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

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 0 ,5 0 -cyclic monophosphate (cGMP) and adenosine 3 0 ,5 0 -cyclic monophosphate (cAMP). Methods: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as L-[3 H]-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 mmol/l calcium ionophore ionomycin (from 0:1094^0:0229 to 0:26850 :0560 pmol=min per mg cell protein, P ¼ 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 ¼ 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 2:710 :10 to 6:80^0:40 pmol=10 6 cells at 30 min, P ¼ 0:0001; cAMP from 1:26^0:06 to 3:020 :30 pmol=10 6 cells at 60 min, P ¼ 0:0001). NOS inhibitor N G -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.

Nitric Oxide Synthesis Leads to Vascular Endothelial Growth Factor Synthesis via the NO/Cyclic Guanosine 3′,5′-Monophosphate (cGMP) Pathway in Human Corpus Cavernosal Smooth Muscle Cells

“…Our primary cultures of CCSMCs expressed eNOS as well as the original cavernosal smooth muscle tissue. Anfossi et al [9] and Filippi et al [10] reported the expression of eNOS in cultured CCSMCs. They also showed NO synthesis indirectly by L-[ 3 H]citrulline assay [9][10].…”

“…Anfossi et al [9] and Filippi et al [10] reported the expression of eNOS in cultured CCSMCs. They also showed NO synthesis indirectly by L-[ 3 H]citrulline assay [9][10]. In the present study, we demonstrated NO synthesis by measuring NOx with an automated high-performance liquid chromatography system after incubation of CCSMCs with the NO precursor, L-arg.…”

Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance

“…To measure NO production in VSMCs incubated for 24 h with 2 nmol L −1 of insulin, we used a technique extensively described in previous papers [28,29], which evaluates L‐[ 3 H]‐citrulline synthesis after incubation with L‐[ 3 H]‐arginine.…”

“…We and others have demonstrated that insulin, via PI3‐K, increases NO synthesis not only in endothelial cells [24,25] and platelets [26] but also in VSMCs and cavernosal SMCs, which express endothelial and inducible nitric oxide synthases (NOS) [27–29]: furthermore, we recently observed that NO mediates relevant biological actions in VSMCs, such as glucose transport [30]. It could therefore be supposed that NO is involved in insulin actions on VEGF.…”

Hemodynamic actions of insulin: beyond the endothelium

“…An integrative pathophysiological view of the relationships between hemodynamic and metabolic functions of insulin has been presented in detail in previous reviews (Muniyappa et al, 2007; Clark, 2008; Muris et al, 2013). Likewise, it has been recognized a similar vascular and metabolic insulin-signaling via insulin receptor/phosphatidylinositol 3-kinase (PI3K) pathways (Kim et al, 2006; Kubota et al, 2011), which are present in both endothelium and vascular smooth muscle (VSM), leading to nitric oxide synthase (NOS) activation, increased nitric oxide (NO) production and subsequent vasodilation (Zeng and Quon, 1996; Anfossi et al, 2002). Most previous research in this field has focused on insulin actions in and/or through the endothelium, assumed as the preponderant sensor/effector organ underlying the vascular effects of insulin (Muniyappa et al, 2007; Kubota et al, 2011).…”