Intravascular Hemolysis Leads to Increased Esophagus Smooth Muscle Contractions in Mice: Implication for Dysphagia and Odynophagia

Introduction:

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway is well established as a regulatory pathway of smooth muscle relaxation and contraction. Intravascular hemolysis is a hallmark of some hemolytic anemia, such as paroxysmal nocturnal hemoglobinuria (PNH). NO scavenging by excess plasma hemoglobin (Hb) has been associated with clinical manifestation of intravascular hemolysis. PNH patients display odynophagia and dysphagia, particularly during hemolytic exacerbations, and appear to be due to esophageal spasm. In this study, since plasma Hb excess leads to lower NO bioavailability, we hypothesized that exaggerated intravascular hemolysis may impair the esophageal function. The aim of this study was to evaluate the esophageal function in phenylhydrazine (PHZ)-treated mice, a murine model of intravascular hemolysis. The magnitude of intravascular hemolysis in sickle cell disease (SCD) is a controversial issue. For comparative purpose, SCD mice were also studied. We have focused on the role of the NO signaling pathway and contractile mechanisms. Moreover, the effects of iron chelator deferoxamine treatment on esophagus contractile mechanisms in PHZ mice were also evaluated.

Methods: C57BL/6 mice (control) and Berkeley SCD transgenic male mice (3-4 mo old) were used. Intravascular hemolysis was induced in C57BL/6 mice by intraperitoneal injection of 2 doses of PHZ at 50 mg/Kg 8h apart. Mice were euthanized in isoflurane chamber 4 days after the second PHZ injection. The esophagus smooth muscle rings were mounted in isolated organ baths. Concentration-response curves to muscarinic agonist carbachol (CCh; 10 nM -100 μM) and potassium chloride (KCl) were obtained in esophagus from control, PHZ and SCD mice. Control and PHZ mice were treated with deferoxamine (150 mg/kg/day) or vehicle (saline) from the second to the 4^th day via i.p. injection.

Results: Hematological parameters (RBC [x 10^-6/μL], Hb [g/dL], HCT [%]) show that PHZ (2.8±0.2, 6±0.4 and 17±1) and SCD mice (5.6±0.5, 6.1±0.3 and 28±3) have severe anemia, compared to control mice (8.8±0.1, 12.4±0.1 and 45±1). Plasma Hb was higher in SCD and PHZ compared to control mice (2.1- and 3.3 fold, respectively, P<0.05). Moreover, plasma Hb was 57% higher (P<0.05) in PHZ compared to SCD mice. CCh induced concentration-dependent esophagus contractions in control, PHZ and SCD mice. The maximal responses (E_max) were higher (P<0.05) in the esophagus of PHZ (0.75±0.3 mN/mg) compared to control mice (0.50±0.03 mN/mg).In order to evaluate the esophagus contractions independently of receptor stimulation, a concentration-response curve to KCl was constructed. Again, E_max induced by KCl were greater (P<0.05) in PHZ esophagus (0.45±0.01 mN/mg) compared to control mice (0.28±0.02 mN/mg). In SCD mice esophagus, contractile responses induced by CCh and KCl remained unchanged. In vitro preincubation of esophagus with the NO-independent heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (1 μM) fully reversed the higher E_max of CCh and KCl in PHZ mice to control values, without changing the responses in control mice. No changes in the contractile responses for CCh and KCl were observed with the preincubation with NO donor sodium nitroprusside (SNP; 300 μM) in the PHZ and control group. Treatment with deferoxamine did not prevent the elevation of contractile responses in PHZ mice esophagus.

Conclusion: Our data show that exaggerated intravascular hemolysis can lead to enhanced esophagus contractions in PHZ mice. BAY 41-2272 is likely to counteract the elevation of contractile responses by stimulating sGC, elevating cGMP levels by stimulating the sGC mostly via NO-independent mechanisms. NO donor failed to modify the increased esophagus contractions in PHZ mice. It is likely that Hb or increased O₂^- production in esophagus of PHZ mice act to inactivate NO before it reaches sGC. Deferoxamine treatment failed to avoid the esophageal dysfunction, suggesting that excess iron may not be associated with this disorder. SCD mice do not display esophageal disorder, indicating that excess of plasma Hb is not abundant enough to cause esophagus damage in SCD. To the best of our knowledge this is the first study in an experimental model that supports the hypothesis that esophageal clinical symptoms in PNH are secondary to impairment of NO-cGMP pathway caused by intravascular hemolysis.

Financial Support: FAPESP.