Role of Protein Kinase C PKC

PKC exists as twelve isoforms that are subdivided into 4 groups according to their cofactor requirements, namely; classical, novel, atypical and recently described PKCs. Activation of classical and novel isoforms of PKC, via the application of phorbol esters, elicits Ca2+-independent contractions in pulmonary arteries (42) and other vascular smooth muscle preparations. PKC elicits Ca2+

sensitization by phosphorylating and activating CPI-17, which in turn inhibits the catalytic domain, PP-1C, ofMLCP (7, 44). An example of PKC-induced Ca2+ sensitization and contraction is shown in Figure 3, where application ofphorbol 12,13-dibutyrate to a rat small intrapulmonary artery elicited a slowly-developing contractile response, whilst [Ca2+]j remained constant. It is noteworthy that the time-course of this contractile response is similar to that of sustained HPV. However, inhibition of all isoforms of PKC with the bisindoylmaleimide PKC inhibitor, Ro-31-8220 (at a concentration that abolished the contraction to phorbol ester), had no effect on the sustained HPV (34). Interestingly, this compound did reduce the transient constrictor response to hypoxia by ~42% (34) consistent with a role for PKC during the transient phase of HPV. However, the latter possibility is complicated by the fact that Ro-31-8220 has subsequently been shown to inhibit several intracellular kinases other than PKC (3, 12). The lack of specificity of this compound does not, however, undermine the important conclusion that the Ca2+ sensitization observed during sustained HPV is independent of activation of PKC in rat isolated intrapulmonary arteries (34).

Figure 3. Activation of PKC elicits Ca2+ sensitization and contraction in pulmonary arteries. Experimental record of changes in [Ca2+]j (upper panel) and tension (lower panel) in a rat pulmonary artery exposed to either 75 mM KPSS (physiological salt solution containing 75 mM KC1, isotonic replacement of NaCl) or phorbol 12,13-dibutyrate (PdBu). Depolarization of the artery with KPSS results in elevations of both tension and [Ca2+];. In contrast, activation of PKC via exposure of the artery to PdBu results in a large sustained contraction that is not associated with any rise in [Ca2+]|.

Figure 3. Activation of PKC elicits Ca2+ sensitization and contraction in pulmonary arteries. Experimental record of changes in [Ca2+]j (upper panel) and tension (lower panel) in a rat pulmonary artery exposed to either 75 mM KPSS (physiological salt solution containing 75 mM KC1, isotonic replacement of NaCl) or phorbol 12,13-dibutyrate (PdBu). Depolarization of the artery with KPSS results in elevations of both tension and [Ca2+];. In contrast, activation of PKC via exposure of the artery to PdBu results in a large sustained contraction that is not associated with any rise in [Ca2+]|.

The lack of specificity of conventional pharmacological agents, not only for PKC vs other intracellular kinases, but also among the various isoforms ofPKC, makes interpretation of the effects of these compounds problematical. While inhibitors of PKC decrease HPV in a variety of preparations including isolated rat arteries (16) and rat (32), rabbit (57) and dog (2) isolated perfused lungs, the inhibitors used in these studies are not selective for PKC. For example, the inhibitors H-7 and staurosporine are compounds that inhibit virtually every kinase they have been tested against (30), and bisindolylmaleimide I and Go-6976 inhibit at least 7-10 intracellular kinases in addition to PKC (3). The extrapolation of the effects of these agents to a role for PKC activation during HPV may not, therefore, be justified. However, it is noteworthy that Barman (2) found that the inhibitor calphostin C, which appears to be less promiscuous in its non-PKC related actions, reduced HPV in isolated perfused dog lungs.

The lack of specificity of PKC antagonists has therefore made definitive studies of the possible role of PKC in HPV virtually impossible. However, the recent development of highly specific, cell-permeable peptide inhibitors of classical, novel and atypical PKC isoforms, namely myristoylated analogs ofthe respective pseudosubstrates for each of these PKC groups, offers promise that the role of PKC isoforms in the generation of HPV may yet be defined. Moreover, the availability ofPKC isoform knockout mice will undoubtedly help in this regard. In a recent study, PKCe knockout mice had a blunted HPV response, whereas other vasoconstrictor responses were unaltered (24). Littler and co-workers reported that the PKCe knockout mice had an increased lung and pulmonary artery expression of the 02-sensitive potassium channel Kv3.1, and they attributed the blunted HPV in isolated perfused lungs to this up-regulation. It is therefore unclear from this study whether an activation of PKCe is directly involved in HPV, or whether the deletion of this isoform leads to blunted HPV because of a functional antagonism associated with the upregulation of potassium channel expression. Nonetheless, the fact that HPV was preferentially blunted by deletion of PKCe is an exciting finding, and the application of such techniques to this field will undoubtedly prove enlightening.

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