Effects of pertussis toxin treatment on the metabolism of rat adipocytes

The protein toxin present in Bordetella pertussis vaccine blocks the inhibition of adenylate cyclase by prostaglandins and adenosine which may be secondary to ADP-ribosylation of an inhibitory guanine nucleotide-binding protein. The stimulatory effects of alpha 1-catecholamine agonists on 32P uptake into phosphatidic acid and phosphatidylinositol in isolated rat adipocytes were virtually abolished by pertussis toxin treatment. In contrast, the stimulatory effects of insulin were increased in adipocytes after pertussis toxin treatment. Pertussis toxin treatment did not alter insulin stimulation of glucose oxidation and actually increased glucose conversion to lipid. Basal lipolysis was elevated in adipocytes by pertussis toxin treatment but not basal cyclic AMP. However, the increases in cyclic AMP and lipolysis due to low concentrations of catecholamines and forskolin were markedly potentiated by pertussis toxin treatment. The inhibitory effects of adenosine on cyclic AMP stimulation due to catecholamines were abolished by pertussis toxin. These data indicate that pertussis toxin selectively interferes with inhibition of cyclic AMP accumulation in rat adipocytes by adenosine, potentiates the increases in cyclic AMP due to catecholamines, increases the stimulatory effects of insulin on adipocyte metabolism, and interferes with alpha 1-catecholamine stimulation of phosphatidylinositol turnover.     

Effect of pertussis toxin on hormonal responsiveness of rat hepatocytes

The ureogenic action of epinephrine in hepatocytes from normal adult rats is mediated through activation of alpha 1-adrenoceptors. beta-Adrenoceptors in addition to alpha 1-adrenoceptors, became involved in mediating this effect in cells from animals treated with pertussis toxin. The accumulation of cyclic-AMP in response to epinephrine or isoproterenol was markedly increased in hepatocytes from pertussis-treated rats as compared to that observed in control cells. The accumulation of cyclic-AMP due to glucagon was also increased. It is suggested that pertussis toxin may release a constraint on adenylate cyclase activity by blocking the inhibitory coupling mechanism (Ni) or some other entity involved in the regulation of the activity of this enzyme.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Effect of hydromechanical forces on the production of filamentous haemagglutinin and pertussis toxin ofBordetella pertussis

Summary The production ofBordetella pertussis extracytoplasmic filamentous haemagglutinin (FHA) and pertussis toxin (PT) in a bioreactor under stirring conditions was studied in order to investigate the effect of hydromechanical forces on yields of both antigens. It was shown that FHA loses its haemagglutinin activity when the power transmitted by the agitator and the aerator per unit volume increases, whereas PT production is not affected. The loss of FHA activity can be explained by the action of shear forces on the filamentous structure of this antigen.Nomenclature C^* dissolved oxygen saturation concentration - C₁ dissolved oxygen concentration - D impeller diameter - power transmitted by the agitator and the aerator per unit of liquid volume - E_m maximum local energy dissipation rate per unit of liquid volume - K_La volumetric oxygen transfer coefficient - N impeller speed - Pg power input in aerated system - qO₂m maximum specific oxygen consumption rate - R_e Reynold number (D²N /) - VVM volume of air per volume of fermentation broth per minute - Xm maximum of biomass concentration - o Kolmogorov-microscale - fermentation broth viscosity - fermentation broth kinematic viscosity - fermentation broth density - expt experiment     

Action of insulin modulated by pertussis toxin in rat adipocytes

We studied the effect of pertussis toxin (PT) treatment on the ability of insulin to inhibit lipolysis and to stimulate glucose oxidation in isolated rat adipocytes. In cells maximally modified by PT (100% ADP ribosylation of a 41-kdalton protein in membranes), the ability of insulin to inhibit lipolysis stimulated either by PT alone or in combination with a catecholamine was abolished. In cells wherein ADP ribosylation was submaximal (about 67% modification), a small but variable antilipolytic action of insulin could still be detected. In cells maximally modified by PT, both basal and insulin-stimulated glucose oxidation were markedly reduced (to 10-15% of control levels). However, relative to the basal oxidation level, the fold stimulation by insulin in PT-treated cells was equivalent to the fold stimulation in control cells. Nonetheless, PT treatment caused a rightward shift in the dose-response curve for insulin-stimulated glucose oxidation as well as a small reduction in insulin binding. Our results point strongly not only to a link between the inhibitory guanine nucleotide regulatory complex (Gi) and the antilipolytic action of insulin but also to a link between the Gi complex and the overall regulation of glucose metabolism in adipocytes.     

Effect of pertussis toxin on the adrenergic regulation of plasma renin activity

Basal plasma renin activity (PRA) was not modified by pertussis toxin administration. On the contrary, the modulation of PRA by adrenergic amines was markedly affected by the toxin. Administration of epinephrine did not modified PRA in the controls but markedly increased it in toxin-treated rats. This effect of epinephrine was reproduced in control rats when yohimbine was given before the catecholamine. Clonidine decreased PRA to a much more significant extent in control rats than in animals treated with the toxin. Isoproterenol stimulated PRA to a greater level in toxin-treated rats. Our data indicates that pertussis toxin blocks the alpha 2-adrenergic modulation of renin release and magnifies the ability of beta adrenergic activation to stimulate PRA.     

Effect of pertussis toxin on the heart muscarinic-cholinergic receptors and their function

Administration of pertussis toxin to rats induced a significant increase in heart rate that was evident as soon as 24 hours after the administration of the toxin and that persisted for at least 15 days. Electrical stimulation of the vagus decreased dramatically the heart rate of control animals but was unable to do it so in rats treated with pertussis toxin. In cardiac membranes muscarinic agonists decreased adenylate cyclase activity (approximately equal to 20-25%); no effect was observed in membranes obtained from toxin-treated animals. Agonist displacement of antagonist binding [( 3H] Quinuclidinyl benzilate) indicated that treatment with pertussis toxin decreased the proportion of receptors in the high affinity state for agonists. All these data suggest that blockade of the parasympathetic tone plays a key role in the induction of tachycardia by pertussis toxin.     

Genetics of pertussis toxin

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis. The cloning and nucleotide sequencing of the PT genes from B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica has elucidated the evolution of the Bordetella species and allowed considerable advances towards the understanding of their gene expression and the development of safer vaccines against pertussis.     

Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Enhancement of differentiation of rat adipocyte precursor cells by pertussis toxin

To examine whether GTP-binding protein(s) is (are) involved in adipocyte differentiation, the effect of pertussis toxin (PT) was studied in rat adipocyte precursor cell culture. PT potentiated adipose conversion induced by dexamethasone, insulin, and 1-methyl-3-isobutylxanthine in a dose- and time-dependent fashion. Attenuation of an inhibitory control of adenylate cyclase was not the mechanism of action of PT. The dose-dependent inhibition of PT-catalyzed ADP-ribosylation of the Mr 40,000 protein of the cell membrane by preincubation of the toxin was inversely related to the potentiating effect on differentiation. PT-sensitive G protein(s) may be involved in adipocyte differentiation in a negative fashion.     

Differential effects of cholera toxin and pertussis toxin on the c-fos and c-jun mRNA expression in rat C6 glioma cells

Cholera toxin (CTX) increased c-fos mRNA level whereas it down-regulated the c-jun mRNA level in rat C6 glioma cells. In contrast to the action of CTX, pertussis toxin (PTX) did not affect either c-fos or c-jun mRNA level. The elevated c-fos mRNA level induced by CTX was significantly inhibited by the co-treatment with dexamethasone (DEX). However, DEX did not affect CTX-induced down-regulation of c-jun mRNA level. Cycloheximide (CHX) increased c-fos and c-jun mRNA levels. CHX caused a super-induction of CTX-induced c-fos mRNA level. Our results suggest that CTX-, but not PTX-, sensitive G-proteins may play an important role for c-fos mRNA up-regulation and c-jun mRNA down-regulation. In addition, DEX appears to have a selective inhibitory action against c-fos mRNA expression regulated by CTX. Ongoing protein synthesis inhibition is required for the superinduction of c-fos, but not c-jun, mRNA induced by CTX.     

The effect of pH on the production of pertussis toxin by Bordetella pertussis.

The production of pertussis toxin by Bordetella pertussis was increased by controlling the pH at 7.0 through the addition of sulfuric acid. The more commonly used hydrochloric acid and Tris buffer were observed to be detrimental to toxin yields.     

Effect of pertussis toxin on the hormonal regulation of cyclic AMP levels in hamster fat cells

Pertussis toxin was purified approx. 1800-fold from pertussis vaccine. Administration of as little as 1 microgram of toxin/100 g body weight to hamsters markedly decreased the sensitivity of their adipocytes to agents that inhibit adenylate cyclase through receptor-mediated, GTP-dependent mechanisms such as alpha 2-adrenergic amines, prostaglandins, phenylisopropyladenosine and nicotinic acid. On the contrary, the inhibitory effect of 2',5'-dideoxyadenosine on cyclic AMP accumulation was not affected by the toxin. Activation of adenylate cyclase by isoproterenol, ACTH or forskolin was not diminished by the toxin but the maximum cyclic AMP accumulation was consistently increased. Furthermore, the dose-response curves for ACTH and forskolin were clearly shifted to the left in adipocytes from toxin-treated hamsters as compared to control adipocytes. It is concluded that pertussis toxin blocks the transfer of inhibitory information from the receptors to adenylate cyclase.     

Effect of ICV pertussis toxin and N-ethylmaleimide on the levels of substance P and serotonin in brain and spinal cord of the rat.

The effects of single intracerebroventricular (icv) injections of either 0.5 microgram pertussis toxin or 5 micrograms N-ethylmaleimide (NEM) on the levels of immunoreactive substance P (ir-SP) and serotonin (5-HT) in the brain and spinal cord of rats have been assessed. At two and six days after pertussis toxin injection, the levels of ir-SP appeared significantly diminished in the spinal cord (about 34%). This reduction was even greater at two days after NEM injection (43%). These two agents did not alter the ir-SP of the midbrain and thalamus, whereas NEM increased the neuropeptide content in the pons-medulla. On the other hand, the thalamic content of serotonin was reduced two days after pertussis toxin (32%) or NEM (20%) injection. The indoleamine levels of the spinal cord were reduced by these treatments (20%), while in the midbrain a slight decrease could be observed. These findings suggest that pertussis toxin and NEM produce these effects by acting upon a common neural substrate.