Tonic suppression of baroreceptor reflex by endogenous neurotensin in the rat

We evaluated the modulatory role of endogenous neurotensin (NT) in baroreceptor reflex (BRR) response in Sprague-Dawley rats anesthetized with pentobarbital sodium. Intracerebroventricular (i.c.v.) administration of NT (15 or 30 nmol) significantly reduced the sensitivity of the BRR response. Blocking the endogenous activity of the tridecapeptide with its specific antagonist, (D-Trp11)-NT (4 or 8 nmol) or antiserum against NT (1:20); or inhibiting the aminopeptidases with bestatin (200 nmol), on the other hand, promoted a potentiation of BRR response. When administered together with bestatin (200 nmol), the suppressive effect of NT (15 nmol) on the BRR response was further enhanced, as was the augmentative action of (D-Trp11)-NT (4 nmol). Upon microinjection into the bilateral nucleus tractus solitarius (NTS), NT (600 pmol) and (D-Trp11)-NT (150 pmol) respectively elicited a reduction and enhancement of the BRR response. These results suggest that neurons that contain NT may participate in central cardiovascular regulation by tonically suppressing the BRR, possibly via an action on the NTS where baroreceptor afferents terminate.     

Mastoparan, a wasp venom, activates platelets via pertussis toxin-sensitive GTP-binding proteins

Mastoparan induced limited release of serotonin from intact human platelets, while neither intracellular calcium ion elevation nor arachidonic acid mobilization was observed. Cytolysis induced by mastoparan was negligible in the concentration range that induced serotonin release. In digitonin-permeabilized cells, mastoparan induced Ca(++)-independent release of serotonin and Ca(++)-dependent arachidonic acid release. Both serotonin release and arachidonic acid release were reduced by pertussis toxin, suggesting that platelet activation induced by mastoparan is mediated by GTP-binding proteins.     

Participation of galaninergic neurotransmission at the paraventricular hypothalamic nucleus in the suppression of baroreceptor reflex response by locus ceruleus in the rat

We evaluated the potential participation of galanin (GAL) at the paraventricular nucleus of hypothalamus (PVN) in the suppression of baroreceptor reflex (BRR) response by locus ceruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection of GAL (100 pmol) bilaterally into the PVN significantly depressed the BRR response. This suppressive effect was appreciably antagonized when GAL (100 pmol) and GAL antiserum (1:20) were coadministered into the bilateral PVN. Whereas bilateral microinjection of GAL antiserum into the PVN by itself elicited minimal effect, it nevertheless significantly attenuated the suppressive effect of either electrical or chemical activation of LC on the BRR response. Pretreatment with the same amount of normal rabbit serum (1:20), on the other hand, was ineffective. These results suggest that a galaninergic projection from the LC to PVN may participate in the suppression of BRR response by this dorsal pontine nucleus.     

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation.

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that two inhibitory sites of action of GTP gamma S can be distinguished by their dependence on GTP gamma S concentration and their sensitivity to pertussis toxin. Ca(2+)-stimulated exocytosis was half-maximally inhibited by 6.8 microM GTP gamma S, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTP gamma S inhibition of Ca(2+)-stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to second messenger generation.     

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.     

Facilitation of baroreceptor reflex response by endogenous somatostatin in the rat.

We evaluated the potential participation of endogenous brain somatostatin-14 (SOM) in central cardiovascular regulation, using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of SOM (2 or 4 nmol) promoted a significant elevation in baroreceptor reflex (BRR) response, induced by phenylephrine (5 micrograms kg, i.v.). Blocking the endogenous SOM activity with its specific receptor antagonist, cyclo-[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] (2 or 4 nmol, i.c.v.) or antiserum against SOM (1:20, i.c.v.), on the other hand, appreciably attenuated the same response. These modulatory effects on the BRR response were essentially duplicated upon bilateral microinjections of SOM (320 pmol), SOM antagonist (320 pmol) or anti-SOM (1:20) into the caudal portion of the nucleus of tractus solitarius (NTS), the terminal site for baroreceptor afferents. These results suggest that neurons that contain SOM may participate in cardiovascular control by tonically facilitating the BRR, possibly by exerting an influence on the neurons at the NTS.     

Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophil degranulation.

Stimulation of human normodense eosinophils with immobilized secretory IgA (sIgA) or IgG, or with the soluble stimulus, FMLP, triggers the exocytotic release of the granule protein, eosinophil-derived neurotoxin (EDN). In this report, we demonstrate that these stimuli also provoke an increase in phospholipase C-mediated phosphoinositide breakdown in eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) for 2 h irreversibly abolished the increases in phospholipase C activity and EDN release induced by immobilized sIgA or FMLP. In contrast, PTX treatment only transiently inhibited eosinophil activation induced by immobilized IgG. Maximal inhibition of IgG-stimulated phosphoinositide hydrolysis and EDN release occurred after 2 h of PTX pretreatment with PTX, followed by a gradual recovery of cellular responsiveness to immobilized IgG as the duration of PTX pretreatment was extended to 16 h. Activated PTX catalyzed the in vitro ADP-ribosylation of 41- and 44-kDa proteins in eosinophil membranes. A 2-h pretreatment of intact cells with PTX markedly reduced the pools of unmodified 41- and 44-kDa substrates available for subsequent ADP-ribosylation in vitro, suggesting that both proteins were substrates for PTX in intact eosinophils. Continuous exposure of eosinophils to PTX for times ranging from 2 to 15 h resulted in the gradual reappearance of unmodified 44-kDa protein, whereas the levels of unmodified 41-kDa protein were persistently reduced in PTX-treated cells. The time course of the decline and reappearance of unmodified 44-kDa substrate in PTX-treated eosinophils closely paralleled the changes in the responsiveness of these cells to immobilized IgG. These results suggest that the receptors for sIgA, FMLP, or IgG transduce activating signals for eosinophil degranulation through differential coupling to at least two PTX-sensitive G proteins.     

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.     

Development of opiate receptors and GTP-binding regulatory proteins in neonatal rat brain

The mu and delta opiate receptors present in rat brain were measured independently during postnatal development. The numbers of delta receptors were almost undetectable at birth and increased substantially during the first few weeks, whereas the numbers of mu receptors remained relatively constant. Activation of either of these receptors caused inhibition of adenylate cyclase, but inhibition coupled to mu receptors was much smaller than that associated with delta receptors at all ages. Attempts to use pertussis toxin-catalyzed ADP-ribosylation as an assay for the GTP-binding proteins Ni and No were hampered by the development of an NADase with age. However, specific antibodies directed against the alpha subunits of Ni or No allowed separate quantitation of these transducer proteins. Both increased with age. No is present at levels at least 5-fold higher than Ni in the adult rat brain. The N proteins are in vast excess over receptors and as such are unlikely to be limiting factors in receptor function. The data further suggest that the number of opiate receptors present throughout neonatal development is in excess over that required for optimal function.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.     

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment.These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.     

A pertussis toxin-sensitive GTP-binding protein plays a role in the G0-G1 transition of rat hepatocytes following establishment in primary culture

Acute spontaneous c-myc gene expression and sustained increase of a GTP-binding protein(s) (G-protein) which is sensitive to islet-activating protein (IAP), pertussis toxin, occurred early during primary culture of adult rat hepatocytes. Following these earlier events, DNA synthesis was demonstrated in response to EGF and insulin. Addition of IAP immediately after plating of primary cultures inhibited c-myc expression and the hormone-induced DNA synthesis. Addition at 24 h or later following cell inoculation, however, produced only weak effects on DNA synthesis, even though the IAP-sensitive G-proteins were completely inactivated. We conclude that the IAP-sensitive G-protein(s) plays a role in the earlier process(es) of the G0-G1 transition, which is essential for the initiation of growth factor-dependent DNA synthesis.     

Involvement of pertussis toxin-sensitive GTP-binding protein in prostaglandin F2 alpha- induced phosphoinositide hydrolysis in osteoblast-like cells

Prostaglandin F2 alpha (PGF2 alpha) stimulated the formation of inositol phosphates in a dose-dependent manner in cloned osteoblast-like MC3T3-E1 cells. This reaction was markedly inhibited dose-dependently by pertussis toxin. In the cell membranes, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGF2 alpha. These results suggest that pertussis toxin-sensitive GTP-binding protein is involved in the coupling of PGF2 alpha receptor to phospholipase C in these cells.     

Tonic enhancement of the sensitivity of baroreceptor reflex response by endogenous substance P in the rat.

Modulation of baroreceptor reflex (BRR) by endogenous substance P (SP) in the brain was investigated in rats anesthetized with pentobarbital sodium. Intracerebroventricular administration of the undecapeptide (15 or 30 nmol) and its antagonist, (D-Pro2, D-Trp7,9)-SP (30 or 60 nmol) or SP antiserum (1:20), respectively, promoted a significant increase and decrease in the sensitivity of BRR response. Prolonging the endogenous activity of SP with the aminopeptidase blocker, bestatin (200 nmol) or with the endopeptidase-24.11 inhibitor, phosphoramidon (200 nmol) significantly augmented the same reflex. Combining the undecapeptide with either peptidase blocker, moreover, promoted additional potentiation of the BRR response. On the other hand, simultaneous administration of bestatin and (D-Pro2, D-Trp7,9)-SP produced a reduction of the augmented effect of bestatin on the sensitivity of BRR response. Bilateral microinjection of SP (600 pmol) or an antiserum against SP (1:20) into the nucleus tractus solitarius (NTS) elicited respectively an enhancement of and reduction in the BRR response. These data suggest that neurons that contain SP may participate in central cardiovascular control by tonically enhancing the sensitivity of the BRR response, possibly via an action on the NTS.     

Properties of 1-methyladenine receptors in starfish oocyte membranes: involvement of pertussis toxin-sensitive GTP-binding protein in the receptor-mediated signal transduction.

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes, suggesting that a guanine nucleotide-binding protein (G protein) serving as the substrate of pertussis toxin is involved in the 1-MA receptor-mediated signal. We thus investigated properties of 1-MA receptors by means of binding of the radiolabeled ligand to the oocyte membranes. There were apparently two forms of 1-MA receptors with high and low affinities in the membranes. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. A 39-kDa protein, which had been identified as the alpha-subunit of the major substrate G protein for pertussis toxin, was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. The ADP-ribosylated 39-kDa alpha-subunit could be immunoprecipitated with antibodies raised against the carboxy-terminal site of mammalian inhibitory G-alpha. These results indicate that 1-MA receptors are functionally coupled with the 39-kDa pertussis toxin-substrate G protein in starfish oocyte membranes.     

Interaction of GTP-binding regulatory proteins with chemosensory receptors

GTP-binding regulatory proteins (G-proteins) were identified in chemosensory membranes from the channel catfish, Ictalurus punctatus. The common G-protein beta-subunit was identified by immunoblotting in both isolated olfactory cilia and purified taste plasma membranes. A cholera toxin substrate (Mr 45,000), corresponding to the G-protein that stimulates adenylate cyclase, was identified in both membranes. Both membranes also contained a single pertussis toxin substrate. In taste membranes, this component co-migrated with the alpha-subunit of the G-protein that inhibits adenylate cyclase. In olfactory cilia, the Mr 40,000 pertussis toxin substrate cross-reacted with antiserum to the common amino acid sequence of G-protein alpha-subunits, but did not cross-react with antiserum to the alpha-subunit of the G-protein from brain of unknown function. The interaction of G-proteins with chemosensory receptors was determined by monitoring receptor binding affinity in the presence of exogenous guanine nucleotides. L-Alanine and L-arginine bind with similar affinity to separate receptors in both olfactory and gustatory membranes from the catfish. GTP and a nonhydrolyzable analogue decreased the affinity of olfactory L-alanine and L-arginine receptors by about 1 order of magnitude. In contrast, the binding affinities of the corresponding taste receptors were unaffected. These results suggest that olfactory receptors are functionally coupled to G-proteins in a manner similar to some hormone and neurotransmitter receptors.