Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes

We have studied the compartmentation of cyclic AMP action in purified ventricular cardiomyocytes prepared by collagenase perfusion of adult rabbit hearts. Incubation of purified adult myocytes with 1 microM isoproterenol causes rapid accumulation of intracellular cyclic AMP in both soluble (2.3 leads to 7.7 pmol/ mg of protein) and particulate (3.0 leads to 9.2) fractions of cell homogenates (3000 X g for 5 min), increases in the total activity and activity ratio of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.66), a decrease in protein kinase activity remaining in the particulate fraction (47 leads to 30%), and an increase in the activity ratio of glycogen phosphorylase (0.15 leads to 0.47). Incubation of myocytes with 10 microM prostaglandin E1 (PGE1) leads to a comparable increase in soluble cyclic AMP (2.3 leads to 5.8 pmol/mg of protein) and activation of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.39) but does not result in any change in cAMP or protein kinase in the particulate fraction and fails to cause an activation of glycogen phosphorylase. PGE1 does not inhibit the effects of isoproterenol; when myocytes are incubated with both isoproterenol and PGE1, the accumulation of cyclic AMP, activation of cAMP-dependent protein kinase and phosphorylase b leads to a conversion are equal to that achieved with isoproterenol alone. Perturbation of cellular calcium using the ionophore A23187, verapamil, or high or low extracellular calcium did not alter the ability of isoproterenol to cause activation of particulate cAMP-dependent protein kinase or influence the inability of PGE1 to do so. Activation of adenylate cyclase by forskolin (30 microM) caused immediate activation of both soluble and particulate cAMP-dependent protein kinase leading to rapid activation of phosphorylase. We conclude that the hormonally specific compartmentation of cyclic AMP and cAMP-dependent protein kinase that occurs in intact heart (Hayes, J. S., Brunton, L. L., Brown, J. H., Reese, J. B., and Mayer, S. E. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1570-1574) is not explained on the basis of cellular heterogeneity but has a subcellular basis within the cardiomyocyte.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Action of the cardiac alpha 1-adrenergic receptor. Activation of cyclic AMP degradation

Using purified rat ventricular myocytes and membranes prepared from them, we have previously found that alpha 1-adrenergic stimulation causes decreased cyclic AMP accumulation and decreased activation of cyclic AMP-dependent protein kinase. We have now analyzed the mechanism by which alpha 1 stimulation is linked to cyclic AMP metabolism. In an adenylate cyclase assay in which carbachol inhibits the stimulatory effect of norepinephrine, the addition of prazosin (alpha 1-antagonist) has no effect on the response to norepinephrine. In membranes prepared from myocytes treated with pertussis toxin, norepinephrine competes for alpha 1-receptors (assessed by [3H]prazosin binding) with two components, binding to the high affinity component being sensitive to exogenous GTP, exactly as in membranes prepared from control myocytes. In intact cells labeled with [3H]adenine in which carbachol antagonizes the norepinephrine response, prazosin enhances accumulation of [3H]cyclic AMP due to norepinephrine. Treatment of cells with pertussis toxin eliminates inhibition by carbachol but does not alter prazosin's capacity to enhance the norepinephrine response. Addition of phosphodiesterase inhibitors eliminates this effect of alpha 1 blockade. In [3H]adenine-labeled cells loaded with [3H]cyclic AMP by prior treatment with isoproterenol, alpha 1-adrenergic stimulation enhances disappearance of [3H]cyclic AMP. Measurements of cellular cyclic AMP give results similar to those obtained with the adenine labeling technic. We conclude that occupation of the myocyte alpha 1-receptor results in stimulation of cyclic AMP phosphodiesterase activity.     

Rapid agonist-induced decrease of 125I-pindolol binding to beta-adrenergic receptors. Relationship to desensitization of cyclic AMP accumulation in intact heart cells

Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment.     

Introduction of cyclic AMP phosphodiesterase into rat submandibular acini prevents isoproterenol-stimulated cyclic AMP rise without affecting mucin secretion

Cyclic AMP phosphodiesterase has been incorporated into isolated rat submandibular acini by hypotonic swelling. This resulted in complete inhibition of the cyclic AMP rise stimulated by isoproterenol (10 microM), but had no effect on the stimulation of mucin secretion. Acini swollen in the absence of cyclic AMP phosphodiesterase showed similar cyclic AMP and mucin secretion responses to those of unswollen acini. The dissociation between cyclic AMP rise and mucin secretion was not due to stimulation of different beta-receptor subtypes since both responses to isoproterenol were inhibited by the beta 1 antagonist atenolol, but not by the beta 2 antagonist, butoxamine. The results are the first to directly demonstrate that a maximally effective concentration of isoproterenol can increase mucin secretion in the absence of a detectable increase in cyclic AMP.     

Effect of the Tricyclic Antidepressant Desipramine on β-Adrenergic Receptors in Cultured Rat Glioma C6 Cells

Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of beta-adrenergic receptors in a time- and dose-dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic beta-adrenergic antagonist (+/-)-[3H]4-(3-tert-butylamino-2-hydroxypropoxyl)benzimidazole-2-o n HCl ([3H]CGP-12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine-treated cells accumulated less cyclic AMP than control cells. The affinity of the beta-adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 microM. The presence in the culture medium of alprenolol or propranolol, potent beta-adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of beta-adrenergic receptors from cells maintained in serum-free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of beta-adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.     

Alterations in activities of cyclic nucleotide systems and in beta-adrenergic receptor-mediated activation of cyclic AMP-dependent protein kinase during progression and regression of isoproterenol-induced cardiac hypertrophy.

Initial and transient increases in the basal levels of cyclic GMP in the heart were noted prior to cardiac hypertrophy in rats administered isoproterenol. Increased levels of cyclic AMP-phosphodiesterase (in both the soluble and particulate fractions) and stimulatory modulator of cyclic GMP-dependent protein kinase, however, were associated with the progression, or the state, of cardiomegaly, with their levels returning to the control values upon regression of the hypertrophy. The levels of cyclic GMP phosphodiesterase in the soluble fraction were lower, whereas those in the particulate fraction were higher, in the hypertrophied heart than the control. In cardiac hypertrophy, the maximal activity ratio(--cyclic AMP/+cyclic AMP) of cyclic AMP-dependent protein kinase in the incubated minced heart caused by isoproterenol was lower, whereas the concentration of isoproterenol required to increase the activity ratio half-maximally was higher than controls; the reduced responsiveness to the drug, however, was reversed when the hypertrophy regressed. These observations, taken collectively, appear to suggest that the desensitization of the beta-adrenergic mechanism seen in the cardiac hypertrophy produced by repeated administration of isoproterenol is associated with adaptive modifications in certain parameters of the cyclic nucleotide systems.     

Modeling beta-adrenergic control of cardiac myocyte contractility in silico

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.     

Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.     

Beta-adrenergic receptors and stimulatory effects of (-) isoproterenol on testosterone production in fetal mouse Leydig cells

Beta-adrenergic receptors were characterized in freshly excised fetal mouse testis using the radioiodinated antagonist iodocyanopindolol (ICYP). [125I]-CYP bound to a single class of high affinity sites with a KD value of 42.2 +/- 7.0 pM. Adrenergic agonists competed for ICYP binding sites with the following order of potency: (-)isoproterenol greater than (-)epinephrine much greater than (-)norepinephrine which is typical for a beta 2-adrenergic receptor. A selective beta 2-receptor antagonist ICI 118-551 showed an approximately 200 fold higher affinity than the beta 1-selective compound, betaxolol. The beta-adrenergic agonist (-)isoproterenol did not or slightly affect testosterone production by freshly isolated fetal Leydig cells. The ability of fetal Leydig cells to respond to (-)isoproterenol increased during culture. This change in responsiveness was not accompanied either by modification of the number of binding sites or by change in the binding affinity. Taken together these data suggest that i) the stimulatory effect of (-)isoproterenol on testosterone production by cultured fetal Leydig cells is mediated through beta 2-adrenergic receptors ii), the inability of freshly Leydig cells to respond to catecholamines is probably due to post receptor events.     

Pre-and Postnatal Developmental Changes of Adrenoceptor Subtypes in Rat Brain

beta-Adrenergic receptor subtypes, beta 1 and beta 2, were studied during pre- and postnatal development in the rat brain. [125I]Iodocyanopindolol (6-300 pmol/L) binding assays in the presence of 5-hydroxytryptamine (0.6-6 mumol/L) were used to measure exclusively beta-adrenergic receptors. In forebrain tissue, saturable and stereoselective binding was detected on gestational day 13. The amount of beta-adrenergic binding increased until postnatal day 23, when adult values were reached. The dissociation constants of [125I]iodocyanopindolol binding remained the same throughout development, as did the affinity of several beta-adrenergic and non-beta-adrenergic compounds. The proportion of the beta 2-adrenergic receptors was determined using the beta 1-selective antagonist ICI-89406 (7-150 nmol/L) and was found to change from 65% in prenatal forebrain tissue to 28% in adulthood. In cerebellum/medulla pons tissue, however, the proportion of beta 2-receptor binding (80%) remained unchanged during the whole developmental period.     

Ca2+, calmodulin and cyclic AMP-dependent modulation of actin-myosin interactions in aorta

Incubation of bovine aortic native actomyosin with cyclic AMP and bovine aortic cyclic AMP-dependent protein kinase produced a rightward shift in the relation between free Ca2+ and both superprecipitation and actomyosin ATPase activity. The relation between free Ca2+ and phosphorylation of myosin light chains was also shifted to the right. The concentration of free Ca2+ required for half-maximal activation of both ATPase activity and myosin light chain phosphorylation was approximately 1.0 microM for control actomyosin and 2.5 microM for actomyosin incubated with cyclic AMP-protein kinase. Neither basal nor maximal activities were significantly affected by incubation with cyclic AMP-protein kinase. Addition of e microM calmodulin to cyclic AMP-protein kinase-treated actomyosin relieved inhibition of both superprecipitation and myosin light chain phosphorylation. These findings suggest that cyclic AMP-protein kinase-mediated inhibition of actin-myosin interactions in vascular smooth muscle involve a shift in the Ca2+ sensitivity of the system. This shift probably involves Ca2+-calmodulin interactions and the control of phosphorylation of the myosin light chains.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase.

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.     

Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues.

The concentrations of the regulatory (R) and catalytic (C) subunits of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase(s) were measured in extracts of skeletal muscle, heart, liver, kidney, and brain. These concentrations were also estimated for the particulate fraction from brain, the only tissue in which a major part of the total activity was not readily extracted in a soluble form. Values for R were determined by measuring the amount of cyclic [3H]amp bound to protein in these tissue fractions under specified conditions; it was assumed that 1 mol of cyclic AMP binds to 1 mol of R. Values for C were determined from measurements of the specific protein kinase activity of the fractions utilizing the turnover number of pure C in the calculations. Turnover numbers for C were found to be identical for this subunit obtained in the pure form from rabbit skeletal muscle, rabbit liver, and beef heart. The methods used for measuring C were evaluated by kinetic studies and through the use of the specific heatstable protein inhibitor of cyclic AMP-dependent protein kinase(s). R and C were found to exist in a 1:1 molar ratio in all of the tissue fractions that were studied. the absolute concentrations of R and C ranged from 0.23 mumol/kg wet weight for liver to 0.78 mumol/kg wet weight for brain. For brain this value was based on the amount of each subunit in the particulate as well as the soluble fraction. For other tissues the values were based solely on the subunit content of the latter fraction. It was noted that the molar concentrations of R are close to those of cyclic AMP under basal conditions in the various tissues.     

Beta-adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

Effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity in bovine coronary artery

The effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity were compared in helical strips of bovine coronary artery. Elevation of cyclic AMP and activation of the protein kinase appeared to be well correlated with relaxation of potassium-contracted arteries by isoproterenol. Forskolin, at 1 microM or higher concentrations, also markedly elevated cyclic AMP levels, activated the kinase, and relaxed the arteries. However, a lower concentration of forskolin (0.1 microM) caused significant increases in both cyclic AMP levels and cyclic AMP dependent protein kinase activity, but did not relax the muscles. Relaxation caused by isoproterenol was accompanied by an apparent translocation of cyclic AMP dependent protein kinase activity from the soluble to the particulate fraction in these preparations. A similar shift in the distribution of the kinase was caused by various concentrations of forskolin, irrespective of whether the arteries were relaxed or not. In contrast to previous results in other tissues, low concentrations of forskolin (less than or equal to 1 microM), which themselves markedly elevated cyclic AMP levels in the arteries, did not potentiate the effects of isoproterenol on cyclic AMP levels or tension in these preparations. These results suggest that either cyclic AMP is not solely responsible for the relaxation caused by these agents, or some form of functional compartmentalization of cyclic AMP and cyclic AMP dependent protein kinase exists in this tissue.     

Characterization of Dopamine and β-Adrenergic Receptors Linked to Cyclic AMP Formation in Intact Cells of the Clone D384 Derived from a Human Astrocytoma

3,4-Dihydroxyphenylethylamine (dopamine) and beta-adrenergic receptor agonists and antagonists were assessed for their effects on cyclic AMP accumulation in human astrocytoma derived clone D384 cells. Dopamine, SKF 38393, and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene increased cyclic AMP content with Ka values of 2.0, 0.2, and 1.6 microM. The D1-selective antagonists SCH 23390 (Ki, 1.2 nM) and SKF 83566 (Ki, 0.8 nM) were over 5,000-fold more potent than the D2-selective antagonist domperidone (Ki, 6.7 microM) at inhibiting dopamine stimulation of cyclic AMP formation. SCH 23388 (Ki, 560 nM; the S-enantiomer of SCH 23390) was 400-fold less potent than SCH 23390. Isoprenaline, adrenaline, salbutamol, and noradrenaline increased cyclic AMP content with Ka values of 0.13, 0.12, 0.22, and 7.60 microM. The beta 2-selective antagonist ICI 118,551 (Ki,0.8 nM) was almost 8,000-fold more potent than the beta 1-selective antagonist practolol (Ki, 5.9 microM) at inhibiting isoprenaline stimulated cyclic AMP accumulation. These results demonstrate that D384 cells express D1-dopamine and beta 2-adrenergic receptors linked to adenylate cyclase. Furthermore, the dopamine receptor expressed by D384 cells exhibits a pharmacological profile typical of a mammalian striatal D1-receptor and therefore the use of this clone represents another approach to studying central D1-receptors.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)