Lipolysis and beta-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats

Adipocytes from spontaneously hypertensive rats demonstrated a blunted lipolytic response to isoproterenol and dibutyryl cyclic AMP. (-)-[3H]Dihydroalprenolol binding was examined in adipocytes from normotensive and spontaneously hypertensive rats. Increasing concentrations of isoproterenol decreased total (-)-[3H]dihydroalprenolol binding to intact cells from normotensive rats, and the efficacy of competition was decreased in adipocytes from spontaneously hypertensive rats. Scatchard analysis indicated that the number of (-)-[3H]dihydroalprenolol binding sites and the affinity of dihydroalprenolol binding were comparable between normotensive and spontaneously hypertensive rats. Isoproterenol- and Gpp(NH)p-stimulated adenylate cyclase activity was consistently depressed in adipocyte membranes from spontaneously hypertensive rats as compared to normotensive rats. No difference in fluoride-stimulated adenylate cyclase activity was observed. The blunted lipolytic and cyclic AMP response to isoproterenol in these cells suggest a postreceptor lesion of the lipolytic pathway (possibly the guanine nucleotide regulatory protein) in adipocytes from spontaneously hypertensive rats. The blunted lipolytic response to dibutyryl cyclic AMP suggests defective regulation of lipolytic enzymes at the protein kinase-hormone-sensitive lipase level.     

Enhanced lipolysis is not evident in adipocytes from exercise-trained SHR

Adipocytes from spontaneously hypertensive rats (SHR) are not as responsive to isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulation compared with Sprague-Dawley or Wistar-Kyoto rats. Lipolytic activity in adipocytes from trained normotensive rats was enhanced in response to 1 microM isoproterenol and 0.5 mM dibutyryl cAMP but not in adipocytes from trained SHR. Decreases in isoproterenol-stimulated (1 microM) cAMP accumulation were evident in adipocytes from trained normotensive rats but not in adipocytes from trained SHR. Basal and agonist-induced lipolysis in fat cells isolated from both normotensive rats and SHR immediately following a 60-min run was increased in both sedentary and trained rats. Adenylate cyclase activity in fat cell membranes was blunted in sedentary and trained SHR both in the absence and presence of 100 microM 5'-guanylyl imidophosphate. No apparent differences existed in antagonist affinity of binding sites for the antagonist dihydroalprenolol in normal rats or SHR. Evidence for a change in affinity of agonist isoproterenol might be indicated based on the enhanced potency of isoproterenol to stimulate lipolysis in trained normal rats. beta-Adrenergic receptor density and antagonist affinity were not different in normotensive rats and SHR in response to training. However, displacement of [3H]dihydroalprenolol in adipocytes from SHR required greater concentrations of isoproterenol compared with adipocytes from normotensive rats, further suggestive of increased agonist affinity of binding sites in normal rats. These data suggest a postreceptor lesion of the lipolytic pathway in adipocytes from spontaneously hypertensive rats, possibly at the guanine nucleotide regulatory protein level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced number of beta-adrenergic receptors in the myocardium of spontaneously hypertensive rats.

Inotropic response to β-adrenergic stimulation of the myocardium is decreased in hypertension. A biochemical basis for this decrease was provided by the observation that the number of β-adrenergic receptors — as reflected in specific [³H]dihydroalprenolol binding — was diminished in the myocardium of spontaneously hypertensive rats without a change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in β-adrenergic receptor numbers is not secondary to blood pressure elevation and may be related to increased sympathetic drive in spontaneously hypertensive rats.     

Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp[NH]p, isoproterenol plus GTP or Gpp[NH]p was significantly greater in the SHR. Beta-receptor density measured by [3H]DHA binding was unaltered. The dissociation constant, Kd, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 +/- 0.45 nM) compared with the WKY (8.53 +/- 0.82 nM). Activity of Gs was assessed by complementing S49 cyc- acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp[NH]p, and isoproterenol plus Gpp[NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary Gs in the hypertensive rats.     

In vitro characterization of skeletal muscle beta-adrenergic receptors coupled to adenylate cyclase.

[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify the adenylate cyclase-coupled beta-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 . 10(7) M-1 . min-1 and 3.21 . 10(-1) min-1, respectively, were obtained. The dissociation constant (Kd) of 15 mM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the Kd derived from the ratio of dissociation and association rate constants (K2/K1). Several beta-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol greater than epinephrine greater than norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (-)-isomers being more potent than (+)-isomers. Phenylephrine, an alpha-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known beta-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Ki values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the beta-adrenergic receptor.     

Effects of local anesthetics on guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on beta-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2--20 mM) reduces the responsiveness of adenylate cyclase to (a) guanyl-5'-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5'-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5'-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10(-7) M. Tetracaine also competitively inhibited binding of both the antagonist [3H]dihydroalprenolol and the agonist [3H]hydroxybenzylisoproterenol to beta-adrenergic receptors. However, it was twice as potent in inhibiting [3H]hydroxybenzylisoproterenol as [3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the beta-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylatecyclase and in dissociating high-affinity agonist-receptor complexes. The other of potency for both processes was dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine which agrees with their relative potencies as local anesthetics. By contrast, a different order of potency was observed for competitive inhibition of [3H]dihydroalprenolol binding: dibucaine greater than tetracaine greater than greater than lidocaine greater than bupivacaine.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands.The specific binding of the labelled beta-adrenergic antagonist [³H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [³H]-dihydroalprenolol compared with normal tissue.Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [³H]dihydroalprenolol binding sites during isoproterenol-induced growth.Previously-described differences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Decreased number of beta-adrenergic receptors in hypertensive vessels.

Responsiveness to inotropic agents is altered in hypertension and may contribute to its initiation and maintenance. A biochemical basis for this change was provided by the observation that the number of beta-adrenergic receptors, as reflected in specific [3H]dihydroalprenolol binding, was diminished in both arteries and veins of spontaneously hypertensive rats. There was no change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in beta-adrenergic receptor numbers is not secondary to blood pressure elevation but may, instead, contribute to the pathogenesis of hypertension.     

Binding of [14,15-3H]14,15-dihydroforskolin to rat liver membranes: comparison with the stimulatory effect of forskolin on adenylate cyclase

The binding of [14,15-3H]14,15-dihydroforskolin ([3H]DHF) to rat liver membranes has been further characterized and was compared with the stimulatory effect of forskolin on adenylate cyclase. The binding equilibrium dissociation constant (KD) for 14,15-dihydroforskolin obtained in inhibition experiments was 0.6 microM, with a maximal binding capacity (Bmax) of 114 pmol/mg protein. A similar KD value (0.5 microM) was derived from kinetics studies that revealed very rapid association and dissociation reactions. For structure-activity relationship studies several forskolin derivatives were synthesized and tested for their ability to inhibit [3H]DHF binding and increase adenylate cyclase activity. Among the tested compounds, forskolin itself was the most potent agonist (K1 = 0.2 microM). Further modification of the molecule in position 7 and (or) 1 decreased or abolished its agonist properties in both adenylate cyclase and binding studies. [3H]DHF binding was not affected by several nucleotides, carbohydrates, lectins, and hormone receptor agonists including isoproterenol, glucagon, and adenosine, but the steroids 17-beta-estradiol, progesterone, and testosterone showed slight inhibitory effects at unphysiologically high concentrations. [3H]DHF binding and forskolin-stimulated adenylate cyclase were sensitive to heat and N-ethylmaleimide treatment. Forskolin protected adenylate cyclase against inactivation by heat but not by N-ethylmaleimide. Preincubation of the membrane with trypsin decreased [3H]DHF binding. The results presented in this study demonstrate that the binding sites identified with [3H]DHF have a high specificity for forskolin and provide evidence that these binding sites are involved in the stimulation of adenylate cyclase by forskolin.     

Decreased number of beta-adrenergic receptors in hypertensive vessels

Responsiveness to inotropic agents is altered in hypertension and may contribute to its initiation and maintenance. A biochemical basis for this change was provided by the observation that the number of β-adrenergic receptors, as reflected in specific [³H]dihydroalprenolol binding, was diminished in both arteries and veins of spontaneously hypertensive rats. There was no change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in β-adrenergic receptor numbers is not secondary to blood pressure elevation but may, instead, contribute to the pathogenesis of hypertension.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Effects of local anesthetics of guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on β-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2–2.0 mM) reduces the responsiveness of adenylate cyclose to (a) guanyl-5′-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5′-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5′-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10^?7 M.Tetracaine also competitively inhibited binding of both the antagonist [³H]-dihydroalprenolol and the agonist [³H]hydroxybenzylisoproterenol to β-adrenergic receptors. However, it was twice as potent in inhibiting [³H]-hydroxybenzylisoproterenol as [³H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the β-adrenergic receptor induced by agonists.Other local anesthetics mimicked the effects of tetracaine on adenylate     

Age-Related Development of γ-Aminobutyric Acid (GABA)BReceptor Functions in Various Brain Regions of Spontaneously Hypertensive Rats

The age-related development of GABA_Breceptors and their coupling to adenylate cyclase were studied in the brains of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, the specific [³H]GABA binding to GABA_Breceptors showed a significant decrease not only in the posterior hypothalamus, midbrain, hippocampus and striatum of eleven-week-old SHR, which maintain a hypertensive state, but also in the posterior hypothalamus of four-week-old normotensive SHR. Similarly, the GABA_Breceptor agonists (baclofen and DN-2327)-induced suppression of adenylate cyclase activity showed a decrease in the posterior hypothalamus of four-week-old SHR as well as in the posterior hypothalamus and striatum of eleven-week-old SHR. These results suggest that the functions of the GABA_Breceptor in the brain of SHR may be decreased independently from the occurrence of blood pressure elevation and that such changes may even be involved in the pathogenesis of SHR.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands.

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands. The specific binding of the labelled beta-adrenergic antagonist [3H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [3H]-dihydroalprenolol compared with normal tissue. Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [3H]dihydroalprenolol binding sites during isoproterenol-induced growth. Previously-described di-ferences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

Beta-adrenergic activity of (+/-)-hydroxybenzylisoproterenol in isolated rat fat cells and hepatocytes

The pharmacology of (+/-)-hydroxybenzylisoproterenol with respect to stimulation of cyclic AMP accumulation by isolated rat fat cells and liver cells was examined. (+/-)-Hydroxybenzylisoproterenol was found to be a full agonist and twice as potent as (-)-isoproterenol in liver cells, and equipotent to (-)-isoproterenol in fat cells with regard to stimulating cyclic AMP accumulation. A study of the ability of this catecholamine to stimulate adenylate cyclase activity of broken-cell preparations revealed that (+/-)-hydroxybenzylisoproterenol was equipotent to (-)-isoproterenol in liver cell homogenates, while 3- to 4-fold more potent than (-)-isoproterenol in fat cell ghost membranes. (+/-)-Hydroxybenzylisoproterenol was also found to be as potent as (-)-isoproterenol in stimulating cyclase activity of S49 mouse lymphoma cell membranes. Competition studies of specific [125I]iodohydroxybenzylpindolol binding to liver cell membranes revealed a Kd of 10 nM for (+/-)-hydroxybenzylisoproterenol and 25 nM for (-)-isoproterenol binding to the liver beta-adrenergic receptor. Competition studies of specific (-)-[3H]dihydroalprenolol binding to fat cell membranes indicated a similar affinity of these sites for both (+/-)-hydroxybenzylisoproterenol and (-)-isoproterenol. The guanyl nucleotide Gpp(NH)p induced a shift in the curve for competition of (-)-[3H]dihydroalprenolol binding by (-)-isoproterenol to the right, but failed to do so when (+/-)-hydroxybenzylisoproterenol was the competing agonist. Properties of (+/-)-[3H]hydroxybenzylisoproterenol binding to fat cell or liver cell membranes were inconsistent with those expected of adenylate cyclase coupled beta-adrenergic receptors.     

Beta-adrenergic receptor-adenylate cyclase alterations during the postnatal development of skeletal muscle

The postnatal development of mammalian skeletal muscle is associated with an increased capacity for glycogenolysis. In the present study rabbit skeletal muscle underwent a 7-fold increase in glycogen synthase and glycogen phosphorylase activity over the postnatal period of 0--8 weeks. An enriched fraction of sarcolemma was prepared from neonatal and adult muscle to examine the development of the beta-adrenergic receptor-adenylate cyclase system. Adult membranes possessed a 2-fold greater Na+K+(Mg2+)-ATPase activity and a 6--8 fold greater sodium fluoride- and epinephrine-stimulated adenylate cyclase activity. The activation ratio (effector activity/basal activity) increased 2--3 fold for epinephrine and sodium fluoride in adult sarcolemma. The activation by catecholamines conformed to the physiological beta 2 type response with isoproterenol (1.8 . 10(-8) M) > epinephrine (1.1 . 10(-7) M) > norinephrine (3.2 . 10(-6) M). In contrast, binding studies employing (-)-[3H]dihydroalprenolol showed little difference between neonatal and adult membranes with respect to (1) number of binding sites, (2) equilibrium dissociation constant and (3) displacement of (-)-[3H]dihydroalprenolol by catecholamine agonists. Protein and lipid components of the sarcolemma were also modified during development. Neonatal membranes possessed two glycopeptides of Mr 80000 and 86000, whereas in the adult only a single Mr 113000 species was evident. The total lipid phosphorus and phospholipid composition was unchanged during development. The content of linoleic acid increased approx. 3-fold during development in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids. The cholesterol content of adult membranes was decreased by 29% compared to neonatal membranes.