Human platelet adenylate cyclase: persistent binding of guanine nucleotide is central to the regulation of both hormone-stimulated and basal activities

Prostaglandin E1 stimulation of human platelet adenylate cyclase, in purified plasma membranes, occurs without the addition of exogenous GTP. Possible contamination of the adenylate cyclase assay mixture by GTP either from nonspecifically bound nucleotide in the plasma membrane or from the substrate ATP was ruled out as follows: (a) variation of the membrane concentration, repeated washing, inclusion of EDTA, GDP beta S, or GMP in the wash step, or UDP in the assay, are all without effect, and (b) analysis of the substrate by high-performance liquid chromatography revealed no contaminating GTP. Other prostaglandins (I2, E2, D2) also activate cyclase without the addition of GTP. In sharp contrast, stimulation of adenylate cyclase in the human neutrophil plasma membrane by prostaglandin E1 shows an obligatory requirement for GTP, under identical assay conditions. GDP beta S pretreatment amplifies the fold cyclase stimulation by GTP in the presence and absence of prostaglandin E1, by lowering the basal activity. This alteration occurs without lowering the GTP-independent prostaglandin E1 activation, and is specific for inhibitory guanine nucleotides (GDP beta S, GMP, GDP) in the pretreatment. Extensive washing with buffer or incubation with other nucleotides, epinephrine, or prostaglandin E1 prior to the assay, is without effect. GTP gamma S treatment of the membrane induces a high-activity state and abolishes the GDP beta S effect on basal activity as well as prostaglandin E1 activation of cyclase. The results suggest distinct patterns of prostaglandin stimulation in platelet and neutrophil cyclase systems, and further imply that guanine nucleotide, prebound to specific sites within the GTP-regulatory proteins, may modify the kinetic characteristics of platelet adenylate cyclase.     

Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.     

Activation of adenylate cyclase in cultured fibroblasts by trypsin

Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.     

Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.     

Spin-labeled stearates as probes for microenvironment of murine thymocyte adenylate cyclase-cyclic adenosine 3':5'-monophosphate system.

The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.     

Roles of GTP and GDP in the regulation of the thyroid adenylate cyclase system

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regeneratign system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP and GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of nucleotide-regenerating system, addition of GDP to the adenylate cyclase assay mixture int he parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparatiosn possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrst to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic componenet of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Cytochemical localization of adenylate cyclase and of calcium ion, magnesium ion-activated ATPases in the dense tubular system of human blood platelets.

Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

Metabolism of arachidonic acid by isolated rat hepatocytes, renal cells and by some rabbit tissues. Detection of vicinal diols by mass fragmentography

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regenerating system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP or GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of a nucleotide-regeneration system, addition of GDP to the adenylate cyclase assay mixture resulted in the parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparations possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrast to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic component of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Endogenous GTP and the regulation of epinephrine stimulation of adenylate cyclase.

Epinephrine increased adenylate cyclase activity 10 to 15 fold in lysates of the cultured human astrocytoma cell line 132-1N1. GTP had little effect on adenylate cyclase activity of lysed cell preparations either with or without added epinephrine. However, the epinephrine stimulation of adenylate cyclase was essentially lost (less than 90%) when a washed nuclei-free membrane preparation of the cyclase was assayed. A 10 to 15 fold epinephrine stimulation of the membrane adenylate cyclase could be demonstrated if cytosol of GTP were added to the assay with the hormone. The criteria of anion exchange, cation exchange, gel exclusion and paper chromatography indicated that the cytosolic agents which acted synergistically with hormones were GTP and GDP. The apparent Kact's for the synergistic action of GDP and GTP were essentially identical (1.0 muM) and of all the other nucleotides examined only GDP had a potency similar to GTP. However, the effect of GDP was apparently due to its rapid conversion to GTP even in the absence of a regenerating system. With epinephrine pretreatment of the intact 132-1N1 cells there was a specific loss of epinephrine stimulation of adenylate cyclase activity. The hormone pretreatment did not alter the capacity of the cytosol from these desensitized cells to potentiate epinephrine stimulation of the cyclase. Rather, the alteration was in the particulate fraction of the lysate. The desensitization of the membranous cyclase was stable and not reversed by GTP.     

Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTP gamma S) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.     

Cross talk between stimulatory and inhibitory guanosine 5'-triphosphate binding proteins: role in activation and desensitization of the adenylate cyclase response to vasopressin

The inhibitory GTP-binding protein (Gi) is known to mediate the effects of a number of hormones that act through specific receptors to inhibit adenylate cyclase. In this study we examined the mechanism whereby Gi modulates the response of adenylate cyclase to a stimulatory hormone and its role in desensitization. In membranes prepared from the cultured renal epithelial cell line LLCPK1, adenylate cyclase activity was stimulated 16-fold by 1-2 microM lysine vasopressin. Addition of GTP (1-100 microM) resulted in stimulation of basal activity but inhibition of hormone-stimulated activity (approximately 40% inhibition at 100 microM GTP). This contrasts with the usual effect of GTP to support or augment activation by stimulatory receptors. The inhibitory effect was abolished by pertussis toxin, which had little effect on basal activity in the absence or presence of added GTP or on vasopressin-stimulated activity in the absence of added GTP. GTP-mediated inhibition was vasopressin concentration dependent. At concentrations of vasopressin below the K1/2 for enzyme activation (approximately 0.6 nM), GTP was stimulatory, and at higher concentrations, GTP was inhibitory. The inhibitory effect of GTP was also observed for a V2-receptor agonist and was not abolished by a V1-receptor antagonist, indicating that a distinct V1 receptor did not mediate inhibition of adenylate cyclase. Using the known subunit structure of adenylate cyclase, we developed the minimal mechanism that would incorporate a modulatory role for Gi in determining net activation of adenylate cyclase by a stimulatory hormone. The predicted enzyme activities for basal and maximal hormone stimulation in the presence and absence of GTP were generated, and model parameters were chosen to match the experimental observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta-adrenergic receptor in the human neutrophil plasma membrane: receptor-cyclase uncoupling is associated with amplified GTP activation

We compared the properties of the adenylate cyclase system in plasma membranes derived from gas cavitation and sonication of human neutrophils. In membranes prepared from cavitated cells, cyclase was stimulated by fluoride ion and Gpp(NH)p. Stimulation by isoproterenol (and PGE1) was absent, although the beta-receptor was present as judged by 125I-HYP binding. Two unusual characteristics of this cyclase system are a) substantial activation of cyclase by GTP in the absence of hormone, and b) reduction in the regulation of the beta-receptor by GTP. By contrast, vesicles isolated from sonicated cells displayed normal GTP-dependent activation by isoproterenol (as well as PGE1) and GTP regulation of the beta-receptor, while hormone-independent stimulation by GTP was negligible. Cholera toxin-catalyzed ADP ribosylation revealed a prominent band at 42K in both membranes, and a minor band at 35K, although the degree of labeling of this protein was lower in the "cavitated" vesicles. Our results suggest that the mode of cell lysis and membrane preparation influence receptor-cyclase interactions and that receptor-cyclase uncoupling is associated with amplified GTP activation and altered receptor regulation by GTP.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

The phorbol ester TPA prevents the expression of both glucagon desensitisation and the glucagon-mediated block of insulin stimulation of the peripheral plasma membrane cyclic AMP phosphodiesterase in rat hepatocytes

The phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) causes a dose-dependent inhibition of the glucagon-stimulated adenylate cyclase activity expressed in plasma membranes isolated from TPA-treated hepatocytes. However, no observable inhibitory effect of TPA on adenylate cyclase activity was observed in cells which had been exposed to glucagon for 5 min, prior to isolation, to desensitise adenylate cyclase. The degree of inhibition of adenylate cyclase elicited by both glucagon desensitisation and TPA treatment of hepatocytes was identical. Pre-treatment of hepatocytes with TPA was also found to prevent glucagon from blocking insulin's activation of the peripheral plasma membrane cyclic AMP phosphodiesterase in intact hepatocytes. TPA treatment also inhibited the ability of cholera toxin to activate the peripheral cyclic AMP phosphodiesterase in intact hepatocytes. It is suggested that in these particular instances TPA and glucagon elicit mutually exclusive processes rather than TPA mimicking glucagon desensitisation per se.     

GTP stimulates and inhibits adenylate cyclase in fat cell membranes through distinct regulatory processes.

GTP and hormones activate, synergistically, adenylate cyclase in purified plasma membranes from rat adipocytes. Addition of chelating reagents (EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or thiol-reducing reagents (dithiothreitol or 2-mercaptoethanol) results in marked inhibition of enzyme activity without altering the synergistic stimulatory effects of GTP and hormones. The inhibitory effects of the reagents required the presence of GTP, indicating that inhibition involves a GTP-dependent process. This process is separate from the GTP-dependent process responsible for activation of the enzyme since it is selectively abolished by pretreatment of fat cell membranes with trypsin. It is suggested that inhibition and activation of fat cell adenylate cyclase by GTP occur through distinct regulatory processes.     

Hormonally stimulated adenylate cyclase and cAMP dependent protein kinase in membranes of rabbit erythroid cells separated according to density

Plasma membranes were prepared after density gradient separation of erythroid cells obtained from bled animals. In a fraction enriched in young reticulocytes (lowest density), the basal and the prostaglandin stimulated adenylate cyclase were greatly augmented if compared with the membranes from unfractionated cells or from the layers of higher densities. Potentiation by GTP or soluble factor(s) of the prostaglandin stimulated adenylate cyclase was found solely in the fractions containing the youngest cells (lowest density). A very significant augmentation of both the basal and the effectors stimulated adenylate cyclase was obtained when the white blood cells and the platelets were removed by filtration through alpha-cellulose prior to density separation. A small population of probably very young reticulocytes was shown to contain a very active adenylate cyclase coupled to the hormonal receptor. Upon short time of maturation this coupling could no longer be detected. The cAMP generated in the fraction enriched in young reticulocytes increased the phosphorylation of some membrane proteins. The presence of a hormonally regulated adenylate cyclase and eventually the phosphorylation of some specific membrane proteins by the cAMP generated in situ permit to envisage possible functions of this system in young reticulocytes.