Visualizing the temporal effects of vasoconstrictors on PKC translocation and Ca²⁺ signaling in single resistance arterial smooth muscle cells

Abstract

Arterial smooth muscle (ASM) contraction plays a critical role in regulating blood distribution and blood pressure. Vasoconstrictors activate cell surface receptors to initiate signaling cascades involving increased intracellular Ca(2+) concentration ([Ca(2+)](i)) and recruitment of protein kinase C (PKC), leading to ASM contraction, though the PKC isoenzymes involved vary between different vasoconstrictors and their actions. Here, we have used confocal microscopy of enhanced green fluorescence protein (eGFP)-labeled PKC isoenzymes to visualize PKC translocation in primary rat mesenteric ASM cells in response to physiological vasoconstrictors, with simultaneous imaging of Ca(2+) signaling. Endothelin-1, angiotensin II, and uridine triphosphate all caused translocation of each of the PKC isoenzymes alpha, delta, and epsilon; however, the kinetics of translocation varied between agonists and PKC isoenzymes. Translocation of eGFP-PKCalpha mirrored the rise in [Ca(2+)](i), while that of eGFP-PKCdelta or -epsilon occurred more slowly. Endothelin-induced translocation of eGFP-PKCepsilon was often sustained for several minutes, while responses to angiotensin II were always transient. In addition, preventing [Ca(2+)](i) increases using 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl) ester prevented eGFP-PKCalpha translocation, while eGFP-PKCdelta translocated more rapidly. Our results suggest that PKC isoenzyme specificity of vasoconstrictor actions occurs downstream of PKC recruitment and demonstrate the varied kinetics and complex interplay between Ca(2+) and PKC responses to different vasoconstrictors in ASM.

Regulation of the ATP-sensitive Potassium Channel Subunit, Kir6.2, by a Ca2+-dependent Protein Kinase C

“…Recent studies have shown a clear translocation of PKC␣ from the cytosol to the membrane following stimulation with a phorbol ester in aortic and arterial smooth muscle cells (43,49). More specifically for our studies, the Ca 2ϩ -dependent isoforms PKC␣, ␤I, and ␤II show a strong endogenous presence in HEK293 cells (44).…”

Role for PKCβ in enhanced endothelin-1-induced pulmonary vasoconstrictor reactivity following intermittent hypoxia

“…Because PKC activity is generally reflected by changes in membrane-association of the enzyme (31,35,45), the effect of ET-1 on levels of membrane-localized PKC␣, ␤I, or ␤II were assessed in intrapulmonary arteries from sham and E-IH rats by Western blotting (3,43). Intrapulmonary arteries were isolated as above and placed in Arterial PO2, PCO2, and pH following 4 wk eucapnic intermittent hypoxia (E-IH) exposure.…”

Ca²⁺ dependent but PKC independent signalling mediates UTP induced contraction of rat mesenteric arteries

“…We have shown previously that UTP causes mobilization of PKCα, δ and ε in these cells (9). However, examination of the PKC dependence revealed that both contraction and the increased Ca 2+ in response to UTP were relatively insensitive to Tat-linked membrane permeable peptide inhibitors of PKC (see Fig.…”

“…We have shown that the vasoconstrictors AngII, ET-1 and UTP all mobilize PKCα, PKCδ and PKCε in mesenteric smooth muscle cells (9). We investigated the extent of various PKC isoform involvement in UTP induced contractions by using Tat-linked membrane permeable peptide inhibitors targeting particular PKC isoforms.…”

“…UTP, which is released in response to cellular injury (5), causes contraction of vascular smooth muscle by activating G-protein coupled P2Y receptors (6, 7); but in rat cerebral arteries UTP induced contraction was unaffected by the general PKC inhibitor bisindolylmaleimide and UTP inhibition of voltage-gated K + channels was not dependent on PKC (8). However, in rat mesenteric artery smooth muscle cells UTP, Ang II and ET-1 all increase intracellular Ca 2+ and induce the translocation of PKCα, PKCδ and PKCε (9). Interestingly, the contraction induced by Ang II works almost exclusively through PKCε while ET-1 works through PKCα (4), but whether specific PKC isoforms are involved in generating UTP induced contraction is not known.…”