The effect of nitric oxide (NO) gas and NO donors may differ due to different amounts of NO, leading to different signalling. Therefore, the present study examined the functional responses to NO in in aqueous solution and compared the effect to a classical NO donor, sodium nitroprusside (SNP) in healthy and inflamed rat urinary bladder (detrusor) smooth muscle preparations.

Twenty-four male rats (285–440 g) of the Sprague-Dawley strain (Charles-River, Calco, Italy) were used in the study. The rats received either no treatment (controls) or an intraperitoneal injection with cyclophosphamide (CYP; 100 mg/kg) in order to chemically induce cystitis. Experiments were conducted 60 hours after induction of cystitis, starting with euthanization of the rat and excision of the urinary bladder, which was kept in oxygenated Krebs solution at all times. 
From each bladder, two full thickness strips were prepared (approximately 6 × 2 mm), excised proximal to the ureters and above the trigone. The strips were mounted in 20 mL organ baths filled with Krebs solution and gassed with 5% CO2 in 95% O2 at a temperature of 37°C. Functional responses were recorded using the MP100WSW data acquisition system and the Acquire software (Biopac Systems, Goleta, USA). After the specimens were mounted, they were stretched to approx. 10 mN and let to equilibrate for 45 minutes, obtaining a basal tension of about 5 mN.
Production of an aqueous solution of NO was conducted in accordance with a published protocol (1), where vials containing, in sequence, pyrogallol (10 mM), NaOH (10 mM) and de-ionized water were set up in an airtight fashion. Argon was employed to remove any traces of oxygen before pure NO was led through the deoxygenated NaOH and water vials. The resulting aqueous solutions contained an NO concentration of 2 mM (1).
Functional responses to NO (4, 10, 20, 40 μM) and the NO-donor SNP (5, 50, 500, 5000 nM) were studied by cumulative addition to the organ baths in methacholine (3 or 10 μM) pre-contracted healthy or inflamed bladder strips. The NO solution (2 mM) was administered close to the surface of the tissue in the organ bath using a 1000 μL gas tight Hamilton syringe in a volume of 40, 100, 200 or 400 μL, whereas SNP was administered in the classical fashion, administering the solution into the bath at a volume of 100 μL using a pipette. At the end of every experiment, methacholine (50 μM) was administered to assess viability. 
Statistical significance was determined by two-way ANOVA followed by Sidak’s test for multiple comparisons using the GraphPad Prism 8 software (GraphPad Software Inc., San Diego, USA). P-values < 0.05 were considered statistically significant. Data are presented as mean ± SEM.

In methacholine pre-contracted rat detrusor strips, aqueous NO solution (4-40 μM) induced concentration-dependent relaxations ranging from -1.83 ± 0.17 mN to -2.65 ± 0.22 mN in the control group compared to -2.32 ± 0.18 mN to -3.42 ± 0.31 mN in the CYP-treated group (Fig 1a). The relaxation to NO in the CYP-treated group was statistically increased at all but the lowest concentration, as compared to controls (p<0.05; Figure 1a).
The response to the NO-donor SNP (5-5000 nM) on the other hand was contractile at low concentrations, followed by a relaxation below baseline in both groups at higher concentrations, ranging from +2.19 ± 0.17 mN to -1.79 ± 0.18 mN in the control group compared to +2.66 ± 0.18 mN to -2.88 ± 0.17 mN in the CYP-treated group (Fig 1b).

While the difference in response between normal and inflamed bladder may seem more evident for NO, compared to SNP, one should bear in mind that the concentrations employed were lower for the NO-donor SNP. In fact, the lowest concentration of NO corresponds to the highest SNP concentration. Also, the routes of administration differed, i.e. NO was injected close to the surface of the strip preparation, whereas SNP was dissolved in the entirety of the bath. This taken into consideration, the results from the two substances are remarkably comparable. 
The observed phenomenon that SNP causes both contraction and relaxation is somewhat puzzling, but similar paradoxical functional responses have been described previously (2). Generally, the functional effects of NO, and NO-donors, seem to vary between species and different tissues. Thus, the present results may be one step forward in further understanding the complex role of NO in the rat urinary bladder, and for interpreting previous studies which utilized NO-donors.

The present study succeeded in demonstrating its main objective; proof of concept that an aqueous solution of NO could be manufactured and used in an organ bath set-up for investigating the role of nitrergic signalling on functional responses in the rat urinary bladder. NO was shown to exert its effect in a manner similar to that of the NO-donor SNP, which was expected but far from obvious. Furthermore, it was shown that nitrergic relaxatory responses were altered during cystitis. Thus, the currently demonstrated way of administering NO may be important for future studies on nitrergic effects and signalling pathways in the rat urinary bladder and, possibly, other tissues as well.

Simonsen, U., R M Wadsworth, N H Buus, and M J Mulvany. "In Vitro Simultaneous Measurements of Relaxation and Nitric Oxide Concentration in Rat Superior Mesenteric Artery." The Journal of Physiology 516 ( Pt 1) (1999): 271-82.
Moon, Annick. "Influence of Nitric Oxide Signalling Pathways on Pre-contracted Human Detrusor Smooth Muscle in Vitro." BJU International 89.9 (2002): 942-49.