Ca²⁺ pathway involved in the refilling of store sites in rat adrenal medullary cells

Abstract

It has been suggested that store-operated Ca(2+) entry (SOC) facilitates catecholamine secretion and synthesis in bovine adrenal medullary (AM) cells. However, there has been no experimental result clearly showing that cation channel activity is enhanced by store Ca(2+) depletion. Thus the present experiments were undertaken to address the issue of whether rat AM cells have SOC channels. Inhibition of the sarco(endo)plasmic reticulum Ca(2+) (SERCA) pump resulted in a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat AM cells. This increase was completely suppressed by 2 mM Ni(2+) but not by 100 muM D600. A bath application of Ni(2+), but not D600, produced an outward current at -60 mV in rat AM cells, whereas exposure to a SERCA pump inhibitor did not affect either the whole cell current level or the Ni(2+)-induced outward current. The refilling of intracellular store sites was suppressed by the addition of Ni(2+) to the perfusate. RT-PCR revealed that transcripts for transient receptor potential channels 1 (TRPC1) and 5 (TRPC5) were present in rat adrenal medullas. Immunocytochemistry showed that TRPC1 channels, which have been implicated in SOC in certain types of cells, were mainly localized in the endoplasmic reticulum (ER) and not in the plasma membrane, and that STIM1, a Ca(2+) sensor in the ER, was not expressed in rat AM cells. On the basis of these results, we conclude that rat AM cells lack the SOC mechanism.

Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

“…7). TRPC1-like immunoreactivities were distributed in a reticular pattern in the cytoplasm (n ϭ 16), as has been noted in rat AM cells (35). TRPC4-like (n ϭ 8) and TRPC7-like immunoreactivities (n ϭ 12) exhibited a punctate distribution.…”

“…TRPC5 channels were immunologically detected at the cell periphery, probably at the cell membrane, and TRPC5-GFP channels exogenously expressed in PC12 cells were also located at the cell periphery (35). On the other hand, TRPC4 channels and exogenously expressed TRPC4-GFP channels were distributed in a punctate pattern in the cytoplasm of guinea-pig AM and PC12 cells (unpublished observations by K. Harada), respectively.…”

“…Immunoblotting of homogenates of the brain, adrenal cortex, and adrenal medulla revealed TRPC4 proteins of 110 kDa and TRPC5 of 106 kDa and TRPC7 of about 96 kDa. TRPC1 proteins, however, could not be detected in immunoblotting of any homogenates (35). Thus immunoprecipitation and immunoblotting were combined to detect TRPC1 channels.…”

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

“…Besides, it is plausible that the scaffolding proteins that have been shown to interact with TRPC1 are still able to interact with TRPC1 anchored in the SR membrane. Very recently, TRPC1 was also found to localize in the endoplasmic reticulum in freshly isolated adrenal medullary cells, and this distribution was confirmed by the finding that exogenous expression of TRPC1-GFP proteins in adrenal medullary cell line resulted in the localization of the fusion protein in the endoplasmic reticulum and not in the plasma membrane (15). As described in these glandular cells, the similar expression patterns we observed for endogenous and exogenous TRPC1 using two different modalities for detection give strong evidence that TRPC1 localizes to the longitudinal SR and not to the plasma membrane in adult skeletal muscle.…”

Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells

“…The reticular pattern of the IR distribution suggested that Syt 9 was located in the endoplasmic reticulum (ER). Therefore, rat chromaffin cells immunostained for Syt 9 were exposed to BODIPY-FL thapsigargin, a chemical composed of the irreversible SERCA pump inhibitor, thapsigargin, conjugated with a fluorescent molecule (Inoue et al 2003;Matsuoka et al 2009). Double-staining clearly showed that Syt 9-like IR was consistent with fluorescent thapsigargin-binding (coincidence rate of Syt 9-like IR with thapsigargin [Thap] binding: Thap/ Syt 9, 73.0±4.3%, n=7).…”