Kinase-dependent activation of voltage-gated Ca 2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

Trevor Luke, Julie Maylor, Clark Undem, J. T. Sylvester, Larissa Shimoda

Research output: Contribution to journalArticle

Abstract

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca 2+]i) occurring through activation of voltage-dependent Ca 2+ channels (VDCC) even though ET-1-induced depolarization via inhibition of K + channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca 2+]i in PASMCs from rats exposed to CH (10% O 2, 3 wk) using the Ca 2+-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca 2+]i by 70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca 2+]i to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca 2+]I in PASMCs occurs via Ca 2+ influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume302
Issue number10
DOIs
StatePublished - May 15 2012

Fingerprint

rho-Associated Kinases
Endothelin-1
Muscle Cells
Phosphotransferases
Lung
Calcium Channels
Protein-Tyrosine Kinases
Smooth Muscle Myocytes
Pulmonary Hypertension
Staurosporine
Hypoxia
Genistein
Fura-2
Vasoconstrictor Agents
Guanosine Triphosphate
Protein Kinase C
protein kinase C kinase
Microscopy
Acetates
Calcium

Keywords

  • Endothelin-1
  • Intracellular calcium concentration
  • Protein kinase C
  • Rho kinase
  • Vascular smooth muscle

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology
  • Physiology

Cite this

Kinase-dependent activation of voltage-gated Ca 2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia. / Luke, Trevor; Maylor, Julie; Undem, Clark; Sylvester, J. T.; Shimoda, Larissa.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 302, No. 10, 15.05.2012.

Research output: Contribution to journalArticle

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abstract = "Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca 2+]i) occurring through activation of voltage-dependent Ca 2+ channels (VDCC) even though ET-1-induced depolarization via inhibition of K + channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca 2+]i in PASMCs from rats exposed to CH (10{\%} O 2, 3 wk) using the Ca 2+-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca 2+]i by 70{\%}. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca 2+]i to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca 2+]I in PASMCs occurs via Ca 2+ influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.",
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AU - Shimoda, Larissa

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AB - Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca 2+]i) occurring through activation of voltage-dependent Ca 2+ channels (VDCC) even though ET-1-induced depolarization via inhibition of K + channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca 2+]i in PASMCs from rats exposed to CH (10% O 2, 3 wk) using the Ca 2+-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca 2+]i by 70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca 2+]i to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca 2+]I in PASMCs occurs via Ca 2+ influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.

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