Norepinephrine infusions increase adenylate cyclase responsiveness in brown adipose tissue

Previous work from our laboratory demonstrated that exposing rats to cold increases interscapular brown adipose tissue (IBAT) adenylate cyclase activity through a postreceptor modification of the adenylate cyclase system. The cold-induced sensitization is correlated with an increase in the activity of the sympathetic innervation of IBAT, and is prevented by prior surgical denervation of this tissue. The present experiments were aimed at identifying the neurogenic signal that mediates cold-induced sensitization. We found that, like cold exposure, infusions of norepinephrine increased adenylate cyclase activity and enhanced the ability of cholera toxin to ADP-ribosylate the stimulatory regulatory protein of adenylate cyclase (Gs) in warm-adapted rats whose IBAT had been denervated surgically. Infusions of isoproterenol increased adenylate cyclase responsiveness more potently than norepinephrine; however, the maximal effect achieved by isoproterenol was less than that produced by norepinephrine. Infusions of phenylephrine and clonidine had no effect on adenylate cyclase responsiveness. The effects of low doses of isoproterenol, however, were greatly potentiated by coinfusion of phenylephrine. Furthermore, the sensitizing effects of norepinephrine could be blocked by either propranolol or prazosin, indicating that the effects of norepinephrine require simultaneous stimulation of beta and alpha-1 adrenergic receptors.     

Transmission blockade and stimulation of ganglionic adenylate cyclase by catecholamines.

Isolated rat superior cervical ganglia treated with isoproterenol and related drugs show an increase in ganglionic cyclic adenosine 3':5'-monophosphate (cAMP) and a block of transmission. For isoproterenol, the maximum increase in cAMP occurred at 1 X 10(-6) M, a concentration without effect on transmission. Approximately 5 X 10(-4) M isoproterenol was required to reduce the ganglionic compound action potential by 50%. Dopamine, in contrast to isoproterenol, had no effect on the content of cAMP but depressed transmission. The maximum increase in cAMP produced by norepinephrine occurred with 5 X 10(-4) M, a concentration that reduced transmission by approximately 35%. The effects of isoproterenol on adenylate cyclase and transmission were prevented either by practolol (10(-4) M) or phentolamine (10(-5) M). Dopamine-induced blockade of transmission was antagonized by phentolamine (10(-5) M). Whereas the blockade of transmission by norepinephrine was antagonized by practolol (10(-5) M) or phentolamine (10(-5) M), the stimulation of adenylate cyclase by norepinephrine was prevented by practolol (10(-4) M) but not by phentolamine (10(-5) M). These results show that the blockade of transmission and stimulation of adenylate cyclase are unrelated in rat ganglia and that adrenergic receptor classification is ambiguous. The role of adenylate cyclase in ganglia is unclear.     

Adrenergic innervation and cyclic adenosine 3':5'-monophosphate levels in response to norepinephrine in stomach of postnatal rats.

Changes in receptor function, innervation and cyclic adenosine 3':5'-monophosphate (cAMP) levels in response to norepinephrine were investigated in the stomach of postnatal developing rats. We found that although the beta adrenergic receptor function and responsiveness of cAMP-generating system to norepinephrine are present in the early postnatal stage (3-day-old), these systems are not fully developed at birth and that increase in sensitivity comes with age. Adrenergic innervation is present at birth in Auerbach's plexus but not in smooth muscle, during the 1st postnatal week. Direct adrenergic innervation to smooth muscle appears 1 week after birth and matures during the first 4 weeks. This maturation of direct adrenergic innervation runs in parallel with the increased sensitivity of beta receptors and is accompanied by an elevated responsiveness of the cAMP-generating system to norepinephrine.     

Interactions of flerobuterol, an antidepressant drug candidate, with beta adrenoceptors in the rat brain.

Interactions of dl-flerobuterol with central beta adrenoceptors were investigated. It inhibited the binding of [3H]CGP 12177, a selective beta adrenoceptor ligand, to membranes prepared from rat cerebral cortex, cerebellum, heart and lung. The affinity of dl-flerobuterol was very close in all tissues (Ki approximately 1 microM). In cerebral cortex, binding inhibition of [3H]CGP 12177 was stereospecific, l-flerobuterol (Ki = 483 nM) being 70-fold more potent than d-flerobuterol (Ki = 34 microM). Moreover, dl-flerobuterol (Ki = 926 nM) was 7-fold less potent than isoproterenol (Ki = 140 nM) to displace [3H]CGP 12177 binding, but 5-fold more potent than salbutamol (Ki = 4600 nM). Flerobuterol did not inhibit the radioligand binding to the other receptors at the highest concentration tested, thus leading to a very high beta adrenergic selectivity. Flerobuterol increased the concentration of cyclic AMP in slices of rat cerebral cortex in a dose-dependent manner; this effect was antagonized by atenolol and propranolol. Compared to isoproterenol or norepinephrine, which produced cyclic AMP maximal increases of 380 and 460%, respectively, it showed a weaker activity with a maximal stimulation obtained at 100 microM, corresponding to a cAMP increase of 140% over basal value (100%). These data revealed that flerobuterol possessed a beta adrenergic agonist activity. Moreover, it antagonized competitively the isoproterenol- or norepinephrine-stimulated accumulation of cAMP. At low concentrations of isoproterenol or norepinephrine, the stimulation of adenylate cyclase was only due to the action of flerobuterol, but at higher concentrations, the response of isoproterenol or norepinephrine was competitively blocked by flerobuterol. At 10 microM, isoproterenol surmounted fully this antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 2,4,6-triaminopyrimidine on the electromechanical properties of guinea-pig myocardium

We studied the relationship between epinephrine-induced increases in automaticity and in the adenylate cyclase-adenosine 3',5'-monophosphate (cAMP) system in canine cardiac Purkinje fibers. In intact Purkinje fiber bundles superfused with Tyrode's solution, epinephrine induced a concentration-dependent increase in automaticity and in cAMP content. Both of these effects were reduced by propranolol. Decreasing the temperature of the superfusate from 37 degrees to 25 degrees C blocked the epinephrine-induced increase in automaticity, but not the increase in cAMP content. Addition of the metabolic blocking agent, iodoacetate, to the superfusate did not block the effect on Purkinje fiber automaticity, but the cAMP content did not increase. In Purkinje fiber bundle whole homogenates adenylate cyclase was stimulated more by isoproterenol than by epinephrine. Low concentrations of phenylephrine decreased adenylate cyclase activity; higher concentrations induced an increase toward control values. Iodoacetate did not significantly alter cyclase activity and did not affect its response to epinephrine. These students have shown that although intact Purkinje fiber bundles respond to epinephrine with increases in automaticity and cAMP content, these two events can be dissociated under appropriate conditions.     

Effect of albuterol and terbutaline, synthetic beta adrenergic stimulants, on the cyclic 3',5'-adenosine monophosphate system in smooth muscle.

The effect of albuterol and terbutaline on the cyclic 3',5'-adenosine monophosphate (cAMP) system was studied in rat uterus, aorta and myocardium and in dog bronchus, and was compared to that of isoproterenol in order to determine whether the tissue specificity observed in their functional effects is reflected in their effect on the cAMP system. Tissue specimens were either homogenized in Tris buffer for enzyme activity measurements or incubated in Krebs-Ringer-bicarbonate medium with the test drugs. Both albuterol and terbutaline produce an increase in cAMP content in the tissues due to a direct effect on adenylate cyclase. This effect can be potentiated by a phosphodiesterase inhibitor and antagonized by a beta adrenergic blocking compound. The cAMP response to each beta adrenergic agonist differs in the tissues examined: in uterus and aorta where the maximal effects are idenitcal, the ED50 values may reflect differences in affinity which may account for the different cAMP response to the compounds at the lower concentrations. In bronchus and myocardium, both the maximum effect and ED50 values of the compounds are different. Albuterol and terbutaline increases cAMP content in bronchus significantly and have only a small effect on cAMP cont in myocardium, whereas isoproterenol increases cAMP level significantly in both tissues. The results indicate that the tissue specificity of albuterol and terbutaline may have its origin at the level of the cAMP system.     

Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes.

Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation.     

Effects of verapamil on myocardial contractility, cardiac adenosine 3,'5'-monophosphate and heart phosphorylase.

The effects of verapamil on myocardial isometric force on contraction, cardiac adenosine 3,'5'-monophosphate (cyclic AMP) and heart phosphorylase alpha activity were studied in the isolated perfused rat heart. When hearts were perfused with verapamil (5.98 times 10- minus 8 M), force of contraction was reduced approximately 50% within 4 to 5 minutes; at this point, the concentration of cyclic AMP was significantly lower than control but phosphorylase alpha activity was unchanged. In hearts perfused continuously for 60 minutes with verapamil, force of contraction and cyclic AMP levels returned to normal within 20 minutes after administration of verapamil was begun. Isoproterenol (0.355 nmol/min) reversed the depressant effect of verapamil on cardiac contractility and restored heart cyclic AMP levels to normal. Methoxamine (35.5 nmol/min) given to verapamil-depressed hearts, caused contractile force to return to normal, but cardiac cyclic AMP levels remained low. Mephentermine (23.0 nmol/min) had no effect on cardiac contraction, cyclic AMP or phosphorylase alpha activity in hearts depressed by verapamil. It was concluded that with the concentration of verapamil used in these experiments, the drug caused a transient decrease in force of contraction and myocardial cyclic AMP. Both the depression in myocardial contractility and in cardiac cyclic AMP caused by verapamil were reversed promptly by isoproterenol, whereas methoxamine overcame acutely only the negative inotropic effect of verapamil. Mephentermine had no effect on hearts depressed by verapamil.     

Hydrocortisone and human lymphocytes: increases in cyclic adenosine 3':5'-monophosphate and potentiation of adenylate cyclase-activating agents

We have investigated the effect of hydrocortisone on the cyclic adenosine 3':5'-monophosphate (cAMP) response of human lymphocytes and polymorphonuclear leukocytes. Hydrocortisone (10(-6)-10(-3)M) caused a dose-dependent increase in the cAMP content of human lymphocytes which occurred rapidly (within 1 min); the cAMP level peaked at about 10 min, remained elevated for 90 min and decreased promptly to base line if the cells were washed free of hydrocortisone. In contrast to its effects on lymphocytes, hydrocortisone caused only a small dose-dependent increase in cAMP content of polymorphonuclear leukocytes which became significant only at high concentrations. In addition to increasing lymphocyte cAMP levels, hydrocortisone (10(-6)-10(-3)M) markedly potentiated the effect of many adenylate cyclase-stimulating agents including beta adrenergic stimuli, histamine, adenosine, prostaglandin E1 and cholera enterotoxin. The biochemical mechanism(s) of these actions of hydrocortisone were explored and it was found that hydrocortisone exerted its effects neither by blocking extracellular cAMP efflux, nor by inducing protein synthesis, nor by activating prostaglandin metabolic pathways, nor by preventing receptor (e.g., beta adrenergic receptor) desensitization. Hydrocortisone probably does not work as a cAMP phosphodiesterase inhibitor, since it did not inhibit lymphocyte phosphodiesterase, and the magnitude of synergistic potentiation by hydrocortisone was greater than that of potent phosphodiesterase inhibitors. Thus, hydrocortisone might act on the adenylate cyclase enzyme system by other, unknown mechanism(s). The ability of hydrocortisone to increase cAMP and especially to potentiate adenylate cyclase-stimulating agonists may partly explain the potent in vivo anti-inflammatory effect of corticosteroids in man.     

Evidence that parathyroid hormone-mediated calcium transport in rat brain synaptosomes is independent of cyclic adenosine monophosphate.

In vivo PTH administration to rats resulted in increased brain synaptosomal Ca++ transport, while parathyroidectomy (PTX) resulted in decreased transport. To determine the mechanism of action of PTH on Ca++ transport in rat brain synaptosomes, we performed transport studies by the Na-Ca exchanger and also measured cAMP generation in synaptosomes from PTX rats. Ca++ transport was studied after in vivo additions of either bovine (b)PTH, cAMP, or forskolin, and adenylate cyclase activity was assessed after additions of either bPTH, forskolin, sodium fluoride (NaF), or isoproterenol. In the presence of 1-34 bPTH [10(-7) M], Ca++ uptake was significantly increased by 55% (P less than 0.001) above control, while 3-34 bPTH [10(-7) M] had no effect on uptake. Both 8br,cAMP [10(-6) M] and dibut,cAMP [10(-6) M] also significantly increased (P less than 0.001) Ca++ uptake above control by 63 and 44%, respectively. Similarly, forskolin [10(-5) M], the adenylate cyclase activator, increased Ca++ uptake by 41%. We next evaluated Ca++ efflux, and found that 1-34 bPTH [10(-7) M], 1-84 bPTH [10(-7) M], and forskolin [10(-5) M] also increased Ca++ efflux by 50, 73, and 120%, respectively, above control. Since Ca++ transport was increased by either PTH, cAMP, or forskolin, we decided to determine if PTH action on Ca++ transport in synaptosomes was dependent on cAMP. This was investigated by measuring cAMP production during the conversion of 32P-ATP to 32P-cAMP in the presence of an ATP regenerating system (30 micrograms creatine phosphokinase, 10 mM creatine phosphate), and the cyclic nucleotide phosphodiesterase inhibitor (1 mM IBMX). Whereas forskolin [10(-4) M] and NaF [100 mM] significantly increased (P less than 0.001) adenylate cyclase activity in synaptosomes by eight- and fourfold, respectively, neither 1-34 bPTH nor 1-84 bPTH increased synaptosomal cyclase activity. However, in canine renal cortical plasma membranes (CRCPM), we observed significant increases in cAMP production with either forskolin, NaF, or PTH. Finally, to determine if synaptosomes contain an intact adenylate cyclase system, we measured cAMP production in the presence of the beta adrenergic agent, isoproterenol. Isoproterenol significantly increased adenylate cyclase activity in both synaptosomes (90%) and CRCPM (50%). These data suggest that although there is an intact adenylate cyclase system in rat brain synaptosomes, PTH-stimulated calcium transport in synaptosomes appears to be independent of this system.     

Increased negative inotropic effect of calcium-channel blockers in hypertrophied and failing rabbit heart.

The effects on ventricular function of calcium channel blockers and isoproterenol were studied in isovolumically beating perfused control rabbit hearts and in hearts subjected to a double pressure plus volume overload studied at the early phase of heart failure. In control hearts, isoproterenol produced an increase of systolic ventricular function and relaxation that was maximal at 10(-7) M. In failing hearts, inotropic state increase in response to isoproterenol was significantly smaller (P less than .01) with no observed lusitropic effect. In control hearts, verapamil and diltiazem produced dose-dependent decreases of ventricular function which were larger with verapamil than with diltiazem (median drug concentration50 of developed pressure was, respectively, 1163 +/- 131 nM and 4524 +/- 451 nM, P less than .001). In failing hearts, contractility decrease was larger than in control hearts (median drug concentration50 of developed pressure was 604 +/- 69 nM and 2691 +/- 580 nM with verapamil and diltiazem, respectively). In contrast, Ro 40-5967, a new calcium-channel blocker, did not produce reductions of inotropic state with concentrations up to 10(-5) M. All three calcium-channel blockers produced a 2-fold increase of coronary flow at 10(-6) M. We conclude that the deleterious effect of verapamil and diltiazem in heart failure is due, at least in part, to a direct depressant effect of these drugs on contractility, which is larger than in control hearts. Additionally, the in vivo sympathetic compensation is probably reduced, as indicated by the decreased ventricular responsiveness to isoproterenol.     

Inhibition of vasopressin-stimulated cyclic AMP accumulation by alpha-2 adrenoceptor agonists in isolated papillary collecting ducts

The effects of selective alpha adrenoceptor agonists and antagonists on vasopressin (VP)-sensitive cyclic AMP (cAMP) formation in microdissected rat papillary collecting ducts were examined. In the presence of 10(-10) M VP, norepinephrine and the selective alpha-2 adrenoceptor agonist, B-HT 933, produced almost total inhibition of VP-stimulated cAMP accumulation. Half-maximal inhibition occurred at 1 x 10(-8) M and 6 x 10(-7) M for norepinephrine and B-HT 933, respectively. Cirazoline, a selective alpha-1 adrenoceptor agonist, had no significant effect on VP-stimulated cAMP accumulation. The inhibitory effects of norepinephrine and B-HT 933 were antagonized by rauwolscine but not by prazosin. The antagonism of B-HT 933-induced inhibition of VP-stimulated cAMP accumulation was competitive with an antagonist dissociation constant (KB) of 10.9 x 10(-9) M. Preincubation of papillary collecting ducts with pertussis toxin (1 microgram/ml for 1 hr at 37 degrees C) attenuated, by 65%, the inhibitory effect of B-HT 933 on VP-stimulated cAMP levels. These results demonstrate that alpha-2 adrenoceptors capable of inhibiting VP action are present on the papillary collecting duct. Furthermore, the alpha-2 adrenoceptor-induced inhibition of VP-stimulated cAMP accumulation is pertussis-toxin sensitive. This suggests that alpha-2 adrenoceptors are coupled negatively to adenylate cyclase, via the guanine nucleotide binding protein, in the collecting tubule.     

Physiological approach of beta receptor coupling to adenylate cyclase in rat airways: ontogenical modification and functional antagonism

The relaxant effects of isoproterenol, forskolin and sodium nitroprusside were studied on tracheal pieces and lung parenchymal strips of Sprague-Dawley and Wistar rats according to age and functional antagonism with carbachol applied previously to induce the contraction. The beta receptor-related maximal relaxant effect of isoproterenol decreased from 4 to 11 weeks in Sprague-Dawley rat airways contracted with 10(-6) M carbachol. This maximal relaxant effect did not change with age in the Wistar strain. When lower carbachol concentrations were applied to Wistar trachea, the maximal relaxant effect of isoproterenol raised with a large decrease of the EC50 values. In the Sprague-Dawley strain, a similar diminution of carbachol concentration also allowed to increase the maximal amplitude of relaxation, but a smaller decrease of EC50 was observed as referred to the Wistar strain. These results suggest that the decrease with age of the maximal relaxation of Sprague-Dawley airways by isoproterenol might be linked to impaired functional antagonism between beta adrenergic and muscarinic stimulation in this rat strain. This hypothesis was strengthened by the observation of the effects of forskolin, an activator of adenylate cyclase, and sodium nitroprusside, a cyclic GMP-related relaxant drug, that did not show any modified effect in function of age in both rat strains. A modified regulation of adenylate cyclase complex with ontogenesis and with rat strain is suggested.     

Biological maturation and beta-adrenergic effectors: pre- and postnatal development of the adenylate cyclase system in the rabbit heart

The relationship between biological maturation and adenylate cyclase activity was studied in membrane preparations of rabbit ventricular muscle. Basal adenylate cyclase activity was lower in the adult than in the 1-day-old neonate or 27-day-old fetus. Maximal stimulation of adenylate cyclase by isoproterenol was 2.5 times greater and the EC50 values were 2-fold higher in the adult than the 27-day-old fetus or 1-, 7- and 12-day-old neonate. No significant differences in isoproterenol- or Mg++-stimulated activity were observed among the younger age groups nor was the Mg++-stimulated Vmax of adenylate cyclase significantly affected by biological maturation. Sodium fluoride, guanyl-5'yl imidodiphosphate and GTP also stimulated adenylate cyclase activity in a dose-dependent fashion similar to isoproterenol. Sodium fluoride (2.5-10 mM) increased adenylate cyclase activity in the adult to a significantly greater extent than the 1-day-old neonate. Guanyl-5'yl imidodiphosphate and GTP (0.1-10.0 microM) augmented adenylate cyclase activity to approximately the same degree (although some small differences were observed) in the fetus, neonate and adult. However, when guanyl-5'yl imidodiphosphate was preincubated with membrane preparations before in vitro assay, adenylate cyclase activity was increased 10-fold in the adult, whereas membranes from 1-day-old animals were unaffected. These data suggest that the processes regulating hormonal and pharmacological activation of adenylate cyclase are modified during biological maturation.     

Norepinephrine and BRL 37344 stimulate adenylate cyclase by different receptors in rat brown adipose tissue

The beta adrenergic activation of adenylate cyclase was examined in membrane homogenates of rat interscapular brown adipose tissue (IBAT). In control membranes, isoproterenol and norepinephrine (NE) stimulated adenylate cyclase with activation constants of about 20 and 300 nM, respectively. Exposure of rats to 4 degrees C for 3 days increased the maximal stimulation of adenylate cyclase to these agonists but did not alter the respective activation constants. The beta 1-selective antagonist 1-(2-cyanophenoxy)-3-beta-(3-phenylureido)ethylamino-2-pr opa nol blocked isoproterenol stimulation of adenylate cyclase in control and cold-exposed membranes at a concentration 100 times lower than did the beta 2-selective antagonist erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobuta n-2-ol. These data indicate that typical adrenergic agonists stimulate IBAT adenylate cyclase via beta 1 receptors. (R*,R*)-4-[2-[2 [9 3-chlorophenyl)-2-hydroxyethyl]amino)propyl) phenyl]phenoxyacetic acid (BRL 37344), an atypical agonist with activity at the beta 3 receptor, stimulated adenylate cyclase in control membranes with an activation constant of approximately 700 nM. Membranes of cold-exposed rats exhibited a high affinity response to BRL 37344 similar to that seen in control membranes and, in addition, a low affinity response. BRL 37344 stimulation of adenylate cyclase was unaffected by 1-(2-cyanophenoxy)-3-beta-(3-phenylureido)ethyl-amino-2-prop anol, whereas stimulation by NE or epinephrine was potently blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of choroid plexus peptide IIF on adenylate cyclase and 3',5'-cyclic adenosine monophosphate in adipose tissue.

Two hypophyseal lipolytic peptides, adrenocorticotropin (ACTH) and beta-melanocyte-stimulating hormone (beta-MSH), and the extrhypophyseal lipolytic peptide IIF, were compared with regard to their effects on free fatty acid production and 3',5'-cyclic adenosine monophosphate (cAMP) concentration in isolated rabbit and rat adipose tissue, and on adenylate cyclase activity in the tissue homogenates. ACTH at concentrations of 0.01 mug/ml or more increased lipolysis and cAMP levels in both tissues. beta-MSH at concentrations of 0.001 mug/ml or more increased lipolysis and cAMP in the rabbit tissue, but a concentration of 10 mug/ml did not stimulate lipolysis and did not alter nucleotide concentration in the rat tissue. Peptide IIF at 0.01 mug/ml or more stimulated lipolysis in rabbit adipose tissue and caused an accumulation of cAMP. A concentration of 100 mug/ml failed to stimulate free fatty acid production in the rat tissue and the cAMP level was also unaffected. In a medium containing 7.6 mEq/l of Mg++ and no Ca++, ACTH at 0.1 mug/ml or more stimulated adenylate cyclase activity in both rabbit and rat adipose homogenates by 6- to 12-fold. This effect was inhibited when Mg++ was replaced by Ca++, Na+ or K+. beta-MSH stimulated adenylate cyclase in rabbit, but not in rat, adipose homogenate in Mg++-containing incubation midium; again, the effect on rabbit adenylate cyclase was suppressed when Mg++ was replaced by Ca++, Na+ or K+. Peptide IIF failed to influence adenylate cyclase in the rabbit tissue homogenate in the Mg++-containing, Ca++-free medium; but when the medium contained 7.6 mEq/l of Ca++ in place of Mg++, 0.1 mug/ml or more of IIF caused a 4- to 15-fold increase in cyclase activity. IIF did not affect cyclase in the rat tissue homogenate in the presence or absence of Ca++. The data are consistent with the conclusion that extrahypophyseal lipolytic peptide IIF, as well as hypophyseal peptides ACTH and beta-MSH, accelerates lipolysis in susceptible adipocytes by stimulating adenylate cyclase to produce cAMP. The effect of IIF on cyclase requires the presence of exogenous Ca++; that of ACTH and beta-MSH requires exogenous Mg++.     

G protein and adenylate cyclase complex-mediated signal transduction in the rat heart during sepsis

Changes in the protein level of various subunits of GTP-binding protein and the activity of adenylate cyclase in the rat heart during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The protein levels of various subunits of GTP-binding protein were determined by Western blot analysis. The activity of adenylate cyclase was measured based on the rate of formation of cAMP from [alpha-32P]ATP. The results show that protein levels of G alphas and G beta remained stable during the early and the late phases of sepsis. The protein levels of G alpha i-2 and G alpha i-3 remained relatively unaltered during the early phase of sepsis, but they were increased by 46.5% (P < 0.05) and 61.3% (P < 0.01), respectively, during the late phase of sepsis. The basal adenylate cyclase activity remained unchanged during the early phase while it was decreased by 25.7% (P < 0.05) during the late phase of sepsis. The isoproterenol-stimulated adenylate cyclase activity was unchanged during early sepsis while it was decreased by 44.6% (P < 0.01) during late sepsis. These data demonstrate that during the late hypodynamic phase of sepsis, myocardial G alpha i-2 and G alpha i-3 protein levels were increased and the increases were coupled with a reduction in adenylate cyclase activity. Because GTP-binding proteins mediate sympathetic control of cardiac function, the present findings may have a pathophysiological significance in contributing to the understanding of the pathogenesis of cardiac dysfunction during the late stage of sepsis.     

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)     

Binding and functional characteristics of beta adrenergic receptors in the intact neutrophil

Beta adrenergic receptors have been previously characterized in human neutrophil sonicates. In the present study the intact neutrophil has been assessed for the number and affinity of beta adrenergic binding sites by using the antagonist DNA. Agonist and antagonist potencies, characterized by their effect on DHA binding and cyclic AMP accumulation, are compared with agonist inhibition of lysosomal enzyme (beta glucuronidase) release. Criteria for beta adrenergic receptor identification were successfully demonstrated. At 30 degrees C, beta adrenergic binding was rapid (t 1/2 2 min) and reversible (t 1/2 9 min). Receptor binding was saturable, revealing approximately 900 high-affinity receptors per neutrophil with DHA concentrations of 0.1 to 10 nM. By utilizing both equilibrium and kinetic techniques, the KD was determined to be approximately 0.6 nM. Agonists and antagonists competed for DHA binding in a manner consistent with their effect on cyclic AMP generation. Rank order potency was suggestive of a beta-2 receptor: isoproterenol greater than epinephrine greater than norepinephrine. Stereoselectivity was shown by the greater potency of L-propranolol compared to the D isomer. A high degree of receptor-adenylate cyclase coupling efficiency was suggested by the observation that with only 1% receptor occupancy isoproterenol stimulated 50% maximal cyclic AMP generation. Finally, there was an excellent correlation between the isoproterenol concentration which resulted in 50% of maximal inhibition of beta glucuronidase release (Ki) and that causing 50% maximal cyclic AMP stimulation (Kact), suggestive of a close relationship between beta adrenergic-induced adenylate cyclase activation and beta adrenergic regulation of neutrophil lysosomal enzyme release. The data presented suggest that the use of the intact neutrophil for study of the beta adrenergic receptor is feasible and may provide information which is considerably more closely related to modulation of physiological function by neurohormones than is possible with disrupted cell preparations.     

Beta adrenergic receptors in lymphocytes and granulocytes from patients with cystic fibrosis.

Intact lymphocytes from patients with cystic fibrosis (CF) produce significantly (P less than 0.001) less adenosine 3':5' cyclic monophosphate (cAMP) than normal lymphocytes in response to isoproterenol (10(-8)-10(-4) M), although the basal cAMP content and the response to prostaglandin E1 are normal. Obligate heterozygotes for CF have significantly (P less than 0.005) reduced cAMP response to isoproterenol as well, suggesting a genetic component in the beta adrenergic deficiency in CF. The number of beta adrenergic receptors, as determined by equilibrium binding of [3H]dihydroalprenolol to lymphocyte particulates, is the same in normal lymphocytes (969 +/- 165 receptors/cell) and lymphocytes from patients with CF (1,333 +/- 263 receptors/cell). Binding properties of the receptor for both antagonist and agonist, as assessed by KD for dihydroalprenolol and Ki for (-)-isoproterenol, are also normal in the CF lymphocytes. Similarly, in granulocytes from patients with CF, the cAMP response to isoproterenol (10(-8)-10(-4) M) is significantly reduced compared with healthy controls (P less than 0.03), as is the response of granulocytes from obligate heterozygotes (P less than 0.05). Again, the basal cAMP levels and the response to prostaglandin E1 are normal. The number of beta adrenergic receptors, as determined by equilibrium binding of [3H]dihydroalprenolol to granulocyte particulates, was the same in normal (1,462 +/- 249 receptors/cell) and CF (1,621 +/- 221 receptors/cell) preparations. Binding properties of the receptor for both agonist and antagonist, as assessed by KD for dihydroalprenolol and Ki for isoproterenol, are normal in CF granulocyte particulates. The lymphocyte and granulocyte beta adrenergic defect in CF cannot be explained by abnormalities of the beta adrenergic receptor or of adenylate cyclase itself. Receptor-cyclase coupling is the most likely site of the heritable beta adrenergic defect in CF.