Evidence that the peripheral cyclic AMP phosphodiesterase of rat liver plasma membranes is a metalloenzyme.

Cyclic nucleotide phosphodiesterase activity in salt extracts of rat liver plasma membranes was progressively inactivated by treatment with the metal chelators 8-hydroxyquinoline and o-phenanthroline, but not the non-chelating m-phenanthroline isomer. Activity at 20 microM-cyclic AMP was lost more slowly than activity at 0.4 microM-cyclic AMP. The activity of treated preparations was partially restored by incubation with Zn2+ or Mn2+ ions (in the presence of 1 mM-MgCl2) but not with Ca2+, Cd2+, Co2+, Cu2+ or Fe2+ ions, nor by MgCl2 alone. The results suggest the presence in the membrane extracts of a cyclic AMP phosphodiesterase containing tightly bound metal, possibly Zn or Mn, that affects the enzyme's affinity for cyclic AMP.     

The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum.

In the absence of cyclic nucleotides heart microsomes have two classes of calcium binding sites with binding constants of 0.69 and 0.071 micron-1 and capacities of 2.2 and 9.7 nmol/mg protein, respectively. Neither cyclic AMP nor monobutyryl cyclic AMP affect binding but cyclic GMP and monobutyryl cyclic GMP cause the complete loss of the high affinity calcium binding sites, Cyclic GMP (but not monobutyryl cyclic GMP) also causes a decrease in the binding constant of the low affinity binding sites. AMP, GMP and Tris-butyrate do not affect calcium binding. The effects of the cyclic nucleotides are direct and are not mediated by protein phosphorylation. Phosphorylation of microsomal proteins increases the binding constant but not the capacity of the high affinity calcium binding sites. The capacity and also, perhaps, binding constant of the low affinity sites is also increased by phosphorylation. In additon to their effects on calcium binding the cyclic nucleotides also affect the movements of calcium into and out of the microsomes. The effects are again direct and not mediated by protein phosphorylation. Cyclic GMP decreases the rate of Ca2+ efflux from preloaded cardiac microsomes and also appears to decrease the rate of uptake of Ca2+ by cardiac microsomes though this effect is less clear cut than the action on efflux. The cyclic nucleotide has a half maximal effect at a concentration of 100 microns. By contrast cyclic AMP increases the rate of influx of Ca2+ into heart microsomes and the rate of efflux of Ca2+ from preloaded preparations. The effect is, however, rather slight. It is suggested that the most obvious interpretation of these results is that cyclic GMP decreases the Ca2+ permeability of the cardiac microsomal membrane while cyclic AMP increases the permeability. In contrast to the results found with membrane preparations from certain other tissues phosphorylation of cardiac microsomal proteins does not appear to alter Ca2+ efflux or influx out of, or into, cardiac microsomal preparations. It is thus concluded that phosphorylation of cardiac microsomal proteins does not affect the Ca2+ permeability of the microsomal membrane.     

The effect of ADP, calcium and some inhibitors of platelet aggregation on protein phosphokinases from human blood platelets.

A protein phosphokinase (ATP: protein phosphotransferase EC 2.7.1.37) which is stimulated by 3',5'-cyclic adenosine monophosphate (cyclic AMP) has been partially purified from both the cytoplasmic and membrane fractions of human platelets. The kinetics of both enzymes preparations are similar in respect to cyclic AMP, ATP, ADP and AMP. 5-10-minus 7 M cyclic AMP stimulated both preparations by approximately 100%. Both ADP and AMP at a concentration of 5-10-minus 5 M inhibited protein phosphokinase activity of the soluble and membrane preparation by between 50% and 70%. The response of the two enzyme preparations to calcium differed. 10 mM Ca-2+ inhibited soluble protein phosphokinase activity approximately 80% both in the presence and absence of 5-10 minus 7 M cyclic AMP whereas the same concentrations of Ca-2+ inhibited the membrane-bound enzyme by approximately 60% in the presence of 5-10-minus 7 M cyclic AMP and 40% in the absence of cyclic AMP. This observation may be of importance in understanding the mechanism of platelet aggregation.     

K+-Evoked Depolarization Stimulates Cyclic AMP Accumulation in Photoreceptor-Enriched Retinal Cell Cultures: Role of Calcium Influx Through Dihydropyridine-Sensitive Calcium Channels

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.     

Effect of cortisol on cyclic AMP production stimulated by 1-24 ACTH in adrenal cortex membranes

The effect of cortisol on cyclic AMP production by crude bovine adrenal cortex membrane preparations has been investigated. Results demonstrate that in the presence of cortisol, cyclic AMP production in response to 1-24 ACTH was enhanced up to a concentration of about 74 microM cortisol. At higher concentrations the effect was reversed. Cortisol had no effect on cyclic AMP production in the absence of 1-24 ACTH, and cyclic AMP production was completely inhibited when 5 mM EDTA was added to the incubation tubes with the cortisol.     

Determination of adenosine 3′:5′-cyclic monophosphate in cerebral tissues by saturation analysis. Assessment of a method using a binding protein from ox muscle

1. The Gilman (1970) procedure for determining cyclic AMP (adenosine 3':5'-cyclic monophosphate) by saturation analysis gave erroneous results when applied to the analysis of extracts of whole brain or preparations of membrane fragments from brain. 2. The extracts contained a non-diffusible factor, which enhanced the binding of cyclic AMP by the muscle protein fraction. 3. Extracts also contained material which inhibited binding, but net inhibition of binding was only observed when relatively concentrated extracts were analysed. 4. The error introduced by the factors modifying binding could be eliminated by incorporation of unlabelled internal standards in the unknowns. The design adopted enables a statistical estimate to be made of the standard error of a single assay. 5. The modified assay was used to determine bound cyclic AMP and adenylate cyclase activity in cerebral membrane fragments. Five preparations of synaptic membrane fragments contained less than 3.5pmol of cyclic AMP/mg of protein; a microsomal fraction from rat contained 65pmol of cyclic AMP/mg of protein.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

Release of norepinephrine from brain vesicular preparations: Effects of an adenylate cyclase activator,forskolin, and a phosphodiesterase inhibitor

1. The calcium-dependent K+-evoked release of [3H]norepinephrine from guinea pig cerebral cortical vesicular preparations is inhibited by norepinephrine, clonidine, and epinephrine. Isoproterenol has no effect and phentolamine prevents the inhibition by norepinephrine. The results indicate that an alpha-adrenergic receptor mediates an inhibitory input to the calcium-dependent release process. The inhibition by norepinephrine is prevented by high concentrations (3.0 mM) of calcium ions. 2. A cyclic AMP phosphodiesterase inhibitor, ZK 62771, slightly elevates [3H]cyclic AMP levels in the guinea pig cerebral cortical preparation and potentiates the marked elevation of [3H]cyclic AMP elicited by the adenylate cyclase activator, forskolin. 3. Neither ZK 62771 nor forskolin alone has significant effects on K+-evoked release of [3H]norepinephrine from the cerebral cortical vesicular preparation; however, a combination of ZK 62771 and forskolin inhibits K+-evoked release by as much as 60%. The inhibition is reversed by high concentrations (2.0 mM) of calcium ions. The results suggest that a marked accumulation of cyclic AMP elicited via both activation of adenylate cyclase and inhibition of phosphodiesterase can be inhibitory to neurotransmitter release from central synaptic terminals.     

Inhibition of the glycogenolytic effects of alpha-adrenergic stimulation and glucagon by cobalt ions in perfused rat liver

Cobalt ions (2 mM) inhibited the glycogenolysis induced by phenylephrine and glucagon in perfused rat liver. Cobalt ions also inhibited 45Ca++ efflux from prelabelled livers induced by phenylephrine and glucagon. In addition, they inhibited the rise in tissue levels of cyclic AMP caused by glucagon, but did not inhibit the stimulation of 45Ca++ efflux or glycogenolysis by cyclic AMP or dibutyryl cyclic AMP. The specific binding of glucagon and alpha-agonist to hepatocytes was not inhibited by cobalt ions. These data suggest that cobalt ions, presumably through their high affinity for calcium binding sites on membranes inhibit the stimulation of glycogenolysis by phenylephrine and glucagon in distinct ways; one by inhibiting calcium mobilization and the other by inhibiting cyclic AMP production. Therefore, it is conceivable that membrane-bound calcium plays an important role in stimulating Ca++ mobilization by phenylephrine, and cyclic AMP production by glucagon.     

Regulation of rat liver microsomal cholesterol 7 alpha-hydroxylase: reversible inactivation by ATP + Mg2+ and a cytosolic activator

Modulation of cholesterol 7 alpha-hydroxylase catalytic activity by adenine nucleotides was studied in rat liver microsomal preparations. Inactivation of cholesterol 7 alpha-hydroxylase showed specific requirements of ATP and ADP. AMP and cyclic AMP were stimulatory and cyclic AMP had no effect in the ATP inactivation. The inactivation reactions by ATP were dependent on Mg2+ ions, a cytosolic factor, and time. Ca2+ ions were less effective whereas Mn2+ ions were highly inhibitory to hydroxylase activity. The inactivation could be reversed in a time-dependent reaction requiring a cytosolic activator that was precipitable by ammonium sulphate of saturation up to 65%. The current data suggest that cholesterol 7 alpha-hydroxylase can exist in two catalytic forms that are reversible.     

The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.     

Adenosine 3'':5''-cyclic monophosphate-dependent and plasma-membrane-associated protein kinase(s) from bovine corpus luteum.

Plasma-membrane fractions FI and FII isolated from bovine corpus luteum by discontinuous sucrose-density-gradient centrifugation, at sucrose-density interfaces of 1.14/1.16 and 1.16/1.18 respectively, contained membrane-associated protein kinases that phosphorylated both the structural proteins of membranes as well as exogenously added protein substrates. Both fractions were characterized with respect to endogenous and exogenous protein substrate specificity, pH-dependence, effect of bivalent metal ions and sensitivity toward cyclic nucleotides. These membrane-associated kinases showed an optimum pH of 6.0 and had an absolute requirement for bivalent metal ions such as Mg2+, Mn2+, or Co2+ that cannot be replaced by Ca2+. Both the activities were stimulated two- to four-fold by cyclic AMP in vitro with an apparent Km of 83 and 50 nM for fractions FI and FII respectively. Other cyclic 3':5'-nucleotides were effective only at higher concentrations, but even the most effective, cyclic IMP, showed a stimulation nearly an order of magnitude lower than that of cyclic AMP. In contrast, stimulation by cyclic dTMP and cyclic dAMP was very weak. Cyclic AMP showed no significant effect on the apparent Km value of both enzymes for histone and MgCl2 but it somewhat decreased the Km value for ATP. Nucleoside triphosphates like GTP, CTP and UTP inhibited the transfer of [32P]Pi from [gamma-32P]ATP into mixed histone catalysed by membrane-associated kinases either in the presence or in the absence of cyclic AMP. In addition to protein kinases, these membrane fractions also possessed cyclic AMP-binding activities. The apparent association constant (Kalpha) for cyclic AMP binding was 1.0 X 10(10) and 2.6 X 10(10) M for FI and FII membrane fractions respectively.     

Mechanism of action of ouabain and cyclic-3',5'-adenosine monophosphate on calcium ion transport in rat cardiac mitochondria]

Ouabain and cyclic AMP action on the Ca2+-accumulating capacity and the efflux of Ca2+ions from the loaded mitochondria of the rat heart was investigated by the tetracycline probe method. On the basis of the experimental results a conclusion was drawn that ouabain failed to act on these processes. Cyclic AMP didn't act on the Ca binding by the mitochondrial membrane; at the same time it induced rapid release of calcium from the loaded mitochondria.     

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Pretreatment of $\text{Balb}\text{c}\text{-3T3}$ cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for ¹²⁵I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.     

An insulin mediator preparation serves to stimulate the cyclic GMP activated cyclic AMP phosphodiesterase rather than other purified insulin-activated cyclic AMP phosphodiesterases

An insulin mediator preparation was obtained from rat hepatocytes which had been treated with insulin. This preparation inhibited adenylate cyclase activity. It stimulated the activity of homogeneous preparations of both the cytosolic and membrane-bound forms of rat liver cyclic GMP-activated cyclic AMP phosphodiesterase. It failed to activate homogeneous preparations of both the peripheral plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases. The insulin mediator preparation stimulated cyclic GMP-activated cyclic AMP phosphodiesterase activity in a dose-dependent fashion with a hill coefficient of 0.46. Insulin caused the dose-dependent production of mediator activity in intact hepatocytes with a Ka of 9 pM, although concentrations of insulin greater than 10 nM progressively reduced stimulatory activity.     

Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay

A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with ¹²⁵I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for ¹²⁵I-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity cAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Effect of hormones and 3', 5'-cyclic AMP on very low density lipoprotein receptors

Human 125I-labelled VLDL interacts with rat adipocytes in vitro, with properties typical of a ligand-receptor interaction. This VLDL-receptor interaction is modulated by hormones which are known to change cyclic AMP levels. Norepinephrine and isoproterenol, both of which elevate cycle AMP, increase the binding of VLDL to adipocytes. Dibutyryl-cyclic AMP, a derivative of cyclic AMP, also increases the VLDL binding to adipocytes. Insulin reverses the catecholamine-induced increase in VLDL binding. This parallels insulin's effect on the catecholamine-induced changes in cyclic AMP. Direct addition of cyclic AMP itself increases VLDL binding to adipocyte membranes, a system in which no lipolysis of new protein synthesis occurs. Based on the competition between unlabelled VLDL and 125I-labelled VLDL, we conclude that catecholamines act on adipocytes, and cyclic AMP on membrane fractions, by increasing their capacity rather than their affinity to bind VLDL.     

Effect of hormones and 3′, 5′-cyclic AMP on very low density lipoprotein receptors

Human ¹²⁵I-labelled VLDL interacts with rat adipocytes in vitro, with properties typical of a ligand-receptor interaction. This VLDL-receptor interaction is modulated by hormones which are known to change cyclic AMP levels. Norepinephrine and isoproterenol, both of which elevate cyclic AMP, increase the binding of VLDL to adipocytes. Dibutyryl-cyclic AMP, a derivative of cyclic AMP, also increases the VLDL binding to adipocytes. Insulin reverses the catecholamine-induced increase in VLDL binding. This parallels insulin's effect on the catecholamine-induced changes in cyclic AMP. Direct addition of cyclic AMP itself increases VLDL binding to adipocyte membranes, a system in which no lipolysis or new protein synthesis occurs. Based on the competition between unlabelled VLDL and ¹²⁵I-labelled VLDL, we conclude that catecholamines act on adipocytes, and cyclic AMP on membrane fractions, by increasing their capacity rather than their affinity to bind VLDL.     

The binding of cyclic nucleotides to membranes of the endoplasmic reticulum of normal and neoplastic rat liver.

Studies are presented which demonstrate that the smooth and rough endoplasmic membranes of normal and neoplastic rat liver possess binding sites for cyclic nucleotides exhibiting a high degree of specificity. In contrast to normal liver, which has only a single binding site for cyclic AMP on membranes of the endoplasmic reticulum, cyclic AMP binding to the intracellular membranes of hepatoma 5123C and 7777 exhibits two apparent binding sites. The binding constant for cyclic AMP of one site on the tumor membranes is comparable to that of the normal liver, whereas the value of the second intrinsic association constant is 4- to 40-fold greater than liver. The possibility that the presence of the second cyclic AMP binding site might be a function of the rapid growth of the tumors was unlikely since membrane preparations from neonatal rats showed a single affinity association constant which was similar to that of normal liver. In addition, membranes of the endoplasmic reticulum of the Morris hepatomas 5123C and 7777 exhibit a binding site for cyclic GMP which is absent from the intracellular membranes of liver.