Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II.
Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (alpha, delta, epsilon, zeta, iota/lambda, and mu) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (alpha, delta, zeta, iota/lambda, and mu) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-alpha from the cytosol to the nuclear envelope and of PKC-delta to the myofilaments. In contrast, cytosolic PKC-zeta did not translocate, but nuclear PKC-zeta was upregulated. Translocation of PKC-alpha and PKC-delta and upregulation of PKC-zeta in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+ concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The alpha and zeta isoforms may act to regulate nuclear events, whereas PKC-delta may be involved in modulating contraction via actions on the myofilaments.
Damron
D S
DS
Center for Anesthesiology Research, Cleveland Clinic Foundation, Ohio 44195, USA.
Nadim
H S
HS
Hong
S J
SJ
Darvish
A
A
Murray
P A
PA
D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D015388:Organelles / Q000201:enzymology; D011493:Protein Kinase C / Q000378:metabolism*; D011651:Pulmonary Artery / Q000201:enzymology*
D007527:Isoenzymes; D000804:Angiotensin II; D011493:Protein Kinase C; D002118:Calcium