Adenylate cyclase 5 and KCa1.1 channel are required for EGFR up‐regulation of PCNA in native contractile rat basilar artery smooth muscle

Abstract

In synthetic phenotype vascular smooth muscle cells (VSMC), activation of epidermal growth factor (EGF) receptor (EGFR) induces a sustained increase in intermediate conductance K(Ca) (int-K(Ca); K(Ca)3.1) channels that is essential for proliferation. However, a comparable mechanism has not been identified in native contractile phenotype VSMC, which express large conductance K(Ca) (maxi-K(Ca); K(Ca)1.1) channels, not int-K(Ca) channels. Using patch clamp of freshly isolated contractile VSMC from rat basilar artery, we found that EGF (100 ng ml(-1)) caused hyperpolarization (7.9 +/- 3.9 mV) due to activation of iberiotoxin-sensitive, maxi-K(Ca) channels. The EGFR ligands EGF (100 ng ml(-1)), transforming growth factor alpha (0.4 ng ml(-1)) and heparin-binding EGF (100 ng ml(-1)) all caused a 20% increase in maxi-K(Ca) channel current that was blocked by AG-1478 or by knock-down of EGFR expression using cisterna magna infusion of antisense oligodeoxynucleotide (AS-ODN). In controls, EGFR knock-down, and EGFR gain-of-expression (angiotensin II hypertension), the increase in maxi-K(Ca) current correlated with the abundance of EGFR protein expressed. The EGFR-mediated increase in maxi-K(Ca) channel activity was blocked by inhibiting cAMP-dependent protein kinase (cAK) using KT-5720 or Rp-cAMP, or by inhibiting adenylate cyclase type 5 (AC-5) using 2',5'-dideoxyadenosine or knock-down of AC-5 expression by intracisternal AS-ODN. Direct infusion of EGF into cisterna magna caused up-regulation of proliferating cell nuclear antigen (PCNA) in VSMC that was prevented by coinfusion of iberiotoxin or of AG-1478. Our data, which are consistent with the hypothesis that hyperpolarization is critical for a proliferative response, are the first to implicate AC-5 and maxi-K(Ca) channels in gene activation related to EGFR signalling in native contractile VSMC.