Walter N. Scott

Partition of Tissue Functions in Epithelia: Localization of Enzymes in " Mitochondria-Rich" Cells of Toad Urinary Bladder

The mucosal epithelium of the toad urinary bladder reabsorbs sodium, acidifies the urine, and is responsive to neurohypophyseal hormnones. Mucosal epithelial cells, consisting of two major morphologic cell types, mitochondria-rich and granular, were removed from the bladder and separated by density gradient centrifugation. The mitochondria-rich cells contained three times as much carbonic anhydrase activity as the granular cells. Oxytocin caused a 235 percent increase in the adenosine 3,5-monophosphate content of mitochondria-rich cells but had no effect on the granular cells. The evidence indicates that the mitochondria-rich cell, which accounts for only 15 percent of the mucosal cells, plays a major role in the mediation of sodium ion and hydrogen ion transport in the toad bladder and is a specific site of action of neurohypophyseal hormones.

Cyclic AMP and Sodium Transport: QUANTITATIVE AND TEMPORAL RELATIONSHIPS IN TOAD URINARY BLADDER

The effects of oxytocin upon tissue cAMP content and short-circuit current (SCC) were measured in the urinary bladder of the toad, Bufo marinus. Tissue cAMP levels doubled before any increment in SCC was observed, the two hormone responses were quantitatively related, and a threshold level for an effect of cAMP upon sodium transport was demonstrated. The period of time over which cAMP levels continued to rise after the threshold level had been attained seemed invariant with hormone concentration. The rate at which cAMP levels rose increased with hormone concentration yielding hormone concentration-dependent maximal levels. The decay of cAMP levels was delayed when sodium influx was curtailed, suggesting a sodium-regulatory effect upon tissue cAMP levels.

Comparison of toad bladder aldosterone-induced proteins and proteins synthesizedin vitro using aldosterone-induced messenger RNA as template

SummaryUsing double-labeled isotope techniques, it can be shown that aldosterone induces the synthesis of several proteins in the mitochondria-rich (MR) cells of the toads urinary bladder. Induced proteins have been identified both in the plasma membrane (mol wt=170,000, 85,000 and 12,000) and the cytosol (mol wt=36,000, 12,000 and 6,000) fractions of these mucosal cells. We have also shown that aldosterone (Aldo) induces the synthesis of a class of RNA having the properties of messenger RNA (mRNA). mRNA isolated from Aldo-treated mucosal cells was used as template in a cell-free protein-synthesis system prepared from rabbit reticulocytes. Preparations charged with mRNA from Aldotreated cells synthesized two proteins that were not labeled when mRNA from control tissues was used as template. The electrophoretic mobility of one of these proteins was similar to an Aldo-induced membrane protein (mol wt=70,000) found in the intact tissue.

Hexose monophosphate shunt activity in compensatory renal hypertrophy.

Summary Hexose monophosphate shunt (HMPS) dehydrogenase activity was measured in the remaining kidney at frequent intervals for 4 days after unilateral nephrectomy of rats. Total HMPS dehydrogenase activity in the residual kidney began to increase 18 hr after nephrectomy and was doubled (1.97) at 72 hr, where it remained at 96 hr (1.97). While the total soluble protein (20,000 g supernate) showed a continuous increase beginning about 18 hr after nephrectomy, HMPS dehydrogenase specific activity exhibited a biphasic pattern with peaks at 36 hr (25% increment) and 72 hr (40% increment). The data indicate that preferential synthesis of the HMPS dehydrogenase enzymes (G6PD and 6PGD) is an early response of the remaining kidney to unilateral nephrectomy.

A proposed role for ascorbate in the transport of amino acids and ions in the cornea.

Abstract Evidence is presented that the cornea actively transports neutral amino acids from the endothelial surface by several carrier mechanisms. One of these mechanisms, responsible for leucine uptake, is markedly stimulated by lactate and to a lesser extent by ascorbate. We are postulating that leucine transport is coupled to an electron transport system that utilizes lactate and ascorbate as substrate or electron donors. We also found a similar acceleration in the SCC and P.D. when ascorbate was added to the medium bathing the cornea and suggest that an electron transport system also supplies energy for the transport of ions by the cornea.

ALDOSTERONE-INDUCED SYNTHESIS OF PROTEINS RELATED TO SODIUM TRANSPORT IN THE TOAD'S URINARY BLADDER*

A number of laboratories have investigated the mechanisms by which mineralocorticoid hormones modulate the active transport of ions by epithelial tissues. CrabbB, observing a significant latent period preceding the onset of the physiologic response to the hormone, hypothesized in 1961 that the synthesis of a mediating substance is a necessary condition for a tissue’s response to aldosterone (Aldo) .l Subsequent observations that inhibitors of protein p, and RNA synthesis block the Aldo-induced increase in sodium transport in epithelial tissues supported the hypothesis that Aldo exerts its effects by initiating the synthesis of messenger RNA (mRNA) coding for specific protein(s) in target cells. Using the toad’s urinary bladder, Rossier et aZ.5 demonstrated that Aldo induces the synthesis of RNA, and Wilce et aleG showed that the incorporation of uridine into poly (A) -containing RNA was increased by the hormone. While there was considerable indirect evidence for a role of protein synthesis in the expression of the hormone’s effect, over a decade elapsed before the direct demonstration of these proteins was achieved. Benjamin and Singer reported Aldo-induced incorporation of amino axid into a low molecular weight protein in the toad bladder.? Scott and Sapirstein showed that Aldo-induced proteins could be demonstrated in the mitochondria-rich cells of the toad bladder, but not in the granular cells, consistent with the localization of the mineralocorticoid binding sites.9 Subsequently, Aldo-induced proteins were also identified in a plasma membrane fraction prepared from mitochondriarich cells following incubation of cells with the hormone.l0 Because there is also considerable evidence that the stimulation by Aldo of sodium transport is associated with significant alterations in lipid metabolism,ll Scott et aZ.12 studied tht effects of a potent inhibitor of fatty acid synthesis upon the the effects of Aldo on the labeling of membrane proteins. They showed that the incorporation of Aldo-induced proteins into the plasma membrane of toad bladder rnucosal cells evidently requires the coordinate synthesis and/or elongation of fatty acids, indicating a key role of fatty acids in the integrated response of the tissue to the hormone. found that there are two proteins whose in vitro synthesis was greatly enhanced by mRNA from AldoUsing a reticulocytc lysate system, Scott et

Irreversible inhibition of sodium transport by the toad urinary bladder following photolysis of amiloride analogs

Active sodium transport was completely and irreversibly inhibited in toad urinary bladders photolyzed in the presence of iodeam loride.

Toad carbonic anhydrase: Purification of the enzyme from erythrocytes of Bufo marinus and comparison with the enzyme activity in the urinary bladder

1. Two forms of carbonic anhydrase, having isoelectric points of 6.1 and 5.8, were purified from erythrocytes of the toad, Bufo marinus, and the presence of a third form, pI = 5.4, was demonstrated. 2. Each of the two purified isozymes catalyzed the hydration of CO2 and the hydrolysis of nitrophenyl acetate esters at rates characteristic of Type C (or high-activity) forms of carbonic anhydrase. 3. Both forms of the erythrocyte enzyme have similar molecular weights (approx 29,000), amino acid composition, sensitivity to acetazolamide, and kinetic properties. 4. The epithelium of the toads urinary bladder also was found to contain significant amounts of carbonic anhydrase, which appears by isoelectric focusing to be indistinguishable from the enzyme isolated from the erythrocyte.

Isolation of Highly Enriched Preparations of Two Types of Mucosal Cells of the Turtles Urinary Bladder

Summary Enriched preparations of the two major cell types of the mucosal epithelium of the turtles urinary bladder can be obtained by density gradient centrifugation. Carbonic anhydrase activity is much greater in the mitochondria-rich than in the granular cell, suggesting the former cell type is the site of acetazolamide-sensitive transport. This technique should be useful in further defining the biochemical and transport properties of epithelial tissues composed of heterogeneous mucosal cells.

Atrial natriuretic factor enhances the hydroosmotic response of toad bladder to submaximal doses of vasopressin

Using the toad urinary bladder, we examined the effects of submaximal levels of atrial natriuretic factor (ANF) upon the hydroosmotic water flux caused by physiologic concentrations of vasopressin (VP). Pretreatment with ANF prior to the addition of VP (10(-9)M) significantly enhanced water transport (123 +/- 23%) compared to tissues exposed to VP alone. Pre-treatment with ANF also significantly enhanced the hydroosmotic response (143 +/- 43%) to cyclic-3,5-adenosine monophosphate (cyclic-AMP). When the concentration of VP was progressively increased during time course experiments, an inhibitory effect of ANF on water transport followed the early stimulatory response to this peptide. The data support a novel, dose-dependent modulatory role for ANF early in the response of transporting epithelia to VP. Moreover, the stimulatory effect of submaximal doses of ANF to cyclic-AMP mediated water transport suggest the possibility that modulation by ANF may occur at a site following the VP receptor-linked adenylate cyclase system.