Inhibitors of protein kinase C selectively enhanced leukotriene D4-induced calcium mobilization in differentiated U-937 cells

U-937 cells differentiated with dimethylsulphoxide for 3-4 days express receptors for leukotriene D4 (LTD4), which are coupled to Ca2+ mobilization and phosphatidylinositol (PI) metabolism. Treatment of U-937 cells with an inhibitor of protein kinase C (PKC) [staurosporine (100 nM)] augmented the Ca2+ mobilized by LTD4. The peak concentration of the LTD4-induced increase in [Ca2+]i was 1500 nM in untreated cells and 3000 nM in cells treated with staurosporine for 30 s. Maximal mobilization responses were observed at 1-10 microM LTD4 in both control and staurosporine-treated cells. The increased Ca2+ response to LTD4 after staurosporine treatment occurred within 30 s and was attributable to both intracellular and extracellular stores. Additionally, a second phase of Ca2+ mobilization occurred after stimulation with LTD4, which was elevated by pretreatment with staurosporine--this effect was maximal after 5-10 min of treatment. Staurosporine either had no effect or decreased the Ca2+ mobilization response of differentiated U-937 cells to other agonists, such as LTB4, platelet activating factor, ATP or the chemotactic peptide f-Met-Leu-Phe. Although staurosporine alone had no effect on basal PI metabolism it increased LTD4-induced PI metabolism. Staurosporine did not prevent the tachyphylaxis observed upon second challenge with LTD4, nor did it prevent LTD4-induced homologous densensitization. Other compounds which inhibit PKC (sphingosine and 1-O-hexadecyl-2-O-methylglycerol), also enhanced the Ca2+ response of U-937 cells to LTD4, but not to other agonists. These data show that inhibition of PKC enhanced responses of LTD4, suggesting that PKC plays a role in determining the responsiveness of LTD4 receptors.     

Two different signal transductions of neuropeptide Y1 receptor in SK-N-MC cells.

The signal transduction systems of the neuropeptide Y (NPY) Y1 receptor were studied in SK-N-MC human neuroblastoma cells. NPY induced an increase in intracellular calcium ion concentration ([Ca2+]i) and inhibition of forskolin-stimulated cyclic AMP accumulation, which were mediated through Y1 receptors. One-min preincubation of cells with phorbol 12-myristate 13-acetate (PMA) inhibited both signal transductions dose-dependently, but its effect on [Ca2+]i was about 100-fold more potent than that on cyclic AMP. PMA had no effect on [125I]BH-NPY binding in SK-N-MC cells and hardly inhibited the endothelin-1-induced increase in [Ca2+]i. Pertussis toxin also inhibited the NPY-induced [Ca2+]i increase 30-fold more effectively than the NPY-mediated inhibition of cyclic AMP accumulation. These results indicate that Y1 receptors in SK-N-MC cells couple to two signal transduction systems that have different sensitivities to phorbol ester and pertussis toxin treatments.     

Transient activation of topoisomerase I in leukotriene D4 signal transduction in human cells.

U937 human monoblast cells incubated with leukotriene D4 (LTD4) rapidly released arachidonic acid metabolites into the culture medium. Release was suppressed by the high-affinity LTD4 receptor antagonist SK&F 104353. Arachidonic acid release induced by LTD4 has been linked to a rapid induction of gene expression, and the propagation of the receptor binding signal is probably associated with enzymes that regulate gene expression. We have studied the participation of DNA topoisomerase I in LTD4 signal transduction. LTD4-specific release of arachidonic acid metabolites was inhibited (60-80%) by the topoisomerase I inhibitor camptothecin. LTD4 increased protein-linked DNA strand breakage induced by camptothecin in U937 cells; this enhancement was prevented by coincubation of the cells with LTD4 plus the receptor antagonist SK&F 104353. In addition, LTD4 produced a rapid transient increase in extractable topoisomerase I activity, which was maximum within the first 10 min after addition of LTD4 to the culture medium. Incubation of cultures for greater than 10 min with LTD4 before the addition of camptothecin resulted in no enhancement of camptothecin-induced DNA strand breakage, consistent with a reversal of topoisomerase I activation. Staurosporine, an inhibitor of protein kinase C, blocked LTD4-induced arachidonic acid release and attenuated the effect of LTD4 on camptothecin-induced DNA strand breakage. These results are consistent with the view that the regulation of topoisomerase I activity is involved in the propagation of LTD4-mediated signals in U937 cells.     

Conjunctival goblet cell secretion stimulated by leukotrienes is reduced by resolvins D1 and E1 to promote resolution of inflammation

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.     

Mediation by platelet-activating factor of 12-hydroxyeicosatetraenoic acid-induced cytosolic free calcium concentration elevation in neutrophils

12(R)-hydroxyeicosatetraenoic acid (HETE) shows biphasic increase in cytosolic free calcium concentration ([Ca2+]i) in rabbit and human neutrophils; the initial transient phase and the continuous falling phase. 12(S)-HETE was less potent in both species. BN50739, a platelet-activating factor (PAF) receptor antagonist, inhibited both phases of 12(R)-HETE-induced [Ca2+]i rise but did not affect leukotriene B4 (LTB4)-induced [Ca2+]i rise. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a PAF synthesis inhibitor, and manoalide, a phospholipase A2 inhibitor, reduced 12(R)-HETE-induced [Ca2+]i rise. These blockers inhibited the continuous phase of [Ca2+]i rise induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with little effect on the initial phase. It had no significant effect on LTB4-induced [Ca2+]i rise. SC-41930, a LTB4-receptor antagonist, did not block 12-HETE-induced [Ca2+]i rise. In 12(R)-HETE-, FMLP- and LTB4-stimulated cells, accumulations of cell-associated PAF and released PAF were detected but not in unstimulated cells. BN50739 did not affect the accumulation of cell-associated PAF and release of PAF in 12(R)-HETE-stimulated cells. These results suggest that 12(R)-HETE-induced and partially, FMLP-induced, but not LTB4-induced [Ca2+]i rise are mediated by PAF, which is produced and released by stimulation of the cells by 12(R)-HETE and FMLP, respectively.     

Co-expression of mCysLT1 receptors and IK channels in Xenopus laevis oocytes elicits LTD4-stimulated IK current, independent of an increase in [Ca2+]i

Addition of LTD4 (10 nM) to Xenopus laevis oocytes expressing the mCysLT1 receptor together with hBK or hIK channels resulted in the activation of both channels secondary to an LTD4-induced increase in [Ca2+]i. In addition, the hIK channel is activated by low concentrations of LTD4 (<0.1 nM), which did not result in any increase in [Ca2+]i. Even though activation of hIK by low concentrations of LTD4 was independent of an increase in [Ca2+]i, a certain "permissive" level of [Ca2+]i was required for its activation, since buffering of intracellular Ca2+ by EGTA completely abolished the response to LTD4. Neither hTBAK1 nor hTASK2 was activated following stimulations with LTD4 (0.1 and 100 nM).     

Cysteinyl leukotriene-dependent [Ca2+]i responses to angiotensin II in cardiomyocytes

With the use of fura 2 measurements in multiple and single cells, we examined whether cysteinyl leukotrienes (CysLT) mediate angiotensin II (ANG II)-evoked increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in neonatal rat cardiomyocytes. ANG II-evoked CysLT release peaked at 1 min. The angiotensin type 1 (AT(1)) antagonist losartan, but not the AT(2) antagonist PD-123319, attenuated the elevations in [Ca(2+)](i) and CysLT levels evoked by ANG II. Vasopressin and endothelin-1 increased [Ca(2+)](i) but not CysLT levels. The 5-lipoxygenase (5-LO) inhibitor AA-861 and the CysLT(1)-selective antagonist MK-571 reduced the maximal [Ca(2+)](i) responses to ANG II but not to vasopressin and endothelin-1. While MK-571 reduced the responses to leukotriene D(4) (LTD(4)), the dual CysLT antagonist BAY-u9773 completely blocked the [Ca(2+)](i) elevation to both LTD(4) and LTC(4). These data confirm that ANG II-evoked increases, but not vasopressin- and endothelin-1-evoked increases, in [Ca(2+)](i) involve generation of the 5-lipoxygenase metabolite CysLT. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] antagonist 2-aminoethoxydiphenyl borate attenuated the [Ca(2+)](i) responses to ANG II and LTD(4). Thus AT(1) receptor activation by ANG II is linked to CysLT-mediated Ca(2+) release from Ins(1,4,5)P(3)-sensitive intracellular stores to augment direct ANG II-evoked Ca(2+) mobilization in rat cardiomyocytes.     

Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression

Cysteinyl leukotrienes (including LTC(4), LTD(4), and LTE(4)), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT(1) and CysLT(2) receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD(4)-induced brain edema. LTD(4) (1ng in 0.5mul PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT(1) receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD(4). In the cultured rat astrocytes, LTD(4) (10(-9)-10(-7)M, for 24h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT(1)/CysLT(2) receptor antagonist, but not by pranlukast. LTD(4) (10(-9)-10(-7)M) also induced the mRNA expression of CysLT(2) (not CysLT(1)) receptor in astrocytes. These results indicate that LTD(4) modulates brain edema; CysLT(1) receptor mediates vasogenic edema while CysLT(2) receptor may mediate cytotoxic edema via up-regulating AQP4 expression.     

Cutting edge: Interleukin 4-dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling

Reactive mastocytosis (RM) in epithelial surfaces is a consistent Th2-associated feature of allergic disease. RM fails to develop in mice lacking leukotriene (LT) C4 synthase (LTC4S), which is required for cysteinyl leukotriene (cys-LT) production. We now report that IL-4, which induces LTC4S expression by mast cells (MCs), requires cys-LTs, the cys-LT type 1 receptor (CysLT1), and Gi proteins to promote MC proliferation. LTD4 (10-1000 nM) enhanced proliferation of human MCs in a CysLT1-dependent, pertussis toxin-sensitive manner. LTD4-induced phosphorylation of ERK required transactivation of c-kit. IL-4-driven comitogenesis was likewise sensitive to pertussis toxin or a CysLT1-selective antagonist and was attenuated by treatment with leukotriene synthesis inhibitors. Mouse MCs lacking LTC4S or CysLT1 showed substantially diminished IL-4-induced comitogenesis. Thus, IL-4 induces proliferation in part by inducing LTC4S and cys-LT generation, which causes CysLT1 to transactivate c-kit in RM.     

Leukotriene D4 and E4 induce transmembrane signaling in human epithelial cells. Single cell analysis reveals diverse pathways at the G-protein level for the influx and the intracellular mobilization of Ca2+

Leukotrienes are recognized as important mediators of the inflammatory process. Recently, increasing attention has been paid to the role of noninflammatory cells in the regulation of the inflammatory process. To further increase our knowledge of this matter we have, in the present study, investigated leukotriene-induced Ca2+ signaling, using a single cell technique in a human epithelial cell line, Intestine 407. It was evident that both LTD4 and LTE4, at physiological concentrations (10 nM), triggered rapid and pronounced cytosolic free Ca2+ transients, due to both influx across the plasma membrane and intracellular mobilization. Preincubation with pertussis toxin (1200 ng/ml) decreased the level of agonist-induced Ca2+ transients to an extent similar to that caused by depletion of extracellular Ca2+, suggesting that the toxin affected the influx but not the intracellular mobilization of Ca2+. Indeed, by using the Mn2+ quenching technique, it could be shown that pertussis toxin totally inhibited the influx of Ca2+. The fact that, even after pertussis toxin treatment, direct G-protein activation by AIF4- was still able to trigger a cytosolic free Ca2+ transient, indicates that, in these cells, G-proteins (GTP-binding proteins) that are insensitive to pertussis toxin are capable of mediating a Ca2+ signal. In order to test the idea that such G-proteins regulate mobilization of intracellular Ca2+ induced by LTD4 and LTE4, we electropermeabilized and preincubated the Intestine 407 cells with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), let them reseal, and, after loading with fura2, investigated the effects on agonist-stimulated Ca2+ signaling. Electropermeabiization and resealing alone did not significantly affect the Ca2+ responses triggered by LTD4 or LTE4. Addition of GDP beta S, in the presence of extracellular Ca2+, reduced the Ca2+ responses by approximately 60-70%. In Ca2(+)-depleted medium, GDP beta S also impaired the LTD4-induced response by 65%, however, it had no effect on the Ca2+ response induced by LTE4. In conclusion, LTD4 and LTE4 trigger cytosolic free Ca2+ signaling in a human epithelial cell line by causing both an influx of Ca2+ and mobilization of intracellular Ca2+. The Ca2(+)-signaling mechanism appears to consist of dual pathways, since the influx is regulated by a pertussis toxin-sensitive G-protein, but, the mobilization of Ca2+ is not. Furthermore, our data suggest that the LTD4-induced mobilization is regulated by a pertussis toxin-insensitive G-protein whereas the LTE4-induced mobilization is relatively insensitive to both pertussis toxin and GDP beta S.     

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.     

Interleukin-13 enhanced Ca2+ oscillations in airway smooth muscle cells

Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca²⁺ signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca²⁺ oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs.LTD4-induced Ca²⁺ oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca²⁺ entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca²⁺ oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP₃R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca²⁺ oscillations. Ca²⁺ oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP₃R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3.We conclude that IL-13 enhances frequency of LTD4-induced Ca²⁺ oscillations in human ASMCs, which may be cooperatively modulated by IP₃R, RyR systems and possibly by SOCE.     

Evidence against a role for a pertussis toxin-sensitive G protein in Ca2+ mobilization in rat parotid acinar cells

Hormone-induced Ca2+ mobilization in rat parotid acinar cells is reportedly mediated via an as yet uncharacterized G protein. We have studied the sensitivity to pertussis toxin (PTx) of this signal transduction mechanism. When rats were treated with Ptx (1.3-1.5 micrograms per animal) for 72 h, a 41 kDa membrane protein was ADP-ribosylated. This PTx treatment regimen, also, resulted in a more than 80% block of the ability of the muscarinic agonist carbachol to inhibit beta-adrenergic receptor-stimulated parotid adenylyl cyclase activity. However, cytosolic Ca2+ levels, in response to either carbachol or AIF-4, were comparable in cells prepared from both untreated or PTx-treated rats, when incubated either in the absence or presence of extracellular Ca2+. Further, both the sensitivity of the Ca2+ response to carbachol and the ability of the agonist-sensitive intracellular Ca2+ stores to be refilled by extracellular Ca2+ were unaffected by PTx treatment. Parotid membranes also contained three low-molecular-weight GTP-binding proteins (25, 22 and 18 kDa) which were unaffected by PTx. These results show that there is only one detectable substrate in parotid membranes for a PTx-catalyzed ADP-ribosylation and that hormone-induced Ca2+ mobilization events in parotid acinar cells are not mediated via PTx-sensitive components.     

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.     

Guinea pig cysteinyl leukotriene receptor 2 (gpCysLT2) mediates cell proliferation and intracellular calcium mobilization by LTC4 and LTD4

We cloned and pharmacologically characterized the guinea pig cysteinyl leukotriene (CysLT) 2 receptor (gpCysLT2). gpCysLT2 consists of 317 amino acids with 75.3%, 75.2%, 73.3% identity to those of humans, mice and rats, respectively. The gpCysLT2 gene is highly expressed in the lung, moderately in eosinophils, skin, spleen, stomach, colon, and modestly in the small intestine. CysLTs accelerated the proliferation of gpCysLT2-expressing HEK293. Leukotriene C4 (LTC4) and Leukotriene D4 (LTD4) enhanced the cell proliferation higher than Bay-u9773, a CysLT2 selective partial agonist and a nonselective antagonist for CysLT receptors. Bay-u9773 did not antagonize the cell proliferation by LTC4 and LTD4. Despite the equipotency of the mitogenic effect among these chemicals, calcium mobilization (CM) levels were variable (LTC4> LTD4> Bay-u9773), and Bay-u9773 antagonized the CM by LTC4. Moreover, the Gi/o inhibitor pertussis toxin perfectly inhibited agonist-induced cell proliferation. These results reveal that cell proliferation via CysLT2 signaling was mediated by Gi/o signaling but independent of calcium mobilization.     

Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones

The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 microM of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 microM thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones.     

Differential effects of protein kinase C activators on carbamylcholine- and high K+-induced rises in intracellular free calcium concentration in cultured adrenal chromaffin cells

Pretreatment of adrenal chromaffin cells with protein kinase C activators, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 1-oleoyl 2-acetyl glycerol (OAG), partially inhibited carbamylcholine (CCh)-induced rise in intracellular free Ca2+ concentration ([Ca2+]i). The apparent IC50 values of TPA and OAG were 3 nM and 25 microM, respectively. The effect of TPA on the CCh-induced rise in [Ca2+]i was overcome by pretreatment of the cells with a protein kinase C inhibitor, 1-(5-isoquinidinesulfonyl)-2-methylpiperazine hydrochloride (H-7). In contrast, KCl-induced rise in [Ca2+]i was not affected by pretreating the cells with TPA or OAG. An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate failed to affect the CCh-induced rise in [Ca2+]i. CCh-induced 45Ca2+ uptake was also partially inhibited by pretreatment of the cells with TPA or OAG, but KCl-induced 45Ca2+ uptake was not affected by these pretreatments. These results indicate that protein kinase C activation causes an uncoupling of signal transduction between the nicotinic receptors and Ca2+ channels.     

Intracellular mechanisms involved in leukotriene C4-stimulated adhesion of U-937 cells.

Leukotrienes C4 and D4 (LTC4 and LTD4) stimulated, 5- to 6-fold, the adhesion of the monoblastoid cell line U-937 to plastic. Half-maximal effects were observed around 1 nM. Leukotrienes E4 and B4 (LTE4 and LTB4) were less effective. The adhesive response to LTC4 was inhibited by pertussis toxin and was completely dependent on the presence of extracellular Ca2+. The LTC4-stimulated increases in inositol-phosphates and in intracellular Ca(2+)-concentration were insensitive to pertussis toxin. Activation of leukocyte adhesion is a novel action of cysteinyl-leukotrienes and the present study suggests that control of U-937-cell adhesion by LTC4 involves two pathways; one pertussis toxin insensitive pathway regulating intracellular Ca2+ in a manner partly dependent on extracellular Ca2+ and one pertussis toxin sensitive pathway not concerned with Cai(2+)-regulation.