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Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations.

ABSTRACT: Store-operated Ca(2+) channels in liver cells have been shown previously to exhibit a high selectivity for Ca(2+) and to have properties indistinguishable from those of Ca(2+)-release-activated Ca(2+) (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938-947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](cyt)) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75 microM), Gd(3+) (1 microM) and 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365; 50 microM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca(2+) oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca(2+) concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 [( R, S )-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl- N, N -di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamide mesylate] (30 microM), used commonly to block Ca(2+) inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca(2+) oscillations. In the absence of added extracellular Ca(2+), 2-APB, Gd(3+) and SK&F 96365 did not alter the kinetics of the increase in [Ca(2+)](cyt) induced by a concentration of adrenaline or vasopressin that induces continuous Ca(2+) oscillations at the physiological extracellular Ca(2+) concentration. Ca(2+) inflow through non-selective cation channels activated by maitotoxin could not restore Ca(2+) oscillations in cells treated with 2-APB to block Ca(2+) inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It is concluded that Ca(2+) inflow through CRAC channels is required for the maintenance of hormone-induced Ca(2+) oscillations in isolated hepatocytes.

SUBMITTER: Gregory RB

PROVIDER: S-EPMC1223201 | biostudies-other | 2003 Mar

REPOSITORIES: biostudies-other

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Project description:Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.

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Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations.

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