PubMed Health⌕ Search

Biomedical subjects

R F Husted

Publications and source records attributed to R F Husted.

35 records · Page 2Linked to original sources
PubMed1988

Characteristics of papillary collecting duct cells in primary culture.

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

Amiloride↗
PubMed1987

Selectivity of basolateral anion exchange in the acidification pathway of turtle bladder.

The turtle urinary bladder in vitro acidifies the solution bathing its luminal surface. Protons are actively extruded across the apical membrane by an H+-ATPase. Bicarbonate ion exits the cell across the basolateral membrane via a stilbene-sensitive, anion exchange for chloride. Chloride then exits the cell via a conductive pathway. The present studies were undertaken to define the specificity of the basolateral anion exchange mechanism for chloride. Turtle bladders were mounted on chambers in vitro, short-circuited, and treated with ouabain. The current remaining after inhibition of sodium transport was used to measure the acidification rate. Ion replacement studies with bromide, isethionate, sulfate, and nitrate indicated that only bromide supported acidification at rates comparable to chloride. In separate experiments, kinetic analysis of anion interaction with the exchanger indicates that maximal acidification rates decrease in the order: Cl greater than Br greater than SO4 greater than methyl sulfate = gluconate. The affinity of the exchanger decreases in the order: Cl greater than SO4 greater than Br greater than HCO3 greater than methylsulfate greater than gluconate. These selectivity sequences indicate "strong" interaction of the anions with the selectivity site. The differences in position of the polyatomic anions in the two sequences indicates that the "binding" site is accessible but that transport is limited by steric factors.

Animals↗
PubMed1986

Variability of functional characteristics of MDCK cells.

We measured several functional parameters of MDCK cells cultured as monolayers in order to more fully characterize their ion transport properties. Most of the present studies were completed with five groups (A-E) of MDCK cells studied from passage 62 to 78. Each group represents the same subline of MDCK cells after having been frozen, stored, and thawed at passage 62 or 64. The median transmonolayer resistances of the groups were 507, 149, 284, 72, and 126 omega X cm2. Addition of amphotericin B to the apical solution induced a oubain-sensitive transepithelial current. The apical membrane voltage and fractional resistance exhibited a wide range of values in two of the groups studied, with mean values of -32 mV and 0.68 in group B and -40 mV and 0.78 in group E. Neither apical nor basolateral membrane displayed significant Na+ conductance. K+ conductance was present in the basolateral but not in the apical membrane. Acidification or alkalinization of the apical solution was dependent on the conditions used to study the cells. The 4,4'-diisothyocyano-2,2'-disulfonic stilbene inhibited acidification (or induced alkalinization), whereas increasing ambient HCO-3 concentration induced alkalinization. The results of these studies indicate qualitatively similar behavior between five groups of MDCK cells but significant quantitative differences between the groups. Analysis of the variability of the measured parameters indicated that there were no differences as a function of passage number within a group. The factors responsible for functional differences between groups are not known but may be related to the cell storage process.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Effects of luminal pH and luminal HCO3 on CI-HCO3 exchange across turtle urinary bladder.

The Cl-HCO3 exchange rate across turtle urinary bladder was studied by measuring the net flux of 36Cl in paired tissues. Serosal bicarbonate concentration and pH were held constant (10 mM and 7.6, respectively) while luminal composition was altered. The rate of net chloride absorption was the same at a luminal pH of 5 or 7 and at luminal HCO-3 concentrations of 0 and 10 mM. These results indicate that the affinity of the exchanger for luminal Cl- is much higher than that for luminal HCO-3 or OH-. Vanadate, an inhibitor of a number of ATPases, had no significant effect on chloride absorption.

Animals↗
PubMed1983

Chloride dependence of the HCO3 exit step in urinary acidification by the turtle bladder.

To characterize the efflux of HCO-3 across the basolateral membrane of the H+-secreting cells of the turtle bladder, we examined the effect of substitution of gluconate or methyl sulfate for Cl- on the rate of acidification (JH). JH was measured as the short-circuit current in bladders in which Na+ transport was abolished with 10(-4) M ouabain. In hemibladders bathed in normal Ringer solution (Cl- = 122 mM) JH was 44.9 microA. Substitution of the Cl- resulted in a marked reduction in JH (12.5 microA with gluconate and 7.5 microA with methyl sulfate). Addition of Cl- to the mucosal surface had no effect on JH. In contrast, serosal addition of Cl- restored JH to control. The apparent Km for Cl- in gluconate Ringer was 0.13 mM. Serosal furosemide (1 mM) inhibited JH by 55% in Cl- Ringer. We conclude that HCO-3 exit across the basolateral membrane of the H+-secreting cell occurs via a Cl-HCO3 exchanger that has a high affinity for chloride.

Animals↗
PubMed1982

Mechanisms of K+ transport in isolated turtle urinary bladder. Induction of active K+ secretion in a K+-absorbing epithelium.

Transepithelial K(+) movement was studied in vitro in the short-circuited turtle bladder by increasing luminal K(+) permeability and by inhibiting the basolateral Na/K pump. Luminal addition of amphotericin B caused net K(+) secretion (180+/-52 nmol/h) compared with net K(+) absorption (42+/-6 nmol/h) in control bladders. Serosal ouabain and luminal amiloride abolished K(+) secretion in amphotericin-treated bladders; ouabain restored net absorption (45+/-16 nmol/h). The direction and rate of net K(+) transport are controlled by the relative K(+) permeabilities and the Na/K pump sites at the two cell membranes of the epithelium.

Amiloride↗
PubMed1981

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

The coupling between H+ transport (JH) and anaerobic glycolysis was examined in 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 less than 0.5 mm Hg) and contained 10 mM glucose. Under these conditions, JH was not inhibited by 3 mM serosal (S) cyanide or by 0.1 mM mucosal (M) dinitrophenol. Control anaerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measured JH. The slope of Jlac on JH was 0.58 0.12 with an intercept for Jlac at JH = 0 of 0.55 micromol/hr. Values for delta Jlac/delta JH were determined in groups of individual bladders when JH 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 delto Jlac/ delta JH indicates a high degree of coupling between JH and Jlac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the delta Jlac/ delta JH values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited by M addition of oligomycin but was inhibited by M addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobic JH without change in delta Jlac/delta JH or basal Jlac and, therefore, acted primarily on the H+ pump. S addition of vanadate also inhibited JH, but the inhibition was associated with an increase in Jlac. 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.

Adenosine Triphosphatases↗
PubMed1981

Surface characteristics of carbonic-anhydrase-rich cells in turtle urinary bladder.

Addition of a disulfonic stilbene, 4-acetamido 4'-isothiocyano-2,2'-disulfonic stilbene (SITS), to the serosal side of the turtle bladder blocks the efflux of bicarbonate ions from the acidifying cells and thereby inhibits hydrogen ion secretion into the luminal solution. Because SITS has little effect on other transport systems, we used it to define the relationship between hydrogen ion secretion (JH) and the different cell types facing the luminal surface. Cells were identified by scanning and transmission electron microscopy (SEM and TEM) and by histochemical localization of carbonic anhydrase (CA). SEM revealed that SITS caused marked alterations in luminal surface characteristics of a cell population with prominent microplicae. Two hours after the serosal addition of SITS, cells with identifiable microplicae had decreased from 12.7 to 0.5% of total cells. TEM studies and CA histochemistry showed that the number of cells rich in CA (CA cells) remained the same, whereas the individual luminal surface areas of a subpopulation of CA cells had decreased markedly. A comparison of the distribution of individual surface areas of cells with microplicae and CA cells revealed that the CA cells with large surface areas corresponded to the cells with microplicae and that both were affected by the serosal addition of SITS. Acetazolamide, which also inhibits JH, caused similar changes. The luminal addition of SITS and an inactive analogue of acetazolamide, which have no effect on JH, did not alter surface morphology, These results indicate that the CA cell with microplicae represents the active state of the hydrogen ion secreting cell.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1981

Potassium absorptive pump at the luminal membrane of turtle urinary bladder.

In the absence of transepithelial electrochemical gradients, the direction of net K transport across the turtle urinary bladder is from the mucosal (M) to the serosal (S) solution. Under control conditions, M leads to S flux was 101 +/- 5 nmol . h-1 . (8 cm2)-1, S leads to M flux was 59 +/- 4, and the net absorptive flux was 42 +/- 6. The K absorptive pump was characterized by examining its dependence on voltage and ambient sodium and its sensitivity to mucosal ouabain. Lumen-negative voltages caused an increase rather than the expected decrease in active K absorption. Thus, the active K flux appeared to be coupled to a flow of positive charge in the opposite direction, possibly representing Na secretion in excess of K absorption. Net K absorption was abolished by removal of Na from the medium and by mucosal, but not serosal, addition of ouabain. The reverse electrogenicity, Na dependence, and ouabain sensitivity of K absorption indicate that the K pump of the mucosal membrane has characteristics of a Na-K-ATPase.

Absorption↗
PubMed1979

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: studies with a disulfonic stilbene.

Bicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO3- generated behind the H+ pump moves this membrane into the serosal solution. This efflux of HCO3- is inhibited by SITS (4-isothiocyano-4'-acetamido-2,2'-disulfonic stilbene). When HCO3- is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl-. This secretory HCO3- flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5 x 10(-4) M SITS on HCO3- secretion and Cl- absorption were examined. The rate of H+ secretion was brought to zero by an opposing pH gradient, and 20 mM HCO3- was added to S. HCO3- secretion, measured by pH stat titration, was equivalent to the increase in M leads to S Cl- flux after HCO3- addition. Neither the S leads to M flux of HCO3- nor the M leads to S flux of Cl- were affected by SITS. In the absence of electrochemical gradients, net Cl- absorption was observed only in the presence of HCO3- in the media; under such conditions, unidirectional and net fluxes of Cl- were not altered by serosal or mucosal SITS. H+ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO3- in series with the H+ pump and the failure of SITS to affect HCO3- secretion and Cl- absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.

Animals↗
PubMed1979

The effects of amiloride and ouabain on urinary acidification by turtle bladder.

To investigate the mechanism by which amiloride inhibits urinary acidification, its effects on H+ secretion were examined in the isolated urinary bladder of the fresh water turtle. In short-circuited turtle bladders amiloride inhibited H+ secretion by 30% and Na+ transport by 100%. Maximal inhibition was reached at 10(-4) M amiloride for both transport systems. In contrast to amiloride, ouabain did not affect H+ secretion despite complete inhibition of Na+ transport. In bladders first treated with ouabain amiloride failed to inhibit H+ secretion and in bladders first treated with amiloride, the inhibition of H+ secretion was partially reversed by ouabain. The inhibition of H+ secretion by amiloride is attributed to hyperpolarization of the luminal cell membrane and the imposition of a voltage opposing the movement of protons in the active transport pathway.

Acid-Base Equilibrium↗
PubMed1977

Regulation of cerebrospinal fluid bicarbonate by the cat choroid plexus.

1. The regulation of cerebrospinal fluid (c.s.f.) bicarbonate concentration was studied using the cat choroid plexus isolated in a chamber in situ. 2. Decreases in plasma bicarbonate concentration caused relatively small changes in the c.s.f. bicarbonate concentration. 3. Alterations in c.s.f. bicarbonate concentration (c.s.f. HCO3-=9 or 28 m-equiv/l.) were countered by changes in the bicarbonate concentration of the fluid produced by the plexus or in the rate of bicarbonate transport which returned c.s.f. bicarbonate towards normal. 4. There was significant regulation of pH in the choroid plexus fluid during hypocapnia and hypercapnia. 5. Alterations of plasma acid-base status did not significantly alter the potential difference across the choroid plexus. However, the potential difference increased when c.s.f. bicarbonate was increased and decreased when c.s.f. bicarbonate was decreased. 6. The data indicate that the bicarbonate concentration in the c.s.f. is actively regulated by the choroid plexus during acid-base disturbances occurring either systemically or in the c.s.f.

Animals↗
PubMed1976

Regulation of cerebrospinal fluid potassium by the cat choroid plexus.

1. The regulation of cerebrospinal fluid (c.s.f.) potassium concentration was studied using the cat choroid plexus isolated in a chamber in situ. 2. Hyperkalaemia (plasma potassium concentration greater than 6 m-equiv/l.) caused relatively small increases in c.s.f. potassium concentration. 3. Alterations in c.s.f. potassium concentration (c.s.f. K = 0-15 or 6-56 m-equiv/l.) were countered by changes in potassium concentration of the produced fluid or in the rate of potassium transport which returned c.s.f. potassium towards normal. 4. The data indicate that potassium concentration in c.s.f. secreted by the choroid plexus is actively regulated by the plexus whether the primary alteration in potassium occurs in plasma or c.s.f.

Animals↗