Protein Kinase C Downregulation Enhanced Extracellular Ca2+-Induced Relaxation of Isolated Mesenteric Arteries from Aged Dahl Salt-Sensitive Rats

Abstract

The Ca²⁺-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca²⁺ _e) concentration ([Ca²⁺]_e) and transduces the signal into modulation of various signaling pathways. Ca²⁺-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca²⁺ _e in wild-type (SS) and CaSR mutant (SS-Casr^em1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca²⁺-sensitive Gӧ 6976 resulted in a >80% increase in relaxation. Significant differences in EC₅₀ values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca²⁺ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.

Fig. 1.

Average ages and body weights of animals. (A) Average ages of SS and SS-Casr^em1Mcwi rats. (B) Average body weights of SS and SS-Casr^em1Mcwi rats. Data are means (±S.E.M.) of 10 SS and 12 SS-Casr^emiMcwi animals.

Fig. 2.

Analysis of expression of CaSR and PKCα in control and PKCα siRNA-treated mesenteric arteries from SS and SS-Casr^em1Mcwi rats. Animals were maintained on 0.1% NaCl diet for 8 months and protein extracts from control siRNA-treated mesenteric arteries were analyzed with monoclonal antibodies. CaSR, PKCα and GAPDH bands were analyzed from the separated proteins in the same samples. After separation, proteins were transferred onto membranes and cut into three strips for blotting with monoclonal antibodies to the CaSR (150 kDa), PKCα (80 kDa) and GPADH (37 kDa). (A) (i) Representative CaSR Western blot, and (ii) bar chart showing densitometric analysis of protein bands from four different Western blot experiments as in (i). Values plotted are means (±S.E.M.) of four experiments. (B) (i) Representative PKCα Western blot, and (ii) bar charts showing densitometric analysis of protein bands from four different Western blot experiments as in (i). Values plotted are means (±S.E.M.) Protein bands were normalized to GAPDH as the loading standard. (*P < 0.05 vs. control; ANOVA followed by multiple comparisons using the Holm-Sidak method).

Fig. 3.

CaSR and PKCα expression in mesenteric arteries from old (≈ 8 months) and young (≤3 months) SS and SS-Casr^em1Mcwi rats. Protein extracts from mesenteric arteries were analyzed with monoclonal antibodies to compare expression levels in young and old rats. CaSR, PKCα, and GAPDH bands were analyzed from the separated proteins in the same samples. After separation, proteins were transferred onto membranes and the latter cut into three strips for blotting with monoclonal antibodies to the CaSR (150 kDa), PKCα (80 kDa), and GPADH (37 kDa). (A) (i) Representative CaSR Western blot; (ii) bar charts showing densitometric analysis of protein bands from four separate experiments as in (i). Values plotted are means (±S.E.M.); (B) (i) Western blot of PKCα; (ii) bar charts showing densitometric analysis of protein bands from four separate experiments as in (i). Values plotted are means (±S.E.M.). Protein bands were normalized to GAPDH as the loading standard. (*P < 0.05 vs. control; ANOVA followed by multiple comparisons using the Holm-Sidak method).

Fig. 4.

Effects of CaSR and PKCα downregulation on normalized and phenylephrine tensions in mounted mesenteric arteries from SS and SS-Casr^em1Mcwi rats. Segments (2-mm) of isolated mesenteric arteries were mounted in a wire myograph chamber in PSS medium (containing 1 mM Ca²⁺ and 100 μM ascorbic acid), equilibrated at 37°C, and gassed with a mixture of 95% air and 5% CO₂. Vessel segments were normalized by step-wise increases in passive force until a resting tension (RT) was achieved. Tensions resulting from the addition of PE were also determined. (A) Normalized resting tensions from SS and SS-Casr^em1Mcwi rats. (B) PE tensions (active force from PE addition minus resting tensions) from SS and SS-Casr^em1Mcwi rats). Statistical analysis of the data showed no significant differences.

Fig. 5.

Effect of downregulation of CaSR and PKCα, and pharmacological inhibition of PKC on Ca²⁺-induced relaxation of PE-contracted mesenteric arteries. Force tracings showing the effect of cumulative additions of Ca²⁺_e to PE-contracted mesenteric arteries, from SS and SS-Casr^em1Mcwi rats, in the absence or presence of 5 μM Gö 6976 on relaxation. (A) SS control; (B) SS + PKCα siRNA; (C) SS-Casr^em1Mcwi control; (D) SS-Casr^em1Mcwi + PKCα siRNA. PKCα siRNA downregulation and PKC inhibition by Gö 6976 resulted in increased relaxation responses to cumulative additions of Ca²⁺_e.

Fig. 6.

Effects of downregulation of CaSR and PKCα and inhibition of PKC on [Ca²⁺]_e-response curves derived from force tracings in Fig. 5. [Ca²⁺]_e-response curves were generated from tension data obtained in arteries from (A) SS and (B) SS-Casr^em1Mcwi rats. (C) Bar charts showing EC₅₀ values determined from response data fitted to sigmoid curves *Significantly different from treated groups; **Significantly different from PKCα siRNA-treated and Go 6976-treated groups (P < 0.05; ANOVA followed by multiple comparisons using the Holm-Sidak method).

Fig. 7.

Proposed mechanism of CaSR-mediated signaling and mesenteric artery relaxation. Mechanism of CaSR-mediated vasodilation and its regulation by PKC. This signaling pathway uses published data from our laboratory (Awumey et al., 2008) and the present studies that focused on PKC and Ca²⁺_i. Cytochrome P450 metabolites EET and GEET play a role in CaSR-mediated mesenteric artery relaxation.