Agonist‐dependent Ca2+ and Mn2+ entry dependent on state of filling of Ca2+ stores in aortic smooth muscle cells of the rat.

1. The properties of intracellular Ca2+ stores of intact‐ and of saponin‐skinned A7r5 (an established cell line from embryonic rat aorta) smooth muscle cells were studied by measuring 45Ca2+ and 54Mn2+ fluxes. 2. Application of 5 microM‐vasopressin to intact cells increased the fractional loss of 45Ca2+ in Ca2(+)‐free solution by a factor of 5.2. This effect was not influenced by a pre‐incubation with 10 microM‐ryanodine. Caffeine (25 mM) did not stimulate the fractional loss of 45Ca2+ from intact cells. 3. In skinned cells 10 microM‐IP3 (inositol 1,4,5‐trisphosphate) and 5 microM‐A23187 (a calcium ionophore) released the same amount of 45Ca2+. This release did not require GTP and was not affected by a pre‐incubation with 10 microM‐ryanodine. Caffeine (25 mM) did not release stored Ca2+. 4. NaF (1 mM) plus 10 microM‐AlCl3 inhibited by 72% the 45Ca2+ uptake by the IP3‐sensitive store of skinned cells at 0.15 microM‐Ca2+. Cyclic AMP‐dependent protein kinase did not stimulate this ATP‐dependent 45Ca2+ uptake, nor could the presence of phospholamban be demonstrated immunologically. 5. The 45Ca2+ uptake by cells which had been depleted of Ca2+ with 5 microM‐vasopressin was 69% higher than the uptake obtained without such proceeding depletion. This enhanced 45Ca2+ uptake did not occur through voltage‐operated Ca2+ channels, because blockade of these channels with verapamil, or depolarization of the plasma membrane by increasing [K+] from 5.9 to 59 mM in the presence of verapamil, did not modify this uptake. 6. A similar increase of the 54Mn2+ uptake occurred in intact cells with a depleted Ca2+ store. If, however, the cells were first skinned and subsequently exposed to 54Mn2+, the ATP‐dependent 54Mn2+ uptake amounted to less than 6% of the ATP‐dependent 45Ca2+ uptake. 7. If intact cells were first exposed to a 45Ca2(+)‐ or 54Mn2(+)‐containing solution, and subsequently skinned in a non‐radioactive intracellular solution, the addition of 10 microM‐A23187 to these cells released stored Ca2+ or Mn2+. The amount of released Ca2+ was only slightly larger than the amount of released Mn2+. If the intracellular store was depleted before loading, the amount of Ca2+ or Mn2+ released by the ionophore increased by 68 and 28%, respectively. 8. It is concluded that A7r5 smooth muscle cells do not express a Ca2(+)‐induced Ca2+ release mechanism, but do contain an IP3‐induced Ca2+ release mechanism which can release approximately all intracellularly accumulated 45Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)