Effects of in vivo lidocaine administration at the time of ischemia and reperfusion on in vitro contractility of equine jejunal smooth muscle.

OBJECTIVE: To determine whether administration of lidocaine during ischemia and reperfusion in horses results in concentrations in smooth muscle sufficient to protect against the negative consequences of ischemia-reperfusion injury on smooth muscle motility. ANIMALS: 12 horses. PROCEDURES: Artificial ischemia and reperfusion injury of jejunal segments was induced in vivo in conjunction with lidocaine treatment during ischemia (IRL) or without lidocaine treatment (IR). Isometric force performance was measured in vitro in IRL and IR smooth muscle preparations with and without additional in vitro application of lidocaine. Lidocaine concentrations in smooth muscle were determined by means of high-performance liquid chromatography. To assess the influence of lidocaine on membrane permeability, activity of creatine kinase and lactate dehydrogenase released by in vitro incubated tissues was determined biochemically. RESULTS: In vivo administration of lidocaine allowed maintenance of contractile performance after an ischemia and reperfusion injury. Basic contractility and frequency of contractions were significantly increased in IRL smooth muscle tissues in vitro. Additionally, in vitro application of lidocaine achieved further improvement of contractility of IR and IRL preparations. Only in vitro application of lidocaine was able to ameliorate membrane permeability in smooth muscle of IR and IRL preparations. Lidocaine accumulation could be measured in in vivo treated samples and serum. CONCLUSIONS AND CLINICAL RELEVANCE: In vivo lidocaine administration during ischemia and reperfusion had beneficial effects on smooth muscle motility. Initiating lidocaine treatment during surgery to treat colic in horses may improve lidocaine's prokinetic features by protecting smooth muscle from effects of ischemia and reperfusion injury.

Altered folding, turnover, and polarized sorting act in concert to define a novel pathomechanism of congenital sucrase-isomaltase deficiency.

Naturally occurring mutants of membrane and secretory proteins are often associated with the pathogenesis of human diseases. Here, we describe the molecular basis of a novel phenotype of congenital sucrase-isomaltase deficiency (CSID), a disaccharide malabsorption disorder of the human intestine in which several structural features and functional capacities of the brush-border enzyme complex sucrase-isomaltase (SI) are affected. The cDNA encoding SI from a patient with CSID reveals a mutation in the isomaltase subunit of SI that results in the substitution of a cysteine by an arginine at amino acid residue 635 (C635R). When this mutation is introduced into the wild type cDNA of SI a mutant enzyme, SI(C635R), is generated that shows a predominant localization in the endoplasmic reticulum. Nevertheless, a definite localization of SI(C635R) in the Golgi apparatus and at the cell surface could be also observed. Epitope mapping with conformation-specific mAbs protease sensitivity assays, and enzymatic activity measurements demonstrate an altered folding pattern of SI(C635R) that is responsible for a substantially increased turnover rate and an aberrant sorting profile. Thus, SI(C635R) becomes distributed also at the basolateral membrane in contrast to wild type SI. Concomitant with the altered sorting pattern, the partial detergent extractability of wild type SI shifts to a complete detergent solubility with Triton X-100. The mutation has therefore affected an epitope responsible for the apical targeting fidelity of SI. Altogether, the combined effects of the C635R mutation on the turnover rate, function, polarized sorting, and detergent solubility of SI constitute a unique and novel pathomechanism of CSID.

A phenylalanine-based folding determinant in intestinal sucrase-isomaltase that functions in the context of a quality control mechanism beyond the endoplasmic reticulum.

Phenotype II of congenital sucrase-isomaltase deficiency in man is characterized by a retention of the brush border protein sucrase-isomaltase (SI) in the ER/cis-Golgi intermediate compartment (ERGIC) and the cis-Golgi. The transport block is due to the substitution of a glutamine by a proline at amino acid residue 1098 that generates a temperature-sensitive mutant enzyme, SI(Q1098P), the transport of which is regulated by several cycles of anterograde and retrograde transport between the ER and the cis-Golgi (Propsting, M. J., Jacob, R. and Naim, H. Y. (2003). J. Biol. Chem. 278, 16310-16314). A quality control beyond the ER has been proposed that implicates a retention signal or a folding determinant elicited by the Q1098P mutation. We have used alanine-scanning mutagenesis to screen upstream and downstream regions flanking Q(1098) and identified a putative motif, F(1093)-x-F(1095)-x-x-x-F(1099) that is likely to be implicated in sensing the folding and subsequent trafficking of SI from the ER to the Golgi. The characteristics of this motif are three phenylalanine residues that upon substitution by alanine generate the temperature-sensitive SI(Q1098P) phenotype. This mutant protein undergoes transport arrest in the ERGIC and cis-Golgi compartments and acquires correct folding and functional activity at reduced temperatures as a consequence of cycles of anterograde and retrograde transport between the ER and cis-Golgi. Other amino acid residues in this motif are not significant in the context of phenotype II. We propose that the phenylalanine cluster is required for shielding a folding determinant in the extracellular domain of SI; substitution of a Q by a P at residue 1098 of sucrase disrupts this determinant and elicits retention of SI(Q1098P) in ERGIC and cis-Golgi in phenotype II of CSID.

A glutamine to proline exchange at amino acid residue 1098 in sucrase causes a temperature-sensitive arrest of sucrase-isomaltase in the endoplasmic reticulum and cis-Golgi.

A striking feature of phenotype II in congenital sucrase-isomaltase deficiency is the retention of the brush border protein sucrase-isomaltase (SI) in the cis-Golgi. This transport block is the consequence of a glutamine to proline substitution at amino acid residue 1098 of the sucrase subunit. Here we provide unequivocal biochemical and confocal data to show that the SI(Q/P) mutant reveals characteristics of a temperature-sensitive mutant. Thus, correct folding, competent intracellular transport, and full enzymatic activity can be partially restored by expression of the mutant SI(Q/P) at the permissive temperature of 20 degrees C instead of 37 degrees C. The acquisition of normal trafficking and function appears to utilize several cycles of anterograde and retrograde steps between the endoplasmic reticulum and the Golgi implicating the molecular chaperones calnexin and heavy chain-binding protein. The data presented in this communication are to our knowledge the first to implicate a temperature-sensitive mutation in an intestinal enzyme deficiency or an intestinal disorder.