| Abstrakt: |
1. To assess the nature of the underlying mechanism of noradrenaline‐induced increase of Cl‐ conductances in hepatocytes, macroscopic and unitary currents through noradrenaline‐induced Cl‐ channels were examined in enzymatically isolated guinea‐pig hepatocytes using whole‐cell, cell‐attached and excised inside‐out configurations of the patch‐clamp technique. 2. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 0.1 microM, bath application of noradrenaline activated the time‐independent membrane currents under whole‐cell voltage‐clamp conditions. The current was similarly activated by phorbol ester (PMA), an activator of protein kinase C (PKC), while a specific protein kinase C inhibitor, H‐9, reversed PMA activation of the current. The inactive phorbol ester, 4 alpha‐phorbol 12‐myristate, 13‐acetate (alpha PMA), failed to activate the channel. 3. The reversal potential of the PMA‐activated current shifted by approximately 60 mV per 10‐fold change in the external Cl‐ concentration, indicating that the current was Cl‐ selective. Bath application of 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS) partially inhibited both the noradrenaline‐ and PMA‐induced currents. 4. In single channel recordings from cell‐attached patches, bath application of noradrenaline or PMA induced unitary current activity, the averaged slope conductance of which was 10.1 +/‐ 1.5 pS (mean +/‐ S.D.; n = 12) in the noradrenaline‐induced current and 9.7 +/‐ 1.3 pS (n = 7) in the PMA‐induced current. The open time distribution was moderately well fitted by a single exponential function with mean open lifetime of 88.5 +/‐ 10.6 ms (n = 10), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 24.4 +/‐ 5.8 ms (n = 10) and for the slow component of 316.9 +/‐ 49.2 ms (n = 10). 5. Bath application of purified PKC to excised inside‐out patches activated the channel. The PKC selective inhibitor, PKC(19‐36), and DIDS inhibited the PKC‐activated channel. 6. These results suggest that PKC can phosphorylate the channel protein or a related structure leading to the activation of Cl‐ channels in guinea‐pig hepatocytes. |
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