Allan Mueller

Characteristics of stimulation of H+ transport by aldosterone in turtle urinary bladder.

Aldosterone stimulates not only Na+ absorption but also urinary acidification. In this investigation the effects of aldosterone on H+ transport are examined in vitro in turtle bladder, a urinary membrane in which several of the factors controlling H+ transport have been defined. H+ transport was increased in bladder halves exposed to aldosterone compared to control halves. Stimulation of H+ secretion was observed as early as 1 h after addition of aldosterone and occurred before that of Na+ transport. In bladders depleted of endogenous substrate addition of glucose increased H+ transport more in aldosterone-treated halves (10.0+/-1.3 nmol/min) than in control halves (6.8+/-2.3). Addition of pyruvate failed to increase H+ transport (--0.3+/-0.7) in control halves but caused significant increments (2.4+/-0.5) in aldosterone-treated halves. In aldosterone-treated bladders glucose caused larger increments (16.5+/-2.7) in H+ transport than pyruvate (9.3+/-2.0) when halves of the same bladders were compared. Na+ transport, however, was equally increased by the two substrates. Despite the differences in time course and substrate requirements between the stimulation of H+ and Na+ transport, both increases were abolished by actinomycin-D. To examine the effect of aldosterone on the force of the H+ pump, protonmotive force, the pH gradient that would nullify the transport rate was determined with and without aldosterone. Aldosterone did not alter protonmotive force but significantly increased the slope of the H+ transport rate on the applied pH gradient. It is concluded that aldosterone stimulates H+ transport independently of Na+ transport. It increases the responsiveness of the transport rate to glucose and to a lesser extent pyruvate, an effect probably secondary to the increased transport rate. Equivalent circuit analysis indicates that aldosterone facilitates the flow of protons through the active transport pathway but does not increase the force of the pump.

Inhibition of the Bicarbonate Exit Step in Urinary Acidification by a Disulfonic Stilbene

Acidification of the luminal solution by the isolated turtle bladder involves H(+) secretion by a pump at the luminal membrane. The OH(-) dissociated in this process reacts with CO(2) and forms HCO(3) (-) which moves passively out of the cell across the serosal cell membrane. In the present study, this exit step for HCO(3) (-) was inhibited by serosal addition of the disulfonic stilbene, SITS, an agent which is thought to bind to a transport protein at the serosal cell membrane. 90 min after serosal addition of 0.5 mM SITS, H(+) secretion decreased by > 80%. In contrast, luminal addition of SITS had no effect. During inhibition of H(+) secretion by serosal SITS, overall cell pH, measured by the 5, 5-dimethyl-2, 3-oxazolidinedione method, increased from 7.48+/-0.03 to 7.61+/-0.02. This increase of 0.13+/-0.02 pH U was associated with a much larger regional pH increase as judged from the decrement in the attainable pH gradient across the epithelium. After serosal SITS, this gradient was reduced from 2.88+/-0.06 to 2.09+/-0.11 pH U. In the absence of evidence for increased H(+) permeability or a change in the force of the H(+) pump, the gradient decrement of 0.79+/-0.08 U reflects a similar pH increment on the cytoplasmic side of the pump.SITS inhibits the exit of bicarbonate across the serosal cell membrane and, thereby, creates a compartment of high alkalinity in series with the pump. The increased electrochemical gradient across the active transport pathway is the primary factor in the inhibition of urinary acidification.

Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: effects of inhibitors of H+ ATPase

SummaryThe coupling between H+ transport (JH) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.JH was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2<0.5 mm Hg) and contained 10mm glucose. Under these conditions,JH was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measuredJH. The slope ofJlac onJH was 0.58±0.12 with an intercept forJlac atJH=0 of 0.55 μmol/hr. Values for δJlac/δJH were determined in groups of individual bladders whenJH was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of δJlac/δJH indicates a high degree of coupling betweenJH andJlac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the δJlac/δJH values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2 H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobicJH without change in δJlac/δJH or basalJlac and, therefore, acted primarily on the H+ pump.S addition of vanadate also inhibitedJH, but the inhibition was associated with an increase inJlac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H+-ATPase characteristics that differ from mitochondrial ATPase in that H+ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H+ and lactate, the H+/ATP stoichiometry of the pump is about 2.

γδ T cell response induced by vaginal Herpes simplex 2 infection

The purpose of the present studies was to determine whether acute vaginal infection with Herpes virus 2 altered the vaginal population of gammadelta T cells, and whether gammadelta T cells influenced the vaginal clearance of HSV-2. BALB/c mice were infected intravaginally with the progressively lethal wild type 333 strain, or the non-lethal thymidine kinase deficient (deltaTK- -HSV-2) mutant strain of HSV-2 virus. Changes in vaginal T cell composition were examined by FACS analysis 4 days after infection. Clearance of vaginal deltaTK- -HSV-2 infection was compared between mice with normal gammadelta T cell populations (BALB/c) and transgenic mice in which all the gammadelta T cells express a receptor that is specific for the b allotype of MHC class Ib T10 antigen (G8/BALB/c). In HSV-2 infected BALB/c mice, but not G8/BALB/c, a subset of gammadelta T cells that express a Vgamma2 TCR accumulated in the vaginal mucosa by the fourth day after infection. Unexpectedly, we found that gammadelta TCR transgenic mice exhibited a more rapid clearance of the virus than control mice (P < 0.05). These findings argue against the hypothesis that the normal populations of vaginal intraepithelial gammadelta T cells play a direct role in the elimination of virally infected epithelial cells.

FUNCTIONAL ORGANIZATION OF PROTON AND BICARBONATE TRANSPORT IN TURTLE URINARY BLADDER

The bladder of the fresh water turtle, Pseudemys scripta, is a relatively “tight” epithelium that resembles amphibian urinary bladders and frogskin in its capacity to absorb sodium by active transport. The major advantage of the turtle bladder preparation is it contains a potent transport system for urinary acidification that lends itself to close examination under simplified and controlled conditions in vitro. Substantial evidence is now available to suggest that the energy-dependent step in acidification is located at the luminal cell membrane and involves the translocation of protons from the cell into the urinary compartment.2-5 The nature of the acidification pump remains to be defined precisely, but recent evidence B. 7 points toward a proton-translocating ATPase that shares some of the characteristics of H+-ATPases in mitochondria, chloroplasts and bacterial membranes. Since net transport across epithelia requires translocation of ions across two cell membranes in series, the organization of the acidification pathway is more complex than in single membrane systems. As shown in FIGURE 1, the extrusion of H+ ions across the luminal membrane results in an accumulation of OHions within the cell. In the presence of CO? and carbonic anhydrase, HC0,will be generated that moves “downhill” across the basolateral cell membrane into the serosal This acidification pathway was first defined in a system free of exogenous CO, and HC0,in which the production and distribution of metabolic CO, and HC0,were measured by means of a conductometric t echn iq~e .~ . Studies by Steinmetz, Omachi and Frazier and Schwartz and Steinmetz s demonstrated that in this system H+ transport is electrogenic and not coupled directly to the transport of sodium. Accordingly in FIGURE I , the two transport systems are shown separately. When net sodium absorption is abolished by addition of ouabain or removal of sodium the electrical potential difference across the tissue reverses so that the lumen becomes positive with respect to the serosa and the reversed short-circuit current becomes equal to the rate of H+ secretion as measured by pH stat titration.8*, lo Aside from the sodium and H+ pumps, FIGURE 1 depicts an exchange trans-

Immunoglobulin (Fc) receptors on murine T- and B-lymphocytes: Investigations using tumor models☆

Lymphoid tumors are productive experimental models for the study of lymphocyte immunoglobulin receptors. Investigations with Fc receptor expressing lymphoid tumor cells have generated much useful information about: (a) the developmental expression of the different classes of Fc receptors on lymphoid cells of the T- and B-lineages; (b) the biochemical steps involved in the regulation of Fc receptor expression on lymphoid cells; (c) the structures of lymphoid cell Fc receptors and their genes; (d) the signals that induce alterations in the expression of Fc receptors on lymphoid cells; and (e) the molecular specificity of the binding of immunoglobulin to lymphoid cells Fc receptors. In addition, tumors that secrete immunoglobulins are providing useful models for analysis of the mechanisms by which B-cells influence Fc receptor expression and function on T-cells. An interesting, bi-directional immunoregulatory circuit involving Fc epsilon R+ host T-cells and IgE-secreting hybridoma cells has been identified that could prove useful in the analysis of the regulation of epsilon heavy chain expression. The studies discussed in this article and elsewhere in this volume serve to emphasize that, in addition to being clonal sources of key molecules such as Fc receptors and their messenger RNAs, lymphoid tumor cells that express Fc receptors are powerful and unique experimental models for investigating the developmental biology, regulation and function of lymphocyte Fc receptors.