Abstract
Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.
Highlights
From the Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892
Role of a Staurosporine-sensitive Protein Kinase in the Ca2ϩdependent Inactivation of store-operated Ca2ϩ entry (SOCE)—Based on our previous studies, we have suggested that the Ca2ϩ-dependent feedback inhibition of SOCE in rat parotid gland cells is mediated via a staurosporine-sensitive protein kinase [12, 14]
To determine whether SOCE in HSG cells is affected by modulators of protein phosphorylation, we studied the effects of okadaic acid and calyculin A on thapsigargin-stimulated [Ca2ϩ]i changes in fura2-loaded HSG cells
Summary
Vol 273, No 50, Issue of December 11, pp. 33295–33304, 1998 Printed in U.S.A. Ca2؉-dependent Inactivation of a Store-operated Ca2؉ Current in Human Submandibular Gland Cells. The data demonstrate that internal Ca2؉ store depletion-activated Ca2؉ current (ISOC) in this salivary cell line is regulated by a Ca2؉-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2؉ pump. We have recently reported [14] that the staurosporine-sensitive protein kinase is involved in the Ca2ϩ-induced feedback inhibition of Ca2ϩ influx in rat parotid acinar cells. This kinase does not appear to be involved in the refill-dependent inactivation of SOCE, i.e. refill-dependent inactivation was not prevented by staurosporine [14]. The present data demonstrate that SERCA modulates the Ca2ϩ-dependent feedback inhibition of the store-operated Ca2ϩ influx channel
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