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.
OBJECTIVE
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 immunofluorescence, 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 micro mol/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/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=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.


CONCLUSIONS
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.
Anfossi
Giovanni
G
Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano, Turin, Italy.
Massucco
Paola
P
Mattiello
Luigi
L
Balbo
Alessandra
A
Russo
Isabella
I
Doronzo
Gabriella
G
Rolle
Luigi
L
Ghigo
Dario
D
Fontana
Dario
D
Bosia
Amalia
A
Trovati
Mariella
M
D000328:Adult; D002118:Calcium / Q000502:physiology; D002455:Cell Division; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000502:physiology; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000008:administration & dosage / Q000494:pharmacology* / Q000502:physiology; D008297:Male; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D010413:Penis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013997:Time Factors
D007328:Insulin; D020030:Nitric Oxide Donors; D009712:Nucleotides, Cyclic; D010726:Phosphodiesterase Inhibitors; D012333:RNA, Messenger; D009569:Nitric Oxide; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium