Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

Giovanni Anfossi, Paola Massucco, Luigi Mattiello, Alessandra Balbo, Isabella Russo, Gabriella Doronzo, Luigi Rolle, Dario Ghigo, Dario Fontana, Amalia Bosia, Mariella Trovati

Research output: Contribution to journalArticle

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Abstract

Aims: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3′,5′-cyclic monophosphate (cGMP) and adenosine 3′,5′-cyclic monophosphate (cAMP). Methods: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluoresence, cNOS activity as L-[3H]-citrulline production from L-[3H]-arginine and cyclic nucleotides by radioimmunoassay. Results: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 μmol/l calcium ionophore ionomycin (from 0.1094±0.0229 to 0.2685±0.0560 pmol/min per mg cell protein, P = 0.011) and to 2 nmol/l insulin (from 0.1214±0.0149 to 0.2045±0.0290 pmol/min per mg cell protein, P = 0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71±0.10 to 6.80±0.40 pmol/106 cells at 30 min, P = 0.0001; cAMP from 1.26±0.06 to 3.02±0.30 pmol/106 cells at 60 min, P = 0.0001). NOS inhibitor NG-monomethyl-L-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. Conclusion: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.

Original languageEnglish
Pages (from-to)689-700
Number of pages12
JournalEuropean Journal of Endocrinology
Volume147
Issue number5
Publication statusPublished - Nov 2002

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Cyclic Nucleotides
Nitric Oxide Synthase
Smooth Muscle Myocytes
Nitric Oxide
Insulin
Phosphatidylinositol 3-Kinase
Adenosine
Proteins
Iloprost
omega-N-Methylarginine
Citrulline
Messenger RNA
Ionomycin
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Nitric Oxide Donors
Calcium Ionophores
Guanosine
Guanylate Cyclase
Phosphoric Diester Hydrolases
Colforsin

ASJC Scopus subject areas

  • Endocrinology

Cite this

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase. / Anfossi, Giovanni; Massucco, Paola; Mattiello, Luigi; Balbo, Alessandra; Russo, Isabella; Doronzo, Gabriella; Rolle, Luigi; Ghigo, Dario; Fontana, Dario; Bosia, Amalia; Trovati, Mariella.

In: European Journal of Endocrinology, Vol. 147, No. 5, 11.2002, p. 689-700.

Research output: Contribution to journalArticle

Anfossi, G, Massucco, P, Mattiello, L, Balbo, A, Russo, I, Doronzo, G, Rolle, L, Ghigo, D, Fontana, D, Bosia, A & Trovati, M 2002, 'Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase', European Journal of Endocrinology, vol. 147, no. 5, pp. 689-700.
Anfossi, Giovanni ; Massucco, Paola ; Mattiello, Luigi ; Balbo, Alessandra ; Russo, Isabella ; Doronzo, Gabriella ; Rolle, Luigi ; Ghigo, Dario ; Fontana, Dario ; Bosia, Amalia ; Trovati, Mariella. / Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase. In: European Journal of Endocrinology. 2002 ; Vol. 147, No. 5. pp. 689-700.
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abstract = "Aims: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3′,5′-cyclic monophosphate (cGMP) and adenosine 3′,5′-cyclic monophosphate (cAMP). Methods: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluoresence, cNOS activity as L-[3H]-citrulline production from L-[3H]-arginine and cyclic nucleotides by radioimmunoassay. Results: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 μmol/l calcium ionophore ionomycin (from 0.1094±0.0229 to 0.2685±0.0560 pmol/min per mg cell protein, P = 0.011) and to 2 nmol/l insulin (from 0.1214±0.0149 to 0.2045±0.0290 pmol/min per mg cell protein, P = 0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71±0.10 to 6.80±0.40 pmol/106 cells at 30 min, P = 0.0001; cAMP from 1.26±0.06 to 3.02±0.30 pmol/106 cells at 60 min, P = 0.0001). NOS inhibitor NG-monomethyl-L-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. Conclusion: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.",
author = "Giovanni Anfossi and Paola Massucco and Luigi Mattiello and Alessandra Balbo and Isabella Russo and Gabriella Doronzo and Luigi Rolle and Dario Ghigo and Dario Fontana and Amalia Bosia and Mariella Trovati",
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T1 - Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

AU - Anfossi, Giovanni

AU - Massucco, Paola

AU - Mattiello, Luigi

AU - Balbo, Alessandra

AU - Russo, Isabella

AU - Doronzo, Gabriella

AU - Rolle, Luigi

AU - Ghigo, Dario

AU - Fontana, Dario

AU - Bosia, Amalia

AU - Trovati, Mariella

PY - 2002/11

Y1 - 2002/11

N2 - Aims: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3′,5′-cyclic monophosphate (cGMP) and adenosine 3′,5′-cyclic monophosphate (cAMP). Methods: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluoresence, cNOS activity as L-[3H]-citrulline production from L-[3H]-arginine and cyclic nucleotides by radioimmunoassay. Results: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 μmol/l calcium ionophore ionomycin (from 0.1094±0.0229 to 0.2685±0.0560 pmol/min per mg cell protein, P = 0.011) and to 2 nmol/l insulin (from 0.1214±0.0149 to 0.2045±0.0290 pmol/min per mg cell protein, P = 0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71±0.10 to 6.80±0.40 pmol/106 cells at 30 min, P = 0.0001; cAMP from 1.26±0.06 to 3.02±0.30 pmol/106 cells at 60 min, P = 0.0001). NOS inhibitor NG-monomethyl-L-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. Conclusion: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.

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