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: 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 05/2014; DOI:10.1016/j.pbb.2014.03.005 · 2.82 Impact Factor
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ABSTRACT: Cisplatin-like chemotherapeutics cause vomiting via release of multiple neurotransmitters (dopamine, serotonin (5-HT), or substance P (SP)) from the gastrointestinal enterochromaffin cells and/or the brainstem via a calcium dependent process. Diverse channels in the plasma membrane allow extracellular Ca2+ entry into cells for the transmitter release process. Agonists of 5-HT3 receptors increase calcium influx through both 5-HT3 receptors and L-type Ca2+ channels. We envisaged that L-type calcium agonists such as FPL 64176 should cause vomiting and corresponding antagonists such as nifedipine would behave as broad-spectrum antiemetics. Administration of FPL 64176 did cause vomiting in the least shrew in a dose-dependent fashion. Nifedipine and the 5-HT3 receptor antagonist palonosetron, potently suppressed FPL 64176-induced vomiting, while a combination of ineffective doses of these antagonists was more efficacious. Subsequently, we investigated the broad-spectrum antiemetic potential of nifedipine against diverse emetogens including agonists of serotonergic 5-HT3- (e.g. 5-HT or 2-Me-5-HT), SP tachykinin NK1- (GR73632), dopamine D2- (apomorphine or quinpirole), and cholinergic M1- (McN-A-343) receptors, as well as the non-specific emetogen, cisplatin. Nifedipine by itself suppressed vomiting in a potent and dose-dependent manner caused by the above emetogens except cisplatin. Moreover, low doses of nifedipine potentiated the antiemetic efficacy of non-effective or semi-effective doses of palonosetron against vomiting caused by either 2-Me-5-HT or cisplatin. Thus, our findings demonstrate that activation of L-type calcium channels causes vomiting, whereas blockade of these ion channels by nifedipine-like antagonists not only provides broad-spectrum antiemetic activity but can also potentiate the antiemetic efficacy of well-established antiemetics such as palonosetron. L-type calcium channel antagonists should also provide antiemetic activity against drug-induced vomiting as well as other emetogens including bacterial and viral proteins.European journal of pharmacology 01/2014; 722:2–12. DOI:10.1016/j.ejphar.2013.08.052 · 2.68 Impact Factor
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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. DOI:10.1016/B978-0-12-407870-3.00007-X · 1.77 Impact Factor