Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BACKGROUND: In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology. OBJECTIVES: To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. ANIMALS: Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. METHODS: Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing. RESULTS: Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu). CONCLUSIONS AND CLINICAL IMPORTANCE: This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH-binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.

[Mechanism of the contracting actions of K, acetylcholine and Ba and of the antispasmodic actions of Cd and Mn in the pyloric antrum strip of the rat, with special reference to their relationship to Ca].

Action mechanisms of the contractile agents, K ACh and Ba and of the antispasmodics, Cd and Mn were investigated. The contractions by K,ACh and Ba are exponential in shape, but consisted of phasic contraction (PC) and the subsequent tonic contraction (TC). PC by K and ACh are inititated by the release and the passive influx of Ca, whereas the PC by BA is due to the release of Ca. On the other hand, TC by these three agents is maintained by the active influx of Ca requiring energy. Since only the contraction by Ba remains constant in Ca(-) bath solution, it is assumed that the direct stimulation to muscle contractile elements without the mediation of Ca mobilization is also partly related to the contracting mechanism of Ba. Storage sites of Ca in the cell membrane of this preparation are distinguished into three divisions, the first, the second and the third, which contains loosely-, less lossely-, and tightly-bound Ca, respectively. K releases Ca to elicit contraction from the first divisions, ACh does so from the first and the second divisions, and Ba does so from all of the three divisions. The following assumption was obtained on the antispasmodic action of Cd and Mn, on the basis of the influence of Ca removed from bath solution and of addition of high K to bath solution and the analysis with concentration-action curves. The antispasmodic mechanism Cd and Mn is due to inhibition of cell membrane (competitive inhibition of influx and then release of Ca and subsequently competitive and non-competitive inhibition of influx and release of Ca) followed by the non-competitive inhibition of muscle contractile system, with the increase of dosage.