Fluoride-stimulated Adenylate Cyclase Activity Research Articles

Intraventricular changes in the beta-adrenoceptor-adenylate cyclase system of the rat heart with the progress of monocrotaline-induced right ventricular hypertrophy.

We investigated the changes in the membranous beta-adrenoceptor-adenylate cyclase system in the right ventricle, left ventricle and interventricular septum during the progress of monocrotaline-induced right ventricular hypertrophy and failure. The beta-adrenoceptor density was decreased in hypertrophied right ventricle 2 to 4 weeks after treatment. When the rats showed symptoms of right ventricular failure 4 weeks after treatment, the beta-adrenoceptor density was decreased in the interventricular septum. Both basal and forskolin-stimulated adenylate cyclase activities were decreased in the right ventricle at 3 and 4 weeks, and in the interventricular septum at 4 weeks, after treatment, which indicates that the catalytic activity of adenylate cyclase is reduced. Changes in isoproterenol plus Gpp (NH) p- or sodium fluoride-stimulated adenylate cyclase activity were generally similar to those in basal activity. These data indicate that a chamber-specific decrease in beta-adrenoceptor density begins in the early stages of right ventricular hypertrophy, and that beta-adrenoceptor density and adenylate cyclase activity in the interventricular septum are decreased in the advanced stages of heart failure in monocrotaline-treated rats.

Cardiac β-adrenoceptors, G-proteins and adenylate cyclase regulation during myocardial hypertrophy

The role of the β-adrenoceptor-G-protein-adenylate cyclase system in the pathogenesis of cardiac hypertrophy was studied. We have used a minipig model of pressure-overload cardiac hypertrophy secondary to aortic banding. Four groups of five animals were used: minipigs made hypertrophic were evaluated 2 months (CH2 group) and 9 months (CH9 group) later and compared to controls (C2 and C9 groups, respectively). A decrease in β-adrenergic receptor density and an increase in antagonist affinity were shown in left ventricular membranes of hypertrophied animals compared with controls. In both groups, CH2 and CH9, an increase in EC 50 for isopreterenol-stimulated adenylate cyclase activity, an increase in forskolin-stimulated adenylate cyclase activity and a diminished inhibition by carbachol of isoproterenol-stimulated adenylate cyclase were observed. In contrast, fluoride-stimulated adenylate cyclase activity was markedly increased only in the end stage of hypertrophy. α s-cholera toxin-catalysed ADP-ribosylation is increased in early hypertrophy and then decreases with late hypertrophy and a similar pattern is observed with α O pertussis toxin-catalysed ADP-ribosylation, whereas α i-ADP-ribosylation remains unchanged. Tissue content of G s-, G i- and G 0-proteins, as assessed by specific antibodies, was found unchanged in CH9 and CH2 groups when compared with that in C9 and C2 control groups, respectively. Modifications in G s functional activity in later hypertrophic stages, expressed as alterations in cholera toxin ADP-ribosylation and adenylate cyclase fluoride responsiveness, may be important in the pathogenesis of decompensation from compensated hypertrophy to cardiac failure.

The mechanisms of action of lithium. II. Effects on adenylate cyclase activity and beta-adrenergic receptor binding in normal subjects.

As part of a study of the effects of lithium carbonate on neurochemical function in man, platelet and lymphocyte adenylate cyclase activity and lymphocyte beta-adrenergic receptor binding characteristics were determined before and after 2 weeks of lithium treatment in 10 normal volunteers. Lithium had differential effects on platelet and lymphocyte adenylate cyclase activity. In platelets, basal and stimulated (guanyl imidodiphosphate [Gpp[NH]p] or cesium fluoride) adenylate cyclase activity was significantly augmented by lithium treatment. By contrast, in lymphocytes, Gpp(NH)p- and cesium fluoride-stimulated adenylate cyclase activity was unaffected, while basal activity was decreased modestly after lithium. These results are consistent with preclinical studies that suggest that lithium's effects on adenylate cyclase activity are specific with respect to tissue and brain region and that lithium may interfere with guanine nucleotide binding (G) protein function. Lithium treatment significantly increased the ratio of low- to high-affinity dissociation constants for agonist displacement of antagonist binding to lymphocyte beta-adrenergic receptors (thought to reflect coupling between the beta-adrenergic receptor and stimulatory G protein). Lithium had significant effects on measures associated with signal transduction that might be contrasted to its more subtle effects on neuronal function (norepinephrine release) and neuroendocrine systems (responses to serotoninergic challenge) in these same subjects (reported in a companion article). Lithium's primary site of action may be on signal transduction mechanisms. These effects subsequently may be manifested in changes in neurotransmitter function that may be important to lithium's mood-stabilizing actions.

Beta-adrenoceptor adenylate cyclase system adaptation to physical training in rat ventricular tissue.

The beta-adrenergic receptor adenylate cyclase system of ventricular tissue was evaluated in a group of rats submitted to a progressive 10-wk running program on a treadmill and compared with that in a group of rats maintained sedentary during the same period. Adequate training was confirmed by a 46% increase in the gastrocnemius isocitrate dehydrogenase activity in the trained group [1.50 +/- 0.04 vs. 1.03 +/- 0.06 (SE) pmol.g-1.min-1; P less than 0.01). Binding studies with [125I]iodocyanopindolol showed a 13% reduction in the density of beta-adrenergic receptors in trained rats (42.6 +/- 2.1 vs. 49.0 +/- 2.1 fmol/mg; P less than 0.05) without any significant modification in the dissociation constant. The amount of [125I]iodocyanopindolol bound to beta-adrenoceptors in the high-affinity state was reduced by 16.6% in trained rats (12.5 +/- 0.9 vs. 15.0 +/- 0.5 fmol/mg; P less than 0.05) without any significant changes for those in the low-affinity state, indicating a decrease in the coupling between the beta-adrenergic receptors and the guanine stimulatory binding protein. Furthermore, although the basal and sodium fluoride-stimulated adenylate cyclase activities were similar in the two groups of rats, the response of adenylate cyclase maximally stimulated by 10(-5) M isoproterenol was reduced by 16% in trained rats (29.7 +/- 1.4 vs. 35.3 +/- 1.3 pmol.mg-1.min-1; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in Cyclic AMP Concentration and Adenylate Cyclase Activity in Specific Brain Areas of Rats with Inherited Hypothalamic Diabetes Insipidus (Brattleboro Rats)

Abstract The concentration of cyclic AMP (cAMP) and the activity of sodium-fluoride-stimulated adenylate cyclase was measured in 29 microdissected brain areas of homozygous Brattleboro rats and their Long-Evans control rats. In ten of the investigated brain areas a decreased cAMP level was measured in Brattleboro rats. It was particularly decreased in the supraoptic nucleus, cingulate and parietal cortex, hippocampus, habenula and organum vasculosum laminae terminalis. Significantly lower cAMP levels were also found in the periventricular nucleus, bed nucleus of the stria terminalis, area postrema and locus coeruleus. An increased cAMP concentration was detected only in the subcommissural organ of Brattleboro rats. In most brain areas, where cAMP was decreased, sodium fluoride-stimulated adenylate cyclase activity was significantly increased (supraoptic nucleus, parietal cortex, periventricular nucleus, bed nucleus of the stria terminalis, locus coeruleus) or unchanged (hippocampus, habenula, organum vasculosum laminae terminalis). The coincidence of alterations in cAMP concentration and adenylate cyclase activity in brain areas of Brattleboro rats with relatively dense vasopressinergic innervation and/or vasopressin receptor population in control rats, suggests an influence of brain vasopressin on the cAMP-adenylate cyclase second messenger system.

Beta-receptors and adenylate cyclase: comparison of nonischemic, ischemic, and postmortem tissue

We compared the effects of myocardial ischemia and postmortem changes on beta-adrenergic receptors and their coupling to adenylate cyclase activity. The effects of 1 h of left circumflex coronary artery occlusion were examined in eight conscious calves, which were then anesthetized with pentobarbital sodium, and the left ventricle was divided into nonischemic and ischemic regions. A crude membrane fraction was prepared from each region and from the nonischemic tissue 1 h postmortem. beta-Adrenergic receptor density increased (152 +/- 55%) and decreases in basal (-21 +/- 6.1%), isoproterenol-stimulated (-25 +/- 8.0%), 5'-guanylylimidodiphosphate [Gpp(NH)p]-stimulated (-17 +/- 5.8%), fluoride-stimulated (-26 +/- 5.8%), and forskolin-stimulated (-31 +/- 8.4%) adenylate cyclase activities were observed in the ischemic myocardium compared with nonischemic myocardium. Similarly, in postmortem samples, beta-adrenergic receptor density rose 58 +/- 16%, whereas decreases in basal (-48 +/- 8.7%), isoproterenol-stimulated (-61 +/- 7.8%), Gpp(NH)p-stimulated (-58 +/- 7.0%), fluoride-stimulated (-64 +/- 6.1%), and forskolin-stimulated (-52 +/- 6.2%) adenylate cyclase activities were observed. Agonist-binding competition curves with isoproterenol were shifted, indicating that beta-adrenergic receptors were binding agonists with low affinity in both the ischemic and postmortem myocardium. The marked, but directionally opposite, changes in receptor density and adenylate cyclase that occur postmortem indicate the importance of prompt processing of tissues. The striking similarity in response of beta-adrenergic receptor agonist and antagonist binding and adenylate cyclase activity in ischemic and postmortem tissue raises the speculation that similar mechanisms may operate under both conditions.

Calmodulin modulates thymocyte adenylate cyclase activity through the guanine nucleotide regulatory unit

We have previously demonstrated in rat thymocyte plasma membranes that adenylate cyclase activity and its stimulation by 3,5,3'-triiodothyronine (T 3) are influenced by Calmodulin, and that these effects of calmodulin require calcium. In the present study, the mechanism by which calmodulin exerts its action was examined, in situ, in fresh plasma membranes isolated from rat thymocytes. Adenylate cyclase activity was potentiated by guanyl nucleotides, NaF and forskolin. Calmodulin did not affect basal adenylate cyclase activity. However, calmodulin influenced the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity, but had no effect on the fluoride-stimulated enzyme activity. This was evident from experiments with inhibitors of calmodulin: trifluoperazine, calmidazolium, and antibodies against calmodulin. The three inhibitors did not change basal adenylate cyclase activity, but all produced a marked decrease in the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity. The inhibitory effect of all three agents was reversed completely by the addition of calmodulin. The three inhibitors, however, failed to affect the fluoride-stimulated adenylate cyclase activity. In addition, T 3, like the calmodulin inhibitors, did not change basal adenylate cyclase activity, increased the guanyl nucleotide- and forskolin-stimulated enzyme activity, but had no effect on the fluoride-stimulated enzyme activity. From these results I suggest that in the rat thymocyte calmodulin activation, and thereby T 3 stimulation of the calcium-sensitive adenylate cyclase system is mediated through the guanine nucleotide regulatory unit.

Effects of low-sodium diet on regulation of platelet alpha 2-adrenergic receptors in young and elderly humans

To examine whether there are age differences in agonist-mediated alpha 2-adrenergic receptor (alpha 2-AR) regulation, we studied the effect of a sustained increase in plasma norepinephrine (pNE) during a 7-day 10-meq Na+/day diet on platelet alpha 2-AR binding and its linked adenylate cyclase (AC) activity in 11 elderly and 11 young healthy subjects. In the young, a 41% increase in mean pNE after a low-sodium diet was correlated with a decline in receptor density (Bmax; r = -0.816; P less than 0.01) and was accompanied by a reduction in the maximal percent inhibition of sodium fluoride-stimulated AC activity by epinephrine (%AC INH; 33 +/- 4 vs. 24 +/- 4%, mean +/- SE; P less than 0.05 vs. normal diet). Despite a comparable 39% increase in mean pNE in the elderly, neither Bmax nor %AC INH was significantly reduced after a low-sodium diet. The amount of pertussis toxin substrate (Gi protein) was similar in both groups before and after dietary sodium restriction. At comparable pNE, %AC INH in the groups was similar (young, 24 +/- 4 vs. elderly, 18 +/- 4%; P = NS). We postulate that higher basal pNE levels in the elderly on normal diet may account for the lack of further downregulation of platelet alpha 2-AR density and response after low-sodium diet.

Characterization of lymphocyte beta-adrenergic receptors at rest and during exercise in ambulatory patients with chronic congestive heart failure

To investigate β-adrenergic receptor dysfunction in congestive heart failure (CHF), the density of lymphocyte β receptors and adenylate cyclase activity was measured at rest and at peak exercise in 30 patients with CHF and 7 age-matched control subjects. At rest, patients with CHF had reduced β-receptor density (normals 33 ± 2; CHF 21 ± 2 fmol/ mg protein; p < 0.01) and isoproterenol-stimulated adenylate cyclase activity (normals 50 ± 9; CHF 28 ± 4 pmol/mg protein/min; p < 0.05). Sodium fluoride-stimulated adenylate cyclase activity was also reduced (normals 98 ± 17; CHF 48 ± 12 pmol/mg protein/min; p < 0.01). In the patients with CHF, there was no significant correlation between receptor density and peak exercise VO 2, ejection fraction or resting plasma catecholamines. In the normal subjects, maximal exercise increased β-receptor density by 100% (rest 33 ± 2; exercise 67 ± 7 fmol/mg protein) and isoproterenol-stimulated adenylate cyclase activity by 66% (rest 50 ± 9; exercise 83 ± 18 pmol/mg protein/min (both p < 0.01)). In contrast, patients with CHF exhibited only a 58% increase in β-receptor density (rest 20 ± 3; exercise 32 ± 6 fmol/mg protein; p < 0.01) and no significant change in isoproterenol-stimulated adenylate cyclase activity (rest 27 ± 5; exercise 24 ± 5 pmol/mg protein/min). These findings suggest that (1) CHF is associated with reduced density of β receptors and either uncoupling of guanine nucleotide regulatory binding protein from adenylate cyclase or defective cyclase; (2) β-receptor density in CHF is not a simple function of ejection fraction, functional capacity or circulating catecholamines; and (3) exercise produces desensitization or uncoupling of the β receptor from adenylate cyclase in CHF.

Dietary cholesterol influences cardiac β-adrenergic receptor adenylate cyclase activity in the marmoset monkey by changes in membrane cholesterol status

The activity of the β-adrenergic receptor/adenylate cyclase system of the marmoset monkey heart was investigated following dietary cholesterol supplementation (0.5%). After 22 weeks, plasma cholesterol levels in the cholesterol group were more than twice that of the control group. In the cholesterol-fed group, the affinity for ICYP binding to cardiac membranes was elevated more than 2-fold, while the receptor number was decreased by 31%. Isoproterenol, norepinephrine and sodium fluoride stimulated adenylate cyclase activity was significantly higher in the cholesterol-fed group although the fold stimulation over basal levels was not affected. The most prominent change in the cardiac membrane lipids was an increase in the cholesterol to phospholipid ratio in marmoset monkeys fed cholesterol. These results indicate that in the marmoset, membrane cholesterol is an important factor in determining various properties of the cardiac β-adrenergic receptor particularly receptor affinity which may impact on the response of the β-adrenergic receptor/adenylate cyclase system of the heart to catecholamines. This result is in agreement with dietary fatty acid supplements designed to increase cardiac membrane cholesterol in this animal species (McMurchie, E.J. et al. (1988) Biochim. Biophys. Acta 937, 347–358). Elevated membrane cholesterol enhances β-adrenergic receptor affinity and certain aspects of adenylate cyclase activity. This is a likely mechanism whereby atherogenic diets could promote cardiac arrhythmia in non-human primates and indeed in man.

Adenylate cyclase and beta-receptors in salivary glands of rats fed diets containing trans fatty acids.

The effects of trans fatty acids on adenylate cyclase were determined in the submandibular salivary glands (SMSG) of rats fed diets containing 20% corn oil, 20% partially hydrogenated soybean oil (PHSBO) or 18% PHSBO + 2% corn oil. The fluoride- and isoproterenol-stimulated adenylate cyclase activities were higher in the SMSG from rats fed 20% PHSBO than in the control group fed 20% corn oil. The feeding of 2% corn oil with the diet containing 18% PHSBO resulted in a complete restoration of isoproterenol-stimulated and a partial restoration of fluoride-stimulated adenylate cyclase activity. There was no significant difference in the concentration of the beta-adrenergic receptor or in the receptor-binding affinity constants among the three dietary groups as measured by [3H]dihydroalprenolol (DHA)-binding. Higher fluorescence polarization of diphenyl hexatriene (DPH) was observed in SMSG membranes of rats fed diet containing 20% PHSBO than in the other two oils, suggesting that membrane fluidity may play a role in adenylate cyclase activity.

Differences in platelet enzyme activity between alcoholics and nonalcoholics.

Blood platelets are an accessible tissue that reflects the activity of many enzymes found in the brain. To investigate the possible effect on such enzymes of long-term consumption of large quantities of ethanol, we assayed the activities of two enzymes, monoamine oxidase and adenylate cyclase, in platelet membranes of men with alcoholism and controls matched for sex and age. We also compared these two groups in terms of the inhibition of platelet monoamine oxidase activity by ethanol in vitro (400 mM), and in terms of the stimulation of adenylate cyclase activity by various agents. There was no significant difference in monoamine oxidase activity between the alcoholics and the controls. However, the inhibition of monoamine oxidase by ethanol was significantly higher in the platelets of alcoholics. The basal activity of adenylate cyclase was the same in platelets from the alcoholics and the controls, but the platelet adenylate cyclase activity after stimulation with guanine nucleotide, cesium fluoride, or prostaglandin E1 was significantly lower in alcoholics. These differences were not associated with age, race, smoking, or illicit drug use, and there was no significant correlation with the duration of problems with alcohol. The changes were long-lasting; cesium fluoride-stimulated adenylate cyclase activity was lower in alcoholic subjects who had abstained from alcohol for one to four years. Discriminant analysis showed that the use of values for the inhibition of monoamine oxidase activity by ethanol and cesium fluoride-stimulated adenylate cyclase activity correctly classified 75 percent of the alcoholics and 73 percent of the controls. These measures may be of value either as indexes of excessive alcohol consumption or as an indication of a predisposition to alcoholism.

Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase.

Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli. Following transformation of either of these plasmids into E. coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera. Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261, 9587-9590). Expression of these cDNAs in E. coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively. Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E. coli expressing either form of Gs alpha. Partial purification of Gs alpha from E. coli lysates yields preparations in which significant and stable activity can be assayed. Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent. As expressed in E. coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with [32P]NAD+ and cholera toxin. However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E. coli appears to be active. Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity. Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed [32P]ADP-ribosylation.

Preserved cardiac beta-adrenergic sensitivity in early renovascular hypertension.

To determine the mechanism of blunted sympathetic reflex responses in early renovascular hypertension, we measured inotropic and chronotropic responses of the heart to beta-adrenergic stimulation in vivo and myocardial beta-adrenergic receptor number and adenylate cyclase activity in 10 dogs during an early stage of one-kidney renal hypertension. Mean aortic pressure was higher in the hypertensive dogs (152 +/- 4 mm Hg) than in eight sham-operated dogs (122 +/- 1 mm Hg; p less than 0.001), but heart rate, cardiac output, and left atrial pressure did not differ between the two groups. Blood pressure reduction with a direct-acting vasodilator, pinacidil, resulted in marked increases in heart rate (+97 +/- 12 beats/min) and rate of change of left ventricular pressure (dP/dt; +1447 +/- 367 mm Hg/sec) in normotensive dogs but only blunted heart rate (+54 +/- 12 beats/min) and minimal left ventricular dP/dt (+376 +/- 264 mm Hg/sec) responses in hypertensive dogs. In contrast, intravenously administered isoproterenol produced similar increases in heart rate and left ventricular dP/dt in the two groups. These two groups also did not differ in either left ventricular beta-adrenergic receptor number and affinity or basal, isoproterenol-stimulated, and fluoride-stimulated adenylate cyclase activity. Thus, despite blunted reflex responses to blood pressure reduction, hypertensive dogs showed neither reduction in chronotropic and inotropic responses to direct beta-adrenergic stimulation nor beta-adrenergic desensitization of the myocardium, as assessed by beta-adrenergic receptor number and adenylate cyclase activity. Blunted reflex responses in this model of early hypertension must be due to factors operating at some locus other than the beta-adrenergic receptor-adenylate cyclase complex.

Lipolysis and β-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats

Adipocytes from spontaneously hypertensive rats demonstrated a blunted lipolytic response to isoproterenol and dibutyryl cyclic AMP. (-)-[3H]Dihydroalprenolol binding was examined in adipocytes from normotensive and spontaneously hypertensive rats. Increasing concentrations of isoproterenol decreased total (-)-[3H]dihydroalprenolol binding to intact cells from normotensive rats, and the efficacy of competition was decreased in adipocytes from spontaneously hypertensive rats. Scatchard analysis indicated that the number of (-)-[3H]dihydroalprenolol binding sites and the affinity of dihydroalprenolol binding were comparable between normotensive and spontaneously hypertensive rats. Isoproterenol- and Gpp(NH)p-stimulated adenylate cyclase activity was consistently depressed in adipocyte membranes from spontaneously hypertensive rats as compared to normotensive rats. No difference in fluoride-stimulated adenylate cyclase activity was observed. The blunted lipolytic and cyclic AMP response to isoproterenol in these cells suggest a postreceptor lesion of the lipolytic pathway (possibly the guanine nucleotide regulatory protein) in adipocytes from spontaneously hypertensive rats. The blunted lipolytic response to dibutyryl cyclic AMP suggests defective regulation of lipolytic enzymes at the protein kinase-hormone-sensitive lipase level.

Phorbol ester induces loss of VIP stimulation of adenylate cyclase and VIP-binding sites in HT29 cells

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected byPMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.

Effect of Essential Fatty Acid Deficiency on Acyl Group Composition, Membrane Fluidity and Adenylate Cyclase Activity in Plasma Membranes of Rat Submandibular Salivary Glands

The effects of a marginal and severe deficiency of essential fatty acids (EFA) on the fatty acid composition, fluorescence polarization of diphenyl-hexatriene (DPH) and adenylate cyclase activity were studied in plasma membranes of rat submandibular salivary glands (SMSG). Three groups of male weanling Sprague-Dawley rats were fed ad libitum EFA-deficient (EFAD), marginally EFA-deficient (MEFAD) or the control diets. Rats were killed after 8–17 wk of feeding and plasma membranes were prepared from the SMSG and the assayed for basal and fluoride-stimulated adenylate cyclase activity. In membranes prepared from EFAD and MEFAD rats, the fluoride-stimulated enzyme activity was 30–35% higher than that of the control group. Lower double-bond index of phospholipids in plasma membranes of the SMSG from EFAD and MEFAD rats was observed, suggesting a lower fluidity compared with the control group. Higher fluorescence polarization of DPH in membranes of EFAD and MEFAD rats was more evidence of lower fluidity. It is suggested that higher adenylate cyclase activity in SMSG of rats fed EFAD or MEFAD diets may be related to lower membrane fluidity. Fluoride-stimulated enzyme activity and fatty acid composition of membrane lipids were restored to normal values after feeding the control diets for 6 wk to the EFAD and MEFAD rats.

Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes

We have studied the effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity in cultured cardiocytes from atria (left and right) and ventricles from neonatal rats. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration dependent manner in cultured atrial (right and left atria) and ventricular cells. However the inhibition was greater in atrial cells as compared to ventricular cells. The maximal inhibition observed in ventricular cells was about 35% with an apparent Ki of about 10 −10 M, whereas about 55% inhibition with an apparent Ki between 5 × 10 −10 M and 65% inhibition with an apparent Ki of 10 −9 M were observed in right and left atrial cardiocytes respectively. The inhibitory effect of ANF was dependent on the presence of guanine nucleotides. Various hormones and agents such as isoproterenol, prostaglandins, adenosine, forskolin and sodium fluoride stimulated adenylate cyclase activities to various degrees in these atrial and ventricular cardiocytes. ANF inhibited the stimulatory responses of all these agonists, however the degree of inhibition varied for each agent. In addition ANF also inhibited cAMP levels in these cells. These data indicate that ANF receptors are present in cardiocytes and are negatively coupled to adenylate cyclase.

Beta adrenoceptor density and adenylate cyclase response in right atrial and left ventricular myocardium of patients with mitral valve disease.

Beta adrenoceptor density, measured by radioligand binding techniques, is reportedly much higher in atrial than in ventricular myocardium of patients with mitral valve disease. In the present study adenylate cyclase activity, both basal and in response to beta adrenergic agonists and sodium fluoride, in biopsy preparations from these same patients was significantly lower in atrial than in ventricular tissue when stimulated with either isoproterenol, noradrenaline, isoproterenol combined with terbutaline, or sodium fluoride. Terbutaline stimulated and basal adenylate cyclase activity was not significantly different in the two cardiac regions. The ratios of receptor density to isoproterenol stimulated and to sodium fluoride stimulated adenylate cyclase activity were 4-5 times higher in atrial than in ventricular biopsy specimens. Thus ventricular beta receptors, although present in comparatively low concentrations, are coupled to considerably more catalytic moieties of the receptor-adenylate cyclase complex than their atrial counterparts. The reason for this is probably a relative lack of coupling proteins (N components) in atrial tissue. A weak positive correlation between receptor density and isoproterenol stimulated adenylate cyclase activity was found in atrial but not in ventricular tissue. This may indicate individual variation in receptor-adenylate cyclase coupling. Furthermore, no correlation was found between atrial and ventricular values for any variable. One reason for this may be the different haemodynamic stresses in the two cardiac chambers.

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.