Mechanisms involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)-derived reactive oxygen species (ROS) modulation of muscle function in human and dog bladders

Abstract

Roles of redox signaling in bladder function is still under investigation. We explored the physiological role of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) in regulating bladder function in humans and dogs. Mucosa-denuded bladder smooth muscle strips obtained from 7 human organ donors and 4 normal dogs were mounted in muscle baths, and trains of electrical field stimulation (EFS) applied for 20 minutes at 90-second intervals. Subsets of strips were incubated with hydrogen peroxide (H2O2), angiotensin II (Ang II; Nox activator), apocynin (inhibitor of Noxs and ROS scavenger), or ZD7155 (specific inhibitor of angiotensin type 1 (AT1) receptor) for 20 minutes in continued EFS trains. Subsets treated with inhibitors were then treated with H2O2 or Ang II. In human and dog bladders, the ROS, H2O2 (100μM), caused contractions and enhanced EFS-induced contractions. Apocynin (100μM) attenuated EFS-induced strip contractions in both species; subsequent treatment with H2O2 restored strip activity. In human bladders, Ang II (1μM) did not enhance EFS-induced contractions yet caused direct strip contractions. In dog bladders, Ang II enhanced both EFS-induced and direct contractions. Ang II also partially restored EFS-induced contractions attenuated by prior apocynin treatment. In both species, treatment with ZD7155 (10μM) inhibited EFS-induced activity; subsequent treatment with Ang II did not restore strip activity. Collectively, these data provide evidence that ROS can modulate bladder function without exogenous stimuli. Since inflammation is associated with oxidative damage, the effects of Ang II on bladder smooth muscle function may have pathologic implications.

Fig 1. Exogenous hydrogen peroxide (H₂O₂, 100μM) enhances EFS-induced muscle strip contractions in human and dog bladders.

The maximal responses are expressed in milli Newtons (mN). EFS = electrical field stimulation. Data is presented as mean ± 95% CI. *: p < 0.05, comparing pre- versus post-H₂O₂ treatment.

Fig 2. Bladder muscle strip responses to direct stimulation by 100μM H₂O₂.

Representative tracings of H₂O₂-induced direct muscle strip contraction in human (A) and dog (B). Maximal responses to 100μM H₂O₂ in muscle strips from bladders of both species (C), comparing pre- versus post-H₂O₂ treatment. The maximal responses in (C) are expressed in milli Newtons (mN). Data is presented as mean ± 95%CI. *: p < 0.05, comparing pre- versus post-H₂O₂ treatment.

Fig 3. Nox inhibitor and ROS scavenger, apocynin, attenuated EFS-induced bladder muscle strip contractions in both species.

The H₂O₂ (100μM, second treatment) was added to the muscle baths without washout of the apocynin (100μM, first treatment). (A) The maximal responses are expressed in milli Newtons (mN). (B) Representative tracing of the apocynin effect and then the H₂O₂ effect on EFS-induced human bladder muscle strip contraction. EFS = electrical field stimulation. Tx = treatment. Data is presented as mean ± 95%CI. *: p < 0.05 and **: p < 0.01, comparing post-apocynin versus either pre-apocynin, or H₂O₂ treatments.

Fig 4. Angiotensin II (Ang II) enhances EFS-induced muscle strip contractions in dog bladders, but not humans.

The maximal responses are expressed in milli Newtons (mN). EFS = electrical field stimulation. Data is presented as mean ± 95%CI. *: p < 0.05, comparing pre- versus post-Ang II treatment.

Fig 5. Bladder muscle strip responses to a direct stimulation by 1μM angiotensin II (Ang II).

Representative tracing of Ang II-induced direct muscle strip contraction in human (A) and dog (B). Maximal responses to 1μM Ang II in muscle strips from human and dog bladders, comparing pre- versus post-Ang II treatment. The maximal responses in (C) are expressed in milli Newtons (mN). Data is presented as mean ± 95%CI. *: p < 0.05 and **: p < 0.01, comparing pre- versus post-Ang II treatment; ^#: p < 0.01, comparing post-Ang II treatment between humans and dogs.

Fig 6. Angiotensin II treatment, after apocynin, recovered EFS-induced muscle strip contractions in human and dog bladders.

(A) The maximal responses are expressed in milli Newtons (mN). (B) Representative tracing of apocynin effect and then Ang II effect on EFS-induced human bladder muscle strip contraction. (C) Representative tracing of apocynin (first treatment) effect, then H₂O₂ (second treatment), and then Ang II (third treatment) effect on EFS-induced human bladder muscle strip contraction. In each, the subsequent muscle bath treatments occurred without washout of prior treatment(s). EFS = electrical field stimulation. Tx = treatment. Data is presented as mean ± 95% CI. *: p < 0.05, comparing responses to post-apocynin versus either pre-apocynin, or Ang II treatments.

Fig 7. AT1 receptor specific inhibitor, ZD7155, attenuated EFS-induced muscle strip contractions in human and dog bladders.

Angiotensin II (Ang II, 1μM, second treatment) was added to the muscle baths without washout of the ZD7155 (10μM, first treatment). (A) The maximal responses are expressed in milli Newtons (mN). (B) Representative tracing of ZD7155 effect and then Ang II effect on EFS-induced human bladder muscle strip contraction. EFS = electrical field stimulation. Tx = treatment. Data is presented as mean ± 95%CI. *: p < 0.05, comparing post-ZD7155 versus either pre-ZD7155, or Ang II treatments.

Fig 8. NADPH enhanced ROS, and specifically, superoxide levels in dog bladder smooth muscle using lucigenin-enhanced chemiluminescence.

(A) Total muscle homogenates were exposed to dark-adapted lucigenin in balanced salt solution and baseline was measured (Basal). Superoxide production was enhanced in the presence of NADPH (100μM). Superoxide production was attenuated by the addition of 20 mM Tiron. (B) Representative photon emission in response to the 3 different conditions (Basal, NADPH, and Tiron) measured in a luminometer over time (in minutes). MLU = mean light units. RLU = relative light units. Data is presented as mean ± 95%CI. *: p < 0.05, NADPH versus baseline or Tiron.