Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase

Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.     

Natriuretic peptides inhibit adenylyl cyclase activity in dispersed eel gill cells

The effect of natriuretic peptides on forskolin-evoked adenylyl cyclase activity was investigated in dispersed gill cells from the Australian short-finned eel (Anguilla australis). Molecular cloning techniques were employed to identify the putative G-protein-activating motif within the intracellular domain of the eel natriuretic peptide C receptor. Eel ANP, eel CNP and the NPR-C-specific C-ANF inhibited the forskolin-stimulated production of cyclic AMP. This effect was abolished by pretreatment of cells with pertussis toxin. Eel VNP was without effect on adenylyl cyclase activity. PCR and molecular cloning indicated that the intracellular domain of A. australis NPR-C has the same amino acid sequence as Anguilla japonica. Alignment of these sequences with Rattus norvegicus NPR-C indicated conservation of the putative G-protein-activating motif BB...BBXXB (B=basic, X=nonbasic residues). These data suggest that branchially-expressed NPR-C may play a physiological role additional to that of ligand clearance.Abbreviations ANP atrial natriuretic peptide - CNP C-type natriuretic peptide - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - eANP-NH₂ amidated form of eel ANP - GC guanylyl cyclase - G_i inhibitory G-protein - IBMX isobutylmethylxanthine - NP natriuretic peptide - NPR natriuretic peptide receptor - PCR polymerase chain reaction - PTX pertussis toxin - VNP ventricular natriuretic peptideCommunicated by I.D. Hume     

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein.

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 microM 5'-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 +/- 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 +/- 0.7 nM) to one site of intermediate affinity (KD = 0.52 +/- 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cloned platelet thrombin receptor couples to at least two distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase.

Thrombin both stimulates phosphoinositide hydrolysis and inhibits adenylyl cyclase in a variety of cell types. Whether the cloned human platelet thrombin receptor accounts for both of these signaling events is unknown. We report that thrombin receptor agonist peptide causes both phosphoinositide hydrolysis and inhibition of adenylyl cyclase in naturally thrombin-responsive CCL-39 cells. To exclude the possibility that the agonist peptide or thrombin itself may activate these pathways via distinct receptors and to circumvent a lack of suitable thrombin receptor-null cells, we utilized a designed "enterokinase receptor," a thrombin receptor with its thrombin cleavage recognition sequence LDPR replaced by DDDDK, the enterokinase cleavage recognition sequence. Transfection of enterokinase-unresponsive cells with this construct conferred both enterokinase-sensitive phosphoinositide hydrolysis and inhibition of adenylyl cyclase. The phosphoinositide hydrolysis response was largely insensitive to pertussis toxin, whereas the adenylyl cyclase response was completely blocked by pertussis toxin. These data show that the cloned thrombin receptor can effect both phosphoinositide hydrolysis and inhibition of adenylyl cyclase via at least two distinct effectors, most likely Gq-like and Gi-like G-proteins.     

Prostaglandin E2 does not inhibit tumoricidal activity of mouse macrophages against adherent tumor cells

We determined whether endogenously produced PGE2 can down-regulate the tumoricidal properties of macrophages by a negative feedback mechanism. Peritoneal exudate macrophages or resident peritoneal macrophages of mice were incubated in medium (control) or in medium containing IFN-gamma and LPS. Activated macrophages were highly tumoricidal against syngeneic melanoma cells and secreted high levels of PGE2. Treatment with indomethacin or diclofenac sodium (voltaren) completely inhibited the production and secretion of PGE2 but not the tumoricidal activity of activated macrophages measured either immediately after activation or 1 to 3 days thereafter. Finally, the addition of exogenous PGE2 did not alter the ability of peritoneal exudate macrophages to respond to IFN-gamma or of LPS to produce high levels of tumor cell lysis. Collectively, these results show that PGE2 produced by activated macrophages is not a down-regulator of their tumoricidal activity against adherent tumor cells.     

High-affinity prostaglandin E receptors attenuate adenylyl cyclase activity in isolated bovine myometrial membrane

Purified bovine myometrial plasma membranes were used to characterize prostaglandin (PG) E2 binding. Two binding sites were found: a high-affinity site with a dissociation constant (KD) of 0.27 +/- 0.08 nM and maximum binding (Bmax) of 102.46 +/- 8.6 fmol/mg membrane protein, and a lower affinity site with a KD = 6.13 +/- 0.50 nM and Bmax = 467.93 +/- 51.63 fmol/mg membrane protein. Membrane characterization demonstrated that [3H]PGE2 binding was localized in the plasma membrane. In binding competition experiments, unlabelled PGE1 displaced [3H]PGE2 from its receptor at the same concentrations as did PGE2. Neither PGF2 alpha nor PGD2 effectively competed for [3H]PGE2 binding. Adenylyl cyclase activity was inhibited at concentrations of PGE2 that occupy the high-affinity receptor. These data demonstrate that two receptor sites, or states of binding within a single receptor, are present for PGE2 in purified myometrial membranes. PGE2 inhibition of adenylyl cyclase activity support the view that cAMP has a physiological role in the regulation of myometrial contractility by PGE2.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

Sonication as a tool for the study of adenylyl cyclase activity.

The technique of sonication was applied in studying adenylyl cyclase activity of cultured fibroblasts. Exposure of BHK 21 c/13 to brief periods of low power sonication gives cell preparations with greater basal, fluoride and hormone sensitive adenylyl cyclase activites than those of broken cell preparations of homogenized cells. The sonicated cells provide a convenient method to study adenylyl cyclase since they are added directly to the adenylyl cyclase reaction vessels without further processing. Maximal epinephrine stimulated activity in sonicated cells is nearly equivalent to that activated by sodium fluoride, but the apparent affinity of the enzyme system is similar to that of broken cell preparations. Furthermore, broken cell preparations of sonicated cells possess greater adenylyl cyclase activity than broken cell preparations of unsonicated cells. This procedure may provide a useful tool for the analysis of the hormonal regulation of adenylyl cyclase activity of isolated cells.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Electron microscopic cytochemistry of adenylyl cyclase activity in mouse spermatozoa.

We investigated adenylyl cyclase activity of mouse spermatozoa by electron microscopic cytochemistry. Subcellular localization of enzyme activity was determined in the presence and absence of bicarbonate ions. Results confirm the existence in sperm of a bicarbonate-regulated adenylyl cyclase, which suggests microdomain signaling.     

Prostaglandin E2 and alpha 2 adrenoceptor agonists inhibit the pentose phosphate shunt in pancreatic islets

Glucose utilization in isolated pancreatic islets of the rat was inhibited by prostaglandin (PG) E2 and the alpha 2 adrenoceptor agonist, clonidine, to a similar extent; other prostaglandins did not affect glucose utilization. Islet oxidation of [1-14C]glucose and [6-14C]glucose demonstrated that the pentose phosphate shunt was inhibited by PGE2 and clonidine. Pertussis toxin antagonizes the effects of clonidine and PGE2 on total glucose utilization and pentose phosphate shunt activity. The results suggest that PGE2 and alpha 2 adrenoceptor agonists may regulate glucose metabolism through similar transduction mechanisms, and that a guanine nucleotide binding regulatory (G) protein modulates certain metabolic effects of prostaglandins and adrenergic agonists.     

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase.

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2+ channel activity.     

Modulation of adenylyl cyclase activity in young and adult rat brain cortex. Identification of suramin as a direct inhibitor of adenylyl cyclase

Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.     

Prostaglandin E2 and cAMP inhibit B lymphocyte activation and simultaneously promote IgE and IgG1 synthesis.

Macrophage-secreted prostaglandins of the E series inhibit numerous immunologic events, including IgM secretion by B lymphocytes. In this study, we investigated whether PGE also regulates the activation of normal quiescent murine B cells and subsequent isotype differentiation to IgE and IgG1 production. PGE2 and PGE1 were found to inhibit cellular enlargement induced by IL-4 or bacterial LPS, IL-4 and LPS, or anti-mu and IL-4 by approximately 75%, and completely inhibit enlargement in response to anti-mu antibody. PGE2 also suppresses activation-induced class II MHC up-regulation by 35% and expression of the low affinity IgE receptor, Fc epsilon RII/CD23, by 30%. Interestingly, PGE completely inhibits a fraction of cells from these activation events, while other cells fully respond to activation stimuli, even in the presence of high PGE2 concentrations. Therefore, a PGE-resistant subset of B lymphocytes may exist. A closely related PG, PGF2 alpha, had no immunoregulatory effect in these systems. Because PGE induces production of cAMP in B cells, we determined whether other agents that increase cAMP could inhibit B cell activation. Cholera toxin and dibutyryl cAMP mimicked the ability of PGE2 to inhibit B cell enlargement, and class II MHC and Fc epsilon RII induction, suggesting that PGE2 signaling occurs via cAMP. In addition, cholera toxin and dibutyryl cAMP inhibited B cell activation much more potently (90-100% inhibition) than PGE, indicating that whereas all B cells are cAMP-sensitive only some are PGE-sensitive. Although PGE inhibits activation-associated events, we previously reported that PGE enhances IL-4 and LPS-induced differentiation to IgE and IgG1 synthesis. To investigate the relationship between the cells that are activation-inhibited and those that are differentiation-enhanced by PGE, we sorted B cell subsets by FACS and determined their relative abilities to produce IgM, IgG1, and IgE in response to IL-4 and LPS in the presence of PGE. The population of lymphocytes that was unaffected by PGE in terms of class II hyperexpression was also unaffected by PGE for Ig synthesis, again indicating a PGE-resistant subpopulation of B cells. Furthermore, the PGE activation-inhibited subset of B cells was responsive to PGE enhancement of IL-4-induced class switching, reducing IgM synthesis and inducing a sevenfold increase in IgE and IgG1 synthesis compared with other sort groups. These results are consistent with the hypothesis that the B lymphocytes that are PGE activation-inhibited are the same cells that are PGE differentiation-enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Ca2+ and calmodulin on adenylyl cyclase activity in sheep olfactory epithelium

Sheep olfactory epithelium contains an adenylyl cyclase which is stimulated by many but not all odorants. Here we report that this enzyme is activated by calmodulin in a dose-dependent manner, and that calcium ions are required for this response. Odorant stimulation of adenylyl cyclase is unaffected by the complex Ca²⁺/calmodulin, as suggested by the results obtained both in Ca²⁺/calmodulin-depleted membranes and under calmodulin antagonist treatment; this confirms the prediction that the Ca²⁺ binding protein and odorants stimulate the olfactory adenylyl cyclase through parallel mechanisms. The persistent activation of the regulatory component of adenylyl cyclase by GppNHp does not alter the response of the enzyme to either odorant or Ca²⁺/calmodulin. In sheep olfactory epithelium a cAMP-phosphodiesterase activity is also present, which is highly inhibited by IBMX and aminophylline, searcely by RO 20-1724, and unaffected by Ca²⁺/calmodulin. The modulatory role exerted by calcium on cAMP system in sheep olfactory signal transduction is discussed.     

Different roles for Gi and Go proteins in modulation of adenylyl cyclase type-2 activity

The effect of Gi/o protein-coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed alpha2A-adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxin-sensitive manner. Activation of Gs proteins switched the response of the alpha2A-adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gbetagamma scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of Go proteins (alphao1 + beta1gamma2) restored both the inhibition and the potentiation, whereas expression of Gi proteins (alphai1 + beta1gamma2) resulted in a potentiation of both the TPA- and the Gs-stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for Go protein-linked signalling.     

GM1 ganglioside modulates prostaglandin E1 stimulated adenylyl cyclase in neuro-2A cells

This study demonstrates modulation by GM1 ganglioside of prostaglandin E1 (PGE1)-induced cAMP formation in Neuro-2a neuroblastoma cells. Pretreatment of the cells with neuraminidase, an enzyme that increases cell surface GM1, resulted in significant elevation of PGE1-induced cAMP formation, as did preincubation of the cells with nmolar concentrations of GM1. Pretreatment with brain ganglioside mixture lacking GM1 had no effect. Cholera toxin B subunit, a specific GM1-binding ligand, inhibited adenylyl cyclase. When the concentration of exogenous GM1 in which the cells were preincubated was increased from nmolar to molar levels there was a dose-responsive fall off in cAMP elevation, attributed to progressive inhibition of adenylyl cyclase by increasing GM1. These results are interpreted as indicating modulation of this PGE1 receptor in Neuro-2a cells by plasma membrane-localized GM1 in a structure-specific manner.Abbreviations PGE1 prostaglandin E1 - Ctx B B subunit of cholera toxin - BBG bovine brain ganglioside mixture - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - IBMX 3-isobutyl-1-methylxanthine - N'ase neuraminidase - D-PBS Dulbecco's phosphate-buffered saline