Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).     

Analogs of caffeine: antagonists with selectivity for A2 adenosine receptors

Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7-dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3- to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists.     

Activation of adenylate cyclase from rat striatum and tuberculum olfactorium by adenosine

Adenosine caused a dose-dependent stimulation of adenylate cyclase in homogenates from rat striatum and tuberculum olfactorium (200 and 300% stimulation by 100 muM adenosine). The effect of adenosine was not antagonized by haloperidol. Subcellular fractionation suggested that adenosine stimulates a different adenylate cyclase than dopamine. Basal adenylate cyclase activity in freshly prepared homogenates was reduced by dialysis and by the addition of adenosine deaminase. Basal adenylate cyclase activity was enchanced by papaverine and dipyridamole, but reduced by theophylline and isobutylmethylxanthine. The results are compatible with the opinion that endogenous adenosine is capable of activating adenylate cyclase in these areas of the rat brain.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.     

Irreversible inhibition of adenylate cyclase by 5'-(p-bromomethylbenzoyl) adenosine

The effect of 5'-(p-bromomethylbenzoyl) adenosine (pBMBA) on adenylate cyclase from bovine caudate nucleus membranes was studied. Adenylyl-5'-methylenediphosphonate (but not adenosine) protected adenylate cyclase against inactivation by this compound. The degree of pBMBA-induced inhibition of adenylate cyclase increased in the presence of Mg2+. 5'-(p-fluorosulfonylbenzoyl) adenosine (pFSBA) was also a specific irreversible inhibitor of adenylate cyclase. It was demonstrated that the enzyme inactivated by pFSBA completely restored its activity under the action of dithiothreitol. The results obtained are indicative of the presence of the -SH group in the enzyme active site.     

Effects of adenosine and adenosine-analogs on adenylate cyclase activity in the rat adipocyte plasma membrane: comparison of the properties of the enzyme with Mn2+ and Mg2+ as divalent cations

Summary We investigated the influence of Mg²⁺ and Mn²⁺ on the effects of adenosine and some derivatives on basal adenylate cyclase activity in rat fat cell membranes as well as on enzyme activity stimulated by isoprenaline or sodium fluoride. Adenosine and derivatives modified in the ribose function were inhibitory, irrespective of the stimulant used, both in the presence of MgCl₂ or MnCl₂. Inhibition of basal and sodium fluoride stimulated adenylate cyclase activity was more pronounced in the presence of MnCl₂ than in the presence of MgCl₂. N⁶-substituted adenosine analogs proved to be inhibitory in the presence of 5 MM MgCl₂, but in the presence of 1 mM MnCl₂ the fluoride stimulated adenylate cyclase activity was potentiated, while basal and isoprenaline stimulated activity were not significantly inhibited. These effects of adenosine and derivatives could not be blocked by theophylline with or without guanyl nucleotides.The potentiating effect of N⁶-substituted adenosine derivatives on sodium fluoride activated adenylate cyclase is dependent on the structure of the N₆-substitutent and consists of an enhancement of Vrnax in combination with a small decrease of the K_m for MnATP^2–, indicative of an allosteric effect on adenylate cyclase. No potentiation by N⁶-phenylisopropyladeno sine of sodium fluoride stimulated cyclase was found on digitonin solubilized cyclase, while the inhibitory effect of adenosine was retained. The relevance of these findings is discussed in connection with the current hypothesis concerning the presence of two adenosinesensitive sites on rat fat cell membranes.     

Ethanol alters the adenosine receptor-Ni-mediated adenylate cyclase inhibitory response in rat brain cortex in vitro

It has been suggested that ethanol stimulates adenylate cyclase in vitro through an increased function of Ns, the activatory component of adenylate cyclase. Because of the interaction of Ns with Ni, the adenylate cyclase inhibitory component, we have studied the effect of ethanol (0.05-0.2 M) on Ni-mediated adenylate cyclase inhibition caused by the adenosine analog N6-phenylisopropyladenosine (N6-PIA) in brain cortical membranes. Ethanol did not alter N6-PIA binding to the adenosine Ri-receptors, stimulated slightly basal adenylate cyclase activity but abolished adenylate cyclase inhibition due to N6-PIA, suggesting an effect of ethanol on the inhibitory coupling pathway. This was further supported by loss of the adenylate cyclase inhibitory response to GTP (greater than 10(-5) M). It thus seems that, besides its effect on the Ns system, ethanol may also impair Ni-mediated adenylate cyclase responses in rat cerebral cortex.     

Effects of chronic caffeine on brain adenosine receptors: Regional and ontogenetic studies

The effect of chronic caffeine treatment on three different binding sites in five brain areas of mice is characterized. The sites studied were the adenosine receptor, using [³H] diethylphenylxanthine, the benzodiazepine receptor, using [³H] diazepam and the adenosine uptake site, using [³H] nitrobenzylthioinosine. Significant increases were only observed in adenosine receptors with the greatest degree of change seen in the cerebellum and brain stem at both 16 and 23 days of caffeine treatment. The lack of significant effects of chronic caffeine on benzodiazepine receptors and adenosine uptake sites indicates that the caffeine effect is specific. The effect of chronic caffeine treatment on the ontogency of adenosine receptors was also studied with the result showing a significantly accelerated development of the receptor in the caffeine treated animals. The adult adenosine receptor levels were 20–30% higher than those observed in control animals. The observed alterations in adenosine receptor number which occur as a consequence of caffeine consumption may underlie some of the behavioral effects of this cortical stimulant as well as provide insights concerning the mechanisms of tolerance to and dependence on caffeine.     

Regulation of adenylate cyclase by adenosine

Summary Adenosine may well be as important in the regulation of adenylate cyclase as hormones. Sattin and Rall first demonstrated in 1970 that adenosine was a potent stimulator of adenylate cyclase in the brain. However, adenosine is an equally potent inhibitor of adenylate cyclase in other cells such as adipocytes. The concentration of adenosine required for this regulation of adenylate cyclase is in the nanomolar range (10 to 100 nm). Both the inhibitory and stimulatory effects of low concentrations of adenosine on adenylate cyclase are antagonized by methylxanthines. This antagonism of adenosine action may account for all or part of the effects of methyl xanthines on cyclic AMP levels in many tissues. Adenosine appears to be a particularly important endogenous regulator of adenylate cyclase in brain, smooth muscle and fat cells. Under conditions in which intracellular AMP rises, adenosine formation and release is accelerated. In addition to its direct effects on adenylate cyclase, adenosine (at higher concentrations approaching millimolar) exerts multiple effects on cellular metabolism as a result of its intracellular metabolism and especially conversion to nucleotides.The effects of nanomolar concentrations of adenosine on adenylate cyclase are mediated through an adenosine site possessing strict structural specificity for the ribose moiety of the molecule (the R adenosine site) which is presumably located on the external surface of the plasma membrane. In brain, lung, platelets, bone, lymphocytes, skin, adrenals, Leydig tumors, and coronary arteries adenosine stimulates adenylate cyclase via this site. However, in rat adipocytes, brain astroblasts and ventricular myocardium adenosine inhibits adenylate cyclase through the R or adenosine site. Although the R site requires an intact ribose moiety, adenosine analogs modified in the purine ring such as N⁶-phenylisopropyladenosine appear to be potent agonists for this site. All effects of adenosine mediated via the R site are competitively antagonized by methyl xanthines.The effects of micromolar concentrations of adenosine appear to be mediated via a site with strict structural specificity with respect to the purine moiety of the molecule (the P or adenine adenosine site). This P site is postulated to be located on the intracellular face of the plasma membrane and mediates the effects of adenosine due to conversion of adenosine to 5-AMP or perhaps other nucleotides. The effects of high concentrations of adenosine are always inhibitory to adenylate cyclase activity, are readily demonstrated in broken cell preparations, and are unaffected by methylxanthines. An intact purine ring is required for these adenosine effects but modifications of the ribose moiety of the molecule generally increases the potency of the analog. A prime example is 2,5-dideoxyadenosine, which is the most potent known R-site specific adenosine analog.We propose a unitary model which explains both the stimulatory and inhibitory effects of low concentrations of adenosine on adenylate cyclase. In brief, adenylate cyclase is postulated to exist in three interconvertible activity states: (i) an inactive state (E₀); (ii) a GTP-liganded state with high activity (E_GTP); and (iii) a GDP-liganded state (E_GDP) which is inactive in cells where adenosine stimulates adenylate cyclase, but active in cells where adenosine inhibits adenylate cyclase. We postulate that the enzyme cycles through these states in the following manner: the E₀ state binds GTP and forms the E_GTP state; hydrolysis of bound GTP converts the E_GTP to the E_GDP state; and release of bound GDP converts E_GDP to the E₀ state. The E₀ state is the only form of the enzyme which can be stimulated by either hormones or GTP and its formation from the E_GDP state is rate-limiting in this cycle. The conversion of E_GDP to E₀ regulates the ability of hormones and GTP to activate adenylate cyclase and is postulated to be adenosine sensitive.In cells where both E_GDP and E₀ states are inactive, adenosine stimulates adenylate cyclase activity. In cells where E₀ is inactive, but E_GDP is active, adenosine inhibits adenylate cyclase activity. In addition we suggest that in cells where adenosine inhibits adenylate cyclase activity (cells postulated to have an E_GDP state which is active) high concentrations of GTP favor accumulation of the enzyme in E_GDP and thus are inhibitory to activity. Prostaglandins may also regulate adenylate cyclase in a manner similar to that described above for adenosine.We conclude that adenosine is an important regulator of adenylate cyclase whose role has only been appreciated recently. Further studies are warranted on both its binding to cells and mechanisms by which it regulates adenylate cyclase.This work was supported by United States Public Health Service Research Grant AM-10149 from the National Institute of Arthritis, Metabolism and Digestive Diseases.     

Regulation of Adenosine-Sensitive Adenylate Cyclase from Rat Brain Striatum

An adenosine-sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N⁶-phenylisopropyl adenosine (PIA), 2-chloroadenosine, and adenosine N′-oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA-stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg^2-. The degree of stimulation by PIA was greater at a low concentration of Mg²⁺, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg²⁺. Activation of adenylate cyclase by PIA was blocked by theophylline or 3-isobutyl- 1-methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)-stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A₂ treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA-stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.     

Vasopressin affects adenylate cyclase activity in rat brain: a possible neuromodulator

The effect of vasopressin on adenylate cyclase activity was measured in the homogenates of selected rat brain regions. Adenylate cyclase activity in homogenate of the caudate nucleus did not change significantly with various concentrations of vasopressin. Furthermore, vasopressin did not reliably alter adenylate cyclase activity in various brain regions. Vasopressin in low concentrations significantly enhanced the activation of caudate adenylate cyclase activity by dopamine. This effect of vasopressin was dose dependent. Maximal enhancement by vasopressin occurred at 100 microM vasopressin. These results indicate that vasopressin may not have a direct effect on brain adenylate cyclase activity but appears to modulate the action of dopamine on brain adenylate cyclase.     

Bovine Brain Coated Vesicles Contain Adenosine A1 Receptors. Presence of Adenylate Cyclase Coupled to the Receptor

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay.

In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.     

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK1L) grown in defined media

LLC-PK1L cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PK1L cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.     

Chronic caffeine consumption increases the number of brain adenosine receptors

Caffeine, a potent central stimulant, is known to competitively inhibit the specific binding of both adenosine and benzodiazepine receptor ligands to brain membranes in vitro. In mice receiving a diet containing non-toxic doses of caffeine (200 or 400 mg/kg diet) for periods up to 40 days, a dose-related increase in the number of binding sites for [3H]-CHA and [3H] DPX was observed in whole brain membranes without modifications of the receptors' affinity. Furthermore, a transitory increase in the number of [3H]-DZP binding sites was observed. These preliminary data seem to confirm the involvement of the adenosine receptors in the mode of action of caffeine and may be relevant to the development of both tolerance and dependence to some of the central effects of this compound.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Pertussis toxin treatment modifies opiate action in the rat brain striatum

In this report we present evidence that a guanine nucleotide regulatory protein, Gi, mediates opiate action in the rat brain striatum. Opiates inhibit basal adenylate cyclase activity in rat brain striatum. This effect on adenylate cyclase is dose-dependently attenuated by pretreatment of membranes with pertussis toxin, which ADP-ribosylates a protein with a molecular mass of 41,000 daltons. This protein co-migrates with the GTP-binding subunit of Gi, which mediates inhibition of adenylate cyclase. Several brain regions were compared for the extent of radiolabeling and effects on adenylate cyclase activity. Although Gi was found in each region examined, opiate inhibition of adenylate cyclase is clearly seen only in the striatum.     

Effect of forskolin and quinacrine on leukocyte functions under the effect of low-dose radiation

The adenylate cyclase and phospholipase A2 incorporation in the functional responses as well as lipid peroxidation processes and glutathione system homeostasis of animal leukocytes to small doses of ionizing radiation (1-100 mGy) have been estimated. The cells were irradiated by introduction of radioactive isotope 14C-leucine into the incubation medium. It is established that the ionizing radiation has different effects on the modification of cellular functions by the agents, which change adenylate cyclase and phospholipase A2 activity. Neutralization of stimulative irradiation effect on chemokinesis of polymorphonuclear leukocytes by quinacrine (the inhibitor of phospholipase A2) indicates for certain, that metabolism of eicosanoids takes immediate part in the cell response to ionizing radiation. Apparently, adenylate cyclase has no influence on this process, where at indicates the lack of influence of forskolin (the stimulator of adenylate cyclase) on the spontaneous motility, and on the radiation action on this leukocyte function. Rosette forming ability of lymphocytes is regulated by both enzymes because it is modified both by the inhibitor of phospholipase A2, and by the adenylate cyclase stimulant. In this case it is impossible to exclude the action of ionizing radiation both through the adenylate cyclase cascade, and through the eicosanoid metabolism. In all the concentration range the radionuclides do not affect the studied biochemical indexes of the cell, but change the action of the modifiers.