Regulation of pro-opiomelanocortin synthesis by dopamine and cAMP in the amphibian pituitary intermediate lobe

The modulation of pro-opiomelanocortin (POMC) synthesis in Xenopus laevis pituitary intermediate lobe (IL) during background adaptation and the role of dopamine and cAMP in mediating this effect were examined. Neurointermediate lobes (NILs) were pulselabeled in vitro with [3H]arginine and analyzed for POMC synthesis by acid-urea gel electrophoresis. After black background adaptation of the animal (7 days), POMC synthesis increased 5-6-fold, while after white background adaptation (7 days), POMC synthesis decreased by 76%. Dopamine (50 microM) suppressed POMC synthesis in NILs in culture. In the absence of dopamine, POMC synthesis was stimulated. Several experiments were conducted to determine the category of dopamine receptor in the X. laevis IL. A D-2 dopamine receptor agonist inhibited immunoreactive alpha-MSH release from the NIL in a D-2 antagonist-reversible manner. A D-1 receptor agonist or antagonist did not alter the release of immunoreactive alpha-MSH from the NIL. Dopamine (10 microM) inhibited forskolin-stimulated cAMP accumulation. In addition, dopamine inhibition of POMC synthesis in cultured ILs was reversed by 8-Br-cAMP. These studies suggest that white background adaptation results in stimulation of the X. laevis D-2 receptor, which reduces cAMP production and POMC synthesis. Conversely, during black background adaptation the IL D-2 receptor is not stimulated, leading to increased cAMP production and POMC synthesis.     

Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase

We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99–126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition ( 45%) was observed in the presence of 10-30 M GTPS, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na⁺ enhanced the degree of inhibition by about 60%, whereas K⁺ and Li⁺ suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn²⁺ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 mM, and at 2 mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A₂ treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca²⁺-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.Abbreviations ANF Atrial Natriuretic Factor - GTPS Guanosine 5-0-(Thiotriphosphate) - Gi inhibitory guanine nucleotide regulatory protein - NEM N-Ethylmaleimide - PMA Phorbol, 12-Myristate, 13-Acetate, C-kinase, Ca ²⁺, phospholipid-dependent protein kinase - PHL-A₂ Phospholipase A,     

The dopamine receptor in the intermediate lobe of the rat pituitary gland is negatively coupled to adenylate cyclase

The potency of a series of drugs to inhibit cyclic AMP accumulation in cells of the intermediate lobe of the rat pituitary gland in culture is typically dopaminergic. Dopaminergic antagonists reverse the inhibition of cyclic AMP levels according to their known pharmacological activity. The present data show that activation of the dopamine receptor in pars intermedia cells leads to inhibition of basal cyclic AMP accumulation and thus suggest that this receptor is negatively coupled to adenylate cyclase.     

Glycogen synthesis stimulation by adenylate cyclase inhibition in rat epididymal adipocytes

The effects of adenylate cyclase inhibition on the transport of glucose and fructose and their incorporation into glycogen were investigated in order to assess the extent to which lowered cAMP levels can take part in the various components of glycogen synthesis regulation in isolated rat epididymal adipocytes. The dose-response characteristics of (R)-N-(2-phenylisopropyl)adenosine (PIA), a potent and specific adenylate cyclase inhibitor, on glycogen synthesis were compared with those effectively inhibiting lipolysis, a measure of functional cAMP levels. PIA had no effect on basal glucose or fructose transport but stimulated glucose and fructose incorporation into glycogen. Their respective incorporation was 10 and 69% of that achieved in the presence of insulin. These effects of PIA were shown to be in part the result of increased glycogen synthase I activity. PIA was 20% as effective as insulin in this action. Thus, were insulin to lower cAMP levels and/or inhibit cAMP-dependent protein kinase, this action would be irrelevant to glucose transport but would contribute to the stimulation of glycogen metabolism. However, an additional mechanism(s) involving neither increased glucose transport nor lowered cAMP levels is required to account for the full action of insulin. Fat cells in the absence of medium glucose and in the presence of 10(-7) M PIA and adenosine deaminase constitute a system functionally depleted of cAMP where this mechanism can be studied in isolation.     

ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding

Adenylate cyclase in NG108-15 (neuroblastoma X glioma hybrid) cells is responsive to both stimulatory and inhibitory ligands. Bordetella pertussis toxin (PT) catalyzes the ADP-ribosylation of a 41,000-Da peptide believed to be a subunit of the putative guanyl nucleotide-binding protein (Gi) involved in cyclase inhibition and abolishes inhibitory effects of opiate agonists. In studying the effects of PT on opiate receptors, we found that [3H]enkephalinamide binding was reduced by approximately 90% in membranes prepared from cells incubated with PT compared to control membranes. Agonist affinity, assessed by enkephalinamide competition for [3H]diprenorphine-binding sites, was markedly reduced in cells incubated with PT. Furthermore, inhibition by guanylylimidodiphosphate of ligand binding to opiate receptors was reduced following treatment with PT. The number of opiate receptors assessed by [3H]diprenorphine binding was unaltered by PT. These data are consistent with the hypothesis that PT-catalyzed ADP-ribosylation impairs the interaction of Gi with the inhibitory receptor-ligand complex, effectively uncoupling the inhibitory receptor from Gi and the cyclase catalytic unit.     

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

Agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland

The mechanism of agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe (IL) of the rat pituitary gland was investigated. Exposure of neurointermediate lobe to 60 microM (-)apomorphine (APO) for 60 min altered the binding of [125I]-N-(p-aminophenethyl)spiperone (NAPS), a D-2 receptor-specific ligand. The capacity of the tissue to bind the ligand (Bmax) was not significantly altered by the exposure to (-)APO but the affinity for [125I]NAPS was decreased 3.6-fold in (-)APO-exposed tissue. The molar potency of YM-09151-2, a D-2 receptor-specific antagonist, showed a minimal difference between in control and (-)-APO-exposed tissue. However, the molar potency of (-)APO towards the D-2 receptor was diminished. The loss of [125I]NAPS binding in (-)APO-exposed tissue was reversed by the addition of guanyl nucleotide. These data suggest that exposure to agonist causes a persistent occupancy of the high affinity state of the receptor. Exposure to (-)APO had no effect on either basal or forskolin-activated adenylate cyclase activity of the intermediate lobe. However, the inhibitory effect of (-)APO upon adenylate cyclase activity of IL homogenates was diminished when the tissue was exposed to (-)APO before homogenization. Furthermore, the ability of GTP but not 5'-guanylyl imidodiphosphate [Gpp(NH)p] to inhibit enzyme activity diminished in the (-)APO-exposed tissue. These data suggest that an agonist-induced desensitization of D-2 receptor in rat IL is thought to occur by uncoupling the receptor from the inhibitory guanyl nucleotide binding protein (Gi) or potentiating the hydrolysis of GTP by Gi.     

The morphological changes in cultured cells caused by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis is the causative agent for human whooping cough. It was found that Bordetella pertussis infection caused a change in shape from flat to round in L2 cells, which are derived from rat type 2 alveolar cells. This phenomenon was reproduced using the culture supernatant of B. pertussis, and bacterium-free adenylate cyclase toxin (CyaA) was identified as the factor responsible. A purified preparation of wild-type CyaA but not an enzyme-dead mutant caused the cell rounding. It was examined whether CyaA causes similar morphological changes in various cultured cell lines. L2, EBL, HEK293T, MC3T3-E1, NIH 3T3, and Vero cells were rounded by the toxin whereas Caco-2, Eph4, and MDCK cells were not, although all these cells showed a significant elevation of the intracellular cAMP level in response to CyaA treatment, which indicates that there is no quantitative correlation between the rounding phenotype and the intracellular cAMP level. CyaA has been believed to target various immunocompetent cells and support the establishment of the bacterial infection by subverting the host immune responses. The possibility that CyaA may also affect tissue cells such as respiratory epithelial cells and may be involved in the pathogenesis of the bacterial infection is also indicated.     

High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system

The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.     

Attenuation of chick heart adenylate cyclase by muscarinic receptors after pertussis toxin treatment

Pertussis toxin selectively modifies the function of Ni, the inhibitory guanine nucleotide binding protein of the adenylate cyclase complex. In chick heart membranes, guanine nucleotide activation of Ni resulted in a decrease in the apparent affinity of the muscarinic receptor for the agonist oxotremorine, inhibition of basal adenylate cyclase activity, and the attenuation of adenylate cyclase by oxotremorine. Treatment of chicks with pertussis toxin caused the covalent modification of 80-85% of cardiac Ni. After this treatment Gpp(NH)p had no effect on muscarinic receptor affinity and GTP stimulated basal adenylate cyclase activity. In contrast, the GTP-dependent attenuation of adenylate cyclase caused by muscarinic receptors was unaffected.     

Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

Ethyl 2{5(4-chlorophenyl)pentyl}oxiran-2-carboxylate (POCA) is a new hypoglycaemic compound. The POCA-CoA ester strongly inhibits β-oxidation at carnitine palmitoyltransferase I. Chronic administration of POCA to rats decreases plasma concentrations of cholesterol and triacylglycerol and increases the number of hepatic peroxisomes similarly to hypolipidaemic drugs related to clofibrate. Peroxisomal fractions from rats fed a diet containing 0.2% of POCA for 4 weeks were prepared on self-generated Percoll gradients. POCA induced a 4-fold increase in catalase activity and peroxisomal β-oxidation, agreeing with the morphological data. The increase in peroxisomal β-oxidation caused by POCA feeding does not prevent accumulation of lipid following the inhibition of mitochondrial β-oxidation.     

Activation by cholera toxin of adenylate cyclase solubilized from rat liver.

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.     

Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.     

Inhibition by S-adenosyl-L-homocysteine of dopamine dependent adenylate cyclase in rat retina

S-adenosyl-L-homocysteine (S-AH), a potent inhibitor of biological transmethylation, decreased the response of rat retina adenylate cyclase to dopamine and to 2-amino-6, 7-dihydroxytetrahydronaphtalene (ADTN). This effect appeared for 10^?7M of S-adenosyl-L-homocysteine and was linear for concentration ranging to 10^?4M. S-adenosyl-L-homocysteine did not decrease the cyclic AMP accumulation with sodium fluoride, a non specific adenylate cyclase activator. On the other hand, the incorporation of methyl group was reduced in rat retina homogenates by S-adenosyl-L-homocysteine. These findings suggest that the activity of the dopamine dependent adenylate cyclase is linked to a methylation process.     

Iron inhibits D-1 dopamine receptor coupled adenylate cyclase via G-proteins in the caudate nucleus of the rat.

The effects of iron ions (Fe(II)sulfate) on basal, forskolin, and dopamine-stimulated activity of adenylate cyclase in membrane preparations from caudate-putamen of the rat have been studied. Iron dose-dependently inhibited both basal and activated adenylate cyclase activity. In contrast to guanylylimidodiphosphate (Gpp(NH)p), guanosine triphosphate (GTP) was found to enhance this inhibitory effect of iron ions. In addition, cholera toxin was able to antagonize the inhibitory effect of iron on forskolin-activated adenylate cyclase. In our preliminary study we suggest an interaction between iron and the guanine nucleotide regulatory subunit. However, further studies are necessary.     

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

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

Olfactory adenylate cyclase of the rat. Stimulation by odorants and inhibition by Ca2+.

The Ca2+-dependent regulation of the activation of myosin MgATPase by vascular-smooth-muscle thin filaments involves caldesmon. This effect may be due to the direct interaction of caldesmon with a Ca2+-binding protein such as calmodulin or phosphorylation of caldesmon by a Ca2+-dependent kinase. I have found that Ca2+ switches on aorta thin filaments in less than 10 s, whereas the caldesmon in the thin filaments is phosphorylated only slowly (half-time greater than 10 min) and the maximum phosphorylation is very low (1 molecule per 7 molecules of caldesmon). I conclude that the phosphorylation of caldesmon hypothesis is untenable.