PTX-sensitive G-protein Research Articles

Activation of δ-Opioid Receptors Inhibits Neuronal-Like Calcium Channels and Distal Steps of Ca2+-Dependent Secretion in Human Small-Cell Lung Carcinoma Cells

Human small-cell lung carcinoma (SCLC) cells express neuronal-like voltage-operated calcium channels (VOCCs) and release mitogenic hormones such as serotonin (5-HT). Opioid peptides, on the other hand, have been shown to reduce SCLC cell proliferation by an effective autocrine pathway. Here we show that in GLC8 SCLC cells, only delta-opioid receptor subtype mRNA is expressed. Consistently, the selective delta-opioid agonist [D-Pen2-Pen5]-enkephalin (DPDPE), but not mu and kappa agonists, potently and dose-dependently inhibits high-threshold (HVA) VOCCs in these cells. As in peripheral neurons, this modulation is largely voltage-dependent, mediated by pertussis toxin (PTX)-sensitive G-proteins, cAMP-independent, and mainly affecting N-type VOCCs. With the same potency and selectivity, DPDPE also antagonizes the Ca(2+)-dependent release of [3H]serotonin ([3H]5-HT) from GLC8 cells. However, DPDPE inhibits not only the depolarization-induced release, but also the Ca(2+)-dependent secretion induced by thapsigargin or ionomycin. This suggests that besides inhibiting HVA VOCCs, opioids also exert a direct depressive action on the secretory apparatus in GLC8 cells. This latter effect also is mediated by a PTX-sensitive G-protein but, contrary to VOCC inhibition, it can be reversed by elevations of cAMP levels. These results show for the first time that opioids effectively depress both Ca2+ influx and Ca(2+)-dependent hormone release in SCLC cells by using multiple modulatory pathways. It can be speculated that the two mechanisms may contribute to the opioid antimitogenic action on lung neuroendocrine carcinoma cells.

Thapsigargin activates univalent- and bivalent-cation entry in human neutrophils by a SK&F I3 96365- and Gd3+-sensitive pathway and is a partial secretagogue: involvement of pertussis-toxin-sensitive G-proteins and protein phosphatases 1/2A and 2B in the signal-transduction pathway

The Ca2+-ATPase inhibitor thapsigargin (TG) activates bivalent-cation early in human neutrophils via depletion of intracellular Ca2+ stores bu little is known about the underlying mechanism and the functional role of TG-induced cation entry. We studied the effects of TG on univalent- and bivalent cation entry, lysozyme release and superoxide-anion (O2-) formation in human neutrophils. TG, like the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), stimulated entry of Ca2+, Mn2+, Ba2+, Sr2+ and Na+ in a 1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365)- and Gd3+-sensitive manner. The inhibitors of protein phosphates 1/2A, calyculin A and okadaic acid, diminished TG-induced cation influxes, whereas the inhibitors of protein phosphatase 2B, cyclosporin A and FK-506, were potentiators. Pertussis toxin (PTX) partially inhibited the effects of TG on Ca2+ and Mn2+ entry. TG and fMLP activated inward currents with a linear current-voltage relationship and a reversal potential at about 0 mV. TG activated lysozyme release and potentiated fMLP-induced O2- formation. TG-induced lysozyme release was inhibited by SK&F 96365, PTX and the removal of extracellular Ca2+ or Na+. Our data show that TG activates a non-selective and SK&F 96365- and Gd3+-sensitive cation entry pathway and is a partial secretagogue. TG-stimulated cation entry involves PTX-sensitive G-proteins and protein phosphatases, with protein phosphatases 1/2A and 2B playing opposite roles.

Endothelin-1 inhibitsl-type Ca currents enhanced by isoproterenol in guinea-pig ventricular myocytes

To investigate the action of endothelin-1 (ET-1) on L-type Ca currents (ICa,L) in guinea-pig ventricular cells, whole-cell currents were recorded at approximately 36-37 degrees C in enzymatically isolated myocytes. ET-1 (> or =10 nM) suppressed the basal ICa,L to 79+/-8% of control at 20 nM. Bath application of isoproterenol (ISO; 10 nM) enhanced ICa,L to 192+/-28% with about a -10-mV shift of its relationship with membrane potential. ET-1 concentration dependently inhibited this ISO-enhanced ICa,L with a half-maximally inhibitory concentration (IC50) of 168 pM. The inhibitory actions of ET-1 were antagonised by BQ-123 (300 nM), cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp), a specific ETA receptor antagonist. Histamine-enhanced ICa,L was also suppressed by ET-1, but ICa, L potentiated by internal adenosine 3',5'-cyclic monophosphate (cAMP) was unaffected. Preincubation of myocytes with pertussis toxin (PTX, at 5 microgram/ml for >60 min at 36 degrees C) completely occluded the ET-1 action. Thus, stimulation of ETA receptors by subnanomolar ET-1 inhibits ICa,L via PTX-sensitive G-proteins.

Selective inhibition of high voltage-activated L-type and Q-type Ca2+ currents by serotonin in rat melanotrophs.

1. Whole-cell Ca2+ currents (ICa) from cultured rat melanotrophs were identified by their sensitivity to Ca2+ channel blockers, and their modulation by serotonin (5-HT) was studied. All cells displayed high voltage-activated (HVA; > -30 mV) Ca2+ currents. A low voltage-activated (LVA; > -60 mV) Ca2+ current was detected in 92% of the cells. 2. The whole-cell ICa was insensitive to omega-conotoxin GVIA (0.5-1 microM) indicating the absence of N-type Ca2+ channels. 3. At a holding potential (Vh) of -70 mV, the L-type channel blocker nifedipine reduced ICa in a dose-dependent manner with a half-maximal effective concentration (IC50) of 28 nM. The L-type current represented 39% of the total ICa. 4. omega-Agatoxin IVA (omega-Aga IVA) produced a biphasic dose-dependent inhibition of ICa, with IC50 values of 0.4 and 91 nM, indicating the presence of P-type and Q-type Ca2+ channels, which accounted respectively for 16 and 45% of the total ICa. The P-type current was also blocked by synthetic funnel-web spider toxin (sFTX 3.3; 1-10 microM) and was present only in a subpopulation (60-70%) of cells. 5. All cells possessed a Ca2+ current which was resistant to nifedipine (10 microM) and omega-Aga IVA (50 nM). This current was not affected by Ni2+ (40 microM) but was abolished by a low concentration of Cd2+ (10 microM) and by omega-conotoxin MVIIC (1 microM) indicating that it was a Q-type Ca2+ current. 6. 5-HT (10 microM) inhibited the whole-cell ICa in 70% of the cells tested (n = 120) by activating 5-HT1A and 5-HT2C receptors. 5-HT produced either a kinetic slowing of the activation phase (37% of the cells) or a scaling down (14% of the cells) of ICa. In the majority of cells (49%) both types of inhibition were found to coexist. 7. The effects of 5-HT were voltage dependent, rendered irreversible when GTP-gamma-S (30 microM) was present in the pipette solution and abolished by pretreatment of the cells with pertussis toxin (PTX; 150 ng ml-1, 18 h). 8. Low concentrations of omega-Aga IVA (20 nM), which blocked mainly P-type channels, did not reduce the effect of 5-HT on ICa. The scaling down effect of 5-HT on ICa was eliminated in the presence of nifedipine (10 microM) and the kinetic slowing effect of 5-HT persisted after blockade of L- and P-type channels but was abolished by omega-conotoxin MVIIC (1 microM). 9. We conclude that rat melanotrophs possess functional L-, P- and Q-type Ca2+ channels and that 5-HT inhibits selectively L-type and Q-type Ca2+ currents with different modalities. These effects are voltage dependent and mediated by a PTX-sensitive G-protein.

Serotonin modulates high-voltage-activated CA 2+ channels in rat ventromedial hypothalamic neurons

The modulation of high-voltage-activated (HVA) Ca 2+ channels by serotonin (5-HT) was studied in ventromedial hypothalamic (VMH) neurons acutely dissociated from 12–14-day-old Wistar rats using the whole-cell and nystatin perforated-patch recording configurations. 5-HT inhibited the HVA Ca 2+ channels in a concentration-, time- and voltage-dependent manner. This inhibition was mimicked by the 5-HT 1A agonist 8-hydroxy-dipropylaminotetralin and was prevented by pretreatment with pertussis toxin (PTX). ω-Conotoxin-GVIA, ω-agatoxin-IVA, nicardipine and ω-conotoxin-MVIIC blocked each fraction of HVA Ca 2+ channel currents, suggesting the existence of N-, P-, L- and Q-types of HVA Ca 2+ channels. A component of the current resistant to these Ca 2+ channel antagonists also existed in the VMH neurons. Among these five components of HVA Ca 2+ channel currents, the N- and Q-type currents were significantly inhibited by 5-HT. These findings suggest that the activation of 5-HT 1A receptors produces the selective inhibition of N- and Q-type Ca 2+ channels through a PTX-sensitive G-protein in rat VMH neurons.

Receptor-independent G protein activation may account for the stimulatory effects of first-generation H 1-receptor antagonists in HL-60 cells, basophils, and mast cells

The first-generation histamine H 1-receptor antagonists, chlorpheniramine (CPHE) and diphenhydramine (DPH), may activate histamine release from basophils and mast cells. Because CPHE and DPH are cationic-amphiphilic and because several substances with such physicochemical properties activate heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in a receptor-independent manner, we asked the question of whether or not H 1-receptor antagonists could be G-protein activators as well. In dibutyryl cAMP-differentiated HL-60 cells, CPHE and DPH increased cytosolic Ca 2+ concentration and azurophilic granule release in pertussis toxin (PTX)-sensitive manners. In HL-60 membranes, PTX-sensitive stimulations of GTPase [E.C. 3.6.1.-] and binding of guanosine 5′-[γ-thio]triphosphate by H 1 receptor antagonists were observed. CPHE and DPH also increased GTP hydrolysis by the purified PTX-sensitive G-protein, transducin. In all- trans-retinoic acid-differentiated HL-60 cells and rat basophilic leukemia cells (RBL 2H3 cells), H 1-receptor antagonists induced, unlike in dibutyryl cAMP-differentiated HL-60 cells, Ca 2+ influx without Ca 2+ mobilization from intracellular stores. CPHE and DPH also induced serotonin release from RBL 2H3 cells. Our data indicate that first-generation H 1-receptor antagonists are receptor-independent G-protein activators and that such a mechanism of action accounts for their stimulatory effects in HL-60 cells, basophils, and mast cells.

Multiplicative interaction between intrathecally and intracerebroventricularly administered morphine for antinociception in the mouse: Effects of spinally and supraspinally injected 3-isobutyl-l-methylxanthine, cholera toxin, and pertussis toxin

1. 1. Either intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration of morphine alone at the dose of 0.2 μg slightly increased inhibition of the tail-flick response. However, combined i.t. and i.c.v. injections of morphine at the same dose increased the inhibition of the tail-flick response in a synergistic manner. 2. 2. Cholera toxin (CTX, 0.05 to 0.5 μg) pretreated i.t. or i.c.v. for 24 hr or pertussis toxin (PTX, 0.05 to 0.5 μg) for 6 days dose-dependently attenuated inhibition of the tail-flick response induced by combined i.t. and i.c.v. injection of morphine. 3. 3. 3-Isobutyl-l-methylxanthine (IBMX, 0.001 to 0.1 ng) pretreated i.t. for 10 min dose-dependently attenuated the inhibition of the tail-flick response induced by combined i.t. and i.c.v. injections of morphine. However, IBMX pretreated i.c.v. for 10 min was not effective in attenuating the inhibition of the tail-flick response induced by combined i.t. and i.c.v. injections of morphine. 4. 4. It is concluded that both spinal and supraspinal CTX- and PTX-sensitive G-proteins are involved in the antinociception produced by morphine-induced multiplicative interaction between spinal and supraspinal sites. However, only spinal but not supraspinal cAMP phosphodiesterase is involved in mediating antinociception induced by morphine-induced multiplicative interaction.

Withdrawal of somatostatin augments L-type Ca2+ current in primary cultured rat somatotrophs.

It is known that withdrawal of somatostatin (SRIF) augments the growth hormone (GH) releasing hormone (GRF)-induced GH secretion. To investigate the mechanism of this augmentation in GH secretion, effects of GRF and SRIF on L-type Ca2+ current (Ba2+ was used as a charge carrier) or primary cultured rat somatotroph were studied by perforated patch clamp technique. The reason is that GRF-induced GH secretion is thought to be causally related to the influx of Ca2+ through L-type Ca2+ channels. 10 mM GRF augmented maximum amplitude of L-type Ba2+ current by 12.2% (n = 12). Subsequent application of SRIF slightly suppressed the currents but the suppression never exceeded the control level of the current. Removal of SRIF, however, promptly augmented the L-type Ba2+ current by 26.8%. Such off-response of SRIF was not observed in cells treated overnight with 100 ng/ml pertussis toxin. Further, specific inhibitor of protein kinase A, H-89 at 1 microM reversibly suppressed the augmentation of L-type Ba2+ current to control level. At 10 microM, H-89 suppressed L-type Ba2+ current by more than 40% from control level. These results suggest that (1) L-type Ca2+ channel of somatotroph is probably phosphorylated in a basal condition and may be slightly modulated by GRF through increased level of cAMP; (2) SRIF only slightly suppress the channel activity; (3) Withdrawal of SRIF facilitates the activity of L-type Ca2+ channel via PTX-sensitive G-protein, although the precise mechanism of this facilitation is unknown. The augmentation by SRIF-pretreatment of GRF-induced GH secretion may be at least partly due to the facilitation of the activity of L-type Ca2+ channel.

Role of pertussis toxin-sensitive G-proteins in the analgesic and anesthetic actions of alpha 2-adrenergic agonists in the rat.

alpha 2 Adrenoceptors are coupled to G-proteins sensitive to pertussis toxin (PTX) in the locus coeruleus. At this site, the hypnotic response to dexmedetomidine, an alpha 2 agonist, can be blocked by pretreatment with PTX. G-proteins sensitive to PTX may also be involved in the transduction of anesthetic and analgesic responses to alpha 2 agonists at supraspinal or spinal sites. To address this question the effects of pretreatment with PTX administered intracerebroventricularly, intrathecally, or a combination of the two were examined on the MAC for halothane, and the anesthetic-sparing and analgesic effects of a systemically administered alpha 2 agonist, dexmedetomidine. Rats were cannulated intracerebroventricularly, intrathecally, and with a combination of intracerebroventricular/intrathecal and treated with PTX (0 and 2.5 micrograms intracerebroventricularly; 0 or 0.5 microgram intrathecally; 0 + 0 or 2.5 + 0.5 intracerebroventricular-intrathecal)). After 7 days, either the analgesic (tail-flick latency) or the MAC-sparing effects of a calculated 50% effective dose of dexmedetomidine were measured. To confirm that intracerebroventricularly administered PTX was effective, ribosylation of G-proteins was assessed in periventricular brain tissue. The analgesic action of dexmedetomidine was blocked by PTX intrathecally but not by PTX via the intracerebroventricular route. The MAC-sparing action of dexmedetomidine was not blocked by PTX via the intrathecal or intracerebroventricular routes alone or in combination. Yet, intracerebroventricularly administered PTX effectively ribosylated the G-proteins. Taken together with the authors' previous report, these data suggest that the hypnotic and the analgesic responses to dexmedetomidine are transduced via PTX-sensitive G-protein-coupled alpha 2 adrenoceptors but at separate sites (analgesic-spinal; hypnotic-locus coeruleus). Further studies are needed to localize the precise site(s) for the MAC-sparing effect of dexmedetomidine and to establish whether PTX-sensitive G-proteins are involved in this response.

A comparison of post-receptor signal transduction events in Jurkat cells transfected with either IL-8R1 or IL-8R2 Chemokine mediated activation of p42/p44 MAP-kinase (ERK-2)

The CXC chemokine, IL-8, is a potent chemoattractant of neutrophils and binds to two distinct receptors, termed IL-8R1 and IL-8R2. These receptors share high affinity for IL-8, however, only IL-8R1 is specific for IL-8 whereas IL-8R2 binds other related chemokines, including GROα with high affinity. Stable Jurkat transfectants were generated expressing either functional IL-8R1 or IL-8R2 (J-IL8R1 and J-IL812). Both J-IL8R1 and J-IL8R2 exhibited high affinity IL-8 binding ( K d 3–5nM) with respective receptor densities of23,000 ± 3,000 and18,500 ± 1,500. Pre-treatment of both transfectants with 1.0 μg/ml B. pertussis toxin (PTx) resulted in inhibition of IL-8 mediated intracellular Ca 2+ mobilisation and chemotaxis, without altering the receptor's affinity for its ligand. This indicates that both receptors couple to a PTx-sensitive G-protein. Further studies showed that IL-8R1 and IL-8R2 could mediate time-dependent phosphorylation of p42/p44 MAP-kinase. In both transfectants, phosphorylation was maximal at 1–2 min after IL-8 stimulation and could be inhibited by PTx. Stimulation of J-IL8R1 and J-IL8R2 with GROα revealed that this chemokine was a more potent activator of MAP-kinase in J-IL8R2, an observation reflected in the high affinity binding of GROα to IL-8R2. These studies indicate that chemokines are capable of activating protein kinases and with regards to PTx-sensitivity and MAP-kinase stimulation, no significant differences between IL- 8R1 and IL-8R2 post-receptor signalling occur during cell activation by IL-8.

Muscarine increases a voltage-independent potassium conductance through an M 4 receptor in rat dorsolateral septal nucleus neurons

The direct effect of triuscarine on neurons of the rat dorsolateral septal nucleus (DLSN) was examined by using conventional microelectrode and voltage-clamp techniques. Muscarine (1–50μM) caused a hyperpolarization accompanied by an increase of a voltage-independent potassium conductance. Pirenzepine competitively antagonized the muscarine-induced hyperpolarization with an apparent dissociation constant ( K d) value of 54 nM. Furthermore, intracellular loading with GTPyS, a non-hydrolyzable GTP analog, blocked irreversibly the muscarine-induced hyperpolarization. In addition, pretreatment of neurons with pertussis toxin (PTX) prevented the hyperpolarization produced by muscarine. These results suggest that muscarine hyperpolarizes DLSN neurons via a voltage-independent potassium conductance by acting at M 4 subtype receptors which are coupled to a PTX-sensitive G-protein in DLSN neurons.

Cationic-amphiphilic arpromidine-derived guanidines and a histamine trifluoromethyl-toluidide derivative may activate pertussis toxin-sensitive G-proteins by a receptor-independent mechanism

Formyl peptides activate superoxide anion (O2-) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O2- formation via H2-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H2-receptor agonists in the guinea pig atrium, on O2- formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O2- formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mumol/l inhibited and, at concentrations above 30 mumol/l, enhanced formyl peptide-induce O2- formation in neutrophils. In HL-60 cells, guanidines per se activated O2- formation. The stimulatory effects of guanidines on O2- formation were not inhibited by H1- or H2-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and Gi/G(o)-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O2- formation.

Histamine receptor-dependent and/or -independent activation of guanine nucleotide-binding proteins by histamine and 2-substituted histamine derivatives in human leukemia (HL-60) and human erythroleukemia (HEL) cells

In dibutyryl cAMP-differentiated human leukemia (HL-60) cells, the potent histamine H 1-receptor agonist, 2-(3-chlorophenyl)histamine, activates pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins) of the G i-subfamily by a mechanism which is independent of known histamine receptor subtypes (Seifert et al. Mol Pharmacol 45: 578–586, 1994). In order to learn more about this G-protein activation, we studied the effects of histamine and various 2-substituted histamine derivatives in various cell types and on purified G-proteins. In HL-60 cells, histamine and 2-methylhistamine increased cytosolic Ca 2+ concentration ([Ca 2+] i) in a clemastine-sensitive manner. Phenyl- and thienyl-substituted histamines increased [Ca 2+] i as well, but their effects were not inhibited by histamine receptor antagonists. 2-Substituted histamines activated high-affinity GTPase in HL-60 cell membranes in a PTX-sensitive manner, with the lipophilicity of substances increasing their effectiveness. Although HEL cells do not possess histamine receptors mediating rises in [Ca 2+] i, 2-(3-bromophenyl)histamine increased [Ca 2+] i in a PTX-sensitive manner. It also increased GTP hydrolysis by G i-proteins in HEL cell membranes. All these stimulatory effects of 2-substituted histamine derivatives were seen at concentrations higher than those required for activation of H 1-receptors. In various other cell types and membrane systems, 2-substituted histamine derivatives showed no or only weak stimulatory effects on G-proteins. 2-Substituted histamine derivatives activated GTP hydrolysis by purified bovine brain G i/G o-proteins and by pure G i2 (the major PTX-sensitive G-protein in HL-60 and HEL cells). Our data suggest the following: (1) histamine and 2-methylhistamine act as H 1-receptor agonists in HL-60 cells; (2) incorporation of bulky and lipophilic groups results in loss of H 1-agonistic activity of 2-substituted histamine derivatives in HL-60 cells but causes a receptor-independent G-protein-stimulatory activity; (3) the effects of 2-substituted histamine derivatives on G-proteins are cell-type specific.

Expression of human platelet-activating factor receptor gene in EoL-1 cells following butyrate-induced differentiation.

Platelet-activating factor (PAF) is a potent lipid mediator of allergic inflammation through its interaction with eosinophils. Expression of the PAF receptor is modulated by many agents, including those responsible for cell differentiation. We report here that differentiation of a human eosinophilic leukaemia cell line, EoL-1, by sodium n-butyrate is associated with induction of PAF receptor gene expression, as indicated by: PAF receptor mRNA accumulation; increases in the binding of [3H]WEB 2086, a PAF antagonist; analysis of cell-surface expression of PAF receptor protein using a monoclonal anti-(PAF receptor) antibody; and augmentation of PAF-induced increase in the intracellular concentration of calcium. Using cDNA cloning, the receptor expressed in EoL-1 cells was identified as 'Transcript 1', one of two transcripts which was previously reported from human genomic analysis (Mutoh, Bito, Minami, Nakamura, Honda, Izumi, Nakata, Kurachi, Terano and Shimizu (1993) FEBS Lett. 322, 129-134). The PAF-induced calcium response and phosphoinositide turnover were decreased by pertussis toxin (PTX) treatment, suggesting that these signals are coupled largely with PTX-sensitive G-protein(s) in EoL-1 cells. These systems may provide a useful experimental model with which to investigate the relationship between eosinophilic differentiation and PAF receptor induction, and the role of eosinophils in allergic responses.

Muscarinic receptors modulate N-, P-, and L-type Ca2+ currents in rat striatal neurons through parallel pathways

Muscarinic modulation of calcium currents was studied in acutely isolated striatal neurons from the adult rat using the whole-cell configuration of the patch-clamp technique. Muscarinic agonists reduced calcium currents through two distinct signaling pathways. One pathway depended upon PTX-sensitive G-proteins and targeted N- and P-type currents. The other pathway depended upon PTX-insensitive G-proteins and was rendered inactive by high intracellular concentrations of BAPTA and targeted L-type currents. The modulation of N- and P-type currents was relieved by strong depolarizing prepulses, whereas the modulation of L-type currents was not. These findings support the proposition that parallel signaling pathways exist between muscarinic receptors and calcium channels.

β-Amyloid peptide, substance P, and SEC receptor ligand activate cytoplasmic Ca2+ in neutrophil-like HL-60 cells: Effect of chemotactic peptide antagonist BocMLF

It has been reported that a discrete peptide fragment of beta-amyloid protein, beta A(25-35), and neuropeptide substance P (SP) possessed sequence homology and could bind to the serine protease inhibitor (serpin) enzyme complex (SEC) receptor. Thus, it has been thought that these peptides and SEC receptor ligand might have similar biological activities. In the present study, we found that C-terminal amidated beta A(25-35)-NH2, SP, and the SEC receptor ligand, Phe-Val-Phe-Leu-Met(FVFLM), could induce an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neutrophil-like human leukemic (HL-60) cells. Pretreatment with pertussis toxin (PTX) potently inhibited the increase in [Ca2+]i stimulated by these peptides, suggesting that these responses might be mediated by PTX-sensitive G-proteins. Furthermore, we examined the effect on these responses of t-butyloxycarbonyl-methionyl-leucyl-phenylalanine (BocMLF), which is a competitive antagonist of chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF) at its receptor. BocMLF scarcely inhibited the [Ca2+]i increase stimulated by beta A(25-35)-NH2. However, the increase in FVFLM-induced [Ca2+]i was potently inhibited by BocMLF. The results suggest that the [Ca2+]i activation of beta A(25-35)-NH2 may have a different mechanism from that of FVFLM in neutrophil-like HL-60 cells, which is not mediated by the SEC-receptor.

ET-1 Stimulates Ca2+ Currents in Cardiac Cells

Using the whole-cell voltage-clamp technique, endothelin (ET-1) was found to stimulate T- and L-type Ca2+ currents in a dose-dependent manner in both human and chick ventricular single cells. However, ET-1 had no effect on both the fast sodium current and the delayed outward K+ current in these cells. The effect of ET-1 on both Ca2+ currents was blocked by the ET(A) receptor antagonist BQ123. Treatment of single ventricular cells with pertussis toxin (PTX) prevented stimulation of T- and L-type calcium currents by ET-1. These results suggest that there are functional ET(A) receptors in both human and chick ventricular cells. The stimulation of both types of Ca2+ currents appears to be mediated by a PTX-sensitive G-protein.

G-protein involvement in muscarinic receptor-stimulation of inositol phosphates in longitudinal smooth muscle from the small intestine of the guinea-pig.

1. Aluminium fluoride (AlF), pertussis toxin (PTX) and cholera toxin (ChTX) have been used to examine the involvement of G-proteins during muscarinic acetylcholine receptor (AChR) stimulation of inositol phospholipid hydrolysis in fragments of longitudinal smooth muscle from the small intestine of the guinea-pig. 2. Carbachol (CCh) induced time- and concentration-dependent increases in [3H]-inositol monophosphates, [3H]-inositol (1,4) bisphosphate, [3H]-inositol (1,3,4) trisphosphate, [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins (1,4,5)P3) and [3H]-inositol tetrakisphosphates measured by h.p.l.c. These increases were inhibited > 95% in the presence of the muscarinic AChR antagonist atropine (0.5 microM). 3. AlF transiently increased the basal levels of [3H]-Ins (1,4,5)P3 but increases in the levels of the other [3H]-inositol phosphates occurred more slowly. CCh-induced increases in the levels of all the [3H]-inositol phosphates were strongly inhibited in the presence of AlF. 4. PTX had no effect on basal levels of any of the [3H]-inositol phosphates but reduced the effects of CCh on these; ChTX had no effects on either basal or CCh-stimulated levels. 5. It was concluded that muscarinic AChR-stimulated increases in the levels of [3H]-inositol phosphates occur via both a PTX-sensitive G-protein and a PTX-insensitive mechanism. The actions of AlF may suggest the involvement of an inhibitory G-protein in the regulation of muscarinic AChR-stimulated inositol phospholipid turnover.

ET-1 Stimulates Ca2+ Currents in Cardiac Cells

Summary: Using the whole-cell voltage-clamp technique, endothelin (ET-1) was found to stimulate T- and L-type Ca2+ currents in a dose-dependent manner in both human and chick ventricular single cells. However, ET-1 had no effect on both the fast sodium current and the delayed outward K+ current in these cells. The effect of ET-1 on both Ca2+ currents was blocked by the ETA receptor antagonist BQ123. Treatment of single ventricular cells with pertussis toxin (PTX) prevented stimulation of T-and L-type calcium currents by ET-1. These results suggest that there are functional ETA receptors in both human and chick ventricular cells. The stimulation of both types of Ca2+ currents appears to be mediated by a PTX-sensitive G-protein.

Mastoparan may activate GTP hydrolysis by Gi-proteins in HL-60 membranes indirectly through interaction with nucleoside diphosphate kinase.

The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX)-sensitive G-proteins in a receptor-independent manner. We studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cells and a reconstituted system and on nucleoside diphosphate kinase (NDPK)-catalysed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an EC50 of 1-2 microM and a maximum at 10 microM. Unlike the effects of the formyl peptide receptor agonist, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only partially PTX-sensitive. MP-induced rises in cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells were also only incompletely PTX-sensitive. N-Ethylmaleimide inhibited MP-stimulated GTP hydrolysis to a greater extent than that stimulated by fMet-Leu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalysed ADP-ribosylation of, Gi-protein alpha-subunits in HL-60 membranes. By contrast to fMet-Leu-Phe, MP did not or only weakly stimulated binding of guanosine 5'-[gamma-thio]triphosphate to Gi-protein alpha-subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted Gi/G(o)-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [3H]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (1) MP activates Gi-proteins in HL-60 cells, but (2) the venom does not simply mimic receptor activation. (3) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (4) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective.