The CRF-related peptide sauvagine stimulates and the GABAB receptor agonist baclofen inhibits cyclic-AMP production in melanotrope cells of Xenopus laevis.

Release of alpha-MSH from the pars intermedia melanotrope cells of Xenopus laevis is regulated by various classical neurotransmitters and neuropeptides. We have examined the effect of two of these regulatory substances, the neurotransmitter GABA and the CRF-related peptide sauvagine, on the adenylate cyclase system of the melanotrope cells. Sauvagine treatment, which stimulates alpha-MSH release, lead to an elevation in the level of cyclic-AMP, an effect which was potentiated by cholera toxin. Treatment with baclofen, a GABAB receptor agonist, gave a pertussis toxin-sensitive decrease in the cyclic-AMP level and an inhibition of alpha-MSH release. We conclude that sauvagine stimulates alpha-MSH secretion through activation of adenylate cyclase and that GABAB receptor activation inhibits secretion through inhibition of cyclic-AMP production. Baclofen treatment sensitized melanotrope cells to the stimulatory action of 8-bromo-cyclic-AMP on the secretion of alpha-MSH. This observation supports the conclusion that GABAB receptor activation inhibits cyclic-AMP production.     

Angiotensin II and dopamine modulate both cAMP and inositol phosphate productions in anterior pituitary cells. Involvement in prolactin secretion

Despite their opposite effects on prolactin secretion, both dopamine and angiotensin II inhibit adenylate cyclase activity in homogenates of anterior pituitary cells in primary culture. Dopamine and angiotensin II inhibition of adenylate cyclase was not additive, suggesting that both neurohormones inhibit the adenylate cyclase of the lactotroph cells. Pretreatment with Bordetella pertussis toxin (islet activator protein) completely suppressed the dopamine-induced inhibition of both adenylate cyclase and prolactin secretion. The islet activator protein also reversed the angiotensin II-induced inhibition of the adenylate cyclase activity. In contrast, angiotensin II stimulation of prolactin release was not affected by the toxin. Angiotensin II also induced a dose-dependent stimulation of inositol phosphates (250%) with an EC50 of 0.1 nM, close to that observed for prolactin secretion. Islet activator protein pretreatment did not block the stimulation of inositol phosphate production. Dopamine inhibited the angiotensin II-stimulated prolactin release and the production of inositol phosphates induced by angiotensin II. It is concluded that angiotensin II and dopamine receptors of lactotroph cells are able to modulate both cAMP and inositol phosphate production. The dopamine receptor of lactotrophs appears to be the first example of a receptor which is negatively coupled to the production of inositol phosphates.     

Dynamics of cyclic-AMP efflux in relation to alpha-MSH secretion from melanotrope cells of Xenopus laevis.

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of alpha-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike alpha-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on alpha-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of alpha-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABAB receptor agonist baclofen both inhibited cyclic-AMP efflux and alpha-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of alpha-MSH secretion.     

Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor

Mast cells are critical for allergic reactions, but also for innate or acquired immunity and inflammatory conditions that worsen by stress. Corticotropin-releasing hormone (CRH), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. We investigated the expression of CRH receptors and the effects of CRH in the human leukemic mast cell (HMC-1) line and human umbilical cord blood-derived mast cells. We detected mRNA for CRH-R1alpha, 1beta, 1c, 1e, 1f isoforms, as well as CRH-R1 protein in both cell types. CRH-R2alpha (but not R2beta or R2gamma) mRNA and protein were present only in human cord blood-derived mast cells. CRH increased cAMP and induced secretion of vascular endothelial growth factor (VEGF) without tryptase, histamine, IL-6, IL-8, or TNF-alpha release. The effects were blocked by the CRH-R1 antagonist antalarmin, but not the CRH-R2 antagonist astressin 2B. CRH-stimulated VEGF production was mediated through activation of adenylate cyclase and increased cAMP, as evidenced by the fact that the effect of CRH was mimicked by the direct adenylate cyclase activator forskolin and the cell-permeable cAMP analog 8-bromo-cAMP, whereas it was abolished by the adenylate cyclase inhibitor SQ22536. This is the first evidence that mast cells express functional CRH receptors and that CRH can induce VEGF secretion selectively. CRH-induced mast cell-derived VEGF could, therefore, be involved in chronic inflammatory conditions associated with increased VEGF, such as arthritis or psoriasis, both of which worsen by stress.     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.     

Adrenergic regulation of adipocyte metabolism

Adipocytes can be readily isolated from intact adipose tissue. In adipocytes from hamster and human white adipose tissue it is possible to demonstrate beta, alpha 1, and alpha 2 adrenoceptors. Alpha 2 adrenoceptor activation inhibits while beta adrenoceptor activation stimulates cyclic AMP accumulation and lipolysis. The effects of catecholamines on cyclic AMP accumulation are mediated through regulation of adenylate cyclase activity, which is activated through beta adrenoceptors and inhibited through alpha 2 adrenoceptors. Activation of alpha 1 adrenergic receptors has been shown to be associated with elevations of cytosol calcium and increased turnover of phosphatidylinositol. In white adipocytes, the only known alpha 1 adrenergic effects are inhibition of glycogen synthase and stimulation of glycogen phosphorylase via mechanisms distinct from those by which cyclic AMP produces similar end effects. In brown adipocytes, alpha 1 adrenoceptor activation stimulates respiration. Thyroid hormones primarily regulate the sensitivity of adipocytes to beta-adrenergic amines while having little effect on alpha adrenoceptor sensitivity.     

Reducing reagent-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum.

Binding of an intrinsic agonist (cAMP) to specific receptors on the cell surface induces transmembrane signals for activation and desensitization (adaptation and down regulation) of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. It is generally believed that dithiothreitol (DTT) induces the activation through interaction between the receptor and gradually accumulated cAMP, since DTT is known to inhibit cAMP-phosphodiesterase which degrades cAMP. In the present paper, we investigated the mechanism of activation of adenylate cyclase by the thiol-reducing agents, DTT and 2,3-dimercapto-1-propanol (BAL). We found that BAL activated adenylate cyclase transiently even under conditions where the intrinsic agonist supersaturated the cAMP-receptors and competitively inhibited phosphodiesterase. This result is inconsistent with the generally accepted notion. We conclude that BAL has an independent effect from those of the intrinsic agonist (cAMP) and phosphodiesterase in activation of adenylate cyclase. Since BAL could induce activation just after the activation induced by a supersaturating concentration of the intrinsic agonist had ceased, the independent effect of BAL is not a simple enhancement of the cAMP-induced activation. Our result also suggests that the cAMP-induced adaptation (but not down regulation) suppresses the BAL-induced activation while BAL itself does not induce adaptation to cAMP or BAL. We propose that the thiol-reducing reagent induces or modifies the transmembrane activation signal for adenylate cyclase.     

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.     

P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol trisphosphate in rat hepatocytes

Stimulation of P2-purinergic receptors by ATP resulted in activation of phosphorylase, which was associated with marked production of inositol trisphosphate (Ins-P3), in rat hepatocytes. ATP also inhibited forskolin-induced accumulation of cAMP in the presence of a phosphodiesterase inhibitor. On the contrary, adenosine or AMP never inhibited the cAMP accumulation, but increased hepatocyte cAMP; the stimulation was antagonized by a methylxanthine. Thus, P1-purinergic receptors are linked to adenylate cyclase in a stimulatory fashion in hepatocytes. Various kinds of purine nucleotides stimulating P2-receptors can be divided into two groups on the basis of their relative abilities to stimulate Ins-P3 production and to inhibit cAMP accumulation; the first group including adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, 5-adenylyl imidodiphosphate, GTP, and guanosine 5'-O-(3-thiotriphosphate) has an efficacy similar to that of ATP, and the second group of nucleotides including alpha, beta-methyleneadenosine 5'-triphosphate, beta, gamma-methyleneadenosine 5'-triphosphate (App(CH)2)p), and GDP exerts considerable inhibitory effects on cAMP accumulation, but only slight effects on inositol lipid metabolism. Treatment of hepatocytes with islet-activating protein, pertussis toxin, blocked the nucleotide-induced inhibition of cAMP accumulation, but exerted only a small effect on Ins-P3 production. In membranes prepared from hepatocytes, forskolin-stimulated adenylate cyclase was inhibited by GTP. This GTP-induced inhibition of the enzyme was susceptible to islet-activating protein and dependent on the concentration of ATP (or its derivatives, ATP gamma S or App(CH2)p). It is concluded that there are two types of P2-purinergic receptors: one is linked to adenylate cyclase via an inhibitory guanine nucleotide regulatory protein (Gi) and the other is linked to phospholipase C.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

Homologous Regulation of the MSH Receptor in Melanoma Cells

Abstract

We have examined the mechanism of homologous regulation of MSH receptor binding and receptor-mediated adenylate cyclase activation in three human and two mouse melanoma cell lines. Pretreatment with α-MSH resulted in a time- and dose-dependent up-regulation of MSH receptors in human D10 and 205 melanoma cells whereas in human HBL and in mouse B16–F1 and Cloudman S91 cells α-MSH induced receptor down-regulation. Up-regulation of receptors was maximal after a 24–h incubation period and an α-MSH concentration of 100 nM (EC₅₀ = 2.4 nM). The increase in α-MSH binding was independent of adenylate cyclase activation and protein synthesis and appeared to be caused by recruitment of spare receptors. The structural requirements of the peptide for triggering this process differed from those found in receptor-binding analyses. Receptor down-regulation was maximal after 12 h and hence more rapid than up-regulation. In B16–F1 cells, 10 nM α-MSH caused the disappearance of 85–90% of the MSH receptors, the EC₅₀ of 0.23 nM lying exactly between that for α-MSH-induced melanogenesis (0.027 nM) and the dissociation constant of receptor binding (1.31 nM). Down-regulation in B16–F1 cells appears to be the consequence of receptor internalization following MSH binding and seems to be initiated during an early step in MSH signalling, preceding the activation of adenylate cyclase and the cAMP signal. Receptor up- and down- regulation were not accompanied by an alteration in affinity to a-MSH, as demonstrated by Scatchard analysis of the binding curves.     

Control of human T-lymphocyte interleukin-2 production by a cAMP-dependent pathway

Cyclic AMP has long been proposed to be the intracellular second messenger that conveys the inhibitory signal for T-cell activation and clonal T-cell proliferation. The present study further explores the mechanism by which the cAMP pathway regulates human T-lymphocyte interleukin-2 (IL-2) production and T-cell blastogenesis. Activation of adenylate cyclase, inhibition of cAMP-dependent phosphodiesterase, or the direct addition of the cell-permeable cAMP analog, 8-N3-cAMP, increased occupancy of intracellular cAMP receptors, inhibited IL-2 production, and reduced T-cell proliferation. However, inhibition of cAMP-dependent protein phosphorylation by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, partially restored IL-2 production. Our data support the conclusion that the cAMP pathway conveys an inhibitory signal for IL-2 production and T-cell proliferation via an integral protein phosphorylation step.     

Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.     

Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase

The affinity of the chemoattractant receptor for N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) on human polymorphonuclear leukocytes (PMNs) is regulated by guanine nucleotides, and chemoattractants stimulate increased intracellular cAMP levels in PMNs. Our data, however, indicate that this receptor does not activate membrane-bound adenylate cyclase via direct nucleotide regulatory protein (N) coupling but instead raises cAMP levels indirectly via a mechanism which appears to require Ca2+ mobilization. This conclusion is based on the following data: 1) prostaglandin E1 (PGE1) activated and alpha 2-adrenergic treatment inhibited adenylate cyclase activation in PMN plasma membranes; fMet-Leu-Phe, however, neither activated nor inhibited adenylate cyclase in these membranes; 2) depletion of extracellular Ca2+ had no effect on isoproterenol and PGE1 elicited cAMP responses in intact PMNs while peak fMet-Leu-Phe and A23187-induced responses were reduced by approximately 50 and 80%, respectively; 3) 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate, a purported Ca2+ antagonist, caused almost complete inhibition of fMet-Leu-Phe and ionophore-induced cAMP responses in intact cells but had no effect on PGE1 and isoproterenol; 4) alpha 2-adrenergic agonists inhibited PGE1 but not chemoattractant- or A23187-elicited cAMP responses in intact PMNs; and 5) pretreatment of cells with a phosphodiesterase inhibitor (isobutylmethylxanthine) greatly potentiated the PGE1 and isoproterenol cAMP responses but nearly abolished the peak fMet-Leu-Phe response. Thus, chemoattractants appear to utilize a novel mechanism to raise cAMP levels which appear to require Ca2+ mobilization and could be mediated in part through a transient inhibition of phosphodiesterases. We suggest that stimulation of PMN functions by chemoattractants may utilize an N-coupled process to generate a Ca2+ signal which could in turn raise intracellular cAMP levels indirectly and thereby provide negative regulation.     

Interferon-gamma down-regulates adenosine 2b receptor-mediated signaling and short circuit current in the intestinal epithelia by inhibiting the expression of adenylate cyclase

Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.     

Membrane transduction pathway in the neuronal control of protein secretion by the albumen gland in Helisoma (Mollusca)

The albumen gland in Helisoma secretes a perivitelline fluid which surrounds each egg and is made up of several 66 kDa protein subunits and polysaccharide complexes. Forskolin, an adenylate cyclase activator, stimulated the secretion and release of the perivitelline fluid. An acidic extract of the central nervous system increased the intracellular concentration of cAMP in the albumen gland and this results in the release of the 66 kDa molecule and other proteins. Digestion of the brain extract with proteases abolished this activity, suggesting that the factor is a peptide. Cyclic AMP analogues and [BMX also stimulated the protein secretion in dose-dependent manner. Forskolin when added with the brain factor had an additive response. SQ22536, a non-competitive inhibitor of adenylate cyclase, inhibited brain extract dependent adenylate cyclase activity whereas aluminum fluoride, a G protein activator, was found to stimulate adenylate cyclase. Dopamine also stimulates protein secretion by the albumen gland and through the application of various agonists and antagonists of dopamine, it was established that the neurotransmitter acts via D1-like receptors by stimulating adenylate cyclase.     

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

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

Role of a guanine nucleotide binding protein, G alpha 2, in regulation of adenylate cyclase in Dictyostelium discoideum

Two substances, cAMP and 2,3-dimercapto-1-propanol (BAL) are known to induce transient activation of adenylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a deletion in a gene for G alpha 2, a guanine nucleotide binding protein and cannot activate the cyclase in response to cAMP. We found that BAL induced activation in the frigid mutant. This result suggests that the BAL-induced activation is independent of G alpha 2 and that BAL mimics a role of activated G alpha 2. We also found that cAMP promoted the BAL-induced activation. This result suggests that cAMP plays a role in activation through a mechanism in which G alpha 2 is not involved. We lastly showed that continuous cAMP stimulation could not inhibit the BAL-induced activation in the frigid mutant. Since the cAMP-induced inhibition observed in the wild type strain (NC4) proceeds with the time course identical to the cAMP-induced adaptation (Oyama, submitted), this result suggests that G alpha 2 is involved in adaptation of adenylate cyclase.     

3H]GDP release from rat and hamster adipocyte membranes independently linked to receptors involved in activation or inhibition of adenylate cyclase. Differential susceptibility to two bacterial toxins

When rat adipocyte membranes had been labeled with [3H]GTP in the presence of a beta-adrenergic agonist, the subsequent [3H]GDP release was stimulated by beta-agonists or agonists (e.g. glucagon and secretin) of other "activatory" receptors involved in activation of adenylate cyclase, but was not stimulated by agonists (e.g. prostaglandin E1 and adenosine) of "inhibitory" receptors involved in cyclase inhibition. On the contrary, agonists of inhibitory receptors were effective in stimulating GDP release from hamster adipocyte membranes that had been labeled via inhibitory alpha 2-adrenergic receptors, but an activatory receptor agonist such as isoproterenol was not. Thus, the guanine nucleotide regulatory protein (Ni) involved in adenylate cyclase inhibition is an entity distinct from the regulatory protein (Ns) involved in cyclase activation, and multiple activatory or inhibitory receptors are coupled to a respective common pool of Ns or Ni. Preactivated cholera toxin added together with NAD enhanced GDP release from rat adipocyte membranes prelabeled with isoproterenol but was without effect on the release from hamster adipocyte membranes that had been labeled with an alpha-agonist. In sharp contrast, the active subunit of islet-activating protein, pertussis toxin, failed to alter GDP release from the former membrane but completely abolished inhibitory agonist-induced stimulation of GDP release from the latter membrane preparation in the presence of NAD. Thus, the site of action of cholera toxin is Ns, while that of islet-activating protein is Ni. The function of Ni to communicate between inhibitory receptors and adenylate cyclase was lost when it was ADP-ribosylated by islet-activating protein.