Pertussis toxin inhibition of anti-immunoglobulin-stimulated proliferation and inositol phosphate formation

A variety of receptor agonists activate cells by stimulating polyphosphoinositide hydrolysis. Increasing evidence supports the concept that receptor-stimulated phosphoinositide hydrolysis is mediated by a guanosine triphosphate binding protein, which in some cell systems is inhibited by pertussis toxin through ADP-ribosylation. The cross-linking of membrane immunoglobulin by antigen or anti-Ig stimulates phosphoinositide hydrolysis resulting in the formation of inositol phosphate and diacylglycerol which act as second messengers in initiating B lymphocyte activation. In this report, we demonstrate that anti-Ig-stimulated inositol phosphate formation is enhanced by the nonhydrolyzable guanosine triphosphate analogue, GppNHp, in permeabilized B lymphocytes and also inhibited by pretreatment of intact cells with pertussis toxin. This latter effect is associated with the pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa membrane protein which is of the same molecular weight as the guanosine triphosphate binding protein reported to mediate receptor-stimulated phosphoinositide hydrolysis in other cellular receptor systems. B lymphocyte proliferation induced by agents such as lipopolysaccharide and PMA plus calcium ionophore, which activate cellular proliferation without stimulating phosphoinositide breakdown, is not inhibited by pertussis toxin. We conclude that anti-Ig activation of B lymphocytes contains pertussis toxin- and guanosine triphosphate-sensitive components which are involved in regulating phosphoinositide breakdown and initiating cellular activation.     

Comparison of calcium ionophore and receptor-activated inositol phosphate formation in primary glial cell cultures.

The possible role of Ca2+ influx in alpha 1-adrenoceptor-stimulated [3H]inositol phosphate [( 3H]InsP) formation was examined in primary cultures of glial cells from 1-day-old rat brain. The Ca2+ ionophore A23187 caused a concentration- and time-dependent increase in [3H]InsP formation similar in magnitude to that caused by norepinephrine (NE). Responses to A23187 and NE were both completely dependent on extracellular Ca2+, with a similar concentration dependence. However, cadmium was more potent in blocking the response to A23187 than to NE. Lanthanum (1 mM) blocked the response to NE, although cobalt (5 mM) did not. The [3H]InsP response to A23187 was not additive with the response to NE or to the muscarinic agonist carbachol, although responses to NE and carbachol were addictive Both A23187 and ionomycin inhibited the additive stimulation caused by a combination of NE and carbachol, and this inhibition was potentiated by cadmium. Ionomycin stimulated [3H]InsP formation at concentrations lower than those inhibiting receptor-mediated responses, and this stimulation was not additive with responses to NE or carbachol. High-performance liquid chromatography separation showed similar patterns of [3H]InsPs formed in response to both Ca2+ ionophore and receptor agonists. These results raise the possibility that receptor-activated Ca2+ influx may be involved in stimulation of [3H]InsP formation in these cells.     

Pertussis toxin inhibits signal transduction at a specific metabolotropic glutamate receptor in primary cultures of cerebellar granule cells

In primary cultures of cerebellar granule cells, glutamate receptors have been classified into metabolotropic (GP1 and GP2) and ionotropic (GC1 and GC2). The GP1 and GC1 receptors are negatively modulated by magnesium and noncompetitively inhibited by phencyclidine; GP2 and GC2 receptors are insensitive to inhibition by magnesium and phencyclidine (Costa, Fadda, Kozikowski, Nicoletti and Wroblewski, 1988). Exposure of cultured cerebellar granule cells to pertussis toxin (PTX, 1 microgram/ml for 14-16 hr) reduced the stimulation of the hydrolysis of inositol phospholipids (PI) by the GP2 receptor agonists, glutamate and quisqualate in the presence of magnesium, but did not inhibit the stimulation of the hydrolysis of PI by GP1 receptor agonists. The stimulation of the hydrolysis of PI by the muscarinic cholinergic receptor agonist, carbamylcholine, remained unchanged after pretreatment with pertussis toxin. In membranes prepared from cerebellar granule cells in primary culture, the addition of guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-s), a nonhydrolyzable analogue of GTP, enhanced the hydrolysis of PI and reduced the Bmax of quisqualate-displaceable binding of [3H]glutamate. These results indicate that, in primary cultures of cerebellar granule cells, a specific class of metabolotropic glutamate receptors (the GP2 receptor) is coupled with the hydrolysis of PI through a pertussis toxin-sensitive GTP-binding protein.     

Pertussis toxin inhibits colchicine-induced DNA synthesis in human fibroblast

Several lines of evidence indicate that microtubule depolymerization initiates DNA synthesis or enhances the effects of serum or purified growth factors in many types of fibroblasts. Yet little is known about the intracellular events responsible for the mitogenic effect of microtubule disrupting agents. The effects of antitubulin agents on DNA synthesis in sparse and dense cultures in the presence or absence of serum and possible involvement of G-proteins in their mitotic action were examined. In these studies, colchicine by itself appeared to be mitogenic only for confluent quiescent human lung fibroblasts. In sparse culture, however, colchicine inhibited serum-stimulated DNA synthesis. Colcemid, another antitubulin agent, showed similar effects of growth inhibition and stimulation in sparse and confluent cultures while lumicolchicine, inactive colchicine, did not. The mitogenic effect of two antitubulin agents, colchicine and colcemid, was partially inhibited by pertussis toxin. These data suggest that microtubular integrity is associated with the expression of either negative or positive controls on DNA synthesis and mitogenic effect of antitubulin agents may be partially mediated by pertussis toxin-sensitive G protein.     

Pertussis toxin inhibits endothelium-dependent relaxations evoked by fluoride

Fluoride is a direct activator of G-proteins. In isolated rings of canine coronary artery, fluoride caused relaxation of rings with endothelium, but only slight contraction of rings denuded of endothelium. The endothelium-dependent relaxations to fluoride were inhibited by pertussis toxin, an inhibitor of G-proteins, or by methylene blue, an inhibitor of soluble guanylate cyclase. Therefore, fluoride causes endothelium-dependent relaxations in part by activating a pertussis toxin-sensitive G-protein in the endothelial cells.     

Effect of desipramine on inositol phosphate formation and inositol phospholipids in rat brain and human platelets.

To examine the mechanism of action of antidepressant drugs, we studied the effect of desipramine (DMI) in vitro on agonist-stimulated inositol phosphate formation and inositol phospholipids in rat brain and human platelets. We observed that DMI inhibited thrombin-stimulated 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) but not 3H-inositol monophosphate (IP1) formation in human platelets. DMI also inhibited norepinephrine (NE) and serotonin (5-HT) stimulated 3H-IP1 formation in rat cerebral cortex. DMI increased levels of all three 3H-inositol phospholipids, 3H-phosphatidyl inositol (PI), 3H-PI-4-phosphate (PIP), and 3H-PI 4,5-bisphosphate (PIP2), in both platelets and rat cortex. The decreased formation of inositol phosphates and increased levels of [3H]-PI, [3H]-PIP, and [3H]-PIP2 by DMI appears to be due to the inhibition of the enzyme phospholipase C rather than its effects on receptors. It is thus possible that interaction of tricyclic antidepressant drugs with the PI-signaling system may be related to their mechanism of action.     

Pertussis toxin inhibits morphine analgesia and prevents opiate dependence

Six days after intracerebroventricular pretreatment of rats with pertussis toxin (PTX 0.5 microgram/rat) there was a marked decrease in the antinociceptive effect of morphine, regardless of the route of opioid administration (into the periaqueductal gray matter, intrathecally or intraperitoneally) or the analgesic test used (tail flick and jaw opening reflex). PTX pretreatment also partially attenuated the naloxone-precipitated withdrawal syndrome in morphine-dependent rats, significantly reducing teeth chattering, rearing and grooming. These in vivo findings indicate that G-protein-dependent mechanisms are involved in morphine analgesia and dependence. The biochemical mechanism could be related to ADP ribosylation of Gi coupled to the adenylate cyclase system, but an interaction of PTX with other G-proteins linked to different second messengers or directly to ionic channels cannot be excluded.     

Ototoxic and nephrotoxic drugs inhibit agonist-induced inositol phosphate formation in rat brain synaptoneurosomes

Neomycin (an aminoglycoside antibiotic), ethacrynate (a loop diuretic), cisplatin (an anticancer drug) and mercuric chloride are chemically unrelated drugs which present similar ototoxic and nephrotoxic properties. We have found that all these molecules inhibit inositol phosphate turnover induced by carbachol or glutamate in rat brain synaptoneurosomes. Since this second messenger system appears to be a key mechanism for cell functioning and even survival, our observations raise the possibility that the expression of the specific toxicity of these compounds may result from excessive inhibition of the phosphoinositide cascade.     

Orexins/hypocretins stimulate accumulation of inositol phosphate in primary cultures of rat cortical neurons

BackgroundOrexins A and B (also named hypocretins 1 and 2) are hypothalamic peptides with pleiotropic activity. They signal through two G protein-coupled receptors: OX₁R and OX₂R. We have previously demonstrated that both types of orexin receptors are expressed in cultured rat cortical neurons, and stimulation of the predominant OX₂R inhibits cyclic AMP synthesis. In the present work, we examined effects of orexins on inositol phosphate (IP) accumulation in rat cortical neurons.MethodsExperiments were performed on primary neuronal cell cultures prepared fromWistar rat embryos on day 17 of gestation. Following 1 h incubation with orexins, IP levels were measured using the ELISA IP-One assay kit.ResultsOrexins A and B increased, in a concentration-dependent manner, IP accumulation in primary neuronal cell cultures from rat cerebral cortex. Both peptides acted with a similar potency. The calculated EC₅₀ values were 6.0 nM and 10.4 nM for orexin A and orexin B, respectively.ConclusionThe results indicate that in cultured rat cortical neurons orexin receptors are also coupled to inositol phosphates signaling pathway.     

Ethanol potentiates serotonin stimulated inositol lipid metabolism in primary astroglial cell cultures

Serotonin-stimulated activation of phospholipase C in primary astroglial cell cultures was studied as a mean of evaluating the effect of acute ethanol exposition on this signal transduction system. The addition of 50-150 mM ethanol prior to stimulation with 10(-5) M serotonin led to a potentiation of the serotonin-induced [3H]-inositol phosphate formation and an increased incorporation of [3H]-inositol into the three phosphoinositides studied. This potentiating effect of ethanol was observed only when ethanol was added together with serotonin. No stimulatory effect of ethanol per se was found. Furthermore, ethanol had no effect on arginine-vasopressin, bradykinin or phenylephrine stimulated inositol lipid metabolism.     

Pertussis toxin inhibits the antinociceptive action of morphine in the rat

The influence of pertussis toxin (PTX) injected intracerebroventricularly (i.c.v., 0.5 micrograms) on the analgesic effect induced in the rat by i.c.v. injection of morphine (5 micrograms) was studied. Morphine analgesia was unaffected 24 h after toxin administration, but there was a significant decrease after 6 days. Therefore a PTX-sensitive substrate, probably a guanine nucleotide regulatory protein could be involved in the coupling of opiate receptors to cellular effectors responsible for the expression of the antinociceptive action of morphine.     

Characterization of cysteinyl-leukotriene formation in primary astroglial cell cultures

The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1). The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.     

Pertussis toxin inhibits alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase without affecting muscarinic regulation of [Ca2+]i or inositol phosphate generation in SH-SY5Y human neuroblastoma cells

The present study reports the differential effects of pertussis toxin on muscarinic regulation of intracellular Ca2+ and inositol phosphate generation and alpha 2-adrenoceptor-mediated inhibition of cAMP formation in SH-SY5Y human neuroblastoma cells. Carbachol caused a biphasic increase in intracellular Ca2+ (release of internal stores and Ca2+ entry) and a dose-dependent increase in inositol phosphate formation. Pertussis toxin pretreatment did not affect either of these components of the signal transduction pathway but did completely reverse the alpha 2-adrenoceptor-mediated inhibition of forskolin-stimulated cAMP formation. These data indicate that muscarinic regulation of inositol phosphate generation occurs via a pertussis toxin-insensitive G-protein and that Ca2+ entry in these cells may not occur via a G-protein.     

Pertussis toxin inhibits noradrenaline accumulation by bovine adrenal medullary chromaffin cells

Summary Bovine adrenal medullary chromaffin cells maintained in tissue culture accumulated [³H]-noradrenaline by a high affinity, Na⁺-dependent, desipramine-sensitive process. The accumulation was linear with time (1–90min) and had an apparent K_m of 0.52±0.24 mol/l and V_max of 1.70±0.48 pmol/(10⁵ cells · 15 min). Pretreatment of the cells with the ADP-ribosylating agent pertussis toxin resulted in a reduction in the V_max, [0.81±0.39 pmol/(10⁵cells · 15 min)] but no significant change in the apparent affinity (K_m = 0.42±0.07 mol/l). This inhibition of [³H]noradrenaline accumulation was distinct from that produced by the vesicular transport inhibitor reserpine. Pertussis toxin inhibition probably did not arise through an indirect action on the Na⁺-gradient because while, as expected, Na⁺, K⁺-ATPase inhibition reduced [³H]noradrenaline accumulation, pertussis toxin pretreatment always caused a further significant reduction even in the presence of maximally effective concentrations of ouabain. Stimulation of the CAMP-protein kinase A system by forskolin or 8-bromocyclic AMP also caused a reduction in [³H] noradrenaline accumulation but again pertussis toxin pretreatment always resulted in a further reduction. Thus, the data provide evidence for a pertussis toxin-sensitive element in the catecholamine accumulation process and are consistent with an action at a site directly associated with the transporter itself rather than with an indirect action via secondary processes.Correspondence to S. J. Bunn at the above address     

Inhibition of inositol phospholipid synthesis and norepinephrine-stimulated hydrolysis in rat brain slices by excitatory amino acids

Inhibition by excitatory amino acid agonists of norepinephrine (NE)-stimulated phosphoinositide hydrolysis was studied in rat brain slices. Inhibition was not observed in cortical slices prelabeled with [3H]inositol but was observed when slices were incubated simultaneously with [3H]inositol, glutamate, and NE. Therefore, we hypothesized that glutamate inhibits the synthesis of inositol phospholipids available to the alpha 1-adrenergic receptor, thereby reducing NE-stimulated phosphoinositide hydrolysis. To test this hypothesis, the distribution of [3H]inositol in cortical slices was measured after 5, 10, 20, 40 and 60 min of incubation, with some slices being exposed to 200 microM NE, 1 mM glutamate, 1 mM N-methyl-D-aspartate (NMDA), 1 mM kainate, 1 mM quisqualate, or to NE in the presence of each of the excitatory amino acid agonists. Glutamate had little effect on the slice content of free [3H]inositol, but it severely reduced the synthesis of [3H]inositol phospholipids, in the presence or absence of NE. Glutamate also abolished NE-induced production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Quisqualate mimicked the effects of glutamate, whereas NMDA and kainate caused less inhibition of the synthesis of [3H]inositol phospholipids and did not inhibit the response to NE. Glutamate produced similar inhibitory effects in slices from hippocampus and striatum. To test if the inhibitory effect of glutamate was the result of irreversible cell damage, cortical slices were incubated with 1 mM glutamate for 60 min prior to exposure to [3H]inositol and NE. Preincubation with glutamate did not reduce the synthesis of [3H]inositol phospholipids or inhibit NE-stimulated [3H]inositol monophosphate production. These results indicate that glutamate impairs the synthesis of inositol phospholipids. Each of the excitatory amino acid agonists, quisqualate, NMDA and kainate, inhibited [3H]inositol phospholipid synthesis, but only quisqualate affected [3H]inositol phospholipids available to the alpha 1-adrenergic receptor.     

Effect of stress on norepinephrine-stimulated cyclic AMP formation in brain slices.

Experiments were conducted to determine if stressful procedures, which increase brain norepinephrine (NE) release in rats, lower the responsiveness of central noradrenergic receptors as measured by the catecholamine (CA)-induced cAMP accumulation in hypothalamic and cerebral cortical slices. No conclusive evidence of subsensitivity was found after either acute or chronic electric footshock or continuous restraint. Failure to find a significant reduction after stress may have resulted from several methodological problems. These include (a) the inhibition of phosphodiesterase activity with isobutylmethylxanthine, which may have obscured possible adaptive changes in cAMP degradation and/or adenosine-dependent adrenergic receptors; (b) a low initial responsiveness to NE in these animals as suggested by the greater case in inducing supersensitivity with reserpine than subsensitivity with amphetamine; and (c) the use as a test agent of exogenous NE which may stimulate a far broader population of receptive sites in brain slices than are activated during stress by the local release of endogenous NE.     

5-Hydroxytryptamine stimulates the formation of inositol phosphate in astrocytes from different regions of the brain

5-Hydroxytryptamine (5-HT) stimulated the turnover of phosphoinositide in primary cultures of astroglia from the cerebral cortex, striatum, hippocampus and brain stem. Ketanserin and ritanserin, selective antagonists for the central 5-HT2 receptor, inhibited the 5-HT-stimulated formation of inositol monophosphate. In contrast, there was no statistically significant accumulation of cyclic AMP after incubation with different concentrations of 5-HT in any of the cultures studied. The results indicate that astrocytes from various regions of the brain possess 5-HT2 receptors coupled to the formation of inositol phosphates.     

Selective dopaminergic mechanism of dopamine and SKF38393 stimulation of inositol phosphate formation in rat brain.

We have previously reported that dopamine and the D1 receptor-selective agonist, SKF38393, stimulate the formation of inositol phosphates in rat brain slices (Undie and Friedman, 1990, J. Pharmacol. Exp. Ther. 253, 987). The present experiments were conducted to determine if actions at alpha-adrenoceptors or at serotonergic sites may contribute to, or interact with, the observed stimulation of phosphoinositide hydrolysis by dopamine receptor agonists. Rat striatal slices prelabeled with [3H]inositol were treated with up to 500 microM dopamine, norepinephrine, serotonin (5-HT), or the dopamine D1 receptor agonist, SKF38393, and accumulated inositol phosphates determined. The action of norepinephrine was dose-dependently blocked by the selective alpha 1-adrenoceptor antagonist, prazosin, but not by SCH23390. The actions of dopamine and SKF38393 were dose-dependently blocked by the dopamine D1 receptor antagonist, SCH23390, but not by prazosin. The effects of 5-HT were blocked by the nonselective 5-HT antagonist, methiotepin, the selective 5-HT2 antagonist, ketanserin, the mixed 5-HT2/5-HT1C antagonist, mianserin, and, with much less potency, by the selective 5-HT1C antagonist, mesulergine. On the contrary, the serotonin receptor antagonists did not block the response to SKF38393, and there was no dose-dependent blockade of the 5-HT response by SCH23390. These observations indicate that the actions of dopamine and SKF38393 in stimulating inositol phosphate formation are selectively mediated through a D1-like dopamine receptor.     

Receptor-mediated inositol phosphate formation in relation to calcium mobilization: a comparison of two cell lines

Previous studies indicated that activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells (S. K. Ambler and P. Taylor, J. Biol. Chem. 261:5866-5871, 1986) and muscarinic receptors in 1321N1 astrocytoma cells (S. B. Masters, T. K. Harden, and J. H. Brown, Mol. Pharmacol. 27:325-332, 1985) resulted in the rapid mobilization of Ca2+ from internal stores of both cell types. Paradoxically, alpha 1-adrenergic agonists did not rapidly increase inositol trisphosphate (Ins-P3) formation in BC3H-1 cells, in distinction to the rapid increase in Ins-P3 accumulation observed in 1321N1 cells after muscarinic stimulation. To determine whether the variations observed in the Ins-P3 response could be ascribed to differences in the relative amounts of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate (respectively, Ins-1,4,5-P3, Ins-1,3,4-P3, and Ins-P4), we have separated the individual inositol phosphates by high-performance liquid chromatography and examined the rates of conversion of individual inositol phosphates in the two types of cells. Muscarinic stimulation of 1321N1 cells resulted in increased Ins-1,4,5-P3 production, as well as the rapid production of Ins-1,3,4-P3 and Ins-P4. Application of alpha 1-agonist to BC3H-1 cells produced a modest but delayed increase in accumulation of Ins-1,4,5-P3. Adrenergic stimulation also resulted in a smaller and even slower production of Ins-1,3,4-P3, and Ins-P4 could not be detected in BC3H-1 cells under any conditions employed. Thus, over a 30-sec interval in which Ca2+ is mobilized to a maximum extent, increases in Ins-1,4,5-P3, Ins-1,3,4-P3, or Ins-P4 amounted to less than 10% over basal values in BC3H-1 cells. These results indicate that the regulation of Ins-P3 isomer formation and conversion may vary substantially between different cell types. In addition, if inositol 1,4,5-trisphosphate is the sole mediator of intracellular Ca2+ release, it is necessary to propose that an increase in Ins-1,4,5-P3 sufficient to mobilize Ca2+ rapidly may occur only within discrete cellular localities in some cell types. According, it may not be possible to detect the increases in Ins-1,4,5-P3 over basal concentrations when measuring total cellular inositol phosphates.