Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C81
Protein kinase C delta isoform shRNA plasmid construct restores vascular disorders in spontaneously hypertensive rats
T. Novokhatska1, O. Boldyrev2, S. Tishkin1, V. Dosenko2, A. Soloviev1
1. Experimental therapy, Institute of pharmacology and toxicology of AMS, Kiev, Ukraine. 2. Bogomoletz Institute of Physiology of NAS, Kiev, Ukraine.
It is known that protein kinase C (PKC) family involved in arterial hypertension development due to its overexpression and related decrease in BKCa channels activity. The vascular smooth muscle force development is clously coupled to membrane potential and changes in conductivity for K+ ions carried in rat thoracic aorta mainly through BKCa channels which, in turn, appear to be PKC dependent. The goal of this study was to clarify δPKC role in hypertension development in SHRs and to make an attempt to reduce vascular abnormalities related to ionic channelopathy and vascular hypercontactivity in SHRs using siRNA and plasmid-based siRNA. Experimental design of the study comprised RT-PCR, patch-clamp technique and systolic blood pressure measurement in non-anesthetized rats using calf tail Sphyngomanometer S-2 (Hugo Sachs Elektronik, Germany). Vector system pSilencer-siδPKC was constructed and used for suppressing δPKC expression in a target-specific manner. siRNA and pSilencer-siδPKC were injected intravenously via the tail vein in conscious SHRs. The rats were then killed on the 7th day by cervical dislocation following ketamine (45mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia. It was found that protein kinase C delta isoform gene silencing using both siRNA and pSilencer-siδPKC in SHRs led to significant decrease in arterial blood pressure by 16 +/- 5 mmHg and 17 +/- 4 mmHg, respectively, on the first intravenous post-injection day and persisted for 7 days after. To investigate the effect of post-transcriptional gene silencing on BKCa, the patch-clamp technique was used. It was shown that outward ionic currents was 52 ± 5 pA/pF in healthy rats, and 25 ± 2 pA/pF in SHRs. In SHRs treated with δ-PKC siRNA BKCa current density was 35 ± 3 pA/pF, and in SHRs treated with pSilencer-siδPKC it was 36 ± 3 pA/pF ( P<0.05, n=24). In conclusion, the silencing of δPKC gene expression using both siRNA and plasmid-based siRNA led to an increment in BKCa channels activity and arterial blood pressure normalization.
Where applicable, experiments conform with Society ethical requirements