Effect of cyclic 3,5-adenosine monophosphate on the virulence of Salmonella typhimurium

To study the role of cAMP in the virulence of S. typhimurium, cAMP-producing plasmid pTG 4 was transferred to cAMP-deficient S. typhimurium mutant. The transfer of the plasmid enhanced the virulence of the microorganisms due to the increased destruction of macrophages and the intensified multiplication of salmonellae in the spleen of mice.     

Involvement of adenylyn cyclase signaling mechanisms in relaxing effect of relaxin and insulin on the rat uterus, trachea and human myometrium

Participation of adenylyl cyclase signaling mechanisms of relaxin and insulin action in their regulating influence on the process of relaxation of the rat uterine and tracheal smooth muscles and human myometrium was shown. The study was based on the discovery of novel adenylyl cyclase signaling mechanisms of relaxin and insulin action in the muscle of vertebrates which involve: receptor --> Gi protein (betagamma dimer) --> phosphatidylinositol-3-kinase --> protein kinase Csigma (zeta) --> Gs protein --> adenylyl cyclase --> cAMP. In the rat uterus, trachea and human myometrium, relaxin, insulin and isoproterenol induced relaxation of KCl-contraction. The order of efficiency of the agents based upon their ability to induce the inhibiting effect on the KCl-contraction was as follows: relaxin = insulin > isoproterenol. The hormones induce activating effect on adenylyl cyclase leading to production of cAMP in the rat uterine and tracheal smooth muscles and human myometrium. It is shown that cAMP reproduces relaxing effect of the hormones under study. Thus, the involvement of novel adenylyl cyclase signaling mechanisms of relaxin and insulin action in realization of their relaxation effect on rat uterus, trachea and human myometrium was revealed for the first time.     

Selective regulation by pertussis toxin of insulin-induced activation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes

Incubation of 3T3-L1 adipocytes with insulin or isoproterenol for 10 min increased particulate "low Km" cAMP phosphodiesterase activity by 42% and 50%, respectively. Pertussis toxin catalyzed the [32P]-ADP ribosylation of a 41,000 dalton protein in adipocyte particulate fractions; prior incubation of adipocytes with toxin markedly reduced incorporation of radiolabel. Exposure of adipocytes to pertussis toxin (0.3 microgram, 18 hr) increased glycerol production and inhibited activation of cAMP phosphodiesterase by insulin, but not by isoproterenol. These results suggest that pertussis toxin can interfere with receptor-mediated processes that stimulate cAMP hydrolysis as well as those that inhibit cAMP formation.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

Effect of cAMP modifiers on the cytotoxic activity of macrophages

The effects of various cAMP modifiers on the changes in the intracellular cAMP level and on the coupling of the cAMP system with realization of macrophage cytotoxicity depending on their functional activity were studied. Nonactivated and activated by E. coli polysaccharides peritoneal macrophages of BALB/c mice and macrophage-like cells of J744 mice were incubated in the presence of cAMP modifiers and further assayed for cytolytic and cytostatic activities. Cells of tetraploid strain of Ehrlich carcinoma G10 were used as target cells. Among other modifiers only dibutyryl-cAMP caused a steady increase of the intracellular nucleotide content, whereas methylisobutylxanthine and isoproterenol in combination with methylisobutylxanthine caused only a temporary increase of the cAMP level. Isoproterenol did not induce any appreciable changes in the intracellular cAMP level. All modifiers under study suppressed the cytotoxic activity of macrophages irrespective of the nature of changes in the intracellular cAMP content. It was assumed that cAMP accomplishes a triggering function in the regulation of the cytotoxic activity of macrophages and that the cAMP system is universal in the regulation of cytotoxicity at various functional states of macrophages.     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

Phospholipid methyltransferase activity in diabetic rat fat cells: effect of isoproterenol and insulin

The effects of isoproterenol and insulin on phospholipid methyltransferase (PLMT) activity were investigated in adipocytes from control and streptozotocin-diabetic rats. PLMT activity was assayed by measuring the rate of incorporation of ³H-methyl groups from S-adenosyl-L-[methyl-³H] methionine into phospholipids. Basal PLMT activity was higher in adipocytes from diabetic animals. Treatment of adipocytes with isoproterenol induced a concentration-dependent stimulation of PLMT activity. In control adipocytes, the maximal effect was obtained at 100 nM isoproterenol with 2.3 fold increase in PLMT activity and a half maximal effect at 25 nM. In adipocytes from diabetic rats, a lower dose of isoproterenol (10 nM), caused 1.2 fold increase with a half maximal effect at 4 nM. Addition of 100 nM insulin inhibited basal PLMT activity and the stimulatory effect of isoproterenol in both types of adipocytes. The -adrenergic blocking agent propranolol inhibited the stimulatory effect of isoproterenol on PLMT activity in control and diabetic adipocytes. Intracellular concentration of cAMP was higher in diabetic adipocytes but decreased to normal values after incubation in the presence of insulin.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.     

Interaction of T-activin with peritoneal macrophages

The action of T-activin on peritoneal macrophages of CBA mice after its introduction into the animals has been studied. In intact mice the phagocytic activity of macrophages and their resistance to the cytopathogenic action of Salmonella typhimurium live cells remains unchanged. The injection of corpuscular pertussis vaccine into mice leads to a decrease in the resistance of macrophages to the action of salmonellae. The simultaneous injection of T-activin into mice in doses of 0.1 and 1.0 microgram per animal abolishes the damaging action of the vaccine. The analysis of the in vitro action of T-activin on macrophages of intact mice revealed that the preliminary incubation of cells with the preparation sharply increases their resistance to the action of salmonellae, while its introduction simultaneously with bacteria or after them rapidly leads to the death of macrophages. The action of T-activin is supposed to be linked with triggering the biosynthetic processes mediating the resistance of macrophages to the cytopathogenic action of salmonellae.     

Defective Fc-mediated phagocytosis in C3H/HeJ macrophages. II. Correction by cAMP agonists

Peritoneal macrophages from LPS hyporesponsive C3H/HeJ mice lose the capacity to bind and phagocytose opsonized sheep erythrocytes (EA) over a 48-hr culture period. This loss in Fc receptor capacity is markedly different from the progressive increase in phagocytic ability exhibited by cultured macrophages derived from LPS-responsive C3H/HeN mice. Since dibutyryl-cyclic adenosine monophosphate (DBcAMP) has previously been reported to modulate membrane receptor expression in lymphocytes and certain macrophage-like cell lines, we examined its effects on EA binding and phagocytosis by C3H/HeJ macrophages. DBcAMP not only reverses the binding defect in C3H/HeJ macrophages but also restores EA phagocytosis to the level of control C3H/HeN cultures. 8-Bromo-cAMP, as well as other agents known to elevate intracellular cAMP (i.e., isoproterenol plus isobutylmethylxanthine or prostaglandin E2) also corrected the phagocytic defect. Since the C3H/HeJ macrophage phagocytic defect can also be reversed by in vitro stimulation with a lymphokine-rich culture supernatant, we examined the effect of this treatment on intracellular cAMP levels. Lymphokine treatment produced a 60% increase in the levels of macrophage intracellular cAMP. These findings suggest that the C3H/HeJ differentiation defect may be secondary to some abnormality in a cAMP dependent pathway.     

Regulation of adenylyl cyclase signaling system by insulin, biogenic amines and glucagon at their separate and combined action in muscle membranes of mollusc Anodonta cygnea

In the smooth muscles of mollusc Anodonta cygnea the regulatory action of hormones on adenylyl cyclase signaling system (ACSS) are realized through the receptors of serpentine type (biogenic amines, isoproterenol, glucagon) and receptor tyrosine kinase (insulin) type. Intracellular mechanisms of their interaction are interconnected. Application of hormones, their antagonists and pertussis toxin in combination with insulin and biogenic amines or glucagon on adenylyl cyclase (AC) activity allows revealing the possible sites of cross-linking in the mechanisms of their action. Combined influence of insulin and serotonin or glucagon leads to decreased stimulation of adenylyl cyclase (AC) by these hormones, whereas combined application of insulin and isoproterenol suppresses AC-stimulating effect of insulin, but AC-inhibiting effect of isoproterenol is maintained in the presence and absence of non-hydrolysable analog of GTP—guanylyl imido diphosphate (GIDP). The specific blockage of AC-stimulating effect of serotonin by cyproheptadine—antagonist of serotonin receptors, did not change AC stimulation by insulin. Beta-adrenoblockers (propranolol and alprenolol) prevent inhibition of AC activity by isoproterenol, but did not change AC stimulation by insulin. Pertussis toxin blocked AC-inhibiting effect of isoproterenol and weakened AC-stimulating action of insulin. Thus, in the muscles of Anodonta cygnea negative interaction between ACS have been revealed, which are realized under combined influence of insulin and serotonin or glucagon, most probably, at the level of receptor of serpentine type (serotonin, glucagon), whereas under action of insulin and isoproterenol at the level of G_i protein and AC interaction.     

Inhibition of lipolysis in hamster adipocytes by the cation ionophor X537A.

The present study reports the effects of the lipophylic ionophore X537A on lipolysis and accumulation of cAMP in isolated hamster epidiymal adipocytes. X537A inhibited lipolysis activated with norepinephrine, isoproterenol, dibutyryl cAMP or theophylline but failed to influence basal lipolysis. The minimum effective concentration of X537A required to inhibit lipolysis was between 1 and 3 micrograms/ml; at a concentration of 10 micrograms/ml, X537A inhibited lipolysis by approximately 50%. The antilipolytic effect of X537A does not result from decreased formation of cAMP because the accumulation of cAMP in response to isoproterenol or theophylline was significantly potentiated in the presence of the ionophore. Most of the additional cAMP that accumulated in the presence of X537A was found to be intracellelular, the distribution of cAMP between cells and incubation medium not being influenced by X537A. Neither the basal activity of cAMP dependent protein kinase nor the activity in the presence of isoproterenol or theophylline was influenced by X537A. The effects of X537A on lipolysis and on accumulation of cAMP were found to persist in the absence of extracellular calcium, but adipocytes that were preincubated in a calcium free media containing 4.0 mM EGTA failed to respond to X537A with an increase in cAMP levels. It is concluded that X537A inhibits lipolysis by uncoupling cAMP accumulation from activation of triglyceride lipase by a mechanism unrelated to activation of protein kinase.     

The antilipolytic effect of insulin in human adipocytes requires activation of the phosphodiesterase

Human fat cells were incubated with two different cAMP analogues, 8-bromocAMP and 6N-monobutyrylcAMP. The former analogue is an excellent substrate for the phosphodiesterase while the latter is resistant to hydrolysis. In the presence of adenosine deaminase, isoproterenol (10(-6)M) stimulated lipolysis 8-10 fold which was similar to the effect exerted by the cAMP analogues. Basal lipolysis and lipolysis activated by 6N-monobutyrylcAMP was not inhibited by insulin even at high concentrations, whereas the effect of 8-bromocAMP was virtually completely inhibited. This effect of insulin was completely prevented by the addition of IBMX. Thus, activation of phosphodiesterase by insulin is necessary to elicit the antilipolytic effect in human adipocytes.     

Possible involvement of pertussis toxin-sensitive GTP-binding protein(s) in c-fos expression during differentiation of 3T3-L1 fibroblasts to adipocytes.

Following the differentiation of 3T3-L1 fibroblasts by insulin/dexamethasone/methylisobutylxanthine, marked increases in cAMP levels by isoproterenol but not forskolin and in 2-deoxyglucose uptake by insulin occurred. Pertussis toxin-pretreatment prior to addition of insulin/dexamethasone/methylisobutylxanthine and exposure of cells to pertussis toxin during differentiation attenuated glycerophosphate dehydrogenase activity as a differentiation marker enzyme and the responses to isoproterenol and insulin by approximately 50% of those in pertussis toxin-untreated cells. On the other hand, insulin/dexamethasone/methylisobutylxanthine caused induction of c-fos proto-oncogene in confluent 3T3-L1 fibroblasts. This induction was also reduced in pertussis toxin-pretreated cells. These results suggested that pertussis toxin-sensitive GTP-binding protein(s) is involved in expression of c-fos mRNA accompanied by differentiation. In addition, accumulation of c-fos mRNA by insulin/dexamethasone/methylisobutylxanthine was enhanced in protein kinase C-depleted cells pretreated with phorbol 12-myristate 13-acetate, indicating that protein kinase C may negatively regulate c-fos expression induced by insulin/dexamethasone/methylisobutylxanthine.     

Insulin-stimulated protein synthesis in submandibular acinar cells: interactions with adrenergic and cholinergic agonists

The effects of insulin and secretory agonists on amino acid incorporation into submandibular gland proteins were studied using isolated acinar cell aggregates. Insulin stimulated the incorporation of 3H-leucine into TCA-precipitable proteins in a rapid, dose-dependent manner (half-maximal response at 1 nM). Isoproterenol, a beta-adrenergic agonist, also stimulated amino acid incorporation, and this effect was mimicked by both dibutyryl cAMP and IBMX, a phosphodiesterase inhibitor. Although insulin further stimulated incorporation in the presence of isoproterenol and IBMX, no additional increase in the rate of synthesis was observed after stimulation by dibutyryl cAMP. High concentrations of carbamylcholine, a cholinergic agonist, inhibited both basal and insulin-stimulated incorporation. At low concentrations, however, carbamylcholine stimulated synthesis, and the effects of insulin and carbamylcholine were additive. A23187, a calcium ionophore, also inhibited 3H-leucine incorporation and insulin stimulation, but in contrast to carbamylcholine, low concentrations of A23187 neither inhibited nor enhanced the rate of synthesis. Thus, protein synthesis in the rat submandibular gland is regulated by both insulin and neurotransmitters. Whereas beta-adrenergic stimulation appears to be mediated through cAMP, the intracellular signals mediating the actions of insulin and cholinergic agonists remain to be elucidated.     

Influence of isoproterenol on net potassium uptake in whole pigeon erythrocytes in vitro

Isoproterenol increases net uptake of potassium in whole pigeon erythrocytes $\text{in vitro}$; effect of 10^?5 M isoproterenol is blocked by 10^?4 M propranolol. Pentifylline, a potent inhibitor of cAMP-phosphodiesterase, significantly amplifies effect of isoproterenol, indicating that isoproterenol-effect is mediated by cAMP. cAMP alone has no direct influence on net potassium uptake, while dibuturyl-cAMP has a very weak effect. Isoproterenol-effects are also mediated by the cell membrane protein-phosphorylation.