Renal dopamine and salt-retaining states

Although generally regarded as a neurotransmitter, dopamine is also known to be secreted by the kidney whereby it promotes sodium excretion in its role as a natriuretic honnone. Peripheral dopamine may be formed by two alternative pathways; the decarboxylation of circulating L-Dopa by L-aromatic amino acid decarboxylase (LAAAD), and the desulphation of dopamine sulphate by arylsulphatase A (ASA), the latter being poorly represented in the literature. In many conditions and diseases with which sodium retention is associated, a reduced urinary excretion of dopamine has been noted implicating the involvement of dopamine in the maintenance of sodium homeostasis.This study investigates renal dopamine production via the desulphation of dopamine sulphate in a sample cohort during normal unregulated dietary sodium intake and following a low sodium regimen. After dietary salt restriction urinary dopamine sulphate levels were significantly increased, indicating that dopamine sulphate is indeed a physiological reservoir of active free dopamine, the necessity for which is reduced during self depletion. This confirmed the dopamine/dopamine sulphate pathway as one which may be relevant to the maintenance of sodium homeostasis. The activity of urinary ASA was investigated in diabetes mellitus as an example of a sodium-retaining state, and compared with that in a matched normal control group. A decreased ASA activity was anticipated, given the blunted dopamine excretion observed in many sodium-retaining states, however an unexpected increase in activity in the diabetic group was observed. Enzyme kinetic analysis of ASA showed that this was not due to the existence of an isoform having an altered affinity for dopamine sulphate. This rather paradoxical situation, that urinary-dopamine is decreased while ASA activity is increased, may be explained by the sequestering of free dopamine by autoxidation to 6-hydroxydopamine as has been hypothesised recently to occur in diabetes mellitus. To confirm the homogeneity of ASA in the normal and diabetic groups, four amplicons spanning the 3637bp intronic and exonic regions of the gene were generated by PCR. These were sequence utilising a fluorescent-dye terminator reaction using the forward PCR primer as sequencing primer. Although single nucleotide polymorphisms were observed between the two groups these occurred either in intronic regions or, when exonic, generated silent mutations, supporting the enzyme kinetic data. The expression of ASA was investigated to determine the basis of the increased activity observed in diabetes mellitus. Although a validated comparative RT-PCR assay was developed for amplification of arsa transcripts from fresh blood samples, expression analysis from archived paraffin-embedded renal tissue was complicated by the low yield and degradation of unprotected mRNA. Suggestions for the development of this work using renal cell-culture are discussed.

Phenotypic and genotypic analysis of intestinal spirochacies

Pulsed field gel electrophoresis of 82 intestinal spirochaete isolates showed specific differentiation of Serpulina pilosicoli and Serpulina hyodysenteriae although considerable heterogeneity was observed, especially amongst S. pilosicoli isolates. In several cases genotypically similar isolates originated from different animals suggesting that cross-species transmission may have occurred. The Caco-2 and Caco-21HT29 cell models have been proposed as potentially realistic models of intestinal infection. Quantitation of adhesion to the cells showed isolate 3 82/91 (from a bacteraemia) to adhere at significantly greater numbers than any other isolate tested. This isolate produced a PFGE profile which differed from other S. pilosicoli isolates and so would be of interest for further study. Comparison of bacteraemic and other S. pilosicoli isolates suggested that bacteraemic isolates were not more specifically adapted for adhesion to, or invasion of the epithelial cell layer than other S. pilosicoli isolates. Genotypically similar isolates from differing animal origins adhered to the Caco-2 model at similar levels. Generation of a random genomic library of S. pilosicoli and screening with species specific monoclonal antibody has enabled the identification of a gene sequence encoding a protein which showed significant homology with an ancestral form of the enzyme pyruvate oxidoreductase. Immunoscreening with polyclonal serum identified the sequences of two gene clusters and a probable arylsulphatase. One gene cluster represented a ribosomal gene cluster which has a similar molecular arrangement to Borrelia burgdorjeri, Treponema pallidum and Thermatoga maritima. The other gene cluster contained an ABC transporter protein, sorbitol dehydrogenase and phosphomannose isomerase. An ELISA type assay was used to demonstrate that isolates of S. pilosicoli could adhere to components of the extracellular matrix such as collagen (type 1), fibronectin, laminin, and porcine gastric mucin.