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.
OBJECTIVE
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 cGKIalpha-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 micromol/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 cGKIalpha-EGFP, the [Ca2+]i dynamic of SHR-transfected cells regained sensitivity to the nitric oxide/cyclic GMP pathway.


CONCLUSIONS
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.
Di Cesare Mannelli
Lorenzo
L
Department of Pharmacology, Viale Pieraccini, 6-University of Florence, Florence, Italy.
Nistri
Silvia
S
Mazzetti
Luca
L
Bani
Daniele
D
Feil
Robert
R
Failli
Paola
P
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D001483:Base Sequence; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006973:Hypertension / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D011994:Recombinant Proteins / Q000235:genetics; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D014162:Transfection
D017931:DNA Primers; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D003995:Dibutyryl Cyclic GMP; D026423:S-Nitroso-N-Acetylpenicillamine; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases