Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications

Abstract

1 To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO-mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L-['4C]-arginine to L-['4C]-citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2 NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg-' protein min-'): urethra (33 + 3.3), detrusor (13.1 + 1.1) and ureter (1.5 + 0.2). No activity was detected in the particulate fraction of any region. 3 NOS activity was dependent on Ca2+-calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4 NOS activity was inhibited to a greater extent by NG-nitro-L-arginine (L-NOARG) and its methyl ester (L-NAME) than by NG-monomethyl-L-arginine (L-NMMA), while 7-nitroindazole (7-NI) was a weak inhibitor and L-cannavine had no effect. 5 Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin-sensitive manner, suggesting feedback inhibition of NOS by NO. 6 EFS induced prominent NO-mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L-NOARG = L-NAME>7-NI> L-NMMA. 7In conclusion, we have demonstrated the presence of NO-synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NOmediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely. Keywords: Nitric oxide; nitric oxide synthase; urethra; detrusor; ureter; nitrergic relaxation; urinary tract Introduction It has been suggested that nitric oxide (NO) has important regulatory functions in the urinary tract acting as a nonadrenergic, non-cholinergic (NANC)-relaxant transmitter. Specially relevant is the proposed role of NO in mediating the decrease in outlet resistance which accompanies micturition (Andersson, 1993). A rich supply of nitrergic nerves has been identified by NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry in the outflow region (urethra, bladder neck and bladder base or trigone) of several species including rat (McNeill et al., 1992), pig (Persson et al., 1993), sheep (Triguero et al., 1993 and human (Sme...

Does Cyclic AMP Mediate Rat Urinary Bladder Relaxation by Isoproterenol?

“…Whereas ODQ did not significantly attenuate the isoproterenol or forskolin effect, L-NNA lacked inhibition of maximum isoproterenol effects but may have enhanced its potency. These findings are difficult to interpret since the overall role of NO for detrusor function remains controversial (Garcia-Pascual et al, 1996;Liu and Lin-Shiau, 1997).…”

Effects of diabetes on neurotransmission in rat vaginal smooth muscle

“…NOS-activity was determined by measuring the formation of L-[ 14 C]citrulline from L-[ 14 C]arginine. 12 The tissue was homogenized in ice-cold buffer using a glass pestle homogenizer. Homogenization buffer (pH 7.2) contained: 20 mM HEPES, 0.5 mM EDTA and a protease inhibitor cocktail.…”

Morphological and Biochemical Investigation of Nitric Oxide Synthase and Related Enzymes in the Rat and Pig Urothelium

“…NOS activity was determined by measuring the formation of l -[ 14 C]-citrulline from l -[ 14 C]-arginine according to the procedure described by Garcia-Pascual et al (1996), with slight modifications. This NOS assay requires a substantial amount of tissue and was therefore performed using pig bladders.…”