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ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery

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Abstract

Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 +/− and Rock2 +/− mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used.

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Abbreviations

ACh:

Acetylcholine

eNOS:

Endothelial nitric oxide synthase

ET-1:

Endothelin-1

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

MLC:

Myosin light chain

MLCK:

MLC kinase

MLCP:

MLC phosphatase

MYPT1:

Myosin phosphatase targeting subunit 1

PDBu:

Phorbol 12,13-dibutyrate

PE:

Phenylephrine

ROCK:

Rho-associated kinase (Rho-kinase)

SNP:

Sodium nitroprusside

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Acknowledgments

We thank Drs. James Sherley and Albert Wang of the BBRI for their comments on the manuscript. This work was supported by National Institute of Health grant R01 HL070881 to TK and HL052233, HL080187, and DK085006 to JKL.

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Authors and Affiliations

  1. Boston Biomedical Research Institute, 64 Grove St, Watertown, MA, 02472, USA

    Yang Hoon Huh & Toshio Kitazawa

  2. Brigham & Women’s Hospital, 65 Landsdowne Street, Cambridge, MA, 02139, USA

    Qian Zhou & James K. Liao

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  1. Yang Hoon Huh
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  2. Qian Zhou
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  3. James K. Liao
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  4. Toshio Kitazawa
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Correspondence to Toshio Kitazawa.

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Huh, Y.H., Zhou, Q., Liao, J.K. et al. ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery. J Muscle Res Cell Motil 32, 65–76 (2011). https://doi.org/10.1007/s10974-011-9252-y

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  • DOI: https://doi.org/10.1007/s10974-011-9252-y

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