Our studies in intact pulmonary arteries have suggested that capacitative Ca²⁺ entry (CCE) may play a role in the initial rise in [Ca²⁺]_i associated with hypoxia in small intrapulmonary arteries (IPA) of the rat. We have examined CCE in more detail in enzymatically dispersed IPA smooth muscle cells (PASMC). Male Wistar rats (250-350 g) were anaesthetized with sodium pentobarbitone (55 mg kg^-1 I.P.) and killed by cervical dislocation as approved by the Home Office Inspector. PASMC loaded with fura PE-3 exhibited a measurable rate of fluorescence quench with extracellular Mn²⁺. This was considerably diminished by 10 µM La³⁺ and completely suppressed with 10 µM Gd³⁺. Emptying of intracellular calcium stores with thapsigargin (Thg, 100 nM) significantly increased the rate of quench. In whole-cell recording occasional opening of non-selective cation channels with a conductance of ~35 pS occurred. Thg dramatically increased their frequency of opening, generating a noisy inward current with an amplitude of 6.2 ± 2.4 pA pF^-1 (mean ± S.E.M., n = 17). This current was carried mainly by Na⁺, but could also be sustained in various degrees with K⁺, Cs⁺ and Ca²⁺. La³⁺, Gd³⁺ (both 1 µM) and 2-aminophenylborate (2-APB, 75 µM) blocked the Thg-induced inward current, but did not block an additional cation current induced by 50 µM ATP. In small IPA re-introduction of Ca²⁺ after pre-incubation with Thg in Ca²⁺-free solution caused a significant contraction that was sensitive to La³⁺ and 2-APB. This response was not observed when Sr²⁺ was substituted for Ca²⁺. However, the constrictor response to ATP could be observed with Sr²⁺, and, unlike the Thg-induced response, was resistant to 2-APB and low concentrations of La³⁺.

Thus we have found that emptying of intracellular Ca²⁺ stores greatly increases Ca²⁺ entry into PASM. Such entry occurs via non-selective cation channels that are highly sensitive to lanthanides, blocked by 2-APB, and are not permeable to Sr²⁺. Agonists such as ATP activate an additional Ca²⁺ entry pathway that is relatively resistant to lanthanides and 2-APB, but permeable to Sr²⁺. These data are consistent with the description of two distinct Ca²⁺ entry pathways in TRP3-transfected HEK293 cells (Ma et al. 2000). Interestingly, both pathways are coupled with substantial functional contractile responses in IPA.This work was supported by The Wellcome Trust (062554).

Ma, H.T., Patterson, R.L., Van Rossum, D.B., Birnbaumer, L., Mikoshiba, K. & Gill, D.L. (2000). Science 287, 1647-1651.