Hypothesis / aims of study
Functional effects of nitric oxide (NO) have in the urinary bladder been suggested to be mediated through a signaling pathway involving soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP), in turn modulating Ca2+-channels (1). The present study examines cGMP-dependent responses to aqueous NO in healthy and inflamed rat urinary bladder (detrusor) smooth muscle preparations.
Study design, materials and methods
Thirty-six male Sprague-Dawley rats (210–510 g; Charles-River, Calco, Italy) were used, receiving either no treatment (serving as controls) or an intraperitoneal injection with cyclophosphamide (CYP; 100 mg/kg) 60 h before the experiment, in order to chemically induce cystitis. Rats were euthanized with an overdose of pentobarbitone, and the bladders were excised and stored in oxygenated Krebs solution at all times.
Two full-thickness strips, approximately 6×2 mm, excised proximal to the ureters and above the trigone, were prepared from each bladder. The preparations were mounted in 20 mL organ baths filled with Krebs solution (gassed with 5% CO2 in 95% O2 at a temperature of 37°C), stretched to approximately 10 mN, and let to equilibrate for 45 minutes, obtaining a basal tension of about 5 mN. Functional responses were recorded using the MP100WSW data acquisition system and the AcqKnowledge software (Biopac Systems, Goleta, USA).
Argon was used to remove traces of oxygen before pure NO was led through deoxygenated NaOH and water. The production of aqueous NO solution was done as per a published protocol (2), where vials containing, in sequence, pyrogallol (10 mM), NaOH (10 mM), and de-ionized water were set up in an airtight manner. The resulting solutions contained a calculated NO concentration of 2 mM (2).
Functional responses to NO (4, 10, 20, 40 μM) were studied in the absence and presence of the sGC inhibitor ODQ by cumulative addition to the organ baths in methacholine (3 μM) pre-contracted healthy or inflamed bladder strips. Viability was assessed by addition of methacholine (50 μM) or high K+ Krebs solution (124 mM) at the beginning and end of every experiment. Aqueous NO (2 mM) was administered in close proximity of the tissue in the organ bath using a 1000 μL gas-tight Hamilton syringe in volumes of 40, 100, 200 or 400 μL. When employing ODQ, it was added to the baths and let to equilibrate for approximately 20 minutes before adding the NO solution.
Statistical significance was determined by two-way ANOVA followed by Sidak’s test for multiple comparisons using the GraphPad Prism 9 software (GraphPad Software Inc., San Diego, USA). p-values < 0.05 were considered statistically significant. Data are presented as mean ± SEM.
Results
Aqueous NO solution (40, 100, 200, and 400 µL, corresponding to final concentrations of 4, 10, 20, and 40 μM in the baths) induced concentration-dependent relaxations in methacholine (3 μM) pre-contracted rat detrusor strips, e.g., ranging from -0.85 ± 0.18 mN to -1.47 ± 0.29 mN in the control group, and -0.84 ± 0.22 mN to -1.42 ± 0.29 mN in the CYP-treated group (Fig 1a, b). The relaxation to NO was statistically significantly abolished in the presence of a high concentration of the sGC inhibitor ODQ (25 µM), and a trend showing a shift towards a contractile response was instead observed, most prominently in the control group.
In contrast, in the presence of a low concentration of ODQ (0.25 µM), the relaxatory responses to NO were not abolished. Instead, a slightly greater NO relaxation was observed (p = 0.026 at 10 μM in the CYP-treated group; Fig 1d). The median difference between relaxatory responses in the absence vs. presence of the low concentration of ODQ was 10% in the control group (Fig 1c) and 14% in the CYP group (Fig 1d).
Interpretation of results
The present results confirm previous findings and demonstrate that the method to produce aqueous NO solution is robust and can be used to study direct functional nitrergic effects. While no statistical comparisons were presently made between controls and the CYP (inflamed) group, it was evident that both groups displayed relaxatory responses to NO and that the sGC inhibitor, ODQ, abolished these responses. This finding was further strengthened by the lack of effect seen when adding the sGC inhibitor at a lower concentration, displaying a pharmacological concentration dependency of this effect.
The fact that ODQ was shown to block the relaxatory response evoked by NO confirms the hypothesis that nitrergic relaxations in the detrusor to a large extent are mediated through a cGMP-dependent pathway. These findings are in accordance with previous studies where ODQ has been shown to block NO donor responses in the rat urinary bladder (3). Thus, the present results strengthen the conclusion that aqueous NO can be readily produced and used for functional pharmacological studies, acting in a specific manner on sGC. Work remains to further investigate the details of which downstream pathways that are involved. Finally, the current study suggests that the sGC-mediated pathway is important also during cystitis, although further investigations should be conducted to scrutinize possible changes in detail.