Regulation of the novel Mg2+ transporter transient receptor potential melastatin 7 (TRPM7) cation channel by bradykinin in vascular smooth muscle cells

J Hypertens. 2009 Jan;27(1):155-66. doi: 10.1097/hjh.0b013e3283190582.

Abstract

Background: Transient receptor potential melastatin 7 (TRPM7) channels have been identified in the vasculature. However, their regulation and function remain unclear.

Methods: Here, we tested the hypothesis that bradykinin and its second messenger cAMP upregulate TRPM7, which stimulates activation of annexin-1 (TRPM7 substrate) and increases transmembrane Mg2+ transport and vascular smooth muscle cell (VSMC) migration. Human and rat VSMCs were studied. TRPM7 phosphorylation was assessed by immunoprecipitation:immunoblotting using antiphospho-serine/threonine and anti-TRPM7 antibodies. [Mg2+]i was measured by mag-fura-2. TRPM7 was downregulated by small interfering RNA and 2-aminoethoxydiphenyl borate. Annexin-1 activity was assessed by cytosol-to-membrane translocation. Cell migration and invasion, functional responses to bradykinin, were assessed in transwell chambers.

Results: Bradykinin increased expression of TRPM7 and annexin-1. TRPM7 was rapidly (5 min) phosphorylated on serine/threonine but not on tyrosine residues by bradykinin. [Mg2+]i was increased in bradykinin-stimulated cells (0.53 versus 0.68 mmol/l, basal versus bradykinin, P < 0.01). Annexin-1 activation was increased by bradykinin and inhibited by 2-aminoethoxydiphenyl borate. Although Hoe 140 (B2 receptor antagonist), U-73122 (phospholipase C inhibitor), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (c-Src inhibitor) and chelerythrine (protein kinase C inhibitor) blocked bradykinin actions, dibutyryl-c-AMP was without effect. In small interfering RNA-transfected and in 2-aminoethoxydiphenyl borate-treated cells, bradykinin-induced Mg2+ influx and VSMC migration were reduced.

Conclusion: Our results demonstrate that bradykinin regulates TRPM7 and its downstream target annexin-1 through phospholipase C-dependent, protein kinase C-dependent and c-Src-dependent and cAMP-independent pathways; effects are mediated through bradykinin type 2 receptor; and bradykinin regulates VSMC [Mg2+]i and migration through TRPM7. These data identify TRPM7/annexin-1/Mg2+ as a novel pathway in bradykinin signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Annexin A1 / metabolism
  • Bradykinin / pharmacology*
  • Cell Movement / drug effects
  • Cells, Cultured
  • Cyclic AMP / physiology
  • Humans
  • Ion Transport / drug effects
  • Magnesium / metabolism*
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / metabolism*
  • Myocytes, Smooth Muscle / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases
  • Rats
  • Rats, Inbred WKY
  • Receptor, Bradykinin B2 / physiology
  • TRPM Cation Channels / physiology*

Substances

  • Annexin A1
  • Receptor, Bradykinin B2
  • TRPM Cation Channels
  • Cyclic AMP
  • Protein-Serine-Threonine Kinases
  • TRPM7 protein, human
  • Magnesium
  • Bradykinin