Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84)

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

Oxidant inhibition of epithelial active sodium transport.

The purpose of this study was to quantify the effects of extracellularly generated partially reduced oxygen species on active sodium (Na+) transport across the ventral toad skin, a well-studied epithelium. Sections of skin from decapitated toads were mounted in an Ussing chamber, bathed on both sides with electrolyte solution containing 500 microM xanthine and bubbled continuously with room air. The tissues were short-circuited, and short-circuit current (Isc) and tissue resistance (Rt) were monitored continuously with an automatic voltage clamp apparatus. Fifteen mU/ml of xanthine oxidase (XO), either purchased from Calbiochem or purified from cream, were instilled in either the apical (mucosal) or basolateral (serosal) baths at t = 0 and t = 10 min. Hydrogen peroxide (H2O2) concentrations increased to 200 microM within the first 20 min and then decreased, reaching a value of 40 microM by 60 min. Mean [H2O2] was 90 microM. Instillation of XO in the apical bath resulted in a large decrease in Isc and an increase in Rt, their values being 43% and 160% of their corresponding controls 85 min after the first instillation. Addition of superoxide dismutase and catalase completely prevented these changes. Instillation of XO in the basolateral bath had no effect. Similar physiological responses were obtained using the Calbiochem XO or the purified XO, which contained no measurable protease activity. It was concluded that extracellularly generated partially reduced oxygen species may interfere with active Na+ transport by possibly damaging apical Na+ channel proteins.

Enantio- and diastereoselective reductive aldol reactions with iridium-pybox catalysts.

[see reaction]. A catalytic amount of [(cod)IrCl]2 and indane-pybox converts diethylmethylsilane, methyl acrylate, and certain aldehydes to the derived reductive aldol adduct with good enantio- and diastereocontrol.

Interaction of heptanol and pressure on sodium and chloride transport by toad skin.

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.

Etiologic factors associated with anterior knee pain in distance runners.

PURPOSE: The objectives of this study were 1) to examine the differences between a noninjured (C) cohort of runners (N = 70) and runners afflicted with anterior knee pain (AKP) according to selected training, anthropometric, rearfoot motion, ground reaction force, and muscular strength and endurance variables; 2) to explore multivariate relationships among these measures in the well and injured groups; and 3) to develop specific hypotheses concerning risk factors for injury that will later be tested in a prospective clinical study. METHODS: High speed videography (200 frames x s(-1)), a force platform (500 Hz), and a Cybex II+ isokinetic dynamometer were used to assess rearfoot motion, ground reaction forces, and knee muscular strength and endurance, respectively. A linear discriminant function was performed on each of the five categories of variables and revealed 19 significant (P < or = 0.05) predictors. These variables were then combined and a final discriminant function analysis was performed. RESULTS: Pronation through the first 10% of stance, arch index, shoe mileage, and extension peak torque were the best overall (P < or = 0.05) predictors. The AKP group had smaller mean values on all four significant predictors. CONCLUSION: With the exception of shoe mileage, which is likely a response to rather than a risk factor for AKP, these results should prove useful to clinicians in identifying runners at risk for anterior knee pain.

Effect of partial ureteral obstruction on results of renal scintigraphy in dogs.

OBJECTIVE: To use scintigraphy to determine the effects of partial ureteral obstruction on renal transit time and induction of diuresis in dogs. ANIMALS: 8 adult dogs. PROCEDURE: Scintigraphy was performed, using technetium Tc 99m diethylenetriaminepentacetic acid (Tc 99m-DTPA), before and within 2 weeks after surgical induction of unilateral partial ureteral obstruction. Time of peak (TOP) for the parenchyma (pTOP) and whole kidney (wTOP) and mean-transit time (MTT) for the parenchyma (pMTT) and whole kidney (wMTT) were determined by evaluation of renal time-activity curves before and after deconvolution analysis. Percentage uptake for each kidney between 1 and 3 minutes after injection of Tc 99m-DTPA was determined and used to indicate glomerular filtration rate. The effect of diuresis was determined by measuring the slope of decrease in activity after i.v. administration of furosemide. Obstruction was documented by direct inspection of the ureter. RESULTS: There was a concomitant increase in pTOP, wTOP, pMTT, and wMTT of the kidney with the partially obstructed ureter in all dogs at various times between 2 and 9 days after surgery. Concurrently, renal time-activity curves changed shape. Percentage renal uptake of the affected kidney was decreased in 2 dogs. Response to furosemide injection was inconsistent for kidneys before surgery and for kidneys with obstructed and nonobstructed ureters after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Scintigraphy may be a useful procedure for the evaluation of renal function in dogs with ureteral obstruction. Induction of diuresis appears to be of little value for differentiating renal function in dogs with obstructed and nonobstructed ureters.

Incomplete urethral duplication with cyst formation in a dog.

Incomplete urethral duplication with cyst formation was diagnosed in a dog that had soft, fluctuant, subcutaneous swellings in the ventral perineal and penile areas and a history of nocturia and incontinence during recumbency that were unresponsive to treatment with antibiotics. Retrograde urethrocystography, voiding urethrography, double-contrast cystography, radiography after direct administration of contrast medium into cystic structures, and excretory urography were performed to evaluate the urinary tract. Communication between the cysts and the urethra was demonstrated radiographically only after intralesional injection of contrast medium. Nocturia and incontinence resolved after surgical removal of the urethral duplication and cysts. The dog was clinically normal 1 year after surgery.

An evidence-based project for evaluating strategies to improve knowledge acquisition and critical-thinking performance in nursing students.

This longitudinal, quasi-experimental study with 142 junior nursing students focused on measurement of learning outcomes in two areas: acquisition of knowledge and development of critical thinking skills. The variation in clinical teaching strategy (structured versus unstructured health pattern assessment) was the independent variable. Results indicated significant gains in both knowledge and critical thinking performance from the beginning to the end of the semester. The significant gains in critical thinking performance provides support to the assertion that domain-specific measures of critical thinking are needed in nursing education. Additionally, our results suggested that it was the interaction between learning strategy and the characteristics of the learner that was more significant in determining knowledge improvement than the particular strategy. As a result of this study, it is recommended that faculty develop and use an evidence-based model to support their decision making regarding teaching methodologies. This seems especially relevant for large, introductory clinical courses that use team teaching to achieve educational goals related to improvement in critical thinking or knowledge.

The evolution of a profession: where do boards of nursing fit in?

Differentiation between the structure and functions of Boards of Nursing and the professional association is important to understanding the evolution of nursing as a profession. This descriptive exploratory study revealed that Boards of Nursing usually have 5 members who are appointed by and can be removed by the governor of the state. All Boards are responsible for education and licensing at the professional entry-level but may not be responsible beyond that level. In contrast to the professional association, Boards report appreciable variability in structure and administration. Boards differ from the professional association in source of power and focus on the protection of the public rather than improvement of practice.

Intracellular pH and bicarbonate activities in rabbit colon.

The short-circuited epithelium of rabbit colon is thought to actively absorb chloride ion by a mechanism in the mucosal cell membrane that exchanges chloride for bicarbonate ion. If this model is correct bicarbonate may be accumulated above electrochemical equilibrium across the mucosal cell membrane. To test this model intracellular pH was measured using a new hydrogen ion-selective liquid membrane microelectrode that is fast, highly selective, and easy to fabricate with a very small tip diameter. These measurements show that the average intracellular pH in this epithelium is 6.9 +/- 0.1. The mucosal cell membrane electrical potential difference, measured by conventional open-tipped microelectrodes, averaged -52 +/- 3 mV. Intracellular pH is above a value predicted for an equilibrium distribution of hydrogen ion across both cell membranes, implying that a mechanism exists for "uphill" extrusion of this ion from the cell. Intracellular bicarbonate activity calculated from these measurements averaged 8 +/- 1 mM. The electrochemical potential gradient for bicarbonate across the mucosal membrane averaged -28 +/- 2 mV, demonstrating that intracellular bicarbonate is concentrated above an equilibrium distribution across the mucosal membrane. Thus energy in the "downhill" electrochemical potential gradient for bicarbonate exit from the cell may drive the entrance of chloride into this epithelium and energize transepithelial chloride absorption.

Discriminant analysis to predict graduation--nongraduation in a master's degree program in nursing.

Discriminant analysis was used to predict graduation and two categories of nongraduation from readily available admissions data at the University of Kansas nursing master's degree program. The traditional admissions indices, baccalaureate grade point average (GPA) and Graduate Record Examination (GRE)-verbal and -quantitative scores, were used as predictors. Criterion categories were composed of 102 graduates, 103 individuals who dropped out of the program, and 65 individuals who were not accepted. The first discriminant function was, chi 2 (6) = 87.567, p less than .0001, and extracted 98% of the variance of the discriminant space. Follow-up procedures using one-way ANOVA's and Scheffé multiple comparisons indicated that the baccalaureate GPA and GRE-verbal and -quantitative scores independently differentiated the graduate and dropout groups from the not-accepted group at a statistically significant level (p less than .05). Practical significance of the independent contribution of these variables to group differentiation, as measured by omega 2 was 22% for the baccalaureate GPA, 13% for the GRE-verbal scores, and 10% for the GRE-quantitative scores. Implications for future research are discussed.

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study.

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

Chloride activities in epithelia.

In Cl-absorbing epithelia, intracellular Cl activities have only been determined in tissues possessing a NaCl cotransport process at the apical (lumen-facing) membrane, which appears to be responsible for active Cl absorption. In these tissues, cell Cl activities average 2-4 times the values predicted for a passive distribution of Cl across the apical membrane, but this cellular Cl accumulation is abolished if Na in the luminal solution is replaced by non-transported cations. Thus, the energy for cell Cl accumulation and transepithelial transport appears to be derived from the interaction between Na and Cl entry into the cell and the electrochemical potential difference for Na across the apical membrane. In epithelia that actively secrete Cl, cell Cl activities are also significantly higher than the predicted equilibrium values, so that the uphill step in transepithelial Cl transport lies at the basolateral (plasma-facing) membrane. Available evidence suggests that NaCl cotransport may also be responsible for cell Cl accumulation by Cl-secreting tissues. In both absorbing and secreting epithelia, the exit of Cl from the cell is directed down a favorable electrochemical potential difference for the anion, but the mechanisms responsible for Cl exit require further study.

Intracellular pH and membrane potassium conductance in rabbit distal colon.

Intracellular pH (pHc) was measured in the short-circuited epithelium of rabbit distal colon using H(+)-selective microelectrodes. pHc was 6.91 +/- 0.02 (SE) when the bath pH was 7.4. Intracellular HCO3- activity (acHCO3-) was estimated from these measurements to be 8 +/- 0 mM. When we replaced all Cl- in the tissue bathing solutions with the impermeant anion gluconate, pHc rose to 7.44 +/- 0.08 and acHCO3- increased to 30 +/- 6 mM. These results demonstrate that this tissue contains a Cl(-)-HCO3- exchange mechanism. During the Cl- replacement the apical membrane electrical potential difference hyperpolarized from -55 +/- 1 to -74 +/- 3 mV, suggesting that membrane ionic conductance had changed. Elevation of either the apical or basolateral membrane bathing solution K+ concentration produced a greater depolarization of membrane potential during Cl- replacement than when tissues were bathed in normal electrolyte solutions. In additional experiments, pHc was raised by lowering the bath CO2 concentration while the bath Cl- concentration was kept normal. Under these conditions, membrane potential hyperpolarized and was more sensitive to the elevation of bath K+ concentration than when pHc was normal. These results suggest that membrane K+ conductance in this tissue is increased by intracellular alkalinization.

G proteins activate ionic conductances at multiple sites in T84 cells.

We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K+ equilibrium potential (E(K)) or Cl- equilibrium potential (E(Cl)) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K+ current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K+ current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), to pipettes stimulated large oscillating K+ and inward currents. This occurred even when Ca2+ was absent from the bath but not when the Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K+ was replaced with Na+ and cells were voltage clamped between E(Na) and E(Cl), GTPgammaS activated oscillating Na+ and Cl- currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to pipettes activated large oscillating K+ currents but only small inward currents. These results suggest that a carbachol-induced release of Ca2+ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P3 signaling pathway. In addition, this or another G protein activates Cl- current by directly gating Cl- channels to increase their sensitivity to Ca2+.