Islet-activating Protein Treatment Research Articles

Acceleration of desipramine-induced changes on the dopamine receptor-coupled adenylate cyclase system by pertussis toxin.

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in striatal membranes from desipramine (DMI)-treated rats (10 mg/kg, b.i.d., for 5 days). GPT exerted the same biphasic effect on basal and DA-stimulated enzyme activity in membranes from DMI-treated rats as on saline-treated rats. Rats were injected intraventricularly once with islet activating protein (IAP), pertussis toxin, and given extended treatment with DMI in order to study the effects on the inhibitory GTP-binding protein (Gi). Gi loses its function as a signal transducer on being ADP-ribosylated selectively by the IAP. D2 inhibition of adenylate cyclase by DA was attenuated by the IAP treatment in both DMI-and saline-treated rats; peak levels of DA plus GTP stimulation shifted from 1 microM to 100 microM GTP. D1 stimulation of adenylate cyclase by DA was also attenuated by the IAP in the DMI-treated rats. Since long-term treatment with DMI (15 mg/kg, once a day, for 3 weeks) resulted in suppression of D1 stimulation similar to that seen in the present findings, uncoupling between D2 receptors and Gi due to IAP treatment might accelerate DMI-induced adaptive changes of dual control of adenylate cyclase system by DA.

An Islet-Activating Protein-Sensitive G-Protein Is Involved in Dopamine Inhibition of Both Angiotensin-Stimulated Inositol Phosphate Production and Human Placental Lactogen Release in Human Trophoblastic Cells*

In isolated human trophoblastic cells, dopamine (DA) significantly inhibited the angiotensin-II (AII)-stimulated inositol phosphate (IP) accumulation by 44 +/- 8% (EC50, 0.5 +/- 0.2 microM) and human placental lactogen (hPL) release by 85 +/- 5% (EC50, 1.0 +/- 0.8 microM). These effects were blocked by sulpiride, a specific D2 antagonist. On the contrary, scherring 23390 (a specific D1 antagonist) and propranolol (a specific beta-adrenergic antagonist) were ineffective, suggesting that these DA effects are mediated through a DA receptor of the D2 subtype. The mechanism by which DA inhibited AII-stimulated inositol phosphate production implicates a GTP-binding protein sensitive to the islet-activating-protein (IAP), since DA's effects on IP accumulation and hPL release were blocked by this toxin. To further characterize this GTP-binding protein, particulate fractions of placental cells were incubated with [alpha-32P]NAD and IAP. Solubilized extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins of 40 and 41 kDa mol wt were specifically ADP ribosylated. They were probably involved in the DA inhibitory processes, since IAP treatment, known to suppress the effects of DA, also reduced the labeling of these two molecules by around 40%. The effects of AII and DA on hPL release appear to be insensitive to the external calcium concentration, since the results were not significantly different in normal (1.8 mM Ca2+) and low Ca2+ (10(-5) M Ca2+) concentrations. On the other hand, increasing the intracellular concentration of cAMP by adding forskolin did not modify the effect of DA on either IP accumulation or hPL release, suggesting that cAMP is not implicated in hPL release from freshly isolated human trophoblastic cells.

The effects of islet activating protein on oral glucose tolerance in the genetically obese fa/fa rat

When tested in insulin-deficient animal models of diabetes, islet activating protein (IAP) has been shown to increase the secretion of insulin and to improve glucose intolerance. The genetically obese fa/fa rat is an animal model of impaired oral glucose tolerance that does not have reduced insulin secretion. In this model IAP treatment increases basal insulin levels, resulting in lower basal glycemia. However, glucose tolerance following an oral glucose load was worsened by IAP. This was found to be due to an exaggerated stimulation of hepatic glucose production (HGP) following glucose, a defect that is already present in the absence of IAP. IAP has been reported to inhibit (by ADP ribosylation) the inhibitory regulatory protein (Ni) of adenylate cyclase. It is therefore suggested that the increased HGP following oral glucose in fa/fa rats either in the absence or in the presence of IAP treatment may result from a cAMP-mediated mechanism. A beta adrenergic activation or a stimulation of glucagon output could therefore be potential candidates responsible for glucose intolerance in obese fa/fa rats.

Coupling of inhibitory GTP binding protein to somatostatin receptors on rat cerebrocortical membranes

We studied the interaction between somatostatin receptors and inhibitory GTP binding protein in rat cerebrocortical membranes. Guanine nucleotides reduced [125I-Tyr1] somatostatin binding to cerebrocortical membranes in a dose-dependent manner with rank order of potency being guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) greater than GTP greater than GMP. Maximum reduction of the binding to 32% of control was observed in the presence of 10(-5) M Gpp(NH)p. Scatchard analysis of the labeled somatostatin binding revealed that the decrease in the binding by Gpp(NH)p was due to the decrease in the binding affinity for somatostatin. Divalent cations, such as Mg++, Mn++, and Ca++, caused an increase in labeled somatostatin binding to membranes with the maximum binding observed at a concentration of 10, 10, 1 mM, respectively. However, Na+ decreased a labeled somatostatin binding in a dose-dependent manner, and half maximum inhibition of the binding was observed at 10 mM Na+. Moreover, Gpp(NH)p and Na+ lowered labeled somatostatin binding in an additive fashion. When cerebrocortical membranes were treated at 37 degrees C for 40 min with various concentrations of Islet-Activating-Protein (IAP), which had been preactivated with dithiothreitol, subsequent labeled somatostatin binding to the membranes was decreased in a dose-dependent manner. 30 micrograms/ml IAP treatment caused a decrease in the binding to 50% of control, which was characterized by the decreased binding affinity without a significant change in the binding capacity. Furthermore, exposure of IAP plus NAD to cerebrocortical membranes caused ADP-ribosylation of a membrane protein with Mr = 41,000 on autoradiogram. Such an IAP treatment of cerebrocortical membranes abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. These results suggest that somatostatin receptors in the brain couple to inhibitory GTP binding protein, which mediates adenylate cyclase inhibition by somatostatin.

Tonic inhibition of renal response to vasopressin by a pertussis toxin substrate

The putative role of the inhibitory guanine nucleotide binding protein (Gi) in modulating the renal response to vasopressin was investigated using islet activating protein (IAP). IAP treatment in rats in vivo abolished the capacity of alpha 2-adrenoceptors to reverse vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in microdissected cortical collecting tubule (CCT) segments. IAP pretreatment also caused a marked upward shift in the dose-response curve of vasopressin (10(-10) to 10(-4) M)-induced cAMP accumulation. Augmentation of the response to vasopressin in rat CCT was dependent on the in vivo dose of IAP and paralleled the loss in alpha 2-adrenoceptor responsiveness. In the isolated perfused kidney the antinatriuretic and antidiuretic effects of the V2-receptor agonist desamino-8-D-arginine vasopressin (DDAVP) (1 pM) were enhanced following IAP pretreatment. alpha 2-Adrenoceptor stimulation (30 nM epinephrine) inhibited the renal effects of DDAVP (1 pM) in kidneys from control but not IAP-pretreated rats. Interestingly, IAP pretreatment alone caused increased urine flow rate and enhanced excretion of sodium and chloride without affecting potassium excretion or renal hemodynamics in vitro. Our results suggest that an IAP substrate, probably Gi, 1) is required for signal transduction by renal alpha 2-adrenoceptors, 2) may tonically modulate the response to vasopressin in the CCT but not of parathyroid hormone in the proximal convoluted tubule, and 3) participates in renal water and electrolyte reabsorption independent of exogenous adenylate cyclase stimulation.

Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells.

Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

Possible involvement of pertussis toxin substrates (Gi, Go) in desipramine-induced refractoriness of adenylate cyclase in cerebral cortices of rats.

To evaluate the efficiency of coupling between beta-receptor and adenylate cyclase catalyst via a GTP-binding protein, Gs, in the brain membrane two parameters were employed: a beta-agonist-induced increase in the membrane GTP-dependent adenylate cyclase activity and a beta-agonist-induced shortening of the lag time preceding the onset of the steady-state activation by guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p] of the membrane cyclase. Both parameters showed lower values in membranes from desipramine-treated rats compared with untreated rats. Thus, coupling of beta-adrenergic receptors to adenylate cyclase in the brain membrane was impaired by the desipramine treatment. Rats once injected intraventricularly with islet-activating protein (IAP), pertussis toxin, were subjected to desipramine treatment, for the purpose of studying effects of another kind of the GTP-binding protein (Gi), which loses its function as a signal transducer on being ADP-ribosylated selectively by the toxin. IAP treatment did not impair the beta-receptor coupling by itself, since neither of the above two parameters for the coupling were reduced by IAP treatment. Moreover, the first parameter was normalized, though the second one was not, by superimposition of the IAP treatment upon the desipramine-treated rats. It seems likely, therefore, that Gi interacts with a Gs-adenylate cyclase coupling in an inhibitory fashion in brain membranes. The desensitization might be overcome when the inhibitory interaction of Gi on the subsequent process is attenuated by IAP treatment.

Modification of neuronal muscarinic receptor-mediated responses by islet-activating protein

Islet-activating protein (IAP) was used to investigate the role of the guanosine triphosphate binding proteins G i and/or G o in muscarinic acetylcholine receptor-mediated responses in neuroblastoma cells (clone N1E-115). Incubation of intact cells for 24 h with 20 ng/ml IAP resulted in inhibition of subsequent IAP catalyzed incorporation of [ 32P]ADP-ribose into a membrane protein doublet of molecular weight 40,000 (G iα and G oα). IAP treatment fully blocked muscarinic receptor-mediated inhibition of cAMP accumulation. Incubation of intact cells with carbachol for 8 h resulted in the concentration dependent loss of membrane muscarinic receptor. Pretreatment of cells with IAP prior to carbachol exposure partially blocked the subsequent decrease in receptor number. Pretreatment of cells with IAP had no effect on the ability of carbachol to stimulate phosphoinositide hydrolysis in neuroblastoma cells. Thus, while the guanosine triphosphate binding proteins G i and/or G o are involved in coupling the muscarinic receptor to some of the physiological responses in these cells, it is clear that activation of phospholipase C by the muscarinic receptor is a G i/G o independent response.

Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability

The involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.

Inhibition by islet-activating protein of a chemotactic peptide-induced early breakdown of inositol phospholipids and Ca2+ mobilization in guinea pig neutrophils.

Receptors for a chemotactic peptide (fMet-Leu-Phe) in guinea pig neutrophils were primarily coupled to phospholipase C catalyzing breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate, which was in turn responsible for intracellular Ca2+ mobilization. These early responses of neutrophils to fMet-Leu-Phe, eventually leading to O2- generation, were abolished by prior exposure of cells to islet-activating protein (IAP), pertussis toxin, which had been reported to bring about ADP-ribosylation of a membrane Mr = 41,000 protein (Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13863-13871). The IAP substrate, probably the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (Ni) or an analogous protein, is hence proposed to mediate fMet-Leu-Phe receptor-linked activation of the phospholipase C. In support of this proposal, A23187 and phorbol myristate acetate which stimulate arachidonate release or O2- generation by-passing these early processes of signaling were effective in IAP-treated cells as well. Release of arachidonic acid and accumulation of inositol 1-monophosphate in delayed response to fMet-Leu-Phe were also abolished by the IAP treatment of cells, despite the fact that slowly-onset inflow of Ca2+ which must be responsible for these delayed responses was observed in these IAP-treated cells. Thus, the IAP substrate may play an additional role in Ca2+-dependent activation of somehow compartmentalized phospholipases.

Attenuation of muscarinic cholinergic inhibition by islet-activating protein in the heart.

Experiments were carried out to investigate the influence of islet-activating protein (IAP), a pertussis toxin, on the autonomic regulation of cardiac function. In atria isolated from rats given intravenous IAP (0.125-1.0 micrograms/100 g body wt) 12-72 h prior to experiments, the negative chronotropic and inotropic actions of carbachol were attenuated in a dose- and time-dependent manner. The inhibitory action of carbachol in the absence of isoproterenol and its attenuation by IAP were not associated with any changes in tissue adenosine 3',5'-cyclic monophosphate (cAMP) levels. In addition, carbachol decreased the isoproterenol-induced elevation of cAMP levels and inhibited the positive chronotropic and inotropic responses to isoproterenol. The inhibitory action of carbachol on the isoproterenol-induced functional and cAMP responses was also reduced by the IAP treatment. The increase in tissue guanosine 3',5'-cyclic monophosphate (cGMP) levels produced by carbachol was likewise abolished by the IAP treatment. These results indicate that IAP treatment attenuates the inhibitory actions of carbachol elicited via muscarinic receptors through both cAMP-dependent and cAMP-independent subcellular processes.

Islet-activating protein discriminates between different inhibitors of thyroidal cyclic AMP system

TSH-induced cyclic AMP accumulation in dog thyroid slices is inhibited by norepinephrine through an α 2-adrenergic receptor, by carbamylcholine through a muscarinic cholinergic receptor, and by iodide. The inhibitory effect of iodide bears on the adenylate cyclase, but the exact mechanism of its action is still unknown. It is known that norepinephrine acts through activation of the N 1 subunit of the cyclase, and that carbamylcholine, activating a phosphodiesterase, acts independently of N i IAP (islet-activating protein) has been shown to inactivate the N i subunit. We studied the effect of IAP on the inhibitory action of iodide, norepinephrine, and carbamylcholine on cyclic AMP accumulation in TSH-stimulated thyroid slices. Incubations of 15 or 22 h, and relatively high concentrations of IAP (250 ng ml ) were necessary to demonstrate an effect of IAP on thyroid slices. We report here that, under those conditions, inhibition of cyclic AMP accumulation by norepinephrine, but not by carbamylcholine or iodide, was suppressed by IAP treatment. These results indicate that the cyclase inhibition by iodide, is either not mediated by N i or if mediated by N i involves a mode of regulation of this coupling protein that is different from that by which the other 'N i-mediated' inhibitory hormones act on the enzyme.

Does the guanine nucleotide regulatory protein Ni mediate progesterone inhibition of Xenopus oocyte adenylate cyclase?

In Xenopus laevis oocytes progesterone is able to inhibit directly the plasma membrane adenylate cyclase activity and induce reinitiation of meiotic maturation. To determine whether progesterone inhibition is mediated by the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase, Ni, the effect of the Bordetella pertussis toxin (IAP) and limited proteolysis on progesterone action in oocytes was investigated. Treatment of oocyte membranes with islet activating protein (IAP) in the presence of [32P]NAD led to incorporation of radiolabel into a 41 000-dalton membrane protein. However, exposure of isolated oocytes to 100 ng/ml IAP for up to 24 h, or oocyte membranes with concentrations of toxin as high as 100 micrograms/ml, had no effect on either progesterone inhibition of adenylate cyclase or induction of maturation. Similarly, limited alpha-chymotrypsin proteolysis of oocyte membranes failed to modify progesterone-induced inhibition of adenylate cyclase. In contrast, inhibition of human platelet adenylate cyclase by epinephrine, acting via a GTP-dependent, alpha 2-adrenergic receptor-mediated pathway, is almost completely abolished by both IAP treatment and limited proteolysis of platelet membranes. These data indicate that unlike attenuation of platelet enzyme activity, the inhibition of adenylate cyclase in oocyte membranes by progesterone does not occur via a classical Ni-mediated pathway.

Mechanism of pertussis toxin action on the adenylate cyclase system. Inhibition of the turn-on reaction of the inhibitory regulatory site.

The activation reaction of the inhibitory guanine nucleotide-binding regulatory site of the adenylate cyclase system was studied in membranes of rat adipocytes, S49 lymphoma wild-type cells and their cyc- variants, pretreated without and with the Bordetella pertussis toxin, islet-activating protein (IAP), by measuring the kinetics of adenylate cyclase inhibition by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The IAP treatment, which caused a loss of GTP and hormone-induced adenylate cyclase inhibition, did not prevent enzyme inhibition by the stable GTP analogue. However, in either cell type studied, pretreated with IAP, the lag phase of GTP[S] inhibitory action was largely increased by about fivefold compared to control membranes. Similar to the controls, the lag phase of GTP[S] inhibition of adenylate cyclase in membranes of IAP-pretreated cells was shortened in the presence of an inhibitory hormone. Furthermore, the lag phase of inhibition by GTP[S] was decreased with increasing concentrations of Mg2+. The data indicate that the pertussis toxin does not principally prevent an interaction of the inhibitory guanine nucleotide regulatory site of the adenylate cyclase system with either the catalytic moiety or an inhibitory hormone receptor. The data, furthermore, suggest that the toxin inhibits the activation reaction (turn-on reaction) of the inhibitory coupling component. This inhibition, which may take place at a Mg2+-binding site, can account for the observed functional loss of GTP and hormone-induced adenylate cyclase inhibition after IAP treatment.

Effect of islet activating protein on glucose tolerance, insulin secretion and insulin responsiveness in the NZO mouse.

The effect of islet activating protein on glucose tolerance, insulin secretion and insulin responsiveness was studied in the NZO mouse, a model of non-insulin dependent diabetes and obesity. A single IV injection of 5 ng/g body weight islet activating protein markedly lowered plasma glucose and the glucose response to IP glucose administration, measured 5 days later (mean +/- SEM, plasma glucose levels 0, 10, 30 and 60 min after glucose 6.0 +/- 0.9, 14.6 +/- 1.3, 14.1 +/- 1.3 and 13.2 +/- 1.7 mmol/l in islet activating protein-treated NZO mice versus 12.8 +/- 1.6, 27.8 +/- 3.4, 34.7 +/- 4.1, 39.1 +/- 3.8 mmol/l in carrier-treated NZO mice). There was no difference in fasting plasma insulin levels between islet activating protein and carrier-treated mice. No response of plasma insulin to glucose occurred in the carrier-treated mice, but a highly significant insulin response to glucose was seen in the islet activating protein-treated mice. The in vitro responsiveness of pancreatic islets of islet activating protein-treated NZO mice to glucose was improved, and the inhibitory effect of adrenaline on insulin secretion was reduced. The in vivo hypoglycaemic response to exogenous insulin was not improved by islet activating protein and a demonstrated defect in the insulin sensitivity and responsiveness of glucose utilization by isolated soleus muscle was not reversed by islet activating protein treatment. It is concluded that islet activating protein is highly effective in improving glucose tolerance and insulin secretion in NZO mice, and that the improvement in glucose tolerance occurs without demonstrable improvement in the responsiveness to exogenous insulin or sensitivity of soleus muscle to insulin.

Effect of islet-activating protein (IAP) on contractile responses of rat vas deferens: Evidence for participation of Ni(inhibitory GTP binding regulating protein) in the α2-adrenoceptor-mediated response

The effects of islet-activating protein (IAP) on the contractile responses of vas deferens to exogenous norepinephrine (NE) and electric stimulation (ES) were investigated. IAP treatment had no effect on the contractile response to NE. Pretreatment of the vas deferens with IAP (2.0 μg/ml) for 2 h however significantly attenuated the inhibitory effect of clonidine on ES-induced contractile responses, suggesting that feedback inhibition of presynaptic α 2-adrenoceptors is mediated by the Ni subunit of presynaptic adenylate cyclase.

Islet-activating protein prevents nicotinic acid-induced GTPase stimulation and GTP but not GTPγS-induced adenylate cyclase inhibition in rat adipocytes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTPγS, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells.

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.

Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP ribosylation by islet-activating protein, pertussis toxin, in adipocyte membranes.

Adenylate cyclase of rat adipocyte membranes exhibited dual responses in a strictly GTP-dependent manner; an activation took place in the presence of certain receptor agonists such as isoproterenol or secretin, whereas an inhibitory phase was observed with other agonists such as prostaglandin E1 or purine-modified adenosine as well as with the stimulatory agonists at higher GTP concentrations. Treatment of membrane donor cells with islet-activating protein (IAP), pertussis toxin, abolished the inhibitory phase while preserving the activatory phase. This unique action of IAP was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. In contrast, the inhibitory phase was preserved in membranes from cholera toxin-treated cells. Monophasic and persistent activation of the cyclase was provoked by guanyl-5'-yl beta,gamma-imidodiphosphate. The time lag normally observed for the guanyl-5'-yl beta,gamma-imidodiphosphate activation was decreased by isoproterenol or cholera toxin but was not altered by IAP treatment. Our conclusion is that the sole site of IAP action is the guanine nucleotide regulatory protein (Ni) that is required for transmission of inhibitory signals from receptors to the catalytic unit of adenylate cyclase; the function of Ni is lost upon IAP-catalyzed ADP ribosylation of the Mr = 41,000 protein which appears to be an active subunit of Ni. A possibility is discussed that rather diverse effects of IAP so far reported with various cell types are accounted for in terms of such interference with the function of Ni.

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane M r = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.