Adenylate cyclase 5 and KCa1.1 channel are required for EGFR up-regulation of PCNA in native contractile rat basilar artery smooth muscle.
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.
Ivanov
Alexander
A
Department of Neurosurgery, 22 S. Greene Street, Suite 12SD, Baltimore, MD 21201-1595, USA.
Gerzanich
Volodymyr
V
Ivanova
Svetlana
S
Denhaese
Ryan
R
Tsymbalyuk
Orest
O
Simard
J Marc
JM
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001488:Basilar Artery / Q000201:enzymology; D049109:Cell Proliferation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D016048:Dideoxyadenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005260:Female; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D016211:Transforming Growth Factor alpha / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D007527:Isoenzymes; D051036:Large-Conductance Calcium-Activated Potassium Channels; D016376:Oligonucleotides, Antisense; D018809:Proliferating Cell Nuclear Antigen; D016211:Transforming Growth Factor alpha; D016048:Dideoxyadenosine; D004815:Epidermal Growth Factor; C021008:2',5'-dideoxyadenosine; D066246:ErbB Receptors; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; C419510:adenylyl cyclase type V