Adenylyl cyclase regulates signal onset via the inhibitory GTP-binding protein, Gi

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G(s) and G(i), respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Galpha(s) and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G(s) (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331). This latter action of ACV would facilitate the rapid onset of signaling via G(s). Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Galpha(i), we investigated whether the receptor-mediated activation of heterotrimeric G(i) was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G(i) such that the amount of peptide required to stimulate G(i) in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) to G(i) was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPgammaS binding to G(i) in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc(-) cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G(i) and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G(i).     

Immunocytochemical localization of muscarinic acetylcholine receptors in the rat endocrine pancreas

Summary Immunocytochemical application of the antimuscarinic acetylcholine receptor antibody M35 to pancreas tissue revealed the target areas for the parasympathetic nervous system. Immunoreactivity in the endocrine pancreas was much higher than that in the exocrine part. Moreover, the endocrine cells at the periphery of the islets of Langerhans displayed the highest level of immunoreactivity. Based on these findings in the mantle of the islets, two types of islets have been distinguished: type-I islets with intensely stained mantle cells, and type-II islets with a much lower concentration of these cells. On average, type-I islets were larger (244.8 m±6.1 SEM) than type-II islets (121.5 m±3.8 SEM). M35-immunoreactivity was present on the majority of D cells, which were characterized by their immunoreactivity to somatostatin [of 446 D cells 356 (79.8%) were M35-immunopositive]. However, only a small proportion of the intensely stained mantle cells belonged to the D cell population. Therefore, it is concluded that the majority of the intensely stained mantle cells represent glucagon-secreting A and/or pancreatic polypeptide-secreting F cells. The intensity of M35-immunoreactivity at the periphery and central core of the islets paralleled the density of cholinergic innervation, suggesting a positive correlation between the intensity of cholinergic transmission and the number of muscarinic acetylcholine receptors at the target structures. The present study further revealed some striking parallels for the muscarinic acetylcholine receptor characteristics between the (endocrine) pancreas and the central nervous system.     

Immunocytochemical studies of vasotocin and mesotocin in the hypothalamo-hypophysial system of the chicken

Summary The hypothalamo-hypophysial system of the adult chicken has been studied with a monoclonal antibody that cross-reacts with arginine vasotocin and mesotocin. We have used this antibody on thick (100 m) sections in conjunction with a peroxidase-conjugated rabbit antimouse antibody that permits the visualization not only of entire perikarya, but also of long portions of their axons and dendrites. Our results confirm older concepts based on classical methods, but the more sensitive immunocytochemical method reveals that the system is more extensive than previously recognized. Immunostained neurons in the chicken are widely scattered in the hypothalamus. In the rostral preoptic region, there are three immunostained neuronal cell groups: 1) a prominent closely packed group that extends along the ventromedial surface, 2) a diffusely distributed lateral group, and 3) an external group that surrounds the lateral aspect of the septomesencephalic tract. Caudally in the preoptic area and in the anterior hypothalamus, the same groups are present; but there are also conspicuous periventricular perikarya. Many of them have processes that project to the lumen of the third ventricle, as well as parallel axons that arch lateroventrally in the hypothalamus. In the midhypothalamic area, the periventricular perikarya and processes are particularly numerous at the level of the palliai commissure. The dorsal periventricular group located at the level of the dorsomedial anterior nucleus of the thalamus are the most caudal perikarya. They extend laterally in a wing-like formation. The immunostained axons from all of these perikarya form a compact hypothalamo-hypophysial tract as they run from the midhypothalamus to the median eminence and converge beneath the third ventricle. Axons branching from this tract innervate the zone externa of the anterior median eminence; another group of axons running in the fibrous layer of the zona interna proceeds to the neural lobe.     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The coloned 5-HT_1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT_1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the -subunits of G_i1, G_i2, G_i3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the G_i proteins, but preferentially G₃, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same C protein can regulate several distinct effector molecules.     

Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.     

Pertussis toxin-mediated ADP-ribosylation of rabbit luteal Gi uncouples enkephalin inhibition of adenylyl cyclase

1. Some of the actions of pertussis toxin on the rabbit luteal adenylyl cyclase system were analyzed. 2. Incubation of luteal membranes with pertussis toxin and [32P]NAD resulted in the [32P]ADP-ribosylation of a 40,000 Da protein that is distinct from the proteins ADP-ribosylated by cholera toxin. 3. Pertussis toxin specific [32P]ADP-ribosylation was time-dependent and dependent upon the concentration of pertussis toxin present during the incubation. 4. Pertussis toxin mediated [32P]ADP-ribosylation was enhanced by ATP, ADP, adenylyl imidodiphosphate, GTP, guanosine-5'-O-(2-thiodiphosphate), guanosine-5'-O-(3-thiotriphosphate), and NaF but not AMP or guanylyl imidodiphosphate [GMP-P(NH)P]. 5. Treatment of luteal membranes with NAD and pertussis toxin prevents GTP and enkephalin but not GMP-P(NH)P mediated inhibition of forskolin stimulated adenylyl cyclase, demonstrating the existence of a functional Gi in the rabbit corpus luteum.     

Immunocytochemical study of the hypothalamo-neurohypophysial system

Summary An antiserum cross-reactive against ovine neurophysins-I-II and -III has been used in conjunction with the immunoperoxidase histochemical procedure to localize the cells of the sheep paraventricular (PVN) and supraoptic nuclei (SON). In order to describe the topographical distribution of the SON and PVN a study was made on the serial sections cut (a) transversely from rostral to caudal positions and (b) sagittally from lateral to medial positions of the hypothalamus.The cells of the SON, when examined in the transverse aspect, extended approximately 1900 caudally and when examined in the sagittal plane were contained within a lateral-medial distance of 4830 . In each case the SON cells lay adjacent to the optic chiasm.As sections were cut transversely, the cells of the PVN first appeared in a rostral position defined as 0 and close to the ventral lining of the third ventricle. This general ventral and ventro-lateral distribution of cells maintained up to a caudal distance of approximately 840 . From positions 1260–2310 there was a dramatic dorsal shift of the PVN cells which by this time had also extended laterally. The total rostral-caudal distance occupied by the PVN cells was 3150 . That the lateral-medial distance occupied by the PVN was small (1050 ) was determined on examining the magnocellular nuclei in sagittal section.This work was financed by a grant awarded by the Medical Research Council of New Zealand     

Go but not Gi2 or Gi3 is required for muscarinic regulation of heart rate and heart rate variability in mice

Muscarinic receptor-mediated cardiac parasympathetic activity is essential for regulating heart rate and heart rate variability (HRV). It has not been clear which G(i)/G(o) protein is responsible for these effects. We addressed this question using knockout mice that lack G protein alpha(i2), alpha(i3), or alpha(o) specifically. Unlike previously reported, our alpha(o)-null mice had significantly more survivors with normal life span. Isolated hearts from alpha(o)-null mice demonstrated much less sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol to lower heart rate at baseline and a more profound effect under the stimulation of the beta-adrenergic agonist isoproterenol. In the presence of parasympathetic activation indirectly produced by methoxamine, an alpha(1)-adrenergic agonist, alpha(o)-null mice showed markedly decreased HRV compared with wild-type control mice. These differences in heart rate and HRV were not observed in alpha(i2)-null or alpha(i3)-null mice. Our findings establish an essential role for alpha(o) G protein in the anti-adrenergic effect of carbachol on heart rate regulation.     

Immunocytochemical study of the hypothalamo-neurohypophysial system

Summary Antibodies raised against porcine neurophysin-I and porcine neurophysin-II using an injection regime in rabbits over a short time period, were used to localize neurophysin-I and neurophysin-II in hypothalamic neurosecretory elements of the domestic pig.In transverse section, neurophysin-II containing cells were more abundant in the dorsal medial region of the rostral supraoptic nucleus (SON) as compared with the distribution of neurophysin-I neurons. The main bulk of the cells of the SON were heavily stained for neurophysin-I with neurophysin-II containing cells positioned dorsal from the edge of the optic chiasma.Neurosecretory cells of the SON as seen in sagittal section also showed a differential staining for neurophysins-I and -II.Rostral regions of the pig paraventricular nucleus (PVN) contained magnocellular elements near the third ventricle which were stained predominantly for neurophysin-II. In regions corresponding to the caudal PVN there appeared two populations of neurosecretory neurons: (a) an area of cells adjacent to the third ventricle which contained neurophysin-II antigen and (b) a group of densely populated cells in the dorsal-lateral region which was stained for neurophysin-I.The results support the existence in the pig of at least two distinct populations of neurosecretory neurons corresponding to the neurophysin-I and neurophysin-II neurosecretory system.This work was financed by the Medical Research Council of New Zealand     

Immunocytochemical study of the hypothalamo-neurohypophysial system

Summary Antisera, with cross reactive antibodies removed by affinity chromatography, were used in the immunoperoxidase-bridge technique to study the distribution of oxytocin and vasopressin together with neurophysin in the hypothalamo-neurohypophysial system of the rat. The hormones were demonstrated in different areas of the supraoptic nucleus (SON) and paraventricular nucleus (PVN), in neurosecretory fibres of the hypothalamoneurohypophysial tract, median eminence, and in nerve terminals of the neurohypophysis. Intact normal and rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain), and rats dehydrated by the administration of oral hypertonic saline were studied. In dehydrated rats the hormone concentration in the neurons, and the number of neurons containing hormone varied according to the time of dehydration stress.The observations support the hypotheses that: 1) oxytocin and oxytocinneurophysin, and vasopressin and vasopressin-neurophysin are synthesised in different neurons and are transported along different axons; 2) the SON and PVN are functionally indistinguishable in that neurons containing oxytocin or vasopressin are present in both nuclei; and 3) the two types of neurons respond to osmotic stimulation in a way that is qualitatively the same but quantitatively different.This work was supported by a grant from the Medical Research Council of New Zealand     

Immunocytochemical studies of hamster oocyte activation

By indirect immunofluorescence, using rabbit anti-heparin-binding placental protein (HBPP) antiserum, we studied HBPP expression by physiologically and non-physiologically (microsurgically) activated hamster gametes. Whereas mature gametes (sperm, metaphase II oocytes) were negative, in vivo conceived preimplantation embryos, from pronuclear to two- and four-cell stages, were HBPP positive. No HBPP was demonstrated in the zona pellucida, but HBPP-dependent immunofluorescence was localized in the perivitelline space. Oocytes incubated with hyaluronidase demonstrated variable responses from negative to positive. (Diluent or sperm) microinjected oocytes were all activated and HBPP positive within 4 h after stimulation. Thus neither activation by microinjection nor HBPP expression required paternal gametes. These kinetics suggest that HBPP may be a cortical granule secretogogue which can be applied to monitor oocyte responses during in vitro manipulations.     

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.     

Immunocytochemical studies of the evolution of islet hormones.

By using both immunofluorescence and peroxidase-anti-peroxidase procedures to detect cells producing the four islet hormones, supplemented by biochemical, biological, and radioimmunological assays of tissue extracts, it has been shown that insulin seems to be the most original hormone, apparently occurring already in invertebrates in cells of open type in the alimentary tract mucosa. Insulin cells also predominate in the first islet organ, namely that of the cyclostomes. The order of appearance in the endocrine pancreas during the subsequent evolution is: somatostatin; glucagon; and the pancreatic polypeptide. Even in lower vertebrates pancreatic polypeptide cells occur in those parts of the pancreas situated in close proximity to the gut.     

Hemoglobin mediated nitrite activation of soluble guanylyl cyclase

Nitrite has long been known to be vasoactive when present at large concentrations but it was thought to be inactive under physiological conditions. Surprisingly, we have recently shown that supraphysiological and near physiological concentrations of nitrite cause vasodilation in the human circulation. These effects appeared to result from reduction of nitrite by deoxygenated hemoglobin. Thus, nitrite was proposed to play a role in hypoxic vasodilation. We now discuss these results in the context of nitrite reacting with hemoglobin and effecting vasodilation and present new data modeling the nitric oxide (NO) export from the red blood cell and measurements of soluble guanylate cyclase (sGC) activation. We conclude that NO generated within the interior of the red blood cell is not likely to be effectively exported directly as nitric oxide. Thus, an intermediate species must be formed by the nitrite/deoxyhemoglobin reaction that escapes the red cell and effects vasodilation.     

Immunocytochemical localization studies of myelin basic protein

The location of myelin encephalitogenic or basic protein (BP) in peripheral nervous system (PNS) and central nervous system (CNS) was investigated by immunofluorescence and horseradish peroxidase (HRP) immunocytochemistry. BP or cross-reacting material could be clearly localized to myelin by immunofluorescence and light microscope HRP immunocytochemistry. Fine structural studies proved to be much more difficult, especially in the CNS, due to problems in tissue fixation and penetration of reagents. Sequential fixation in aldehyde followed by ethanol or methanol provided the best conditions for ultrastructural indirect immunocytochemical studies. In PNS tissue, anti-BP was localized exclusively to the intraperiod line of myelin. Because of limitations in technique, the localization of BP in CNS myelin could not be unequivocally determined. In both PNS and CNS tissue, no anti-BP binding to nonmyelin cellular or membranous elements was detected.     

Immunocytochemical studies on the zona pellucida of cow blastocysts

Labeling of the zona pellucida of cow blastocysts with zona-specific anti-serum shows that antigenicity is unaffected by abnormal cleavage, in vitro culture, or frozen storage. The uniform labeling in thin sections indicates that the zona pellucida is homogeneous antigenically. Heavier labeling of the inner and outer surfaces of the zona pellucida in thick sections appers to be due to greater porosity of these regions, in which the zona material becomes highly dispersed, or even partly solubilized, thereby permitting the formation of an antigen-antibody matrix.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.