Abstract
In rat aorta rings (RA) and in gastric circular muscle (CM) and gastric longitudinal muscle (LM) preparations maintained in vitro, inducible nitric oxide synthase (iNOS) induction was monitored functionally (1 mM l-arginine-induced relaxation), biochemically (appearance of iNOS mRNA), and immunohistochemically. Functional iNOS (l-arginine-mediated relaxation) was induced in RA and CM tissues (but NOT in the LM preparation) over 2 to 5 h. iNOS induction was detected by immunocytochemistry in RA smooth muscle elements and in macrophage-like cells in CM. Functional iNOS induction correlated with iNOS mRNA induction. In the RA and CM, functional iNOS induction was blocked by both actinomycin D and cycloheximide; actinomycin D also blocked the appearance of iNOS mRNA in both tissues. In contrast, cycloheximide blocked CM (but not RA) iNOS mRNA induction. In CM tissue, functional iNOS induction was not affected by genistein, tyrphostin 47/AG213, or vanadate. But, in the RA, both genistein and tyrphostin 47/AG213 blocked the appearance of functional iNOS; neither inhibitor prevented the appearance of RA iNOS mRNA. Vanadate, in the RA tissue, blocked both the appearance of iNOS mRNA and the induction of functional iNOS. In RA tissue, but not in the CM, inhibitors of NF-κB activation blocked the appearance of both functional iNOS and iNOS mRNA. We conclude that in different smooth muscle preparations (aorta versus gastric), there can be a differential induction of iNOS mRNA and “functional” iNOS not only in different cellular elements but also in terms of different signaling pathways.
Footnotes
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Send reprint requests to: Dr. Morley D. Hollenberg, Departments of Pharmacology & Therapeutics and Medicine, The University of Calgary, Faculty of Medicine, 3330 Hospital Drive, Calgary, AB Canada T2N 4N1. E-mail: mhollenb{at}acs.ucalgary.ca
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↵1 These studies were supported primarily by a grant from the Alberta Heart and Stroke Foundation (M.D.H.), with ancillary support from the Medical Research Council of Canada (M.D.H. and K.A.S.). X.-L.Z. was supported in part by a William H. Davies Research Scholarship and a Graduate Studentship from the Canadian Hypertension Society in conjunction with Pfizer and the Canadian Medical Research Council.
- Abbreviations:
- ACTD
- actinomycin D
- AG
- aminoguanidine
- AG213
- tyrphostin 47
- CHX
- cycloheximide
- CM
- gastric circular muscle
- GS
- genistein
- I-κB
- NF-κB inhibitory subunit
- iNOS
- inducible nitric oxide synthase (NOS2)
- LM
- gastric longitudinal muscle
- LR
- l-arginine
- PDTC
- pyrrolidinedithiocarbamate
- RA
- rat aorta rings
- RT-PCR
- reverse transcriptase-polymerase chain reaction
- TPCK
- tosyl-phenylalanyl-chloromethyl ketone
- VAN
- vanadate
- LPS
- lipopolysaccharide
- SNP
- sodium nitroprusside
- Received May 28, 1998.
- Accepted November 25, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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