Guanine Nucleotide Regulatory Protein Research Articles

Khellin, but not 8-methoxypsoralen, inhibits adenylyl cyclase system in HeLa cells

Until recently, the therapeutic effects of furocoumarins and furochromones plus UV-A light were thought to be due to their ability to form photoadducts with DNA in the cell nuclei; now it appears that membrane effector systems may be involved as targets. Here we show that in HeLa cells khellin at 1 and 5 μM final concentration, in combination with UV-A light, inhibits NaF-stimulated adenylyl cyclase activity and Pertussis Toxin (PT)-catalyzed ADP-ribosylation of α-subunits of inhibitory guanine nucleotide regulatory proteins (Gi) and increases GTPase activity. In the same experimental conditions, 8-methoxypsoralen (8-MOP), either alone or plus UV-A, does not affect adenylyl cyclase and GTPase activities. Our results suggest that in HeLa cells, through an interaction with a receptor and the mediation of Gi proteins, the adenylyl cyclase system is a target for khellin but not for 8-MOP.

Transmitter release by non-receptor activation of the alpha-subunit of guanine nucleotide regulatory protein in rat striatal slices.

The effects of 5 mM NaF + 10 microM AlCl3, a direct activator of guanine nucleotide-binding proteins (G proteins), on the release of [3H]dopamine ([3H]DA), [3H]gamma-aminobutyric acid ([3H]GABA), and [3H]acethylcholine ([3H]ACh) were investigated in slices of rat striatum. When the tissue was exposed to NaF + AlCl3 the release of [3H]DA, [3H]GABA, and [3H]ACh was enhanced significantly. In a calcium-free solution the release of [3H]GABA and [3H]DA was increased by NaF+AlCl3 much more than in the presence of [Ca2+]o. In slice preparations taken from reserpinized animals, in which the vesicular storage of [3H]DA was therefore prevented, NaF + AlCl3 had no effect on [3H]DA release. HPLC analysis of the radioactivity of the perfusate showed that, in the presence of NaF + AlCl3, the content of dihydroxyphenylacetic acid (DOPAC) in perfusate samples increased significantly, while in pargyline-treated animals only the DA content was increased. Inhibition of DA carriers by nomifensine or low temperature prevented the effect of NaF + AlCl3. N-ethylmaleimide (NEM) preincubation did not modify the effect of NaF + AlCl3 on [3H]DA release Neomycin (0.1 mM), a phospholipase C (PLC) inhibitor, significantly decreased the effect of NaF + AlCl3 on [3H]DA and [3H]GABA release. The internal concentration of Ca2+ in synaptosomes was enhanced by NaF + AlCl3 in normal solution. However, [Ca2+]i was not influenced by NaF + AlCl3 in Ca(2+)-free medium. It is concluded that a non-receptor-mediated activation, by NaF + AlCl3, of the alpha-subunit of a G protein, results in a [Ca2+]o-independent release of DA and GABA, but not that of ACh.

Age-related changes in [ 3H]Baclofen binding in mouse cerebellum

1. 1. Specific [ 3h]baclofen binding to cerebellar membranes from 1-month-old (young), 8-month-old (older) and 20-month-old (aged) mice was lower than that to membranes from 3-month-old mice (mature adult), whereas in cerebral cortical membranes there were no age-related changes in [ 3h]baclofen binding among the four age groups. 2. 2. Scatchard analysis revealed that the density of cerebellar GABA B receptors significantly decreased during aging. 3. 3. There were no age-related changes in the inhibitory effect of Gpp(NH)p on [ 3H]baclofen binding. 4. 4. These results suggest that the characteristics of GABA B receptors in the cerebellum change during aging without any alteration in the coupling of the receptor to guanine nucleotide regulatory proteins.

Differentially expressed mRNAs as a consequence of oxidative stress in intact cells

Intracellular redox conditions influence the activity of several transcription factors leading to a modulation of the expression of the genes controlled by these factors. We examined the changes in cell transcription patterns after oxidative stress induced by diethylmaleate (DEM). Using the differential display technique we identified several differentially expressed sequence tags, four of which are identical or highly homologous to sequences contained in the human cDNAs encoding vimentin, c-fos, cytochrome oxidase IV and ribosomal protein L4; another one corresponds to a transcript of the mitochondrial genome of unknown function. The remaining five cDNAs are not recorded in any sequence data bank. One of these, named Rox3, lights up two mRNA species of ∼3400 and 3600 bp, significantly increased after treatment with DEM or with other oxidizing agents. This increase appears precociously after exposure to DEM and it is completely prevented by pretreatment with N-acetylcysteine. The Rox3 fragment was used to screen a cDNA library; one fully sequenced clone showed 100% homology with the putative human guanine nucleotide regulatory protein nep1.

Alternatively spliced human type 1 angiotensin II receptor mRNAs are translated at different efficiencies and encode two receptor isoforms.

The peptide hormone angiotensin II (AngII) plays a principal role in regulating blood pressure and fluid homeostasis. Most of its known effects are mediated by a guanine nucleotide-regulatory protein (G protein)-coupled receptor pharmacologically defined as the type-1 AngII receptor or AT1. Characterization of cDNA and genomic clones shows that the human AT1 gene contains five exons and encodes two receptor isoforms as a result of alternative splicing. Exon 5 contains the previously characterized open reading frame for AT1, and exons 1 to 3 are alternatively spliced upstream of it to generate several mRNA species, while transcripts containing exon 4 are of minor abundance. In an in vitro translation system, the presence of exon 1 was found to be extremely inhibitory to translation, probably because it can form a stable secondary structure at the RNA level. The alternatively spliced second exon also had a strong inhibitory effect on translation, presumably because it contains a minicistron commencing with an ATG in an optimal context for translation initiation. Exon 2 was similarly inhibitory to protein production in transfected cells, but exon 1 was found to enhance protein synthesis in this system. Transcripts containing exon 3 and 5, which comprise up to one-third of AT1 mRNAs in all tissues examined, encode a receptor with an amino-terminal extension of 32-35 amino acids. These transcripts were translated into a larger receptor isoform in vitro and produced a functional receptor with normal ligand binding and signaling properties in transfected cells.

C-type natriuretic peptide and brain natriuretic peptide inhibit adenylyl cyclase activity: interaction with ANF-R2/ANP-C receptors

C-type natriuretic peptide (CNP) and brain natriuretic peptide (BNP) are members of the natriuretic peptide family, which have been shown to interact with ANP-C/ANF-R2 receptors in addition to ANP-B receptor subtypes. The present study was undertaken to investigate if the interaction of CNP and BNP with ANP-C receptors results in the inhibition of adenylyl cyclase activity. CNP and BNP inhibited adenylyl cyclase activity in heart and brain striatal membranes in a concentration dependent manner with an apparent K i between 0.1 and 1.0 nM. Maximal inhibition observed in heart membranes were about 25% and 35% for BNP and CNP respectively, however the inhibitions in brain striatal membranes were smaller (∼2%). The inhibition was dependent on the presence of guanine nucleotides and was attenuated by pertussis toxin treatment. In addition, CNP inhibited the stimulatory effect of isoproterenol on adenylyl cyclase, whereas CNP as well as BNP showed an additive effect with the inhibitory response of angiotensin II on adenylyl cyclase activity. When the combined effect of C-ANF 4−23/BNP, C-ANF 4−23/CNP and BNP/CNP at optimal concentrations was studied together on adenylyl cyclase activity, the percent inhibition remained the same for C-ANF 4−23 and BNP or C-ANF 4−23 and CNP, however, an additive inhibitory effect was observed for BNP and CNP. These results suggest that CNP and BNP like C-ANF 4−23 interact with ANP-C receptors and result in the inhibition of adenylyl cyclase activity. On the other hand, CNP and BNP interact with the ANP-C receptor, however, the interaction may be at different sites or there may be two subpopulations of ANP-C receptors specific for each of the peptides. These results indicate that BNP and CNP, like ANP and C-ANF 4−23, inhibit the adenylyl cyclase/cAMP signal transduction system through an inhibitory guanine nucleotide regulatory protein, by interacting with ANP-C receptor subtypes.

Inositol phosphates modulate human red blood cell Ca 2+—adenosine triphosphatase activity in vitro by a guanine nucleotide regulatory protein

d- myo-inositol 1,4,5-trisphosphate [lns(1,4,5)P 3) inhibits human red blood cell (RBC) Ca 2+-stimulable, Mg 2+-dependent adenosine triphosphatase (Ca 2+-ATPase) activity in vitro. Because we have previously shown that adrenergic receptors exist on the human mature RBC membrane and can modulate Ca 2+-ATPase activity, we examined the possibility that a guanine nucleotide regulatory protein (G protein) mediated the lns(1,4,5)P 3 effect. Guanosine 5′- O-(3-thiotrisphosphate) (GTP γyS) 10 −4 mol/L also inhibited RBC Ca 2+-ATPase activity. Pertussis toxin 200 ng/mL blocked the effects of both lns(1,4,5)P 3 and GTP γS on Ca 2+-ATPase activity. In separate studies, pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation was shown to occur in RBC membranes under conditions in which measurements of Ca 2+-ATPase activity were performed. When lns(1,4,5)P 3 10 −7 mol/L and GTP γS 10 −6 mol/L were added to membranes concurrently, their inhibitory actions on the enzyme were additive. At greater concentrations of lns(1,4,5)P 3 (10 −6 to 10 −5 mol/L) and GTP γS (10 −4 mol/L), the inositol phosphate reversed the inhibitory effect of GTP γS. These observations indicate that the novel effect of lns(1,4,5)P 3 on the activity of a plasma membrane Ca 2+-ATPase depends at least in part on the action of a pertussis toxin-susceptible G protein.

Molecular mechanisms underlying the sensing of extracellular Ca2+ by parathyroid and kidney cells.

Mineral ion homeostasis in mammalian species is maintained by a complex mechanism comprising sensors of the extracellular calcium concentration (Ca2+0) (i.e. parathyroid cells) as well as effectors that modify their translocation of mineral ions into and out of the extracellular fluid (e.g. kidney) in response to calciotropic hormones. Indirect evidence accumulated over the past decade suggested that parathyroid cells sense Ca2+0 through a cell surface receptor coupled to intracellular second messenger systems via one or more guanine nucleotide regulatory (G) proteins. More recently, Brown et al. employed expression cloning in Xenopus laevis oocytes to isolate a cDNA encoding a Ca2+0-sensing receptor from bovine parathyroid. The expressed receptor activates phospholipase C in a G-protein dependent manner and shows pharmacological properties almost identical to those of the native parathyroid receptor. Agonists for the receptor include not only divalent cations (e.g. Ca2+ and Mg2+) but also trivalent cations and even organic polycations such as neomycin. The deduced amino acid sequence of the cloned receptor confirms that it is a member of the superfamily of G-protein-coupled receptors. Receptor transcripts are present in parathyroid as well as in kidney, thyroid and brain. Therefore, this receptor may mediate the sensing of Ca2+0 not only by parathyroid cells but also by other tissues directly regulated by Ca2+0 (e.g. the thyroidal C cells and certain kidney cells) as well as those not currently known to be involved in calcium homeostasis (viz. in the brain).(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin receptor subtype 3 mediates the inhibitory action of somatostatin on gastric smooth muscle cells.

Previous functional studies show that somatostatin (SS) interacts with specific receptors to inhibit relaxation in gastric smooth muscle cells. There are no ligand binding studies, and it is unknown which of the five subtypes of SS receptors mediates the action. Dispersed gastric smooth muscle cells from guinea pig bound both 125I-labeled SS-14 and 125I-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Nal-NH2 (where Nal indicates N-naphthylalanine) (cyclo-SS-8), a synthetic peptidase-resistant octapeptide SS analogue. SS-28 and SS-14, cyclo-SS-8, and SS analogue D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol [SMS-(201-995) (octreotide)] inhibited 125I-cyclo-SS-8 binding with relative potencies of SS-28 = cyclo-SS-8 = SMS-(201-995) (octreotide), and the binding was not affected by the addition of protease inhibitors. SS-14 caused inhibition only in the presence of protease inhibitors. Ligand analysis demonstrated a two-binding-site model. Analysis of the relationship between biological function and binding suggested the high-affinity sites mediated the relaxant action of SS. 5'-Guanylylimidodiphosphate [Gpp-(NH)p] inhibited binding by reducing the affinity of the high-affinity site. Six SS-8 analogues that distinguish SS subtypes showed that 125I-SS-14 bound to somatostatin receptor subtype 3 (SSTR3). The results demonstrated that gastric smooth muscle cells possess distinct receptors for SS of the SSTR3 subtype. Occupation of these sites inhibits relaxation in gastric smooth muscle cells. Modulation between the high- and low- affinity binding states of SSTR3 is at least partially mediated by activation of guanine nucleotide regulatory proteins.

Chronic ethanol feeding increases the quantity of Gα s-protein in rat liver plasma membranes

The liver is a primary target for both acute and chronic effects of ethanol. Because ethanol is known to alter the function of guanine nucleotide regulatory proteins (G-proteins), changes in hepatic G-proteins could contribute to the adverse effects of ethanol on liver function. Male Wistar rats were fed a liquid diet containing 36% of calories as ethanol for 4 weeks. Control rats were pair-fed or allowed free access to a diet that isocalorically substituted maltose dextrins for ethanol. Liver plasma membranes were isolated and separated into basolateral and canalicular fractions by sucrose-density gradients. Enrichment of marker enzymes (5′-nucleotidase for canalicular membranes and forskolin-stimulated adenylyl cyclase activity for basolateral membranes) was not affected by ethanol feeding. Quantity of Gα s and Gα i proteins in membrane fractions was determined by immunoblot. After ethanol feeding, immunoreactive Gα s protein was increased in liver plasma membranes compared with pair-fed controls. Gα i and Gα s were present in both the basolateral and canalicular fractions of the plasma membrane in control and ethanol-fed rats. Gα s quantity in the basolateral membrane was greater in ethanol-fed rats compared with controls, with no differences in Gα s observed in canalicular membranes. The quantity of Gα i did not change in response to ethanol feeding in any of the membrane fractions. Treatment of isolated plasma and basolateral membranes with 10 μmol/L 5′-guanylimidophosphate, a non-hydrolyzable guanosine triphosphate analogue that activates G-proteins, increased cAMP production to a greater extent in ethanol-fed rats compared with controls. These data indicate that ethanol increases the quantity and function of Gα s protein in rat liver plasma membranes.

Chronic ethanol feeding increases the quantity of Gαs-protein in rat liver plasma membranes

The liver is a primary target for both acute and chronic effects of ethanol. Because ethanol is known to alter the function of guanine nucleotide regulatory proteins (G-proteins), changes in hepatic G-proteins could contribute to the adverse effects of ethanol on liver function. Male Wistar rats were fed a liquid diet containing 36% of calories as ethanol for 4 weeks. Control rats were pair-fed or allowed free access to a diet that isocalorically substituted maltose dextrins for ethanol. Liver plasma membranes were isolated and separated into basolateral and canalicular fractions by sucrose-density gradients. Enrichment of marker enzymes (5'-nucleotidase for canalicular membranes and forskolin-stimulated adenylyl cyclase activity for basolateral membranes) was not affected by ethanol feeding. Quantity of G alpha s and G alpha i proteins in membrane fractions was determined by immunoblot. After ethanol feeding, immunoreactive G alpha s protein was increased in liver plasma membranes compared with pair-fed controls. G alpha i and G alpha s were present in both the basolateral and canalicular fractions of the plasma membrane in control and ethanol-fed rats. G alpha s quantity in the basolateral membrane was greater in ethanol-fed rats compared with controls, with no differences in G alpha s observed in canalicular membranes. The quantity of G alpha i did not change in response to ethanol feeding in any of the membrane fractions. Treatment of isolated plasma and basolateral membranes with 10 mumol/L 5'-guanylimidophosphate, a nonhydrolyzable guanosine triphosphate analogue that activates G-proteins, increased cAMP production to a greater extent in ethanol-fed rats compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic receptor function in rat proximal tubule epithelial cells in culture.

The adrenergic system is important in regulating proximal tubule sodium reabsorption. Although alpha-adrenergic receptors have been identified in proximal tubules, the presence and function of beta-adrenergic receptors (BAR) in proximal tubules is less certain. The purpose of our study was to determine whether functional BAR are present on apical or basolateral surfaces of proximal tubule epithelial cells (PTEC) of rat kidney. We specifically focused on BAR coupling to adenylate cyclase and on differences between requirements for apical and basolateral receptor coupling to adenylate cyclase. To determine BAR expression and function, primary cultures of rat PTECs were grown on permeable supports. Scatchard analysis of 125I-labeled cyanopindolol binding revealed a single class of receptors on both apical and basolateral surfaces. Apical isoproterenol (ISO) resulted in time- and concentration-dependent increases in adenosine 3',5'-cyclic monophosphate (cAMP) that were 50% of responses after basolateral ISO. Apical BAR-cAMP coupling was mediated by B1-adrenergic receptors (B1AR), since apical cAMP responses were abrogated with apical (but not basolateral) B1 but not B2 antagonists. Apical B1AR required endocytosis prior to adenylate cyclase activation, since increases in cAMP were prevented by phenylarsine oxide or colchicine. B1AR-adenylate cyclase coupling was independent of intra- or extracellular calcium, cyclooxygenase metabolites, and protein kinase C (PKC) and dependent on Gs guanine nucleotide regulatory protein. Prolonged exposure to ISO resulted in time- and concentration-dependent homologous desensitization of cAMP responses. Desensitization was independent of receptor sequestration, PKA, or PKC. We conclude the following: B1AR are present on both apical and basolateral surfaces of rat PTECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cyclic AMP in cultured hepatocytes.

Cellular responses to adenosine depend on the distribution of the two adenosine receptor subclasses. In primary cultures of rat hepatocytes, adenosine receptors were coupled to adenylate cyclase via A1 and A2 receptors which inhibit and stimulate cyclic AMP production respectively. R-(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA), the adenosine A1 receptor-selective agonist, inhibited glucagon-stimulated cyclic AMP production with an IC50 of 19 nM. This inhibition was blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPDX). 5'-N- Ethylcarboxamidoadenosine (NECA), an agonist which stimulates A2 receptors, increased cyclic AMP production with an EC50 of 0.6 microM. Treatment of primary cultures of rat hepatocytes with 100 mM ethanol for 48 h decreases the quantity and function of the inhibitory guanine-nucleotide regulatory protein (G(i)), resulting in a sensitization of receptor-stimulated cyclic AMP production [Nagy and deSilva (1992) Biochem. J. 286, 681-686]. When cells were cultured with 2 units/ml adenosine deaminase, to degrade extracellular adenosine, ethanol-induced increases in cyclic AMP production were completely prevented. Moreover, the specific A1-receptor antagonist, CPDX, also blocked the chronic effects of ethanol on receptor-stimulated cyclic AMP production. Treatment with adenosine deaminase or CPDX also prevented the decrease in quantity of the alpha subunit protein of G(i) observed in hepatocytes after chronic treatment with ethanol. Taken together, these results suggest that activation of adenosine A1 receptors on primary cultures of hepatocytes is involved in the development of chronic ethanol-induced sensitization of receptor-stimulated cyclic AMP production.

Multi-site phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 occurs in intact rat hepatocytes.

A phosphorylated form of alpha-Gi-2 (the alpha-subunit of Gi-2), immunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI approximately 5.7, the labelling of which increased approximately 2-fold in cells challenged with either vasopressin or phorbol 12-myristate 13-acetate (PMA); agents which activate protein kinase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP produced a more acidic species of phosphorylated alpha-Gi-2 having a pI of approximately 5.4 and whose labelling was increased approximately 3-fold. Trypsin digestion of labelled alpha-Gi-2 isolated from hepatocytes under basal conditions identified, on two-dimensional peptide analyses, three positively charged phosphoserine-containing peptides (C1, C2 and C3), with only peptides C1 and C2 being evident upon less extensive digestion with trypsin. These are suggested to reflect a single site of phosphorylation, with proteolysis by trypsin being incomplete, and where C2 is larger than C1, which is larger than C3. An identical pattern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptides was increased by approximately 2-fold compared with the basal state. In contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the 'basal' sites approximately 3-fold, but identified a novel positively charged tryptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-Gi-2 from basal and PMA-treated cells produced a pattern of peptides which was identical with those found when the tryptic phosphopeptide was treated with V8 protease. We tentatively suggest that, on alpha-Gi-2, Ser144 is phosphorylated through the action of protein kinase C and Ser207 is phosphorylated upon elevation of the intracellular concentrations of cyclic AMP.

Altered adenylyl cyclase activities and G-protein abnormalities in portal hypertensive rabbits.

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. We hypothesized that alterations in the activity of a guanine-nucleotide regulatory protein (G-protein) might be partially responsible for the marked circulatory disturbances observed in PHT. We, therefore, determined alterations in adenylyl cyclase/cAMP system in prehepatic portal hypertensive rabbits and correlated these changes to the activity of a G-protein. Basal and G-protein-stimulated adenylyl cyclase activities were lower in the PHT superior mesenteric artery (22-26%) and thoracic aorta (31-46%) membranes, but higher (178-321%) in portal vein. The functional activity of Gi alpha proteins (pertussis toxin-catalyzed ADP-dependent ribosylation) increased in the PHT superior mesenteric artery and thoracic aorta, but decreased in portal vein. Immunodetection revealed an increase in the Gi alpha protein subunits (Gi alpha 1/Gi alpha 2 and Gi alpha 3/Go alpha) in PHT thoracic aorta, without any change in Gs alpha proteins; and a decrease in the amount of Gi alpha proteins in PHT portal vein. There was no change in the amount of Gs alpha/Gi alpha in the PHT superior mesenteric artery. We conclude the hemodynamic alterations of PHT are associated with intrinsic alterations in G-protein-enzyme effector systems. These alterations are vessels specific and suggest a possible unique global derangement underlying the vasculopathy of PHT.

Isoprenylated proteins in myelin.

Incubation of rat brainstem slices with [3H]-mevalonate ([3H]MVA) in the presence of lovastatin resulted in the incorporation of label into three groups of myelin-associated proteins with molecular masses of 47, 21-27, and 8 kDa, as revealed on sodium dodecyl sulfate-polyacrylamide rod gel electrophoresis. Although the gel patterns of [3H]MVA-derived prenylated proteins were similar, the relative level of 3H incorporated into each protein species differed between myelin and the brainstem homogenate. Immunoprecipitation studies identified the 47-kDa prenylated protein as a 2'-3'-cyclic nucleotide phosphodiesterase, whereas the 8-kDa protein proved to be the gamma subunit of membrane-associated guanine nucleotide regulatory protein. The 3H-labeled 21-27-kDa group in myelin corresponds to the molecular mass of the extensive Ras-like family of monomeric GTP-binding proteins known to be prenylated in other tissues. Increase in lovastatin concentration resulted in reduced levels of [3H]MVA-labeled species in myelin and concomitantly increased levels in the cytosol. A cold MVA chase restored to normality the appearance of [3H]MVA-labeled proteins in myelin. Furthermore, a high lovastatin concentration in the brainstem slice incubation mixture altered the appearance of newly synthesized nonprenylated myelin proteins, including proteolipid protein and the 17-kDa subspecies of myelin basic protein. Because other myelin proteins were unaffected by the high lovastatin concentration, restricting the availability of MVA in myelin-forming cells may selectively alter processes required for myelinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10 −4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10 −2 M). Isoporterenol (10 −4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E 1 (10 −5 M) produced similar responses in the two strains. G i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G i2α and G qα/G 11α were similar in the two strains, while the levels of G sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ∼20% in MHS membranes. The α-subunit of G i3 was dramatically reduced by ∼80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.

Heparin inhibits melatonin binding and signal transduction in chick brain.

The high-affinity receptor for melatonin is coupled to a pertussis toxin-sensitive, inhibitory guanine nucleotide regulatory protein, Gi, which mediates inhibition of adenylate cyclase activity in the chick and other species. Heparin has been found to uncouple alpha 2-adrenoceptors from a similar Gi; therefore it was of interest to examine the effect of this agent on melatonin binding and signal transduction in chick brain. In competition studies, melatonin inhibited the binding of 2-[125I]iodo-melatonin with high- and low-affinities of KH = 20 pM and KL = 15 nM, respectively. In the presence of heparin (100 units/ml), a single site with an affinity of about 6 nM was detected. The monovalent cations, Na+ and Li+, produced a greater rightward shift of the agonist competition curve than heparin and converted all high-affinity sites to a low affinity of approximately 15-18 nM. In saturation studies, heparin reduced the affinity of high-affinity sites and caused a significant decrease in the density of low-affinity sites. In keeping with its ability to reduce high-affinity binding, heparin blocked melatonin's inhibitory effect on forskolin-stimulated adenylate cyclase activity in chick brain synaptosomal membranes. These findings indicate that heparin impairs the coupling between the melatonin receptor and Gi, with a consequent decrease in binding affinity and a loss of receptor-mediated signalling.

Coupling of endothelin B receptors to the calcium pump and phospholipase C via Gs and Gq in rat liver.

We have demonstrated in liver from male rats that both endothelin A (ETA) and ETB receptors coexist in equal proportion and that ETA receptors mediate a calcium-dependent activation of glycogenolysis. We describe here a sex difference in endothelin action in hepatocytes because, in female rats, 80% of the ET receptors are of ETB type and, accordingly, activation of glycogenolysis is an ETB-mediated process (EC50 = 0.03 pM). ET-1 stimulation of glycogenolysis in female rats was consecutive to activation of phosphatidylinositol 4,5-bisphosphate hydrolysis (EC50 = 0.03 pM) and to inhibition of the calcium extrusion pump (IC50 = 0.03 pM) in plasma membranes, with ET-1 approximately sarafotoxin S6C approximately ET-3. Endothelin regulation of each effector was potentiated by GTP gamma S. ET-1 did not stimulate adenylyl cyclase activity. To identify the nature of the guanine nucleotide regulatory proteins (G protein(s)) coupling ETB receptors to each effector, we used antibodies against the COOH terminus of different G protein alpha subunits. Antibodies reactive with Gs alpha (RM) blocked ET-1 inhibition of the calcium pump, while they did not affect ET-1 stimulation of phospholipase C. Antibodies reactive with Gq alpha (QL) dose-dependently antagonized stimulation of phospholipase C by ET-1 and vasopressin, without affecting ET-1 inhibition of the calcium pump. Antibodies reactive with Gi1 alpha/Gi2 alpha (AS) had no effect on either system. We conclude that the calcium signal provoked by endothelins in hepatocyte is not only consecutive to activation of phospholipase C but also to inhibition of the plasma membrane calcium pump, each effector being coupled to ETB receptors by different G proteins, Gq, and Gs.

Characterization of mammalian Gs-alpha proteins expressed in yeast.

The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization.