Effects of beta-adrenoceptor-related agents on hypoxic pulmonary vasoconstriction and relation with cyclic AMP and prostaglandins]

Effects of a beta-agonist (isoproterenol) and beta-antagonists (propranolol and pindolol) on hypoxic pulmonary vasoconstriction (HPV) and on changes in some chemical mediators were compared between HPV-responsive lobes and non-responsive lobes in which HPV was induced by aspirin DL-lysine (ASA groups). Hypoxic ventilation (4 min) was repeated in 56 of isolated, blood-perfused dog lung lobes. Each drug was administrated in a bolus dose of 0.2 mg in the intermittent period between hypoxia. In HPV-responsive lobes, the first hypoxia increased pulmonary vascular resistance by 33% or more in all groups. Both isoproterenol and pindolol inhibited the elicitation of HPV completely, but propranolol induced almost the same degree of HPV as control. In ASA groups, HPV was completely inhibited by isoproterenol, but was not influenced by propranolol. However, pindolol's inhibitory effect on HPV was less than that in HPV-responsive lobes. Isoproterenol significantly increased cyclic AMP from 17.0 to 76.7 pmol/ml in HPV-responsive lobes (n = 7). Pindolol increased prostaglandin E2 from 87.0 to 1015.4 pg/ml in HPV-responsive lobes (n = 7), and from 93.4 to 361.3 pg/ml when ASA was treated. Propranolol did not show the different results from the control group whether ASA was present or not. The different mechanisms among beta-adrenoceptor-related agents in HPV and pulmonary circulation were investigated.     

Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

Thrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca(++) mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y(12) inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.     

Effects of hyperosmolarity on the cyclic AMP concentration and lipolysis of the adipocyte stimulated by adrenocorticotropic hormone.

The effects of ACTH on 3',5'-cyclic AMP (cAMP) levels and lipolysis were examined on isolated adipocytes incubated in either isosmolar or hyperosmolar media. The ability of ACTH to induce intracellular cAMP accumulation was greatly enhanced by incubating cells in hyperosmolar sucrose (100 to 400 mM) solutions. Hyperosmolar solutions prepared by the addition of either NaCL, glucose or mannitol enhanced the ACTH effect on cAMP to the same extent as did the hyperosmolar sucrose solution, but hyperosmolar urea solutions did not have such an effect. The effect of hyperosmolarity was shown only in cells stimulated by lipolytic hormones, and the effects were still evident in the presence of high concentrations of theophylline, indicating the effect of hyperosmolarity is to facilitate hormone action on the receptor-coupler system of the adipocyte membrane. The action of glucagon on cAMP was augmented much less than the actions of ACTH and isoproterenol. Basal as well as ACTH or exogenous cAMP stimulated lipolysis was lower in hyperosmolar sucrose solutions. Some mechanism by which hyperosmolarity interferes with the metabolic sequence beyond the accumulation of cAMP was suggested.     

Effects of betazole hydrochloride and cyclic AMP on the pepsinogen secretion by rabbit gastric mucosa in organ culture

The effects of betazole hydrochloride, dibutyryl cyclic AMP (DB-cyclic AMP) and betazole hydrochloride plus aminophylline on pepsinogen secretion by rabbit gastric mucosa were studied in organ culture. Betazole hydrochloride alone did not stimulate pepsinogen secretion at the concentrations of 10(-8), 10(-6), 10(-5) and 10(-4) M. However, 10(-3) M DB-cyclic AMP produced a significant stimulation of pepsinogen secretion into the culture medium when compared with the control. In the presence of 3 x 10(-3) M aminophylline, betazole hydrochloride in the concentration of 10(-5) and 10(-4) M stimulated pepsinogen secretion into the culture medium, and the magnitude of this increase was 2.0- and 2.8-fold, respectively, compared with the control. Aminophylline alone could not change pepsinogen secretion into the culture medium. These results suggested that the pepsinogen secretion, stimulated by betazole hydrochloride, was mediated by cyclic AMP in the chief cells.     

Epithelial cell death and cyclic AMP increase during palatal development.

Cyclic AMP levels increase abruptly in the secondary palatal shelf (mesenchyme plus a two- to three-cell-layered epithelium) between day 15 and 16 of gestation in the rat embryo. The addition of dibutyryl cyclic AMP to the media in which immature palatal shelves (day 14) are cultured causes a precocious decrease in DNA synthesis, but increased glycoprotein synthesis and epithelial adhesiveness. These changes are most evident in the medial-edge epithelial cells and resemble the developmental events that occur normally on day 15 and 16 during formation of the secondary palate. The initiation of epithelial cell death and adhesiveness in the medial edge of the palatal shelf may be mediated through an increase in cyclic AMP.     

Effects of bradykinin and indomethacin on cyclic GMP and cyclic AMP in lung slices

Bradykinin, 1-100 mug/ml, produced a rapid rise in the concentration of 3':5'-guanosine monophosphate (cyclic GMP) and 3':5'-adenosine monophosphate (cyclic AMP) in slices of guinea pig lung. Concentrations of both nucleotides reached a maximum in about 2 min, then declined to a basal levels in 6-12 min. The transient nature of the effect was presumbaly due to the rapid destruction of bradykinin as evidenced by (1) reaccumulation of nucleotides when bradykinin was added a second time, and (2) potentiation of the bradykinin effects by pyroGlu-Lys-Trp-Ala-Pro, a peptide that inhibits inactivation of bradykinin by kininase. It has been reported elsewhere that histamine, prostaglandins E(1) and E(2), and beta-adrenergic stimulation can cause accumulation of cyclic AMP in lung slices without affecting cyclic GMP concentration, whereas acetylcholine increases the concentrations of both cyclic GMP and cyclic AMP. Thus it was possible that the effects of bradykinin were indirect, i.e., secondary to release of one or more of these compounds. Promethazine (an antihistamine), propranolol (a beta-adrenergic blocking agent) and atropine (an anticholinergic agent) did not alter basal cyclic nucleotide concentrations or the effects of bradykinin. Two inhibitors of prostaglandin synthesis, indomethacin and aspirin, which alone were without effect, in the presence of bradykinin completely prevented the rise in cyclic AMP but did not interfere with cyclic GMP accumulation. Similarly, the effect of acetylcholine on cyclic AMP was abolished by indomethacin while that on cyclic GMP was unaltered. We suggest that in lung and probably in other tissues, bradykinin, acetylcholine, and perhaps other stimuli enhance the synthesis and release of prostaglandins as one of the consequences of their effects on cyclic GMP metabolism.     

Effect of isoproterenol on myocardial mechanical function and cyclic AMP content in the fetal rabbit

The effect of isoproterenol on mechanical function was studied in the isolated arterially perfused heart of the fetal (21st and 28th day of gestation) and newborn rabbits. The inotropic effect of isoproterenol in the fetus was less than in the newborn. In contrast, myocardial cyclic AMP levels after isoproterenol infusion in the fetus were greater than in the newborn. The inotropic effects of dibutyryl cyclic AMP and of calcium in the fetus were less than in the newborn. These data suggest that the process from beta-receptor to cyclic AMP in the fetus was equally or even more responsive to isoproterenol than in the newborn. The diminished inotropic effect of isoproterenol in the fetus may be due, at least in part, to the decreased inotropic effect of calcium.     

Evidence that platelet density depends on the alpha-granule content in platelets

The relation between platelet buoyant density and beta-thromboglobulin (beta-TG), a marker for platelet alpha-granule content, was assessed by three independent approaches. (1) Platelets were separated on iso- osmolar discontinuous Stractan density gradients into five fractions, ranging in density from 1.061 g/ml to 1.091 g/ml (20 degrees C). The beta-TG content (mean +/- SD, n = 17) increased with the platelet density from 27.8 +/- 8.6 micrograms beta-TG/10(9) cells (20% less- dense platelets) up to 65.6 +/- 15.5 micrograms beta-TG/10(9) cells (15% most-dense platelets). (2) Activation of platelets in platelet- rich plasma with thrombin, adenosine diphosphate, collagen, or epinephrine resulted in a decreased density of the platelets. This was only seen when there was simultaneous secretion of beta-TG. (3) The less-dense and the more-dense platelet fractions, after isolation by density gradient centrifugation, were separately treated with thrombin. After complete degranulation, the density distribution of the originally less-dense and more-dense platelets were identical and were much narrower than the density distribution of resting platelets.     

Evidence that cyclic GMP may regulate cyclic AMP metabolism in the isolated frog ventricle

Both acetylcholine and 8-bromo cyclic GMP depress the contractile response of the isolated frog ventricle. An investigation has been made of the effects of both substances on the metabolism of endogenous 3,5 cyclic nucleotides. The levels of adenosine 3′, 5′ cyclic monophosphate (cyclic AMP) and guanosine 3′, 5′ cyclic monophosphate (cyclic GMP) were measured after superfusing preparations with varying concentrations (10^?10 to 10^?4m) of acetylcholine and 8-bromo cyclic GMP. The decline in contractile force was found to be accompanied by a progressive fall in intracellular cyclic AMP and a rise in cyclic GMP levels. Both the decline in contractility and the reduction in endogenous cyclic AMP are attenuated by 10^?4 theophylline. The decline in isometric twitch tension was paralleled, under all conditions, by a quantitatively equivalent reduction in the ratio cyclic AMP: cyclic GMP. The possibility that endogenous cyclic GMP may accelerate the conversion of cyclic AMP to 5 AMP, by stimulating a cyclic GMP-sensitive form of cyclic AMP phosphodiesterase, is discussed.     

Forskolin stimulates prostaglandin synthesis in rabbit heart by a mechanism that requires calcium and is independent of cyclic AMP

Infusion of forskolin, an adenylate cyclase activator, in concentrations (2 microM) that do not alter basal prostaglandin (PG) synthesis inhibit synthesis of PG elicited by isoproterenol in rabbit heart. This inhibitory action of forskolin appears to be dependent on cyclic AMP (cAMP). Bolus injection of forskolin (75 nmol), however, was found to stimulate PG synthesis in rabbit heart. The purpose of this study was to elucidate the mechanism of the stimulatory action of forskolin on PG synthesis (prostaglandin I2 measured as 6-ketoprostaglandin F1 alpha [6-keto-PGF1 alpha]) in isolated perfused rabbit heart. Forskolin enhanced PG production in a dose-dependent manner. 1,9-Dideoxyforskolin, a forskolin analogue devoid of adenylate cyclase-stimulating activity, also enhanced PG synthesis. The cAMP analogue chlorophenylthio-cAMP failed to stimulate output of 6-keto-PGF1 alpha, although this agent produced dose-related changes in mechanical function in rabbit heart. Furthermore, the adenylate cyclase inhibitor (-)-N6-(R-phenylisopropyl)adenosine potentiated, whereas the phosphodiesterase inhibitor cilostamide attenuated, forskolin-stimulated PG production. (-)-N6-(R-Phenylisopropyl)adenosine and cilostamide had no effect on the mechanical actions of chlorophenylthio-cAMP, suggesting selectivity of these agents for adenylate cyclase and phosphodiesterase, respectively. 6-Keto-PGF1 alpha output elicited by forskolin was abolished by reduction of calcium in the perfusion fluid as well as by the calcium channel blocker diltiazem. The intracellular calcium antagonists TMB-8 and ryanodine also abolished forskolin-stimulated PG synthesis in rabbit heart. PG synthesis stimulated by 1,9-dideoxyforskolin was also prevented by reduced extracellular calcium, diltiazem, and ryanodine. The calmodulin antagonists trifluoperazine, W-7, and calmidazolium failed to significantly alter PG production in response to forskolin. These results indicate that forskolin-stimulated PG synthesis in rabbit heart is independent of cAMP and requires calcium from both extracellular and intracellular sources.     

Parathyroid hormone does not increase nephrogenous cyclic AMP excretion by the dog

The renal excretion of nephrogenous cyclic AMP (NcAMP) increases in response to parathyroid hormone (PTH) in man, and thereby serves as a test of parathyroid function. However, during studies of calcium-PTH (PTH) in man, and thereby serves as a test of parathyroid function. However, during studies of calcium-PTH interrelationships in dogs we observed no change in total urinary cAMP (UcAMP) excretion when endogenous plasma PTH levels were increased up to 10-fold. This study was designed to investigate the effects of physiologic and pharmacologic levels of PTH on NcAMP and UcAMP excretion in the dog. Maintaining plasma Ca 2 mg/dl below normal for 40 minutes caused an 8-fold increase in plasma PTH concentration, a 50% increase in the urinary fractional excretion of phosphate (FEP) but no changes in plasma cAMP levels or in UcAMP or NcAMP excretion. Infusion of a pharmacologic amount of parathyroid extract (15 U/min for 20 min) increased plasma cAMP 5-fold, UcAMP excretion 3-fold and FEP by 50% but was without effect on NcAMP excretion. We conclude that NcAMP excretion is not stimulated by PTH in the dog and thus cannot be used as an index of PTH action in vivo. The increase in UcAMP excretion by pharmacologic amounts of PTH results from glomerular filtration of increased plasma cAMP, which may be generated in bone.     

The quantitation of platelet-associated IgG on cohorts of platelets separated from healthy individuals by buoyant density centrifugation

Evidence suggests that as platelets age in the circulation they become progressively smaller and less dense through the loss of protein. The smallest, least dense platelets have a significantly shortened survival, but the mechanism of clearance of these platelets is not known. To evaluate whether the binding of IgG could play a role in the clearance of senescent platelets, we measured platelet size, total protein, and platelet-associated IgG on subpopulations of platelets isolated from 6 healthy individuals using a discontinuous iso-osmotic arabinogalactan (stractan) gradient. There was a close correlation between density, size, and total protein content (r greater than 0.9) for all platelet fractions. There was also a relationship between the amount of platelet-associated IgG (PAIgG), total protein, and platelet size (r greater than 0.9) for the first 3 progressively less dense platelet factions. However, the fourth platelet fraction containing the smallest, least dense, and on current evidence, oldest platelets had very elevated amounts of IgG. This amount was approximately 10 times higher than the mean platelet IgG for the same individual and was similar to the amount of PAIgG found on platelets from patients with immune thrombocytopenia. A progressive increase in the ratio of PAIgG measured after platelet solubilization to PAIgG measured on intact platelets was also noted for the first three populations, indirectly suggesting that platelets clear IgG from their surface during aging. Increased binding of IgG to senescent platelets may mediate their destruction.     

Agents that increase cellular cyclic AMP inhibit proliferative activity and decrease lipid content in cells cultured from atherosclerotic human aorta

Cholera toxin, methylisobutylxanthine and prostacyclin (PGI2) analogues as well as dibutyryl cyclic AMP inhibit by 2-7-fold 3H-thymidine uptake into intimal cells isolated from atherosclerotic human aorta in primary culture. These agents also decrease cholesteryl ester and triglyceride levels and do not affect content of phospholipids and free cholesterol in cells cultured from atherosclerotic lesions.