Isolation of GTP-binding Gs-protein liberated from human erythrocyte membranes under the action of fluoride ions

Human erythrocyte membranes were incubated in the presence of sodium fluoride. After centrifugation at 30,000 g for 30 min the supernatant was able to stimulate the catalytic subunit of adenylate cyclase. The stimulatory factor was purified from the supernatant of fluoride-treated membranes by three subsequent chromatographic steps including DEAE-Sephacel ion-exchange chromatography in the absence of detergent, gel-filtration on Ultrogel AcA 44 in the presence of 1% sodium cholate and phenyl-Sepharose CL/4B hydrophobic chromatography. The final preparation showed approximately 120-fold purification in stimulatory activity over the initial extract and contained two polypeptides (Mr 42 kDa and 36 kDa). The stimulator activity of the preparation was inhibited by 60% by beta gamma-subunits of the GTP-binding protein of bovine brain membranes, G0. The data obtained suggest that the regulatory GTP-binding stimulatory protein of adenylate cyclase, GS, dissociates from human erythrocyte membranes as a result of fluoride-ion treatment.     

Activation of rat brain adenylate cyclase by proteases: involvement of distinct protein components in the activation by α-chymotrypsin and trypsin

Adenylate cyclase in synaptic plasma membranes from rat brain is activated by α-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with α-chymotrypsin (100 μg/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10–30 μg/ml) much more weakly enhances the cyclase activity. (2) α-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not α-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of α-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5′-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified βγ-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the βα-subunits with α-chymotrypsin (> 10 μg/ml). (6) Trypsin (1–10 μg/ml) inactivates the GTPase of the α-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin.We conclude that two distinct protein components are involved in the activation of adenylate cyclase by α-chymotrypsin and trypsin. One component sensitive to α-chymotrypsin is probably the βγ-subunits of the GTP-binding proteins. The other component sensitive to trypsin may be the α-subunit of Gi.     

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.     

Possible Involvement of Inhibitory GTP Binding Regulatory Protein in α2-Adrenoceptor-Mediated Inhibition of Adenylate Cyclase Activity in Cerebral Cortical Membranes of Rats

Influences of alpha 2-adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet-activating protein and NAD resulted in a significant increase in basal activity as well as in GTP- or forskolin/GTP-induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet-activating protein and NAD. It is suggested that alpha 2-receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.     

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.     

Modulation by Monoamines of Somatostatin-Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.     

Domoic acid inhibits adenylate cyclase activity in rat brain membranes

Adenylate cyclase activity measured by the formation of cyclic AMP in rat brain membranes was inhibited by a shellfish toxin, domoic acid (DOM). The inhibition of enzyme was dependent on DOM concentration, but about 50% of enzyme activity was resistant to DOM-induced inhibition. Rat brain supernatant resulting from 105,000×g centrifugation for 60 min, stimulated adenylate cyclase activity in membranes. Domoic acid abolished the supernatant-stimulated adenylate cyclase activity. The brain supernatant contains factors which modulate adenylate cyclase activity in membranes. The stimulatory factors include calcium, calmodulin, and GTP. In view of these findings, we examined the role of calcium and calmodulin in DOM-induced inhibition of adenylate cyclase in brain membranes. Calcium stimulated adenylate cyclase activity in membranes, and further addition of calmodulin potentiated calcium-stimulated enzyme activity in a concentration dependent manner. Calmodulin also stimulated adenylate cyclase activity, but further addition of calcium did not potentiate calmodulin-stimulated enzyme activity. These results show that the rat brain membranes contain endogenous calcium and calmodulin which stimulate adenylate cyclase activity. However, calmodulin appears to be present in membranes in sub-optimal concentration for adenylate cyclase activation, whereas calcium is present at saturating concentration. Adenylate cyclase activity diminished as DOM concentration was increased, reaching a nadir at about 1 mM. Addition of calcium restored DOM-inhibited adenylate cyclase activity to the control level. Similarly, EGTA also inhibited adenylate cyclase activity in brain membranes in a concentration dependent manner, and addition of calcium restored EGTA-inhibited enzyme activity to above control level. The fact that EGTA is a specific chelator of calcium, and that DOM mimicked adenylate cyclase inhibition by EGTA, indicate that calcium mediates DOM-induced inhibition of adenylate cyclase activity in brain membranes. While DOM completely abolished the supernatant-, and Gpp (NH)p-stimulated adenylate cyclase activity, it partly blocked calmodulin-, and forskolin-stimulated adenylate cyclase activity in brain membranes. These results indicate that DOM may interact with guanine nucleotide-binding (G) protein and/or the catalytic subunit of adenylate cyclase to produce inhibition of enzyme in rat brain membranes.     

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.     

Tosyl-lysyl chloromethylketone detects conformational changes in the catalytic unit of adenylate cyclase induced by receptor and G-protein stimulation

Incubation of striatal membranes with tosyl-lysyl chloromethylketone (TLCK) led to the irreversible inactivation of adenylate cyclase. However, under conditions where an interaction between the catalytic unit of adenylate cyclase and the alpha-subunit of the stimulatory G-protein GS were promoted, then the ability of TLCK to inhibit adenylate cyclase was markedly attenuated. The potency of stimulatory ligands, functioning through GS, to attenuate the sensitivity of adenylate cyclase to inactivation by TLCK was paralleled by their potency to activate adenylate cyclase. The local anaesthetic and membrane-fluidizing agent benzyl alcohol amplified GS-mediated stimulation of adenylate cyclase activity, whilst diminishing the ability of GS-mediated coupling to attenuate inactivation of adenylate cyclase by TLCK. In the absence of GS-mediated coupling, benzyl alcohol exerted only a small stimulatory effect on adenylate cyclase activity and had little effect on the ability of TLCK to inactivate this enzyme. We suggest that TLCK modifies a reactive group at or near the active site of adenylate cyclase which causes the functional inactivation of this enzyme. The reactivity of this group appears to be markedly affected by conformational changes elicited through coupling of adenylate cyclase to GS.     

Adenylate cyclase activity in crude plasma membranes of subcutaneous adipocytes during ontogenetic development of rats

In order to explain the differences in the hormone stimulated lipolysis during ontogenic development of rats, the activity of adenylate cyclase was determined in crude plasma membranes of subcutaneous adipocytes of 5, 14, 21 and 45 to 55-day-old animals. Stimulatory effects of nonhormonal and hormonal agents were expressed as the increment in percentage of basal values which were not significantly changed in the age groups studied. The highest stimulatory effect was observed after sodium fluoride in 14 and 21-day-old rats. Guanylylimidodiphosphate and GTP revealed the lowest stimulatory effects in adult animals (greater than 45-day-old). The beta-adrenergic agent isoproterenol revealed the highest stimulatory effect in the 5 and 45-day-old group while in the preparation from 14-day-old rats the adenylate cyclase activity was significantly lower. On the other hand, tetracosactide (beta 1-24-corticotropin) revealed the smallest stimulatory effect on the preparation from 5-day-old rats; its stimulatory effect steadily increased and reached the highest value in adenylate cyclase preparations from adult animals. It can be concluded that the adenylate cyclase system in subcutaneous adipocytes is already basically mature at early ontogenic stages of development in rats. Nevertheless, the explanation for the small variations of the enzyme activity in different age groups requires further study.     

Calmodulin regulation of adenylate cyclase activity in human platelet membranes

The mechanism of calmodulin dependent regulation of adenylate cyclase has been studied in human platelet membranes. Calmodulin activated adenylate cyclase exhibited a biphasic response to both Mg2+ and Ca2+. A stimulatory effect of Mg2 on adenylate cyclase was observed at all Mg2+ concentrations employed, although the degree of activation by calmodulin was progressively decreased with increasing concentrations of Mg2+. These results demonstrate that the Vmax of calmodulin dependent platelet adenylate cyclase can be manipulated by varying the relative concentrations of Mg2+ and Ca2+. The activity of calmodulin stimulated adenylate cyclase was always increased 2-fold above respective levels of activity induced by GTP, Gpp(NH)p and/or PGE. The stimulatory influence of calmodulin was not additive but synergistic to the effects of PGE1, GTP and Gpp(NH)p. GDP beta S inhibited GTP-and Gpp(NH)p stimulation of adenylate cyclase but was without effect on calmodulin stimulation. Since the inhibitory effects of GDP beta S have been ascribed to apparent reduction of active N-protein-catalytic unit (C) complex formation, these results suggest that the magnitude of calmodulin dependent adenylate cyclase activity is proportional to the number of N-protein-C complexes, and that calmodulin interacts with preformed N-protein-C complex to increase its catalytic turnover. Our data do not support existence of two isoenzymes of adenylate cyclase (calmodulin sensitive and calmodulin insensitive) in human platelets.     

Liver membrane adenylate cyclase. Synergistic effects of anions on fluoride, glucagon, and guanyl nucleotide stimulation.

Some effects of salts on the adenylate cyclase of partially purified plasma membranes from rat liver have been studied. Under conditions where cyclic adenosine 3':5'-monophosphate formation was linear with respect to time and protein concentration, the enzyme was stimulated 3- to 6-fold by 10 mM NaF, 10- to 30-fold by 1 muM glucagon, 4- to 5-fold by 0.1 mM 5'-guanylylimidodiphosphate, and in the presence of 3 muM GTP, 2-fold by 10 mug/ml of prostaglandin E1. Various salts were found to stimulate basal activity slightly, but enhanced the response to NaF 3- to 4-fold, to glucagon 1.5- to 2-fold, to 5'-guanylylimidodiphosphate 2- to 3-fold, and to prostaglandin E1 1.5-fold. This enhancement was observed at maximally effective concentrations of each of the respective activators. Of the salts tested, NaN3 and the Na- or K-halides were most effective. Their action appeared to be due to the respective anions. Stimulation was detectable with 1.5 mM NaN3 or 3 mM NaCl and was maximal with 30 mM NaN3 or 60 mM NaCl. The stimulatory effect of NaN3 was not due to ATP-sparing, nor to an altered cyclic adenosine 3':5'-monophosphate recovery. It was independent of the chromatography and assay methods used, and was therefore not due to procedural artifact. Fluoride-stimulated cyclase activity was enhanced by salts to a greater degree than were 5'-guanylylimidodiphosphate-, glucagon-, or (prostaglandin E1 + GTP)-stimulated activities. The effects of NaN3 were not the result of significant changes in the enzyme's responses to GTP, which increased basal and glucagon-stimulated activities but inhibited F--stimulated activity. The effects of NaN3 were greater when cyclase was assayed with Mn2+ than with Mg2+. The facilitatory effect of NaN3 or NaCl on fluoride-stimulated adenylate cyclase activity was partially reversible as was the stimulatory effect of fluoride in the presence of NaN3. Enhancement of hormonal stimulation by NaN3 was also demonstrable with cardiac and adipose tissue adenylate cyclase. However, NaN3 did not stimulate detergent-dispersed adenylate cyclases from either liver plasma membranes or brain. The data suggest that stimulation of adenylate cyclase by salts may require the added presence of other stimulatory agents and an intact membrane structure.     

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.     

Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of Muscarinic-Stimulated Adenylate Cyclase Activity in Rat Olfactory Bulb

The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg-ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l-isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the beta-adrenergic stimulation, the muscarinic effect disappeared when guanosine 5'-O-(3'-thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin-sensitive GTP-binding protein different from the stimulatory GTP-binding protein.     

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.     

Cholesterol modulation of membrane fluidity and VIP receptor/effector system in rat prostatic epithelial cells.

Treatment of rat prostatic epithelial cells with cholesteryl hemisuccinate (ChH) resulted in a time- and dose-dependent inhibition of the stimulatory effect of the neuropeptide vasoactive intestinal peptide (VIP) on cyclic AMP accumulation, with a 40% decrease in the response to a maximally effective VIP concentration. Cell treatment with ChH led also to a similar blocking of isoproterenol (a beta-adrenergic agonist) action but did not modify forskolin (which is assumed to act directly on the catalytic unit of adenylate cyclase) activity upon cyclic AMP levels. The levels of the transduction protein Gs were similar in membranes from both control and ChH-treated cells as suggested by experiments on cholera toxin-catalyzed ADP-ribosylation. The inhibitory effect of ChH was accompanied by an increase of membrane microviscosity as estimated by measurements of fluorescence polarization. Experiments on VIP binding indicated that increasing cholesterol concentration in the plasma membrane led to a higher VIP binding capacity without changes in the affinity of VIP receptors. These data suggest that membrane cholesterol incorporation diminishes the coupling efficiency between adenylate cyclase and the VIP-receptor complex or other receptor systems (i.e., desensitization) due to an increase of plasma membrane rigidity.     

ADP-Ribosylation by Cholera Toxin of Membranes Derived from Brain Modifies the Interaction of Adenylate Cyclase with Guanine Nucleotides and NaF

We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.     

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.     

Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.