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M J Welsh

Publications and source records attributed to M J Welsh.

At least 181 records · Page 10Linked to original sources
PubMed1993

Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cytosolic nucleotides and by cAMP-dependent phosphorylation. In excised membrane patches, CFTR Cl- channel activity requires hydrolyzable nucleotides and Mg2+, and is inhibited by ADP. We examined the interactions between CFTR and nucleotides using 8-azidoadenosine 5'-triphosphate (8-N3-ATP), a photoactivatable ATP analog. Because CFTR functions as a membrane ion channel, we studied CFTR in membranes of Sf9 insect cells. We found that [alpha-32P]8-N3ATP specifically photolabeled CFTR, with half-maximal labeling at 10 microM 8-N3ATP in the presence of Mg2+ and 100 microM in the absence of Mg2+. The 8-N3ATP also substituted for ATP in activating CFTR Cl- channels, indicating that it interacts with the active site(s). Both ATP and GTP prevented photolabeling with half-maximal inhibition at 1 mM. ADP and adenyl-5'-yl imidodiphosphate (AMP-PNP) prevented photolabeling but at much higher concentrations, whereas AMP did not inhibit photolabeling at concentrations of up to 100 mM. Phosphorylation of CFTR was not a prerequisite for nucleotide binding. These results demonstrate that CFTR interacts directly with nucleotides at concentrations that regulate CFTR Cl- channel activity.

Adenine Nucleotides↗
PubMed1993

Identification of revertants for the cystic fibrosis delta F508 mutation using STE6-CFTR chimeras in yeast.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (delta F508). We constructed STE6-CFTR chimeras with portions of the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBD1. The chimeras were functional in yeast, but mating efficiency decreased when delta F508 was introduced into NBD1. We isolated two delta F508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBD1 sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and Cl- channel gating defects caused by the delta F508 mutation. These results suggest that the NBD1s of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by delta F508 can be partially corrected by additional alterations in the protein.

ATP-Binding Cassette Transporters↗
PubMed1993

Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel located in the apical membrane of epithelia. Although cystic fibrosis (CF) is caused by mutations in a single gene encoding CFTR, the disease has a variable clinical phenotype. The most common mutation associated with cystic fibrosis, deletion of a phenylalanine at position 508 (frequency, 67%), is associated with severe disease. But some missense mutations, for example ones in which arginine is replaced by histidine at residue at 117 (R117H; 0.8%), tryptophan at 334 (0.4%), or proline at 347 (0.5%), are associated with milder disease. These missense mutations affect basic residues located at the external end of the second (M2) and in the sixth (M6) putative membrane-spanning sequences. Here we report that, when expressed in heterologous epithelial cells, all three mutants were correctly processed and generated cyclic AMP-regulated apical Cl- currents. Although the macroscopic current properties were normal, the amount of current was reduced. Patch-clamp analysis revealed that all three mutants had reduced single-channel conductances. In addition, R117H showed altered sensitivity to external pH and had altered single-channel kinetics. These results explain the quantitative decrease in macroscopic Cl- current, and suggest that R117, R334 and R347 contribute to the pore of the CFTR Cl- channel. Our results also suggest why R117H, R334W and R347P produce less severe clinical disease and have implications for our understanding of cystic fibrosis.

Animals↗
PubMed1993

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.

3T3 Cells↗
PubMed1993

Localization and characterization of the 86- and 84-kDa heat shock proteins in Hepa 1c1c7 cells.

The 90-kDa heat shock protein (hsp90) is present in cells at high levels in the cytoplasm and is composed of two separate gene products, hsp86 and hsp84. Rabbit polyclonal antibodies to the murine N-terminal sequences of the 86- and 84-kDa heat shock proteins were isolated from serum by peptide affinity chromatography. Antibodies against each form of hsp90 are capable of immunoprecipitating hsp90. Each antibody preparation is specific against either hsp86 or hsp84 when tested on a protein blot of Hepa 1c1c7 cytosol. The over-all ratio of hsp84/hsp86 in Hepa 1 cytosol was estimated to be 2 to 1. Each antibody preparation was used to immunoprecipitate hsp84 or hsp86 from Hepa 1 cytosol to test whether hsp86/84 exists as a homo- and/or heterodimer. After electrophoresis, silver staining revealed that anti-hsp86 antibodies immunoprecipitated both hsp86 and hsp84. This result would suggest that hsp86 forms heterodimers with hsp84. In contrast, the anti-hsp84 antibodies immunoprecipitated almost entirely hsp84, suggesting that hsp84 exists largely as homodimers. Both anti-hsp86 and hsp84 antibodies were able to immunoprecipitate the 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dixoin-labeled Ah receptor from Hepa 1 cytosol, indicating that these antibodies are able to bind to hsp90 when it is complexed with other proteins. Both antibody preparations recognize hsp90 in mouse, rat, and human cell lines. Immunofluorescence and confocal microscopy were performed using both antibody preparations, and the results indicated that both hsp86 and hsp84 were located in the cytoplasm and nucleus of Hepa 1 cells. Hsp86 was found to localize unevenly in the cytoplasm, while hsp84 was found evenly dispersed throughout the cytoplasm. Hsp86 also appeared to be localized to a greater degree than hsp84 in the vicinity of the nuclear envelope.

Amino Acid Sequence↗
PubMed1993

A single low cadmium dose causes failure of spermiation in the rat.

Ten-week-old male Sprague-Dawley rats were injected ip with cadmium chloride solution in a single dose of 0, 0.5, or 1.0 mg/kg body wt. At 4, 24, 48, and 72 hr after injection, testes of the animals were perfusion fixed, embedded in plastic, and sectioned. Tissue sections were stained and examined under the light microscope. These amounts of cadmium did not result in visible vascular lesion in the testes. However, at a dose of 1 mg/kg, the cadmium treatment resulted in failure of spermiation from stage IX through later stages of spermatogenesis in the seminiferous epithelium. Detailed statistical analysis revealed failed spermiation 24 hr after dosing. As the exposure time increased, failure of spermiation was observed with increasing frequency within an affected stage, and was seen at later stages of spermatogenesis as well. Testes of the rats treated with a cadmium chloride dose of 0.5 mg/kg showed no change in the frequencies of tubules having unreleased spermatids when compared to the controls. There was no difference in the stage frequencies between all the treatment groups and the controls. These results indicate that a single cadmium chloride dose of 1 mg/kg results in failed spermiation in rat seminiferous tubules, without discernible change to the surrounding endothelium. We conclude that cadmium begins to act during early stage VIII of spermatogenesis to induce failure of spermiation, and the action of cadmium is spermatogenic stage-specific.

Animals↗
PubMed1993

Cystic fibrosis transmembrane conductance regulator splice variants are not conserved and fail to produce chloride channels.

In the human CFTR only the rare exon 4- splice variant is conserved in mice. We have discovered two novel murine variants, exon 5- and exon 11b+. The exon 5- variant represents up to 40% of mRNA in all CFTR-expressing tissues and leaves the reading frame intact. The exon 11b+ variant inserts a novel exon between exons 11 and 12 with expression restricted to the testis. Two variants of 11b have been found and both introduce premature stop codons. When we expressed human CFTR variants lacking either exon 5 or exon 9 in HeLa cells, they failed to generate cAMP-mediated chloride transport, due to defective intracellular processing. The lack of conservation of splice variants between species and the inability of the more abundant splice variants to generate protein that is correctly processed argue against a physiological role and may simply represent aberrant splicing that is tolerated by the cell and organism.

Alternative Splicing↗
PubMed1993

Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis.

A new adenovirus-based vector (Ad2/CFTR-1) has been constructed in which the cDNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, replaces the early region 1 coding sequences, E1a and E1b. The virus retains the E3 region. Ad2/CFTR-1 and a related construct encoding beta-galactosidase replicate in human 293 cells which provide E1 gene functions in trans. Replication of these recombinant viruses was not detected in a variety of other cells, although very limited viral DNA synthesis and transcription from the E4 and L5 regions could be measured. These E1-deletion vectors were also deficient in cellular transformation, shut-off of host cell protein synthesis, and production of cytopathic effects, even at high multiplicities of infection. Ad2/CFTR-1 produced CFTR protein in a variety of cells including airway epithelia from CF patients. Expression of functional CFTR protein in a CF airway epithelial monolayer was detected by correction of the Cl- transport defect characteristic of CF. Surprisingly low multiplicities of infection (0.1 moi) were sufficient to generate CFTR Cl- current across a CF epithelial monolayer in vitro. These data, together with the lack of obvious toxicity, suggest that Ad2/CFTR-1 should be suitable for CF gene therapy.

Adenoviruses, Human↗
PubMed1993

A severe phenotype in mice with a duplication of exon 3 in the cystic fibrosis locus.

To develop an animal model for cystic fibrosis (CF), targeted gene disruption in embryonic stem (ES) cells was used to generate a duplication of exon 3 (cftrm1Bay allele) of the mouse CF gene. ES cells containing this mutation were used to generate chimeric animals that transmitted the mutant allele through the germline. Homozygous mutant animals display a severe phenotype, with approximately 40% dying within 1 week from intestinal obstruction. RNAase protection analysis of the cftrm1Bay allele did not detect any normal mRNA (< 1-2% of wild-type) in mutant animals. Pathologic changes in the intestines from mutant mice included mucus accumulation in the crypts and intestinal lumen, dilatation of the bases of the crypts, enlargement of goblet cells, and the presence of concretions in the crypts or between the villi. Changes were also present in the mucosal glands of the pharynx and the minor sublingual glands, where dilatation of acini and accumulation of eosinophilic material were evident. Atrophy of acinar cells that may be secondary to nutritional deficiency and mild inflammation in the main pancreatic duct were present in the pancreas of mutant animals. No changes were noted in the lung, trachea, liver, or male reproductive tract of mutant animals, and mutant males were fertile. Homozygous mutant mice showed defects in cAMP-mediated ion transport both in ileum and in cultured fetal tracheal explants. Thus, an additional mouse model for CF has been generated that should prove useful for the understanding of the pathogenesis and the development of treatments for CF.

Alleles↗
PubMed1993

Cadmium in vivo causes disruption of tight junction-associated microfilaments in rat Sertoli cells.

Ten-week-old male Sprague-Dawley rats were injected i.p. with cadmium chloride solution in a single dose of 0 or 1.0 mg/kg BW. At 4, 24, 48, and 72 h after injection, testes of the animals were collected, detunicated, and fixed in 10% formalin. Individual seminiferous tubules were isolated and the stages of the cycle of the seminiferous epithelium were determined through use of transillumination under a stereomicroscope. The tubules were stained with rhodamine-phalloidin, mounted on glass slides, and examined via a confocal microscope. This cadmium dose did not cause visible vascular lesion in the testes. The cadmium treatment resulted in changes in the pattern produced by microfilaments in the basal region of Sertoli cells. The observed change in basal Sertoli cell microfilaments consisted of fragmentation of the microfilament bundles as compared to those in seminiferous tubules from control animals. This apparent lesion was first observed in stages VIII through XI at 24 h after the cadmium exposure. As the time after exposure increased, the lesion within a stage occurred with increasing severity, and later stages of the cycle of the seminiferous epithelium were also affected. At 48 h after exposure, disorganization of microfilament bundles was seen in stages VIII through XIII/XIV. At 72 h after exposure, severe fragmentation of microfilament bundles was observed from stage VIII through stages II/III. The microfilament bundles in several stages prior to stage VIII remained unaffected. No change was observed in the microfilaments of peritubular cells. At 4 h after exposure, testes showed no change in the organization of microfilament bundles at the basal region of Sertoli cells or microfilaments in the peritubular cells. We conclude that a single cadmium chloride dose of 1 mg/kg results in the disruption of basal Sertoli cell microfilament bundles in the rat seminiferous epithelium, and that the action of cadmium is cell-specific and stage-specific.

Actin Cytoskeleton↗
PubMed1993

5'-Adenylylimidodiphosphate does not activate CFTR chloride channels in cell-free patches of membrane.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel requires both phosphorylation of its R domain and the presence of nucleoside triphosphates for activation. Our previous work suggested that hydrolysis of nucleoside triphosphates may be required to support channel activity. However, recent studies have suggested that the nonhydrolyzable adenosine triphosphate analogue, 5'-adenylylimidodiphosphate (AMP-PNP), may support some Cl- channel activity in sweat gland duct epithelia in the presence of low ATP concentration and in Cl- channels associated with expression of the P-glycoprotein multidrug resistance transporter. To examine the effect of AMP-PNP, we applied it to the cytosolic surface of phosphorylated CFTR Cl- channels contained in excised, cell-free patches of membrane. We found that preparations of 10 mM AMP-PNP opened phosphorylated CFTR Cl- channels. However, this effect was due to contaminating ATP: high-pressure liquid chromatography analysis of AMP-PNP demonstrated that 10 mM AMP-PNP could contain up to 50 microM ATP, which could account for the observed stimulation of CFTR Cl- channel activity. When contaminating ATP was hydrolyzed with hexokinase, AMP-PNP was unable to support CFTR channel activity. AMP-PNP (10 mM) also failed to attenuate or potentiate the current induced by 0.3 mM ATP. These results suggest that AMP-PNP has no direct effect on CFTR Cl- channels.

3T3 Cells↗
PubMed1993

Expression of CFTR and a cAMP-stimulated chloride secretory current in cultured human fetal alveolar epithelial cells.

During development the fetal lung secretes fluid that is osmotically linked to chloride (Cl-) transport. One possible pathway for Cl- secretion across the fetal pulmonary epithelium is through the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in epithelia and functions as a Cl- channel regulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and intracellular ATP. Previous studies have shown that CFTR mRNA is expressed throughout the human fetal pulmonary epithelium and CFTR protein can be immunoprecipitated from human fetal lung homogenates. In cultured fetal lung tissue explants, CFTR mRNA was localized to alveolar epithelial cells. To test the hypothesis that fetal alveolar epithelial cells express functional CFTR, we immunolocalized CFTR in human fetal lung and looked for evidence of Cl- secretion in cultured alveolar epithelial cell monolayers. Monoclonal anti-CFTR antibodies localized CFTR in cultured lung explants to the epithelial cells, predominantly at the apical surface. Bioelectric properties of cultured monolayers of midgestation fetal alveolar epithelial cells were measured in modified Ussing chambers. In unstimulated monolayers, transepithelial electrical potential difference (psi t) = -1.1 +/- 0.1 mV, transepithelial resistance (Rt) = 768 +/- 58 omega.cm2, and short-circuit current (Isc) = 1.9 +/- 0.2 microA/cm2 (mean +/- SE, n = 17). Addition of amiloride to the apical surface significantly decreased basal Isc. Apical diphenylamine-2-carboxylate (DPC), a Cl- channel inhibitor, caused no significant change in basal Isc. In the presence of apical amiloride, isoproterenol significantly increased Isc, a response that was inhibited by apical DPC and submucosal bumetanide. The cAMP agonists forskolin and IBMX also stimulated Isc.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Fluid and electrolyte transport by cultured human airway epithelia.

An understanding of the fluid and electrolyte transport properties of any epithelium requires knowledge of the direction, rate, and regulation of fluid transport and the composition of the fluid. Although human airway epithelial likely play a key role in controlling the quantity and composition of the respiratory tract fluid, evidence for such a role is not available. To obtain such knowledge, we measured fluid and electrolyte transport by cultured human nasal epithelia. Under basal conditions we found that epithelia absorbed Na+ and fluid; both processes were inhibited by addition of amiloride to the mucosal surface. These data suggest that active Na+ absorption is responsible for fluid absorption. Interestingly, Na+ absorption was not accompanied by the net absorption of Cl-; some other anion accompanied Na+. The combination of cAMP agonists and mucosal amiloride stimulated the secretion of NaCl-rich fluid. But surprisingly, the response to cAMP agonists in the absence of amiloride showed substantial intersubject variability: cAMP stimulated fluid secretion across some epithelia, for others, cAMP stimulated fluid absorption. The explanation for the differences in response is uncertain, but we speculate that the magnitude of apical membrane Na+ conductance may modulate the direction of fluid transport in response to cAMP. We also found that airway epithelial secrete H+ and absorb K+ under basal conditions; both processes were inhibited by cAMP agonists. Because the H+/K(+)-ATPase inhibitor, SCH 28080, inhibited K+ absorption, an apical membrane H+/K(+)-ATPase may be at least partly responsible for K+ and H+ transport. However, H+/K+ exchange could not entirely account for the luminal acidification. The finding that cAMP agonists inhibited luminal acidification may be explained by the recent finding that cAMP increases apical HCO3- conductance. These results provide new insights into how the intact airway epithelium may modify the composition of the respiratory tract fluid.

Amiloride↗
PubMed1993

Early SFAS 106 adopters provide revealing healthcare data.

Healthcare executives should have a strong interest in the recently released Statement of Financial Accounting Standards No. 106, Employers' Accounting for Postretirement Benefits other than Pensions (SFAS 106). The requirements of SFAS 106 focus heavily on estimating the healthcare benefits of retirees and forces corporations to project the magnitude of expected increases in healthcare costs. Estimates for recording these retiree healthcare benefits for all U.S. companies range from $200 billion to $400 billion, imposing significant financial statement implications for all firms that provide healthcare benefits to retirees. The requirements of SFAS 106 will also have an impact on the way corporations design and implement healthcare cost controls, which may directly affect many healthcare entities. Understanding SFAS 106, and the reactions of firms to this standard, will allow healthcare executives to better manage their organizations to take advantage of new opportunities and meet new challenges that may arise.

Accounting↗
PubMed1993

Dysfunction of CFTR bearing the delta F508 mutation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in patients with cystic fibrosis (CF). The most common CF-associated mutation is deletion of phenylalanine at residue 508, CFTR delta F508. When expressed in heterologous cells, CFTR bearing the delta F508 mutation fails to progress through the normal biosynthetic pathway and fails to traffic to the plasma membrane. As a result, CFTR delta F508 is mislocalized and is not present in the apical membrane of primary cultures of airway epithelia. Consequently, the apical membrane of CF airway epithelia is Cl- -impermeable, a defect that probably contributes to the pathogenesis of the disease.

Cell Membrane↗
PubMed1993

Effect of deletion mutations on the function of CFTR chloride channels.

Mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a unique R domain; CFTR functions as a Cl- channel regulated by phosphorylation and by nucleoside triphosphates. To study the domains of CFTR involved in Cl- channel function, we expressed mutants lacking various domains and assayed cAMP-stimulated Cl- channel activity using the halide-sensitive fluorophore, 6-methoxy-N-(3'-sulfopropyl)-quinolinium. We previously reported that deletion of part of the R domain (residues 708-835) produced Cl- channels that were constitutively open. Here we show that more extensive deletions within the R domain failed to generate functional CFTR Cl- channels; the portion of protein that could be deleted without destroying function corresponds to sequences that are not conserved in related proteins. In contrast, when we deleted the two NBDs (either alone, together, or in combination with the R domain), we did not observe functional Cl- channels. CFTR has a unique carboxyl terminus that is conserved across species. However, truncation of the carboxyl terminus (up to, but not including, NBD2) produced a regulated anion permeability similar to that of wild-type CFTR, suggesting that this region is not essential for channel function. Expression of two CF-associated nonsense mutants (G542X and W1316X) also failed to generate functional CFTR Cl- channels. These results help define structure:function relationships for CFTR and identify the domains that are required for Cl- channel function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗