Bani, Daniele

Chiappini, L.

Failli, Paola

Mazzetti, L.

Nistri, Silvia

English

Florence Research

311Ann. N.Y. Acad. Sci. 1041: 311–313 (2005). © 2005 New York Academy of Sciences.doi: 10.1196/annals.1282.048Effects of Relaxin on Vascular Smooth Muscle and Endothelial Cells in Normotensive and Hypertensive RatsPAOLA FAILLI,a SILVIA NISTRI,b LUCA MAZZETTI,a LAURA CHIAPPINI,b AND DANIELE BANIbaDepartment of Preclinical and Clinical Pharmacology and bDepartment of Anatomy, Histology & Forensic Medicine, University of Florence, Florence, ItalyABSTRACT: We tested the effects of relaxin on [Ca2+]i response to angiotensinII in smooth muscle (vSMC) and endothelial cells isolated from hypertensive(SHR) and normotensive (WKY) rats. Relaxin markedly reduced the [Ca2+]iresponse of vSMCs from WKY, but not from SHR rats. Western blots showedthat cGMP-dependent protein kinase G was reduced in vSMCs from SHR ascompared with WKY rats. Relaxin also blunted the [Ca2+]i response in endo-thelial cells from WKY, but not from SHR rats. However, in endothelial cellsfrom SHR and WKY rats, protein kinase G was nearly unexpressed, thusaccounting for an alternative pathway of the intracellular response to nitricoxide and relaxin. Hence, vSMCs and endothelial cells in SHR rats show adeficiency response to nitric oxide that may render them insensitive to relaxin.KEYWORDS: endothelial cells; smooth muscle cells; SHR; calcium; nitric oxide;protein kinase GINTRODUCTIONRelaxin has striking vasodilatory properties on reproductive and nonreproductiveorgans.1,2 When given systemically to spontaneously hypertensive rats (SHR strain),relaxin reduces systemic blood pressure and blunts the vascular response to vasocon-strictors.3,4 These properties of relaxin likely rely on multiple mechanisms. In vitrostudies have shown that relaxin increases the expression of inducible nitric oxidesynthase (NOS II) in rat endothelial cells (ECs) and vascular smooth muscle cells(vSMCs). By this pathway, relaxin reduced vasoconstrictor-induced increases inintracellular calcium concentration [Ca2+]i and promoted cell relaxation.5,6This in vitro study was designed to compare the effects of relaxin (porcine) on the[Ca2+]i response to angiotensin II (AT II) by vSMCs and ECs isolated from sponta-neously hypertensive (SHR) rats or normotensive Wistar-Kyoto (WKY) rats.Address for correspondence: Prof. Daniele Bani, Department of Anatomy, Histology andForensic Medicine, Viale G. Pieraccini 6, I-50139, Florence, Italy. Voice: (+39) 0554271390;fax: (+39) 0554271385.daniele.bani@unifi.it312 ANNALS NEW YORK ACADEMY OF SCIENCESMATERIAL AND METHODSWe used eight SHR and nine WKY rats 12–14 wk old. Endothelial cells were iso-lated from rat hearts by enzymatic digestion and selected from other cardiac celltypes by centrifugation and adhesion to plastic surfaces. Aortic vSMCs were isolatedby elastase-collagenase digestion of endothelium-free thoracic aortas and usedbetween the second and fourth culture passage. Cells plated on glass coverslips wereloaded with fura-2 AM and used for intracellular calcium [Ca2+]i image analysisassay in control conditions or after a 24-h pretreatment with porcine relaxin (10−8M). Angiotensin II (AT II, 10−6 M) was used as the vasoactive stimulus. Untreatedand relaxin-treated cell cultures seeded in 25 cm2 culture flasks were also used forwestern blot analysis of protein kinase G expression.Vascular Smooth Muscle CellsResting [Ca2+]i  and AT II-induced [Ca2+]i peak and decay times were similar inSHR and WKY rats. In vSMCs from WKY rats, relaxin (10 nM) decreased the decaytime and peak values of [Ca2+]i transiently. This effect was prevented when the cellswere pretreated with the NOS inhibitor L-NAME (10 mM), suggesting the involve-ment of endogenous nitric oxide generation. Conversely, vSMCs from SHR ratswere insensitive to relaxin as well as to L-NAME. By western blotting, vSMCs fromWKY rats fairly expressed protein kinase G, whereas those from SHR rats onlyshowed very weak protein kinase G expression.Endothelial CellsResting [Ca2+]i was higher in SHR than in WKY rats (144 ± 7.4 vs 109 ± 4.0 nM;P <0.01). The AT II-induced increase in [Ca2+]i was characterized by a fast rise fol-lowed by a slower decrease in both WKY and SHR rats. [Ca2+]i peak and decaytimes (time needed to return to basal values) were similar in SHR and WKY rats.In ECs from WKY rats, relaxin (10 nM) significantly decreased the decay timeand peak values of [Ca2+]i transiently. This effect was prevented when the cells werepretreated with L-NAME (10 mM), suggesting involvement of endogenous nitric ox-ide generation. Conversely, ECs from SHR rats were insensitive to relaxin as well asto L-NAME. By western blotting, ECs from either WKY or SHR rats did not expressdetectable protein kinase G.RESULTS AND CONCLUSIONSThis study shows that ECs and vSMCs from SHR rats have a deficiency responseto nitric oxide that renders them insensitive to the relaxant action of relaxin. Thisappears to depend on a defect in the expression of cGMP-dependent protein kinaseG by vSMCs, the cells chiefly involved in the regulation of vascular tone, whichleads to a breakdown of the nitric oxide-cGMP signaling pathway. These findingssupport the pivotal role of nitric oxide-cGMP signaling in the vasorelaxant effects ofrelaxin. Conceivably, the reduction of arterial blood pressure in SHR rats givenrelaxin systemically reported in previous studies may rely on alternative, nitricoxide-independent mechanisms.313FAILLI et al.: RELAXIN AND VASCULAR CELLS IN WKY AND SHR RATSREFERENCES1. BANI, D. 1997. Relaxin: a pleiotropic hormone. Gen. Pharmacol. 28: 13–22.2. SHERWOOD, O.D. 2004. Relaxin's physiological roles and other diverse actions. Endocr.Rev. 25: 205–234.3. ST. LOUIS, J. & G. MASSICOTTE. 1985. Chronic decrease of blood pressure by ratrelaxin in spontaneously hypertensive rats. Life Sci. 37: 1351–1357.4. MASSICOTTE, G., A. PARENT & J. ST. LOUIS. 1989. Blunted responses to vasoconstric-tors in mesenteric vasculature but not in portal vein of spontaneously hypertensiverats treated with relaxin. Proc. Soc. Exp. Biol. Med. 190: 254–259.5. FAILLI, P., S. NISTRI, S. QUATTRONE, et al. 2001. Relaxin up-regulates inducible nitricoxide synthase expression and nitric oxide generation in rat coronary endothelialcells. FASEB J. 16: 252–254.6. BANI, D., P. FAILLI, M.G. BELLO, et al. 1998. Relaxin activates the L-arginine-nitricoxide pathway in vascular smooth muscle cells in culture. Hypertension 31: 1240–1247.

EFFECTS OF RELAXIN ON VASCULAR SMOOTH MUSCLE AND ENDOTHELIAL CELLS IN NORMOTENSIVE AND HYPERTENSIVE RATS

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EFFECTS OF RELAXIN ON VASCULAR SMOOTH MUSCLE AND ENDOTHELIAL CELLS IN NORMOTENSIVE AND HYPERTENSIVE RATS

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