Biomedical subjects

M J Caplan

Publications and source records attributed to M J Caplan.

58 records · Page 4Linked to original sources

PubMed1987

Monoclonal antibody localization of Na+-K+-ATPase in the exocrine pancreas and parotid of the dog.

A monoclonal antibody specific to the beta-subunit of the canine Na+-K+-ATPase has been characterized and used to directly localize the enzyme in thin frozen sections of dog pancreas and parotid. The antibody, 7-2M, recognizes only the beta-subunit of the sodium pump as determined by immunoprecipitation and immunoblot and is not directed against an oligosaccharide determinant. 7-2M immunolocalizes to the same cellular and subcellular domains of renal tubular cells as do other, previously characterized, antibodies directed to the alpha-subunit of the sodium pump. In the pancreas the preponderance of the Na+-K+-ATPase is found on the basolateral membranes of centroacinar and intralobular duct cells. Interlobular duct cells also express a large component of basolaterally located enzyme, although comparatively little pump is seen on acinar cells. In the parotid a large amount of Na+-K+-ATPase is seen on the striated duct cells, with high levels also noted on cells of the intercalated ducts and serous demilunes. Again the acinar cells show comparatively low levels of Na+-K+-ATPase. In no instance is Na+-K+-ATPase found on the apical membranes of pancreas or parotid cells. These data suggest that Na+-K+-ATPase, located on the basolateral plasmalemma of duct-derived cells, may be involved in water and electrolyte secretion from the pancreas and parotid.

Animals↗

PubMed1986

Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes.

Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two-chamber culture system were used to study the postsynthetic sorting of the alpha-subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB-ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting.

Ammonium Chloride↗

PubMed1986

Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells.

Recently published data indicate that the alpha-subunit of Na,K-ATPase, a transmembrane protein of animal cell plasma membranes, is synthesized as a soluble precursor. In the present experiments we demonstrate that an apparent "soluble" form can indeed be detected in crude cytosolic fractions prepared by centrifugation from MDCK cells disrupted by sonication. We find, however, that this form has no precursor-product relationship with membrane-associated alpha-subunit. The quantity of unsedimentable alpha-subunit can be greatly diminished by increasing the centrifugal field employed to remove membranous vesicles from the cytosol fraction. Sonication of membrane pellets generates alpha-subunit which, like the "soluble" form, resists pelleting. Finally, cytosol fractions prepared from cells disrupted by sonication contain membrane-bound vesicles which can be immunoadsorbed on Staphylococcus aureus cells coated with a monoclonal antibody directed against alpha-subunit. We find, therefore, that the previously observed soluble precursor of alpha-subunit is actually a component of small unpelleted membrane vesicles generated by harsh disruption conditions. When cells are disrupted by less violent techniques no newly synthesized alpha-subunit can be detected in the cytosol fraction. We calculate that to escape detection under our experimental conditions a bona fide soluble precursor of alpha-subunit must have a cytosolic t1/2 less than 20 s. We conclude that the alpha-subunit is most probably inserted into the bilayer cotranslationally.

Animals↗

PubMedDate not supplied

Dependence on pH of polarized sorting of secreted proteins.

The plasma membranes of epithelial cells are divided into apical and basolateral domains. These two surfaces are characterized by markedly different protein compositions, reflecting the ability of the cell to target newly synthesized membrane proteins to specific regions of the cell surface. This targeting capability is also apparent in the polarized release of secretory products. Recent studies using canine renal tubule (MDCK) cells have suggested that distinct sets of secretory proteins are released from their apical and basolateral poles. We report experiments designed to examine secretory protein sorting by MDCK cells. We have shown that secretion of basement membrane components (laminin and heparan sulphate proteoglycan (HSPG] takes place from the basolateral cell surface and that this polarized release results from active sorting. The sorting process which mediates this polarized secretion requires an acidic intracellular compartment. MDCK cells treated with NH4Cl to raise the pH of their intracellular compartments, secrete laminin and HSPG by a default pathway which leads to their release in roughly equal quantities into the medium of both the apical and basolateral compartments.

Ammonium Chloride↗