TRPC channels: integrators of multiple cellular signals.

TRPC channels are ubiquitously expressed among cell types and mediate signals in response to phospholipase C (PLC)-coupled receptors. TRPC channels function as integrators of multiple signals resulting from receptor-induced PLC activation, which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 depletes Ca2+ stores and TRPC3 channels can be activated by store-depletion. InsP3 also activates the InsP3 receptor, which may undergo direct interactions with the TRPC3 channel, perhaps mediating store-dependence. The other PLC product, DAG, has a direct non-PKC-dependent activating role on TRPC3 channels likely by direct binding. DAG also has profound effects on the TRPC3 channel through PKC. Thus PKC is a powerful inhibitor of most TRPC channels and DAG is a dual regulator of the TRPC3 channel. PLC-mediated DAG results in rapid channel opening followed later by a slower DAG-induced PKC-mediated deactivation of the channel. The decreased level of PIP2 from PLC activation also has an important modifying action on TRPC3 channels. Thus, the TRPC3 channel and PLCgamma form an intermolecular PH domain that has high specificity for binding PIP2. This interaction allows the channel to be retained within the plasma membrane, a further operational control factor for TRPC3. As nonselective cation channels, TRPC channel opening results in the entry of both Na+ and Ca2+ ions. Thus, while they may mediate Ca2+ entry signals, TRPC channels are also powerful modifiers of membrane potential.

Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes.

Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

Mast cells stimulated by membrane-bound, but not soluble, steel factor are dependent on phospholipase C activation.

The steel factor (SLF) and c-Kit growth factor/receptor pair are key molecules governing mast cell development and survival. SLF is expressed on stromal cells as a membrane-bound molecule (mSLF) which can be cleaved by proteases to release a soluble form (sSLF). We investigated the importance of phospholipase C (PLC) activation in mast cells stimulated by sSLF and mSLF. PLC antagonists U73122, neomycin sulfate and oleic acid inhibited mast cell thymidine incorporation stimulated by mSLF, but not by sSLF. These antagonists suppressed sSLF-induced Ca2+ transients but did not significantly interfere with c-Kit phosphorylation or PLC-gamma2 recruitment. p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase), was found to be efficiently recruited to c-Kit following stimulation by sSLF or mSLF. However PKB/Akt, a kinase activated by PI3-kinase products, was phosphorylated following sSLF stimulation, but not with mSLF. Taken together, these studies demonstrate the importance of PLC activation by mSLF in supporting mast cells.

Follicular stage-dependent tumor necrosis factor alpha-induced hen granulosa cell integrin production and survival in the presence of transforming growth factor alpha in vitro.

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNF alpha) and transforming growth factor alpha (TGF alpha) have been suggested. TGF alpha stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNF alpha and its possible interaction with TGF alpha in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNF alpha and TGF alpha interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGF alpha (0.1 or 10 ng/ml), but not TNF alpha (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin beta(3) subunit was also stimulated by TGF alpha in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNF alpha in the presence of maximal stimulatory concentrations of TGF alpha potentiated this response. TGF alpha increased both F5,6 and F1 granulosa cell [(3)H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNF alpha had no effect on [(3)H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNF alpha and TGF alpha than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGF alpha-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNF alpha alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF alpha but not of TNF alpha. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNF alpha and TGF alpha was necrosis and not apoptosis. The current study demonstrates that TNF alpha and TGF alpha interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF alpha; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.

Acyl chain length-specific ceramide-induced changes in intracellular Ca2+ concentration and progesterone production are not regulated by tumor necrosis factor alpha in hen granulosa cells.

Although tumor necrosis factor alpha (TNF-alpha) has long been known to be a potent inhibitor of gonadotropin-induced cytodifferentiation in the ovaries of a variety of mammalian species, its early signal transduction events are poorly understood. We previously demonstrated that TNF-alpha induces a small, delayed follicular stage-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in hen granulosa cells and promotes carbachol (Cch)-induced mobilization of Ca2+ from intracellular stores in cells otherwise unresponsive to the cytokine. The focus of the current study was to examine the role of ceramide in TNF-alpha-induced Ca2+ regulation. Treatment with exogenous sphingomyelinase (SMase; 50 mU/ml) failed to influence basal [Ca2+]i but increased the magnitude of Cch-induced Ca2+ transients. While C8-ceramide (0.03-30 microM), but not C2-ceramide (0.03-30 microM), mimicked this effect of SMase, challenge with sphingosine (3 microM) resulted in a slow and delayed increase in basal [Ca2+]i. In order to determine whether SMase is activated by TNF-alpha action, changes in sphingomyelin and ceramide concentrations in F1 and F5,6 granulosa cells were determined. SMase activation was not observed after 1-, 5-, 15-, and 60-min incubations with TNF-alpha (1-50 ng/ml) in either F1 or F5,6 cells. Exogenous SMase and C2-ceramide both inhibited LH-induced progesterone production in F1 and F5,6 cells; however, incubation with C8-ceramide resulted in increases in both basal and LH-induced progesterone. In contrast, incubation with TNF-alpha had no effect on either basal or LH-induced steroidogenesis. In conclusion, our findings indicate that although ceramide regulates [Ca2+]i and progesterone secretion, the sphingolipid does not appear to play a role in the action of TNF-alpha in avian granulosa cells. Furthermore, ceramide-mediated responses are highly dependent on acyl chain length, potentially reflecting differences in the abilities of these ceramides to access, bind to, and/or activate ceramide-dependent signal transduction mechanisms. Nonetheless, since TNF-alpha did not increase the production of ceramide, the physiological regulator(s) of these responses remain unknown.

Muscarinic activation inhibits T-type Ca2+ current in hen granulosa cells.

The effect of the muscarinic agonist carbachol on Ca2+ currents in hen granulosa cells isolated from the largest ovarian follicle was studied. The major Ca2+ current observed using the perforated patch technique with a recording solution containing 10 mM Ca2+, but no Na+ or K+, exhibited characteristics typical of T-type Ca2+ current: maximal amplitude at -20 mV, rapid inactivation (half-time of 42 +/- 3 msec at -20 mV), inhibition by 100 microM Ni2+, and insensitivity to the dihydropyridine Ca2+ channel antagonist, nifedipine. In all cell studied, carbachol (0.5 mM) caused an inhibition of this current (elicited by depolarizing pulses from -70 to -20 mV) to an average maximal decrease of 90 +/- 2% below basal values. In some 50% of the cells, the Ca2+ current also partially recovered during the 10-min exposure to the muscarinic agonist. These effects were prevented by the muscarinic antagonist atropine (1 microM). To test whether this inhibition was due to increases in intracellular free Ca2+ concentrations ([Ca2+]i), [Ca2+]i was simultaneously measured in fura-2-loaded cells. For cells incubated in normal solution, [Ca2+]i was 0.15 +/- 0.02 microM, but increased to 0.25 +/- 0.6 microM in cells exposed to the recording solution. Under these conditions, carbachol failed to produce the expected [Ca2+]i transients, but, rather, caused a small decrease (8 +/- 2%) in basal [Ca2+]i attributable to its diminution of Ca2+ current. Thus, the results demonstrated an important muscarinic inhibition of the T-type Ca2+ current not related to [Ca2+]i fluctuations. They indicate, on the other hand, that [Ca2+]i can strongly modulate carbachol-induced mobilization of Ca2+ from the intracellular stores.

Influence of tumor necrosis factor alpha on intracellular Ca2+ in hen granulosa cells in vitro during follicular development.

In the present study, we have determined the influence of tumor necrosis factor alpha (TNF alpha) on both basal and carbamylcholine chloride (Cch)-induced [Ca2+]i in granulosa cells from the largest (F1) and smallest (F5,6) preovulatory follicles. TNF alpha (10 ng/ml) induced a small (51-63 nM) and delayed (approximately 1 min) transient increase in [Ca2+]i. The percentage of cells that responded to the cytokine was greater in F5,6 granulosa cells (48%; n = 48) than F1 granulosa cells (24%; n = 41). These responses were completely abolished in Ca(2+)-free media containing 5 mM EGTA and 2.5 mM Mg2+ or 1 mM Mn2+. Cch induced large increases (> 250 nM) in [Ca2+]i via mobilization of Ca2+ from intracellular stores in approximately 50% of Cch-responsive F1 granulosa cells but only about 15% of Cch-responsive F5,6 cells. Pretreatment with TNF alpha (4-5 min) increased the magnitude of the Cch response in both F1 and F5,6 granulosa cells previously incapable of producing large Cch-induced changes in [Ca2+]i. In F1 cells, the effects of TNF alpha on Cch-induced [Ca2+]i were far more extensive, such that the Cch response in the presence of TNF alpha was indistinguishable from the fast Cch-induced Ca2+ transient reported previously. Furthermore, the TNF alpha effect was reversible, as subsequent challenge with Cch in the absence of TNF alpha failed to produce the large Ca(2+)-transients observed earlier with the cytokine present. In conclusion, TNF alpha induces transient increases in [Ca2+]i by transmembrane Ca2+ flux, which are suppressed during cytodifferentiation. In addition, TNF alpha appeared to enhance Cch-induced mobilization of Ca2+ from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the muscarinic agonist carbachol on intracellular Ca2+ in chicken granulosa cells: I. Dependence on follicular maturation.

The present study addressed the influence of follicular development on carbamylcholine chloride (Cch)-induced Ca2+ transients and inositol 1,4,5-trisphosphate (IP3) production in granulosa cells isolated from the first (F1), third (F3), and fifth and sixth (F5,6) largest follicles. Intracellular free calcium, [Ca2+]i, was measured in fura-2-loaded cells about 20-36 h after their isolation. The percentage of cells responding to a maximal stimulatory concentration of Cch (0.2 mM) was higher in the F1 (89%) granulosa cells than in cells from the F3 (68%) and the F5,6 (72%) follicles. Most of the Ca2+ transients that were elicited in F1 granulosa cells were characterized by large (696 +/- 119 nM), fast (260 +/- 55 nM/sec) increases in [Ca2]i followed by a slow, uneven decrease in [Ca2+]i to the resting concentration. In contrast, Cch-induced changes in [Ca2+]i in F3 and F5,6 granulosa cells were generally both smaller (154 +/- 37 nM and 165 +/- 37 nM, respectively) and slower (36 +/- 25 nM/sec and 46 +/- 16 nM/sec, respectively) than those observed in cells from the largest follicle. Removal of external Ca2+ did not alter the large, fast increases in [Ca2+]i; however, it nearly blocked the slow responses observed in F3 and F5,6 cells. IP, production was elevated in 3H-myo-inositol-loaded F1 granulosa cells after 1 min of Cch (0.2 mM) treatment, whereas inositol bisphosphate (IP2) production and inositol monophosphate (IP) production were elevated only after longer incubations.(ABSTRACT TRUNCATED AT 250 WORDS)