Involvement of protein kinase C-CPI-17 in androgen modulation of angiotensin II-renal vasoconstriction

Abstract

Aims: Previous studies suggested that androgens augmented renal vascular responses to angiotensin II (Ang II). The protein kinase C (PKC)-CPI-17 pathway is involved in vascular constriction. We tested the hypothesis that this pathway may contribute to androgenic amplification of Ang II-renal vasoconstriction in the New Zealand genetically hypertensive (NZGH) rat.

Methods and results: NZGH underwent sham operation, castration, or castration with testosterone replacement at 5 weeks of age. When the rats were 16-17 weeks of age, mean arterial pressure (MAP) and renal vascular resistance (RVR) responses to intravenous Ang II infusion (20, 40, and 80 ng/kg/min) were recorded before and after treatment with a PKC inhibitor, chelerythrine. mRNA expression of PKC isoforms and CPI-17 protein expression were analysed in renal cortex. MAP and RVR responses to Ang II were enhanced in androgen-replete NZGH. The Ang II-induced increase in RVR was significantly lower in castrated NZGH (ranged from 100 +/- 8% to 161 +/- 9% of baseline) than in sham-operated NZGH (ranged between 123 +/- 3% and 237 +/- 19% of baseline). Testosterone treatment restored RVR responses to Ang II in castrated rats. Chelerythrine treatment markedly reduced the MAP and RVR responses to Ang II in each group and attenuated the differential MAP and RVR responses to Ang II amongst the three groups. PKCdelta and PKCepsilon mRNA levels were significantly reduced by castration and increased by testosterone treatment. In contrast, no significant differences in protein expression were detected for these PKC isoforms. Castration decreased while testosterone treatment increased CPI-17 and phospho-CPI-17 expression.

Conclusion: Collectively, these results suggest that androgens modulate renal vascular responses to Ang II in part via an effect on the PKC-CPI-17 signalling pathway.

Figure 1

Mean arterial blood pressure (MAP) responses to Ang II (20, 40, and 80 ng/kg/min) in anaesthetized sham-operated (Sham n = 5), castrated (Cas n = 5), and castrated + testosterone replacement (Cas + T n = 5) NZGH at 16–17 weeks of age. (A) MAP responses in the absence of chelerythrine. (B) MAP responses in the presence of chelerythrine. Values are mean ± SEM. *P < 0.05 and **P < 0.01 Sham vs. Cas. ^#P < 0.05 and ^##P < 0.01 Cas + T vs. Cas.

Figure 2

Renal vascular resistance (RVR) responses to Ang II (20, 40, and 80 ng/kg/min) in anaesthetized sham-operated (Sham n = 5), castrated (Cas n = 5), and castrated + testosterone replacement (Cas + T n = 5) NZGH at 16–17 weeks of age. (A) RVR responses in the absence of chelerythrine. (B) RVR responses in the presence of chelerythrine. Values are mean ± SEM. *P < 0.05 and **P < 0.01 Sham vs. Cas. ^#P < 0.05 and ^##P < 0.01 Cas + T vs. Cas.

Figure 3

Normalized relative mRNA expression for the PKC isoforms (n = 8/group). Upper panel: PKCα; middle panel: PKCδ; lower panel: PKCε. Kidney tissue samples were obtained from male NZGH at 16–17 weeks of age. Sham, sham-operated; Cas, castrated; Cas + T, castrated + testosterone replacement. Values are means ± SEM. *P < 0.05 compared with sham.

Figure 4

Protein expression for PKCδ and PKCε in punches of renal cortex obtained from NZGH (n = 8/group) at 16–17 weeks of age. The top panel shows an example of PKCδ bands (∼81 kDa), β-actin bands (∼42 kDa) and quantification of the blots expressed as the ratio of PKCδ band and actin band density. The lower panel shows an example of PKCε bands (∼88 kDa), β-actin bands (∼42 kDa) and quantification of the blots expressed as the ratio of PKCδ band and actin band density. Sham, sham-operated; Cas, castrated; Cas + T, castrated + testosterone replacement. Values are means ± SEM.

Figure 5

CPI-17 and phospho-CPI-17 protein expression as assessed by western blot in the renal cortex of NZGH (n = 8/group) at 16–17 weeks of age. The top part of each panel shows an example of CPI-17 (left panel) and phospho-CPI-17 bands (right panel) (∼17 kDa) and β-actin bands (∼42 kDa). The lower part of each panel shows the quantification of the blots expressed as the phospho-CPI-17/actin ratio. Sham, sham-operated; Cas, castrated; Cas + T, castrated + testosterone replacement. Values are means ± SEM. *P < 0.05 compared with sham.