Receptor-adenylate Cyclase Research Articles

Calcium metabolism in Williams-Beuren syndrome

Increased 1,25-dihydroxyvitamin D levels and decreased basal and calcium-stimulated calcitonin serum levels have been found in children with Williams-Beuren syndrome (WBS). To determine whether isolated or combined disturbances of secretion or action of the calcium-regulating hormones may cause the tendency to hypercalcemia in WBS, we investigated several aspects of calcium metabolism in 27 normocalcemic children and adults, aged 2 to 47 years, with WBS. With the exception of slightly decreased 25-hydroxyvitamin D and slightly increased calcitonin in serum, all measured basal indexes of calcium and bone metabolism, including the serum levels of intact parathyroid hormone and 1,25-dihydroxyvitamin D, were comparable to control values. Total and extractable calcitonin, the latter representing the monomeric and biologically important form of the hormone, showed the same relative increase after a low-dose calcium infusion in patients and control subjects, indicating a normal capacity of the calcitonin-producing C cells of the thyroid gland in WBS. Furthermore, exogenous parathyroid hormone induced a normal response of 1,25-dihydroxyvitamin D, cyclic adenosine monophosphate, and phosphate excretion, indicating a normal response of the renal 25-hydroxyvitamin D-1 alpha-hydroxylase and the renal receptor-adenylate cyclase system to parathyroid hormone. These findings suggest that neither deficient calcitonin secretion nor increased renal sensitivity to parathyroid hormone is a feature of WBS in normocalcemic patients.

Binding sites of atrial natriuretic peptide (ANP) in the mammalian cochlea and stimulation of cyclic GMP synthesis

The distribution of binding sites for atrial natriuretic peptide (ANP) has been examined in frozen sections of the guinea pig inner ear by means of autoradiography. The highest density was found in the stria vascularis of all cochlear turns. In membrane preparations of stria vascularis in vitro, the production of the second messenger cGMP was strongly stimulated by synthetic ANP in a dose dependent manner. Adenylate cyclase was neither stimulated nor inhibited by ANP. thus suggesting, that the binding sites coincide with an ANP receptor, which is coupled to guanylate cyclase but not negatively coupled to an adenylate cyclase molecule. The production of cyclic GMP could not be reduced by GDP-βS, a strong inhibitor of the G s protein. We conclude the existence of an ANP receptor-guanylate cyclase signal transfer system, similar to the β2 receptor-adenylate cyclase system in the inner ear, without coupling to a G protein. ANP might play a role in sodium and water regulation of the endolymph and might antagonize the action of vasopressin.

Effects of beta-adrenergic blockade on papillary muscle function and the beta-adrenergic receptor system in noninfarcted myocardium in compensated ischemic left ventricular dysfunction.

beta-Adrenergic receptor blockade has been reported to improve hemodynamics and beta-adrenergic receptor-adenylate cyclase function in idiopathic dilated cardiomyopathy. The purpose of this study was to determine the effects of beta-adrenergic receptor blockade on the beta-adrenergic receptor system and myocardial function in a model of compensated ischemic heart failure. We examined the effects of propranolol treatment on the beta-adrenergic receptor-adenylate cyclase system and isolated papillary muscle isometric function in noninfarcted left ventricular myocardium in rats after coronary artery ligation. In untreated rats with large myocardial infarction (MI), developed tension (DT) (3.0 +/- 0.7 versus 5.1 +/- 1.1 g/mm2), peak rate of tension rise (+dT/dt) (40.3 +/- 9.5 versus 71.2 +/- 12.0 g/mm2/sec), and peak rate of tension fall (-dT/dt) (24.4 +/- 5.0 versus 38.2 +/- 6.0 g/mm2/sec) were decreased (p < 0.05). In addition, DT, +dT/dt, and -dT/dt of untreated MI rats demonstrated an impaired response to isoproterenol stimulation compared with controls. beta-Adrenergic receptor density (Bmax) measured by [125I]iodocyanopindolol (ICYP) binding was decreased 23% after infarction (9.3 +/- 0.6 versus 12.0 +/- 1.8 fmol/mg protein [prot]) (p < 0.05); however, the dissociation constant (Kd) for ICYP was not changed (24.1 +/- 5.7 versus 33.2 +/- 12.1 pM). Adenylate cyclase activity in the presence of 10(-2) M MgCl2 was reduced (p < 0.05) in MI rats (30.3 +/- 10.8 versus 45.9 +/- 12.5 pmol cAMP/min/mg prot). Maximal isoproterenol (52.5 +/- 7.3 versus 79.9 +/- 10.0 pmol cAMP/min/mg prot), guanyl-5'-imidodiphosphate (GppNHp) (95 +/- 8 versus 141 +/- 25 pmol cAMP/min/mg prot) and forskolin (503 +/- 76 versus 753 +/- 157 pmol cAMP/min/mg prot) stimulation of adenylate cyclase was also decreased (p < 0.05). In addition, manganese-stimulated adenylate cyclase activity was depressed (p < 0.05) in MI rats compared with controls (23.5 +/- 2.8 versus 52.1 +/- 9.0 pmol cAMP/min/mg prot). Chronic propranolol treatment in MI rats improved DT (4.1 +/- 0.9 versus 3.0 +/- 0.7 g/mm2) and +dT/dt (54.4 +/- 11.3 versus 40.5 +/- 9.5 g/mm2/sec) (p < 0.05); however, isoproterenol-stimulated isometric function remained impaired. Propranolol treatment normalized Bmax (11.9 +/- 1.7 versus 9.3 +/- 0.6 fmol/mg prot) (p < 0.05), whereas adenylate cyclase activity remained depressed. After large MI in rats, there is impaired papillary muscle function with decreased beta-adrenergic receptors and adenylate cyclase activity in the noninfarcted myocardium. Propranolol treatment improved basal isometric muscle function and beta-adrenergic receptor density in rats after myocardial infarction but did not improve adenylate cyclase activity or isoproterenol-stimulated muscle function. These data suggest that there is a primary defect in adenylate cyclase function that persists despite upregulation of receptors with propranolol treatment.

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Persistent defective coupling of dopamine-1 receptors to G proteins after solubilization from kidney proximal tubules of hypertensive rats.

The natriuretic effect of dopamine-1 (DA-1) agonists is reduced in spontaneously hypertensive rat (SHR), partly because of defective DA-1 receptor-adenylate cyclase (AC) coupling in renal proximal convoluted tubules. To investigate this defective coupling, DA-1 dopamine receptors from renal proximal tubules were solubilized and reconstituted into phospholipid vesicles. The binding of DA-1-selective ligand [125I]SCH 23982 was specific and saturable, with no differences in receptor density or Kd between SHR and normotensive rats (Wistar-Kyoto rats; WKY). Competition experiments of the reconstituted DA-1 dopamine receptors in WKY with a DA-1-selective agonist, SKF R-38393, revealed the presence of high- (Kh = 350 +/- 209 nM) and low-affinity (Kl = 70,500 +/- 39,500 nM) binding sites. 100 microM Gpp(NH)p abolished the agonist high-affinity sites, converting them to a low-affinity state (Ki = 33,650 +/- 10,850 nM). In SHR, one affinity site was noted (Ki = 13,800 +/- 500) and was not modulated by Gpp(NH)p (Ki = 11,505 +/- 2,295). The absence of guanine nucleotide-sensitive agonist high-affinity sites may explain the defective DA-1/AC coupling mechanism in the SHR.

Diminished phospholipase C activation by dopamine in spontaneously hypertensive rats.

It is reported that a defect in dopamine-1 (DA-1) receptor adenylate cyclase coupling in the proximal convoluted tubule in the spontaneously hypertensive rat may contribute to the diminished natriuretic response to DA-1 receptor agonists. Since the tubular DA-1 receptor is also coupled to phospholipase C, and both of these cellular signaling processes are involved in DA-1 receptor-mediated diuresis and natriuresis, it is important to know whether a similar defect is also present in DA-1 receptor-coupled phospholipase C pathway. The present study was therefore designed to determine the functional status of DA-1 receptor-phospholipase C coupling system of adult spontaneously hypertensive rats using a renal cortical slice preparation. In addition, the renal response to exogenously administered dopamine (1 microgram/kg/min i.v.) was also determined. We found that basal phospholipase C activity was significantly higher in hypertensive rats than in age-matched Wistar-Kyoto rats (7.36 +/- 0.32% versus 5.61 +/- 0.27%, p less than 0.05). However, compared with the normotensive controls, dopamine-induced increases in phospholipase C activity were significantly attenuated in the preparations of hypertensive rats in a concentration-dependent manner (13 +/- 6% versus 38 +/- 6% for 1 mM dopamine, p less than 0.05; 49 +/- 6% versus 71 +/- 9% for 3 mM dopamine, p less than 0.05; 50 +/- 16% versus 106 +/- 22%, p less than 0.05 for 10 mM dopamine).(ABSTRACT TRUNCATED AT 250 WORDS)

P03 Desensitization of coronary adenosine receptor-adenylate cyclase system

Adenosine agonists, N6-cyclohexyladenosine (CHA), N6-cyclopentyladenosine (CPA), N6-phenylisopropyladenosine (PIA), 5′-N-ethylcarboxamindoadenosine (NECA), 5′-N-methylcarboxamidoadenosine (MECA) and 2-chloroadenosine (CADO), produced a dose-related inhibition of acetylcholine (ACh)-induced writhing in mice. The antinociceptive potency of adenosine agonists was comparable to that of morphine. Adenosine agonists were 10–1000 times more potent when given i.c.v. than p.o., suggesting a central site of action. Theophylline antagonized the antinociceptive activity of R-PIA in the writhing assay, suggestive of an adenosine receptor-mediated event. The sedative/ataxic properties of adenosine agonists were evaluated using a rotorod assay. Adenosine agonists impaired performance on the rotorod in doses comparable to and in some cases lower than those active in the ACh writhing assay. The results of the present study suggest that adenosine agonists attenuate nociceptive responding to a chemical stimulus through a central purinergic mechanism. The ability of adenosine agonists to inhibit ACh-induced writhing may be secondary to their sedative/ataxic properties.

Regulation by chloride ion of astroglial cell functions and morphological transformation

Recently, several lines of evidence have indicated the important roles of glial cells, especially astrocytes, in the regulation of neuronal functions. The neuron-glia interaction is one of the most important issues in neuroscience, including neuropharmacology. I reviewed the present status and perspectives on the physiologic and pathologic functions of astrocytes in relation to the roles of intracellular Cl-. Astrocytes have different types of Cl- transport systems, such as voltage-sensitive and ligand-gated channels; HCO3(-)-Cl- exchange; and Na+, K+, Cl- cotransport systems. Anion exchange and cotransport systems are responsible for intracellular pH regulation and astrocytic volume regulation, respectively. Especially, astrocytic volume regulation is physiologically important for reducing the concentrations of K+ and glutamate in the extracellular space by their uptake systems. Disturbance of astrocytic volume regulation is expressed as astrocytic swelling, which is usually observed in various brain pathologic states including ischemia. Experimentally, glutamate caused a typical swelling of astrocytes in culture by Cl- and Ca(++)-dependent processes. Glutamate-induced swelling is qualitatively different from reversible swelling induced by hypoosmotic medium. Recently, we found that Cl- is intracellular factor for modulating the receptor-adenylate cyclase system in brain slices. Similarly, the receptor- and forskolin-stimulated adenylate cyclase of astrocytes showed a clear Cl- dependence. This was functionally confirmed by astrocytic morphological transformation induced by the cyclic AMP system.

Regulation of corticotropin responsiveness in human fetal adrenal cells

The human fetal adrenal gland exhibits a high rate of steroidogenesis during fetal development and produces the majority of steroids used by the placenta for estrogen synthesis. Corticotropin appears to be the principal hormonal regulator of steroidogenesis in the fetal adrenal gland. However, little is known concerning the regulation of corticotropin receptors. In this study we examined the long-term regulation of corticotropin responsiveness as measured by the ability of human fetal adrenal gland cells to produce cyclic adenosine monophosphate after corticotropin treatment for 3 hours. We also examined the regulation of corticotropin receptors as determined by iodine 125-labeled corticotropin binding to fetal adrenal cells. Fetal adrenal glands were obtained from second-trimester abortuses. The two distinct zones of the fetal adrenal gland, the definitive zone and the fetal zone, were separated and the tissue mechanically dispersed. Freshly isolated cells responded to corticotropin with a sevenfold to tenfold increase in the production of cyclic adenosine monophosphate, indicating a functional corticotropin receptor-adenylate cyclase coupling. However, when either fetal zone or definitive zone cells were grown and passed in monolayer culture, corticotropin stimulation of cyclic adenosine monophosphate production dropped to only twofold. The loss of corticotropin stimulation of cyclic adenosine monophosphate production occurred with a loss of the steroid-metabolizing enzyme 17α-hydroxylase (P-45017α). Because P-45017α expression can be stimulated after treatment of fetal adrenal gland cells with corticotropin or forskolin, we attempted to increase the ability of corticotropin to stimulate cyclic adenosine monophosphate production in a similar manner. After cells were pretreated with corticotropin (0.1 to 100 nmoi/L) or forskolin (0.1 to 100 μmol I L) for 4 days, their ability to produce cyclic adenosine monophosphate in response to corticotropin was examined. Pretreatment with both corticotropin and forskolin caused a dose-dependent increase in the ability of corticotropin to stimulate the production of cyclic adenosine monophosphate. Cells stimulated with corticotropin after pretreatment with forskolin exhibited a 35- to 50-fold increase in cyclic adenosine monophosphate production compared with nontreated cells (≃twofold). Corticotropin pretreatment increased responsiveness to a lesser extent than forskolin pretreatment. The increase in corticotropin responsiveness occurred along with an induction of P-45017α enzyme levels. The effect of pretreatment with corticotropin and forskolin on the binding of iodine 125-labeled corticotropin to definitive zone cells was also investigated. Corticotropin pretreatment increased corticotropin receptor binding 2.8 times; forskolin pretreatment increased corticotropin binding by seven times. These data indicate that corticotropin responsiveness and corticotropin receptors in human fetal adrenal cells are regulated by a cyclic adenosine monophosphate-dependent mechanism. This observation is similar to findings in bovine and ovine adrenal cells where the number of corticotropin receptors is increased by corticotropin or cyclic adenosine monophosphate treatment. (AM J OBSTET GYNECOL 1991;165:1649-54.)

Beta-adrenergic receptor-G protein-adenylate cyclase complex in experimental canine congestive heart failure produced by rapid ventricular pacing.

Changes in the beta-adrenergic receptor-G protein-adenylate cyclase complex were investigated in an experimental canine model of low-output heart failure produced by chronic rapid ventricular pacing. The contractile response occurring after exposure to the beta-adrenergic agonist dobutamine, measured as peak left ventricular + dP/dt, was decreased after 3 weeks of pacing. To further characterize the diminished functional responsiveness to beta-adrenergic receptor stimulation, beta-adrenergic receptor-adenylate cyclase coupling was investigated using membranes prepared from both control and paced animals. The density of beta-adrenergic receptors was decreased by 40% with a selective downregulation of the beta 1-subtype. The affinity of the receptor for the antagonist radioligand [125I]iodocyanopindolol remained unchanged. A defect in coupling was suggested by a decreased ability of isoproterenol, fluoride, and forskolin to stimulate adenylate cyclase in membranes prepared from failing hearts. Determination of the levels of Gi alpha (the alpha-subunit of Gi) by immunoblotting and pertussis toxin labeling revealed modest increases of approximately 30%. Furthermore, Mn2+ and purified Gs failed to stimulate adenylate cyclase in membranes prepared from failing hearts, indicating an impairment in the catalytic moiety of adenylate cyclase itself or in the ability of adenylate cyclase to couple to Gs. In contrast, complementation assay did not reveal differences in the functional activity of Gs alpha (the alpha-subunit of Gs). Taken together, these data demonstrate a selective decrease in the beta 1-subtype of adrenergic receptors and an increase in a 40-kd G1-like protein in the failing heart. Similar changes have been described in human idiopathic dilated cardiomyopathy. In addition to these changes, we identified a possible defect at the level of the catalytic subunit of adenylate cyclase.

Mu and delta opiate receptors in neuronal and astroglial primary cultures from various regions of the brain—coupling with adenylate cyclase, localisation on the same neurones and association with dopamine (D 1) receptor adenylate cyclase

Primary cultures, enriched in neurones or astroglial cells, from three phylogenetically different regions of the brain of the rat, the cerebral cortex, the striatum and the brain stem, were used to investigate the presence of opiate receptors, coupled to adenylate cyclase. Morphine was used as a μ-receptor agonist and [ d-Ala 2, d-Leu 5]-enkephalin (DADLE) was used as a δ -receptor agonist. In the neuronal cultures, both ligands inhibited the prostaglandin (PG)E 1 -stimulated intracellular accumulation of cyclic AMP dose-dependently, with the most prominent effects seen in the cultures of striatum and with DADLE being more potent than morphine. The opiate receptor antagonist, naloxone reversed the effects. Morphine and DADLE, added together, inhibited the PGE 1-stimulated accumulation of cyclic AMP, less than the sum of the effects of each drug. Therefore, it might be that these opioid receptors are localized together on the same neurone. Striatal neurones contained dopamine receptors coupled to cyclic AMP, as second messenger. It was shown that the D 1 (dopamine) receptor-stimulated activity of adenylate cyclase was inhibited by the μ and δ opioid receptor ligands. Thus, interactions at the level of adenylate cyclase seem to exist between D 1, μ and δ opiate receptors. In the astroglial enriched cultures, DADLE inhibited the PGE 1-induced accumulation of cyclic AMP, however, with a less prominent effect in the brain stem cultures. Morphine did not influence the basal or the PGE 1-stimulated intracellular accumulation of cyclic AMP in the cultures used and DADLE was less effective in inhibiting the PGE 1-induced accumulation of cyclic AMP in the astroglial cultures, than in the neuronal enriched cultures, indicating less developed δ receptor-second messenger efficacy in the astroglial cultures, than in the neuronal cultures. These results thus suggest the presence of δ receptors on astroglial cells in culture, while no μ receptors with cyclic AMP as second messenger were identified.

Reduced responsiveness of adenylate cyclase in alveolar macrophages from patients with asthma

Alveolar macrophages from patients with asthma accumulated less cyclic adenosine monophosphate when these macrophages were exposed to isobutyl methylxanthine, salbutamol, or prostaglandin E 2, compared to cells from control subjects without asthma, and the degree of the hyporesponsiveness was related to the severity of asthma. In addition, a significantly lower adenylate cyclase activity was observed in crude membrane fractions of macrophages from the group with asthma in the presence of salbutamol and prostaglandin E 2. The refractoriness observed in patients with asthma is thus not accounted for by a specific β-adrenergic desensitization at the adenylate cyclase receptor level but should rather be explained by a cyclic adenosine monophosphate-dependent postreceptor mechanism.

Effects of Isomazole on Force of Contraction and Phosphodiesterase Isoenzymes I-IV in Nonfailing and Failing Human Hearts

The phosphodiesterase (PDE) inhibitor isomazole increased the force of contraction to 278.3 +/- 89.1% (n = 7) of the predrug value in ventricular trabeculae carneae isolated from nonfailing human hearts. This effect can be attributed mainly to a PDE III or a combined PDE III/IV inhibition since at the concentration of the maximal positive inotropic effect of isomazole, PDE III and PDE IV were completely inhibited. In explanted failing human hearts (end-stage myocardial failure, NYHA IV), isomazole increased the force of contraction only marginally to 110.1 +/- 10.7% of the predrug value. The lack of a distinct positive inotropic efficacy of isomazole in failing human hearts could not be explained by an impairment of PDE inhibition since the properties of the PDE I-IV isoenzymes separated by DEAE-Sepharose chromatography and the inhibitory effects of isomazole did not differ in both preparations. The positive inotropic effect of the beta-adrenoceptor agonist isoprenaline was also reduced in failing hearts. However, in the presence of isomazole, the diminished positive inotropic effect of isoprenaline was restored to values obtained with isoprenaline alone in nonfailing hearts. Thus, the decreased effect of inotropic drugs like isoprenaline or isomazole in preparations from failing human heart might be explained mainly by a diminished cAMP formation due to a defect in receptor-adenylate cyclase coupling.

Thyroid hormones transcriptionally regulate the beta 1-adrenergic receptor gene in cultured ventricular myocytes

Exposure of ventricular myocytes in primary culture to triiodothyronine (T3) increased the number of beta 1-adrenergic receptors per cell by 2.1 +/- 0.3-fold (n = 7) within 48 h. Immunoblots of membranes prepared from myocytes revealed a marked increase by T3 in the 64-kDa species of the beta 1-adrenergic receptor. Steady-state levels of beta 1- and beta 2-adrenergic receptor mRNAs quantified by DNA-excess solution hybridization were 0.26 +/- 0.06 and 0.81 +/- 0.05 amol of beta-adrenergic receptor mRNA/micrograms of total cellular RNA, respectively (n = 4). beta 1-Adrenergic receptor mRNA increased to 0.90 +/- 0.07 amol/micrograms RNA by 2-4 h after exposure to T3 and then declined by 6 h to twice that of control cells. beta 2-Adrenergic receptor mRNA levels were unaffected by T3. Northern blot hybridization also showed a rapid and sustained increase of 2.2 +/- 0.4-fold (n = 4) in beta 1-adrenergic receptor mRNA. The rate of beta 1-adrenergic receptor gene transcription assessed by nuclear run-on transcription assays increased by 3.4 +/- 0.4-fold in cells treated for 30 min with T3. Ventricular cells contained nuclear T3 receptors and mRNAs of c-erbA genes that encode T3 binding proteins. These studies indicate that thyroid hormones regulate the cardiac beta-adrenergic receptor-adenylate cyclase system by controlling the rate of transcription of the beta 1-adrenergic receptor gene. This regulation involves nuclear T3 receptors and appears to be exerted in a tissue-specific manner.

Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations

The present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the beta-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydro-ouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors. To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I-III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I-III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor-adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored. These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypoxia and glucose independently regulate the beta-adrenergic receptor-adenylate cyclase system in cardiac myocytes.

We explored the effects of two components of ischemia, hypoxia and glucose deprivation, on the beta-adrenergic receptor (beta AR)-adenylate cyclase system in a model of hypoxic injury in cultured neonatal rat ventricular myocytes. After 2 h of hypoxia in the presence of 5 mM glucose, cell surface beta AR density (3H-CGP-12177) decreased from 54.8 +/- 8.4 to 39 +/- 6.3 (SE) fmol/mg protein (n = 10, P less than 0.025), while cytosolic beta AR density (125I-iodocyanopindolol [ICYP]) increased by 74% (n = 5, P less than 0.05). Upon reexposure to oxygen cell surface beta AR density returned toward control levels. Cells exposed to hypoxia and reoxygenation without glucose exhibited similar alterations in beta AR density. In hypoxic cells incubated with 5 mM glucose, the addition of 1 microM (-)-norepinephrine (NE) increased cAMP generation from 29.3 +/- 10.6 to 54.2 +/- 16.1 pmol/35 mm plate (n = 5, P less than 0.025); upon reoxygenation cAMP levels remained elevated above control (n = 5, P less than 0.05). In contrast, NE-stimulated cAMP content in glucose-deprived hypoxic myocytes fell by 31% (n = 5, P less than 0.05) and did not return to control levels with reoxygenation. beta AR-agonist affinity assessed by (-)-isoproterenol displacement curves was unaltered after 2 h of hypoxia irrespective of glucose content. Addition of forskolin (100 microM) to glucose-supplemented hypoxic cells increased cAMP generation by 60% (n = 5; P less than 0.05), but in the absence of glucose this effect was not seen. In cells incubated in glucose-containing medium, the decline in intracellular ATP levels was attenuated after 2 h of hypoxia (21 vs. 40%, P less than 0.05). Similarly, glucose supplementation prevented LDH release in hypoxic myocytes. We conclude that (a) oxygen and glucose independently regulate beta AR density and agonist-stimulated cAMP accumulation; (b) hypoxia has no effect on beta AR-agonist or antagonist affinity; (c) 5 mM glucose attenuates the rate of decline in cellular ATP levels during both hypoxia and reoxygenation; and (d) glucose prevents hypoxia-induced LDH release, a marker of cell injury.

Adrenergic receptor responsiveness and congestive heart failure

Our knowledge and understanding of the structure, mechanism of action and regulation of receptor-adenylate cyclase systems have increased dramatically in the last few years. A family of receptors (including the β-adrenergic receptors) and guanine nucleotide regulatory proteins (G proteins) have been purified and cloned. Structure-function studies are beginning to provide insight into how the various components of the transmembrane signaling apparatus interact to promote alterations in the activity of the effector systems. Much effort has been devoted to understanding how various pathophysiologic conditions, such as ischemia or congestive heart failure, and the therapeutic methods used to treat such conditions perturb or regulate receptor systems. It has become abundantly clear that such regulation does occur but is not restricted to simple alterations in receptor number, and may well involve covalent modification (phosphorylation) of receptors or alteration in the ability of receptors to interact with G proteins. In addition, regulation of the quantity or functionality of the various G proteins and the catalytic unit of adenylate cyclase itsett appear to occur. For example, recent evidence suggests that congestive heart failure in humans is associated with a decreased number of β-adrenergic receptors as well as an increased quantity of the inhibitory G protein (G i). These alterations may provide important insight into how to develop new therapeutic methods. Mechanisms generally responsible for transmembrane signaling, how the components are regulated by pathophysiologic conditions, and drugs used to treat disease states are discussed in detail.

?-Adrenergic receptor activity of cerebral microvessels is reduced in aged rats

The effect of age on beta- (beta) adrenergic receptor number (Bmax) and adenylate cyclase (AC) activity was determined in microvessels isolated from male F-344 rats at 3, 18, and 24 months of age. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated reduced Bmax (fmol/mg) of microvessels isolated from 24 month old rats (27.2 +/- 4.9) compared with 3 month old (50.4 +/- 5.2) and 18 month old rats (p less than 0.01) (61.4 +/- 7.6). The basal AC activity (pmol cAMP/mg) in 24 month old rats (32.0 +/- 6.7) and in 18 month old rats (30.4 +/- 2.1) were significantly reduced compared to the basal activity in the young (50.1 +/- 4.2). The net isoproterenol or NaF stimulated AC activity in 24 month old rats (zero and 15.6 +/- 8.5 respectively) was also reduced compared to young rats (10.1 +/- 3.9 and 166.0 +/- 21.2 respectively). It is concluded that aging is associated with reduced isoproterenol stimulated AC activity of cerebral microvessels. This reduction is the product of reduced beta-adrenergic receptor number and reduced activity of AC in aged rat cerebral microvessels.

Effect of physical training on ventricular β-adrenergic receptor adenylate cyclase system of diabetic rats

This study was designed to assess the effect of physical training on the ventricular β-adrenergic receptor adenylate cyclase system of diabetic rats. Mild diabetes mellitus was induced by an intravenous (IV) injection of streptozotocin (45 mg/kg). Rats were randomized into a group submitted to a progressive 10-week running program on a treadmill, while another group was kept sedentary. A group of sedentary nondiabetic rats was used as normal controls. Results showed a similar reduction in the density of β-adrenergic receptors in sedentary diabetic ( P < .05) and trained diabetic rats ( P < .01) compared with controls, without any significant alteration in the dissociation constant. The basal and the sodium fluoride-stimulated maximal adenylate cyclase activities were similar in the three groups. However, the maximal response of adenylate cyclase to isoproterenol was significantly reduced in the two diabetic groups compared with controls ( P < .01). The decrease in adenylate cyclase response to isoproterenol observed in the diabetic groups appeared to be associated with a reduction in the total number of β-adrenergic receptors and more specifically in those existing in the high-affinity state. On the other hand, the hyperglycemia and hyperglucagonemia present in sedentary diabetic rats was improved by training. These data suggest that the beneficial effects observed in response to training in experimental diabetes are not associated with changes in β-adrenergic receptor adenylate cyclase system on membranes from ventricular tissue.

Beta-adrenoceptor adenylate cyclase system adaptation to physical training in rat ventricular tissue.

The beta-adrenergic receptor adenylate cyclase system of ventricular tissue was evaluated in a group of rats submitted to a progressive 10-wk running program on a treadmill and compared with that in a group of rats maintained sedentary during the same period. Adequate training was confirmed by a 46% increase in the gastrocnemius isocitrate dehydrogenase activity in the trained group [1.50 +/- 0.04 vs. 1.03 +/- 0.06 (SE) pmol.g-1.min-1; P less than 0.01). Binding studies with [125I]iodocyanopindolol showed a 13% reduction in the density of beta-adrenergic receptors in trained rats (42.6 +/- 2.1 vs. 49.0 +/- 2.1 fmol/mg; P less than 0.05) without any significant modification in the dissociation constant. The amount of [125I]iodocyanopindolol bound to beta-adrenoceptors in the high-affinity state was reduced by 16.6% in trained rats (12.5 +/- 0.9 vs. 15.0 +/- 0.5 fmol/mg; P less than 0.05) without any significant changes for those in the low-affinity state, indicating a decrease in the coupling between the beta-adrenergic receptors and the guanine stimulatory binding protein. Furthermore, although the basal and sodium fluoride-stimulated adenylate cyclase activities were similar in the two groups of rats, the response of adenylate cyclase maximally stimulated by 10(-5) M isoproterenol was reduced by 16% in trained rats (29.7 +/- 1.4 vs. 35.3 +/- 1.3 pmol.mg-1.min-1; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)