepatectomized 3 days before experimentation, the receptor involved in the stimulation of these metabolic pathways by epinephrine was of the beta-adrenergic type. Interestingly, the adrenergic receptor involved in the metabolic actions of epinephrine, in hepatocytes from rats partially hepatectomized 7 days before experimentation was again of the alpha 1-subtype. Thus, it appears that during the process of liver regeneration which follows partial hepatectomy there is a transition in the type of adrenergic receptor involved in the hepatic actions of catecholamines from beta in the initial stages to later alpha 1. A similar transition seems to occur as the animal ages. Cyclic AMP accumulation in response to beta-adrenergic stimulation was significantly enhanced in hepatocytes obtained from rats partially hepatectomized 3 days before the experiment, as compared to control hepatocytes or cells obtained from animals operated 7 days before experimentation. This enhanced beta-adrenergic sensitivity is probably related to the increased number of beta-adrenergic receptors observed at this stage. However, a clear dissociation between cyclic AMP levels and metabolic effects was evidenced when the different conditions were compared. The number and affinity (for epinephrine or prazosin) of alpha 1-adrenergic receptors did not change at any stage of the process, which indicates that the markedly diminished alpha 1-adrenergic sensitivity observed in hepatocytes obtained from rats partially hepatectomized 3 days before experimentation is probably due to defective generation or intracellular processing of the alpha 1-adrenergic signal, rather than to changes at the receptor level.     

Homologous and heterologous desensitization of one of the pathways of the α1-adrenergic action. Effects of epinephrine,vasopressin, angiotensin II and phorbol 12-myristate 13-acetate

Activation of protein kinase C blocks the α₁-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or mepinephrine + propranolol markedly diminished the α₁-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the α₁-adrenergic responsiveness was assayed in buffer containing calcium no effect of the preincubation with vasopressin, angiotensin II or PMA was observed. Preincubation with epinephrine diminished the α₁-adrenergic responsiveness of the cells. In hepatocytes from hypothyroid rats the preincubation with the activators of protein kinase C (vasopressin, angiotensin II, phorbol 12-myristate 13-acetate and epinephrine) reduced markedly the α₁-adrenergic responsiveness of the cells, whereas in identical experiments using cells from adrenalectomized rats only the preincubation with epinephrine diminished the responsiveness. It is concluded that activation of protein kinase C induces desensitization of the α₁-adrenergic action in hepatocytes and that the calcium-independent pathway of the α₁-adrenergic action (predominant in cells from hypothyroid animals) resensitizes more slowly than the calcium-dependent pathway (predominant in cells from adrenalectomized rats). Epinephrine in addition to inducing this type of desensitization (through protein kinase C) leads to a further refractoriness of the cells towards α₁-adrenergic agonists.     

Inositol administration restores the sensitivity of liver cells formed during liver regeneration to alpha1-adrenergic amines,vasopressin and angiotensin II

Hepatocytes obtained from rats partially hepatectomized 72 h before experimentation are insensitive to alpha₁-adrenergic amines, vasopressin, angiotensin II and ionophore A23187. However, if inositol is administered to the rats 24 and 48 h after surgery, the above-mentioned hormones stimulate glycogenolysis, gluconeogenesis and ureogenesis in the newly formed hepatocytes. Furthermore, ionophore A23187 is able to stimulate glycogenolysis in these cells, the mechanism through which inositol produces this effect being unknown. A rôle for phosphatidylinositol is suggested.     

Possible involvement of two mechanisms of signal transduction in α1-adrenergic action. Selective effect of cycloheximide

We have previously suggested that the effects of α₁-adrenergic agents on hepatocyte metabolism involve two pathways: (a) a calcium-independent, insulin-sensitive process which is modulated by glucocorticoids; and (b) a calcium-dependent, insulin-insensitive process which is modulated by thyroid hormones. Cycloheximide stimulated ureogenesis through a prazosin-sensitive mechanism in liver cells (α₁-adrenergic). The effect of cycloheximide was insulin-insensitive and calcium-dependent. Furthermore, a clear effect of cycloheximide was observed in hepatocytes obtained from adrenalectomized animals, whereas no effect was observed in cell from hypothyroid rats. The effects of epinephrine and cycloheximide were blocked by phorbol esters in all the conditions tested. Binding competition experiments indicated that cycloheximide interacts with only a fraction of the α₁-adrenergic sites labeled with [³H]prazosin. It is suggested that cycloheximide activates preferentially one of the pathways involved in the α₁-adrenergic action in liver cells.     

Cholinergic and adrenergic receptors on mouse cardiocytes in vitro

The effects of adrenergic and cholinergic receptor agonists and antagonists on single and clustered mouse cardiocytes in culture have been studied. Cardiocytes were obtained from mice, ranging in ages from 9 days in utero to 1 day postpartum, and were grown in culture for 2–14 days. Single isolated cells of every age tested possessed the ability to respond both via a muscarinic cholinergic receptor to the cholinergic agonist, carbamylcholine, and via α- and β-adrenergic receptors to norepinephrine and epinephrine. Thus, cholinergic and adrenergic receptors are simultaneously present on the same cell. Cardiocyte clusters had considerably higher sensitivity to both autonomic agents, but, because of the extensive functional specializations between cells, the localization of functional receptors to specific cells could not be made. [³H]Alprenolol, a potent β-adrenergic receptor antagonist, and [³H]quinuclidinyl benzilate ([³H]QNB), a potent muscarinic cholinergic receptor antagonist, were used to localize β-adrenergic and muscarinic cholinergic receptors by autoradiography. Quantitation of the muscarinic ACh receptor gave ～800 sites/μm², a value comparable to that for the nicotinic ACh receptor on primary skeletal muscle in culture. Electrophysiological and fine-structural studies confirmed the myocardial nature of these cells.     

Immunoanalogue of Vertebrate β-adrenergic Receptor in the Unicellular Eukaryote Paramecium

Cell fractionation, SDS-PAGE, quantitative Western blot, confocal immunolocalization and immunogold labelling were performed to find an interpretation of the physiological response of the unicellular eukaryote Paramecium to β-adrenergic ligands. The 69?kDa polypeptide separated by SDS-PAGE in S2 and P2 Paramecium subcellular fractions cross-reacted with antibody directed against human β₂-adrenergic receptor. This was detected by Western blotting followed by chemiluminescent detection. Quantitative image analysis showed that β-selective adrenergic agonist (–)-isoproterenol – previously shown to enhance phagocytic activity – evoked redistribution of the adrenergic receptor analogue from membraneous (P2) to cytosolic (S2) fraction. The relative increase in immunoreactive band intensity in S2 reached 80% and was paralleled by a 59% decrease in P2 fraction. Confocal immunofluorescence revealed β₂-adrenergic receptor sites on the cell surface and at the ridge of the cytopharynx – where nascent phagosomes are formed. This localization was confirmed by immunoelectron microscopy. These results indicate that the 69?kDa Paramecium polypeptide immunorelated to vertebrate β₂-adrenergic receptor appeared in this evolutionary ancient cell as a nutrient receptor.     

Insulin-like effect of epidermal growth factor in isolated rat hepatocytes: Modulation of the α-1-adrenergic stimulation of ureagenesis

Insulin and epidermal growth factor (EGF) inhibit the stimulation of ureagenesis induced by adrenaline (α₁-adrenergic effect) in hepatocytes from control rats incubated in medium without calcium and in cells from hypothyroid rats. In hepatocytes from euthyroid rats incubated in normal buffer neither insulin or EGF diminished the α₁-adrenergic stimulation of ureagenesis. No effect of EGF or insulin on the α₁-adrenergic stimulation of phosphatidylinositol labeling was observed under any conditions. It is suggested that EGF mimics the action of insulin on one of the pathways of the α₁-adrenergic action: the calcium-independent, insulin-sensitive pathway which predominates in hepatocytes from hypothyroid rats.     

Inositol administration restores the sensitivity of liver cells formed during liver regeneration to alpha 1-adrenergic amines, vasopressin and angiotensin II

Hepatocytes obtained from rats partially hepatectomized 72 h before experimentation are insensitive to alpha 1-adrenergic amines, vasopressin, angiotensin II and ionophore A23187. However, if inositol is administered to the rats 24 and 48 h after surgery, the above-mentioned hormones stimulate glycogenolysis, gluconeogenesis and ureogenesis in the newly formed hepatocytes. Furthermore, ionophore A23187 is able to stimulate glycogenolysis in these cells, the mechanism through which inositol produces this effect being unknown. A r?le for phosphatidylinositol is suggested.     

α-Adrenergic inhibition of cyclic AMP accumulation in epithelial cells isolated from rat small intestine

The present work shows that α-adrenergic agonists induce the suppression of basal and hormone-stimulated cyclic AMP levels in rat intestinal epithelial cells. Epinephrine (100 μM) suppresses by 35% the cyclic AMP levels evoked by the vasoactive intestinal peptide (VIP). The adrenergic agent induces a similar percentage of inhibition at 15, 30 and 37°C. Addition of epinephrine 20 min prior to, on 5 or 20 min after VIP yields the same magnitude of inhibition as when performed together with the stimulus. The α-adrenergic agent does not alter the K_0.5 of VIP in stimulating cyclic AMP production but reduces its efficacy. Epinephrine also suppresses prostaglandin E₁- and E₂-stimulated cyclic AMP levels by about 35%. The lowest effective concentration of epinephrine required to suppress VIP-stimulated cyclic AMP levels is 0.1 μM, half-maximal (K_0.5) and maximal effects being observed at 5 and 100 μM, respectively. Norepinephrine has the same efficacy but a slightly lower potency (K_0.5 = 18 μM) than epinephrine. Phenylephrine acts as a partial agonist of very low potency; clonidine has very little intrinsic activity and antagonizes the inhibition by epinephrine. The inhibition of VIP-stimulated cyclic AMP levels is observed in the absence of any blocking agents. It is not affected by the β blocker propranolol, but is completely reversed with α blockers with the following order of potency: dihydroergotamine>yohimbine>phentolamine. Yohimbine is much more potent than prazosin, which only partially reverses the inhibition induced by epinephrine. It is concluded that α-adrenoreceptors of the α₂ subtype mediate the suppression of VIP-stimulated cyclic AMP levels in intestinal epithelial cells. This effect is likely to be due to the inhibition of adenylate cyclase within intact cells as epinephrine is able to reduce adenylate cyclase activity of intestinal epithelial cell plasma membranes.     

Evidence for two types of β-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [³H]adenine labeling and was found to be coupled to a β₁-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [³H]ATP was stimulated via β₂-adrenergic receptors.     

Development of clonidine-tolerance in the rat vas deferens: Cross tolerance to other presynaptic inhibitory agents

Possibility of the development of clonidine-tolerance in the peripheral nervous tissue was examined using vas deferens isolated from rats chronically treated with clonidine. Rats were treated with clonidine for 10 days by adding the drug to drinking water (10 μg/ml). For the control rats, drug-free tap water was provided. Electrically evoked twitch response of vas deferens was suppressed by adenosine, β-endorphine and α₂-adrenergic agonists, such as clonidine and B-HT 933, both in control and clonidine-treated groups. Vas deferens isolated from clonidine-treated rats showed significantly lower responsiveness to the inhibitory effects of clonidine and B-HT 933 compared to those from control rats. Vas deferens from clonidine-treated rats also was less responsive to adenosine and β-endorphin, both of which interact with presynaptic inhibitory receptors other than α₂-adrenergic and muscarinic cholinergic stimulation responsiveness of the postsynaptic smooth muscle to both α-adrenergic and muscarinic cholinergic stimulation did not change after 10 days of treatment with clonidine. These results suggest that clonidinetolerance can be induced in the peripheral nervous system by chronic treatment of this drug and that the tolerance is not specific to α₂-adrenergic agonists. Some common pathway in the inhibitory mechanisms of various agents or possible interactions between the different types of presynaptic inhibitory receptors may be involved in this phenomenon.     

The beta-adrenergic receptor in human lymphocytes: subclassification by the use of a new radio-ligand, (+/-)-125 Iodocyanopindolol

(±)?¹²⁵Iodocyanopindolol (ICYP), a new radio-ligand with high affinity and specificity to β-adrenoceptors was used to identify and characterized β-adrenergic receptors in human lymphocytes. Binding of ICYP was saturable with 1.56 ± 0.2 fmol ICYP specifically bound/10⁶ cells at maximal occupancy of the sites and of high affinity (K_D=57 ± 7.1pM, N=4. In contrast to ¹²⁵Iodohydroxybenzylpindolol ICYP-binding was not affected by phentolamine (up to 10^?4M) or serotin (up to 10^?5M). Analysis of inhibition of ICYP-binding via a pseudo-Scatchard-plot (“Hofstee-plot”) by β_1-selective (practocol, metaprolol) and β_2-selective (IPS 339, zinterol) adrenergic drugs resulted in linear plots suggesting the existence of a homogeneous population of β-adrenergic receptorsin human lymphocytes. From the resulting K_D-values for practolol (16.8 μM), metoprolol (4.11 μM), zinterol (0.08 μM) and IPS 339 (0.002 μM) is concluded that the β-adrenergic receptor present in human lymphocytes is of the β₂-subtype. According to its low non-specific binding and its high specificity to β-adrenergic receptors ICYP appears to be an ideal ligand for long-term studies on the regulation of β-adrenergic receptors of human lymphocytes.     

Effect of adrenalectomy on cellular calcium metabolism and on the response to adrenergic stimulation of hepatocytes isolated from male and female rats

The effects of adrenalectomy on cell calcium metabolism and on the effects of epinephrine on cAMP, phosphorylase a activity, and calcium efflux were studied in hepatocytes isolated from adult male and female rats. Adrenalectomy increased the total calcium of hepatocytes, all exchangeable calcium pools, and all calcium fluxes between the cellular pools in both sexes. After adrenalectomy, basal cAMP was elevated, phosphorylase a + b was decreased, but basal phosphorylase a activity was not changed. In adrenalectomized males and at all concentrations of epinephrine studied (1·10^?8?1·10^?5M) stimulation of calcium efflux was decreased and cAMP accumulation was enhanced, while the resulting phosphorylase a activation was depressed. In hepatocytes from adrenalectomized females there was a similar increase in cAMP accumulation induced by epinephrine, and a decrease in the stimulation of calcium efflux; however, the depression in phosphorylase a activation was much less and was significant only at 1·10^?8 and 1·10^?5M epinephrine. In the male, while activation of phosphorylase a shifted from a pure α-adrenergic response mediated by calcium to one also involving a cAMP-mediated β-adrenergic response, the contribution of the attenuated calcium signal was still significant. Hepatocytes from female rats did not show a comparable α- to β-shift, since the relative contribution of calcium and cAMP to phosphorylase activation was similar in sham-operated and adrenalectomized animals.     

Influence of adenosine or adrenergic agonists on growth hormone stimulated lipolysis by chicken adipose tissue in vitro

In vitro lipolysis by chicken adipose explants was stimulated by growth hormone (GH) or glucagon. Adenosine or the adenosine agonist, N⁶-phenylisopropyladenosine (PIA), inhibited GH stimulated lipolysis, the effect of adenosine not being observed in the presence or adenosine deaminase. Glucagon induced lipolysis was also reduced by PIA. It is suggested that adenosine may act by G_i linked to either adenylate cyclase (for glucagon) or the signal transduction mechanism for GH. Lipolysis was not stimulated by GH in the presence of phenylephrine (α₁ adrenergic agonist), isoproterenol (β adrenergic agonist), adrenaline or glucagon. Although the presence of p-amino clonidine (α₂ adrenergic agonist) depressed basal lipolysis, a response to GH was still present. Either glucagon or β-adrenergic agonists (isoproterenol, adrenaline) stimulated lipolysis. In both cases, GH attenuated the lipolytic response to these hormones, which act via a cyclic adenosine monophosphate signal transduction mechanism.     

Beta-adrenergic receptors on leukocytes of the channel catfish, Ictalurus punctatus

Commercial rearing conditions expose teleost fish to numerous acute and chronic stressors that may precipitate dramatic production losses due to infectious diseases. Chemical mediators released in response to acute stress include the catecholamines, epinephrine and norepinephrine. Mammalian lymphocytes and macrophages express beta-adrenergic receptors (AR) that can bind catecholamines, leading to changes in cell function. In this study, radioligand binding assays demonstrated the presence of β-AR in membranes isolated from head kidney and spleen leukocytes of the channel catfish, Ictalurus punctatus. Competition with subtype selective antagonists CGP-20,712 (β₁) and ICI-118,551 (β₂) suggested that the β₂-adrenergic receptor is the primary receptor subtype present on these membranes. These data along with the HPLC-quantification of catecholamines in plasma of I. punctatus lend further support to the contention that crosstalk between the neuroendocrine and immune systems in lower vertebrates is mediated in part by stress-related biogenic amines like epinephrine and norepinephrine.     

Conversion of adrenergic regulation of glycogen phosphorylase and synthase from an alpha to a beta type during primary culture of rat hepatocytes

Adrenergic regulation of glycogen phosphorylase and synthase was studied with adult rat hepatocytes either immediately after isolation (fresh hepatocytes) or after 24-h maintenance in culture (cultured hepatocytes). In fresh hepatocytes, an α-adrenergic agonist caused stronger activation of phosphorylase than a β agonist, and the effect of epinephrine to activate phosphorylase and to inactivate synthase was suppressed by an α antagonist more efficiently than by a β antagonist. In cultured hepatocytes, however, the relative activities of α- and β adrenergic agents were reversed; a β agonist was much more effective than an α-agonist in activating phosphorylase, and the action of epinephrine on phosphorylase, synthase, and cyclic AMP generation was almost totally blocked by a β antagonist but not by an α antagonist. Such a reciprocal change in hepatic α- and β-adrenergic responses occurred progressively during culture; the change was interfered with by cycloheximide, an inhibitor of protein synthesis, added to the culture medium. Thus, β-adrenergic functions became predominant over α functions when hepatocytes were maintained in primary culture. Physiological significance of this phenomenon is discussed.     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.