Hormone sensitivity of adenylate cyclase activity in cardiac sarcolemma and sarcoplasmic reticulum preparations.

The functional significance of the adenylate cyclase activity found in cardiac microsomal preparations enriched in sarcoplasmic reticulum vesicles was examined by comparing the effects of agents known to modify this enzyme in sarcolemmal and sarcoplasmic reticulum fractions prepared from guinea pig ventricles. The sensitivity of the sarcolemmal adenylate cyclase to adrenergic agonists resembled that of a typical β-receptor. Although the response to these agonists was similar in the case of the sarcoplasmic reticulum enzyme, the extent of stimulation was much less than in the sarcolemma. The pH optimum of the sarcoplasmic reticulum enzyme of 7.5 was slightly lower than that of the sarcolemmal enzyme, which was 8.0; and NaF shifted the pH optimum of the latter, but not the former. The sarcoplasmic reticulum adenylate cyclase was less sentitive to alkali metal salts and GTP than was the sarcolemmal enzyme. While these studies cannot exclude the possibility that the lesser response of the sarcoplasmic reticulum enzyme to catecholamines, alkali metal salts, and GTP arose from contaminating sarcolemmal fragments that were modified during the preparation of these subcellular fractions, they are consistent with the view that the cardiac cell sarcoplasmic reticulum contains an adenylate cyclase that is less regulated, or regulated by different factors, than those which modulate sarcolemmal adenylate cyclase.     

Subcellular localization and partial characterization of adenylate cyclase from rat lung

The subcellular localization of adenylate cyclase (ATP pyrophosphate lyase [cyclizing] EC 4.6.1.1) from predominantly alveolar tissue of rat lung was studied using isotonic and hypotonic homogenization and differential centrifugation. The fractions prepared under hypotonic conditions were further subfractionated by nonlinear sucrose gradient centrifugation. All fractions were assayed for adenylate cyclase, marker enzymes, and DNA. Only 5′-nucleotidase (EC 3.1.3.5, a plasma membrane marker enzyme for rat lung) paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (co-efficient of correlation = 0.94). It is concluded that this adenylate cyclase to a large extent but not exclusively is associated with the plasma membranes. Basal adenylate cyclase activity of plasma membrane fractions was stimulated 3.7 times by NaF (0.01 M). It was also stimulated by isoproterenol (55 µM), epinephrine (55 µM), prostaglandin E₁ (0.03 mM), and prostaglandin E₂ (0.03 mM).     

Cardiac adenylate cyclase. I. Preparation and characterisation of a subcellular fraction containing catecholamine-sensitive adenylate cyclase.

Differential centrifugation of homogenates of rat ventricles yielded subcellular fractions which were assayed for adenylate cyclase activity, as well as enzymes associated predominantly with certain intracellular structures. The distribution of adenylate cyclase activity paralleled only that of the marker enzyme for sarcolemma, 5′-AMPase. However, the heterogeneity of the particulate fractions made it impossible to ascertain the exact intracellular localization of adenylate cyclase. Histological examination of the fraction (P₁) containing the bulk of the adenylate cyclase activity revealed large numbers of isolated myofibres. Purification of this fraction was effected by homogenizing it in a large volume of isotonic sucrose to disperse and dissolve the myofibrillar matrix. The resultant homogenate was then subjected to differential centrifugation. A pellet (P₄) was obtained containing only slight activity of marker enzymes for mitochondria and sarcoplasmic reticulum, but which was enriched in 5′-AMPase activity, plasma membranes (as revealed by its cholesterol/phospholipid ratio) and possessed a reduced muscle protein content. Histological examination of this fraction revealed an absence of myofibres. The specific activity of the basal, isoprenaline—and fluoride—stimulated adeynlate cyclase in P₄ was doubled relative to P₁ and this fraction contained approximately half of the total adeynlate cyclase activity of P₁. It was concluded that the bulk of the catecholamine-sensitive adenylate cyclase activity of the cardiac muscle homogenate was localized in the sarcolemma. The described procedure for the isolation of a purified sarcolemma fraction is rapid and does not require the use of an ultracentrifuge or density gradients.     

Subcellular localization and partial characterization of adenylate cyclase from rat lung

The subcellular localization of adenylate cyclase (ATP pyrophosphate lyase [cyclizing] EC 4.6.1.1) from predominantly alveolar tissue of rat lung was studied using isotonic and hypotonic homogenization and differential centrifugation. The fractions prepared under hypotonic conditions were further subfractionated by nonlinear sucrose gradient centrifugation. All fractions were assayed for adenylate cyclase, marker enzymes, and DNA. Only 5′-nucleotidase (EC 3.1.3.5, a plasma membrane marker enzyme for rat lung) paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (co-efficient of correlation = 0.94). It is concluded that this adenylate cyclase to a large extent but not exclusively is associated with the plasma membranes. Basal adenylate cyclase activity of plasma membrane fractions was stimulated 3.7 times by NaF (0.01 M). It was also stimulated by isoproterenol (55 µM), epinephrine (55 µM), prostaglandin E₁ (0.03 mM), and prostaglandin E₂ (0.03 mM).     

Regulation of adenylate cyclase by β-melanotropin in the M2R melanoma cell line

We have examined adenylate cyclase (AC) in the M2R melanoma cell line, a novel clone of transplantable B16 melanoma cells. It has been found that activity of this enzyme is highly responsive to β-melanotropin (β-MSH) and other hormones possessing melanotropic activity (e.g., α-melanotropin (α-MSH) and adrenocorticotrophic hormone (ACTH_1/224)). β-MSH stimulation of adenylate cyclase, both in the intact cell and in a plasma membrane-enriched fraction derived thereof, was shown to be saturable and dose-dependent. In addition, prostaglandin E₁ (PGE₁) was found to be a potent stimulator of AC activity in these cells. Hormone stimulation of enzyme activity in the intact cell was strongly potentiated by forskolin which not only enhanced maximal AC activity 3-fold, but lowered by 40-fold the concentration of β-MSH required for half-maximal stimulation. Using biologically active [¹²⁵I]iodo-β-MSH prepared in our laboratory we have examined the specificity of β-MSH binding to its receptor in both intact M2R cells and plasma membranes derived thereof. Among a series of hormones tested only α-MSH and ACTH_1–24 competed with [¹²⁵I]iodo-β-MSH for binding to the melanotropin receptor in accordance with the results obtained with AC. In contrast to the strong effect on cyclic 3',5'-adenosine monophosphate (cAMP) accumulation in M2R cells forskolin has no effect on [¹²⁵I]iodo-β-MSH binding. It appears that the kinetic properties of β-MSH binding and β-MSH stimulation of adenylate cyclase activity are essentially identical, the half-maximal effects of which are demonstrated at approximately 20 nM β-MSH.     

Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary.

The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.     

Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

Prostaglandins as hormones

In recent years, the concept of a hormone has been greatly changed, and the term ‘cybernin’ is used to describe a substance which possesses not only endocrine activity but also has autocrine and paracrine effects. The cytoprotective effects of prostaglandins are reviewed with respect to the relationship between prostaglandins and cyclic AMP, and to the effects of prostaglandins on ion transport. Prostaglandins are produced by cell membranes of many tissues and are found in the vasculature. However, the metabolic degradation of prostaglandins is rapid and their significance as circulatory hormones has not been clarified. Yet it is clear that prostaglandins have important physiological activity and it is possible that the effects of prostaglandins are mediated by paracrine or autocrine mechanisms. In order to classify prostaglandins as hormones, it is necessary to clarify their biological activities, to identify a specific and saturable receptor, and to determine a second messenger. This paper discusses the extent to which prostaglandins conform to our present concept of hormones. The existence of a prostaglandin receptor and the role of adenylate cyclase have been confirmed using cultured cell clones. The following observations have been made. (i) For a series of compounds, potency in competing for (³H)PGE₁ binding sites correlated with their ability to stimulate adenylate cyclase activity. (ii) There was a relationship between rates of binding and change in enzyme activity. (iii) The presence or absence of PGE₁-sensitive adenylate cyclase corresponded to (³H)PGE₁ binding capacity. The presence of a prostaglandin receptor has been identified in rat liver, bovine thyroid, bovine corpus luteum, frog erythrocyte, hamster adipocyte, and human adipocyte. Guanine nucleotides and monovalent cations are also involved. The impact of these studies on our present concept of a hormone's plasma membrane receptor is discussed. There is little information regarding the relationship between Ca⁺⁺ and the effects of prostaglandins on adrenal cortical cells. However, it has been clearly shown that after the administration of prostaglandins in vitro, aldosterone secretion is dependent on Ca⁺⁺, secretion being affected by cyclic AMP concentration, aldosterone concentration, Ca⁺⁺ blockers, and calmodulin inhibitors. These results indicate that prostaglandin receptors are involved. The latest experimental studies concerning the effect of prostaglandins on stomach parietal cells are reviewed.     

Properties of LH-sensitive adenylate cyclase in purified plasma membranes from rat ovary.

A procedure for the purification of ovarian plasma membranes (PM) is described and applied to ovaries from immature (25-day-old) rats stimulated with pregnant mare serum gonadotropin (PMSG). Luteinizing hormone (LH)-sensitive adenylate cyclase, 5′-nucleotidase and the binding of ¹²⁵I-labeled human chorionic gonadotropin (hCG) served as PM markers. Judged by these three criteria, 8–15-fold purification of PM was achieved, with a yield of 30–40% of the activity present in the crude homogenate. Optimal conditions for the response of rat ovarian adenylate cyclase to LH and hCG were defined with respect to time, pH and the concentrations of Mg²⁺, ATP, GTP and β,γ-imidoguanosine-5′-triphosphate [Gpp(NH)p].     

Interactions of gonadotropins with corpus luteum surface membranes. V. Differential effects of digitonin on the buoyant densities of light and heavy rat ovarian membrane fractions.

Previous studies have indicated that rat luteal cells at certain stages of development can be fractionated so as to obtain two plasma membrane fractions with different densities and different profiles of marker enzymes. The light membrane fractions (density 1.13) contain the majority of hCG-binding sites and little or no cyclase enzyme, while the heavy membranes (density 1.17) contain the majority of cyclase enzyme and lesser quantities of hormone-binding sites. These membrane fractions were further compared with respect to their susceptibility to perturbation by digitonin. The buoyant density of luteal cell light membrane fractions, as marked by [125I]iodo-hCG binding, Mg2+-dependent ATPase, and 5'-nucleotidase, were highly perturbable by digotonin (delta density, greater than 0.05), while adenylate cyclase activity and phosphodiesterase activity associated with this fraction were only slightly perturbed (delta density, less than 0.02). The buoyant density of luteal cell heavy membrane fractions, as marked by adenylate cyclase, ATPase, and nucleotidase, was not significantly perturbed by digotonin. The hCG binding associated with the heavy membrane fraction was not perturbed by digitonin. From these studies, we conclude that the adenylate cyclase activity associated with light membrane fractions is due to contamination by heavy membranes, while the hCG-binding activity in heavy membrane fractions is intrinsic to that membrane. Except for the lysosomal marker (glucuronidase), which was solubilized by digitonin, the detergent had no significant effect on the density of mitochondrial, Golgi, GERL (Golgi, endoplasmic reticulum, and lysomal), or endoplasmic reticulum membranes. Plasma membranes from isolated granulosa cells and ovaries obtained 24 h after priming with PMS gonadotropin-hCG behaved as heavy membranes (density, 1.17) which contained hCG-binding sites, adenylate cyclase, nucleotidase, and Mg2+-dependent ATPase. These were not significantly perturbed by digitonin. The appearance of light membranes and the segregation of adenylate cyclase from the majority of hCG-binding sites is a development feature of the luteal cell.     

Thyrotrophin-responsive adenylate cyclase activity in thyroid toxic adenoma.

The adenylate cyclase system was studied in hyperfunctioning autonomous nodules in comparison with normal thyroid tissue. The basal, TSH- and NaF-stimulated adenylate cyclase activities were tested in purified plasma membrane preparations. Basal enzyme activity in membranes from hyperfunctioning nodules was variable and the response to TSH was either normal, low or absent. The present study demonstrates that an intact adenylate cyclase activity, hyporesponsive to TSH, may exist in the cell membrane of the adenoma.     

Myocardial Phosphodiesterases and Regulation of Cardiac Contractility in Health and Cardiac Disease

Phosphodiesterase (PDE) inhibitors are potent cardiotonic agents used for parenteral inotropic support in heart failure. Contractile effects of these agents are mediated through cAMP-protein kinase A-induced stimulation of I _Ca2+ which ultimately results in increased Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. A number of additional effects such as increases in sarcoplasmic reticulum Ca²⁺ stores, stimulation of reverse mode Na⁺–Ca²⁺ exchange, direct or cAMP-mediated effects on sarcoplasmic reticulum ryanodine receptor, stimulation of the voltage-sensitive sarcoplasmic reticulum Ca²⁺ release mechanism, as well as A₁ adenosine receptor blockade could contribute to positive inotropic responses to PDE inhibitors. Moreover, some PDE inhibitors exhibit Ca²⁺ sensitizer properties as they could increase the affinity of troponin C Ca²⁺-binding sites as well as reduce Ca²⁺ threshold for thin myofilament sliding and facilitate cross-bridge cycling. Inotropic responses to PDE inhibitors are significantly reduced in cardiac disease, an effect largely attributed to downregulation of cAMP-mediated signalling due to sustained sympathetic activation. Four PDE isoenzymes (PDE1, PDE2, PDE3 and PDE4) are present in myocardial tissue of various mammalian species, of which PDE3 and PDE4 are particularly involved in regulation of cardiac myocyte contraction. PDE cAMP-hydrolysing activity is preserved in compensated cardiac hypertrophy but significantly reduced in animal models of heart failure. However, clinical studies have not revealed any changes in distribution profile as well as kinetic and regulatory properties of myocardial PDEs in failing human hearts. A reduction of PDE inhibitors-induced contractile responses in heart failure has therefore been ascribed to reduced cAMP synthesis due to uncoupling of adenylyl cyclase from β-adrenoreceptor. In cardiac myocytes, PDEs are targeted to distinct subcellular compartments by scaffolding proteins such as myomegalin, mAKAP and β-arrestins. Over subcellular microdomains, cAMP hydrolysis by PDE3 and PDE4 allows to control the activity of local pools of protein kinase A and therefore the extent of protein kinase A-mediated phosphorylation of cellular proteins.     

Mitochondria and sarcoplasmic reticulum as model targets for neurotoxic and myotoxic phospholipases A2

Certain neurotoxins and myotoxins from snake venoms have phospholipase A₂ activity (phosphatide 2-acylhydrolase, EC 3.1.1.4), which appears to be necessary for their toxicity. Several of these toxins inhibit the net uptake of Ca²⁺ into sarcoplasmic reticulum vesicles and brain mitochondria. We have obtained evidence that the ability to inhibit this Ca²⁺ uptake is a mechanistically relevant correlate of the toxicity of these proteins rather than being just a nonspecific consequence of their phospholipase A₂ activity. Two of the toxins, β-bungarotoxin and notexin, had 5% and 50%, respectively, of the phospholipase A₂ activity of IVa phospholipase A₂(a nontoxic enzyme), but β-bungarotoxin was as effective as IVa in inhibiting Ca²⁺ uptake into brain mitochondria and notexin was more effective. Each of the myotoxic enzymes substantially inhibited Ca²⁺ uptake into sarcoplasmic reticulum, notexin being the most effective in this regard. This ability correlated better with their myotoxic potency than with their phospholipase A₂ activity. β-Bungarotoxin lost its toxicity but not its measurable phospholipase A₂ activity after modification with ethoxyformic anhydride in the presence of dihexanoylphosphatidylcholine. The modified toxin also lost most of its ability to inhibit Ca²⁺ uptake into sarcoplasmic reticulum and brain mitochondria. Sarcoplasmic reticulum vesicles reconstituted from solubilized sarcoplasmic reticulum retained their sensitivity to notexin.     

Catecholamine and divalent cation effects on frog liver adenylate cyclase

Frog liver adenylate cyclase was characterized with respect to divalent cation interaction and hormonally stimulated activities. The enzyme catalyzed the synthesis of cyclic [³²P]3′,5′-AMP from α-³²P-labeled ATP. The activity of the enzyme was linear with time and00 protein concentration. The K_m for ATP was 0.5 mM, in the presence or absence of stimulators. The temperature optimum was 25°. GTP (10^?4M) increased the stimulation of adenylate cyclase by epinephrine. Similar activities were obtained using 5 mM Mg²⁺ or Mn²⁺. At higher concentrations, both ions inhibited epinephrine-stimulated, but not basal or fluoride-stimulated activities. Approximately equivalent hormonal stimulation was obtained with maximal stimulating concentrations of epinephrine, isoproterenol, glucagon, and prostaglandin E₁. Norepinephrine was less stimulatory. Only catecholamine-stimulated activities were inhibited by propranolol (10^?5M). The data suggest that catecholamines stimulate frog liver adenylate cyclase through interactions with β adrenergic receptors. The adenylate cyclase in frog liver differs from its mammalian counterpart in its response to temperature and maximally stimulatory concentrations of hormones.     

Interactions of gonadotropins with corpus luteum membranes. II. The identification of two distinct surface membrane fractions from superovulated rat ovaries.

Fractions enriched in hCG-binding activity were prepared by differential rate centrifugation of superovulated rat ovarian homogenates and were applied to continuous sucrose density gradients (20-55%). After centrifugation at 63,000 x gav for 3.5 h, fractions of each gradient were collected and assayed for a range of marker enzyme activities characteristic of surface membranes and subcellular organelles. Mitochondria, lysosomes, and rough and smooth endoplasmic reticulum membranes accumulated in the gradient between 38-41% sucrose (1.165-1.180 g/cm3). Nuclei passed through the gradient. However, the various surface membrane markers concentrated in two distinct regions of the gradient. Alkaline phosphatase, phosphodiesterase, (Na+ + K+)ATPase I, and hCG-binding activity concentrated at 29-32% sucrose (1.120-1.135 g/cm3), whereas 5'-nucleotidase, Mg2+-dependent ATPase, and adenylate cyclase activities (and minor peaks of hCG-binding and phosphodiesterase activities) were enriched at 36-38% sucrose (1.16-1.17 g/cm3). A second ATPase, [(Na+ + K+)ATPase II], was also observed in this region of the gradient, which could be distinguished from (Na+ + K+)ATPase I of the light membrane fraction by its sensitivity to the Ca2+-chelating agent, ethylene glycol bis-(aminoethyl)tetraacetic acid (EGTA). The kinetics of binding of radioiodinated hCG to the gonadotropin receptors of the light and heavy membrane fractions were very similar. It is suggested that fractionation of superovulated rat ovaries yields two distinct populations of surface membrane material which have distinct densities and marker enzyme profiles. Furthermore, in contrast to the heavy membrane fraction, light membranes seem to possess considerable amounts of hCG receptor activity but very little adenylate cyclase.     

Changes in microsomal membrane phospholipids and fatty acids and in activities of membrane-bound enzyme in diabetic rat heart

Diabetes mellitus is associated with alterations in lipid metabolism and cardiac dysfunction despite an absence of coronary arteriosclerotic changes. To investigate mechanisms of cardiac dysfunction in diabetic cardiomyopathy, we studied the relation between activities of membrane-bound enzymes and surrounding phospholipids in rats with diabetes induced with a single intravenous injection of streptozotocin (65 mg/kg). We found that total phospholipid content of sarcoplasmic reticulum membrane increased significantly 8 weeks after treatment with streptozotocin owing to increases in phosphatidylcholine and phosphatidylethanolamine, a decrease in arachidonic acid, and an increase in docosahexaenoic acid in the early stage of diabetes. Sarcolemmal Na⁺/K⁺-ATPase activity and the number of receptors decreased in isolated cardiomyoctes of diabetic rats 8 weeks after streptozotocin administration. The Ca²⁺ uptake of both sarcoplasmic reticulum and mitochondria decreased simultancously in permeabilized, isolated cardiomyocytes from diabetic rats. The depression of membrane-bound enzyme activities was correlated with alterations in phospholipids, which are closely related to the microenvironment of membrane-bound enzymes and influence intracellular Ca²⁺ metabolism. Because these changes in phospholipids and fatty acids were reversible with insulin therapy, they are diabetes-specific and might be a cause of cardiac dysfunction in diabetes.     

Effects of prostaglandin H2, prostaglandin E2, and arachidonic acid on parathyroid hormone and antidiuretic hormone activation of rat kidney adenylate cyclase

These experiments examine interactions of arachidonic acid; the substrate for prostaglandin cyclooxygenase, prostaglandin (PG)H₂, a key endoperoxide intermediate in prostaglandin synthesis; and prostaglandin (PG)E₂, an important prostaglandin produced within the kidney; with adenylate cyclase activity in renal cortex, outer medulla, and inner medulla. In addition, the effects of arachidonic acid, PGH₂, and PGE₂ on parathyroid hormone (PTH) activation of adenylate cyclase in cortex, and of antidiuretic hormone (ADH) activation of that enzyme in outer and inner medulla are examined. Arachidonic acid elicited a concentration-dependent inhibition of basal and PTH-stimulated adenylate cyclase activity in renal cortex. Concentration-dependent inhibition by arachidonic acid of basal and ADH-stimulated adenylate cyclase activity was observed in outer and inner medulla. PGH₂ inhibited basal activity in all three areas of the kidney. There was also inhibition by PGH₂ of medullary ADH and cortical PTH stimulation. PGE₂ stimulated adenylate cyclase in all three areas. PGE₂ had no effect upon PTH stimulation in cortex and was additive with ADH in outer and inner medulla. PGE₂ stimulation was inhibited by arachidonic acid, and this inhibition seemed competitive. Inhibition by both arachidonic acid and PGH₂ was not destructive. Experiments with [1-¹⁴C]arachidonic acid and indomethacin suggest that the inhibition by arachidonic acid was actually mediated by arachidonic acid and not a metabolite. Both PGH₂ and arachidonic acid inhibition was independent of phosphodiesterase. This activation by product, PGE₂, and inhibition by its precursors, arachidonic acid and PGH₂, provide a possible mechanism by which the prostaglandin system could modulate adenylate cyclase responsiveness to hormonal activation.     

Effects of Plasmalemmal V-ATPase Activity on Plasma Membrane Potential of Resident Alveolar Macrophages

The acid-base status and functional responses of alveolar macrophages (m) are influenced by the activity of plasmalemmal V-type H⁺-pump (V-ATPase), an electrogenic H⁺ extruder that provides a possible link between intracellular pH (pH_i) and plasma membrane potential (E_m). This study examined the relationships among E_m, pH_i, and plasmalemmal V-ATPase activity in resident alveolar m from rabbits. E_m and pH_i were measured using fluorescent probes. E_m was –46 mV and pH_i was 7.14 at an extracellular pH (pH_o) of 7.4. The pH_i declined progressively at lower pH_o values. Decrements in pH_o also caused depolarization of the plasma membrane, independent of V-ATPase activity. The pH effects on E_m were sensitive to external K⁺, and hence, probably involved pH-sensitive K⁺ conductance. H⁺ were not distributed at equilibrium across the plasma membrane. V-ATPase activity was a major determinant of the transmembrane H⁺ disequilibrium. Pump inhibition with bafilomycin A₁ caused cytosolic acidification, due most likely to the retention of metabolically generated H⁺. V-ATPase inhibition also caused depolarization of the plasma membrane, but the effects were mediated indirectly via the accompanying pH_i changes. V-ATPase activity was sensitive to E_m. E_m hyperpolarization (valinomycin-clamp) reduced V-ATPase activity, causing an acidic shift in baseline pH_i under steady-state conditions and slowing pH_i recovery from NH₄Cl prepulse acid-loads. The findings indicate that a complex relationship exists among E_m, pH_i, and pH_o that was partially mediated by plasmalemmal V-ATPase activity. This relationship could have important consequences for the expression of pH- and/or voltage-sensitive functions in alveolar m.     

The Reaction of Glucagon with Its Receptor: Evidence for Discrete Regions of Activity and Binding in the Glucagon Molecule

Des-histidine-glucagon (DH-glucagon, glucagon_2-29) does not activate the glucagon-sensitive adenylate cyclase system present in either liver plasma membranes or in fat-cell “ghosts”, but inhibits the response of these systems to submaximal concentrations of glucagon. DH-glucagon also inhibits, competitively, the binding of [¹²⁵I]glucagon to its receptor in liver plasma membranes. Amino-terminal fragments of glucagon (glucagon_1-21, glucagon_1-23) and carboxy-terminal fragments (glucagon_20-29, glucagon_22-29) failed to activate adenylate cyclase, to inhibit the response of the enzyme to glucagon, or to compete with labeled glucagon at its receptor.     

Inhibition of histamine-sensitive adenylate cyclase from guinea pig fundic gastric mucosa by arachidonic acid and by an analog of prostaglandin endoperoxide PGH2

The effects of prostaglandin precursors, namely an analog of prostaglandin endoperoxide PGH₂ [(15S)-hydroxy-9α,11α-(epoxymethano)prosta-5, 13-dienoic acid] and arachidonic acid, were assessed on gastric adenylate cyclase activity from cell-free preparations of guinea pig fundic mucosa. The two precursors were tested against basal adenylate cyclase activity and that stimulated by histamine (10^?4M), by PGE₂(10^?4M), by 5′-guanylylimidodiphosphate [Gpp(NH)p] (10^?4M) and by NaF(10^?2M). PGH₂ analog (10^?4M) and arachidonic acid (10^?4M) both inhibited to a similar extent adenylate cyclase stimulated by histamine or by NaF, but not that stimulated by PGE₂ or by Gpp(NH)p. Neither agent significantly affected basal adenylate cyclase levels. In the presence of indomethacin (10^?4M), basal adenylate cyclase activity remained unchanged but the inhibitory effect of arachidonic acid was almost entirely abolished, suggesting that such inhibitory effect may be caused by prostaglandin endoperoxides generated from arachidonic acid in the course of assay. Moreover, indomethacin did not attenuate PGH₂ inhibition of histamine action. Unlike arachidonic acid, which is a natural metabolic precursor of PGE₂, arachidic acid did not significantly influence histamine-stimulated adenylate cyclase activity. These results suggest that the prostaglandin endoperoxides may have an inhibitory effect on histamine-sensitive ciclic AMP generation in gastric mucosa.