Tissue Concentration Of Cyclic AMP Research Articles

Effects of prostaglandin F2α and other potential secretagogues on oxytocin secretion and second messenger metabolism in the ovine corpus luteum in vitro

Release of oxytocin by sliced or minced sheep luteal tissue in vitro was stimulated up to 1.6- and 2.3-fold by arachidonic acid and the calcium ionophore A23187 respectively. Prostaglandin (PG) F2 alpha and the PGF2 alpha analogue cloprostenol, and other potential agonists known to be active in vivo, including noradrenaline and acetylcholine, were ineffective, as was the phorbol ester tetradecanoylphorbol acetate (TPA). The ineffectiveness of PGF2 alpha was not due to a general unresponsiveness of the tissue in vitro, as PGF2 alpha reduced LH stimulation of tissue concentrations of cyclic AMP and activated inositol lipid hydrolysis. The effect of arachidonic acid was accompanied by release from the tissue of the cytosolic enzyme lactate dehydrogenase (at arachidonic acid concentrations below those required to release oxytocin) and its effect on oxytocin and lactate dehydrogenase release was mimicked by oleic and linolenic acids; arachidonic acid was concluded to act by a non-physiological physicochemical effect without conversion to an eicosanoid. As PGF2 alpha in vitro is known to raise intracellular Ca2+ concentrations in the large luteal cells that secrete oxytocin, and as A23187 stimulates oxytocin release in vitro in the presence and absence of TPA, it is concluded that in-vitro incubation results in an artifactual blockade of the oxytocin-releasing action of PGF2 alpha at an unidentified point distal to the effect on intracellular Ca2+.

Glucagon and insulin regulate in Vitro hepatic glycogenolysis in the axolotl Ambystoma mexicanum via changes in tissue cyclic AMP concentration

Glucagon increases the rate of glycogenolysis in in vitro cultures of hepatic tissue from the axolotl Ambystoma mexicanum. The hormone causes an increase in the concentration of cyclic AMP in the tissue which is followed by activation of glycogen phosphorylase and subsequent breakdown of glycogen and release of glucose from the tissue. Insulin counteracts the glycogenolytic effect of glucagon by inhibiting the increase in tissue cyclic AMP concentration brought about by glucagon. This inhibitory effect of insulin is not seen in the presence of the phosphodiesterase inhibitor IBMX and so it appears that the initial action of insulin is a stimulation of cyclic AMP phosphodiesterase activity which lowers the tissue concentration of cyclic AMP and so counters the actions of hormones that act by raising the tissue concentration of cyclic AMP. This model for the mode of action of insulin is supported by the finding that insulin also interferes with the glycogenolytic actions of adrenaline, a second hormone which acts by raising tissue cyclic AMP concentrations.

A possible mechanism of action of a beta-adrenergic partial agonist (carteolol) in guinea pig taenia caecum.

Carteolol, 5-(3-tert-butylamino-2-hydroxy)propoxy-3, 4-dihydrocarbostyril hydrochloride was found to be a beta-adrenergic partial agonist in the taenia caecum of guinea pig. Concentration response curves of carteolol and isoprenaline were parallelly shifted by propranolol (10(-7) M) suggesting that site of action of both the drugs was the same beta-adrenoceptor. The pD2-values of carteolol obtained from the concentration action curves in mechanical response and increase of tissue concentration of cyclic AMP were significantly different from its pA2-value against isoprenaline and its pKi-value (negative log of its apparent dissociation constant). A possible explanation of our observation would seem to be as follows. Carteolol and isopranaline interact with the same beta-adrenoceptor where there may be two different sites: a binding site for agonist and binding site for antagonist.

Cyclic nucleotides and contractility in human and sheep umbilical arteries

Strips of human umbilical artery (HUA) and sheep umbilical artery (SUA) were freeze-clamped at selected times during drug-induced contraction or relaxation. Tissue concentrations of cyclic AMP and cyclic GMP were measured. 5-Hydrpxytryptamine (10 μM) contracted HUA and SUA, but had no detectable effect on cyclic GMP; cyclic AMP rose in SUA (but not in HUA) after contractions had begun. Histamine (4 gmM) elevated cyclic GMP in HUA with a time course that lagged behind contraction. Prostaglandin E 1 (PGE 1), PGE 2 and PGF 2α (2.8 μM with 0.1 % v/v ethanol) contracted HUA and decreased cyclic GMP. Nitroglycerin (3 μM) relaxed KCl-contracted HUA; this relaxation was preceded by an initial 29-fold elevation of cyclic GMP. Our data shows no consistent correlation of cyclic nucleotide changes with contraction of unbilical arteries and suggest that such changes are not essential for contraction of vascular smooth muscle. In contrast, the temporal relationship between cyclic GMP elevation and relaxation induced by nitroglycerin suggests a possible role for cyclic GMP.

Peripheral resistance to the cellular action of insulin in obese diabetic subjects in vivo.

1. The concentrations of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) in human adipose tissue were studied after intravenous insulin administration (0.3 unit/kg body weight) in obese diabetic and obese non-diabetic subjects. 2. The declines in plasma glucose, glycerol and free fatty acids in the two groups after insulin were similar if allowances were made for the fasting hyperglycaemia in the diabetic subjects. 3. Insulin in vivo lowered the concentrations of cyclic AMP in adipose tissue from non-diabetic subjects. 4. Insulin administration in vivo did not alter the tissue concentrations of cyclic AMP in adipose tissue of obese diabetic subjects. This lack of effect of insulin provides evidence for tissue resistance in vivo to the action of insulin in diabetes mellitus.

Ethanol inhibition of antigen-induced histamine release from guinea pig lung: Correlation with intracellular levels of cyclic nucleotides

The effect of ethanol on histamine release from lungs of sensitized guinea pigs was studied in conjunction with measurements of tissue concentrations of cyclic AMP and cyclic GMP. Addition of antigen in vitro elicited a rapid increase in cyclic AMP and cyclic GMP and stimulated release of histamine. Ethanol (2%) inhibited antigen-induced release of histamine over 95% and completely inhibited the increase in both cyclic nucleotides. The activity of cyclic AMP-dependent protein kinase was only slightly affected by ethanol. Metiamide blocked the ovalbumin stimulated increase in cyclic AMP but not cyclic GMP. Pyrilamine did not prevent the rise in either cyclic nucleotide. This suggests that the antigen-induced rise in cyclic AMP is an indirect result of histamine released from the tissue. The inability of H 1 and H 2 receptor antagonists to affect antigen-induced elevation of cyclic GMP in sensitized lung fragments suggests that an elevation in cyclic GMP might be either a primary event in the mediator release sequence or secondary to the release of a mediator other than histamine. The ability of ethanol to inhibit mediator release might be due to its capacity to attenuate the antigen-induced elevation of cyclic GMP in sensitized lung.

Coordinate control of intermediary metabolism in rat liver by the insulin/glucagon ratio during starvation and after glucose refeeding: Regulatory significance of long-chain acyl-CoA and cyclic AMP

The levels of serum insulin, glucagon, and free fatty acids (FFA) and the tissue concentrations of hepatic cyclic AMP, long-chain acyl-CoA (LCA), adenine nucleotides, inorganic phosphate, the intermediates of the Embden-Meyerhof pathway, the citric acid cycle (including acetyl-CoA and free CoA), and the cytoplasmic and mitochondrial redox couples were determined in the rat 12, 24, and 48 h after food withdrawal and 5, 10, 20, 40, 60, and 120 min after the refeeding of glucose. Using the measured metabolite contents in the liver, the alterations in the concentration of malate, oxaloacetate, citrate, and α-ketoglutarate and the changes in the energy state of the adenine nucleotide system and the redox state of the NAD system were attributed to the cytoplasmic and mitochondrial compartments by applying established calculation methods. Glucose refeeding provoked significant alterations in all parameters investigated. These changes occurred within minutes, reversing the hormone and metabolite pattern which had developed within 24 h in response to food withdrawal. Particularly, glucose refeeding resulted in a drastic increase in the insulin/glucagon ratio. Simultaneously, the level of serum FFA and the concentration of LCA in the liver declined. The latter alteration was accompanied by an increase in the cytoplasmic and a decrease in the mitochondrial ATP ADP × P ratios. Moreover, the redox state of the cytoplasmic NAD system was shifted toward the oxidized state. When the appropriate data were plotted against each other, highly significant correlations were obtained (i) between the insulin/glucagon ratio and the serum FFA concentration, (ii) between the serum FFA concentration and the concentration of hepatic LCA, (iii) between the hepatic LCA concentration and the cytoplasmic energy state, and (iv) between the cytoplasmic energy state and the redox state of the cytoplasmic NAD system. These findings are interpreted to support the hypothesis derived from experiments carried out in vitro that the insulin/glucagon ratio via the FFA-dependent concentration of hepatic LCA might affect the translocation of adenine nucleotides between the cytoplasmic and the mitochondrial compartment, thereby regulating the cytoplasmic energy state and the redox state of the cytoplasmic NAD system, consequently. Glucose refeeding provoked rapid coordinate changes in the concentration of the intermediates of both the citric acid cycle and the Embden-Meyerhof chain, indicating the altered substrate flow through these pathways. Those metabolites, known to modulate the activity of key regulatory enzymes in vitro, were analyzed with respect to their suggested regulatory function. As to the established shift from pyruvate carboxylation to pyruvate decarboxylation after glucose refeeding, the data revealed that the decrease in pyruvate carboxylase activity can be attributed to the decrease in the intramitochondrial ATP ADP ratio and the simultaneous fall in acetyl-CoA concentration, while the coordinate increase in pyruvate dehydrogenase activity can be ascribed to the decline in the concentration of LCA and, consequently, in the ratios of ATP ADP , NADH NAD , and acetyl- CoA CoA within the mitochondria. As for the citric acid cycle, increased citrate synthesis from acetyl-CoA and oxaloacetate was supported by the rapid drop in the concentration of the established inhibitor of citrate synthesis, LCA. In contrast, the concentration of succinyl-CoA, an inhibitor of the enzyme in vitro, remained practically constant, questioning its regulatory function under the present experimental conditions. In addition to the activation of citrate synthase, the coordinate activation of isocitrate dehydrogenase was indicated by the LCA-mediated decline in both the mitochondrial ATP ADP and the NADH NAD ratios. Glucose refeeding immediately reduced urea excretion to basal values. This alteration was preceded by a drastic fall in the tissue concentration of cyclic AMP, supporting the physiological role of the nucleotide in the control of hepatic gluconeogenesis. In contrast, the observed changes in the concentration of the effectory acting metabolites (ATP, AMP, fructose 1,6-diphosphate, citrate, and alanine) were incompatible with the suggested function of these intermediates in switching over the substrate flow through the Embden-Meyerhof pathway from gluconeogenesis to glycolysis. The results are discussed in reference to the known rapid stimulation of fatty acid biosynthesis in the liver and to the transfer of reducing equivalents by the different shuttles of the inner mitochondrial membrane. In summary, it can be concluded that the insulin/glucagon ratio in a moment-to-moment fashion controls the glucose balance across the liver by regulating hepatic intermediary metabolism via the concentration of both LCA and cyclic AMP.

The inhibition of histamine release from guinea-pig lung by compound I.C.I. 74917 and isoprenaline.

Compounds that increase tissue concentrations of cyclic AMP inhibit the release of histamine from sensitized tissues when they are challenged with antigen. It has been proposed that the anti-asthmatic agent disodium cromoglycate exerts its anti-allergic properties by inhibiting cyclic AMP phosphodiesterase. This hypothesis was based on experiments where it was shown that the inhibition of antigen-induced mast-cell degranulation by isoprenaline and disodium cromoglycate was synergistic (Taylor et al., 1974), and experiments that showed that disodium cromoglycate was a phosphodiesterase inhibitor (Ray & Warren, 1974). Compound I.C.I. 74917, Bufrolin (6-n-butyl-2,8-dicarboxy-4,10-dioxo-1,4,7,10tetrahydro-l,7-phenanthroline) is a potent inhibitor of immediate hypersensitivity reactions in guinea-pig lung and rat mast cells (Evans & Thomson, 1975). It demonstrates inhibition characteristics in lung similar to those of disodium cromoglycate in mast cells, including the critical time of addition relative to antigen (see Fig. la) and the development of tolerance (K. J. Barrett-Bee, unpublished work). However, it is active at onehundredth the dose that disodium cromoglycate is active in vitro. The work described below investigated the allergic release of histamine from guineapig lung and its inhibition by compound I.C.I. 74917 and isoprenaline, alone and in combination, to assess the nature of the inhibition.

Relationship between Intracellular Cyclic AMP and Lipolysis in Human Adipose Tissue

Human subcutaneous adipose tissue has been incubated in vitro in the presence and absence of isoprenaline (ISNA). The tissue concentration of cyclic AMP (cAMP) and the release of glycerol into the incubation medium were measured after various incubation periods. In the presence of ISNA (6 X 10(-5) mol/l), the tissue concentration of cAMP reached a peak after around 10 min and then declined to a level significantly lower than that at the start of the incubation. In contrast, the ISNA-induced rate of lipolysis was a linear function of the incubation time. The addition of propranolol (13 mumol/l) at different times after ISNA did not influence the rate of lipolysis, although it resulted in a decrease in the tissue level of cAMP. There was a positive correlation between the maximal increase in tissue cAMP and the rate of lipolysis in adipose tissue exposed to ISNA, both in individual experiments and in a group of 23 persons. No correlation was found between the rate of lipolysis and the tissue level of cAMP in adipose tissue incubated under basal conditions. The findings are compatible with the theory that the beta-adrenergic-induced lipolysis by human adipose tissue is a function of the maximal rise in the concentration of tissue cAMP. It is concluded that this peak level of cAMP represents single compartment of the nucleotide.

Mechanisms regulating adipose-tissue pyruvate dehydrogenase

1. Isolated rat epididymal fat-cell mitochondria showed an inverse relationship between ATP content and pyruvate dehydrogenase activity consistent with competitive inhibition of pyruvate dehydrogenase kinase by ADP. At constant ATP concentration pyruvate rapidly activated pyruvate dehydrogenase in fat-cell mitochondria, an observation consistent with inhibition of fat-cell pyruvate dehydrogenase kinase by pyruvate. Pyruvate dehydrogenase in fat-cell mitochondria was also activated by nicotinate (100mum) and by extramitochondrial Na(+) (replacing K(+)) but not by ouabain or insulin. 2. In rat epididymal fat-pads incubated in vitro pyruvate dehydrogenase was activated by addition of insulin in the absence of substrate or in the presence of glucose (10mm) or fructose (10mm). Glucose and fructose activated the dehydrogenase in the absence or in the presence of insulin, and pyruvate also activated in the absence of insulin. It is concluded that extracellular glucose, fructose and pyruvate may activate the dehydrogenase by raising intracellular pyruvate and that insulin may activate the dehydrogenase by some other mechanism. 3. Ouabain (300mum) and medium in which K(+) was replaced by Na(+), activated pyruvate dehydrogenase in epididymal fat-pads. Prostaglandin E(1) (1mug/ml), 5-methylpyrazole-3-carboxylate (10mum) and nicotinate (10mum), which are as effective as insulin as inhibitors of lipolysis and which like insulin lower tissue concentration of cyclic AMP (adenosine 3':5'-cyclic monophosphate), did not activate pyruvate dehydrogenase. Higher concentrations of prostaglandin E(1) (10mug/ml) and nicotinate (100mum) produced some activation of the dehydrogenase. 4. It is concluded that the activation of pyruvate dehydrogenase by insulin is not due to the antilipolytic effect of the hormone and that the action of insulin in lowering adipose-cell concentrations of cyclic AMP does not afford an obvious explanation for the effect of the hormone on pyruvate dehydrogenase. The possibility that the effects of insulin, ouabain and K(+)-free medium may be mediated by Ca(2+) is discussed.

Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones.

1. In epididymal adipose tissue synthesizing fatty acids from fructose in vitro, addition of insulin led to a moderate increase in fructose uptake, to a considerable increase in the flow of fructose carbon atoms to fatty acid, to a decrease in the steady-state concentration of lactate and pyruvate in the medium, and to net uptake of lactate and pyruvate from the medium. It is concluded that insulin accelerates a step in the span pyruvate-->fatty acid. 2. Mitochondria prepared from fat-cells exposed to insulin put out more citrate than non-insulin-treated controls under conditions where the oxaloacetate moiety of citrate was formed from pyruvate by pyruvate carboxylase and under conditions where it was formed from malate. This suggested that insulin treatment of fat-cells led to persistent activation of pyruvate dehydrogenase. 3. Insulin treatment of epididymal fat-pads in vitro increased the activity of pyruvate dehydrogenase measured in extracts of the tissue even in the absence of added substrate; the activities of pyruvate carboxylase, citrate synthase, glutamate dehydrogenase, acetyl-CoA carboxylase, NADP-malate dehydrogenase and NAD-malate dehydrogenase were not changed by insulin. 4. The effect of insulin on pyruvate dehydrogenase activity was inhibited by adrenaline, adrenocorticotrophic hormone and dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate). The effect of insulin was not reproduced by prostaglandin E(1), which like insulin may lower the tissue concentration of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and inhibit lipolysis. 5. Adipose tissue pyruvate dehydrogenase in extracts of mitochondria is almost totally inactivated by incubation with ATP and can then be reactivated by incubation with 10mm-Mg(2+). In this respect its properties are similar to that of pyruvate dehydrogenase from heart and kidney where evidence has been given that inactivation and activation are catalysed by an ATP-dependent kinase and a Mg(2+)-dependent phosphatase. Evidence is given that insulin may act by increasing the proportion of active (dephosphorylated) pyruvate dehydrogenase. 6. Cyclic AMP could not be shown to influence the activity of pyruvate dehydrogenase in mitochondria under various conditions of incubation. 7. These results are discussed in relation to the control of fatty acid synthesis in adipose tissue and the role of cyclic AMP in mediating the effects of insulin on pyruvate dehydrogenase.

On the similarity of effects of aldosterone and adenosine 3′,5′-phosphate on Na + transport and glucose metabolism in toad bladder

The stimulation of Na + transport in the toad bladder by aldosterone is associated with an increased utilization of oxygen and an increase in the evolution of 14CO 2 from [6- 14C]glucose in response to the increased energy requirement of stimulated Na + transport. These are secondary effects of aldosterone and are abolished by the removal of Na + from the mucosal bathing medium and consequent absence of Na + transport. At the same time, however, aldosterone decreases the evolution of 14CO 2 from [1- 14C]glucose. This effect persists even in the absence of Na + and has similar characteristics of time of onset, concentration response, steroid specificity, and sensitivity to actinomycin D and spirolactone. Both the effect of aldosterone to decrease the evolution of 14CO 2 from [1- 14C]-glucose and its ability to stimulate Na + transport can be reproduced by adenosine 3′,5′-phosphate (cyclic AMP). This has been shown for endogenous cyclic AMP produced by the action of theophylline or vasopressin in combination with theophylline and for exogenous cyclic AMP as seen with N 6-2′- O-dibutyryl adenosine 3′,5′-phosphate (dibutyryl cyclic AMP) or with cyclic AMP in the presence of low concentrations of theophylline. Although these results suggest that cyclic AMP might mediate the action of aldosterone, no change in tissue concentration of cyclic AMP was found even after prolonged incubation with the hormone.

The influence of insulin and epinephrine on adenosine 3′,5′-phosphate and glycogen transferase in muscle

1. 1. The role of adenosine 3′,5′-phosphate (cyclic AMP) in the mechanism of action of insulin on the enzyme glycogen transferase (UDP glucose: α-1,4-glucan α-4-glucosyltransferase, EC 2.4.1.11) was investigated in rat diaphragm muscle. 2. 2. Incubation of intact rat diaphragms with insulin alone did not alter the cyclic AMP content of the muscle. When the diaphragms were exposed to both insulin and epinephrine simultaneously, the increase in the cyclic AMP concentration was the same as with epinephrine alone. Under these conditions, insulin produced the previously observed increase in the proportion of glycogen transferase in the glucose-6-phosphate-independent ( I) form, but had no significant effect on glycogen phosphorylase (α-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1). 3. 3. When diaphragms were pe-incubated with insulin, there was a diminished rise in cyclic AMP levels in the first few minutes after the addition of epinephrine. An increase in activity of the I form of glycogen transferase was observed, but even under these conditions, phosphorylase activity was not altered significantly by insulin. 4. 4. These results suggest that the effect of insulin on glycogen transferase activity in skeletal muscle does not depend upon changes in the tissue concentration of cyclic AMP.