Regulatory Protein Of Adenylate Cyclase Research Articles

Escherichia coli cytotoxins and enterotoxins.

Vero cell cytotoxins and cytotonic enterotoxins produced by E. coli are toxic proteins, which have been implicated in a number of specific diseases in humans and animals. Nomenclature for these toxins is complicated by the existence of different names for the same toxin. The Vero cell cytotoxins are called verotoxins because they are lethal for Vero cells in culture; they are also known as Shiga-like toxins (SLTs) because they are clearly related to Shiga toxin in structure, amino acid sequence, mechanism of action, and biological activity. SLTs belong to two classes. SLT-I is identical with Shiga toxin and is in a class by itself (class I). The other SLTs are closely related to each other and form a second class (class II). Class II SLTs include SLT-II, SLT-IIv, SLT-IIvha, SLT-IIvhb, and SLT-IIva. All SLTs that have been investigated are A-B subunit protein toxins, whose A subunits possess N-glycosidase activity against 28S rRNA and cause inhibition of protein synthesis in eukaryotic cells. These toxins are enterotoxic as well as cytotoxic. SLTs produced in the intestine are absorbed into the blood stream and affect vascular endothelial cells in target organs. They may also have a direct toxic effect on enterocytes. Diseases in which E. coli SLTs have been implicated include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans and edema disease in pigs. Variation in receptor specificities among SLTs may be the reason for different disease syndromes in different host species. The E. coli enterotoxins belong to three distinct classes: heat-labile enterotoxin (LT), heat-stable enterotoxin type I or type a (STI, STa), and heat-stable enterotoxin type II or type b (STII, STb). There is clear evidence that these cytotonic enterotoxins play an essential role in diarrheal disease. LT is an A-B subunit protein toxin, closely related to cholera toxin. Following binding of LT to receptors in enterocytes the A subunit is internalized. The enzymatically active A subunit transfers ADP-ribose from NAD to a GTP-dependent adenylate cyclase regulatory protein, thereby elevating intracellular levels of adenylate cyclase. The increased levels of cyclic AMP cause stimulation of A kinase and lead to hypersecretion of electrolytes and fluid. STI is a small peptide of 18 or 19 amino acids. It binds to receptors in enterocytes and stimulates particulate guanyl cyclase. Elevated intracellular cyclic GMP stimulates G kinase, resulting in increased Cl- secretion and impaired absorption of Na+Cl-. STII is a peptide toxin whose mechanism of action is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that the epidermal growth factor receptor and non-tyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways

We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.

The Functional Expression of Recombinant Human Thyrotropin Receptors in Nonthyroidal Eukaryotic Cells Provides Evidence that Homologous Desensitization to Thyrotropin Stimulation Requires a Cell-Specific Factor*

TSH desensitization involves decreased coupling of the TSH receptor to the adenylate cyclase regulatory protein, Gs. There is evidence that a desensitization protein in thyroid cells plays a role in this process. The molecular cloning of the human TSH receptor and its stable expression in Chinese hamster ovary (CHO-TSHR) cells allowed us to test whether or not TSH desensitization can occur in a nonthyroidal cell. Similar to human thyroid cells, maximal stimulation of cAMP levels in CHO-TSHR cells was attained after 30-60 min of exposure to bovine TSH. Unlike in human thyroid cells, however, preincubation of CHO-TSHR cells with TSH for 12-16 h did not decrease the subsequent cAMP response to a 1-h pulse of TSH stimulation. That is, the human TSH receptor in CHO-TSHR cells does not undergo functional desensitization. Scatchard plot analysis of specific TSH binding to the CHO-TSHR cells revealed high and low affinity sites (Ka of 1.8 +/- 0.4 x 10(9) M-1 and 1.4 +/- 0.3 x 10(7) M-1, respectively), with approximately 10(5) TSH receptors per cell. This is 10- to 100-fold greater than the number of TSH receptors estimated to be present on human thyroid cells. Untransfected CHO cells exhibited only the low affinity binding site. Prior exposure of CHO-TSHR cells to bovine TSH or to (Bu)2cAMP for periods up to 24 h did not reduce [125I]TSH binding to these cells. In summary, desensitization of the adenylate cyclase response to TSH stimulation does not occur in nonthyroidal cells expressing a human TSH receptor with normal functional and TSH binding characteristics. These data support the concept that a cell-specific protein may be involved in homologous TSH desensitization.

Neuropeptide Y inhibits vasoactive intestinal peptide- and dopamine-induced cyclic AMP formation in human Ewing's sarcoma WE-68 cells

Neuropeptide Y (NPY) regulation of intracellular cyclic AMP accumulation was studied in human Ewing's sarcoma cell line, WE-68. NPY inhibited vasoactive intestinal peptide (VIP)- and dopamine-stimulated but not basal cyclic AMP formation. The peptide effect was rapid (<2 min), concentration-dependent with a half-maximal effective concentration (EC 50) of 8 nM NPY, and maximal inhibition reaching 60–70% with 100 nM NPY. Prior exposure of WE-68 cells to pertussis toxin completely abolished the inhibitory action of NPY. It is concluded that NPY attenuates agonist-stimulated cyclic AMP formation in Ewing's sarcoma WE-68 cells, and may do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.

GTP binding regulatory proteins of adenylate cyclase in Schistosoma mansoni at different stages of development

Serotonin-stimulated adenylate cyclase activity in the trematode parasite Schistosoma mansoni increases 40–50-fold during its development from newly transformed schistosomulum to adult. The role of GTP in activation of the enzyme at different stages of development was investigated. Adenylate cyclase can be activated by the non-hydrolyzable GTP analogs 5′-guanylylimidodiphosphate (GppNHp) and guanosine-5′-(3- O-thiotriphosphate) (GTPγS), as well as by sodium fluoride. Activation by GTPγS is competitively antagonized by GTP. Cholera toxin catalyzes the ADP-ribosylation of several proteins in both early schistosomula and adults. Proteins of 93 and 53 kDa are labeled in both stages, but the other proteins labeled appear to be different in the two stages. Adult schistosomes exhibit autoribosylation by cholera toxin in a broad band at 41 kDa, and this is not seen in schistosomula. The effect of cholera toxin on cyclase activity was to reduce activation by serotonin, GTPγS, and fluoride, all agents which act through the GTP binding protein. Cholera toxin treatment also inhibits activation by optimal levels of forskolin, a diterpene that acts at the catalytic subunit. Pertussis toxin had little effect on cyclase activity, although it catalyzed the ADP-ribosylation of a single protein band at 45 kDa in both stages. The results suggest that the GTP binding protein that mediates adenylate cyclase activation by serotonin is somewhat different from G s in the adrenergic adenylate cyclase system. The pertussis toxin substrate in S. mansoni does not appear to function as G i. During development the increase in activation of adenylate cyclase by serotonin appears to be due to an increase in all components of the enzyme system.

Expression of histamine and vasoactive intestinal peptide (VIP) receptors in immortalized rat fetal intestinal cells.

Four intestinal cell lines derived from rat fetuses at 19 days of gestation were successfully propagated after electroporation in the presence of different recombinant DNAs containing the viral oncogenes E1A from Adenovirus 5 and large T from SV40 or Polyoma. These immortalized intestinal cells, designated SLC, possess several properties observed in the parent cells of this tissue, including the expression of cytoplasmic villin, enkephalinase and retention of VIP receptors. In contrast, histamine elevated cAMP levels in the SLC cell lines only. The data suggest that the transfection of fetal rat intestinal cells by E1A and large T is associated with the induction of functional histamine receptors coupled with the Gs/Gi regulatory proteins of adenylate cyclase.

Dual effects of calcitonin and calcitonin gene-related peptide on intracellular cyclic 3',5'-monophosphate in a human breast cancer cell line.

The effects of salmon calcitonin (sCT) and human calcitonin (hCT) and of rat (r) and human (h) calcitonin gene-related peptide (CGRP) on intracellular cAMP accumulation were tested in human breast cancer cells (MCF7). In addition to the well known stimulatory effect, each showed a significant inhibitory effect on cAMP accumulation at low doses. cAMP concentrations in response to sCT, hCT, and rCGRP decreased to 47 +/- 2, 45 +/- 4, and 56 +/- 2% (mean +/- 1 SE) of baseline. The potency ratios for the inhibitory action of sCT, hCT, and rCGRP (1:0.25:0.005, respectively) were similar to the potency ratios for stimulatory action (1:0.3:0.005). The inhibition of cAMP accumulation developed at 300-fold lower peptide concentrations than the stimulation. Preincubation with pertussis toxin or with manganese completely abolished the inhibitory effect of the peptides, suggesting that this is mediated by an inhibitory adenylate cyclase regulatory protein. sCT, hCT, and CGRP each showed unique patterns with regard to time course of inhibition of cAMP accumulation. We conclude that 1) CT can activate an inhibitory adenylate cyclase regulatory protein and a stimulatory adenylate cyclase regulatory protein, and 2) CT effect on an inhibitory adenylate cyclase regulatory protein in MCF 7 cells is evident at far lower hormone concentrations than its effect on a stimulatory adenylate cyclase regulatory protein.

Evidence that growth hormone depletion and uncoupling of the regulatory protein of adenylate cyclase (Ns) both contribute to the desensitization of growth hormone responses to growth hormone-releasing factor

Recent data suggest that the response of GH to GH-releasing factor (GRF) is reduced following prior exposure to high concentrations of GRF. However, it is unknown whether this is due to alterations in GRF receptors, adenylate cyclase activity or the size of a GRF-releasable storage pool of GH. In order to clarify these questions we have compared the effects of pretreatment with GRF (10 nmol/l every 2 h for 12 h) with those of pretreatment with somatostatin (SRIF; 1 mumol/l), forskolin (10 mumol/l) and GRF plus SRIF (10 nmol/l and 1 mumol/l added together) on the subsequent responses of GH to GRF (1 pmol/l-10 nmol/l), cholera toxin (10 nmol/l), 3-isobutyl-l-methylxanthine (IBMX) (100 mumol/l) and forskolin (10 mumol/l). Experiments were performed on 4-day monolayer cultures of rat anterior pituitary cells. The cells were pretreated with test substances every 2 h for 12 h and incubated with GRF or forskolin for 3 h. Per cent maximal (Bmax) GH responses to GRF (10 nmol/l) were reduced after pretreatment with both GRF (control, 173% of basal; GRF, 25% of basal; P less than 0.001) and forskolin (98% of basal; P less than 0.001), but were increased after pretreatment with SRIF (246% of basal; P less than 0.02). However, GH responses after pretreatment with GRF plus SRIF were not significantly different from those of the control.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of GTP-binding proteins from myeloid HL-60 cells. Identification of two pertussis toxin substrates.

We have isolated the major GTP-binding proteins from myeloid HL-60 cell plasma membranes. Two pertussis toxin substrates with similar apparent molecular masses of 40 and 41 kDa, respectively, are contained in these preparations, with both proteins being ADP-ribosylated to a similar extent. Partial chymotryptic proteolysis of fractions containing the [32P]ADP-ribosylated 40-kDa GTP-binding protein alpha subunit demonstrated production of 32P-labeled peptides of 28 and 16 kDa which were not observed after partial proteolysis of fractions containing solely the 41-kDa protein. Similarly, mild acid hydrolysis produced an additional 28-kDa fragment only from fractions containing the 40-kDa protein. The results presented here indicate the presence of two distinct pertussis toxin substrates in myeloid cells. The 41-kDa pertussis toxin substrate is likely to represent the alpha subunit of the inhibitory GTP-binding regulatory protein of adenylate cyclase, whereas the 40-kDa substrate may represent the alpha subunit of the GTP-binding protein which is coupled to chemoattractant receptors. In addition to the pertussis toxin substrates, an additional major peak of guanosine 5'-(3-O-thio)triphosphate-binding activity closely corresponded to the appearance of a 23-kDa protein.

Hydroxylamine-stable covalent linkage of myristic acid in G oα, a guanine nucleotide-binding protein of bovine brain

G oα, a guanine nucleotide-binding protein with a strong homology to the G iα and G sα regulatory proteins of adenylate cyclase, is shown to contain myristic acid. The attachment of myristate to the protein is stable to hydroxylamine treatment, and since the amino-terminal sequence of G oα is typical of proteins with amino-terminal myristate, the inference is strong that G oα is also myristylated at its amino-terminal glycine.

Prevention of alpha 2-adrenergic inhibition on ADH action by pertussis toxin in rabbit CCT.

The present studies were performed to investigate the mechanism whereby alpha 2-adrenergic receptor occupancy inhibits the hydrosmotic action of antidiuretic hormone (ADH) in isolated cortical collecting tubules (CCT). The ADH-ribosyltransferase activity of pertussis toxin (PT) was used to promote covalent modification in CCT Ni, the inhibitory regulatory protein of adenylate cyclase, which presumably mediates the alpha 2-adrenergic inhibition of water flow. Tubules preincubated with PT were studied after the addition of ADH and then after the superimposition of clonidine. In these studies, the inhibition of Jv (water absorption, nl X mm-1 X min-1) and Pf (water permeability coefficient, cm/s), by the addition of 10(-4) M clonidine to the bath, was attenuated by PT in a concentration-dependent manner. Reversal of the inhibitory action of clonidine was accomplished with a concentration of 1.0 micrograms/ml PT. To further elucidate the molecular basis of Ni-mediated transduction of the alpha 2-adrenergic signal, ADP-ribosylation studies were undertaken in membrane preparations of dissected CCT segments. PT ADP ribosylated a 40,000 Mr peptide which was proportional to the amount of membrane protein added. Furthermore, pretreatment of CCT during dissection with 0.5 micrograms/ml PT dramatically decreased the susceptibility of the subunit of Ni (alpha i) to be subsequently ADP ribosylated by PT, when compared with CCT preparations not previously treated with PT. Cholera toxin ADP ribosylated a 42,000 Mr peptide from CCT membranes and PT pretreatment did not interfere with the reaction. We conclude that CCT segments have both the pertussis and cholera toxin substrates and the effect of clonidine to attenuate ADH action is mediated through Ni.

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin.

Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.

Polyphosphoinositide labeling in rat liver plasma membranes is reduced by preincubation with cholera toxin.

Incubation of a crude rat liver plasma membrane preparation with [gamma-32P]ATP resulted in a rapid Mg2+-dependent incorporation of 32P into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Preincubation of the membranes with cholera toxin under ADP-ribosylating conditions reduced the labeling of the polyphosphoinositides. This action of cholera toxin required NAD+ and guanine nucleotides, was dose-dependent with respect to cholera toxin, and could not be mimicked by cAMP. It therefore appears that ADP-ribosylation of the stimulatory guanine nucleotide-binding regulatory protein of adenylate cyclase, or another G-protein, in rat liver plasma membranes affects the activity of enzymes in the polyphosphoinositide pathway.

α 2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest that α 2-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.

Phosphatidylserine vesicles increase rat brain synaptosomal adenylate cyclase activity

Phosphatidylserine vesicles incubated in hypotonic conditions with rat brain synaptosomes increased basal adenylate cyclase activity but did not modify the response of the enzyme to norepinephrine. Moreover, phosphatidylserine antagonized the stimulation of adenylate cyclase activity by NaF. We suggest that in present experimental conditions the effect of phosphatidylserine vesicles is at the level of the G S regulatory protein of adenylate cyclase.

Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents.

This paper examines the modulation of insulin-stimulated glucose transport activity in rat adipose cells by ligands for receptors (R) that mediate stimulation (Rs; lipolytic) or inhibition (Ri; antilipolytic) of adenylate cyclase. The changes in glucose transport activity and cAMP, as assessed by 3-O-methylglucose uptake and (-/+) cAMP-dependent protein kinase (A-kinase) activity ratios, respectively, were monitored under conditions that maintain steady-state A-kinase activity ratios (Honnor, R. C., Dhillon, G. S., and Londos, C. (1985) J. Biol. Chem. 260, 15122-15129). Removal of endogenous adenosine with adenosine deaminase decreased insulin-stimulated glucose transport activity by approximately 30%, which was prevented or restored with Ri agonists such as phenylisopropyladenosine, nicotinic acid, and prostaglandin E1. These changes in transport activity were not accompanied by changes in A-kinase activity ratios, indicating that Ri-mediated effects on transport are independent of cAMP changes. Addition of an Rs ligand, isoproterenol, in the presence of adenosine increased kinase activity but did not change glucose transport activity. Conversely, upon removal of adenosine, addition of Rs ligands such as isoproterenol, adrenocorticotropic hormone, or glucagon strongly inhibited transport (approximately 50%) and stimulated kinase activity. However, subsequent addition of phenylisopropyladenosine nearly restored transport activity without alteration of A-kinase activity. These data and additional kinetic experiments suggest that Rs-mediated glucose transport modulations are also independent of cAMP. The interchangeability of ligands for both Rs and Ri receptors in modulating transport activity suggests that these cAMP-independent effects are mediated by the stimulatory (Ns) and inhibitory (Ni) guanyl nucleotide-binding regulatory proteins of adenylate cyclase. All Rs-and Ri-induced changes in transport activity occurred without a change in glucose transporter distribution, as assessed by D-glucose-inhibitable cytochalasin B binding, suggesting that Rs and Ri ligands modulate the intrinsic activity of the glucose transporter present in the plasma membrane.

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate.

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of catalytic unit of adenylate cyclase and activation of GTPase of N i protein by βγ-subunits of GTP-binding proteins

A protein factor which inhibited adenylate cyclase was purified to apparent homogeneity from rat brain and identified as the βγ-subunits of the GTP-binding regulatory proteins of adenylate cyclase. (i) The βγ-subunits (protein factor) inhibited the partially purified catalytic unit of adenylate cyclase in the presence of an activator, forskolin or the stimulative regulatory protein (N s), to 60 and 40% of the control, respectively; inhibition of the catalytic unit in the presence of forskolin required no guanine nucleotides. (ii) The subunits enhanced the GTPase activity of the purified α-subunit of the inhibitory regulatory protein (N iα) 3.8-fold, (iii) The subunits stimulated ADP-ribosylation of n iα catalyzed by islet-activating protein (pertussis toxin). ADP-ribosylation had no effect on the GTPase activity of N iα in the presence of the βγ-subunits. The results suggest that direct inhibition of the catalytic unit by the βγ-subunits liberated from N i is essential for the receptor-mediated inhibition of adenylate cyclase.

Mapping second messenger systems in the brain: differential localizations of adenylate cyclase and protein kinase C.

[3H]Forskolin and [3H]phorbol 12,13-dibutyrate have been used to map the adenylate cyclase and phosphatidylinositol systems respectively in brain slices by light-microscopic autoradiography. [3H]Forskolin binding to brain sections is displaced potently by forskolin (KD approximately equal to 15 nM) and is enhanced by fluoride and GTP analogs, agents which activate the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Highest [3H]forskolin binding occurs in the corpus striatum, substantia nigra, hippocampus, and molecular layer of the cerebellum. Lesion studies demonstrate that binding sites in the substantia nigra are associated with striatal afferents, while hippocampal sites are localized to granule cell dendrites and mossy fiber terminals, and the intense binding in the cerebellar molecular layer is largely associated with granule cell axons and terminals. Protein kinase C mediates the activity of hormones and neurotransmitters, which act through the phosphatidylinositol cycle, and is labeled with high affinity by [3H]phorbol 12,13-dibutyrate. At many synapses, maps of adenylate cyclase and protein kinase C reveal reciprocal distributions, which may have implications for second messenger regulation of synaptic transmission.

PGE2, forskolin, and cholera toxin interactions in rabbit cortical collecting tubule.

To define further the mechanism whereby prostaglandin (PG) E2 inhibits the hydroosmotic response to ADH, we studied the interactions of PGE2 with ADH and two nonhormonal activators of adenylate cyclase, forskolin and cholera toxin, in the isolated perfused rabbit cortical collecting tubule. Forskolin increased hydraulic conductivity (LP) in a dose-dependent fashion and to a degree comparable with ADH-stimulated LP. Forskolin also augmented maximal ADH-stimulated LP, from 135 +/- 15 (SE) to 174 +/- 7 . 10(-7) cm . s-1 . atm-1. Following a 45-min lag phase, 10(-9) M cholera toxin at 37 degrees C increased LP to 107 +/- 12 . 10(-7) cm . s-1 . atm-1, a response that was stable with time. In paired studies at both 25 and 37 degrees C, PGE2 reversibly inhibited ADH-stimulated LP by 45 and 47%, respectively. However, the same protocols with PGE2 and forskolin failed to reveal any inhibitory effect of PGE2 on forskolin-stimulated LP. PGE2 reversibly inhibited cholera toxin-stimulated LP, from 124 +/- 15 to 100 +/- 15 . 10(-7) cm . s-1 . atm-1. These results support the view that PGE2 inhibits ADH-stimulated LP by inhibiting the synthesis of cAMP and suggest that this inhibition occurs at a functional site at or distal to the nucleotide regulatory protein of adenylate cyclase.