Anti-FIM and Anti-FHA Antibodies Inhibit Bordetella pertussis Growth and Reduce Epithelial Cell Inflammation Through Bacterial Aggregation.

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction of B. pertussis with A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Our in vitro results indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survival in vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction of B. pertussis with host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such as in vitro potency assays.

Heterotrimeric G-protein and signal transduction in the nematode-trapping fungus Arthrobotrys dactyloides.

The fungus Arthrobotrys dactyloides produces specialized constricting rings to trap and then consume nematodes. The signal transduction pathway involved in the nematode-trapping process was examined. Mastoparan, an activator of G-protein, had a stimulatory effect on the inflation of ring cells, whereas a G-protein inhibitor, pertussis toxin, prevented ring-cell expansion. The 40-kDa G alpha of heterotrimeric G-proteins was specifically ADP-ribosylated by pertussis toxin. Using an antibody specific to the 35-kDa subunit G beta, we showed that immunogold-labeled G beta was more concentrated in ring cells than in the hyphae. In the absence of nematodes, the rings could be inflated by either pressurizing the culture in a syringe, raising intracellular Ca2+ concentrations, or adding warm water. We used these methods to reveal differences in responses to antagonists. The results support a model in which the pressure exerted by a nematode on the ring activates G-proteins in the ring cells. The activation leads to an increase in cytoplasmic Ca2+, activation of calmodulin, and finally the opening of water channels. The ring cells expand to constrict the ring and thus immobilize the nematode.

Reversal of pertussis toxin-induced thermal allodynia by muscarinic cholinergic agonists in mice.

The intrathecal administration of pertussis toxin (PTX) not only blocks the antinociceptive effects of the muscarinic cholinergic receptor agonist oxotremorine administered systemically, but also produces a long-lasting thermal allodynia in mice. The purpose of the present studies was to determine both the antinociceptive effects in normal mice and the antiallodynic effects in PTX-treated mice of systemically administered muscarinic cholinergic receptor agonists and cholinesterase inhibitors. In normal mice, antinociceptive effects were tested using a 55 degrees C water-bath tail-flick test. In mice treated 7 days previously with PTX (0.3 microg i.t.), antiallodynic effects were tested using a 45 degrees C water-bath tail-flick test. The nonselective high-efficacy muscarinic agonists oxotremorine, H-TZTP (3-(1,2, 5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine oxalate), and methylthio[2.2.1], (exo (+)3-(3-methylthio-1,2, 5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane oxalate), as well as vedaclidine, a mixed M(2)/M(4) muscarinic receptor partial agonist and M(1)/M(3)/M(5) muscarinic receptor antagonist, the nonselective partial agonists RS86 and pilocarpine, and the cholinesterase inhibitors physostigmine and tacrine all produced dose-related antinociception. Oxotremorine, H-TZTP and methylthio[2.2.1] produced dose-related reversals of PTX-induced thermal allodynia whereas vedaclidine produced a partial reversal and RS86 and pilocarpine, as well as physostigmine and tacrine, failed to reverse the allodynia. The present results provide further evidence that decrements in PTX-sensitive G(i/o)-protein functioning may be involved in initiating and/or maintaining some persistent or neuropathic pain states. Moreover, the present results suggest that muscarinic receptor agonists such as vedaclidine may be useful in the treatment of persistent pain states that are due at least in part to dysfunction of inhibitory second messenger systems.

Mepyramine but not cimetidine or clobenpropit blocks pertussis toxin-induced histamine sensitization in rats.

The effects of pertussis toxin (PT) and the role of histaminergic H(1), H(2) and H(3) receptor blockade on the actions of histamine on blood pressure, heart rate, blood gas values, and mortality were studied in anaesthetized rats. Four days after treatment with PT, histamine dose-dependently decreased mean arterial blood pressure (MAP) and PT enhanced the histamine-induced decrease in MAP. In the PT but not in the inactivated PT (IPT) or saline treated group three out of six animals died after the highest dose of histamine (300 mg kg(-1), i.v.) In order to determine the type of histamine receptor that mediates HS, 4 days after PT the selective antagonists mepyramine (H(1)), cimetidine (H(2)) and clobenpropit (H(3)) were administered 20 min before the challenge with histamine. Mepyramine completely inhibited both the enhanced histamine-induced decrease in MAP and mortality brought about by PT. Cimetidine and clobenpropit had no protective effects, but rather enhanced the histamine-induced mortality elicited by PT. The present study shows that PT caused HS in rats which is primarily mediated via H(1) and secondarily via H(2) and H(3) receptors. These results are considered to be a first step in the elucidation of the mechanism(s) of the HS test used in the quality control of acellular pertussis vaccine.

Calcitonin reverts pertussis toxin blockade of the opioid analgesia in mice.

The aim of this paper is to study the influence of salmon calcitonin (SCT) on opioid analgesia when opioid transduction pathways are functionally uncoupled from Gi/o proteins by treatment with pertussis toxin (PTX). The antinociceptive effect of morphine and three selective opioid agonists, [D-Ala2,N-Me-Phe2,Gly5-ol]enkephalin (DAMGO) (OP(3-mu receptor agonist), [D-Pen2.5]-enkephalin (OP-1-delta receptor agonist) and trans-( +/- )-3,4-dichloro-N-methyl-N-[2-1(-pyrrolidinyl)-cyclohexyl]-benzene-acetam ide methane sulfonate (U-50, 488H) (OP1-kappareceptor agonist) was evaluated, using the tail flick test, in mice treated with PTX or with PTX and SCT. PTX blocked the antinociceptive effect of the opioids, being the antinociception similar in control animals and in mice treated with PTX and SCT. Thus, SCT prevents the effect of the blockade of Gi/o-proteins. From this it could be suggested that calcitonin activates alternative antinociceptive mechanisms that are not dependent on Gi/o-proteins.

[Inactivation and toxoiding of biologically-active components of Bordetella pertussis by tea catechins].

An ability of tea catechins known as agents for the disinfection to bacteria and viruses were tested on application for toxoiding biologically active components of Bordetella pertussis. The effects on the activities and antigenicity of filamentous hemagglutinin (FHA) and pertussis toxin (PT) were investigated. The activities of FHA and PT were inactivated by catechins at approximately 10(3) times lower dose (0.2 mM) compared with that of formalin. The activity of inactivated FHA was recovered by dialysis against Tris-HCl buffer, pH 8.0, containing glutathione or Tris-HCl buffer, pH 6.0. But the activity of inactivated PT was not recovered. Antigenicity of catechin-treated antigens were investigated by immunization to mice. The sera from mice immunized by catechin-treated FHA or PT were contained antibody against not only catechin-treated but also non-treated FHA or PT. These results suggest that antigenicity of FHA or PT was not destroyed by the treatment with catechin. We prepared pertussis-component vaccines by treatment of several catechins on the condition that FHA or PT activity was not recovered. Higher efficacy were found on the vaccines made by treatment of epicatechin, epicatechin gallate, or epigallocatechin than those by formalin. The vaccine prepared by using epigallocatechin gallate had significant efficacy as well as that by formalin treated one. From these results, it is suggested that tea leaf catechins were effective agents for toxoiding of vaccine components.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

Inhibition of G-protein betagamma-subunit functions by phosducin-like protein.

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.

Agonist occupation of serotonin1A receptors in the rat hippocampus prevents their inactivation by pertussis toxin.

The present study was undertaken to investigate the nature of the effect of pertussis toxin on the responsiveness of two potentially distinct subgroups of postsynaptic serotonin1A (5-HT1A) receptors of rat hippocampus CA3 pyramidal neurons: those located at the level of the cell body, which can be activated by microiontophoretically-applied 5-HT1A receptor agonists, and those located on dendrites, which can be activated by endogenous serotonin released by the stimulation of the ascending serotoninergic pathway. The former receptors (denoted as extrasynaptic) have been previously demonstrated to be sensitive to pertussis toxin, whereas the latter (denoted as intrasynaptic) have been shown to be pertussis toxin-insensitive. Rats treated with the 5-HT1A receptor agonists flesinoxan or BMY 42568 were used to determine whether tonic activation of extrasynaptic 5-HT1A receptors would prevent their inactivation by pertussis toxin. A pretreatment with p-chlorophenylalanine was used to determine whether a serotonin depletion would render the intrasynaptic 5-HT1A receptors sensitive to pertussis toxin. The responsiveness of CA3 pyramidal neurons to the suppressant effects of microiontophoretically-applied serotonin, 8-hydroxy-2-(di-n-propylamin)-tetralin, baclofen and GABA or to endogenously-released serotonin, elicited by the stimulation of the ascending serotoninergic pathway, was studied one to 10 days after the intrahippocampal injection of pertussis toxin. When compared to control saline-treated rats, the treatments with flesinoxan (5 mg/kg/day, s.c.) and BMY 42568 (5 mg/kg/day, s.c.) delivered for 14 days by osmotic minipumps, starting three days prior to the injection of pertussis toxin, significantly attenuated the effect of pertussis toxin on the responsiveness of CA3 pyramidal neurons to microiontophoretic applications of serotonin and 8-hydroxy-2-(di-n-propylamino)-tetralin, as well as baclofen, an agonist of GABAB receptors, which share the same G proteins with 5-HT1A receptors. The two-day pretreatment with p-chlorophenylalanine (350 mg/kg/day, i.p.) did not render the intrasynaptic 5-HT1A receptors sensitive to pertussis toxin, as indicated by the unchanged efficacy of the stimulation of the ascending serotonin pathway in the suppressing the firing activity of CA3 dorsal hippocampus pyramidal neurons. Our results suggest that the sustained activation of extrasynaptic 5-HT1A receptors prevents the pertussis toxin-induced ADP ribosylation of G protein alpha subunit, and thereby protects an amount of G proteins sufficient to maintain the function, not only of 5-HT1A, but also of GABAB receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Pertussis toxin inhibition of T-cell hybridoma invasion is reversed by manganese-induced activation of LFA-1.

Pertussis toxin (PT) inhibits invasiveness of T-cell hybridomas in vitro and metastasis formation in vivo. We present evidence for the hypothesis that PT interferes with functional activation of LFA-1. Invasion by TAM2D2 T-cell hybridoma cells of fibroblast monolayers was completely blocked by PT pretreatment, but the cells regained invasiveness in the presence of Mn2+, which activates LFA-1. This invasion was blocked by anti-LFA-1 mAb, and Mn2+ did not stimulate invasiveness of LFA-1-deficient TAM2D2 mutants. TAM2D2 cells did not adhere to surfaces coated with the LFA-1 counterstructure ICAM-1, but Mn2+ induced adhesion. Hence, LFA-1 on TAM2D2 cells requires activation before it can participate in the invasion process. The hypothesis is further supported by the slightly different results obtained with the TAM8C4 T-cell hybridoma. PT inhibited invasion strongly but not completely. This reduced invasion was increased by Mn2+. TAM8C4 cells did adhere to ICAM-1, but Mn2+ enhanced adhesion. Thus, part of LFA-1 on TAM8C4 cells is constitutively active, allowing for some PT-insensitive invasion, but further activation is required for optimal adhesion and invasion. PT blocks G-protein-mediated signals, suggesting that an extracellular factor is involved. This is not a serum component or an autocrine motility factor, since the PT effect was serum-independent, and PT did not inhibit motility. Therefore, it is probably produced by the fibroblasts, and either secreted or associated with the cell surface. These results are in line with the hypothesis that a fibroblast constituent activates LFA-1 via a PT-sensitive G-protein and thus stimulates invasion of T-cell hybridomas into the fibroblast monolayer.

Guanine nucleotide binding regulatory proteins in liver from obese humans with and without type II diabetes: evidence for altered "cross-talk" between the insulin receptor and Gi-proteins.

A novel pathway for physiological "cross-talk" between the insulin receptor and the regulatory Gi-protein has been demonstrated. We tested the hypothesis that a coupling defect between Gi and the insulin receptor is present in the liver of obese patients with and without type II diabetes. Insulin 1 x 10(-9) M (approximately ED50) and 1 x 10(-7) M (Max) inhibited pertussis toxin-catalyzed ADP ribosylation of Gi in human liver plasma membranes from lean and obese nondiabetic patients. However, 1 x 10(-7) M insulin was without effect in membranes from patients with type II diabetes. This coupling defect was not intrinsic to Gi, since Mg2+ and GTP gamma S inhibited pertussis toxin-catalyzed ADP ribosylation in both diabetic and nondiabetic patients. Binding of insulin of the alpha-subunit and activation of the tyrosine kinase intrinsic to the beta-subunit of the insulin receptor are not responsible for the coupling defect. 125I insulin binding is the same in obese patients with or without diabetes. Tyrosine kinase of the insulin receptor is decreased in diabetes. However, a monoclonal antibody to the insulin receptor (MA-20) at equimolar concentrations with insulin equally inhibits pertussis toxin-catalyzed ADP ribosylation of Gi without activating tyrosine kinase or insulin receptor autophosphorylation. Immunodetection of G-proteins suggested that Gi3 alpha was normal in diabetes and Gi1-2 alpha was decreased by 40% in the diabetic group as compared to the obese nondiabetic group but was normal when compared to the lean non diabetic group. We conclude that the novel pathway of insulin signaling involving the regulatory Gi proteins via biochemical mechanisms not directly involving the tyrosine kinase of the insulin receptor is altered in obese type II diabetes and offers a new target for the search of the mechanism(s) of insulin resistance.

Cycloheximide abolishes pertussis toxin-induced increase in glutamate release from cerebellar granule neurones.

Release of glutamate from cerebellar granule neurones was stimulated either by adding 50 mM K+ to normal Krebs medium, or by adding 5 mM Ca2+ to neurones continuously depolarised with 50 mM K+ in the absence of Ca2+. Pre-incubation of neurones for 16 h with pertussis toxin (PTX) increased the stimulated glutamate release in both K(+)-stimulated and continuously depolarised neurones. Under both conditions, the PTX-induced increase in release was abolished by cycloheximide. In contrast, in the presence of cycloheximide, PTX still prevented the GABAB agonist (-)-baclofen from inhibiting glutamate release. These results suggest that G-protein ADP-ribosylation by PTX in cerebellar granule neurones may increase synthesis of a protein associated with the L-type calcium channel.

Neuropeptide Y inhibits pertussis toxin-catalyzed ADP-ribosylation in bovine adrenal chromaffin cell membranes.

Evidence is presented that the neuropeptide Y receptor is directly coupled to an inhibitory G protein existing in cultured bovine adrenal chromaffin cell membranes. Pertussis toxin catalyzes the [32P]ADP-ribosylation of a 41 kDa plasma membrane protein. 5'-Guanylylimidodiphosphate inhibited the [32P]ADP labelling of this protein in a dose-dependent manner whereas GTP had no effect. Preincubation of the plasma membranes with high concentrations of neuropeptide Y followed by a brief exposure to a low concentration of 5'-guanylylimidodiphosphate significantly inhibited ADP-ribosylation beyond that observed with 5'-guanylylimidodiphosphate alone. These results suggest that the neuropeptide Y receptor in bovine adrenal chromaffin cells is directly coupled to a 41 kDa PTX substrate (presumably the alpha subunit of an inhibitory G protein).

Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins.

Pertussis toxin (PTX) ADP-ribosylates alpha subunits of GTP-binding proteins (G proteins) when they are in association with beta gamma dimers, and free alpha subunits are thought not to be substrates under standard assay conditions. We now report the rather unexpected discovery that synthetic peptides encompassing the last 10-20 amino acids of alpha subunits of PTX-sensitive G proteins are substrates for PTX by themselves and in the absence of beta gamma dimers. As determined for G13, the Km of PTX for the 20-amino acid carboxyl-terminal peptide is 10-fold higher than that for the trimeric G protein. Interestingly, PTX ADP-ribosylates the free full length alpha 13 subunit with a Km not different from that of the trimer but with a Vmax that is only 1% of that with which it ADP-ribosylates the trimer. It follows that the primary role of beta gamma dimers in ADP-ribosylation of G proteins is one of increasing the Vmax of the reaction without affecting the Km of the substrate for the toxin. Mutant peptides lacking the ADP-ribose acceptor site act as competitive inhibitors.

Peptidergic modulation of G-protein coupled cyclic-AMP accumulation in the rat caudate nucleus.

Somatostatin, substance P, and vasoactive intestinal polypeptide were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of the neuropeptides on G-protein coupled adenylate cyclase in vitro. Somatostatin induced an enhancement of cyclic AMP formation in presence of guanine nucleotides and cholera toxin but inhibited pertussis toxin and forskolin enzyme stimulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation as described previously. Somatostatin was able to antagonize these inhibitory effects of both toxins. On the contrary, substance P reduced GTP and cholera toxin stimulated striatal adenylate cyclase, without affecting forskolin activation. In our preparation, VIP did not influence basal adenylate cyclase activity or the stimulation by guanine nucleotides, cholera toxin, and pertussis toxin. VIP potently inhibited the enhancement of cyclic AMP formation by forskolin and completely antagonized the inhibitory effect of cholera toxin on forskolin activation. These results suggest that neuromodulatory effects of somatostatin, substance P, and VIP are mediated by the inhibitory as well as stimulatory guanine nucleotide proteins G-i and G-s coupled to an adenylate cyclase system.

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells.

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABAb) receptor activation on K(+)-depolarization-induced signals and on voltage-activated Ca2+ currents were investigated. Both GABA (20 microM) plus bicuculline (20 microM) and (-)baclofen (20-100 microM), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10-20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1 micrograms/ml, for 4-6 h at 37 degrees C). Baclofen (50 microM) inhibited Ca2+ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5'-triphosphate GTP gamma S (100 microM), led to complete inhibition of the Ca2+ conductance and of voltage-evoked intracellular Ca ([CA]i) transients within 2 min. In paired cells intracellularly perfused with GTP gamma S-free solution, the Ca2+ current amplitude decreased by only about 30% for 5-6 min. It is concluded that bovine chromaffin cells have functional GABAb receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterization of genetically inactivated pertussis toxin.

Pertussis toxin has been shown to be an important virulence factor and an antigen which will probably be essential to a pertussis vaccine. Inactivation of the pertussis toxin was required due to the pharmacological properties associated with this toxin. However, chemical inactivation has the potential of altering important epitopes or of failing to inactivate the toxin. Cloning and sequencing of the pertussis toxin operon has permitted the introduction of specific mutations in the S1 gene which have been shown to have a profound effect on the subsequent enzyme activity. Various mutations were constructed, re-assembled into the pertussis toxin operon and returned to the Bordetella pertussis chromosome for expression. Pertussis toxin, with lysine substituted for arginine at position 9 in the S1 subunit (PTA-K9) was assembled and expressed to wild type levels. Substitution of codons for aspartic acid, glycine and glutamine, for that of glutamic acid at position 129 were incorporated into the PTA-K9 construction. Virulence of these constructed B. pertussis strains and ADP-ribosylation by their toxoids were greatly reduced relative to that found with the wild type. Additionally, PTA-K9 was found to have reduced leukocytosis promotion and histamine sensitization activities. Finally, PTA-K9 was shown to be a protective immunogen in both intracerebral and aeorosol challenge assays.

Inhibition of endothelium-dependent relaxations by phorbol myristate acetate in canine coronary arteries: role of a pertussis toxin-sensitive G-protein.

Activation of protein kinase C by phorbol esters inhibits the endothelium-dependent relaxations evoked by certain stimuli. The release of endothelium-derived relaxing factor can be evoked by a number of distinct subcellular processes, including activation of a pertussis toxin-sensitive G-protein. The aim of the present study was to determine whether or not the inhibitory effect of phorbol esters on endothelial function was associated with inhibition of the pertussis toxin-sensitive pathway. Rings of canine coronary artery were suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution, gassed with 95% O2-5% CO2 (37 degrees C). Treatment of arterial rings with pertussis toxin (100 ng/ml) or with phorbol myristate acetate (PMA, 10(-8) M) inhibited the endothelium-dependent relaxations produced by UK 14,304, an alpha-2 adrenergic agonist, leukotriene C4 or by NaF, a direct activator of G-proteins, but did not affect the endothelium-dependent relaxations produced by bradykinin or by A23187. If the arterial rings were first treated with pertussis toxin, PMA (10(-8) M) no longer inhibited the endothelium-dependent relaxations to NaF. Increasing the concentration of PMA (to 3 X 10(-8) and 10(-7) M) caused inhibition of responses to bradykinin. At higher concentrations, PMA (3 X 10(-7) and 10(-6)) also inhibited the relaxations evoked by A23187. The endothelium-independent relaxations evoked by nitroglycerin were not affected by PMA (10(-8) to 10(-6)).(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin acts through G-proteins on dopaminergic adenylate cyclase in the caudate-putamen of the rat.

Somatostatin was incubated in an adenylate cyclase assay of a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of the peptide on D-1 receptor coupled adenylate cyclase in vitro. Somatostatin was able to enhance cyclic AMP formation in the presence of guanylylimidodiphosphate and guanosine-triphosphate. In contrast to this, somatostatin inhibited both dopamine and forskolin-stimulated cyclic AMP accumulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation. Somatostatin was found to antagonize these inhibitory effects of pertussis toxin and cholera toxin. The results suggest that somatostatin acts through a stimulatory as well as an inhibitory guanine nucleotide regulatory protein subtype to affect dopaminergic adenylate cyclase activity.