Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi alpha 2) and beta-adrenergic receptors in a porcine model of chronic episodic myocardial ischemia.

Regional myocardial ischemia is associated with increased levels of adenosine and norepinephrine, factors that may alter activation of the beta-adrenergic receptor (beta AR)-G protein-adenylyl cyclase pathway in the heart. We have used the ameroid constrictor model to determine whether alterations in myocardial signal transduction through the beta AR-G protein-adenylyl cyclase pathway occur in the setting of chronic episodes of reversible ischemia. Pigs were instrumented with ameroid occluders placed around the left circumflex coronary artery. 5 wk later, after ameroid closure, flow and function were normal in the ischemic bed, but flow (P = 0.001) and function (P < 0.03) were abnormal when metabolic demands were increased. The ischemic bed showed a reduction in myocardial beta AR number (P < 0.005). Despite regional downregulation of myocardial beta AR number, adenylyl cyclase activity was similar in the ischemic and control beds. Quantitative immunoblotting showed that the cardiac inhibitory GTP-binding protein, Gi alpha 2, was decreased in the ischemic bed (P = 0.02). In contrast, the cardiac stimulatory GTP-binding protein, Gs alpha, was increased in endocardial sections from the ischemic bed (P = < 0.05). Decreased Gi alpha 2 content was associated with decreased inhibition of adenylyl cyclase. Reduced Gi alpha 2 content, in conjunction with increased Gs alpha content in the endocardium, may provide a means by which adrenergic activation is maintained in the setting of chronic episodic myocardial ischemia.     

Decreased stimulatory guanosine triphosphate binding protein in dogs with pressure-overload left ventricular failure.

Alterations in the level and function of the stimulatory guanyl nucleotide binding protein (Gs) from the cardiac sarcolemma were examined in a canine model of heart failure. The present study is based on our previous investigations that demonstrated both a loss of beta-adrenergic agonist high-affinity binding sites and a decreased adenylate cyclase activity in sarcolemma from failing hearts. Using cholera toxin and [32P]NAD, we labeled the alpha subunit of Gs (Gs alpha) and found a 59% reduction in the level of this protein. Further, a 50% reduction in Gs activity was noted in a reconstitution assay utilizing membranes from the mouse S49 lymphoma cell line cyc-, which is deficient in Gs. These data suggest that, in this model of pressure-overload left ventricular failure, the acquired defect in the beta-adrenergic receptor/adenylate cyclase system involves a deficiency in the coupling protein Gs. Such an abnormality may explain the decreased adrenergic responsiveness of the failing left ventricle.     

Overexpression of myocardial Gsalpha prevents full expression of catecholamine desensitization despite increased beta-adrenergic receptor kinase.

Inotropic and chronotropic responses to catecholamines in young adult transgenic mice overexpressing myocardial Gsalpha are enhanced. One might predict that over the life of the animal, this chronically enhanced beta-adrenergic receptor stimulation would result in homologous catecholamine desensitization. To test this hypothesis, old transgenic Gsalpha mice and age-matched controls were studied physiologically in terms of responsiveness of left ventricular function (ejection fraction) to isoproterenol in vivo and in vitro in terms of beta-adrenergic receptor signaling. Old transgenic mice still responded to isoproterenol with augmented (P < 0.05) left ventricular ejection fraction (+44+/-3%) compared with age-matched controls (+24+/-1%). Although total beta-adrenergic receptor density was reduced in the old transgenic mice, and G protein receptor kinase 2 (beta-adrenergic receptor kinase) levels were increased, the fraction of receptors binding agonist with high affinity as well as isoproterenol- and G protein-stimulated adenylyl cyclase activities were enhanced. Thus, classical catecholamine desensitization is not effective in attenuation of persistently enhanced responses to sympathetic stimulation in mice overexpressing myocardial Gsalpha. To support this conclusion further, experiments were performed with chronic isoproterenol, which elicited effective desensitization in wild-type controls, but failed to elicit desensitization in overexpressed Gsalpha mice. The results of this study suggest that the lack of protective desensitization mechanisms may be responsible in part for the dilated cardiomyopathy which develops with chronic sympathetic stress over the life of these animals.     

Myocardial beta-adrenergic receptor function during the development of pacing-induced heart failure.

The development of pacing-induced heart failure was studied in chronically instrumented, conscious dogs paced at a rate of 240 beats/min for 1 d (n = 6), 1 wk (n = 6), and 3-4 wk (n = 7). Left ventricular (LV) dP/dt was decreased (P < 0.0125) at 1 d, LV end-diastolic pressure and heart rate were increased (P < 0.0125) at 1 wk, but clinical signs of heart failure were only observed after 3-4 wk of pacing. Plasma norepinephrine rose (P < 0.0125) after 1 d of pacing, whereas LV norepinephrine was reduced (P < 0.0125) only after 3-4 wk of pacing. Both the fraction of beta-adrenergic receptors binding agonist with high affinity and adenylyl cyclase activity decreased (P < 0.0125) after 1 d of pacing. Total beta-adrenergic receptor density was not changed at any time point, but beta 1-adrenergic receptor density was decreased (P < 0.0125) after 1 wk. The functional activity of the guanine nucleotide binding protein, Gs, was not reduced, but the Gi alpha 2 isoform of the alpha subunit of the GTP-inhibitory protein rose after 3-4 wk of pacing. Thus, myocardial beta-adrenergic signal transduction undergoes change shortly (1d) after the initiation of pacing, before heart failure develops. The mechanism of beta-adrenergic receptor dysfunction in pacing-induced heart failure is characterized initially by elevated plasma levels of catecholamines, uncoupling of beta-adrenergic receptors, and a defect in the adenylyl cyclase catalytic unit. Selective down-regulation of beta 1-adrenergic receptors, increases in Gi alpha 2, and decreases in myocardial catecholamine levels occur as later events.     

Adenylate cyclase and beta adrenergic receptor development in the mouse heart

We have demonstrated previously a postnatal peak for the beta adrenergic receptor in the heart and detected the appearance of a beta adrenergic receptor before an (-)-isoproterenol inducible increase in heart rate. The present study examined 1) agonist displaceable [3H] dihydroalprenolol (DHA) binding in the neonatal and adult mouse heart and 2) adenylate cyclase in fetal, neonatal and adult mouse heart. 3[H]DHA binding displaceable by (-)-isoproterenol gave a similar Ki from 1 day neonate through adult. Similar to the result found for antagonist displacement binding, there was a dramatic increase in the agonist displaceable [3H] DHA binding postnatally. The maximum was achieved in 2 weeks and then gradually declined to adult level. Cyclase activity (basal, (-)-isoproterenol- and NaF- stimulated) paralleled beta adrenergic receptor increases before birth. However, no early postnatal peak was present. In the 13 day fetal mouse heart, there is no (-)-isoproterenol increase in heart rate, but beta adrenergic receptor (13 +/- 4% of adult) and (-)-isoproterenol-stimulated adenylate cyclase activity (15 +/- 5% of adult) are present. It is concluded that 1) no significant difference exists between the agonist and antagonist displaceable [3H] DHA binding during development, 2) adenylate cyclase activity increases significantly during the last third of pregnancy in parallel with the beta adrenergic receptor, 3) both the beta adrenergic receptor and adenylate cyclase activity can be detected before the heart rate responses and 4) total adenylate cyclase activity does not increase in parallel with the early postnatal beta adrenergic receptor peak.     

Cellular mechanisms of impaired adrenergic responsiveness in neonatal dogs.

The myocardial responsiveness of conscious, instrumental dogs to exogenously administered isoproterenol and norepinephrine was investigated in neonatal, 6-wk-old, and adult animals. Comparable base-line values for peak left ventricular derivative of pressure with respect to time were observed in all age categories. However, when compared with adult responses, the sympathomimetic amine-induced increases in neonatal left ventricular dP/dt were significantly blunted at each concentration of adrenergic agonist examined, whereas the 6-wk-old puppies displayed an intermediate inotropic response. To investigate the cellular mechanisms of this blunted neonatal response, we correlated physiologic and biochemical measurements of the myocardial responses to catecholamines in each age category. When compared with adult myocardial membrane preparations, neonatal cardiac membranes were characterized in vitro by an increased density of beta-adrenergic binding sites, comparable affinity for adrenergic agonists and antagonists, and an enhanced coupling of adenylate cyclase activation to receptor occupancy. Simultaneous changes in either the serum catecholamine concentration or the membrane content of other intrinsic proteins failed to account for the observed neonatal increase in beta-adrenergic receptor density. These findings are most consistent with a compensatory mechanism of the cardiac cell membrane, whereby an inherent depression in the adrenergic responsiveness of the immature myocardium appears to induce the increase in receptor density and activation of adenylate cyclase.     

G-protein-coupled receptor kinase activity is increased in hypertension.

Impaired vascular beta-adrenergic responsiveness may play an important role in the development and/or maintenance of hypertension. This defect has been associated with an alteration in receptor/guanine nucleotide regulatory protein (G-protein) interactions. However, the locus of this defect remains unclear. G-Protein-coupled receptor kinases (GRKs) phosphorylate serine/threonine residues on G-protein-linked receptors in an agonist-dependent manner. GRK activation mediates reduced receptor responsiveness and impaired receptor/G-protein coupling. To determine whether the impairment in beta-adrenergic response in human hypertension might be associated with altered GRK activity, we studied lymphocytes from younger hypertensive subjects as compared with older and younger normotensive subjects. We assessed GRK activity by rhodopsin phosphorylation and GRK expression by immunoblot. GRK activity was significantly increased in lymphocytes from younger hypertensive subjects and paralleled an increase in GRK-2 (beta ARK-1) protein expression. In contrast, no alterations in cAMP-dependent kinase (A-kinase) activity or GRK-5/6 expression were noted. GRK activity was not increased in lymphocytes from older normotensive subjects who demonstrated a similar impairment in beta-adrenergic-mediated adenylyl cyclase activation. These studies indicate that GRK activity is selectively increased in lymphocytes from hypertensive subjects. The increase in GRK activity may underlie the reduction in beta-adrenergic responsiveness characteristic of the hypertensive state.     

Differential regulation of right and left ventricular beta-adrenergic receptors in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia secondary to an intracardiac right-to-left shunt alters regulation of the myocardial beta-adrenergic receptor/adenylate cyclase system, we produced chronic hypoxemia in nine newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 wk. Eight lambs served as normoxemic controls. beta-receptor density (Bmax) and ligand affinity (KD) were determined with the radio-ligand [125I]iodocyanopindolol and adenylate cyclase activity determined during stimulation with isoproterenol, sodium fluoride (NaF), and forskolin. During chronic hypoxemia, Bmax decreased 45% (hypoxemic, 180.6 +/- 31.5 vs. control, 330.5 +/- 60.1 fmol/mg) in the left ventricle (exposed to hypoxemia alone) but was unchanged in the right ventricle (exposed to hypoxemia and pressure overload). KD was not different from control in either ventricle. Left ventricular isoproterenol-stimulated adenylate cyclase activity was decreased by 39% (30.0 +/- 4.3% increase vs. 44.1 +/- 9.5% increase) whereas right ventricular adenylate cyclase activity was unchanged. Stimulation of adenylate cyclase with NaF or forskolin was not different from control in either ventricle. Circulating epinephrine was increased fourfold whereas circulating and myocardial norepinephrine were unchanged. These data demonstrate a down-regulation of the left ventricular beta-adrenergic receptor/adenylate cyclase system during chronic hypoxemia secondary to an intracardiac right-to-left shunt.     

Loss of high affinity cardiac beta adrenergic receptors in dogs with heart failure.

We studied the alterations in myocardial beta-adrenergic receptor-adenylate cyclase activity and muscarinic receptor density in a canine model of left ventricular (LV) failure. LV failure was characterized by a doubling of LV weight/body weight ratio (3.3 +/- 0.1 to 6.9 +/- 0.4 g/kg) and an elevation of LV end-diastolic pressure, 32 +/- 4.5 mmHg, compared with 7.7 +/- 0.6 mmHg in normal dogs. Despite a 44% increase in receptor density as measured by antagonist binding studies with [3H]dihydroalprenolol, there was a twofold decrease in receptor affinity, i.e., an increase in the dissociation constant (Kd) (5.6 +/- 0.7 to 12 +/- 1.6 nM) in heart failure. Agonist displacement of [3H]dihydroalprenolol binding with isoproterenol in the presence and absence of 5'-guanylylimidodiphosphate [Gpp(NH)p] demonstrated a striking loss of high affinity binding sites in heart failure (51 +/- 16 to 11 +/- 5%). Beta-Adrenergic receptor-mediated stimulation of adenylate cyclase and maximal stimulation with Gpp(NH)p or sodium fluoride was reduced in heart failure. There was a concomitant marked, P less than 0.01, reduction in muscarinic receptor density (242 +/- 19 vs. 111 +/- 20 fmol/mg). Thus, while muscarinic receptor density fell, beta-adrenergic receptor density actually increased in LV failure. However, a larger portion of the beta-adrenergic receptors are not functionally coupled to the GTP-stimulatory protein (Ns), as evidenced by a decrease in the fraction of receptors that bind agonist with high affinity.     

Beta-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms.

In order to investigate the general cause of beta-adrenergic receptor neuroeffector abnormalities in the failing human heart, we measured ventricular myocardial adrenergic receptors, adrenergic neurotransmitters, and beta-adrenergic receptor-effector responses in nonfailing and failing hearts taken from nonfailing organ donors, subjects with endstage biventricular failure due to idiopathic dilated cardiomyopathy (IDC), and subjects with primary pulmonary hypertension (PPH) who exhibited isolated right ventricular failure. Relative to nonfailing PPH left ventricles, failing PPH right ventricles exhibited (a) markedly decreased beta 1-adrenergic receptor density, (b) marked depletion of tissue norepinephrine and neuropeptide Y, (c) decreased adenylate cyclase stimulation in response to the beta agonists isoproterenol and zinterol, and (d) decreased adenylate cyclase stimulation in response to Gpp(NH)p and forskolin. These abnormalities were directionally similar to, but generally more pronounced than, corresponding findings in failing IDC right ventricles, whereas values for these parameters in nonfailing left ventricles of PPH subjects were similar to values in the nonfailing left ventricles of organ donors. Additionally, relative to paired nonfailing PPH left ventricles and nonfailing right ventricles from organ donors, failing right ventricles from PPH subjects exhibited decreased adenylate cyclase stimulation by MnCl2. These data indicate that: (a) Adrenergic neuroeffector abnormalities present in the failing human heart are due to local mechanisms; systemic processes do not produce beta-adrenergic neuroeffector abnormalities. (b) Pressure-overloaded failing right ventricles of PPH subjects exhibit decreased activity of the catalytic subunit of adenylate cyclase, an abnormality not previously described in the failing human heart.     

Sympathoadrenal modulation of stress-activated signaling in burn trauma

Burn injury stimulates stress-responsive components, p38 mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinases (JNK)/nuclear factor (NF)-kappaB. p38 MAPK plays a role in postburn cardiomyocyte tumor necrosis factor-alpha secretion and cardiac dysfunction. Since burn trauma increases circulating catecholamine levels, which in turn modulate inflammatory cytokine production, we hypothesized that increased sympathetic activity after major burn trauma may trigger postburn cardiac p38 MAPK activation via an adrenergic receptor-mediated phenomenon. We examined adrenergic receptor populations involved in burn-activated cardiac stress signaling. Sprague Dawley rats were divided into six groups: 1) control, 2) control plus alpha1-adrenergic agonist phenylephrine (2 microg/kg, intravenous), 3) control plus beta-adrenergic agonist isoproterenol (1 microg/kg, intravenous), 4) burn (fluid resuscitation with lactated Ringer's 4 ml/kg/% burn), 5) burn plus alpha1-adrenergic antagonist prazosin (1 mg/kg, by mouth), and 6) burn plus beta-adrenergic antagonist propranolol (3.3 mg/kg, by mouth). Phenylephrine, but not isoproterenol, increased cardiac p38 MAPK/JNK/NF-kappaB activation. Burn trauma activated p38 MAPK, JNK, and NF-kappaB, and this stress response was blocked by either prazosin or propranolol. Thus, stimulation of the adrenergic pathway may constitute one upstream activator of stress response in burn.     

Subtype-selective regulation of beta adrenergic receptor-adenylyl cyclase coupling by phorbol esters in 3T3-L1 fibroblasts

To study the epigenetic regulation of beta adrenergic receptor subtypes, we examined the effects of phorbol esters on beta adrenergic receptor coupling to adenylyl cyclase in 3T3-L1 fibroblasts, which express both beta-1 and beta-2 adrenergic receptor subtypes. Pretreatment of intact 3T3-L1 cells with the protein kinase C activator phorbol dibutyrate caused a dose- and time-dependent decrease in subsequent cyclic AMP (cAMP) accumulation mediated by the beta adrenergic agonist isoproterenol. This effect was selective for beta-adrenergic receptor-mediated responses because there was a potentiation of cAMP accumulation caused by other activators such as prostaglandin E1, forskolin or cholera toxin. The inactive phorbol, alpha-phorbol dibutyrate was ineffective at 1 microM in attenuating isoproterenol stimulation, and 25 nM of the protein kinase C inhibitor staurosporine blocked the effects of phorbol ester on beta adrenergic agonist responses. Stimulation of cAMP accumulation by isoproterenol occurred through a greater proportion of beta-2 adrenergic receptors in phorbol dibutyrate-treated cells than in control cells. This was demonstrated using the beta-1 adrenergic selective antagonist ICI 89.406 and the beta-2 adrenergic selective antagonist ICI 118.551 to inhibit competitively isoproterenol-stimulated cAMP accumulation. Beta-2 adrenergic receptor number and subtype in these cells are regulated by glucocorticoids and butyrate. Decreasing the proportion of beta-1 adrenergic receptors and concomitantly increasing beta-2 adrenergic receptors with either glucocorticoids or butyrate decreased the ability of phorbol ester pretreatment to attenuate cAMP accumulation by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct relationship between mononuclear leukocyte and lung beta-adrenergic receptors and apparent reciprocal regulation of extravascular, but not intravascular, alpha- and beta-adrenergic receptors by the sympathochromaffin system in humans.

To examine putative relationships between adrenergic receptors on accessible circulating cells and relatively inaccessible extravascular catecholamine target tissues, we measured mononuclear leukocyte (MNL) and lung beta-adrenergic receptors and platelet and lung alpha-adrenergic receptors in tissues obtained from 15 patients undergoing pulmonary resection. Plasma catecholamine concentrations were measured concurrently to explore potential regulatory relationships between the activity of the sympathochromaffin system and both intravascular and extravascular adrenergic receptors. MNL and lung membrane beta-adrenergic receptor densities were correlated highly (r = 0.845, P less than 0.001). Platelet alpha 2-adrenergic receptor and lung alpha 1-adrenergic receptor densities were not. Lung alpha 1-adrenergic receptor densities were positively related to plasma norepinephrine (r = 0.840, P less than 0.01) and epinephrine (r = 0.860, P less than 0.01) concentrations; in contrast, lung beta-adrenergic receptor densities were not positively related to plasma catecholamine concentrations (they tended to be inversely related to plasma norepinephrine and epinephrine [r = -0.698, P less than 0.05] levels). This apparent reciprocal regulation of alpha- and beta-adrenergic receptors by the sympathochromaffin system was only demonstrable with adrenergic receptor measurements in the extravascular catecholamine target tissue. Neither MNL beta-adrenergic receptor nor platelet alpha-adrenergic receptor densities were correlated with plasma catecholamine levels. Thus, although measurements of beta-adrenergic receptors on circulating mononuclear leukocytes can be used as indices of extravascular target tissue beta-adrenergic receptor densities (at least in lung and heart), it would appear that extravascular tissues should be used to study adrenergic receptor regulation by endogenous catecholamines in humans. These data provide further support for the concept of up regulation, as well as down regulation, of some adrenergic receptor populations during short-term activation of the sympathochromaffin system in humans.     

Rapid desensitization of neonatal rat liver beta-adrenergic receptors. A role for beta-adrenergic receptor kinase.

Exposure of beta-adrenergic receptors (BAR) to agonists often leads to a rapid loss of receptor responsiveness. The proposed mechanisms of such rapid receptor desensitization include receptor phosphorylation by either cAMP-dependent protein kinase or the specific beta-adrenergic receptor kinase (BARK), leading to functional uncoupling from adenylyl cyclase and sequestration of the receptors away from the cell surface. To evaluate the physiological role of such mechanisms, we have investigated whether rapid regulation of BAR occurs in the neonatal rat liver immediately after birth, a physiological situation characterized by a dramatic but transient increase in plasma catecholamines. We have detected a rapid, transient uncoupling of liver plasma membrane BARs from adenylyl cyclase (corresponding to a desensitization of approximately 45%) within the first minutes of extrauterine life, followed by a transient sequestration of approximately 40% of the BARs away from the plasma membrane. In agreement with such pattern of desensitization, we have detected (by enzymatic and immunological assays) rapid changes in BARK specific activity in different neonatal rat liver subcellular fractions that take place within the same time frame of BAR uncoupling and sequestration. Our results provide new evidence on the potential role of BAR desensitization mechanisms in vivo and suggest that they are involved in modulating catecholamines actions at the moment of birth. Furthermore, our data indicate that in addition to its suggested role as a rapid modulator of adrenergic receptor function at synapse, rapid BARK-mediated receptor regulation may have functional relevance in other tissues in response to high circulating or local levels of agonists.     

Beta adrenergic receptor subtypes in the atria: evidence for close coupling of beta-1 and beta-2 adrenergic receptors to adenylate cyclase

The relationship between occupancy of beta adrenergic receptors and stimulation of adenylate cyclase in dog atrial tissue was examined by studying the binding of [125I]iodopindolol and the activation of adenylate cyclase. Computer-assisted nonlinear regression analysis was used to analyze the inhibition of isoproterenol-stimulated adenylate cyclase activity by beta-1- or beta-2-selective antagonists. The Ki values for each subtype of receptor for the selective antagonists resulting from studies of the inhibition of adenylate cyclase activity were similar to those determined in studies of the inhibition of the binding of [125I]iodopindolol. To compare further the occupancy of beta-1 or beta-2 adrenergic receptors with the activation of adenylate cyclase mediated by each class of receptor, computer modeling of the stimulation of adenylate cyclase by the beta-1-selective agonist norepinephrine was carried out. The EC50 values of norepinephrine for each receptor subtype, as measured in studies of norepinephrine-stimulated adenylate cyclase activity, were similar to the Ki values for the inhibition by norepinephrine of the binding of [125I]iodopindolol to each receptor subtype. The data led to the conclusion that beta-1 adrenergic receptors make up about 70% of the total number of beta adrenergic receptors and mediate 70% of the increase in adenylate cyclase activity produced by isoproterenol. These results suggest that the relationship between occupancy of each class of receptor and activation of adenylate cyclase is linear and that, when agonist-stimulated adenylate cyclase activity is used as a functional response, neither spare beta-1 nor spare beta-2 adrenergic receptors exist in the atrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic receptor modulation of basal and beta-adrenergic stimulated function of the failing human left ventricle.

The objective of this study was to evaluate the effect of muscarinic receptor modulation on basal and beta-adrenergic stimulated left ventricular function in patients with heart failure. 21 heart failure patients and 14 subjects with normal ventricular function were studied. In Protocol 1 intracoronary acetylcholine resulted in a 60+/-8% inhibition of the left ventricular +dP/dt response to intracoronary dobutamine in the normal group, and a similar 70+/-13% inhibition in the heart failure group. Acetylcholine also attenuated the dobutamine-mediated acceleration of isovolumic relaxation (Tau) in both groups. Acetylcholine alone had no effect on Tau in the normal group, while it prolonged Tau in the heart failure group. In Protocol 2 intracoronary atropine resulted in a 35+/-10% augmentation of the inotropic response to dobutamine in the normal group, versus a non-significant 12+/-15% augmentation of the dobutamine response in the heart failure group. In Protocol 3, in 6 heart failure patients, both effects of acetylcholine, the slowing of ventricular relaxation and the inhibition of beta-adrenergic responses, were reversed by the addition of atropine. Therefore, in the failing human left ventricle muscarinic stimulation has an independent negative lusitropic effect and antagonizes the effects of beta-adrenergic stimulation.     

Agonist, antagonist, and inverse agonist characteristics of TIPP (H-Tyr-Tic-Phe-Phe-OH), a selective delta-opioid receptor ligand

Recent evidence indicates that the well established delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) also displays agonist activity in several cellular models. Therefore, it is possible that TIPP, and structurally related compounds, might represent a novel class of opioid agonists exhibiting unique characteristics. The purpose of this study was to examine the properties of TIPP at selected points of the signal transduction pathway (i.e., receptor binding, G-protein activation, and effector regulation) in GH(3)DORT cells (GH(3) cells expressing delta-opioid receptors) and compare them with that of an established delta-opioid agonist, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE). DPDPE exhibited properties of an agonist in all assays. In contrast, TIPP demonstrated characteristics of an agonist, antagonist, or inverse agonist, depending on the step in the signal transduction cascade examined and the assay conditions employed. In receptor binding assays, the addition of guanine nucleotides and sodium ions increased the affinity of TIPP for delta-opioid receptors in both membrane preparations and digitonin-permeabilized cells, which is characteristic of an inverse agonist. In assays measuring G-protein activation, TIPP failed to stimulate guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in membrane preparations, which is consistent with an antagonist profile. However, when using cells semi-permeabilized with digitonin, TIPP exhibited properties of an agonist, producing concentration-dependent, antagonist-reversible stimulation of [(35)S]GTPgammaS binding. Finally, in assays examining regulation of the intracellular effector adenylyl cyclase, TIPP exhibited characteristics of an agonist, producing inhibition of enzyme activity in both membrane preparations and whole cells. Therefore, although DPDPE and TIPP act similarly as agonists to regulate the intracellular effector adenylyl cyclase, they demonstrate significant differences in the signal transduction cascade preceding this final point of convergence.     

Alterations in G-proteins and beta-adrenergic responsive adenylyl cyclase in rat urinary bladder during aging

Decreased response of bladder to beta-adrenergic stimulation with aging is related to decreased adenylyl cyclase activity and possibly to changes in guanine nucleotide regulatory protein (G-protein) content or function. G-protein content was quantified by Western blot analysis using antibodies to Gsalpha, Goalpha, and Gialpha in 21-day-old (weanling), 90-day-old (young adult), 6-month-old (adult), and 24-month-old (old) rat bladders. Gi/Go function in bladders with aging was measured by ADP-ribosylation with pertussis toxin. Content of Gsalpha, Goalpha, and Gialpha was lower in 90-day-old bladder than in 21-day-old bladder. Gsalpha content was similar in the 21-day-, 6-month-, and 24-month-old bladders. Gialpha content as well as pertussis toxin-catalyzed ADP-ribosylation was higher in 24-month-old bladders than in 21- and 90-day-old bladders. Pertussis toxin-catalyzed ADP-ribosylation of bladder membranes and treatment of bladder with protein kinase A inhibitors reversed the age-dependent decline in isoproterenol stimulation of adenylyl cyclase. Decreases in beta-adrenergic-induced relaxation response with age in rat bladder are due in part to increases in the content and functional activity of pertussis toxin-sensitive G-protein.     

Hypothyroidism modulates beta adrenergic receptor adenylate cyclase interactions in rat reticulocytes.

We have investigated alterations in beta adrenergic receptor binding sites of rat reticulocytes occurring in animals rendered hypothyroid by thyroidectomy. Beta adrenergic receptor interactions were assessed by measuring the number of (-)[3H]-dihydroalprenolol binding sites and the ability of an agonist to compete for occupancy of the receptors. The number of receptors was significantly reduced in cells from the hypothyroid animals. In addition, there were significant agonist-specific alterations in binding. Using computer assisted curve fitting techniques, it was found that the ability of (-)isoproterenol to stabilize a high affinity guanine nucleotide sensitive "coupled" form of the receptor was impaired. Reticulocytes from hypothyroid animals have, in addition, a reduction in the concentration of the nucleotide regulatory protein as assessed by the number of 42,000 Mr substrates for cholera toxin catalyzed ADP ribosylation. These alterations are associated with reductions in catecholamine and NaF stimulated adenylate cyclase activity. Diminished coupling of beta adrenergic receptors with other regulatory components of the adenylate cyclase system represents a mechanism by which altered thyroid states modulate beta adrenergic receptor function and beta adrenergic responsiveness of tissues.     

Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response.

We have shown recently that norepinephrine stimulates muscle cell hypertrophy in primary cultures from the neonatal rat ventricle and that this stimulation is not blocked by the beta adrenergic antagonist propranolol. The present study was done to define the adrenergic specificity of the myocyte hypertrophic response to norepinephrine. 90% pure, single-cell cultures of nongrowing myocytes were maintained in serum-free medium 199 with transferin and insulin. Myocyte size was quantitated 48 h after addition of adrenergic agents, by measuring cell volume, cell surface area, and cell protein. L-norepinephrine increased myocyte size to a maximum 150% of control; half-maximum effect was obtained at a concentration of 0.2 microM. This increase in cell size was inhibited by the nonselective alpha adrenergic antagonist phentolamine and by the alpha 1 adrenergic antagonists prazosin and terazosin; it was not inhibited by propranolol or by the alpha 2 adrenergic antagonist yohimbine. The beta adrenergic agonist isoproterenol did not increase cell size. Thus, norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response.