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.     

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

Insulin and epidermal growth factor (EGF) inhibit the stimulation of ureagenesis induced by adrenaline (alpha 1-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 alpha 1-adrenergic stimulation of ureagenesis. No effect of EGF or insulin on the alpha 1-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 alpha 1-adrenergic action: the calcium-independent, insulin-sensitive pathway which predominates in hepatocytes from hypothyroid rats.     

Homologous and heterologous desensitization of one of the pathways of the alpha 1-adrenergic action. Effects of epinephrine, vasopressin, angiotensin II and phorbol 12-myristate 13-acetate

Activation of protein kinase C blocks the alpha 1-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or epinephrine + propranolol markedly diminished the alpha 1-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the alpha 1-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 alpha 1-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 alpha 1-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 alpha 1-adrenergic action in hepatocytes and that the calcium-independent pathway of the alpha 1-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 alpha 1-adrenergic agonists.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.     

Phorbol esters, vasopressin and angiotensin II block alpha 1-adrenergic action in rat hepatocytes. Possible role of protein kinase C

The effect of vasopressin, angiotensin II and phorbol myristate acetate on the alpha 1-adrenergic action (induced by epinephrine + propranolol), was studied. We selected three conditions: (a) ureagenesis in medium without added calcium and containing 25 microM EGTA; (b) ureagenesis using cells from hypothyroid animals, and (c) gluconeogenesis from dihydroxyacetone. Under these conditions epinephrine + propranolol produces clear metabolic effects, whereas the vasopressor peptides do not (although they stimulate phosphoinositide turnover). It was observed that the vasopressor peptides and the active phorbol ester inhibited in a concentration-dependent fashion the effect of epinephrine + propranolol. It is suggested that activation of protein kinase C by phorbol esters or physiological stimuli (hormones that activate phosphoinositide turnover, such as vasopressin or angiotensin II) modulate the hepatocyte alpha 1-adrenergic responsiveness.     

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.     

Norepinephrine- and phorbol ester-induced phosphorylation of alpha(1a)-adrenergic receptors. Functional aspects

Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.     

Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes

We investigated the role of arrestins in the trafficking of human alpha2-adrenergic receptors (alpha2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. alpha2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha2c > alpha2b > alpha2a. However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha2c AR was selectively promoted by coexpression of arrestin-3, while alpha2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha2b and alpha2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, alpha2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha2b, alpha2c, and to a lesser extent alpha2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for alpha2-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.     

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.     

Signalling pathways evoked by alpha1-adrenoceptors in human melanoma cells

The biological effects of catecholamines in mammalian pigment cells are poorly understood, but in poikilothermic vertebrates they regulate the translocation of pigment granules. We have previously demonstrated in SK-Mel 23-human melanoma cells the presence of low affinity alpha(1)-adrenoceptors, which mediate a decrease in cell proliferation and increase in tyrosinase activity, with no change of tyrosinase expression. In this report, we investigated the signalling pathways involved in these responses. Calcium mobilization in response to phenylephrine (PHE), an alpha(1)-adrenergic agonist, was investigated by confocal microscopy, and no change of fluorescence during the treatment was observed, suggesting that calcium is not involved in the signalling pathway activated by alpha(1)-adrenoceptors in SK-Mel 23 cells. cAMP levels, determined by enzyme-immunoassay, were significantly increased by PHE (10(-5)-10(-4)M), that could be blocked by the alpha(1)-adrenergic antagonist benoxathian (10(-5)-10(-4)M). Several biological assays were then performed with PHE, for 72 h, in the absence or presence of various signalling pathway inhibitors, in an attempt to determine the intracellular messengers involved in the responses of proliferation and tyrosinase activity. Our results suggest the participation of p38 and ERKs in PHE-induced decrease of proliferation, and possibly also of cAMP and protein kinase A. Regarding PHE-induced increase of tyrosinase activity, it is suggested that the following signalling components are involved: cAMP/PKA, PKC, PI3K, p38 and ERKs.     

Alpha 1- and beta-adrenergic regulation of intracellular Ca2+ levels in brown adipocytes

In order to monitor changes in cytosolic Ca2+ levels, brown-fat cells were incubated with the fluorescent Ca2+-indicator fura-2 and the fluorescence intensity ratio followed. The addition of norepinephrine led to a rapid and persistent increase in the cytosolic Ca2+ level, which was dose-dependent with a maximal effect at about 1 microM. The response was diminished in the absence of extracellular Ca2+ and was inhibited more efficiently by phentolamine and prazosin than by propranolol or yohimbine, indicating alpha 1-adrenergic mediation. Accordingly, selective alpha 1-adrenergic stimulation also increased the cytosolic Ca2+ level. However, selective beta-adrenergic stimulation, as well as the adenylate cyclase activator forskolin, were also able to increase the cytosolic Ca2+ level in these cells to a certain extent. It was concluded that the major part of the increase in cytosolic Ca2+ was mediated, as in other cell types, via alpha 1-adrenergic receptors, but that Ca2+ levels were also positively modulated by a cAMP-mediated process. These observations are discussed in relation to known alpha 1/beta synergisms in brown adipose tissue.     

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.     

Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors.     

Effect of insulin on alpha1-adrenergic actions in hepatocytes from euthyroid and hypothyroid rats. Possible involvement of two pathways in alpha1-adrenergic actions

The effect of insulin on the alpha1-adrenergic stimulation of glycogenolysis and ureogenesis, which is very small or undetectable in hepatocytes from control animals, is marked in hepatocytes from hypothyroid rats; the metabolic actions due to alpha1-adrenergic activation, but not those due to glucagon, were nearly blocked by insulin in cells from hypothyroid rats. The alpha1-adrenergic-mediated stimulation of phosphatidylinositol labelling was not affected by insulin in cells from either control or hypothyroid rats. The data suggest that the alpha1-adrenergic action proceeds through two pathways, one of which is very sensitive to insulin and predominates in cells from hypothyroid rats.     

Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes

We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E.G. Schuetz et al., 1990 J. Biol. Chem. 265, 1188-1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or "phenobarbital-like" inducers to the culture medium inhibited induction of P450b/e mRNA (46-90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with delta-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1(IIIA1) and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1) mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1) mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1) and P450PCN2(IIIA2) are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis.     

Rapid reconstitution of a transmembrane protein into supported planar lipid membranes

In hepatocytes from control rats, the ureogenic action of epinephrine is mainly mediated through alpha 1-adrenoceptors and the effect is independent of the presence of extracellular calcium. In hepatocytes from adrenalectomized rats, both alpha 1- and beta-adrenoceptors are involved in the action of epinephrine. Furthermore, the alpha 1-adrenergic-mediated stimulation of ureogenesis in these cells is dependent on the presence of extracellular calcium. Our results indicate that glucocorticoids modulate the calcium dependency of alpha 1-adrenergic effects and are consistent with our suggestion that two pathways are involved in the transduction of the alpha 1-adrenergic signal.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

Negative control of norepinephrine on the thyroid cyclic AMP system

Negative control on the thyroid cyclic AMP system has been studied. The increase of cyclic AMP levels induced by TSH in dog thyroid slices and its consequent secretion were inhibited by norepinephrine. This effect was different from the previously described activation of cyclic AMP disposal by acetylcholine: it was not calcium-dependent, was observed in the presence of isobutyl methylxanthine and was not inhibited by atropine. The inhibitory action of norepinephrine was abolished by phentolamine but not by L-propranolol. Clonidine and epinephrine also markedly inhibited the elevation of cyclic AMP levels, but phenylephrine did not. The inhibitory effect of norepinephrine and clonidine was abolished by yohimbine but not by prazosin. These results suggest that the effect of adrenergic agents on dog thyroid follicular cells is mediated by alpha 2-receptors. Similar results were obtained on dog thyroid adenylate cyclase activity: norepinephrine diminished the activation of adenylate cyclase induced by TSH, in a sodium-dependent process. This inhibition was abolished by phentolamine and yohimbine, but not by L-propranolol and and prazosin. This shows that the negative alpha 2-adrenergic effect bears directly on adenylate cyclase.