Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes: Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro

Jean Luc Balligand, Dan Ungureanu-Longroisi, William W. Simmons, David Pimental, Tadeusz A. Malinski, Matthias Kapturczak, Ziad Taha, Charles J. Lowenstein, Amy J. Davidoff, Ralph A. Kelly, Thomas W. Smith, Thomas Michel

Research output: Contribution to journalArticle

Abstract

Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocytes, but both interleukin-1β and interferon-γ individually increased iNOS mRNA abundance in primary cultures of ARVM, with maximal expression at 12 h. The half-life of iNOS mRNA in actinomycin C1-treated cells was 4 h. Both dexamethasone and transforming growth factor-β attenuated the induction of iNOS mRNA abundance and enzyme activity by IL-1β and INFγ. Pretreatment with dexamethasone also abolished the induction of iNOS mRNA, but not the increase in GTP cyclohydrolase mRNA in purified cardiac myocytes from lipopolysaccharide-injected rats. In order to further characterize the specific cell type producing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1β and IFNγ in L-arginine-depleted medium. NO release could be detected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjection of D-arginine, and not from ARVM pretreated with L-N-monomethylarginine. Cytokine-pretreated ARVM that had been maintained in L-arginine-depleted medium also exhibited a depressed contractile response to isoproterenol after addition of L-arginine, but not o-arginine. These results indicate that altered contractile function of cardiac myocytes following exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.

Original languageEnglish (US)
Pages (from-to)27580-27588
Number of pages9
JournalJournal of Biological Chemistry
Volume269
Issue number44
StatePublished - Nov 4 1994

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Nitric Oxide Synthase Type II
Cardiac Myocytes
Muscle Cells
Cytokines
Arginine
Rats
Nitric Oxide
Messenger RNA
Interleukin-1
Microsensors
Complementary DNA
Microinjections
Base Pairing
Dexamethasone
Muscle
GTP Cyclohydrolase
In Vitro Techniques
Polymerase chain reaction
RNA-Directed DNA Polymerase
Enzyme activity

ASJC Scopus subject areas

  • Biochemistry

Cite this

Balligand, J. L., Ungureanu-Longroisi, D., Simmons, W. W., Pimental, D., Malinski, T. A., Kapturczak, M., ... Michel, T. (1994). Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes: Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. Journal of Biological Chemistry, 269(44), 27580-27588.

Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes : Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. / Balligand, Jean Luc; Ungureanu-Longroisi, Dan; Simmons, William W.; Pimental, David; Malinski, Tadeusz A.; Kapturczak, Matthias; Taha, Ziad; Lowenstein, Charles J.; Davidoff, Amy J.; Kelly, Ralph A.; Smith, Thomas W.; Michel, Thomas.

In: Journal of Biological Chemistry, Vol. 269, No. 44, 04.11.1994, p. 27580-27588.

Research output: Contribution to journalArticle

Balligand, JL, Ungureanu-Longroisi, D, Simmons, WW, Pimental, D, Malinski, TA, Kapturczak, M, Taha, Z, Lowenstein, CJ, Davidoff, AJ, Kelly, RA, Smith, TW & Michel, T 1994, 'Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes: Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro', Journal of Biological Chemistry, vol. 269, no. 44, pp. 27580-27588.
Balligand, Jean Luc ; Ungureanu-Longroisi, Dan ; Simmons, William W. ; Pimental, David ; Malinski, Tadeusz A. ; Kapturczak, Matthias ; Taha, Ziad ; Lowenstein, Charles J. ; Davidoff, Amy J. ; Kelly, Ralph A. ; Smith, Thomas W. ; Michel, Thomas. / Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes : Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 44. pp. 27580-27588.
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abstract = "Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocytes, but both interleukin-1β and interferon-γ individually increased iNOS mRNA abundance in primary cultures of ARVM, with maximal expression at 12 h. The half-life of iNOS mRNA in actinomycin C1-treated cells was 4 h. Both dexamethasone and transforming growth factor-β attenuated the induction of iNOS mRNA abundance and enzyme activity by IL-1β and INFγ. Pretreatment with dexamethasone also abolished the induction of iNOS mRNA, but not the increase in GTP cyclohydrolase mRNA in purified cardiac myocytes from lipopolysaccharide-injected rats. In order to further characterize the specific cell type producing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1β and IFNγ in L-arginine-depleted medium. NO release could be detected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjection of D-arginine, and not from ARVM pretreated with L-N-monomethylarginine. Cytokine-pretreated ARVM that had been maintained in L-arginine-depleted medium also exhibited a depressed contractile response to isoproterenol after addition of L-arginine, but not o-arginine. These results indicate that altered contractile function of cardiac myocytes following exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.",
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AU - Ungureanu-Longroisi, Dan

AU - Simmons, William W.

AU - Pimental, David

AU - Malinski, Tadeusz A.

AU - Kapturczak, Matthias

AU - Taha, Ziad

AU - Lowenstein, Charles J.

AU - Davidoff, Amy J.

AU - Kelly, Ralph A.

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N2 - Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocytes, but both interleukin-1β and interferon-γ individually increased iNOS mRNA abundance in primary cultures of ARVM, with maximal expression at 12 h. The half-life of iNOS mRNA in actinomycin C1-treated cells was 4 h. Both dexamethasone and transforming growth factor-β attenuated the induction of iNOS mRNA abundance and enzyme activity by IL-1β and INFγ. Pretreatment with dexamethasone also abolished the induction of iNOS mRNA, but not the increase in GTP cyclohydrolase mRNA in purified cardiac myocytes from lipopolysaccharide-injected rats. In order to further characterize the specific cell type producing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1β and IFNγ in L-arginine-depleted medium. NO release could be detected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjection of D-arginine, and not from ARVM pretreated with L-N-monomethylarginine. Cytokine-pretreated ARVM that had been maintained in L-arginine-depleted medium also exhibited a depressed contractile response to isoproterenol after addition of L-arginine, but not o-arginine. These results indicate that altered contractile function of cardiac myocytes following exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.

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