Role of Trpc channels, Stim1 and Orai1 in PGF(2α)-induced calcium signaling in NRK fibroblasts.
ABSTRACT Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F(2α) (PGF(2α))-induced Ca(2+)-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF(2α)-induced Ca(2+) oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF(2α)-induced Ca(2+) oscillations.
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ABSTRACT: Store-operated calcium entry (SOCE) is activated in response to depletion of the endoplasmic reticulum-Ca(2+) stores following stimulation of plasma membrane receptors that couple to PIP2 hydrolysis and IP3 generation. Search for the molecular components of SOCE channels led to the identification of mammalian transient receptor potential canonical (TRPC) family of calcium-permeable channels (TRPC1-TRPC7), which are all activated in response to stimuli that result in PIP2 hydrolysis. While several TRPCs, including TRPC1, TRPC3, and TRPC4, have been implicated in SOCE, the data are most consistent for TRPC1. Extensive studies in cell lines and knockout mouse models have established the contribution of TRPC1 to SOCE. Furthermore, there is a critical functional interaction between TRPC1 and the key components of SOCE, STIM1, and Orai1, which determines the activation of TRPC1. Orai1-mediated Ca(2+) entry is required for recruitment of TRPC1 and its insertion into surface membranes while STIM1 gates the channel. Notably, TRPC1 and Orai1 generate distinct patterns of Ca(2+) signals in cells that are decoded for the regulation of specific cellular functions. Thus, SOCE appears to be a complex process that depends on temporal and spatial coordination of several distinct steps mediated by proteins in different cellular compartments. Emerging data suggest that, in many cell types, the net Ca(2+) entry measured in response to store depletion is the result of the coordinated regulation of different calcium-permeable ion channels. Orai1 and STIM1 are central players in this process, and by mediating recruitment or activation of other Ca(2+) channels, Orai1-CRAC function can elicit rapid changes in global and local [Ca(2+)]i signals in cells. It is most likely that the type of channels and the [Ca(2+)]i signature that are generated by this process reflect the physiological function of the cell that is regulated by Ca(2+).Current Topics in Membranes 01/2013; 71:149-79. · 1.06 Impact Factor
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ABSTRACT: Recent findings indicate that histidine triad nucleotide-binding protein 1 (HINT1) is implicated in the pathophysiology of certain psychiatric disorders, and also exhibits tumor suppressor properties. However, the authentic functions of HINT1 in cellular physiology and especially its role in Ca(2+) signaling remain unclear. Here, we studied Ca(2+) signaling in cultured embryonic fibroblasts derived from wild type control and HINT1 knockout (KO) mice. Resting cytosolic Ca(2+) level (measured with fura-2) was not altered in fibroblasts lacking HINT1. The stored Ca(2+) evaluated by measuring peak amplitude of ATP (10µM)-induced Ca(2+) transients in Ca(2+)-free medium was significantly larger in HINT1 KO fibroblasts than in wild-type cells. Ca(2+) influx after external Ca(2+) restoration, likely via store- and receptor-operated channels (SOCs and ROCs, respectively), was greatly (by 2-fold) reduced in HINT1 KO fibroblasts. This correlated with a down-regulated expression of Orai1 and stromal interacting molecule 1 (STIM1), essential components of store-operated Ca(2+) entry pathway. Expression of C-type transient receptor potential TRPC3 and TRPC6, which function as ROCs, was not altered in HINT1 KO fibroblasts. Immunoblots also revealed that Orai1 was down-regulated by 2-fold in brain lysates of HINT1 KO mice in comparison with the wild-type littermates. Importantly, silencer RNA knockdown of HINT1 in Neuro-2A cells markedly down-regulated Orai1 and STIM1 protein expression and significantly (by 2.5-fold) reduced ATP-induced Ca(2+) influx, while ATP-evoked Ca(2+) release was not changed. Thus, the study demonstrates a novel function of HINT1 that involves the regulation of SOC-mediated Ca(2+) entry pathway (Orai1, STIM1), essential for regulation of cellular Ca(2+) homeostasis.AJP Cell Physiology 04/2013; · 3.71 Impact Factor
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ABSTRACT: The dihydropyridine L-type calcium (Ca2 +) channel blockers such as nifedipine and amlodipine reduce extracellular Ca2 + entry into cells and therefore affect intracellular Ca2 + dynamics. They are widely used for the treatment of hypertensive disorders. We have recently demonstrated that extracellular Ca2 + entry via L-type Ca2 + channels is involved in emesis and that nifedipine has broad-spectrum antiemetic activity. The aim of this study was to evaluate the antiemetic efficacy of the longer-acting L-type Ca2 + channel blocker, amlodipine. Fully effective emetic doses of diverse emetogens such as the L-type Ca2 + channel agonist (FPL 64176) as well as selective and/or nonselective agonists of serotonergic 5-HT3 (e.g. 5-HT or 2-Me-5-HT)-, dopamine D2 (e.g. apomorphine or quinpirole)-, cholinergic M1 (e.g. pilocarpine or McN-A343)- and tachykininergic NK1 (e.g. GR73632)- receptors, were administered intraperitoneally (i.p.) in the least shrew to induce vomiting. The broad-spectrum antiemetic potential of amlodipine was evaluated against these emetogens. Subcutaneous (s.c.) administration of amlodipine (0.5 - 10 mg/kg) attenuated in a dose-dependent and potent manner both the frequency and percentage of shrews vomiting in response to intraperitoneal (i.p.) administration of FPL 64176 (10 mg/kg), 5-HT (5 mg/kg), 2-Me-5-HT (5 mg/kg), apomorphine (2 mg/kg), quinpirole (2 mg/kg), pilocarpine (2 mg/kg), McN-A343 (2 mg/kg), or GR73632 (5 mg/kg). Combination of non-effective doses of amlodipine (0.5 mg/kg, s.c.) and the 5-HT3 receptor antagonist palonosetron (0.05 mg/kg, s.c.) were more effective against FPL 64176-induced vomiting than their corresponding doses tested alone. Amlodipine by itself suppressed the frequency of acute cisplatin (10 mg/kg, i.p)-induced vomiting in a dose-dependent manner. Moreover, combination of a non-effective dose of amlodipine (1 mg/kg) potentiated the antiemetic efficacy of a semi-effective dose of palonosetron (0.5 mg/kg, s.c.) against acute vomiting caused by cisplatin. In conclusion, this study further confirms that influx of extracellular Ca2+ ion underlies vomiting due to diverse causes and demonstrates that L-type Ca2 + channel blockers are a promising new class of broad-spectrum antiemetics.Pharmacology Biochemistry and Behavior 01/2014; · 2.61 Impact Factor