Mechanisms of hormonal modulation of ion transport in the toad's urinary bladder. Subcellular localization of aldosterone-induced proteins.

A dual-label isotope technique was used to study the effects of aldosterone upon the incorporation of amino acids into proteins of the in vitro toad urinary bladder. Following labeling, the mucosal cells were disaggregated and the mitochondria-rich and granular cells were separated. Proteins with an elevated isotope ratio were found in a plasma membrane fraction (170 000, 110 000 and 85 000 daltons) and in the cytosol (36 000 and 6 000 daltons) of the preparations enriched in mitochondria-rich cells. These effects of aldosterone were blocked by cycloheximide. There was no evidence that aldosterone had induced the incorporation of labeled amino acids into carbonic anhydrase isolated from the soluble fraction by affinity chromatography. The results suggest that the physiologic response of the toad bladder to aldosterone is related to the synthesis of both soluble and plasma membrane proteins.

Cell-free translation of RNA isolated from aldosterone-treated toad bladder mucosal cells.

The stimulation of sodium transport by aldosterone in target tissues requires the synthesis of both mRNA and proteins. Aldosterone-induced mRNA and proteins have been demonstrated in toad urinary bladder and rat kidney. We have isolated total RNA and poly(A)-containing RNA from hormone-treated and untreated toad bladder mucosal cells for translation in a rabbit reticulocyte lysate system. Aldosterone-induced proteins synthesized in this system have physical properties similar to those of aldosterone-induced proteins synthesized in the intact toad bladder.

Role of induced proteins in insulin-stimulated sodium transport.

Insulin increases active sodium transport by the toad urinary bladder, an effect that begins with 15 minutes and persists for at least 20 hours. Although pretreatment of bladders with inhibitors of RNA or protein synthesis has no effect on the response to insulin within the first hour, these agents block the long-term component of insulin-stimulated sodium transport. To examine the relationship of protein synthesis to the sustained increase in sodium transport elicited by insulin, we have studied the effects of the hormone upon the incorporation of radioactive amino acids into mucosal cell proteins. There is no detectable effect of insulin on the uptake of aminoisobutyric acid into mucosal cells or on the incorporation of labeled precursors into total protein; however, using a dual-label technique, we find that insulin increases the incorporation of amino acids into specific soluble and plasma-membrane proteins of granular mucosal cells. Insulin has no discernible effect upon the incorporation of amino acids into proteins of mitochondria-rich mucosal cells. Thus the effects of insulin upon sodium transport appear to be the result of two separate mechanisms, (1) a short-term response that is independent of protein synthesis and (2) a long-term response that is expressed after the first hour of hormone treatment and that requires the synthesis of one or more specific proteins in the granular cell.

Age-related changes in carbonic anhydrase of toad lens.

Carbonic anhydrase in the toad lens is immunologically identical to the (high activity) enzyme form present in the erythrocyte. As with the erythrocyte carbonic anhydrase, electrofocusing of the freshly-extracted lens enzyme separated three variants with isoelectric points of 6.1, 5.7 and 5.4, all of which exhibited inhibition properties characteristic of the high activity type of carbonic anhydrase. No evidence of the low activity form of the enzyme was found. In contrast to the erythrocyte isoelectric variants, which are stable in vitro, the three variants of lens carbonic anhydrase exhibited progressive anodization in the course of purification and during storage, and could not be isolated in the original form. Acidification of lens carbonic anhydrase appears to be part of the in vivo aging process of the enzyme protein: the still-active enzyme from the oldest lens region--the nucleus--exhibited a considerably lower isoelectric point than the enzyme extracted from the younger, soft fibers of the lens. Although the specific activity of the carbonic anhydrase from the fibers of lens nucleus was considerably lower than the activity of the enzyme from the younger fibers, the observed modification (acidification) of the aged enzyme did not appear to affect substantially its binding properties towards acetazolamide nor the heat lability of the active protein.

Insulin-induced proteins in the toad urinary bladder.

Insulin increases active sodium transport by the toad urinary bladder within 15 min, an effect which persists for an least 20 h. In tissues pre-treated with inhibitors of transcription or translation, sodium transport briefly increases after insulin addition but returns to basal levels within 60-90 min. We have studied the effects of insulin on the incorporation of radioactive amino acids into mucosal cell proteins. Insulin had no detectable effect on the uptake of amino acids nor on their incorporation into total protein; however, using a dual label technique, we found that insulin increases the incorporation of amino acids into specific soluble and plasma membrane proteins of the granular mucosal cell. The time course and dose-response relationship of the induction of protein synthesis by insulin suggest that these proteins may play a role in the sustained elevation of sodium transport by insulin.