Altered nitric oxide calcium responsiveness of aortic smooth muscle cells in spontaneously hypertensive rats depends on low expression of cyclic guanosine monophosphate-dependent protein kinase type I

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Abstract

Objectives

The nitric oxide/cyclic guanosine monophosphate (GMP)/cyclic GMP-dependent protein kinase type I (cGKI) pathway has been extensively investigated in the spontaneously hypertensive rat (SHR) as a possible pathogenetic factor. Therefore, we investigated the role of nitric oxide/cGKI on intracellular calcium dynamics ([Ca2+]i) of aortic smooth muscle cells isolated from control normotensive Wistar Kyoto rats (WKY) and SHR.

Methods

Rat aortic smooth muscle cells (RASMCs) were obtained from 12 to 16-week-old WKY and SHR. [Ca2+]i dynamics were monitored by imaging analysis of fura-2-loaded RASMCs. cGKI mRNA and cGKI protein expression were evaluated by reverse transcription-PCR and western blot. Plasmids codifying for enhanced green fluorescent protein (EGFP) or cGKIα–EGFP were transfected on SHR RASMCs.

Results

Angiotensin II similarly increased [Ca2+]i in WKY and SHR RASMCs. In WKY RASMCs, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1–100 μmol/l) reduced the decay time of angiotensin II-induced [Ca2+]i transient. On the contrary, in SHR cells, SNAP was ineffective. Dibutyryl cyclic GMP (1–100 nmol/l), a membrane-permeable cyclic GMP analogue, behaved similarly to SNAP. In naive SHR RASMCs, cGKI mRNA and cGKI protein were low or absent. After transfection of a plasmid encoding for cGKIα–EGFP, the [Ca2+]i dynamic of SHR-transfected cells regained sensitivity to the nitric oxide/cyclic GMP pathway.

Conclusion

The low expression of cGKI determines the lack of nitric oxide/cyclic GMP-dependent regulation on [Ca2+]i transient in SHR RASMCs. This alteration may contribute to the development of hypertension and explain suboptimal responses to nitroglycerin and other nitric oxide-releasing molecules in patients.

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