Biomedical subjects

L N Schmidt

Publications and source records attributed to L N Schmidt.

7 recordsLinked to original sources

PubMed1993

Rabbit esophageal cells show regulatory volume decrease: ionic basis and effect of pH.

BACKGROUND: Regulatory volume decrease (RVD) after osmotic cellular swelling has been shown in several gastrointestinal epithelia but not in esophageal cells. In acid reflux disease, esophageal injury may be related in part to loss of RVD. METHODS: Isolated basal esophageal cells were exposed to an external hyposmolar solution, and changes in relative cell size were assessed using a Coulter counter (Hilaleah, FL) in the presence of K+ and Cl- transport inhibitors and at varying extracellular pH (pHo). RESULTS: At pHo 7.4, a 30% hyposmotic dilution of the external solution caused an initial peak cell swelling (1.15 +/- 0.05-fold) followed by a return to starting cell size by 5 minutes (RVD). RVD was inhibited by Ba2+ (4 mmol/L), quinine (1 mmol/L), or increasing the [K+]o > or = 10 mmol/L. RVD was also inhibited by depleting [Cl-]i or in the presence of 0.5 mmol/L 4,4'-diisothiocyanastostilbene-2,2'-disulfonic acid disodium salt (DIDS) or 50 mumol/L diphenylamine-2-carboxylate, a Cl- conductance inhibitor. To test the effect of pH on RVD, cells in solutions at pHo 7.4, 7.0, or 6.8 were subjected to hyposmotic stress; RVD was significantly inhibited at pHo 6.8. This pH-dependent inhibition of RVD was reversed in the presence of valinomycin, a K+ ionophore. CONCLUSIONS: These studies show that isolated esophageal cells possess RVD mechanisms that are mediated by Cl(-)- and pH-dependent K+ effluxes. RVD appears to be inhibited by a decrease in pHo, suggesting the possibility that acid-induced esophageal injury results from inhibition of normal volume regulatory mechanisms.

Animals↗

PubMed1993

Rabbit esophageal cells possess K+ channels: effect of hyposmotic stress on channel activity.

BACKGROUND: In many cell types, basolateral K+ channels are important in maintaining transepithelial Na+ absorption and regulatory volume decrease (RVD) after hyposmolar stress. However, in the esophagus the effect of K+ transport in maintaining baseline short-circuit current (SCC) (Na+ absorption) and RVD is unknown. METHODS: Ussing chambers were used to evaluate changes in SCC of rabbit esophageal mucosa in response to serosal Ba2+ (4 mmol/L), quinine (1 mmol/L), and increasing serosal [K+]. To determine whether K+ channel(s) are activated in RVD, changes in SCC in response to serosal hyposmolarity (156 mOsm) were assessed in the presence or absence of serosal quinine. RESULTS: Serosal Ba2+, quinine, or increased serosal [K+] caused a decline in baseline SCC. Serosal hyposmolarity caused an increase in SCC that was not blocked by mucosal application of amiloride (10(-4) mmol/L). In contrast, serosal quinine completely blocked the hyposmolar-induced increase in SCC. CONCLUSIONS: These studies suggest that rabbit esophageal cells possess Ba(2+)- and quinine-sensitive basolateral K+ channel(s) that are active under baseline conditions. Potassium conductance(s) also appear to be activated by external serosal hyposmolarity and may be involved in the process of RVD.

Amiloride↗

PubMed1991

Isolation, characterization, and attachment of rabbit distal colon epithelial cells.

The authors investigated various enzymatic digestion procedures for isolating epithelial cells from the distal colon of New Zealand White male rabbits. Rabbit mucosa was washed, diced, and digested for 90 minutes in one of five different solutions, including a new combination consisting of 0.03% collagenase IV and 0.1% pronase (solution V). Solution I (0.3% dispase) yielded 14.2 +/- 8.2 x 10(6) colonocytes/g mucosa, solution II (0.15% dispase and 0.03% collagenase) yielded 7.7 +/- 2.8 x 10(6) colonocytes/g mucosa, and solution III (0.03% collagenase IV) yielded 15.4 +/- 10(6) cells/g mucosa. Solutions I-III have previously been described for the isolation of colonocytes. Solution IV (0.1% pronase and 325 U/mL DNAase) was originally described for the isolation of nasal epithelial cells but yielded only 2.5 +/- 1.2 x 10(6) cells/g mucosa when applied to the isolation of colonocytes. The new combination of pronase and collagenase, solution V, yielded significantly more colonocytes, 34.5 +/- 3.0 x 10(6) cells/g mucosa, than previously described methods (P less than 0.01). Inclusion of 5 mmol/L ethylenediaminetetraacetic acid in any of the solutions enhanced neither viability nor yield. The digestion product of solution V could be enriched for crypts by serial low-speed centrifugations. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining for cytokeratins. Functional viability was tested by determining the presence of a Na+/H+ exchanger, using the pH fluorescent dye bis(carboxymethyl)-5(6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH. The authors document that sodium-dependent restoration of intracellular pH in colonocytes acid-loaded to a pH of 6.30 occurred at a rate of 0.19 +/- 0.02 pH U/min. Amiloride at concentrations of 1 mmol/L completely inhibited operation of the exchanger, as did sodium substitution with choline or tetramethylammonium. Lineweaver-Burke analysis at this intracellular pH showed a Michaelis constant of 10.71 mmol/L Na+ and a maximum velocity of 0.12 pH U/min. Exposing the colonocytes to 100 nmol/L phorbol 12,13-dibutyrate increased antiporter activity by 62.0%. Finally, the authors describe the synthesis of a new biomatrix composed of the basement membrane of 3T3 NIH fibroblasts that permits significantly improved colonocyte attachment than to glass, plastic, collagen types I or IV, or matrigel.

Animals↗

PubMed1990

Rabbit esophageal cells possess an Na+,H+ antiport.

The development of esophagitis is the result of hydrogen ion diffusion into the mucosa leading to cellular acidification and necrosis. In these studies, whether esophageal cells possess transport system(s) that can respond to cytoplasmic acidification was assessed; specifically, whether esophageal cells possess an Na+,H+ antiport was determined. Nucleated esophageal cells were isolated from rabbit esophagi using a trypsin-digestion technique that yielded 5-8 x 10(6) cells per esophagus, of which 74% +/- 3% were basal and 26% +/- 8% were squamous. Trypan blue was excluded by 95% +/- 2% of the cells. Cytoplasmic pH (pHi) was measured using the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester. Cells were acidified to the desired pHi by suspension in solutions with varying external pH (pHo) in the presence of nigericin. When cells acidified to pHi 6.3 were suspended in a choline chloride solution (pHo 7.4), cytoplasmic pHi did not increase. In contrast, Nao+ caused a concentration-dependent increase in the rate of cytoplasmic alkalinization with saturation occurring above 50 mmol/L Nao+. The transporter behaved according to first-order Michaelis-Menten type kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to cytoplasmic hydrogen ion concentration. Amiloride (10(-4) mol/L) caused a reversible inhibition of Na(+)-dependent alkalinization. Amiloride-sensitive cytoplasmic alkalinization was not observed when either cholineo or Ko+ was substituted for Nao+, while Lio+ resulted in alkalinization that was 60% +/- 8% of that seen with equimolar concentrations of Nao+. The basal pHi of cells suspended in a bicarbonate-free 130 mmol/L NaCl solution (pHo 7.4) averaged 7.42 +/- 0.03 (n = 10); amiloride (10(-4) mmol/L caused the basal pHi to decrease to 7.26 +/- 0.05 (n = 10; P less than 0.0025). When cells were suspended in a choline chloride (pHo 7.4) solution, pHi averaged 7.29 +/- 0.06 (n = 10) (P less than 0.0025 compared with Nao+). These studies indicate that nucleated esophageal cells obtained from rabbits possess an amiloride-sensitive Na+,H+ antiport that functions to regulate basal pHi and responds to intracellular acidification.

Amiloride↗

PubMed1986

Effects of taurine-conjugated bile salts on ionic transport of the rabbit esophageal mucosa.

Bile salts have been implicated as a cause of esophageal injury. We examined in vitro the changes in ionic transport of the rabbit esophagus resulting from taurine-conjugated bile salts at neutral pH to define and characterize their actions independent of hydrogen ion. In an Ussing chamber changes in potential difference (PD, mV), short-circuit current (SCC, microA X cm-2) and resistance (R, omega X cm2) resulting from taurocholate (TC), taurodeoxycholate (TDC) and taurochenodeoxycholate (TCDC) were studied. Transport properties were unaffected by TC at 5 and 10 mM. With TDC (5 mM) there was an initial rise in SCC and PD. After 60 min PD and R declined in association with an increase in paracellular permeability as measured by [14C]sucrose flux. TCDC (5.0 and 7.5 mM) caused a sustained rise in PD and SCC with a greater rise seen at 7.5 mM. The rise in SCC after TCDC was secondary to an increase in net sodium absorption as basal net sodium absorption increased threefold from 0.15 +/- 0.03 to 0.44 +/- 0.10 mu eq X cm-2 X h-1. Increased net sodium absorption accounted for 73% of the TCDC-induced rise in SCC. Amiloride (10(-4) M) added to the mucosal solution significantly inhibited this bile salt-induced rise in SCC. We conclude that bile salts alter ionic transport in the esophageal mucosa independent of hydrogen ion. These changes are characterized by an early selective increase in apical membrane cation conductance and with longer exposure, particularly in the presence of more hydrophobic bile salts, there is a marked increase in paracellular conductance.

Amiloride↗

PubMed1979

Adherence of Escherichia coli to human urinary tract epithelial cells.

The adherence of Escherichia coli to human uroepithelial cells obtained from midstream urine specimens of healthy women was studied. Bacteria labeled with [(3)H]uridine were used, and unattached organisms were separated from the epithelial cells by vacuum filtration with 5-mum-pore-size Nucleopore membrane filters. These techniques allowed adherence to be measured in large numbers of epithelial cells and overcame the problem of distinguishing experimental bacteria from the indigenous organisms present on uroepithelial cells. Adherence was not appreciably affected by temperature. Adherence was maximal at pH 4 to 5 and at bacterial-to-epithelial-cell ratios of 5,000 or more. The latter observation suggested that there are a limited number of receptors on the epithelial cell surface, an idea which was supported by competition experiments. Adherence occurred within 1 min and then decreased gradually or quickly, depending on the type of bacterial growth medium, to a stationary level of adherence, approximately 50% of that observed initially. The ability of epithelial cells from a single individual to bind E. coli varied in a cyclical and repetitive pattern. Adherence tended to be higher during the early phase of the menstrual cycle and diminished shortly after the time of expected ovulation; adherence frequently correlated with the value obtained on the same day of the menstrual cycle during the preceding months. Adherence was markedly enhanced by bacterial incubation in broth for 72 h and inhibited by alpha-d-mannose. These results suggest that adherence is a complex phenomenon perhaps mediated in part by bacterial pili and mannose residues on uroepithelial cells.

Culture Media↗

PubMedDate not supplied

Metered-dose inhaler technique and quality of life with airways disease: assessing the value of the Vitalograph in educational intervention.

The efficacy of delivering medicines by metered-dose inhaler (MDI) is well established, and the patient's technique with MDIs is related directly to achieving the desired clinical outcome. The present study was designed to assess and improve MDI technique by using a Vitalograph Aerosol Inhalation Monitor (VAIM) in an airways disease education programme. Baseline measurements were made immediately prior to educational intervention incorporating feedback from a VAIM unit. At 6 weeks' follow-up, MDI technique was found to have regressed to the sub-optimal measures recorded at baseline prior to educational intervention. However, patients reported a significant improvement in physical function between baseline and follow-up as measured by the Rand 36-Item Health Survey (SF-36), Version 1.0. The results reinforce the need for a longitudinal educational programme for patients prescribed medications delivered by MDI. The VAIM unit provided health educators and patients with both a visual and a quantitative assessment of patients' MDI technique, and was thus of positive value as part of the intervention process.

Adult↗