Nitric Oxide-dependent Parasympathetic Signaling Is Due to Activation of Constitutive Endothelial (Type III) Nitric Oxide Synthase in Cardiac Myocytes (*)
- Jean-Luc Balligand(1)(§),
- Lester Kobzik(2),
- Xinqiang Han(1),
- David M. Kaye(1),
- Laurent Belhassen(1),
- Donald S. O'Hara(1),
- Ralph A. Kelly(1),
- Thomas W. Smith(1) and
- Thomas Michel(1)
- From the (1) Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and the
- (2)Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, and Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115
- § To whom correspondence should be addressed: Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-6732; Fax: 617-732-5132.
Abstract
Nitric oxide synthase (NOS) isoforms are discovered in an increasing variety of cell types with different roles in signaling. The inducible NOS (i.e. iNOS or NOS II) is expressed in cardiac myocytes in response to specific cytokines. Independent of iNOS induction, however, receptor-dependent signaling is modulated by a constitutive nitric oxide (NO) synthase isoform in these cells (Balligand, J. L., Kelly, R. A., Marsden, P. A., Smith, T. W., and Michel, T.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 347-351). We now show that cardiac myocytes constitutively express the endothelial isoform of NO synthase (ecNOS or NOS III). Transcripts for NOS III were detected by Northern blot in myocyte extracts using as a probe a polymerase chain reaction-generated cDNA amplified with isoform and species-specific primers. In subcellular fractionation experiments, a calcium-sensitive NO synthase activity was present primarily in the particulate fraction, coinciding with the distribution of NOS III analyzed by protein immunoblotting. The localization of NOS III within cardiac myocytes was further demonstrated by immunohistochemistry. The functional role of NOS III was explored by analyzing the effects of NOS inhibitors on single myocyte L-type calcium current and contractility. Inhibition of NOS blocked the attenuation by carbamylcholine of the increases in both parameters induced by β-adrenergic stimulation. We conclude that NO-dependent parasympathetic signaling is mediated by NOS III in cardiac myocytes.
Footnotes
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↵* This work was supported by National Institutes of Health Grants HL 36141 (to T. W. S.) and HL 46457 (to T. M.) and an Established Investigator Award (to T.M.) from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- NO
-
nitric oxide
- NOS
-
nitric oxide synthase
- bp
-
base pair(s)
- ecNOS
-
endothelial constitutive NO synthase
- ncNOS
-
neuronal constitutive NO synthase
- iNOS
-
inducible NO synthase
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- PCR
-
polymerase chain reaction
- CCh
-
carbamylcholine
- ISO
-
isoproterenol.
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↵2Similar immunohistochemical detection of NOS III in cardiac myocytes was observed in sections of human heart ventricle (L. Kobzik and J.-L. Balligand, unpublished observations).
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↵3This is in contrast to cardiac microvascular endothelial cells maintained in serum-containing culture medium for 2 weeks, in which no detectable constitutive NOS activity can be measured (30).
