Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Preparation of hormone-sensitive airway smooth muscle adenylate cyclase from dissociated canine trachealis cells

Conventional homogenizing methods produced membrane preparations of canine trachealis airway smooth muscle which contained adenylate cyclase activity that was stimulated by fluoride but not by isoproterenol. We have devised methods using collagenase digestion of minced trachealis which destroy most of the tough connective tissues but leave dissociated canine trachealis cells in suspension. Gentle homogenization of these cells permitted preparation of a particulate fraction containing adenylate cyclase that was readily stimulated by beta-adrenergic agonist of prostaglandin E2. Isoproterenol stimulation was 2.34 +/- 0.58 (S.E.) times basal and 122 +/- 25% of the stimulation induced by NaF. The beta-adrenergic blocking agent propranolol prevented isoproterenol-induced stimulation of the cyclase but had no effect on prostaglandin E2 stimulation. Catecholamine order of potency was isoproterenol greater than epinephrine greater than norepinephrine. These methods enable demonstration of stimulatory effects of hormones in broken cell preparations of airway smooth muscle that are comparable to those when hormone-stimulated cyclic AMP formation is measured in intact muscle strips.     

Effect of prostaglandins and their analogues on hormone-stimulated glycogenolysis in primary cultures of rat hepatocytes

Hepatocytes were isolated by collagenase perfusion method from adult male rats, cultured and then prelabeled with [14C]glucose. The [14C]glycogen-labeled cells were used in experiments for effect of prostaglandins on hormone-stimulated glycogenolysis. Prostaglandin E1, prostaglandin E2 and 16,16-dimethylprostaglandin E2, but not prostaglandin D2 or prostaglandin F2 alpha, inhibited glycogenolysis stimulated by glucagon, epinephrine, isoproterenol (beta-adrenergic agonist) or epinephrine in the presence of propranolol (beta-antagonist) in primary cultured hepatocytes. The inhibitory effects on day 2 of cultures were approx. twice those on day 1. Dimethylprostaglandin E2 (10(-6)M) caused 60-70% inhibitions of the stimulations by these substances. In the case of the stimulation by glucagon, the inhibition further increased by 80-100% on day 3 of culture. Prostaglandin E1 and prostaglandin E2 caused less inhibition than dimethylprostaglandin E2 of all these stimulations. Dinorprostaglandin E1 (9 alpha,13-dihydroxy-7-ketodinorprost-11-enoic acid), which is a hepatocyte-metabolite of prostaglandin E1 and prostaglandin E2, and arachidonic acid did not have any inhibitory effects. These data indicate that the E series of prostaglandins may function as the regulation of hepatic glycogenolysis stimulated by epinephrine and glucagon, and that their rapid degradation system may contribute to the modulation of the action in liver.     

Beta-adrenergic regulation of contractility and protein phosphorylation in spontaneously beating isolated rat myocardial cells

Spontaneously beating heart myocytes were prepared from adult rat ventricular tissues to study the correlation between beta-adrenergic receptor-stimulated changes in contractile performance and protein phosphorylation in vitro. The plasma membrane of isolated myocardial cells was permeabilized by saponin in the presence of EGTA and Mg-ATP. The permeabilized myocytes, which formed a homogeneous cell population, retained the rod-cell morphology of heart cells in situ and showed spontaneous cyclic contractions. Their contractile activity in response to extracellularly added cAMP mimicked the effects caused by beta-adrenergic stimulation of the whole heart: both the frequency and longitudinal velocity of free contraction and relaxation of the cells increased. Similar increases were observed when beta-agonist, isoproterenol, and GTP were added to suspending medium. In addition, isoproterenol maximally enhanced the adenylate cyclase activity of the cells in the presence of GTP. Both of these effects of isoproterenol were completely blocked by the beta-antagonist propranolol. cAMP-mediated phosphorylation of proteins in the permeabilized myocytes was investigated under conditions in which the beating frequency increased. cAMP elevated the phosphorylation level of five proteins; three of them with apparent molecular masses of 24, 15, and 12 kDa were membrane proteins and the other two with apparent molecular masses of 150 and 28 kDa were myofibrillar proteins. The 24-kDa phosphoprotein dissociated into 12-kDa molecules when boiled in sodium dodecyl sulfate, suggesting that these proteins are oligomeric and monomeric forms of phospholamban. The phosphorylation of these five proteins was stimulated by isoproterenol. The effect of isoproterenol was enhanced by GTP but completely blocked by propranolol. The time course of their phosphorylation correlated well with that of the increase in the beating frequency of the cells; both were measured after the administration of isoproterenol and GTP. When propranolol was added after the start of the stimulation by isoproterenol, only phospholamban and the 15-kDa protein were rapidly dephosphorylated in close correlation with the decrease of the beating frequency. These results demonstrate for the first time that the permeabilized myocytes retain the functional beta-adrenergic receptor and cellular responses to beta-adrenergic stimulation. They also suggest that cAMP-mediated phosphorylation of proteins, possibly phospholamban and/or the 15-kDa protein, is involved in the increased contractile activity of permeabilized heart cells.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Hormones and Neurotransmitters Control Cyclic AMP Metabolism in Choroid Plexus Epithelial Cells

The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 micrograms/ml cholera toxin, a 50-fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10(-5) M isoproterenol, 10(-4) M prostaglandin E1, 10(-5) M histamine, and 10(-5) M serotonin caused increases of intracellular cyclic concentrations of 100-, 50-, 20-, and 4-fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol-mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin-dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained in vitro were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E2. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E2 release on the concentration of isoproterenol were similar, each response was maximal at 10(-6) M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E2 from amnion discs. Although prostaglandin E2, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, may be regulators of prostaglandin production by the amnion.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells.

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Biological activity of agarose-immobilized catecholamines.

Catecholamines substituted to agarose were synthesized in various ways. Norepinephrine and isoproterenol were linked to p-aminobenzamidohexyl agarose by an azo linkage to the catechol ring. Norepinephrine was also couple to hexyl agaros via the amino group, forming an amino, guanidino or amido bond. Biological activity of the immobilized catecholamines was determined by assessing their abilities to interact with adenylate cyclase in several membrane preparations and intact preparations of erythrocytes. In dog heart membranes, stimulation of adenylate cyclase by the catecholamine-gels could be accounted for by leached hormone which had been released from the gels. In frog erythrocyte membranes, leaching was minimal and no significant stimulation of adenylate cyclase was observed. Agarose-immobilized catecholamines, however, competitively inhibited isoproterenol stimulation of adenylate cyclase in these erythrocyte membranes indicating that catecholamines which are bound to agarose interact with the beta-adrenergic receptors as antagonists rather than agonists. When tested on intact frog erythrocytes, agarose immobilzed catecholamines did not increase the intracellular levels of cyclic AMP, although isoproterenol caused as 8-10 fold rise in these levels. Similarly, when tested for antagonist activity in the intact cells the agarose-catecholamines failed to inhibit the stimulation of cyclic AMP caused by isoproterenol. The difference observed in the beta-adrenergic antagonist activity of the agarose-bound catecholamines in membrane preparations and intact cells can be attributed to steric factors which could have prevented the access of the bead-bound ligands with the surface of the cell or to the possibility that receptors might be buried in the membrane matrix.     

Development of the linkage of beta-adrenergic receptors to cardiac hypertrophy and heart rate control: neonatal sympathectomy with 6-hydroxydopamine

Presynaptic neural projections are thought to participate in the maturation of postsynaptic sensitivity to neurotransmitters. In the current study, we have examined the effects of sympathectomy with 6-hydroxydopamine on the ontogeny of the linkage of beta-adrenergic receptors to cardiac growth and heart rate control in the rat. Destruction of sympathetic projections at birth compromised the ability of beta-receptor stimulation to evoke cardiac hypertrophy, a defect which persisted into young adulthood. The chronotropic response to beta-receptor activation, assessed by acute challenge with a submaximally-effective dose of isoproterenol, also exhibited a slowed development, but did eventually achieve normal sensitivity. In contrast, neonatal sympathectomy had only minor effects on resting heart rate, basal heart rate (the intrinsic rate in the absence of autonomic input) or maximal heart rate; these animals also showed beta-receptor desensitization of chronotropic action in response to chronic isoproterenol treatment. Chronic isoproterenol treatment itself lowered the basal heart rate, regardless of whether animals were normal or sympathectomized. Thus, during critical developmental periods, sympathetic input to beta-receptors selectively programmes the linkage between postsynaptic receptors, tissue growth and heart rate.     

Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters

The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.     

Interaction between increased SERCA2a activity and beta -adrenoceptor stimulation in adult rabbit myocytes

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a overexpression and phospholamban depletion have been shown to have beneficial effects on contractility in heart failure. However, the high sympathetic tone during development of failure may interact with increases in SERCA2a activity in potentially deleterious ways. We used adenoviral vectors to overexpress SERCA2a or partially downregulate phospholamban in adult rabbit ventricular myocytes in culture and studied the responses of these cells to beta-adrenoceptor stimulation. SERCA2a overexpression and phospholamban depletion had quantitatively similar effects on basal contraction amplitude and in accelerating relaxation. Increasing SERCA2a activity by either strategy had little effect on the increase in contraction amplitude or incidence of arrhythmias with increasing isoproterenol. Maximum acceleration of relaxation by beta-adrenoceptor stimulation was similar to that produced by SERCA2a overexpression. Isoproterenol treatment of SERCA2a-overexpressing or phospholamban-deficient myocytes produced a further modest decrease in relaxation time, with similar final values in both groups. We find no evidence for Ca(2+) overload induced by SERCA2a overexpression alone or in combination with catecholamines.     

Mechanisms of gonadotropin-releasing hormone and prostaglandin action on luteal cells

Both gonadotropin-releasing hormone (GnRH) and prostaglandin F2 alpha (PGF2 alpha) can inhibit cAMP and progesterone production in the corpus luteum; however, their mechanism of action is not known. GnRH or PGF2 alpha causes a rapid and marked increase of labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min into PA and PI, respectively. The labelling of the other phospholipids is not affected. GnRH and PGF2 alpha exert their stimulatory effects on PA-PI turnover at a mean effective dose value of ca. 15 and 100 nM, respectively. Their effects appeared to be additive when both agents were present in the same incubations. Interestingly, addition of the calcium ionophore A23187 also causes a dramatic increase of PA-PI turnover in luteal cells. By contrast, human chorionic gonadotropin and isoproterenol, agents that stimulate cAMP and progesterone production in luteal cells, as well as PGE2 (1 microM), all fail to alter phospholipid labelling; dibutyryl or 8-bromo-cAMP (2-5 mM) actually attentuates the GnRH or PGF2 alpha effect on PI and PA. A very similar PA-PI response to GnRH and PGF2 alpha has also been observed using rat granulosa cells in culture. It seems that following their binding to membrane receptors, GnRH and PGF2 alpha may share a common mechanism in the ovarian cell, possibly involving the stimulation of PA-PI metabolism.     

Potential role for prostaglandin F2 alpha, D2, E2 and thromboxane A2 in mediating the metabolic and hemodynamic actions of sympathetic nerves in perfused rat liver

In isolated rat liver perfused at constant pressure perivascular nerve stimulation caused an increase of glucose and lactate output and a reduction of perfusion flow. The metabolic and hemodynamic nerve effects could be inhibited by inhibitors of prostanoid synthesis, which led to the suggestion that the effects of nerve stimulation were, at least partially, mediated by prostanoids [Iwai, M. & Jungermann, K. (1987) FEBS Lett. 221, 155-160]. This suggestion is corroborated by the present study. 1. Prostaglandin D2, E2 and F2 alpha as well as the thromboxane A2 analogue U46619 enhanced glucose and lactate release and lowered perfusion flow similar to nerve stimulation. 2. The extents, the kinetics and the concentration dependencies of the metabolic and hemodynamic actions of the various prostanoids were different. Prostaglandin F2 alpha and D2 caused relatively stronger changes of metabolism, while prostaglandin E2 and U46619 had stronger effects on hemodynamics. Prostaglandin F2 alpha elicited greater maximal alterations than D2 with similar half-maximally effective concentrations. Prostaglandin F2 alpha mimicked the nerve actions on both metabolism and hemodynamics best with respect to the relative extents and the kinetics of the alterations. 3. The hemodynamic effects of prostaglandin F2 alpha could be prevented completely by the calcium antagonist nifedipine without impairing the metabolic actions of the prostanoid. Apparently, prostaglandin F2 alpha influenced metabolism directly rather than indirectly via hemodynamic changes. The present results, together with the previously described effects of prostanoid synthesis inhibitors, suggest that prostanoids, probably prostaglandin F2 alpha and/or D2, could be involved in the actions of sympathetic hepatic nerves on liver carbohydrate metabolism. Since prostanoids are synthesized only in non-parenchymal cells, nervous control of metabolism appears to depend on complex intra-organ cell-cell interactions between the nerve, non-parenchymal and parenchymal cells.     

Effects of prostaglandin E1 and isoproterenol on cyclic AMP content of human fibroblasts modified by time and cell density in subculture

In confluent cultures of “young” (< 30 generations) human fibroblasts, maximally effective concentrations of prostaglandin E₁ (5.6 μM) and isoproterenol (2 μM) increased cyclic AMP content several hundred-fold and approximately 30-fold, respectively. On the first day after initiation of cultures at either low (approx. 3 · 10⁵ cells) or high (approx. s · 10⁶ cells) cell density the magnitude of the isoproterenol effect was similar to that in confluent cultures. It increased during the next few days, reaching a maximum around day 2–3, and then declined. On any day during the period of subculture, the magnitude of the isoproterenol effect was inversely related to cell density. Alterations in response to prostaglandin E₁ as a function of time in subculture or cell density were less dramatic. The effects of prostaglandin E₁ were, however, smaller at some point during the first few days of subculture than after day 7, and when effects of prostaglandin E₁ were minimal, those of isoproterenol were maximal and approached those of prostaglandin E₁. On any day of subculture, cells in cultures of higher density tended to accumulate more cyclic AMP in response to prostaglandin E₁ than did those in low density cultures. The effects of prostaglandin E₁ and isoproterenol on cyclic AMP content were qualitatively similar in “young” and in “old” (> 60 generations in culture) human fibroblasts although the changes associated with duration of subculture and cell density tended to be less marked with “old” cells. In the “young” fibroblasts responsiveness to isoproterenol and prostaglandin E₁ appears to correlate with cell morphology and with the fractional rate of growth in subcultures. It is suggested that the capacities of the fibroblasts to respond to these two agents may be altered independently during growth of human fibroblasts.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Effects of vasoactive intestinal polypeptide, monoamines, prostaglandins, and 2-chloroadenosine on adenylate cyclase in rat cerebral microvessels

Abstract: Adenylate cyclase in microvessels isolated from rat cerebral cortex was stimulated by guanine nucleotides, catecholamines, prostaglandin E₁, prostaglandin E₂, and 2-chloroadenosine. Catecholamine stimulation was mediated by interaction with β-adrenergic receptors. The order of relative potency was: isoproterenol > epinephrine > norepinephrine. Activation of microvessel adenylate cyclase by prostaglandins E₁ and E₂ as well as by 2-chloroadenosine was dose related. Twenty-two peptides were tested for possible effects on the microvessel adenylate cyclase. Only vasoactive intestinal polypeptide (VIP) was stimulatory. No inhibitory action was observed. Activation by VIP required guanosine triphosphate and was dose dependent from 10 n M to μ M (ED₅₀= 0.1 μ M ). At 30°C, stimulation of adenylate cyclase by the peptide increased linearly with time for up to 15 min. The effect of VIP was not inhibited by phentolamine or propranolol, suggesting that its action was not elicited by interaction with α- or β-adrenergic receptors. Activation achieved by VIP and isoproterenol, prostaglandin E₁, or 2-chloroadenosine was the sum of the individual stimulations, suggesting that receptors for VIP were distinct from those for isoproterenol, prostaglandin E₁, and 2-chloroadenosine.     

Endorphin-regulated protein phosphorylation in brain membranes

This study was initiated to determine whether opioid peptides exert direct effects on the phosphorylation of specific proteins in membranes from rat neostriatum. It was found that low concentrations of β-endorphin (0.1–10nM) inhibit the phosphorylation of specific proteins designated F and H (M.W. 47,000 and 10–20,000 respectively). In addition, β-endorphin produced an overall stimulation of phosphate incorporation into other membrane proteins, the phosphorylation of which is dependent on calcium ions. The stimulatory effects were blocked by naloxone, but the inhibitory effects were not. The regulation of membrane protein-phosphorylation by endorphins may constitute a biochemical mechanism mediating for some of the physiological affects of these peptides on neuronal function.     

Effect of catecholamines on Na/H exchange in vascular smooth muscle cells

Catecholamines were found to activate Na/H exchange in a concentration-dependent manner in primary cultures of vascular smooth muscle cells (VSMC). The potency order was found to be epinephrine greater than norepinephrine greater than isoproterenol. The major pathway for catecholamine effects appeared to be via interaction with an alpha 1 adrenergic receptor. In addition, it was found that alpha 1 receptor-mediated Na/H exchange in VSMC was increased by angiotensin II and inhibited by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Adrenergic receptors have been shown to be coupled to both adenylate cyclase and to inositol phosphate release (Leeb-Lundberg, L. M. F., S. Cotecchia, J. W. Lomasney, J. F. DeBernadis, R. J. Lefkowitz, and M. G. Caron, 1985, Proc. Natl. Acad. Sci. USA, 82:5651-5655.). It was found that catecholamines increased AMP levels in the potency order isoproterenol greater than norepinephrine greater than epinephrine and the receptor involved was a beta adrenergic receptor. Since these findings did not parallel the results obtained for catecholamine stimulation of Na/H exchange, an increase in AMP levels was probably not the mechanism by which major pathway for catecholamine-stimulated Na/H exchange in VSMC (via the alpha 1 receptor) was activated. When the effects of catecholamines were measured on inositol phosphate release, the potency order for catecholamine stimulation was epinephrine greater than norepinephrine greater than isoproterenol, and the receptor involved was an alpha 1 adrenergic receptor. In addition, angiotensin II increased and TPA inhibited catecholamine-stimulated inositol phosphate release. Since these findings paralleled the results obtained for catecholamine stimulation of Na/H exchange, inositol phosphate release may be the mechanism by which the major pathway for catecholamine-stimulated Na/H exchange in VSMC (via the alpha 1 receptor) was activated.     

Pyruvate potentiates inotropic effects of isoproterenol and Ca(2+) in rabbit cardiac muscle preparations

Catecholamines and elevated extracellular Ca(2+) concentration ([Ca(2+)](o)) augment contractile force by increased Ca(2+) influx and subsequent increased sarcoplasmic reticulum (SR) Ca(2+) release. We tested the hypothesis that pyruvate potentiates Ca(2+) release and inotropic response to isoproterenol and elevated [Ca(2+)](o), since this might be of potential importance in a clinical setting to circumvent deleterious effects on energy demand during application of catecholamines. Therefore, we investigated isometrically contracting myocardial preparations from rabbit hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At a [Ca(2+)](o) of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 1.89 +/- 0.42 to 3.62 +/- 0.62 (SE) mN/mm(2) (n = 8, P < 0.05) and isoproterenol (10(-6) M) alone increased F(dev) from 2.06 +/- 0. 55 to 25.11 +/- 2.1 mN/mm(2) (P < 0.05), whereas the combination of isoproterenol and pyruvate increased F(dev) overproportionally from 1.89 +/- 0.42 to 33.31 +/- 3.18 mN/mm(2) (P < 0.05). In a separate series of experiments, we assessed SR Ca(2+) content by means of rapid cooling contractures and observed that, despite no further increase in F(dev) by increasing [Ca(2+)](o) from 8 to 16 mM, 10 mM pyruvate could still increase F(dev) from 26.4 +/- 6.8 to 29.7 +/- 7. 1 mN/mm(2) (P < 0.05, n = 9) as well as the Ca(2+) load of the SR. The results show that the positive inotropic effects of pyruvate potentiate the inotropic effects of isoproterenol or Ca(2+), because in the presence of pyruvate, Ca(2+) and isoproterenol induced larger increases in inotropy than can be calculated by mere addition of the individual effects.