Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells.

Muscarinic receptor-linked Ca2+ mobilization and changes in cyclic AMP were studied in SH-SY5Y and IMR 32 human neuroblastoma cell lines. Muscarinic agonists acetylcholine, carbachol, methacholine and muscarine induced an increase in cytosolic free Ca2+ in a pertussis toxin (100 ng/ml)-insensitive manner in both cell lines. The ED50 values in IMR 32 cells (8-98 microM) were one order of magnitude higher than in SH-SY5Y cells (0.3-1.6 microM). Oxotremorine and pilocarpine failed to mobilize Ca2+ in IMR 32 cells. Pirenzepine antagonized carbachol-induced Ca2+ mobilization in SH-SY5Y cells with a Ki value in the range of 150-189 nM whereas the corresponding values in IMR 32 cells were 24-28 nM. Atropine inhibited a carbachol-stimulated increase in cytosolic Ca2+ with an equal potency in both cell lines (Ki 2-3 nM). Carbachol stimulated cyclic AMP (cAMP) accumulation in SH-SY5Y cells in a pertussis toxin-insensitive manner. In IMR 32 cells carbachol inhibited prostaglandin E1-stimulated cAMP accumulation. Treatment of IMR 32 cells with pertussis toxin abolished the inhibition of stimulated cAMP accumulation. These results suggest that in SH-SY5Y cells the M3 muscarinic receptor couples to both Ca2+ mobilization and stimulation of cAMP accumulation. In IMR 32 cells the M1 receptor seems to couple to Ca2+ mobilization whereas the inhibition of stimulated cAMP accumulation is coupled to a non-M1 subtype by an inhibitory G-protein.     

Prostaglandin receptors in NIH 3T3 cells: coupling of one receptor to adenylate cyclase and of a second receptor to phospholipase C.

In intact NIH 3T3 murine fibroblasts, prostaglandins (PGs) F2 alpha and E2 induce dose-dependent stimulation of inositol monophosphate generation. PGF2 alpha is greater than 50-fold more potent than PGE2 in eliciting this response. In streptolysin O-permeabilized NIH 3T3 cells, PGF2 alpha and PGE2 induced dose-dependent accumulations of inositol bis- and trisphosphates, which were dependent on the presence of the guanine nucleotide guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) (10 microM). Pretreatment of cells for 16 hr with 100 nM PGF2 alpha resulted in a significant reduction of not only subsequent PGF2 alpha- and PGE2-induced but also GTP gamma S-induced stimulation of inositol phosphate formation in permeabilized cells. PGF2 alpha-induced accumulation of inositol phosphates was partially inhibited by pretreatment with pertussis toxin (1 microgram/ml, 4 hr). The inhibition by pertussis toxin was small but was not related to cyclic AMP formation, because forskolin, which activates adenylate cyclase, did not mimic pertussis toxin-induced inhibition. In the same cell line, PGF2 alpha and PGE2 induced a dose-dependent accumulation of cAMP and a dose-dependent potentiation of 0.5 microM forskolin-stimulated cAMP formation. PGF2 alpha and PGE2 were almost equipotent in eliciting both responses. However, PGF2 alpha was less efficacious than PGE2 and, in the presence of forskolin, PGF2 alpha at 10 microM induced an inhibitory effect on cAMP accumulation. Such inhibition may be related to PGF2 alpha-mediated phospholipase C activation and subsequent stimulation of protein kinase C, because the phorbol ester phorbol 12-myristate-13-acetate, which directly activates protein kinase C, also inhibited forskolin- and PGE2-induced cAMP accumulation. Pretreatment with PGF2 alpha for 16 hr did not reduce subsequent stimulation of cAMP accumulation by PGF2 alpha or PGE2. The results indicate that in NIH 3T3 cells two receptors for PGs are present, one that couples to adenylate cyclase, probably through Gs, and does not exhibit selectivity between PGF2 alpha and PGE2 and a second receptor that couples to phospholipase C through a guanine nucleotide-binding protein that is not sensitive to pertussis toxin pretreatment. The latter shows at least 40-fold selectivity towards PGF2 alpha over PGE2. Because long treatment with PGF2 alpha resulted in desensitization of the GTP gamma S-induced response, it is possible that long exposure to PGF2 alpha may down-regulate the guanine nucleotide-binding involved in phospholipase C signal transduction.     

Activation of beta-adrenergic receptors inhibits Ca2+ entry-mediated generation of inositol phosphates in the guinea pig myometrium, a cyclic AMP-independent event.

In the guinea pig myometrium, carbachol, oxytocin, and fluoroaluminates stimulated the breakdown of phosphatidylinositol 4,5-bisphosphate, which was insensitive to pertussis toxin [Biochem. J. 255:705-713 (1988)]. We now demonstrate that an increased accumulation of inositol phosphates, with an early production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], could also be obtained with K+ (30 mM) and the Ca2+ ionophore ionomycin. Removal of extracellular Ca2+ or addition of the Ca2+ channel antagonists nifedipine and verapamil almost totally abolished stimulations elicited by high K+ and partially attenuated receptor- and fluoroaluminate-mediated increases in inositol phosphates. Isoproterenol similarly attenuated the accumulation of inositol phosphates elicited by carbachol, oxytocin, and fluoroaluminates (maximal inhibition, 35%; EC50, 0.5 nM), with no change in the rate of Ins(1,4,5)P3, inositol bisphosphate, and inositol monophosphate generation. The beta-adrenergic receptor-induced inhibition was prevented by pertussis toxin and could not be reproduced by forskolin, indicating that cAMP was not involved. Experimental findings were, rather, consistent with a predominant role for Ca2+. Thus, inhibition due to isoproterenol was lost in a Ca(2+)-depleted medium and was not additive with that caused by nifedipine. Accumulation of inositol phosphates triggered by high K+ was insensitive to the beta-adrenergic receptor inhibition. The inhibitory effect of isoproterenol, similar to that of nifedipine, was counteracted by ionomycin and also by the Ca2+ channel agonist Bay K 8644. These data indicate that in the myometrium 1) phospholipase C can be activated through a voltage-gated Ca2+ entry-dependent process that contributes at least partially to the stimulations triggered by receptor- and/or guanine nucleotide-binding protein-mediated activation and 2) beta-adrenergic receptor activation is linked via a cAMP-independent, pertussis toxin-sensitive pathway to an inhibition of voltage-gated Ca2+ channels, resulting in an attenuation of the Ca(2+)-associated generation of inositol phosphates.     

NPY and carbachol raise Ca2+ in SK-N-MC cells by three different mechanisms

Summary We have compared the mechanism of NPY-and carbachol-stimulated Ca²⁺ increases in SK-N-MC cells. NPY stimulated Ca²⁺ mobilization via a pertussis toxin-sensitive mechanism. Carbachol stimulated Ca²⁺ mobilization and influx via pertussis toxin-insensitive and -sensitive mechanisms, respectively. Carbachol but not NPY stimulated inositol phosphate accumulation by a pertussis toxin-insensitive mechanism. We conclude that carbachol promotes Ca²⁺ influx via a pertussis toxin-sensitive G protein and Ca²⁺ mobilization via a pertussis toxin-insensitive G-protein coupling to inositol phosphate generation; NPY stimulates Ca²⁺ mobilization via a pertussis toxin-sensitive G protein without apparent involvement of inositol phosphates.     

Ca^2＋ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

AIM: To investigate the alpha1B-adrenoceptor (alpha1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. METHODS: Full-length cDNA encoding alpha1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and alpha1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. RESULTS: Under agonist stimulation, alpha1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin (PTX) had little effect on basal cAMP accumulation as well as norepinephrine (NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin (CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdCl2 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%+/-0.47% to 1.00%+/-0.31%, 0.78%+/-0.23%, and 0.90%+/-0.40%, respectively. CONCLUSION: By coupling with a PTX-insensitive G protein, alpha1BAR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC), thereby increasing the cAMP production in HEK293 cell lines.     

Effects of GTP gamma S on muscarinic receptor-stimulated inositol phospholipid hydrolysis in permeabilized smooth muscle from the small intestine.

1. Smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig were permeabilized with Staphylococcus aureus alpha toxin (alpha-toxin) and used to investigate the role of G-protein activation in the regulation of muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis. 2. The efficiency of alpha-toxin permeabilization was estimated by the release of [3H]-2-deoxyglucose ([3H]-2DG) after prior loading or lactate dehydrogenase (LDH) enzyme release from the smooth muscle fragments. 3. In alpha-toxin-permeabilized smooth muscle, but not in non-permeabilized muscle, GTP gamma S induced time- and concentration-dependent increases in labelled inositol phosphates. Carbachol (CCh) increased labelled inositol phosphates in both permeabilized and non-permeabilized muscle, although the increases were greater in non-permeabilized smooth muscle. The response to 100 microM CCh was severely reduced by 0.5 microM atropine. 4. In permeabilized muscle the effects of GTP gamma S or CCh on inositol phosphate levels were reduced by treatment with pertussis toxin (PTX) and completely inhibited by GDP beta S. 5. GTP gamma S caused a concentration-dependent inhibition of the CCh-induced increases in the levels of labelled inositol phosphates. Dibutyryl cyclic AMP or Sp-cAMPs (adenosine-3',5'-cyclic phosphorothiolate-Sp) reduced the effects of CCh on inositol phosphate levels. 6. The results suggest that muscarinic AChR activation induces inositol phospholipid hydrolysis via more than one G-protein in this smooth muscle and that several mechanisms may contribute to the modulation of both stimulatory and inhibitory responses observed.     

Muscarinic receptors in canine colonic circular smooth muscle. II. Signal transduction pathways.

We have, in the accompanying work, demonstrated the coexistence of M2 and M3 muscarinic receptors in the circular smooth muscle of canine colon. In the present study, the effects of muscarinic receptor stimulation on phosphoinositide turnover and adenylate cyclase activity were examined. In myo-[3H]inositol-labeled circular smooth muscle strips, carbachol caused a concentration-dependent (EC50 = 5 microM) increase in [3H]inositol phosphate production. The more M3 receptor-selective muscarinic antagonist pirenzepine (KB = 53 nM) was approximately 60 times more potent than the more M2-selective agent AF-DX 116 (KB = 3 microM) in blocking carbachol-elicited accumulation of [3H]inositol phosphates. The carbachol-stimulated increase in [3H]inositol phosphate accumulation was not affected by pretreatment of the tissue with pertussis toxin (200 ng/ml, 3 hr). Within the first minute, carbachol (100 microM) caused a rapid and transient increase of [3H]inositol 1,4,5-trisphosphate production that oscillated continuously in the presence of agonist (120 min). The accumulation of [3H]inositol 1,3,4-trisphosphate was also extremely rapid, reaching a peak at 15 sec. The accumulation of [3H]inositol monophosphate was delayed and progressively increased over 30 min. [3H]inositol 1,3,4,5-tetrakisphosphate, although not detectable in the first minute, accumulated to significant levels over 30 min in the presence of agonist. Addition of carbachol in the adenylate cyclase assay caused inhibition of forskolin-stimulated [32P]cAMP production and blocked forskolin-stimulated cAMP accumulation in the intact tissue. The inhibitory effects of carbachol on adenylate cyclase were blocked by atropine, AF-DX 116, and 4-diphenylacetoxy-N-methylpiperidine methobromide but were unaffected by the more M3-selective agent pirenzepine (1 microM). Pretreatment of tissues with pertussis toxin completely eliminated M2 receptor-mediated inhibition of adenylate cyclase activity, without altering inositol 1,4,5-trisphosphate accumulation. We conclude that muscarinic receptor stimulation of inositol trisphosphate production is mediated by the M3 receptor coupled to a pertussis toxin-insensitive GTP-binding protein and results in the rapid formation of inositol tetrakisphosphate, whereas inhibition of adenylate cyclase activity is mediated by the M2 subtype of muscarinic receptor coupled to the pertussis toxin-sensitive GTP-binding protein Gi.     

Cyclic AMP counteracts mitogen-induced inositol phosphate generation and increases in intracellular Ca2+ concentrations in human lymphocytes.

1. The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on mitogen-induced generation of inositol phosphates and increases in intracellular Ca2+ concentration were investigated in human peripheral blood mononuclear leukocytes (MNL). 2. The mitogens concanavalin A (Con A), pokeweed mitogen (PWM) and phytohaemagglutinin (PHA) concentration-dependently stimulated generation of inositol phosphates. Catecholamines inhibited this process with an order of potency: isoprenaline greater than adrenaline greater than noradrenaline indicating involvement of beta 2-adrenoceptors. This order of potency was also consistent with the catecholamine potencies for stimulating the generation of cyclic AMP. 3. In addition to catecholamines, the cyclic AMP formation-stimulating agents prostaglandin E1 (PGE1) and forskolin concentration-dependently inhibited mitogen-induced inositol phosphate generation, too. Moreover, the inhibitory effect of isoprenaline was potentiated by co-incubation with the phosphodiesterase inhibitor isobutylmethylxanthine demonstrating that these inhibitory effects were mediated by cyclic AMP. 4. Con A and PHA concentration-dependently increased the intracellular Ca2+ concentration in human MNL (assessed by the fluorescent indicator dye Fura-2). This increase was almost completely blocked by chelation of extracellular Ca2+, demonstrating influx rather than mobilization from intracellular stores. 5. The elevation of intracellular Ca2+ was not blocked by pretreatment with pertussis toxin, 100 ng ml-1, for 16 h. 6. Isoprenaline, PGE1, and forskolin, however, inhibited the mitogen-stimulated elevation of intracellular Ca2+. This inhibition was enhanced by the phosphodiesterase inhibitors isobutylmethylxanthine and Ro 20-1724, demonstrating mediation by cyclic AMP. 7. We conclude that catecholamines and other cyclic AMP increasing agents can inhibit mitogen-stimulated generation of inositol phosphates and elevation of intracellular Ca2+ in resting human MNL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP potentiates receptor-stimulated phosphoinositide hydrolysis in human neuroepithelioma cells.

A stimulatory role for cAMP in the regulation of receptor-activated phosphoinositide hydrolysis has been examined in human SK-N-MCIXC and SK-N-MCIIE neuroepithelioma cells. The addition of optimal concentrations of oxotremorine-M, norepinephrine, endothelin-1, and ATP enhanced the release of inositol phosphates by 2-9-fold after activation of muscarinic, alpha 1-adrenergic, endothelin, and P2 nucleotide receptors, respectively. All combinations of these agonists elicited a release of inositol phosphates that was at least additive. However, the combined presence of oxotremorine-M and norepinephrine resulted in a phosphoinositide hydrolysis that was 30% greater than additive. This potentiation of inositol lipid hydrolysis resulted from an increased activity of the muscarinic receptor after the addition or norepinephrine and persisted after alpha 1-adrenergic receptor blockade. The enhancement of muscarinic receptor-stimulated inositol phosphate release could be quantitatively mimicked by inclusion of the beta-adrenergic agonist isoproterenol (EC50 approximately 0.1 microM), but not by alpha 1- or alpha 2-adrenergic agonists. Potentiation of oxotremorine-M-stimulated inositol lipid hydrolysis observed in the presence of either norepinephrine or isoproterenol was reduced in the absence of added Ca2+. Addition of either norepinephrine or isoproterenol to SK-N-MCIXC cells also resulted in a 16-fold increase in cAMP concentration. Although the cell-permeant 8-chloro-4-phenylthio-cAMP had a small inhibitory effect on basal inositol phosphate release, its inclusion resulted in a 19-31% enhancement of muscarinic, endothelin, ATP, and alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis. We conclude 1) that, in SK-N-MCIXC cells, the addition of beta-adrenergic agonists selectively enhances muscarinic receptor-stimulated phosphoinositide hydrolysis through a cAMP-dependent process and 2) that the ability of exogenously added cAMP to enhance the activation of all four inositol lipid-linked receptors indicates that the effects of cAMP on inositol lipid hydrolysis are compartmentalized in these cells.     

Second messengers involved in genetic regulation of the number of calcium channels in bovine adrenal chromaffin cells in culture.

Bovine adrenal chromaffin cells in culture show an increased formation of [3H]inositol phosphates (after preloading with [3H]inositol) on depolarisation with increased extracellular K+. This increased breakdown of inositol lipid is further increased by the dihydropyridine Ca2+ channel activator BAY K 8644 at nM concentrations, implying that proteins which bind dihydropyridines are involved in this mechanism. Further, pretreatment of adrenal cells with pertussis toxin (100 ng ml-1) prevented the K(+)-induced breakdown of inositol lipids, arguing the involvement of a pertussis toxin-sensitive G protein in the effect. Chronic exposure of bovine adrenal chromaffin cells to a concentration of ethanol which inhibits K(+)-induced breakdown of inositol phospholipid, caused a 70-100% increase in the binding of [3H]DHP sites. In these experiments it was found that excess extracellular Ca2+ would considerably reduce this up-regulation, whereas growth of cells in pertussis toxin closely mimicked the up-regulation obtained by growth of cells in ethanol. These experiments suggest that inhibition of membrane Ca2+ flux, through a G protein-associated channel, is closely involved in the ethanol-induced regulation of [3H]dihydropyridine binding sites. The inositol lipid-protein kinase C second messenger system is also implicated in this regulation, by experiments in which inhibitors of protein kinase C (chronic treatment with phorbol myristyl acetate, or with sphingosine) up-regulated binding sites for [3H]dihydropyridine to a similar extent as that seen with growth in ethanol.     

Two different signaling mechanisms involved in the excitation of rat sympathetic neurons by uridine nucleotides

UTP stimulates transmitter release and inhibits M-type K(+) channels in rat superior cervical ganglion neurons via G protein-coupled P2Y receptors. To investigate the underlying signaling mechanisms, we treated the neurons with either pertussis or cholera toxin; neither treatment altered the inhibition of M-type K(+) channels by 10 microM UTP. However, pertussis toxin reduced UTP-evoked [(3)H]noradrenaline release by 66%. UTP, UDP, ATP, and ADP caused accumulation of inositol trisphosphate in a pertussis toxin-insensitive manner. Pharmacological inhibition of inositol trisphosphate-induced Ca(2+) release (by inhibition of phospholipase C, of inositol trisphosphate receptors, and of the endoplasmic Ca(2+)-ATPase) prevented the UTP-dependent inhibition of M currents but failed to alter UTP-evoked [(3)H]noradrenaline release. Chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid also reduced the inhibition of M currents by UTP. In addition, all these manipulations attenuated the inhibition of M currents by bradykinin, but hardly affected the inhibitory action of oxotremorine M. These results demonstrate that UTP inhibits M-type K(+) channels via an inositol trisphosphate-dependent signaling cascade that is also used by bradykinin but not by muscarinic acetylcholine receptors. In contrast, the secretagogue action of UTP is largely independent of this signaling cascade but involves pertussis toxin-sensitive G proteins. Thus, UTP-sensitive P2Y receptors excite sympathetic neurons via at least two different signal transduction mechanisms.     

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca(2+) (SOC) entry was investigated. C1CP evoked a modest increase in [Ca(2+)](i). The increase was inhibited by the SOC channel antagonist 1-(beta-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca(2+)](i) was enhanced in comparison to control cells. When Ca(2+) was added to cells treated with both C1CP and thapsigargin in a Ca(2+)-free buffer, the increase in [Ca(2+)](i) was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential (E(m)) of the cells and attenuated the thapsigargin-invoked depolarization of the E(m). The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl(-) ions, as C1CP in a Cl(-)-free solution also enhanced Ca(2+) entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca(2+)](i) nor did it modulate the E(m). In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E(m) and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca(2+) ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.     

Influence of receptor number on functional responses elicited by agonists acting at the human adenosine A(1) receptor: evidence for signaling pathway-dependent changes in agonist potency and relative intrinsic activity

Activation of A(1) adenosine receptors leads to the inhibition of cAMP accumulation and the stimulation of inositol phosphate accumulation via pertussis toxin-sensitive G-proteins. In this study we have investigated the signaling of the A(1) adenosine receptor in Chinese hamster ovary (CHO) cells, when expressed at approximately 203 fmol/mg (CHOA1L) and at approximately 3350 fmol/mg (CHOA1H). In CHOA1L cells, the agonists N(6)-cyclopentyladenosine (CPA), (R)-N(6)-(2-phenylisopropyl)adenosine, and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited cAMP production in a concentration-dependent manner. After pertussis toxin treatment, the agonist NECA produced a stimulation of cAMP production, whereas CPA and (R)-N(6)-(2-phenylisopropyl)adenosine were ineffective. In CHOAIH cells, however, all three agonists produced both an inhibition of adenylyl cyclase and a pertussis toxin-insensitive stimulation of adenylyl cyclase. All three agonists were more potent at inhibiting adenylyl cyclase in CHOA1H cells than in CHOA1L cells. In contrast, A(1) agonists (and particularly NECA) were less potent at stimulating inositol phosphate accumulation in CHOA1H cells than in CHOA1L cells. After pertussis toxin treatment, agonist-stimulated inositol phosphate accumulation was reduced in CHOA1H cells and abolished in CHOA1L cells. The relative intrinsic activity of NECA in stimulating inositol phosphate accumulation, compared to CPA (100%), was much greater in the presence of pertussis toxin (289.6%) than in the absence of pertussis toxin (155.2%). These data suggest that A(1) adenosine receptors can couple to both pertussis toxin-sensitive and -insensitive G-proteins in an expression level-dependent manner. These data also suggest that the ability of this receptor to activate different G-proteins is dependent on the agonist present.     

Polyphosphoinositide hydrolysis and protein kinase C activation in guinea pig tracheal smooth muscle cells in culture by leukotriene D4 involve a pertussis toxin sensitive G-protein.

Leukotriene D4 (LTD4) at concentrations greater than 1 nM induced phosphatidylinositol bisphosphate (PIP2) hydrolysis and protein kinase C (PKC) activation in primary culture of airway smooth muscle cells. Within seconds of activation, an increase in inositol 1,4,5-trisphosphate (IP3) was observed reaching a maximum at 5 min. The level of IP3 decreased after 5 min and was followed by an increase in inositol 1,4-bisphosphate (IP2) and inositol 1-monophosphate (IP1). LTD4-induced PIP2 hydrolysis was inhibited by 1 h pretreatment of cells with 10 micrograms/ml of pertussis toxin (PTX). LTD4 activated both soluble and particulate forms of PKC by 2-3-fold. The LTD4-induced PKC activation was blocked by treatment of cells with PTX, suggesting the involvement of a PTX-sensitive G-protein. To assess the involvement of G(i) in smooth muscle cell receptor activation, the modulation of adenylyl cyclase activity was investigated. LTD4 did not stimulate cAMP formation in smooth muscle cells, and did not inhibit forskolin-induced cAMP formation. These data suggest that the LTD4 receptor in airway smooth muscle cells is coupled to a PTX-sensitive G-protein, possibly G(o).     

Muscarinic receptor regulation of cytoplasmic Ca2+ concentrations in human SK-N-SH neuroblastoma cells: Ca2+ requirements for phospholipase C activation

The relationship between muscarinic receptor-mediated inositol lipid hydrolysis and the generation of Ca2+ signals has been examined in human SK-N-SH neuroblastoma cells. The resting cytoplasmic calcium concentration [( Ca2+]i) as determined by fura-2 fluorescence measurements was 59 +/- 2 nM. Upon the addition of oxotremorine-M, there was a 4-fold increase in [Ca2+]i (293 +/- 18 nM), with half-maximal stimulation obtained at an agonist concentration of 8 microM, a value similar to that previously observed for the enhancement of phosphoinositide hydrolysis. Addition of partial muscarinic agonists for phosphoinositide turnover (bethanechol, oxo-2, and arecoline) elicited correspondingly smaller increases in [Ca2+]i than did oxotremorine-M. Inclusion of EGTA lowered the basal [Ca2+]i within 2 min and markedly reduced (greater than 60%) the magnitude of the agonist-induced rise in [Ca2+]i. Addition of muscarinic agonists to SK-N-SH cells that had been prelabeled with [3H]inositol led to the rapid (5-15 sec) release of inositol mono-, bis-, and triphosphates. When assayed under conditions similar to those employed for the fluorescence measurements, EGTA also inhibited both the basal and oxotremorine-M-stimulated release of inositol phosphates by 45-61%. Conversely, ionomycin both elevated [Ca2+]i and stimulated the release of inositol phosphates. The addition of Ca2+ (10 nM-2 microM) to digitonin-permeabilized cells directly stimulated the release of labeled inositol mono-, bis-, and trisphosphates by 3-4-fold with a half-maximal effect (EC50) observed at 145 nM free Ca2+ (Ca2+f). A further (6-fold) calcium-dependent increase in inositol phosphate release was obtained by inclusion of either guanosine-5-O-(3-thio)-trisphosphate (GTP gamma S) or oxotremorine-M. In the combined presence of agonist and GTP gamma S, a synergistic release of all three inositol phosphates occurred, with half-maximal stimulation observed at 35-40 nM Ca2+f, a value similar to the [Ca2+]i in quiescent cells. These results indicate (i) that the magnitude of the initial rise in [Ca2+]i is directly related to the production of phosphoinositide-derived second messenger molecules and (ii) that the phospholipase C-mediated breakdown of inositol lipids in SK-N-SH cells is particularly sensitive to regulation by physiologically relevant Ca2+ concentrations. It is concluded that, in SK-N-SH cells, either an elevation above or reduction below basal [Ca2+]i can modulate the extent of hydrolysis of inositol lipids and the subsequent generation of calcium signals.     

Propofol regulation of calcium entry pathways in cultured A10 and rat aortic smooth muscle cells.

1. We have investigated the effect of propofol, an intravenous anaesthetic, on the intracellular calcium concentration ([Ca2+]i), Ca2+ entry pathways and on inositol phosphate formation in vascular smooth muscle cells. [Ca2+]i and Ca2+ flux were monitored with the Ca(2+)-sensitive fluorescent dye, fura-2, and by 45Ca2+ uptake. Production of labelled inositol phosphates was analysed by anion-exchange chromatography. 2. Treatment of the cells with endothelin-1 (ET-1) increased formation of inositol phosphates and elevated [Ca2+]i due to both release of Ca2+ from intracellular pools and prolonged entry of Ca2+ from outside the cell. Propofol reduced production of inositol phosphates mediated by ET-1 and arginine vasopressin which activate phospholipase C. 3. The sustained Ca2+ entry stimulated by ET-1 was found to occur through the activation of L-type Ca channels. This was inhibited by propofol in a dose-dependent manner. 4. Activation of protein kinase C (PKC) by phorbol esters activated a pharmacologically-similar channel and produced a similar change in [Ca2+]i due to Ca2+ entry. The entry was blocked by an L-type channel antagonist, nicardipine and by the anaesthetic drug, propofol. 5. Treatment of the cells with thapsigargin, a selective inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, also elevated [Ca2+]i by inducing the release of intracellular Ca2+ and the continued entry of extracellular Ca2+ through a nicardipine-insensitive Ca channel. Neither release nor entry induced by thapsigargin was affected by propofol. 6. These findings suggest that propofol selectively inhibits Ca2+ entry through the L-type channel induced by ET-1 and phorbol esters but has no effects on Ca2+ entry via the nicardipine-insensitive channel and on Ca2+ release from intracellular pools initiated by thapsigargin. This may represent one of the mechanisms responsible for propofol-induced vasodilatation.     

Role of a protein regulating guanine nucleotide binding in phosphoinositide breakdown and calcium mobilization by bradykinin in neuroblastoma X glioma hybrid NG108-15 cells: effects of pertussis toxin and cholera toxin on receptor-mediated signal transduction

The addition of bradykinin to NG108-15 cells resulted in an increase in the intracellular Ca2+ concentration [( Ca2+]i) and the formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in these cells. The bradykinin-stimulated formation of inositol polyphosphates in plasma membrane preparations was dependent on the presence of GTP or guanosine-5'-O-thiotriphosphate (GTP gamma S) but not of GDP. GTP gamma S, unlike GTP, increased the basal formation of inositol polyphosphate in NG108-15 membranes. Iontophoretic injection of GTP gamma S into single cells induced increases in [Ca2+]i. These effects of bradykinin and GTP gamma S on [Ca2+]i and the formation of inositol phosphates in the intact cells and membranes were not affected by treatment of the cells with pertussis toxin or cholera toxin. Data on binding of bradykinin to membrane preparations indicated the presence of two classes of binding sites with Kd values of 0.80 +/- 0.26 and 9.63 +/- 0.13 nM. Approximately 74% of the receptors were in the high affinity state. In the presence of guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], the high affinity sites in the membrane preparations were converted to low affinity sites with no change in the total receptor number. These toxin treatments had no effect on binding of bradykinin to its receptors. Thus, these results indicate that a guanine nucleotide regulatory protein, which is not a substrate of pertussis toxin or cholera toxin, is involved in mediating the effects of bradykinin on membrane-bound phosphoinositide-specific phospholipase C to induce the increase of cytosolic calcium.     

Histamine increases cytosolic Ca2+ in dibutyryl-cAMP-differentiated HL-60 cells via H1 receptors and is an incomplete secretagogue.

Human neutrophils and dibutyryl-cAMP (Bt2cAMP)-differentiated HL-60 cells possess receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), which mediate activation of phospholipase C, with subsequent increase in cytosolic Ca2+ concentration ([Ca2+]i) and activation of specific cell functions. In many cell types, histamine, via H1 receptors, activates phospholipase C, but it is unknown whether neutrophilic cells possess functional H1 receptors. We compared the effects of histamine with those of fMet-Leu-Phe on activation of these cells. In Bt2cAMP-differentiated HL-60 cells, substances increased [Ca2+]i in the effectiveness order fMet-Leu-Phe greater than histamine greater than betahistine. Pertussis toxin diminished fMet-Leu-Phe-induced rises in [Ca2+]i to a greater extent than those induced by histamine. H1 but not H2 antagonists inhibited histamine- and betahistine-induced rises in [Ca2+]i. fMet-Leu-Phe and histamine activated phospholipase C and increased [Ca2+]i through release of Ca2+ from intracellular stores and sustained influx of Ca2+ from the extracellular space. The substances also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). The stimulatory effects of histamine on [Ca2+]i were more sensitive to inhibition by 4 beta-phorbol 12-myristate 13-acetate than were those of fMet-Leu-Phe. Unlike fMet-Leu-Phe, histamine did not activate superoxide anion formation, release of beta-glucuronidase, and tyrosine phosphorylation. In neutrophils, histamine and betahistine did not induce rises in [Ca2+]i. Our data show that (i) in Bt2cAMP-differentiated HL-60 cells, histamine increases [Ca2+]i via H1 receptors coupled to pertussis toxin-sensitive and possibly, pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (ii) histamine activates nonselective cation channels, and (iii) unlike fMet-Leu-Phe, histamine is an incomplete secretagogue.     

Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells.

1. The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 microM) in a concentration-dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells. 2. In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol-induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1-0.2 mM. The higher concentrations of extracellular Ca2+ depressed the influx. 3. Pertussis toxin did not affect mastoparan-induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan-induced inhibition of phosphoinositide hydrolysis. 4. Pertussis toxin augmented mastoparan-induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan. 5. Verapamil, nifedipine and diltiazem (each 10 microM), L-type Ca2+ antagonists, did not affect mastoparan-induced Ca2+ influx. However, verapamil (10 microM) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan. 6. The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action.     

Thrombin signalling in U937 human monocytic cells is coupled to inositol phosphate formation but not to thromboxane B2 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 used 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 (IP3) and the mobilisation of intracellular Ca2+ [( Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formyl-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB2 synthesis under conditions where it evoked increases in IP3 formation and [Ca2+]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 (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. These 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 IP3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB2 synthesis or cause Gi-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.