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The protein kinase C (PKC) familiy was first described in 1977 by Nishizuka and colleagues, the pioneers in PKC research, and more than 30 years ago its role in human diseases was recognized. From that time onward, researchers worldwide endeavored to unravel regulatory functions of PKC isozymes in numerous major disorders. PKC-mediated phosphorylation of serine/threonine residues controls the activation of multiple downstream proteins. This central role in signal transduction as well as ubiquitous expression of most of the 11 PKC members makes it challenging to define the complexity of isozyme specific PKC functions. Dependent on their structure and consequent way of activation, the PKC family is classified into conventional (PKCa, PKCßI, PKCßII, PKC?), novel (PKCd, PKCe, PKC?, PKC?, PKCµ) and atypical (PKC?, PKC?/PKC?) PKC isozymes. The conventional PKCa is the most studied and best characterized isozyme, especially due to its crucial role in cancerogenesis. PKCa-evoked cellular proliferation, differentiation and apoptosis have been also proven and demonstrated in the pathogenesis of cardiovascular disorders. However, the exact role of PKCa in various pathologic conditions is still incompletely understood.
The human lung disease pulmonary arterial hypertension (PAH) is characterized by progressive changes in the morphology and function of pulmonary arteries. Several PKC isozymes including PKCa are known to modulate vascular smooth muscle function. Hence, the aim of the present work was to investigate PKC isozyme specific properties in the pulmonary vasculature following stimulation with vasoactive mediators that are important in the pathogenesis of PAH. For this purpose, effects of acute hypoxia, endothelin-1 (ET-1), serotonin and the thromboxane A2 (TXA2) analog U46619 were studies in isolated perfused and ventilated mouse lungs of PKCa deficient (PKCa-/-) and corresponding wildtype (Dempsey et al.) mice. Broad spectrum PKC inhibition in WT mice was achieved with the non-selective PKC inhibitor bisindolymaleimide I (BIM), while usage of Gö6976 evoked selective PKC inhibition of conventional PKC isozymes PKCa and PKCß.
In this study, acute hypoxic pulmonary vasoconstriction (HPV) was unaffected by loss of PKCa, whereas PKC inhibition with BIM significantly reduced HPV in WT mice. ET-1-induced vasopressor response, an important feature of PAH, was markedly attenuated by PKCa deficiency, non-selective and selective PKC inhibition suggesting a crucial role of PKCa in this scenario. In contrast, serotonin-evoked vasoconstriction was not affected by the absence of PKCa or by PKC inhibition with BIM. Notably, loss of PKCa caused pulmonary vascular hyperresponsiveness to the TXA2 analog U46619. mRNA expression analysis revealed increased thromboxane A2 receptor levels in microdissected intrapulmonary arteries from naïve PKCa-/- mice. Besides, mRNA expression of atypical PKC? was upregulated in intrapulmonary arteries from PKCa-/- mice. However, since PKC? inhibition with sodium aurothiomalate hydrate (ATM) had no effect on TXA2-induced vasopressor response, the observed hyperresponsivness to TXA2 analog U46619 might not involve PKC? but certainly TP receptor upregulation.
Due to the random observation of esophageal dilation in PKCa-/- mice, further analyses were performed in 9-10-week-old and 14-month-old PKCa-/- and WT mice. Megaesophagus is a characteristic of human achalasia. The hallmark of achalasia is impaired smooth muscle cell relaxation of the lower esophageal sphincter (LES) due to neuronal degeneration of the esophageal myenteric plexus. Here, juvenile PKCa-/- mice showed a 60% prevalence of megaesophagus. With a prevalence of 10%, megaesophagi were present in aged PKCa-/- but also in WT mice independent of age. Histopathological investigations revealed an increased portion of the distal esophagus lined by smooth muscle cells in mice displaying meagaesophagus. However, achalasia-like inflammation, fibrosis or neuronal degeneration of the LES was not present. It was therefore hypothesized that loss of PKCa leads to delayed esophageal maturation due to failed replacement of embryonic smooth muscle cells by striated muscle cells. Since PKC isozyme expression profiles in LES of PKCa-/- mice showed decreased expression levels of four out of seven PKC isozymes, changes in the LES basal tone might have evoked smooth muscle cell hyperplasia at the distal esophagus leading to megaesophagus formation. Anyhow, PKCa deficiency appears to be associated with functional-muscular rather than neuronal achalasia-like pathological conditions.
In conclusion, the results gained from this work substantially increase the understanding of PKC isozyme selective properties in the pulmonary vasculature and provide new data on PKCa involvement in smooth muscle cell function of lung and esophagus. Future work should verify the functional role of PKCa in PAH pathogenesis by PAH modeling and LES function by manometric studies.