Soluble guanylate cyclase mediates the relaxation of healthy and inflamed bladder smooth muscle by aqueous nitric oxide

Abstract

Introduction: Due to its chemical properties, functional responses to nitric oxide (NO) are often difficult to examine. In the present study, we established a method to produce NO in an aqueous solution and validated its capacity to evoke functional responses in isolated rat bladders. Furthermore, we compared the NO responses to the commonly used NO donor sodium nitroprusside (SNP). We also investigated the impact of ongoing inflammation on the involvement of soluble guanylate cyclase (sGC) dependent signaling in NO relaxation. Methods: A setup to produce an aqueous NO solution was established, allowing the production of an aqueous solution containing a calculated NO concentration of 2 mM. Sixty male Sprague-Dawley rats received either no treatment (controls) or cyclophosphamide (CYP; 100 mg*kg^-1 i.p., 60 h prior to the experiment) to induce experimental cystitis. Bladder strip preparations were mounted in organ baths and studied at basal tension or pre-contracted with methacholine (3 μM). Aqueous NO solution (40-400 μL; 2 mM corresponding to 4-40 μM) or SNP (1-1,000 μM) was added cumulatively in increasing concentrations. Relaxation to aqueous NO was also studied in the presence of the sGC inhibitor ODQ (0.25-25 μM). The expression of sGC was investigated by immunohistochemical analysis. Results: The NO solution caused functional relaxations in both controls and inflamed bladder preparations. NO-induced relaxations were significantly greater in inflamed bladder strips at basal tension, whereas no differences were seen in methacholine pre-contracted strips. In the presence of the sGC inhibitor ODQ in a high concentration, the NO-evoked relaxations were abolished in both control and inflamed preparations. At a lower concentration of ODQ, only NO relaxations in inflamed preparations were attenuated. Immunohistochemical analysis showed that sGC was expressed in the detrusor and mucosa, with a significantly lower expression in the inflamed detrusor. Conclusion: In the present study, we found that aqueous NO solution induces relaxation of the rat detrusor by activating soluble guanylate cyclase in both control and inflamed bladder strips. Induction of inflammation conceivably leads to decreased sGC expression in the detrusor, which may explain the different susceptibility towards inhibition of sGC in inflamed versus control tissue. The use of an aqueous NO solution should be further considered as a valuable complement to the pharmacological tools currently used.

Keywords: NO donor; animal study; bladder; cystitis; nitric oxide; relaxation; sGC (soluble guanylate cyclase); sodium nitroprusside (SNP).

FIGURE 1

Outline of the NO-sGC-cGMP pathway. The formation of nitric oxide (NO) from L-arginine and molecular oxygen is catalyzed by nitric oxide synthase (NOS). NO binds to soluble guanylate cyclase (sGC), forming cyclic GMP (cGMP) and, in turn, leading to smooth muscle relaxation. Production of cGMP can similarly be induced by NO donors and sGC stimulators. Oxidative stress, for instance during inflammation, leads to oxidation of sGC. A similar effect is achieved by sGC inhibitors such as ODQ. Oxidized sGC cannot be activated by NO, but can instead be activated by sGC activators. Pharmacologically active drugs are illustrated in bold blue, and enzymes in green italics.

FIGURE 2

Schematic illustration of the setup for producing the aqueous NO solution. Five 20 mL glass vials with septum closure were set up in a fume hood. The first was filled with 18.5 mL of pyrogallol (10 mM) to remove any traces of oxygen from the argon gas led through it. The next contained 18.5 mL of NaOH (10 mM) to remove higher nitrogen species from any gas flowing through it. Thereafter, an empty vial was placed to ensure no spillover into the final vials containing pure Milli-Q water. All vials were connected with Teflon tubing (blue arrows), entering each vial into the liquid and exiting above. The final vial had a Teflon tube leading into the open air, functioning as a “chimney” for the supplied gas. To minimize oxidization, all solutions were prepared directly in the glass vials, and the pyrogallol vial was covered with aluminum foil due to its photosensitive nature. Argon gas, pushing out oxygen from the solutions and vials, was first led through the system for 60 min. Then the gas was switched, without interruption, to pure NO gas, entering through the NaOH vial, for an additional 20 min. The end result was two vials of Milli-Q water saturated with pure NO.

FIGURE 3

Nitrergic relaxation of basal tension in bladder preparations. Functional relaxatory responses at mechanically attained basal tension to increasing volumes of (A) aqueous NO solution or concentrations of (B) the NO donor SNP in full-thickness urinary bladder strip preparations from healthy controls (•) or rats with cyclophosphamide-induced cystitis (▪). * and ** denotes p < 0.05 and p < 0.01, respectively. n = 8 in each group. Vertical bars indicate the SEM.

FIGURE 4

Nitrergic relaxation of pre-contracted bladder preparations. Functional relaxatory responses to increasing volumes of (A) aqueous NO solution or concentrations of (B) the NO donor SNP in methacholine pre-contracted full-thickness urinary bladder strip preparations from healthy controls (•) or rats with cyclophosphamide-induced cystitis (▪). n = 8 in each group. Vertical bars indicate the SEM.

FIGURE 5

Effects of the sGC inhibitor ODQ on nitrergic relaxatory responses in pre-contracted bladder preparations. Nitrergic relaxations to increasing volumes of aqueous NO solution in the absence (•) or presence (▪) of ODQ in full-thickness urinary bladder strip preparations from healthy controls (A,C) or rats with cyclophosphamide-induced cystitis (B,D). In the upper panels (A,B), the ODQ concentrations used were high (2.5 × 10⁻⁵ M), and in the lower panels (C,D), the concentrations were low (2.5 × 10⁻⁶ M) in the lower. *, **, and *** denotes p < 0.05, p < 0.01, and p < 0.001, respectively. n = 11 in each group. Vertical bars indicate the SEM.

FIGURE 6

Expression of sGC in the rat detrusor and urothelium. Comparison of fluorescent sGC expression in healthy and inflamed (A) detrusor and (B) urothelium. Expression was graded on a relative scale from 0–3 where 0 denoted no expression and 3 denoted maximum fluorescent intensity. n = 6 in each group; each individual grade is indicated by a black point in the figure. *p < 0.05.