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内容記述 |
Since extracellular Ca2+ has been reported to modulate swelling-activated Cl- currents, I examined an involvement of G protein-coupled Ca2+-sensing receptor (CaR) in the regulation of the volume-sensitive Cl- channel by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblotting and whole-cell patch-clamp techniques, in a human epithelial cell line (Intestine 407). <br />RT-PCR confirmed that the Intestine 407 cell contains mRNAs cording for the CaR, and expression of the CaR protein was evidenced by immunoblotting analysis. The swelling-activated whole-cell Cl- current was augmented by addition of Ca2+ to the bathing solution in a concentration-dependent manner. The total Ca2+ concentration for half-maximal stimulation (EC50) was 6.5 mM. A rise in the extracellular Mg2+ concentration also concentration-dependently increased the amplitude of volume-sensitive Cl- currents, though less effective (EC50 of around 22 mM) than Ca2+. In addition, other CaR agonists, La3+ (3 μM), neomycin (500 μM) and spermine (1 mM), significantly augmented the Cl- current. To further confirm an involvement of the CaR in the upregulating effect of extracellular Ca2+ on the volume-sensitive Cl- current, I examined the effects of GDPβS, which is a G protein inhibitor, and GTPγS, which is a G protein activator, on the Cl- current. Incorporation of GDPβS in the pipette (intracellular) solution abolished extracellular Ca2+-induced enhancement of the Cl- current. Under Ca2＋ - and Mg2+ -free conditions, the amplitude of volume-sensitive Cl- currents became increased by the presence of intracellular GTPγS. Further augmentation was never induced by addition of extracellular Ca2+ in the presence of intracellular GTPγS. These results demonstrate that the G protein-coupled CaR mediates Ca2+-induced upregulation of the volume-sensitive Cl- channel in Intestine 407 cells.<br />I then investigated the signal transduction pathway of CaR-mediated regulation of volume-sensitive Cl- channel. The augmenting effect of extracellular Ca2+ on the Cl- current could be abolished neither by application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA: 5 mM) to the pipette (intracellular) solution nor by 24 h-pretreatment with purtussis toxin (PTX: 100 ng/ml). When the intracellular cAMP concentration was elevated by application of a cocktail of forskolin (10 μM), dibutylyl cAMP (1 mM) and 3-isobutyl-1-methylxanthine (IBMX: 400 μM), the amplitude of volume-sensitive Cl- current was markedly enlarged. Under the cAMP stimulation, extracellular Ca2+ failed to increase the Cl- current. These results suggest that the CaR is coupled to Gs and modulates the volume-sensitive Cl- channel via an increase in intracellular cAMP level.<br />Effects of CaR stimulation on volume sensitivity of swelling-activated whole-cell Cl- current was then assessed. When the whole-cell Cl- current density was plotted against the relative cell surface area measured simultaneously, the data points were well fitted to the Boltzmann function. Elevation of extracellular Ca2+ shifted the curve to the left and increased the slope. These results indicate that CaR-mediated augmentation of the Cl- channel is due to increased sensitivity of the channel (or its accessory volume sensor) to cell volume expansion. <br />Extracellular Ca2+ or Mg2+ exhibited an additional effect on the volume-sensitive Cl- current: facilitation of its depolarization-induced <br />inactivation kinetics. The inactivation time course of the Cl- current at large positive potentials became faster in the presence of extracellular Ca2+ or Mg2+. The relative half inactivation time at + 100 mV was maximally decreased to 60.4 and 40.7% by Ca2+ and Mg2+, respectively. In contrast to the effects on the Cl- current amplitude, EC50 of the Mg2+ effect on the inactivation kinetics (2.1 mM) was smaller than that of the Ca2+ effect (2.5 mM). In addition, all other CaR agonists examined failed to accelerate the inactivation time course. Furthermore, the extracellular Ca2+ effect on inactivation kinetics was not affectcd by GDPβS or GTPγS. These results indicate that the CaR does not mediate the effect of extracellular Ca2+ or Mg2+ on the depolarization-induced inactivation kinetics. <br />Taken together, it is concluded that stimulation of CaR induces upregulation of volume-sensitive Cl- channels by enhancing the volume expansion sensitivity in human epithelial Intestine 407 cells. The second messenger is likely to be cAMP but not Ca2+ in the cytosol. The accelerating effect of extracellular divalent cations on inactivation time course of the Cl- current is induced by a different mechanism without mediation by the CaR. |
要旨・審査要旨 / Abstract, Screening Result (307.5 kB)
