Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Histamine (H2) receptor-adenylate cyclase system in pig skin (epidermis).

Histamine activated adenylate cyclase in pig skin (epidermal) slices, resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of cyclic AMP-phosphodiesterase inhibitors (theophylline, papaverine). A specific H2 receptor inhibitor (metiamide) inhibited the effect of histamine completely, while other antihistamines (diphenhydramine, acetophenazine, perphenazine, fluphenazine, promethazine) inhibited the effect of histamine to various lesser degrees. It has been shown that both epinephrine and prostaglandin E stimulate epidermal adenylate cyclase. Our data using specific blocking agents indicate that histamine, epinephrine and prostaglandin E2 act independently on the epidermal adenylate cyclase system.     

Histamine (H2) receptor-adenylate cyclase system in pig skin (epidermis)

Histamine activated adenylate cyclase in pig skin (epidermal) slices, resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of cyclic AMP-phosphodiesterase inhibitors (theophylline, papaverine). A specific H₂ receptor inhibitor (metiamide) inhibited the effect of histamine completely, while other antihistamines (diphenhydramine, acetophenazine, perphenazine, fluphenazine, promethazine) inhibited the effect of histamine to various lesser degrees. It has been shown that both epinephrine and prostaglandin E stimulate epidermal adenylate cyclase. Our data using specific blocking agents indicate that histamine, epinephrine and prostaglandin E₂ act independently on the epidermal adenylate cyclase system.     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Mechanism of action of ATP on intestinal epithelial cells. Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH2)p. ATP also increased the formation of cyclic [14C]AMP in enterocytes that had been preincubated with [14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and augmenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the Isc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Effects of exogenous adenosine deaminase and erythro-9-(2-hydroxy-3-nonyl) adenine on intracellular cyclic AMP levels in C1300 murine neuroblastoma in tissue culture

The cyclic AMP content of dense cultures of C1300 murine neuroblastoma cells (clone N2a) was elevated after incubation for short periods of time in minimal volumes of serum-free medium (SFM) containing Ro 20 1724, a potent nonxanthine phosphodiesterase inhibitor. This elevation was prevented by theophylline, an adenosine antagonist, and was retarded by dipyridamole or benzylthioinosine, inhibitors of nucleoside transport. Cyclic AMP was also elevated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a potent adenosine deaminase inhibitor. This effect of EHNA was more pronounced in dense cultures, in small volumes of bathing medium, and was antagonized by dipyridamole. The addition of adenosine deaminase to growth medium or SFM lowered the cyclic AMP levels attained after the addition of Ro 20 1724. We conclude that N2a cells continually release adenosine into the growth or bathing medium $\text{via}$ the nucleoside transport system and that sufficient concentrations may be achieved to tonically stimulate adenylate cyclase and influence processes controlled by the cyclic AMP:cyclic AMP-dependent protein kinase system.     

Two mechanisms for inhibition of ADP-induced platelet shape change by 5'-p-fluorosulfonylbenzoyladenosine. Conversion to adenosine, and covalent modification at an ADP binding site distinct from that which inhibits adenylate cyclase

The interaction of ADP with platelets leads to shape change, exposure of fibrinogen binding sites, and aggregation, all of which have been shown to be inhibited by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an alkylating analogue of adenine nucleotides which binds covalently to a 100-kDa polypeptide in intact platelet membranes (Figures, W. R., Niewiarowski, S., Morinelli, T., Colman, R. F., and Colman, R. W. (1981) J. Biol. Chem. 256, 7789-7795). In plasma, FSBA can break down to adenosine which stimulates adenylate cyclase. To distinguish between direct effects of FSBA and the actions of adenosine, we have used washed platelet suspensions and adenosine deaminase. We studied the effects of FSBA on shape change and cyclic AMP metabolism, and on the binding of 2-methylthio-ADP, which mimics the effects of ADP on cyclic AMP metabolism at concentrations too low to activate platelets. Inhibition of ADP-induced shape change of platelets incubated with FSBA for 2 min in platelet-rich plasma was greatly reduced by adenosine deaminase. In the presence of a phosphodiesterase inhibitor, 100 microM FSBA increased platelet cyclic AMP to the same extent as did 10 microM adenosine. These effects were inhibited by theophylline, an adenosine receptor antagonist, and by adenosine deaminase. Incubation of washed platelets for 60 min with FSBA and adenosine deaminase caused a concentration-dependent inhibition of ADP-induced shape change. Inhibition closely paralleled the covalent incorporation of 3H from tritiated FSBA into platelet membranes. Under these conditions, FSBA did not block inhibition of cyclic AMP accumulation by ADP, nor did it block the binding of 2-methylthio-ADP. We conclude that part of the inhibition of shape change caused by brief exposure to FSBA is due to adenosine, but at longer times shape change is inhibited in association with covalent incorporation of sulfonylbenzoyladenosine. This effect of FSBA is independent of adenosine and occurs at a site distinct from that at which ADP inhibits adenylate cyclase.     

Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP.

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.     

Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes.

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

Excitation-secretion coupling in exocrine glands. Properties of cyclic AMP phosphodiesterase and adenylate cyclase from the submaxillary gland and pancreas.

1. The cyclic AMP phosphodiesterase in homogenates of the submaxillary gland and pancreas was found to be associated mainly with the 300,000 times g supernatant fraction. A Lineweaver-Burk plot showed a high-affinity (Km app. = 1.6 muM) and a low-affinity (Km app. greater than 100muM) component for the cyclic AMP substrate. The enzyme was magnesium dependent, and strongly inhibited by papaverine, theophylline and caffeine. Cyclic GMP inhibited cyclic AMP phosphodiesterase, but only in concentrations greatly exceeding that of the cyclic AMP. Calcium did not alter the activity of the enzyme. The activity of the submaxillary cyclic AMP phosphodiesterase was not influenced by noradrenaline, dopamine, histamine, 5-hydroxytryptamine or gamma-amino butyric acid, and that of the pancreatic enzyme by acetylcholine, pancreozymin or secretin. 2. Adenylate cyclases from guinea-pig submaxillary gland and cat pancreas are particulate enzymes. The highest specific activity was recovered from the 1500 times g pellet. Guineo-pig submaxillary adenylate cyclase was activated by fluoride, noradrenaline, isoprenaline and adrenaline. The noradrenaline activation was blocked by the beta-adrenoceptor blocker, propranolol, but not by the alphs-adrenoceptor blocker, phentolamine. Neither acetylcholine nor carbachol had any effect on the adenylate cyclase activity. The apparent Km value for the 10- minus 4 M noradrenaline activated adenylate cyclase activity was completely aboliched by 5 mM calcium. Cat pancreatic adenylate cyclase was clearly and consistently activated by secretin, but not by pancreozymin or carbachol.     

Neuroblastoma cell adenylate cyclase: direct activation by adenosine and prostaglandins.

—Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the conversion of [α³²P]ATP into labelled cyclic AMP. Adenosine (10^?6 - 10^?4m ) induced a dose-dependent increase in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However, none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent K_m of the adenylate cyclase for adenosine was 5 ± 10^?6-10^?5m . Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/10 min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE₁ had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at 10^?6m ). Dopamine and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It seems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in response to adenosine.     

Desensitization of the epidermal adenylate cyclase system: agonists and phorbol esters desensitize by independent mechanisms.

Exposure of pig epidermis to adenylate cyclase stimulators results in receptor-specific desensitization. We investigated the nature of the agonist-induced desensitization, which was compared with the phorbol ester-induced, receptor-nonspecific desensitization. Both phorbol ester-induced desensitization and the agonist-induced desensitization were accompanied by an increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. The magnitude of the increase in the agonist-induced desensitization was parallel to the degree of the initial cyclic AMP accumulation; histamine and adenosine, which increase more cyclic AMP than epinephrine, resulted in a more marked increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. Similarly, epidermis desensitized to multiple receptors revealed more marked forskolin- and cholera toxin-induced cyclic AMP accumulations than epidermis desensitized to a single receptor. In contrast to the phorbol ester-induced desensitization, agonist-induced desensitization was not affected by the protein kinase C inhibitors H-7 and staurosporin. Further, agonist-induced desensitization was still inducible in phorbol ester-desensitized epidermis and vice versa. In contrast to the agonist-induced desensitization, which is accompanied by the preceding adenylate cyclase stimulation, no evidence for the stimulation of the adenylate cyclase during phorbol ester treatment was obtained. Neither agonist-induced desensitization nor phorbol ester-induced desensitization affected the content of inhibitory guanine nucleotide binding protein of the epidermis, which was monitored by the pertussis toxin (IAP)-catalyzed ADP ribosylation reaction. Our results indicate that agonist-induced desensitization and the phorbol ester-induced desensitization are independent of each other. Although both processes are characterized by increased forskolin- and toxin-induced cyclic AMP accumulations, the former is accompanied by initial cyclic AMP accumulation; the latter is not.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation.

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2--3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low Km (0.17 muM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.     

A novel site of action of a high affinity A1 adenosine receptor antagonist

XAC, a high affinity antagonist of the A1 adenosine receptor, enhances adenylate cyclase activity by 1.3-2 fold with an EC50 of approximately 47 nM in adipocyte membranes pretreated with adenosine deaminase to eliminate adenosine and in the presence of total phosphodiesterase inhibition by 100 microM papaverine. This effect of XAC is observed only at concentrations of GTP sufficient to activate Gi (approximately 5 x 10(-6) M GTP) and is not evident in the absence or presence of lower GTP concentrations. ADP ribosylation of Gi by pertussis toxin treatment also abolishes this stimulatory action of XAC. Furthermore, in the presence of GTP activation of inhibitory prostaglandin E1 receptors diminishes the stimulatory effect of XAC on adenylate cyclase. In addition, XAC interferes with GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity in a noncompetitive manner. Finally, XAC is only a weak inhibitor of the low Km cyclic AMP phosphodiesterase, producing approximately 40% inhibition of phosphodiesterase activity at a concentration of 100 microM. These data suggest that XAC increases adenylate cyclase activity in absence of endogenous adenosine by inhibiting tonic Gi activity in a reversible manner.     

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.     

Formation of adenosine 3′,5′-monophosphate from adenosine in mouse thymocytes

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E₁, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E₁ and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E₁, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [¹⁴C] adenine exhibited dramatic increases in cyclic [¹⁴C]AMP 10 min after addition of adenosine or prostaglandin E₁ which corresponded to simultaneously determined increases in total cyclic AMP. Using [¹⁴C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2–3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low K_m (0.17 μM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study. Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.