Species differences in actions of islet-activating protein, pertussis toxin

Islet-activating protein (IAP) is one of the pertussis toxins. The ability of IAP to cause potentiation of insulin secretory responses and promotion of leukocytosis was studied in six animal species (hamsters, rats, guinea pigs, rabbits, dogs and monkeys). The action of IAP on insulin secretion in the animals was estimated by three kinds of tests: effects on epinephrine hyperglycemia, plasma insulin and blood glucose concentrations following the injection of stimuli, and glucose tolerance. Of all animals tested, IAP was most effective in hamsters. Marked hyperinsulinemia was also shown in IAP-treated dogs, rats and monkeys in response to insulin secretagogues, but their sensitivity to IAP was inferior to that of hamsters. In rabbits, IAP was markedly toxic, and the effect on insulin secretion was observed only slightly at a dose close to its minimal lethal dose. In addition, no significant effects of IAP were shown in guinea pigs in the present experiment. On the other hand, leukocytosis promoting activity of IAP appeared in a dose-dependent manner in all animal species; rabbits were the most sensitive to IAP in this regard. It is concluded that both actions of IAP appear differently in different animal species, and the species difference of the effect on insulin secretory responses is in agreement with that on histamine sensitizing activity.     

Suppression of passive cutaneous anaphylaxis by pertussis toxin, an islet-activating protein, as a result of inhibition of histamine release from mast cells

Passive cutaneous anaphylaxis (PCA) produced by antigen challenge to antibody-sensitized rats were interfered with by prior treatment with pertussis toxin, an islet-activating protein (IAP). The degree of interference was dependent on the dose and injection time of IAP; the effect of IAP developed slowly, with a maximal effect being observed 3 days later. Inhibition of PCA by IAP was associated with a decrease in histamine release from peritoneal mast cells, making it very likely that the process affected was mast cell secretion. Much less histamine was discharged in vitro, in response to certain membrane receptor (e.g. IgE receptor) stimulation, from mast cells that had been exposed to IAP than from the cells not exposed. Such an inhibitory effect of IAP was not observed when histamine release was provoked by a calcium ionophore without mediation of membrane receptors. IAP was a stronger inhibitor of histamine release than beta-adrenergic agonists. Further inhibition was produced when a beta-agonist was added to IAP-treated mast cells. The increase in the cellular content of cyclic AMP was associated with beta-agonist-induced, but not with IAP-induced, inhibition of histamine release. Thus, IAP inhibited histamine release by a mechanism in which metabolism of cyclic AMP was not directly involved.     

Effects of pertussis toxin on macrophage activation

The aim of this study was to evaluate capability of pertussis toxin(PT) to activate mouse macrophages. The investigations were undertaken to determine whether the role played by this toxin required the A-protomer of the toxin to ADP-ribosylate a guanine nucleotide binding protein (a Class I activity) or was dependent on the binding if B-oligomer of the toxin to the surface of target cells (a Class II activity). The results of these experiments have established that the mechanism of macrophage activation with PT seems to be dependent upon a Class II activity of the toxin.     

Effects of pertussis toxin on the alpha 2-adrenoceptor-inhibitory GTP-binding protein-adenylate cyclase system in rat brain: pharmacological and neurochemical studies

Behavioral excitement and the increase in locomotion were observed in male adult rats four days after an intraventricular injection of 5 micrograms pertussis toxin (IAP). Clonidine (100 micrograms/kg s.c.)-induced locomotor hypoactivity was not observed in animals pretreated with 1 and 5 micrograms IAP. IAP caused a significant (P less than 0.05) decrease in the KD value of [3H]clonidine binding and enhanced GTP (1 microM)-induced decrease in the binding to cortical membranes from rat brain. In addition, the inhibition of adenylate cyclase induced by alpha 2-receptor stimulation (100 microM adrenaline plus 100 microM propranolol) was completely suppressed in the cerebral cortical membranes by IAP pretreatment. It is suggested that the system consisting of alpha 2-receptor, the inhibitory GTP-binding protein (Ni) and adenylate cyclase inhibits some animal behaviors and cyclic AMP formation. Moreover, IAP seems to inactivate Ni, subsequently producing behavioral excitement and it inhibits clonidine-induced sedation.     

Effects of pertussis toxin on inhibition of synaptosomal tyrosine hydroxylase activity by apomorphine

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase (TH) activity and on the inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, but attenuates apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation of apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (-)-sulpiride, suggesting that G proteins are involved in the dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40-50% inactivation of Gi and Go proteins, which is evident from the reduction of ADP ribosylation with [32P]NAD of the remaining G proteins following preincubation with the toxin. The present study also demonstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors.     

Effect of islet-activating pertussis toxin on the binding characteristics of Ca2+-mobilizing hormones and on agonist activation of phosphorylase in hepatocytes

Islet-activating protein (IAP, a Bordetella pertussis toxin) was employed to test the hypothesis that the inhibitory GTP-binding regulatory protein of adenylate cyclase (Ni) mediates GTP effects on the binding of Ca2+-mobilizing hormones to liver plasma membranes and is involved in calcium mobilization stimulated by these agonists. IAP added to normal liver plasma membranes catalyzed the incorporation of radioactivity from [32P]NAD into a 41,000-Da peptide (presumably the alpha-subunit of Ni). However, no such incorporation was observed in liver membranes prepared from rats 24 hr after intraperitoneal injection of IAP. Angiotensin II attenuated glucagon-stimulated increases in cAMP in hepatocytes prepared from control but not IAP-treated rats. In contrast, following IAP treatment, no changes were observed in the ability of glucagon, vasopressin, angiotensin II, or epinephrine to activate phosphorylase; nor did this treatment alter [3H]vasopressin binding or epinephrine displacement of [3H]prazosin binding. However, IAP treatment decreased [3H]angiotensin II binding affinity when studies were performed in the absence but not the presence of 5'-guanylylimidodiphosphate (GppNHp). This shift was small and represented only 5-8% of the shift in apparent Kd elicited by GppNHp in untreated membranes. In vitro studies with IAP confirmed the results of the radioligand binding studies using in vivo IAP treatment. The effects of NaCl on [3H]angiotensin II binding were also tested but were not typical of other receptors which couple to Ni. The data suggest that, although a small population of hepatic angiotensin II receptors couple to Ni and attenuate glucagon-stimulated increases in cAMP, vasopressin, alpha 1-adrenergic, and the majority of angiotensin II receptors do not interact significantly with Ni. Thus, although there is evidence that agonist-induced Ca2+ mobilization requires a GTP-binding regulatory protein, this protein does not appear to be Ni in rat liver.     

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.     

Modification by islet-activating protein of direct and indirect inhibitory actions of adenosine on rat atrial contraction in relation to cyclic nucleotide metabolism

Adenosine decreased the force of contraction of isolated rat left atria (direct action) and inhibited the positive inotropic action of isoproterenol (indirect action). The direct action of adenosine was not accompanied by changes in cyclic nucleotide levels, while the isoproterenol-induced increase in cyclic AMP level was reduced by the nucleoside. Reserpine pretreatment and pindolol did not affect the direct action of adenosine. Both the direct and indirect actions of adenosine were enhanced by dipyridamole and dilazep and inhibited by 1-methyl-3-isobutylxanthine and theophylline. Among adenosine derivatives, N6-phenylisopropyladenosine was most potent, and 2'-deoxyadenosine was least effective in inducing the negative inotropic action in the absence and presence of isoproterenol. In atria pretreated with islet-activating protein (IAP) both the direct and indirect actions of adenosine, and the actions of N6-phenylisopropyladenosine were markedly attenuated compared with those in nontreated atria. The isoproterenol-induced increase in cyclic AMP level was not reduced by adenosine in IAP-treated atria. These results indicate that adenosine produces a dual action on rat atria: it decreases the force of contraction directly by a mechanism independent of cyclic AMP metabolism, and inhibits the positive inotropic action of isoproterenol by reducing the drug-induced accumulation of cyclic AMP. IAP attenuates both of these inhibitory actions of adenosine on rat atria.     

Effects of scorpion toxin on excitatory and inhibitory presynaptic terminals

The effects of scorpion toxin (STX) on both spontaneous and evoked glycinergic and glutamatergic postsynaptic currents were studied by using both the mechanically dissociated single SDCN neuron (synaptic bouton preparation) and the ‘focal electrical stimulation technique’. In the experimental condition where Na⁺ channels on postsynaptic soma membrane were blocked by intracellular perfusion of QX-314, STX increased dose-dependently the frequency of spontaneous glycinergic and glutamatergic postsynaptic currents (sIPSC and sEPSC, respectively) without affecting the amplitude, suggesting STX acts on inhibitory and excitatory presynaptic nerve terminal. Such a facilitatory effect of STX on sIPSC was stronger than that on sEPSC. On the other hand, STX significantly enhanced the averaged current amplitude and decreased the failure rate (Rf) of both evoked inhibitory and excitatory postsynaptic currents (eIPSC and eEPSC, respectively), indicating that STX increases not only the release frequency of glycine and glutamate but also the amount of their release from the both presynaptic nerve endings. These effects of STX were completely removed by adding Na⁺ or Ca²⁺ channel blockers, indicating that STX increases Ca²⁺ influx through Ca²⁺ channels triggered by activating voltage-dependent Na⁺ channels on the nerve terminals. In addition, the difference of STX actions on the amplitude of spontaneous and evoked currents was discussed.     

Type of lymphocytes affected by the islet-activating protein (IAP)

By flow cytometric analysis, we identified the subclass of lymphocytes that proliferates in response to islet-activating protein (IAP), both in vitro (human peripheral blood mononuclear cells, MNC, cultured with IAP) and in vivo (peripheral blood MNC derived from A/J mice treated with IAP). IAP caused a preferential proliferation of CD8+ T cells. These cells expressed the IL-2 receptors on their surface. CD4+ CD8+ T cells could also be detected in these cultures, IAP caused human MNC to produce IL-1 and to induce expression of HLA-DR antigen. These effects may play an important role in the T-cell proliferation induced by IAP, although IAP by itself suppressed the proliferative action of IL-1 in mouse thymocytes. IAP induced proliferation of the purified CD4+ cells but had a smaller effect on the purified CD8+ cells. This suggests that the proliferation of CD8+ cells in IAP-treated MNC depends on the function of other types of cell, e.g. CD4+ cell and macrophage.     

Effect of pertussis toxin on normorphine-dependence and on acute inhibitory effects of normorphine and clonidine in guinea-pig isolated ileum

In guinea-pig isolated ileum from animals pretreated with Pertussis toxin, the acute inhibitory effects of normorphine and clonidine on electrically induced contractions were markedly attenuated whilst responses to acetylcholine and electrical stimulation were unaltered. Pertussis toxin treatment also reduced naloxone-precipitated withdrawal contractures in normorphine-dependent tissues. These results suggest that the acute and chronic effects of normorphine are mediated by the same mechanism, namely that of adenylate cyclase inhibition.     

Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts.

1. The effect of bacterial toxins on bradykinin-triggered release of arachidonic acid was studied in serum-deprived human foreskin (HSWP) fibroblasts prelabelled with [3H]-arachidonic acid. An 18-h exposure of HSWP cells to cholera toxin, pertussis toxin, or forskolin enhanced the bradykinin-stimulated release of arachidonic acid and metabolites. 2. Prolonged treatment of HSWP cells with these agents also caused a 3 to 4 fold rise in cell surface [3H]-bradykinin binding. The rise was inhibited by concurrent incubation with cycloheximide or actinomycin D. In addition, cholera toxin and foreskolin increased [3H]-bradykinin binding in wildtype PC12 cells, but not in mutant PC12 cells with reduced cyclic AMP-dependent protein kinase type II activity. 3. In conclusion, cholera toxin, pertussis toxin and forskolin enhanced arachidonic acid release in response to bradykinin, and increased the number of bradykinin receptors in HSWP fibroblasts. A cyclic AMP-dependent mechanism appears to mediate the actions of the toxins and forskolin.     

Effects of spinally and supraspinally injected 3-isobutyl-1-methylxanthine, cholera toxin, and pertussis toxin on immobilization stress-induced antinociception in the mouse.

The effects of intracerebroventricular (i.c.v.) and intrathecal (i.t.) 3-isobutyl-1-methylxanthine (IBMX), cholera toxin (CTX) and pertussis toxin (PTX) administration on immobilization-induced antinociception were studied in ICR mice. Antinociception was assessed by the tail-flick assay. Immobilization of the mouse increased inhibition of the tail-flick response for at least 1 h. The pretreatment with i.t. IBMX (0.01-1 ng), but not i.c.v. IBMX, significantly attenuated immobilization-induced inhibition of the tail-flick response. The pretreatments with i.c.v. PTX (0.05-0.5 microg) as well as i.t. CTX, but neither i.c.v. CTX (0.05-0.5 microg) nor i.t. PTX, potentiated the inhibition of the tail-flick response induced by immobilization stress. Our results suggest that spinally located phosphodiesterase appears to be involved in the production of immobilization stress-induced antinociception. In addition, inactivation of supraspinally located PTX-sensitive G-proteins and spinally located CTX-sensitive G-proteins may modulate immobilization stress-induced antinociception.     

Alteration of mast cell responsiveness to adenosine by pertussis toxin

Adenosine potentiates mouse bone marrow-derived mast cell mediator release by a mechanism that appears to involve cell surface adenosine receptors. In an attempt to explore possible interactions between G proteins and adenosine receptors, mast cells were incubated with activated pertussis toxin, an agent that ADP-ribosylates and inactivates some G protein subtypes, prior to challenge with specific antigen or the calcium ionophore A23187. Mast cells preincubated with 10 ng/ml pertussis toxin for at least 2 hr exhibited an inhibition of antigen-induced beta-hexosaminidase and leukotriene C4 release. The ability of adenosine to potentiate beta-hexosaminidase release was attenuated to an even greater degree by pertussis toxin. A23187-stimulated mediator release was not altered by pertussis toxin, although a modest inhibition of the ability of adenosine to enhance A23187-induced beta-hexosaminidase release was observed in pertussis toxin-treated mast cells. Although up to 24-hr exposure to 100 ng/ml pertussis toxin did not alter resting mast cell cyclic AMP levels, the ability of adenosine to elevate cell cyclic AMP concentrations was diminished markedly by doses of the toxin higher than those required to affect mediator release. Neither antigen-stimulated intracellular free calcium level augmentation alone nor the additional potentiation of these levels by adenosine was changed by pertussis toxin treatment. Inositol trisphosphate was generated by mast cells stimulated by IgE-mediated mechanisms, but a preincubation with pertussis toxin did not influence its generation. In summary, adenosine appeared to produce some of its alterations in mast cell biochemical events by a mechanism that was partially inhibited by pertussis toxin. The nature of the G protein linked to the mast cell adenosine receptor is yet to be determined.     

Effects of nifedipine, BAY K 8644, and pertussis toxin on pressor response to sarafotoxin-b in pithed rats.

The pressor actions of sarafotoxin-b (SRTX-b) were examined in pithed rats in the presence of the calcium channel antagonist nifedipine or the calcium channel activator BAY K 8644 intraarterially (i.a.) and also after pretreatment with pertussis toxin intravenously (i.v.). SRTX-b produced dose-dependent pressor effects in the pithed rat. The diastolic blood pressure (DBP) recorded in animals treated with the vehicle was 41 +/- 1 mm Hg; administration of BAY K 8644 0.1 or 0.3 mg/kg increased DBP pressure to 50 +/- 1 and 52 +/- 1 mm Hg, respectively, whereas nifedipine 0.1 or 0.3 mg/kg decreased DBP to 39 +/- 1 and 33 +/- 1 mm Hg, respectively. The actions of SRTX-b were significantly inhibited by nifedipine, whereas BAY K 8644 potentiated the pressor actions of SRTX-b. We observed that animals pretreated with pertussis toxin 25 or 50 micrograms/kg 3 days before we conducted the experiments had significantly lower DBP as compared with saline-treated animals. Treatment with pertussis toxin caused the DBP dose-response curve to SRTX-b to be displaced to the right. These results indicate that a nifedipine-sensitive (presumably extracellular) calcium pool is partly responsible for the pressor response induced by SRTX-b. They further suggest that in vascular smooth muscle, at least in some vascular beds, SRTX-b activates a pertussin toxin-sensitive G-protein that is coupled to a receptor-operated calcium or nonspecific cation channel.     

Effects of spinally and supraspinally injected 3-isobutyl1-methylxanthine,cholera toxin,and pertussis toxin on cold water swimming stress-induced antinociception in the mouse

• 1.1. The cold (4°C) water swimming stress (CWSS) for 3 min significantly increased the inhibition of the tail-flick response in ICR mice.
• 2.2. Pertussis toxin (PTX, 0.05–0.5 μg) in mice pretreated intrathecally (IT) for 6 days attenuated the inhibition of the tail-flick response induced by CWSS. However, intracerebroventricular (ICV) pretreatment with PTX at the same doses did not affect CWSS-induced inhibition of the tail-flick inhibition.
• 3.3. 3-Isobutyl-l-methylxanthine (IBMX, 0.01–1 ng) in mice pretreated IT for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by CWSS. However, IBMX in mice ICV pretreated ICV at the same doses was not effective in attenuating the CWSS-induced inhibition of the tail-flick response.
• 4.4. Neither IT nor ICV pretreatment with cholera toxin (CTX, 0.05–0.5 μg) for 24 hr affected the inhibition of the tail-flick response induced by CWSS.
• 5.5. The ICV or IT injection of PTX, CTX, or IBMX did not affect the basal tail-flick response latency.
• 6.6. It is concluded that spinal, but not supraspinal, PTX-sensitive G-proteins and cAMP phosphodiesterase may be involved in the antinociception produced by CWSS. However, neither spinal nor supraspinal CTX-sensitive G-proteins appear to be involved in mediating the antinociception induced by CWSS.

     

Effect of pertussis toxin on baclofen- and diphenhydramine-induced amnesia

The effect of pretreatment with pertussis toxin at the doses of 0.25 and 0.50 μg per mouse ICV on the amnesic effect produced by baclofen (0.1–4 mg kg⁻¹ IP), diphenhydramine (15–30 mg kg⁻¹ IP) and scopolamine (0.5–5 mg kg⁻¹ IP) was investigated in the mouse passive avoidance test. Ten days after a single injection of pertussis toxin, baclofen (2–4 mg kg⁻¹ IP) amnesia was prevented. By contrast, pertussis toxin had no effect on diphenhydramine- and scopolamine-induced amnesia. Pretreatment with pertussis toxin at both doses used did not impair motor coordination of the mice, as revealed by the rota-rod test. The present results indicate that the activation of pertussis toxin-sensitive G-proteins represents an important transduction step in memory impairment induced by GABA_B (γ-aminobutyric acid B) agonists, but not by antihistaminic and antimuscarinic drugs. Received: 3 June 1997/Final version: 16 October 1997     

Involvement of the inhibitory GTP-binding regulatory protein and a low-affinity benzodiazepine receptor in the inhibitory effect of diazepam on rat brain adenylate cyclase system

The effect of diazepam on the adenylate cyclase system was studied in rat synaptosomal membranes. Micromolar concentrations of diazepam inhibited the cyclase activities in the presence or absence of guanylyl-5'-imidodiphosphate (GppNHp). The inhibitory effect of diazepam was greater on the cyclase activity in the presence of GppNHp than on that in the basal state. This effect of diazepam was not antagonized by Ro15-1788, an antagonist of a high affinity benzodiazepine receptor in the central nervous system. Furthermore, micromolar concentrations of Ro15-1788 had no inhibitory effect on cyclase activities in the presence or absence of GppNHp. In addition, the bromide ion enhanced the inhibition by diazepam of the cyclase activity in the presence of GppNHp, but not the basal activity, although the bromide ion had no effect on both activities in the absence of diazepam. On the other hand, the pretreatment of synaptosomal membranes with GppNHp increased the KD value for [3H]diazepam binding from 98 microM to 198 microM. These data led us to conclude that diazepam inhibits rat brain adenylate cyclase through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and the catalytic protein.