In vitro fluoride dose-response study of sterilized enamel lesions.

Currently our intra-oral model uses enamel specimens that have been disinfected by soaking in buffered formalin (pH 6.8). However, because of increasing emphasis on infection control, it is important to identify a way to sterilize these specimens. The aim of this study was to determine if autoclaved, or gas sterilized, lesioned enamel responds to fluoride (F) in the same way alcohol-disinfected enamel lesions do. Seventy-two formalin-disinfected, human enamel specimens (3 mm) were lesioned in demineralizing solution for 96 h and were then divided into three groups. One group was autoclaved; one group was gas sterilized (ethylene oxide), and the remaining 24 specimens were further disinfected in 70% ethanol for 10 min. Specimens in each group were then treated 4 times/day for 4 weeks with 0, 250 or 1,100 ppm F dentifrice slurries in an in vitro cycling, remin/demin model. Following treatment, fluoride uptake was analyzed by microdrill biopsy, and lesion depth and mineral content changes (DeltaM) were determined by transverse microradiography. Data were analyzed by one-way ANOVA analysis. In all three groups of specimens there were significant (p<0.05) differences in fluoride uptake in response to different fluoride treatments. Autoclaved lesions failed to provide dose- response data with regard to changes in lesion mineral content. Because formalin and 70% alcohol are only disinfectants, and autoclaving altered the responsiveness of enamel lesions, results from this study suggest that, of the methods tested, gas sterilization is the preferred method for sterilizing enamel specimens that will be used in intra-oral studies.

Hyperosmotic stress up-regulates amino acid transport in vascular endothelial cells.

Cultured vascular endothelial cells take up L-proline by sodium-dependent transport. Cells incubated in medium made hyperosmotic by addition of sucrose showed a dose-dependent increase in Na+/proline cotransport. Studies with alpha-(methylamino)isobutyric acid revealed that the up-regulation was specific for amino acid transport system A. Up-regulation was blocked by actinomycin D and cycloheximide, indicating roles for gene transcription and protein synthesis. Up-regulation was maximum after five to six hours of hyperosmotic treatment, but returned to control levels when osmotic stress was maintained for 24 hours. The decline at 24 hours was accompanied by a significant increase in Na+/gamma-aminobutyric acid cotransport. The activity of this system, which also transports betaine, remained unchanged after just five hours of hyperosmotic stress. Inclusion of betaine in the hyperosmotic medium reduced up-regulation of system A. Na/Pi cotransport also was up-regulated by five hours of hyperosmotic stress. Up-regulation of system A, but not Na/Pi cotransport, was detected in isolated membrane fractions indicating that increased activity of this membrane transport system may be one mechanism by which vascular endothelial cells accumulate amino acids. The amino acids may act as organic osmolytes to help maintain normal cell volume during the early phase of hyperosmotic stress.

Dual action of colchicine on hypertonic activation of system A amino acid transport in vascular smooth muscle cells.

Amino acid transport system A is present in many cells and tissues and is regulated by hormones and other factors, including hypertonic stress. System A in vascular smooth muscle cells is also activated when microtubules are disrupted by drugs such as colchicine. The present study examined the action of colchicine on hypertonic activation of system A in smooth muscle cells. In serum-free medium, activation of system A by modest (340 mOsm) hypertonicity was not affected by colchicine addition. However, at high osmotic stress (460 mOsm) the addition of colchicine partially blocked the activation of system A. Addition of colchicine alone, at normal osmolarity, produced activation of system A. In the presence of serum, colchicine action was markedly different. Colchicine consistently inhibited hypertonic activation of system A at any degree of hypertonic stress but had no effect on system A at normal osmolarity. The action of colchicine as both an activator and inhibitor of system A implies microtubule involvement at more than one step in the intracellular regulation of system A.