Effects of extracellular pH on receptor-mediated Ca2+ influx in A7r5 rat smooth muscle cells: involvement of two different types of channel

1. The effects of extracellular pH (pH(o)) on receptor (vasopressin or endothelin-1)-mediated Ca2+ entry and Ca2+-permeable channels were investigated in aortic smooth muscle cells (A7r5) from rat embryonic thoracic aorta. Intracellular Ca2+ ([Ca2+](i)) was measured using fura-2 AM and whole-cell voltage clamp techniques were employed. 2. Vasopressin and endothelin-1 (100 nM) in the presence of nicardipine (10 mu M) evoked a sustained rise in [Ca2+](i) due to calcium entry. Extracellular acidosis decreased receptor (vasopressin or endothelin-1)-mediated Ca2+ entry while extracellular alkalosis potentiated it. 3. Depletion of intracellular Ca2+ stores with thapsigargin (1 mu M) also evoked Ca2+ entry activated by emptying of intracellular Ca2+ stores (capacitative Ca2+ entry). Extracellular acidosis decreased this capacitative Ca2+ entry, while extracellular alkalosis potentiated it. 4. Under voltage-clamp conditions with Cs+ internal solution, vasopressin and endothelin-1 activated non-selective cation currents (I-CAT). Ba2+ or Ca2+ were also charge carriers of I-CAT. Reducing the pH(o) inhibited I-CAT, while increasing pH(o) potentiated it in a reversible manner. 5. Intracellular pH (pH(i)) changes did not cause the same marked effects as pH(o) changes, and a high concentration of Hepes (50 mM) in the patch pipette did not inhibit the effects of pH(o) On I-CAT. 6. Similar results were obtained when I-CAT was activated by GTP gamma S (1 mM) applied through the patch pipette, even in the absence of agonists, probably because of direct activation of GTP-binding proteins coupled to the receptors. 7. In cells treated with thapsigargin, addition of Ca2+ to the bath solution induced Ca2+-dependent K+ currents activated by capacitative Ca2+ entry. However, no measurable ionic currents activated by capacitative Ca2+ entry (I-CRAC) were observed under conditions with Cs+ internal solution and EGTA (5 mM), although vasopressin still activated I-CAT. 8. These results suggest that the contractile agonists vasopressin and endothelin-1 evoke Ca2+ entry through two different types of Ca2+-permeable channel (I-CAT and I-CRAC) and pH(o) affects these channels, which may modulate receptor-mediated Ca2+ influx in A7r5 cells. Thus, pH-induced changes of these channels may play a pathophysiological role in the control of receptor-mediated contractions.