Biomedical subjects

M C Harline

Publications and source records attributed to M C Harline.

7 recordsLinked to original sources

PubMed2001

Mineral content of calcified tissues in cystic fibrosis mice.

Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(-) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(-) mice revealed reduced Ca and normal P in the enamel layer--a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(-) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(-) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(-) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption.

Animals↗

PubMed2000

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium.

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.

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

PubMed1999

Desensitization of P2Y2 receptor-activated transepithelial anion secretion.

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

Adenosine Triphosphate↗

PubMed1998

Dual role of CFTR in cAMP-stimulated HCO3- secretion across murine duodenum.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated HCO3- secretion across the murine duodenum was investigated. Serosal-to-mucosal flux of HCO3- (Js-->m, in mu eq.cm-2.h-1) and short-circuit current (Isc; in mu eq.cm-2.h-1) were measured by the pH stat method in duodenum from CFTR knockout [CFTR(-)] and normal [CFTR(+)] mice. Under control conditions, forskolin increased Js-->m and Isc (+1.7 and +3.5, respectively) across the CFTR(+) but not CFTR(-) duodenum. Both the forskolin-stimulated delta Js-->m and delta Isc were abolished by the CFTR channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate, whereas inhibition of luminal Cl-/HCO3- exchange by luminal Cl- removal or DIDS reduced the Js-->m by approximately 18% without a consistent effect on the delta Isc. Methazolamide also reduced the Js-->m by 39% but did not affect the delta Isc. When carbonic anhydrase-dependent HCO3- secretion was isolated by using a CO2-gassed, HCO3(-)-free Ringer bath, forskolin stimulated the Js-->m and Isc (+0.7 and +2.0, respectively) across CFTR(+) but not CFTR(-) duodenum. Under these conditions, luminal Cl- substitution or DIDS abolished the Js-->m but not the delta Isc. It was concluded that cAMP-stimulated HCO3- secretion across the duodenum involves 1) electrogenic secretion via a CFTR HCO3- conductance and 2) electroneutral secretion via a CFTR-dependent Cl-/HCO3- exchange process that is closely associated with the carbonic anhydrase activity of the epithelium.

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

PubMed1996

CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model.

Acute adenosine 3',5'-cyclic monophosphate (cAMP) stimulation of intestinal epithelium induces net transepithelial Cl- secretion and inhibits neutral coupled NaCl absorption. To investigate the role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in these events, we measured bioelectric changes and radioisotopic NaCl flux across jejunal tissues from gene-targeted cftr "knockout" mice [cftr(-/-) homozygotes] and their normal littermates [cftr(+/+) homozygotes and cftr(+/-) heterozygotes]. Before stimulation, the short-circuit current (Isc, an index of Cl- secretion) of the cftr(-/-) jejunum was essentially zero and significantly less than in the cftr(+/+) or cftr(+/-) intestine. Acute cAMP stimulation had little effect on the bioelectric parameters of the cftr(-/-) intestine but induced a marked increase of Isc and decrease of total tissue conductance in both the cftr(+/+) and cftr(+/-) intestine. Differences in the magnitude of the cAMP-induced Isc between the cftr(+/+) and cftr(+/-) intestine were only observed when the cell-to-lumen anion concentration gradient was maximized by removal of permeant anions from the luminal bath. Radioisotope flux measurements revealed that Na+ and Cl- were absorbed equally across the cftr(-/-) jejunum under basal conditions. In cftr(+/+) and cftr(+/-) intestine, Na+ was absorbed at a similar rate, but net Cl- absorption was reduced from that in cftr(-/-) intestine by an amount approximating the Isc. Acute cAMP stimulation of the cftr(+/+) and cftr(+/-) intestine abolished net NaCl absorption and induced electrogenic Cl- secretion. In contrast, net NaCl absorption was unchanged from the preceding flux period in the cftr(-/-) jejunum. The data suggest that CFTR not only mediates cAMP-induced transepithelial Cl- secretion but is also required for cAMP inhibition of neutral NaCl absorption in the intestine.

Animals↗

PubMed1996

Increased survival of CFTR knockout mice with an oral osmotic laxative.

Mouse models of cystic fibrosis that are generated by targeted disruption (knockout) of the cystic fibrosis transmembrane conductance regulator gene, cftr(-/-), typically die shortly after weaning, from intestinal obstruction/rupture caused by an inability to secrete fluid into the bowel lumen. We investigated the use of a commercial osmotic laxative, Colyte, provided continuously in the drinking water, to increase the survival of cftr(-/-) mice. Genotype analysis of 623 offspring surviving at 10 days of age yielded 28.1% cftr(+/+), 59.6% cftr(+/-), and 12.4% cftr(-/-) mice (25% predicted), suggesting that cftr(-/-) mice have a significant perinatal mortality rate. However, of the 77 cftr(-/-) mice alive at 10 days of age, >98% survived weaning and were maintained in apparent health to a minimum of 56 days of age (arbitrary age for experimentation). In intestinal bioelectric studies Colyte-treated drinking water, compared with tap water, had no significant effect on basal short-circuit current, cyclic AMP-stimulated Cl- secretion, Na+-coupled glucose absorption, or electrogenic Na+ absorption across intestinal sections from cftr(+/+ or +/-) mice. Other than a mild dilatation of the distal portion of the colon in the Colyte-treated animals, examination of jejunal and colonic sections revealed no histologic differences between the two treatments. These findings indicate that the chronic use of Colyte osmotic laxative in drinking water is an economical means of greatly increasing the survival of CFTR knockout mice without altering the major electrolyte transport processes or histomorphologic integrity of the intestine.

Animals↗

PubMed1992

Improved method for screening cDNA expression libraries for DNA-binding proteins.

The ability to successfully screen a lambda gt11 cDNA expression library for specific gene products that can bind to selected sequences of DNA depends on radioactive double-stranded DNA probes with high specific activity. We demonstrate here that probes labeled by the PCR are superior to probes made by the Klenow reaction. The use of these PCR-generated probes have facilitated our efforts to isolate recombinant phage containing putative DNA-binding gene products that recognized a 246-base pair transcriptional enhancer region of Rous sarcoma virus long terminal repeat.

Animals↗