Selective inhibition of alpha1B-adrenergic receptor expression and function using a phosphorothioate antisense oligodeoxynucleotide.

To investigate alpha1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the alpha1B-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on alpha1B-adrenoceptor mRNA expression, protein density, and coupling to phospholipase C. Cells treated with either a control oligodeoxynucleotide (CO) or medium alone served as control groups. Confocal microscopy demonstrated that DDT1 MF2 cells internalized carboxyfluorescein-labeled (FAM) AO within 30 min. Analysis of cellular lysates showed that approximately 50% of the intracellular FAM-AO was present as an intact 18-mer for up to 48 hr. Incubation of cells with AO for 48 hr decreased alpha1B-adrenoceptor density ([3H]prazosin Bmax) versus control groups by 12% (1 microM AO) and 72% (10 microM AO). In time course experiments, AO (10 microM) reduced alpha1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. alpha1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 microM AO versus controls. AO pretreatment (10 microM, 48 hr) reduced the maximum response to agonist-stimulated [3H]inositol phosphate accumulation. The maximal response of the full agonist norepinephrine was reduced by 30% after AO treatment, and by 73% for the partial agonist naphazoline. In contrast, AO did not affect histamine-stimulated total [3H]inositol phosphate accumulation. Thus, AO effectively reduced alpha1B-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit alpha1B-adrenoceptor function in vivo.

Enhanced hypo-osmoregulation induced by warm-acclimation in antarctic fish is mediated by increased gill and kidney Na+/K(+)-ATPase activities.

Serum osmolality and serum inorganic ion concentrations were studied in two antarctic fish species, Trematomus bernacchii and T. newnesi, during 5 weeks of acclimation to 4 degrees C and compared with control values for groups acclimated to -1.5 degrees C. Acclimation to 4 degrees C significantly decreased the serum osmolality of both species, thereby increasing their seawater-to-extracellular fluid (ECF) osmotic gradient. The decline in osmolality with acclimation to 4 degrees C was accompanied by significant and rapid losses of Na+ and Cl- during the first 14 days of acclimation and was maintained throughout the study period. At day 35 of acclimation, the lipid composition and microsomal Na+/K(+)-ATPase specific activities at 4 degrees C and 37 degrees C were determined in membranes from gill, kidney, liver and muscle tissues. No warm-induced decrease in fatty acid unsaturation was found in the tissues of either species. In the gills and kidneys of both species, the Na+/K(+)-ATPase activities assayed at 4 degrees C were increased after acclimation to 4 degrees C. The Na+/K(+)-ATPase activities at 37 degrees C increased at the higher acclimation temperature in T. newnesi kidneys and T. bernacchii gills, but in both species there was no compensation to temperature in the liver, regardless of assay temperature. Muscle Na+/K(+)-ATPase activity decreased in response to warm-acclimation in T. bernacchii and T. newnesi assayed at 4 degrees C and 37 degrees C, respectively. During acclimation to 4 degrees C, the discontinuity in the Arrhenius plot of the Na+/K(+)-ATPase activities of T. newnesi gill moved to a lower temperature, whereas that of kidney remained unchanged. The results indicate that acclimation to 4 degrees C induced a decrease in serum osmolality which resulted from the positive compensation of Na+/K(+)-ATPase in osmoregulatory tissues. The enhancement in Na+/K(+)-ATPase activity at 4 degrees C suggests that energy expenditure in antarctic fish may be lessened, in part, by maintaining a reduced seawater-to-ECF osmotic gradient.