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Biomedical subjects

M A Ainsworth

Publications and source records attributed to M A Ainsworth.

At least 19 recordsLinked to original sources
PubMed2002

The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells.

Duodenal epithelial bicarbonate secretion has previously been shown to be greatly impaired in mice deficient of the cystic fibrosis transmembrane conductance regulator (CFTR). It has been proposed that transmembranal bicarbonate transport occurs through the CFTR channel itself. In the present study, the transport of acid/base equivalents across the plasma membrane of proximal duodenal epithelial cells from CFTR deficient mice was compared with that of cells from normal littermates. Mixed epithelial cells from both villi and crypts were isolated from proximal duodenum and intracellular pH was assessed by cuvette-based fluorescence spectrometry using the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The steady state intracellular pH, the acid extrusion rate and the alkaline extrusion rate were unaffected by CFTR deficiency in the presence of CO(2)/HCO(-)(3). Forskolin had no effect on acid extrusion or alkaline extrusion rates. In control experiments without CO(2)/HCO(-)(3), the intrinsic buffering capacities, the steady state intracellular pH and the acid extrusion rates were equivalent in the cells from CFTR deficient mice and normal littermates. The results are consistent with a model where acid/base transport is almost exclusively mediated by the previously described transporters in the murine duodenum (i.e. Na+/H+ exchange, Cl(-)/HCO(-)(3). exchange and Na+:HCO(-)(3). cotransport). There were no evidence for significant CFTR dependent HCO(-)(3). transport in proximal duodenal epithelial cells of mixed villus and crypt origin.

Animals↗
PubMed2001

Molecular and functional evidence for electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters in murine duodenum.

Inward Na(+)-HCO(3)(-) cotransport has previously been demonstrated in acidified duodenal epithelial cells, but the identity and localization of the mRNAs and proteins involved have not been determined. The molecular expression and localization of Na(+)-HCO(3)(-) cotransporters (NBCs) were studied by RT-PCR, sequence analysis, and immunohistochemistry. By fluorescence spectroscopy, the intracellular pH (pH(i)) was recorded in suspensions of isolated murine duodenal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of proximal duodenal villus cells, whereas the crypt cells did not label with the anti-NBC antibodies. DIDS or removal of extracellular Cl(-) increased pH(i), whereas an acidification was observed on removal of Na(+) or both Na(+) and Cl(-). The effects of inhibitors and ionic dependence of acid/base transporters were consistent with both inward and outward Na(+)-HCO(3)(-) cotransport. Hence, we propose that NBCs are involved in both basolateral electroneutral HCO(3)(-) transport as well as basolateral electrogenic HCO(3)(-) transport in proximal duodenal villus cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells.

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.

Amiloride↗
PubMed1998

Acid/base transporters in human duodenal enterocytes.

BACKGROUND: Duodenal mucosal bicarbonate secretion serves as a key defensive factor against mucosal injury. The purpose of the present study was to isolate human proximal duodenal enterocytes and identify their inherent acid/base transporters that participate in duodenal alkaline secretion. METHODS: Biopsy specimens were obtained from the duodenal bulb in 18 healthy volunteers. Individual duodenal epithelial cells were isolated by means of a combination of calcium chelation and collagenase. Intracellular pH (pHi) was measured by the pH-sensitive dye BCECF and dynamic fluorescence ratio imaging. RESULTS: Cytologic and histologic examination confirmed that isolated cells were of epithelial origin. In HCO3--free media, pHi recovery after acidification with NH4Cl was amiloride-sensitive and Na+-dependent, indicating the presence of an Na+/H+ exchanger. pHi recovery after acidification was significantly enhanced by the presence of HCO3-, showing the presence of an HCO3--dependent recovery mechanism (that is, a base loader/acid extruder). HCO3--dependent recovery required external Na+ yet was Cl-- and amiloride-insensitive, characteristic of an NaHCO3 cotransporter. In the presence of HCO3-, a Cl--dependent anion exchanger serving as a base extruder was shown, indicative of a Cl-/HCO3- exchanger. CONCLUSIONS: Human duodenal enterocytes contain at least three acid/base transporters: an Na+/H+ exchanger that serves as to extrude acid, an NaHCO3 cotransporter that functions as base loader, and a Cl-/HCO3- exchanger that operates as a base extruder.

Acid-Base Equilibrium↗
PubMed1998

Creating and evaluating an independent ambulatory internal medicine clerkship.

Patient care is shifting from an inpatient setting to an ambulatory setting. Despite this shift, most internal medicine clerkships provide the majority of medical student training in inpatient settings or in university tertiary care clinics, which are not representative of patient care in a community setting. We created a separate ambulatory clerkship that used volunteer community faculty at local and distant sites. The steps involved are described here, including finding time within the clerkship, reaching consensus within the department, defining the curriculum, identifying sites, and developing preceptors. Various parameters were measured to ensure quality in educational design. Comparisons of the 1-year pilot program, the full implementation program, and the inpatient program revealed that use of community sites does not affect cognitive knowledge acquisition but does influence students' satisfaction level.

Clinical Competence↗
PubMed1997

Acid-stimulated duodenal bicarbonate secretion involves a CFTR-mediated transport pathway in mice.

BACKGROUND & AIMS: Duodenal bicarbonate secretion is an important factor in epithelial protection. The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in acid-induced bicarbonate secretion is unknown. The aim of this study was to determine whether CFTR mediates acid-stimulated duodenal epithelial bicarbonate secretion. METHODS: Basal and stimulated bicarbonate secretion was examined in the cystic fibrosis murine model cftrm1UNC, which displays defective CFTR in various organs including chloride transport abnormalities in epithelia. After anesthesia, the proximal duodenum was cannulated and perfused with isotonic saline, and [HCO3-] was determined. RESULTS: Basal bicarbonate secretion was diminished in cystic fibrosis vs. normal mice, 2.8 +/- 0.7 vs. 4.7 +/- 1.7 mumol.cm-1.h-1, respectively (P < 0.001). Luminal acidification failed to elicit a bicarbonate secretory response in cystic fibrosis compared with normal littermates (peak response, 2.3 +/- 0.2 vs. 9.9 +/- 1.5 mumol.cm-1.h-1, respectively; P < 0.01). Prostaglandin E2- and vasoactive intestinal peptide-stimulated bicarbonate secretion were also significantly impaired in cystic fibrosis. Defective bicarbonate secretion in cystic fibrosis genotypes was due to decreased net fluid secretion and [HCO3-]. CONCLUSIONS: Basal and stimulated proximal duodenal bicarbonate secretion may involve a CFTR-mediated transport pathway. It is likely that CFTR, directly or indirectly, has a major functional role in mediating bicarbonate transport in the proximal duodenum.

Animals↗
PubMed1997

Setting standards and defining quality of performance in the validation of a standardized-patient examination format.

PURPOSE: To evaluate whether written standards increase the reproducibility of a physician-facilitated station in an objective structured clinical examination (OSCE) designed to assess history, physical-examination, and communication skills. METHOD: The OSCE examination at the University of Texas Medical Branch-Galveston consists of ten eight-minute stations. Six of these stations consist of three History, Physical-examination, Problem-solving, and Plan (HPPP) station pairs. Each existing clinical-problem HPPP station was given to two content experts to develop standards for faculty rating scales appropriate for the evaluation of third-year medical students. Three pairs of faculty members were used to determine interrater reliability by scoring videotapes of three HPPP stations' presentation and problem-solving components. Faculty pairs scored tapes of 15 students without using standards and tapes of 15 students using the standards developed. Differences between the reliabilities without and with the standards were tested for significance using Fisher's R to Z transformation. The reproducibility and standard error of measurement (SEM) were extrapolated for increasing amounts of testing time. The HPPP component scores were also correlated with the written examination scores and preceptors' ratings. Data were obtained from the three HPPP stations used in the 1995-96 internal medicine clerkship SP examination. RESULTS: In all, 196 students completed the OSCE examination. The standards developed improved interrater reliability and reached statistical significance (p < .01) for one HPPP station. Reproducibility for the presentation and problem-solving components of the HPPP stations were > .80 after five hours of testing. The problem-solving component correlated at .37 and .19 with written examinations and with ward grades, respectively. CONCLUSION: The data from this study suggest that standards increase the reproducibility of presentation and problem-solving components of an OSCE to a level as high as, or higher than, that associated with the history, physical-examination, and communication components of traditional standardized-patient examinations.

Clinical Clerkship↗
PubMed1997

CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO3- secretion.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in duodenal alkaline secretion has not been directly examined. The aims of this series of experiments were to determine if CFTR mediates basal and stimulated duodenal epithelial HCO3- secretion. Utilizing the cystic fibrosis murine model (cftr(m1UNC)), we compared normal [CFTR(+/+)] littermates (34-46 days old) with CFTR(-/-) animals (34-39 days old). Anesthesia was induced and maintained with intraperitoneal Hypnorm-midazolam. The proximal duodenum (4-7 mm) was cannulated and perfused with 154 mM NaCl. Either forskolin (10(-6)-10(-4) M) or carbachol (10(-6)-10(-3) M) was perfused intraluminally to activate adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2+-mediated HCO3- secretion, respectively. Effluent volumes were weighed and HCO3- quantitated by back titration. Basal HCO3- secretion was diminished significantly (P < 0.01) in CFTR(-/-)vs. normal CFTR(+/+) mice (2.8 +/- 0.5 vs. 5.3 +/- 0.4 micromol x cm(-1) x h(-1)). Moreover, in CFTR(-/-) mice, both forskolin- and carbachol-stimulated peak HCO3- secretions were fourfold less compared with those in CFTR(+/+) littermates (3.7 +/- 0.2 vs. 15.6 +/- 2.1 and 4.7 +/- 0.3 vs. 14.2 +/- 2.5 micromol x cm(-1) x h(-1), respectively; P < 0.01). In conclusion, CFTR plays a significant role in mediating basal, cAMP-, and Ca2+-activated duodenal epithelial HCO3- secretion.

Animals↗
PubMed1996

Dimensions of clinical competence as conceptualized by medical school faculty.

This study identified dimensions of clinical competence underlying faculty ratings of fourth-year student performance in a standardized patient examination and assessed the impact of these dimensions on faculty pass/fail decisions. Content review coupled with exploratory factor analysis was used to group 17 of 25 specific behavioral rating categories into four homogeneous clusters. Confirmatory factor analysis refined the clusters into four unidimensional scales that included 14 of the original ratings. The dimensions reflected relating to the patient, problem-solving skills, medical history skills, and physical examination skills. The factor structure was cross-validated in a separate data set. Logistic regression indicated problem solving was the only independent predictor of a student passing the exercise.

Adult↗
PubMed1996

Cyclic adenosine-3',5'-monophosphate production is greater in rabbit duodenal crypt than in villus cells.

BACKGROUND: Duodenal surface epithelial cells secrete bicarbonate. Agonists of duodenal alkaline secretion (such as vasoactive intestinal polypeptide (VIP), prostaglandin E2 (PGE(2)), and forskolin) increase intracellular cyclic adenosine-3', 5-monophosphate (cAMP), and cAMP stimulates Cl-HCO(3)- exchange in duodenal brush border membrane vesicles. As intestinal villus and crypt cells differ in function, our aims were to contrast cAMP generation in duodenal villus versus crypt cells in response to VIP, PGE(2), and forskolin. METHODS: Villus and crypt rabbit duodenal enterocytes were isolated by calcium chelation. To prevent the degradation of cAMP in vitro, phosphodiesterase activity was inhibited. cAMP production was quantitated in response to VIP (10(-10)-10(-5)M), PGE(2) (10(-10)-10(-4)M), and forskolin (10(-8)-10(-3)M). RESULTS: In crypt cells cAMP generation was approximately 10-fold greater (P < 0.001) in response to VIP, PGE(2), and forskolin than to villus cells. The relative orders of potency (that is, D(50), VIP > PGE(2) > forskolin) and efficacy (that is, V max, forskolin > VIP and PGE(2)) were similar in villus and crypt cells. CONCLUSION: cAMP production is greater in duodenal crypt than in villus enterocytes at rest and in response to forskolin, VIP, and PGE(2), suggesting that alkaline secretion may differ along the villus-to-crypt axis.

Animals↗
PubMed1996

Acid-base transport in isolated rabbit duodenal villus and crypt cells.

BACKGROUND: Duodenal mucosal bicarbonate secretion is an important first line of defense against gastric acid. Studies in the ileum indicate that the secretion originates from the crypt cells, whereas villus cells are mainly absorptive. Data on acid/base transporters along the crypt-villus axis in duodenal epithelia are not available. It was our purpose to identify and compare acid/base transporters in isolated mammalian duodenal villus and crypt cells. METHODS: The proximal duodenum of rabbits was excised, and duodenal epithelial cells were isolated in five fractions by a modified calcium chelation technique. Intracellular pH (pHi) was measured with a pH-sensitive dye and dynamic fluorescence ratio imaging. RESULTS: In both villus and crypt cells incubated in Hepes buffer, removal of Na+ or addition of amiloride decreased basal pHi and pHi recovery after intracellular acidification, indicating an Na+/H+ exchanger in both cell types. In both cell types acid extrusion rates in bicarbonate-buffered Ringer's solution were significantly higher than in Hepes buffer. The bicarbonate-dependent acid extruder was unaffected by removal of Cl- or addition of amiloride but was blocked by removal of Na+, indicating the presence of a NaHCO3 cotransporter in both villus and crypt cells. Removal of external Cl induced a reversible increase in pHi (inhibited by H2DIDS) in both villus and crypt cells, indicating a Cl-/HCO3- exchanger in both. CONCLUSIONS: Mammalian duodenal villus and crypt cells have identical acid-base transporters. These findings tend to negate the theory of a functional difference in acid-base transporters between duodenal villus and crypt cells and instead imply alkaline secretion by both cell fractions. However, as these experiments were performed in unpolarized, single cells, additional studies with either membrane vesicles or polarized cells are needed.

Animals↗