Pertussis Toxin-sensitive Guanine Nucleotide Binding Research Articles

μ-opioid agonist-induced activation of G-protein-coupled inwardly rectifying potassium current in rat periaqueductal gray neurons

The characteristics of the inwardly rectifying K + current activated by a μ-type opioid agonist, d-Ala 2,N-MePhe 4,Gly 5-ol-enkephalin (DAMGO), were examined in the acutely dissociated rat periaqueductal gray neurons using the nystatin-perforated and the conventional whole-cell recording modes under voltage-clamp conditions. DAMGO activated inward currents in a concentration- and voltage-dependent manner. The DAMGO-induced current was an inwardly rectifying K + current ( I DAMGO) which was sensitive to K + channel blockers, quinine and Ba 2+ but insensitive to Cs + and tetraethylammonium. In the conventional whole-cell clamp mode, guanosine 5′- O-(2-thiodiphosphate) trilithium salt (GDP βs, 0.4 mM) inhibited the amplitude of I DAMGO to 28% of that of the initial current. After the intracellular perfusion with guanosine 5′-[loc]O-(3-thiotriphosphate) tetralithium salt (GTP γs, 0.4 mM) for 1 min, the first application of DAMGO irreversibly activated I DAMGO. By the extracellular application of N-ethylmaleimide at a concentration of 50 μM for 2 min, I DAMGO was completely abolished. When a conventional whole-cell patch was made with a patch-pipette containing 1 μg/ml of pertussis toxin together with 1 mM of β-nicotinamide adenine dinucleotide, I DAMGO gradually declined to about 41% of its initial amplitude. The extracellular application of second messenger modulators including protein kinase inhibitor (staurosporin), protein kinase A activators (forskolin, 3-isobutyl-1-methyl-xanthine and dibutyryladenosine 3′5′-cyclic monophosphate) and protein kinase C activators (phorbol-12-myristate-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol) had no effect on I DAMGO. These results suggest that (i) DAMGO-activated inwardly rectifying K + current is mediated by pertussis toxin-sensitive guanine nucleotide binding proteins (G-proteins); (ii) the types of G protein involved in I DAMGO are G i and/or G o; and (iii) the G-proteins exert their roles in I DAMGO without any mediation of the second messenger systems.

Muscarinic regulation of cyclic AMP in bovine trachealis cells.

The goal of this study was to characterize the receptors and coupling mechanisms mediating muscarinic inhibition of adenylyl cyclase activity in bovine tracheal smooth muscle. In radioligand binding experiments, methoctramine and AF-DX 116 competed for approximately 85% of the 3H-quinuclidynyl benzilate (3H-QNB) binding sites on intact cells with high affinities (-log KI of 7.73 +/- 0.16 and 6.67 +/- 0.31, respectively) characteristic of binding to M2 receptors. The antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) bound the receptors on intact cells with an affinity (-log KI = 7.76 +/- 0.21) characteristic of binding at M2 receptors. In experiments measuring 3',5'-cyclic adenosine monophosphate (cAMP) accumulation, methoctramine, AF-DX 116, and 4-DAMP antagonized the inhibitory effect of carbachol on isoproterenol-stimulated cAMP accumulation with potencies consistent with mediation by M2 muscarinic receptors (-log Kb of 8.01 +/- 0.22 to 7.58 +/- 0.25 for methoctramine; 7.43 +/- 0.36 to 7.02 +/- 0.30 for AF-DX 116; and 7.60 +/- 0.21 for 4-DAMP). In other experiments, 24 +/- 3% of the inhibitory effect of carbachol was not reversed by 60 min exposure to atropine. Moreover, pertussis toxin (10, 250, and 1,000 ng/ml) decreased only a portion of the inhibitory effect of carbachol (8 +/- 19%, 32 +/- 10%, and 33 +/- 8%, respectively) on cAMP accumulation. These findings indicated that M2 receptors were coupled to adenylyl cyclase in trachealis cells, but that coupling mechanisms in addition to those of pertussis toxin-sensitive guanine nucleotide binding proteins were involved. Since the inhibitory effect of carbachol (10(-8) M) on isoproterenol-stimulated cAMP accumulation was decreased from 20 +/- 4% to -1 +/- 5% (n = 6) by okadaic acid (1 microM), protein phosphatases may regulate the processes coupling muscarinic receptors to adenylyl cyclase.

Association of guinea pig lung bombesin receptors with pertussis toxin-sensitive guanine nucleotide binding proteins

The possible interaction of bombesin receptors with guanine nucleotide binding protein in guinea pig lung was studied. The non-hydrolysable GTP analogue guanosine-5′-[γ-thio]triphosphate (GTPγS) was shown to decrease [ 125I-Tyr 4]bombesin binding in a concentration-dependent manner. The specificity of this effect was assessed by examining the effects of other guanine nucleotides on this binding at a concentration of 1mM. GMP and GDP weakly inhibited [ 125I-Tyr 4]bombesin binding (2 and 19%, respectively), whereas GTP, guanosine-5′-[β-thio]triphosphate (GDPβS), and 5-guanylylimidodiphosphate (GppNHp) exhibited similar potencies, inducing 52%, 46%, and 43% inhibition of [ 125I-Tyr 4]bombesin binding respectively. Saturation experiments performed in the absence and presence of 100 μM GTPγS indicated the presence of a single population of receptors in both cases. However, the addition of GTPγS induced a marked decrease in the number of receptors (from 1.76 fmol/mg protein to 0.78 fmol/mg protein) without significantly altering the dissociation constant ( K d). These results provide evidence that bombesin receptors are coupled to a G-protein signal transduction pathway in guinea pig lung. We have further characterised this G-protein on the basis of its toxin sensitivity. Pretreatment of the lung membranes with either pertussus (10 μm/ml) or cholera toxin (50 μg/ml) was performed. Cholera toxin treatment did not affect the ability of GTPγS to inhibit [ 125I-Tyr 4]bombesin binding to guinea pig lung membranes. However, pertussis toxin treatment induced a decrease in binding and resulted in the inability of GTPγS to inhibit [ 125I-Tyr 4]bombesin binding in a concentration-dependent manner. These results suggest that bombesin receptors of the guinea pig lung interact with pertussis toxin-sensitive G-proteins.

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction.

In gallbladder smooth muscle, carbachol interacts with M3 receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [3H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [3H]inositol monophosphates, [3H]inositol bisphosphates and [3H]inositol trisphosphates and contraction were measured at various times (0-90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 micrograms/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [32P]ADP-ribosylation of a approximately 40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [3H]inositol monophosphates, [3H]inositol bisphosphates and [3H]inositol trisphosphates. The time course of [3H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [3H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [3H]inositol trisphosphates and on contraction were abolished by atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta modulates the parathyroid hormone-related protein-induced responses in renal epithelial cells.

PTH-related protein (PTHrP), the major mediator of hypercalcemia of malignancy, reduces tubular phosphate (Pi) reabsorption through its PTH-like renotropic actions. Another peptide detected in tumoral cells, transforming growth factor-beta (TGF beta), has been shown to considerably suppress the sodium-dependent Pi transport system present in the apical membrane of renal epithelial cells. The unexplored interactions between TGF beta and PTHrP were examined in opossum kidney (OK) cells. Using confluent OK cells, we showed that TGF beta attenuated the inhibition of Pi transport mediated by PTHrP. Similarly, 18 h TGF beta incubation resulted in a substantial reduction of the cAMP response elicited by PTHrP without apparent involvement of pertussis toxin-sensitive guanine nucleotide binding protein(s). The number of PTHrP(1-34) binding sites in TGF beta-treated cells was decreased with the affinity unchanged. Forskolin- and prostaglandin E2-stimulated cAMP productions were not significantly altered by TGF beta treatment. Therefore, TGF beta reduced Pi transport in OK cells, modulated the actions of PTHrP, and decreased its receptor number. Whether this happens in vivo is as yet unknown.

Transforming growth factor-beta modulates the parathyroid hormone- related protein-induced responses in renal epithelial cells

PTH-related protein (PTHrP), the major mediator of hypercalcemia of malignancy, reduces tubular phosphate (Pi) reabsorption through its PTH-like renotropic actions. Another peptide detected in tumoral cells, transforming growth factor-beta (TGF beta), has been shown to considerably suppress the sodium-dependent Pi transport system present in the apical membrane of renal epithelial cells. The unexplored interactions between TGF beta and PTHrP were examined in opossum kidney (OK) cells. Using confluent OK cells, we showed that TGF beta attenuated the inhibition of Pi transport mediated by PTHrP. Similarly, 18 h TGF beta incubation resulted in a substantial reduction of the cAMP response elicited by PTHrP without apparent involvement of pertussis toxin-sensitive guanine nucleotide binding protein(s). The number of PTHrP(1-34) binding sites in TGF beta-treated cells was decreased with the affinity unchanged. Forskolin- and prostaglandin E2-stimulated cAMP productions were not significantly altered by TGF beta treatment. Therefore, TGF beta reduced Pi transport in OK cells, modulated the actions of PTHrP, and decreased its receptor number. Whether this happens in vivo is as yet unknown.

Immunological characterization of the guanine-nucleotide binding proteins Gi and Go in rat islets of Langerhans.

Inhibition of insulin secretion from rat islets of Langerhans is known to involve at least one pertussis toxin-sensitive guanine-nucleotide binding (G) protein. We have used antisera raised against unique antigenic determinants of different members of the family of pertussis toxin-sensitive G proteins to identify these proteins in rat islets. Antiserum SG1, which recognizes both Gi1 and Gi2, reacted with an islet protein having an approximate Mr of 40,000. Antiserum I1C (Gi1 specific) failed to recognize any islet proteins, suggesting that Gi2 is present in much greater amounts than Gi1. Indeed, Gi1 levels were below the detection limit of a sensitive immunogold/silver-staining method, indicating that it may be absent from the cells of rat islets. Two different antisera were used to identify Go-like G proteins in rat islet homogenates. Both antisera reacted with a protein band which, under appropriate conditions, could be resolved to reveal two separate proteins of Mr 39-40,000. Thus, at least two molecular forms of Go are present in rat islets. Subcellular fractionation indicated that all three G proteins identified in this study (Gi2 and two forms of Go) are localized to islet membranes. No immunoreactivity could be detected in the cytosolic fraction.

Muscarinic regulation of membrane ion channels in airway smooth muscle cells

We have demonstrated that stimulation of airway smooth muscle by muscarinic agonists results in a coordinated modulation of two membrane ion channel proteins. Both channels are modulated in a similar way, although their effects on open-channel probability are opposite. The voltage dependence of channel activity is shifted to more positive potentials in the case of K Ca, and to more negative potentials in the case of the voltage-dependent calcium channels. Similarly, K Ca channel dwell-time kinetics are shifted to short open lifetimes, whereas the long open state is favored for the large-amplitude voltage-dependent calcium channel. Although little is known about the molecular coupling of calcium channels, muscarinic inhibition of K Ca channels is mediated through a pertussis toxin-sensitive guanine nucleotide binding protein.

Angiotensin II induces calcium release in a subpopulation of single ovarian (granulosa) cells

The effects of angiotensin II on cytosolic free Ca 2+ ion concentrations ([Ca 2+] i were studied in single porcine granulosa cells using the calcium-sensitive fluorescent dye fura-2 and high temporal resolution fluorescent videomicroscopy. Angiotensin II initiated specific, rapid, transient and topographically organized increases in [Ca 2+] i in a subpopulation of single swine granulosa cells. The Ca 2+ source for this angiotensin II-mediated [Ca 2+] i transient appeared to be internal stores, and a pertussis toxin-sensitive guanine nucleotide binding protein was implicated in this receptor-mediated Ca 2+ rise. Our single-cell studies also revealed a striking functional heterogeneity among granulosa cells, since folliclestimulating hormone-responsive cells were not angiotensin II responsive. We conclude that single swine granulosa cells are targets of specific angiotensin II action on intracellular pools of Ca 2+.

Thrombin signalling in U937 human monocytic cells is coupled to inositol phosphate formation but not to thromboxane B 2 synthesis nor to inhibition of adenylate cyclase: distinct differences in thrombin signalling between U937 cells and platelets

The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have use the U937 human monocytic cell line to explore the signal transduction mechanisms utilised by thrombin in these cells. In U937 cells differentiated into a macrophage-like phenotype. thrombin stimulated the formation of inositol trisphosphate (IP 3) and the mobilisation of intracellular Ca 2+ ([Ca 2+] i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx)B 2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formy-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB 2 synthesis under conditions where it evoked increases in IP 3 formation and [Ca 2+] i mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thrombin also failed to cause inhibitory guanine nucleotide binding protein (G i)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. Those results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP 3 and the mobilisation of [Ca 2+ i. However, the failure of thrombin to stimulate TxB 2 synthesis or cause G i-mediated inhibition of adenylate cyclase in U937 cells contrasts with its effects in human platelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin receptor or receptor-effector coupling mechanism(s) present in other thrombin-responsive cells.

Signal Transduction in Human Alveolar Macrophages: Diminished Chemotactic Response to FMLP Correlates with a Diminished Density of Gi Proteins and FMLP Receptors

Alveolar macrophages (AM) migrate less well in response to chemotactic ligands than do monocytes and neutrophils. The response of monocytes and neutrophils to chemotactic ligands is mediated at least in part by pertussis toxin-sensitive guanine nucleotide binding proteins (Gi proteins). Whether this is also true in AM is uncertain. We hypothesized that decreased chemotaxis by AM was due in part to diminished Gi protein and/or chemotactic receptor density in AM. G proteins are heterotrimers made up of alpha, beta, and gamma subunits; the predominant pertussis toxin-sensitive Gi proteins are those containing alpha i2 or alpha i3 subunits. Pertussis toxin pretreatment (0.5 microgram/ml) significantly reduced AM, monocyte, and neutrophil chemotaxis to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and human zymosan-activated serum (P less than 0.05). However, as previously noted, AM chemotaxis was much less than that observed in monocytes and neutrophils. Immunoblots using antibodies that are specific for alpha i2 and alpha i3 showed that AM contained approximately 3-fold less alpha i2 and approximately 10-fold less alpha i3 per microgram of plasma membrane protein than did monocytes or neutrophils. Similar results were obtained in immunoblots made using antibodies to common alpha subunit determinants and to the beta 36 subunit. A comparable approximately 4-fold reduction in density of receptors for [3H]FMLP was found in AM compared to neutrophils. The diminished density of Gi proteins and FMLP receptors was not due to a generally decreased density of plasma membrane proteins in AM, since the density of the membrane-associated tyrosine kinase hck was similar in AM, monocytes, and neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation.

We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor, suggesting that the peptide amides may act as ionophores or channel-formers. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

Angiotensin II and Proximal Tubule Sodium Transport

As a target site for angiotensin II (A-II), renal proximal tubule is unique in that it may be equipped with a local A-II generating system and that both basolateral and apical membranes may be accessible for A-II's action. We have recently conducted studies to examine these possibilities. With in vitro cultured proximal tubular cells, we have demonstrated de novo synthesis of angiotensinogen and renin. With isolated renal brush border membrane (BBM), we have confirmed the presence of A-II receptors and found that A-II directly stimulated BBM Na(+)-H+ exchange. In search of the signal transduction mechanism, we have found that A-II also activated BBM phospholipase A2 (PLA) and that BBM contained a pertussis toxin-sensitive guanine nucleotide binding protein (G-protein) which mediates the effects of A-II. Further studies showed that prevention of PLA activation abolished A-II's effect on Na(+)-H+ exchange, and that activation of PLA by mellitin and addition of arachidonic acid similarly enhanced Na(+)-H+ exchange activity, suggesting that PLA activation may mediate the stimulatory effect of A-II on Na(+)-H+ exchange. These results thus indicate that a local signal transduction mechanism involving G-protein mediated PLA activation exists in renal BBM which mediates A-II's effect on Na(+)-H+ exchange. Taken together, we propose that, independent of A-II in the circulation, local luminal A-II may serve as an important regulatory system on sodium transport in renal proximal tubule.

Limited distribution of pertussis toxin in rat brain after injection into the lateral cerebral ventricles

In vivo administration of pertussis toxin is often used to study the involvement of guanine nucleotide binding proteins in signal transduction. Especially when it is administered in the brain the effect is often poor. This could be due to the fact that pertussis toxin does not reach the area of interest. To evaluate the extent to which pertussis toxin is distributed in rat brain after intraventricular injection, different techniques were used. Immunohistochemical studies with an antibody against pertussis toxin showed that immunoreactivity was limited to peri ventricular brain structures less than 0.5 mm from the lumen. The highest immunoreactivity was seen 16–24 h after injection. After 96 h the labeling was very weak. The proportion of guanine nucleotide binding proteins that were ADP-ribosylated by in vivo injection of pertussis toxin into the ventricles as assessed by in vitro [ 32P]-back-ADP-ribosylation was very low 48 h after the injection, in all regions studied. Direct injection of pertussis toxin into the brain caused a marked ADP-ribosylation localized to the region injected that was maximal at 72 h after injection. At 96 h there were also effects after control injections, indicating non-specific effects. Synaptosomal membranes and other membranes were equally affected by pertussis toxin. The results suggest that in studies regarding the effect of pertussis toxin treatment on signal transduction, the toxin must be injected very close to the brain region of interest and, furthermore, that the rats should be killed 48–72 h after injection. In case of lack of effect on the response of interest one should examine whether the ADP-ribosylation of pertussis toxin-sensitive guanine nucleotide binding proteins in the area of concern has been affected.

Ca2+]i rises via G protein during regulatory volume decrease in rabbit proximal tubule cells

Although animal cells swell in hypotonic medium, their volume is subsequently regulated by a net loss of KCl via Ca2(+)-dependent channels. A rise in intracellular free calcium ([Ca2+]i) thus appears to be an initial event in the adaptation of external tonicity, although details of this mechanism are not known. To investigate cell volume regulation, we measured [Ca2+]i (by use of fura-2) and cell diameters in single cells of cultured renal proximal convoluted tubule. We found that a rapid rise in [Ca2+]i occurred after cells were exposed to hypotonic solution (250 mosM) from 95.8 +/- 3.8 to 468.2 +/- 24 nM (n = 16). The rise in [Ca2+]i was not observed in cells exposed to Ca2(+)-free medium, and exposure to isotonic high-K or low-Na medium did not elicit a rise in [Ca2+]i, suggesting that this rise was a result of Ca2+ influx and not via voltage-dependent Ca2+ influx or decrease of Ca2+ efflux via Na(+)-Ca2+ pump. Pretreatment of cells with pertussis toxin dose dependently blocked the rise in [Ca2+]i. The hypotonic solution enhanced accumulations of inositol tris- and tetra-phosphate after a 1-min exposure. Studies that measured cell diameters suggest that recovery of cell volume may include the rise in [Ca2+]i. These data suggest that the regulatory volume decrease of proximal tubule cells involves a pertussis toxin-sensitive guanine nucleotide binding protein-operated Ca2+ influx.

Somatostatin-14 and somatostatin-28 inhibit calcium currents in rat neocortical neurons

The prosomatostatin-derived peptides, somatostatin-14 and somatostatin-28, are believed to function as neurotransmitters or neuromodulators in the cerebral cortex. To investigate the molecular mechanisms by which these peptides induce their physiological effects in the cerebral cortex, we have examined the effects of somatostatin-14 and somatostatin-28 on voltage-dependent Ca 2+ currents in rat neocortical neurons in culture. Ca 2+ currents were recorded using whole-cell patch-clamp techniques under conditions in which K + and Na + currents were blocked. Ca 2+ currents were induced by depolarization from the holding potential of -80 mV. Somatostatin-14 (100 nM) and somatostatin-28 (100 nM) did not significantly affect low-voltage activated Ca 2+ currents, but blocked high-voltage activated Ca 2+ currents and slowed the activation of this current. The effects of both peptides were concentration-dependent and reversible. Furthermore, the effects of somatostatin-14 and somatostatin-28 on the high-voltage activated Ca 2+ currents were not additive, suggesting that both peptides regulate this ionic current through similar cellular mechanisms. When patch pipettes used to record the Ca 2+ currents contained 100 μ M cAMP and 0.5 mM isobutylmethylxanthine, a phosphodiesterase inhibitor, somatostatin-14 and somatostatin-28 still inhibited Ca 2+ currents, indicating that the effects of these peptides on the Ca 2+ currents were cAMP-independent. Inclusion of the non-hydrolysable guanine triphosphate analogue, guanine triphos-phate-gamma-thiol (500 μM) in the recording electrode produced effects similar to those induced by somatostatin-14 or somatostatin-28, suggesting the involvement of guanine nucleotide binding proteins in the actions of the peptides on the Ca 2+ currents. This hypothesis is supported by the finding that pretreatment of the neurons with pertussis toxin greatly reduced the abilities of somatostatin-14 and somatostatin-28 to inhibit Ca 2+ currents. Our results suggest that somatostatin-14 and somatostatin-28 inhibit Ca 2+ currents of rat neocortical neurons through a cAMP-independent mechanism involving pertussis toxin-sensitive guanine nucleotide binding proteins.

Extracellular ATP stimulates polyphosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells: Involvement of a pertussis toxin-sensitive guanine nucleotide binding protein and feedback inhibition by protein kinase C

ATP stimulated a rapid and dose-dependent formation of inositol polyphosphates in rat glomerular mesangial cells. In parallel there was a 80% increase in 1, 2-diacylglycerol (DAG) after 15 s upon stimulation with ATP. The rank order of potency of a series of ATP and ADP analogoues for stimulation of inositol trisphosphate (InsP 3) formation was ATP > ATPγS > βγ-methylene-ATP > βγ-imido-ATP > ADP, while ADPβs, AMP, adenosine and GTP were inactive, indicating the presence of P 2y-purinergic receptors. ATP also stimulated a marked synthesis of prostaglandin E 2 (PGE2). The reank order of potency of different ATP and ADP analogues was identical to that of InsP 3 generation. Pre-treatment of the cells with pertussis toxin strongly attenuated ATP-induced formation of InsP 3 and DAG. Short-term (10 min) pre-treatment of the cells with 12- O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, produced a dose-dependent inhibition of the ATP-stimulated InsP 3 generation. Furthermore, inhibition of protein kinase C by the potent inhibito staurosporin, or downregulation of protein Kinase C by longterm (24 h) incubation of the cells with TPA, resulted in an enhanced formation of InsP 3 towards a stimulation with ATP.

Foetal calf serum enhances cholera toxin-catalysed ADP-ribosylation of the pertussis toxin-sensitive guanine nucleotide binding protein, G i2, in rat glioma C6BU1 cells

The major G-protein of rat glioma C6BU1 cells corresponds immunologically to G i2. In the absence of guanine nucleotides, this protein is shown to be a substrate for ADP-ribosylation catalysed by both cholera and pertussis toxins. Under these conditions, a receptor for a growth factor, which has previously been shown to be activated by foetal calf serum, modulated the effects of both cholera and pertussis toxins on the G-protein. These ligand-mediated alterations of cholera and pertussis toxin-catalysed ADP ribosylation demonstrated that, in this system, the growth factor receptor interacts functionally with G i2.

Carbachol inhibits stimulant-induced increases in fundic D-cell cytosolic Ca2+ concentration.

We undertook these studies to examine the mechanisms by which carbachol inhibits somatostatin release. For these studies, we utilized cultured D-cells isolated from the canine gastric fundus. Carbachol inhibited somatostatin release induced by both pentagastrin and 12-O-tetradecanoyl-phorbol-13-acetate but did not alter the redistribution of protein kinase C induced by these agents. In contrast, carbachol diminished the increase in D-cell cytosolic free calcium levels ([Ca2+]i) induced by pentagastrin, and this effect was no longer evident after pretreatment of D-cells with pertussis toxin. Although carbachol by itself had no effect on [Ca2+]i, after pretreatment of D-cells with pertussis toxin, carbachol both enhanced [Ca2+]i and stimulated somatostatin release. These data indicate that carbachol activates signals in D-cells that result in both increase and decrease in [Ca2+]i. The latter effect, which appears to be mediated via a pertussis toxin-sensitive guanine nucleotide binding protein, may be one mechanism responsible for cholinergic inhibition of somatostatin release.

Somatostatin receptors on canine fundic D-cells: evidence for autocrine regulation of gastric somatostatin

Somatostatin is known to have inhibitory effects on the release and action of a wide variety of gut peptides. Previous studies in vivo have suggested a potential inhibitory role for somatostatin even on its own secretion. To determine whether this autoregulatory effect is the result of a direct action of the peptide on the cell that is responsible for its secretion, we examined the effect of a non-immunoreactive but biologically active analogue of somatostatin ([Leu8-D-Trp22-Tyr25]S28) on release of somatostatin-like immunoreactivity (SLI) from isolated canine fundic D-cells. We identified somatostatin binding sites with dissociation constants of 1.2 X 10(-9) and 3.8 X 10(-8) M that coenriched with D-cells. Somatostatin 14, somatostatin 28, and [Leu8-D-Trp22-Tyr25]S28 were equivalent in displacing 125I-[Leu8-D-Trp22-Tyr25]S28 from the binding sites. [Leu8-D-Trp22-Tyr25]S28 inhibited SLI release from D-cells stimulated with (DBcAMP), and pentagastrin. Pertussis toxin pretreatment prevented the inhibitory effects of [Leu8-D-Trp22-Tyr25]S28 on both SLI secretion and cAMP accumulation by on both SLI secretion and cAMP accumulation by D-cells stimulated with epinephrine and forskolin. In contrast, [Leu8-D-Trp22-Tyr25]S28 inhibition of SLI release induced by DBcAMP and pentagastrin was not altered by pertussis toxin. Our data suggest that somatostatin autoregulates its own secretion via specific receptors on D-cells. This inhibitory effect is mediated by mechanisms that are both dependent on and independent of pertussis toxin-sensitive inhibitory guanine nucleotide binding proteins.