Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

Am J Physiol Lung Cell Mol Physiol. 2000 Jan;278(1):L118-30. doi: 10.1152/ajplung.2000.278.1.L118.

Abstract

We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adrenergic alpha-Agonists / pharmacology
  • Animals
  • Calcium / metabolism*
  • Calcium Channel Blockers / pharmacology
  • Dogs
  • Electric Conductivity
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / metabolism*
  • Oscillometry
  • Osmolar Concentration
  • Phenylephrine / pharmacology
  • Protein-Tyrosine Kinases / metabolism*
  • Pulmonary Artery / cytology
  • Pulmonary Artery / metabolism*
  • Reproducibility of Results
  • Sarcoplasmic Reticulum / metabolism
  • Thapsigargin / pharmacology

Substances

  • Adrenergic alpha-Agonists
  • Calcium Channel Blockers
  • Enzyme Inhibitors
  • Phenylephrine
  • Thapsigargin
  • Protein-Tyrosine Kinases
  • Calcium