Alterations in rat aortic alpha 1-adrenoceptors and alpha 1-adrenergic stimulated phosphoinositide hydrolysis in intraperitoneal sepsis

We investigated the alterations of rat aortic alpha 1-adrenoceptors and alpha 1-adrenergic stimulated phosphoinositide (PI) metabolism in intraperitoneal sepsis. An analysis of [125I]-hydroxyethylaminotetralone (HEAT) binding to alpha 1-adrenoceptors on rat aortic membranes revealed decreased numbers of receptors without changes in affinity. The maximum number of binding sites decreased from 349 +/- 35 fmol/mg to 146 +/- 16 fmol/mg (P less than 0.05 vs. control). PI metabolism was similarly attenuated in aortae from septic rats. The norepinephrine-stimulated hydrolysis of [32P]-phosphatidylinositol-4,5-bisphosphate was significantly decreased in aortae from septic rats as was the alpha 1-adrenoceptor stimulated accumulation of [3H]-inositol monophosphate. Finally, the basal labeling of [32P]-phosphatidylinositol-4,5-bisphosphate but not of [32P]-phosphatidylinositol or [32P]-phosphatidic acid was significantly diminished. These results imply that signal transduction induced by alpha 1-adrenoceptor agonists in rat aorta is significantly altered in intraperitoneal sepsis. These findings may help define the mechanisms of depressed aortic contractility in models of sepsis and endotoxic shock.     

Normal ontogeny of alpha 1-adrenergic receptor in rat liver is thyroid hormone dependent

Postnatal ontogeny of rat liver alpha 1-adrenergic receptor was examined using alpha 1-specific radioligand [3H]prazosin in control and propylthiouracil-treated congenital hypothyroid rats at various ages. Partially purified rat liver membranes prepared by the Neville method had 8-fold purification of 5'-nucleotidase from the crude homogenates from postnatal day 5 to adulthood. [3H]Prazosin binding was typical of an alpha 1-adrenergic receptor, and (-)epinephrine affinity for the [3H]prazosin-binding sites was not altered in the presence of 10(-5) M guanylyl-imidodiphosphate. The receptor density was lower in 5- and 15-day-old rats than in 28-day-old or older rats in both control and hypothyroid groups. (P less than 0.01). At 28-34 days of age, hypothyroid pups had significantly lower alpha 1-receptor density than controls (399 +/- 10 vs. 869 +/- 40 fmol mg protein-1; P less than 0.01). Replacement therapy with daily T4 injection from postnatal days 16-27 restored 54% of the deficit in PTU-treated hypothyroid pups at 28 days. The dissociation constant of [3H] prazosin did not change with advancing age or with different treatment and was consistent at 0.1 nM. These findings indicate that the normal ontogeny of plasma membrane alpha 1-adrenergic receptors is dependent upon thyroid hormone and matures postnatally in rat liver.     

Differential acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to alpha 1-adrenergic and phorbol ester stimulation

Both alpha 1-adrenergic agonists (e.g. norepinephrine, NE*) and tumor-promoting phorbol esters (e.g. phorbol myristate acetate, PMA) are known to activate protein kinase C (PKC) (Abdel-Latif, 1986, Niedel and Blackshear, 1986). However, alpha 1 agonists and PMA produce very different effects on cardiac function (see Simpson, 1985; Benfey, 1987; Meidell et al., 1986; Leatherman et al., 1987; Yuan et al., 1987; for examples). PKC activation in heart cells has been studied only for PMA treated perfused heart (Yuan et al., 1987). Therefore, acute activation and chronic regulation of PKC by NE and PMA were compared in cultured neonatal rat heart myocytes. NE acutely and transiently activated PKC, as measured by translocation of PKC activity to the cell particulate fraction (Niedel and Blackshear, 1986). Particulate PKC activity peaked at 23% of total after NE for 30 s, as compared with 8% for control (P less than 0.001). By contrast, acute PKC activation by PMA was more pronounced and persistent, with particulate PKC activity 62% of total at 5 min (P less than 0.001). Calcium/lipid-independent kinase activity increased acutely with PMA, but not with NE. Chronic treatment with NE (24 to 48 h) increased total per cell PKC activity and 3H-phorbol dibutyrate (PDB) binding sites, an index of the number of PKC molecules (Niedel and Blackshear, 1986), by 30 to 60% over control (all P less than 0.05 to 0.01). In contrast with NE, chronic treatment with PMA down-regulated PKC, reducing total per cell PKC activity and 3H-PDB binding sites to 3% and 12% of control, respectively (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

MEK1/2-ERK1/2 mediates alpha1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes

We examined the relative roles of the mitogen-activated protein kinases (MAPK) in mediating the alpha1-adrenergic receptor (alpha1-AR) stimulated hypertrophic phenotype in adult rat ventricular myocytes (ARVM). Norepinephrine (NE; 1 microM) in the presence of the beta -AR antagonist propranolol (Pro; 2 microM) caused activation of Ras (>six-fold), MAPK/ERK kinase 1 and 2 (MEK1/2, >10-fold) and extracellular signal-regulated kinases 1 and 2 (ERK1/2, approximately 30-fold) within 5 min, as determined by kinase activity assays and Western blots using phospho-specific antibodies. Conversely, p38 and c-Jun amino-terminal kinases (JNK) were not activated by NE/Pro. Activated MEK1/2 signals remained detectable at 2 h, and activated ERK1/2 remained detectable at 48 h. The alpha1-AR selective inhibitor prazosin (100 nM) completely inhibited the NE/Pro-stimulated activation of Ras, MEK1/2 and ERK1/2. The MEK inhibitor PD98059 caused a concentration-dependent inhibition of NE/Pro-stimulated protein synthesis (as assessed by [3H]leucine incorporation and cellular protein accumulation) and ERK1/2 activation, with approximately 50% inhibition at a concentration between 10 and 50 microM, which is consistent with the known IC50 values of PD98059 for MEK1 (4 microM) and MEK2 (50 microM). Thus, these data show that alpha1-AR stimulated hypertrophy in ARVM is dependent on the MEK1/2-ERK1/2 signaling pathway.     

Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function.

Regions of the hamster alpha 1-adrenergic receptor (alpha 1 AR) that are important in GTP-binding protein (G protein)-mediated activation of phospholipase C were determined by studying the biological functions of mutant receptors constructed by recombinant DNA techniques. A chimeric receptor consisting of the beta 2-adrenergic receptor (beta 2AR) into which the putative third cytoplasmic loop of the alpha 1AR had been placed activated phosphatidylinositol metabolism as effectively as the native alpha 1AR, as did a truncated alpha 1AR lacking the last 47 residues in its cytoplasmic tail. Substitutions of beta 2AR amino acid sequence in the intermediate portions of the third cytoplasmic loop of the alpha 1AR or at the N-terminal portion of the cytoplasmic tail caused marked decreases in receptor coupling to phospholipase C. Conservative substitutions of two residues in the C terminus of the third cytoplasmic loop (Ala293----Leu, Lys290----His) increased the potency of agonists for stimulating phosphatidylinositol metabolism by up to 2 orders of magnitude. These data indicate (i) that the regions of the alpha 1AR that determine coupling to phosphatidylinositol metabolism are similar to those previously shown to be involved in coupling of beta 2AR to adenylate cyclase stimulation and (ii) that point mutations of a G-protein-coupled receptor can cause remarkable increases in sensitivity of biological response.     

Agonist-induced down-regulation of muscarinic cholinergic and alpha 2-adrenergic receptors after inactivation of Ni by pertussis toxin

Desensitization of the responsiveness to hormones or drugs is often mediated by down-regulation of receptors. The stimulatory coupling protein (Ns) of adenylate cyclase has been shown to be involved in the down-regulation of stimulatory beta-adrenergic receptors. Whether the inhibitory coupling protein (Ni) is involved in the down-regulation of receptors that inhibit adenylate cyclase is not known. We wished to determine whether down-regulation of inhibitory muscarinic cholinergic and alpha 2-adrenergic receptors occurs in neuroblastoma X glioma hybrid cells after the ability of Ni to inhibit adenylate cyclase is inactivated by pertussis toxin. After treatment of cells with pertussis toxin, the ability of carbachol or epinephrine to inhibit prostaglandin E1-stimulated cAMP accumulation in intact cells was either completely prevented or markedly attenuated, respectively, indicating functional inactivation of Ni. Furthermore, pertussis toxin treatment of membrane fragments from these cells did not result in labeling of the 41,000-dalton alpha-subunit of Ni with ADP ribose from [32P] NAD, indicating maximal ADP ribosylation of Ni by prior treatment of cells with pertussis toxin. Carbachol treatment of cells resulted in down-regulation of muscarinic cholinergic receptors to 45.7 +/- 12.5% and 52.5 +/- 13.5% of control values for toxin-untreated and toxin-treated cells, respectively. Epinephrine treatment of cells caused homologous desensitization of alpha 2-receptor-mediated inhibition of cAMP accumulation and down-regulation of alpha 2-adrenergic receptors to 42.9 +/- 11.4% and 53.2 +/- 5.3% of control values for toxin-untreated and toxin-treated cells, respectively. Down-regulation of muscarinic cholinergic receptors by carbachol and of alpha 2-adrenergic receptors by epinephrine was not due to the effect of retained agonist and was agonist specific, since it could be prevented by the antagonists atropine and yohimbine, respectively. We conclude that agonist-mediated down-regulation of both the muscarinic cholinergic receptor and the alpha 2-adrenergic receptor does not require functional inhibitory coupling.     

Alpha1-adrenergic stimulation induced hypertrophy in protein kinase C down-regulated cultured cardiac myocytes

To examine whether protein kinase C (PKC) activation is essential for the induction of cardiac myocyte hypertrophy caused by alpha1-adrenergic stimulation, we investigated the hypertrophic effect of phenylephrine in PKC down-regulated and non-treated cultured cardiac myocytes obtained from neonatal Sprague-Dawley rat ventricles. The treatment with 10 nmol/L 12-tetra decanoylphorbol-13-acetate (TPA) for more than 2 hours decreased PKC activity by approximately 80% without marked hypertrophy. Phenylephrine increased [14C] phenylalanine (Phe) incorporation in both TPA non-treated and treated cells, 1.54- and 1.71-fold as large as control, respectively. The cell surface area also enlarged in both groups, 1.67- and 1.74-fold, respectively. Thus, phenylephrine induced the similar grade hypertrophy in cultured cardiac myocytes even when PKC was down-regulated. These results suggest that conventional PKC activation may not be essential for mediating myocyte hypertrophy by alpha1-adrenergic stimulation.     

Characterization of alpha 1-adrenergic receptors in perfused rat heart

The characteristics of alpha 1-adrenergic receptors were investigated in perfused rat hearts at 37 degrees C. [3H]Prazosin was bound in a time-dependent manner and reached equilibrium at 15 min. Scatchard analysis of the specific binding isotherm for [3H]prazosin indicated a population of high affinity sites (Kd = 0.41 nM, Bmax = 13.2 pmol/g wet wt). Prazosin binding was displaced by epinephrine as well as by the adrenergic antagonists prazosin greater than phentolamine greater than yohimbine greater than propranolol. Specific prazosin binding was defined as that portion of the binding inhibited by 10 microM phentolamine; phentolamine and epinephrine displaced 3H-prazosin to the same level. [3H]Prazosin was not metabolized by the heart. When pre-labelled hearts were perfused at 37 degrees C with prazosin-free medium non-specific binding of [3H]prazosin decreased more rapidly (t0.5 = 4 min) than specific binding (t0.5 = 38 min). Perfusion of the heart at lower temperatures (less than 10 degrees C) decreased the rate of loss of nonspecific binding and prevented the loss of specific binding. Fractionation of [3H]prazosin perfused hearts at 0 degrees C, when dissociation was minimal, led to a loss of binding so that sarcolemma-enriched fractions contained approximately 2% of the binding sites present in the perfused heart. The binding characteristics of sarcolemma-enriched fractions (Kd 0.10 nM, Bmax 300 fmol/mg protein) differed significantly from those of the perfused heart. Exposure of the heart to 10 min of ischaemia prior to binding studies did not alter the characteristics of the [3H]prazosin binding sites. It is concluded that the perfused rat heart contains a population of alpha 1-adrenoceptors which differ from those of isolated sarcolemma preparations perhaps because of alterations that occur during sarcolemma isolation. The perfused heart should be an appropriate model system in which to study the relationship between receptor occupancy and biological response as well as the direct effects of perturbations such as ischaemia.     

Nuclear alpha1-adrenergic receptors signal activated ERK localization to caveolae in adult cardiac myocytes

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.     

Long-chain acylcarnitines mediate the hypoxia-induced increase in alpha 1-adrenergic receptors on adult canine myocytes

To elucidate the mechanisms responsible for the increase in alpha 1-adrenergic receptors during ischemia in vivo, we developed a procedure for measuring alpha 1-adrenergic receptors in isolated, calcium-tolerant adult canine myocytes. Specific [3H]prazosin binding was rapid, saturable, reversible, and demonstrated the expected order of potency and stereospecificity for the alpha 1-adrenergic receptor. Myocytes exposed to 30 minutes of hypoxia at 25 degrees C or only 10 minutes at 37 degrees C exhibited a twofold to threefold increase in the number of alpha 1-adrenergic receptors with no significant change in receptor affinity. This hypoxia-induced increase in receptor number was reversible by 10 minutes of reoxygenation at 37 degrees C. In contrast, more prolonged hypoxia of 80 minutes or hypotonic shock actually decreased receptor number below normoxic, control values. The concentration of long-chain acylcarnitines in myocytes also increased threefold on exposure to 30 minutes of hypoxia. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA, 10 microM), a potent inhibitor of carnitine acyltransferase I, not only abolished the accumulation of long-chain acylcarnitines but also the increase in alpha 1-adrenergic receptor number induced by 30 minutes of hypoxia. Likewise, incubation of normoxic cells with exogenous palmitoyl carnitine (1 microM) for 10 minutes also increased alpha 1-adrenergic receptor number in the presence or absence of POCA. Thus, hypoxia results in an increase in alpha 1-adrenergic receptors associated with an increase in endogenous long-chain acylcarnitines. Furthermore, inhibition of carnitine acyltransferase I prevents not only the sarcolemmal accumulation of long-chain acylcarnitines but also the exposure of the alpha 1-adrenergic receptor, indicating that accumulation of endogenous long-chain acylcarnitines is critical to the hypoxia-induced increase in alpha 1-adrenergic receptors on adult myocytes.     

Endothelin stimulates phosphatidylinositol hydrolysis in rat vascular smooth muscles

The ability of endothelin to stimulate phosphatidylinositol hydrolysis in rings of rat aorta was studied. Endothelin 10(-8)-10(-5) mol/l caused increases of 200-1000% in inositol phosphate levels. However, physiological responses to endothelin have been reported in the concentration range 10(-10)-10(-8) mol/l. Thus phosphatidylinositol hydrolysis as measured in the present study does not correlate directly with functional responses. Phosphatidylinositol hydrolysis in response to endothelin was attenuated but not abolished by removal of endothelium. Attenuation of inositol phosphate production was also observed with time, consistent with the hypothesis that continuous exposure to the agonist can cause desensitization of the endothelin receptor.     

Alpha 1-adrenergic agonists selectively suppress voltage-dependent K+ current in rat ventricular myocytes.

The effects of alpha 1-adrenergic agonists on the waveforms of action potentials and voltage-gated ionic currents were examined in isolated adult rat ventricular myocytes by the whole-cell patch-clamp recording technique. After "puffer" applications of either of two alpha 1 agonists, phenylephrine and methoxamine, action-potential durations were increased. In voltage-clamped cells, phenylephrine (5-20 microM) or methoxamine (5-10 microM) reduced the amplitudes of Ca2+-independent voltage-activated outward K+ currents (Iout); neither the kinetics nor the voltage-dependent properties of Iout were significantly affected. The effects of phenylephrine or methoxamine on Iout were larger and longer-lasting at higher concentrations and after prolonged or repeated exposures; in all experiments, however, Iout recovered completely when puffer applications were discontinued. The suppression of Iout is attributed to the activation of alpha 1-adrenergic receptors, as neither beta- nor alpha 2-adrenergic agonists had measurable effects on Iout; in addition, the effect of phenylephrine was attenuated in the presence of the alpha antagonist phentolamine (10 microM), but not in the presence of the beta antagonist propranolol (10 microM). Voltage-gated Ca2+ currents, in contrast, were not altered measurably by phenylephrine or methoxamine and no currents were activated directly by these agents. Suppression of Iout was also observed during puffer applications of either of two protein kinase C activators, phorbol 12-myristate 13-acetate (10 nM-1 microM) and 1-oleoyl-2-acetylglycerol (60 microM). We conclude that the activation of alpha 1-adrenergic receptors in adult rat ventricular myocytes leads to action-potential prolongation as a result of the specific suppression of Iout and that this effect may be mediated by activation of protein kinase C.     

Isolation and characterization of beta 1-adrenergic receptors from adult, rat cardiac myocytes

The beta-receptors were isolated from rat cardiac myocytes and characterized. Isolated myocytes were prepared from adult rat hearts and characterized for viability. Membrane proteins were solubilized from myocytes with 1% Triton X-102. The solubilized membrane proteins were fractionated by DEAE-Sephacel ion exchange column chromatography. Two major protein peaks were obtained. The second protein peak sample was found to contain beta-receptors to which 125I-15-(4'-azido-3'-iodobenzyl)-carazolol (125I-ABC) was specifically bound. This sample was labeled covalently with 125I-ABC by UV irradiation. The radiolabeled sample was applied to a Sepharose CL-6B gel column. Two radiolabeled protein peaks, one with a molecular weight of approximately 570,000 and the other with a molecular weight of approximately 95,000 were found. When the 570,000-dalton complex was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, it was dissociated into a component with a molecular weight of 66,000. The 95,000-dalton complex was dissociated into a 58,000-dalton component upon SDS-PAGE under reducing conditions. An excess amount of isoproterenol and propranolol decreased photolabeling of the beta-receptors with 125I-ABC by 60% and 40%, respectively.     

Alpha 1-adrenergic and muscarinic cholinergic stimulation of phosphoinositide hydrolysis in adult rat cardiomyocytes

Cardiac alpha-adrenergic receptors mediate cellular responses to norepinephrine through an undefined series of molecular events. We examined the possibility that phosphoinositide hydrolysis was stimulated through alpha-adrenergic receptors in cardiomyocytes purified from adult rat ventricle. Phosphoinositide stores were labeled with [3H]inositol, and [3H]inositol phosphate formation was assessed after the addition of lithium chloride and norepinephrine. Norepinephrine increased the accumulation of [3H]inositol phosphate by approximately 5-fold, giving a maximal response at approximately 30 microM and a half-maximal response at approximately 1 microM. There was a significant increase in [3H]inositol phosphate formation in response to norepinephrine at 5 minutes, and the response was linear over 40 minutes. Norepinephrine-stimulated [3H]inositol phosphate formation was not blocked by propranolol (1 microM) or yohimbine (0.1 microM) but was completely antagonized by the alpha 1-selective antagonist prazosin (0.1 microM). Muscarinic cholinergic receptor activation by carbachol also stimulated [3H]inositol phosphate formation in rat ventricular myocytes. The maximal effect of carbachol (approximately 2-fold) was always less than that of norepinephrine. The combined effects of norepinephrine and carbachol were additive, suggesting that the two hormones do not share a common rate-limiting step. Removal of extracellular calcium and addition of ethylene glycol bis(beta-amino ether)-N,N'-tetraacetic acid, attenuated, but did not abolish, norepinephrine- or carbachol-stimulated [3H]inositol phosphate formation. Neither the calcium ionophore A23187 nor the calcium channel blockers verapamil and nifedipine had any effect on basal or hormone-stimulated [3H]inositol phosphate formation. We suggest that some of the physiological and metabolic effects of adrenergic and cholinergic stimulation on the rat myocardium are secondary to receptor-mediated hydrolysis of phosphoinositides.     

Thyrotropin upregulates alpha 1-adrenergic receptors in rat FRTL-5 thyroid cells.

FRTL-5 rat thyroid cells grown and maintained in a medium containing 0.05 nM thyrotropin have a 10-fold higher number of alpha 1-adrenergic receptors on their cell surface than FRTL-5 cells maintained in the absence of thyrotropin in their medium. The increased number of alpha 1-adrenergic receptors, measured as increased specific [3H]prazosin binding per microgram of DNA, is not associated with any changes in Kd values for prazosin. Thyrotropin increases the number of alpha 1-adrenergic receptors by inducing their biosynthesis, as evidence by the inhibitory effects of cycloheximide or actinomycin D; the effect on biosynthesis is cAMP-mediated, since 8-bromoadenosine 3',5'-cyclic monophosphate, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine can mimic the thyrotropin effect in both extent and time course. The alpha 1-adrenergic receptors on FRTL-5 thyroid cells have been functionally linked to iodide efflux into the follicular lumen and to the iodination of thyroglobulin--i.e., to the formation of thyroid hormones; the alpha 1-adrenergic receptor signal is mediated by Ca2+ rather than by cAMP and involves arachidonic acid intermediates. The present data thus describe a unique upregulation phenomenon wherein the sequential expression of two receptors (thyrotropin and alpha 1-adrenergic) and two distinct signal systems (cAMP and Ca2+) are apparently a necessary prelude to thyroid hormone homoeostasis.     

Hepatic alpha 1-adrenergic receptor alteration in a rat model of chronic sepsis

Catecholamine therapy is often ineffective in reversing the peripheral vasodilatation and hypotension of septic shock. This suggests that catecholamines might not be able to activate alpha 1-adrenergic receptors to cause vasoconstriction. Despite elevations in endogenous catecholamines, hypoglycemia is also a complication of human sepsis, suggesting that among many other causes, hepatic alpha 1-receptors might be altered. To better understand the pathophysiologic basis for this pharmacologic dilemma, we studied the effect of experimental sepsis on alpha 1-adrenergic receptors in hepatic tissue, a rich source of alpha 1-receptors, from septic and control Sprague-Dawley rats. alpha 1-adrenergic receptors were measured with [3H]-prazosin and data analyzed by a computerized nonlinear least-square regression algorithm. Twenty-four hours following cecal ligation with puncture, a decreased number of alpha 1-adrenergic receptors was noted in crude and purified plasma membrane fractions (23 and 40% reductions respectively) from septic animals. No changes in either agonist or antagonist affinity for receptors from septic animals were noted. These data indicate that the catecholamine refractoriness seen in septic shock may be a result of alterations in alpha 1-adrenergic receptor number or receptor-effector coupling.