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Chloride-sensitive nature of the histamine-induced Ca²⁺ entry in cultured human aortic endothelial cells

Kyoichi Ono, Miki Nakao and Toshihiko Iijima

1. Whole-cell currents and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were recorded in cultured human aortic endothelial cells (HAECs) to study the mechanisms underlying Cl^--sensitive Ca²⁺ entry.

2. In the absence of histamine the membrane potential ranged between -90 and +5 mV and showed bimodal distribution with peaks at -17·8 and -67·5 mV.

3. Histamine (1-100 µM) activated an outward current, followed by a sustained inward current at -50 mV. The reversal potential (V_rev) was more negative than -60 mV for the initial outward current, and approximately -30 mV for the sustained inward current with normal Tyrode solution and internal solution containing 30 mM Cl^-.

4. V_rev of the sustained inward current was hardly affected by varying the external concentrations of K⁺, Na⁺ and Ca²⁺, but was greatly changed by varying the external Cl^- concentration ([Cl^-]_o). The relationship between V_rev and log[Cl^-]_o showed a slope of -44·8 mV per tenfold increase of [Cl^-]_o.

5. The Cl^- channel blockers 9-anthracene carboxylic acid (1 mM), N-phenylanthranilic acid (0·1 mM) and niflumic acid (0·1 mM) all depressed the histamine-induced inward current. The non-selective cation channel blocker Gd³⁺ (10 µM) was without effect on the current.

6. In the absence of histamine, [Ca²⁺]_i was not affected by varying the membrane potential. During the continuous presence of histamine, however, hyperpolarization increased and depolarization decreased [Ca²⁺]_i, indicating that Ca²⁺ entry through the plasma membrane was activated by histamine.

7. V_rev of the histamine-induced Cl^- current, measured by the gramicidin-perforated patch clamp method, was -28·4 ± 6·6 mV (n = 8), which gave an intracellular Cl^- concentration of approximately 34 mM. Under the current clamp condition, the membrane potential varied from cell to cell in the control, but application of histamine induced either depolarization or hyperpolarization, depending on the membrane potential before histamine application, and the membrane potential became stable near the equilibrium potential for Cl^-.

8. We conclude that the histamine-induced inward current is carried mainly by Cl^-. Although Ca²⁺ entry was also activated, we consider that its amplitude was too small to be resolved by the patch clamp method. The Cl^- current may play a functional role in the sustained [Ca²⁺]_i elevation by providing a constant driving force for Ca²⁺ entry in the presence of histamine.

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