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A K Mircheff

Publications and source records attributed to A K Mircheff.

At least 73 records · Page 4Linked to original sources
PubMed1990

Analytic subcellular fractionation of acini from rat lacrimal gland.

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na+/K(+)-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.

Acid Phosphatase↗
PubMed1990

Subcellular distribution of muscarinic acetylcholine receptors in rat exorbital lacrimal gland.

The muscarinic acetylcholine receptor (MAChR) is an important mediator of parasympathetic regulation of secretion by the rat exorbital lacrimal gland. In order to survey the subcellular distribution of MAChR in lacrimal acinar cells, we have measured the binding of the specific muscarinic cholinergic antagonist [3H]-quinuclidinyl benzilate ([3H]-QNB) to membrane samples isolated from rat exorbital lacrimal glands by differential and equilibrium density gradient centrifugation. Binding of [3H]-QNB in all membrane fractions was consistent with the presence of a single class of receptor which was muscarinic in nature on the basis of its Kd for [3H]-QNB (0.30-0.35 nM) and its ability to interact with the muscarinic agonists carbachol and methachol and the antagonist atropine. MAChR were present at the highest specific activity in acinar cell basal-lateral plasma membrane-derived populations, where Bmax was as high as 1960 fmole/mg protein. However, the density distributions of MAChR and of other membrane markers indicated that the receptors were present also in membranes derived from cytoplasmic structures, where Bmax values ranged from 50.4 to 152.8 fmole/mg protein. Stimulation with 10 microM carbachol for 30 min led to a 20% (P less than 0.05) increase in the relative MAChR content of a population of membranes derived from the acinar cell basal-lateral membrane; an apparent tendency for MAChR activity to decrease in other membrane populations suggests that stimulation might cause a redistribution of MAChR between cytoplasmic pools and the cell surface membranes.

Animals↗
PubMed1989

Parathyroid hormone-induced translocation of Na-H antiporters in rat proximal tubules.

Parathyroid hormone (PTH) is believed to inhibit bicarbonate reabsorption by inhibiting Na-H antiport activity in proximal tubular brush-border membranes. The sequence of events triggered by PTH was investigated in a crude preparation of proximal tubules obtained by mechanical disruption and filtration through nylon mesh filters. Tubule samples were subjected to analytical subcellular fractionation after 2-, 5-, and 30-min treatments with 1 IU/ml PTH. These PTH-treatment intervals caused 54, 63, and 68% decreases in the Na-H antiport activity of a population of brush-border membrane vesicles that was resolved from a PTH-unresponsive brush-border population by density-gradient centrifugation. The rapid loss of Na-H antiport activity from the responsive population was accompanied by a transient increase in the Na-H antiport activity of a region of the density gradient, designated density window III, which was shown to contain two distinct membrane populations; these populations were both enriched in acid phosphatase activity, and one of them was also an important locus of galactosyltransferase activity. The increase in the Na-H antiport activity of window III accounted for 52% of the activity lost from the PTH-responsive population after 2 min, and for 43% of the activity lost after 5 min, but it was completely abolished after 25 more minutes in the presence of PTH. These observations suggest that PTH triggers a rapid translocation of Na-H antiporters from the microvillus membrane to a distinct membrane domain, where they are subsequently inactivated.

Alkaline Phosphatase↗
PubMed1989

Lacrimal fluid and electrolyte secretion: a review.

Lacrimal gland fluid is an important component of the precorneal tear film. The rate of lacrimal gland fluid secretion is controlled primarily by parasympathetic innervation, and it is, apparently, modulated by sympathetic innervation. Lacrimal gland fluid is produced in two stages, secretion of a primary fluid which resembles an isotonic ultrafiltrate of plasma in the acinus-early intercalated duct region, and secretion of a KCl-rich fluid in subsequent ductal elements. Little is known about the electrolyte transport mechanisms of the ductal epithelia. Recent work using a variety of techniques, including tracer flux measurements, intracellular electrical recording, intracellular ion activity measurements, patch clamping, and analytical subcellular fractionation, supports a model for transcellular Cl-secretion in the acinus which involves Cl--selective channels in the apical plasma membrane and an array of Na+/H+ antiporters, Cl-/HCO3-antiporters, K+ channels, and Na,K-ATPase in the basal-lateral plasma membrane.

Animals↗
PubMed1988

Stimulation-associated redistribution of Na,K-ATPase in rat lacrimal gland.

To test the possibility that stimulation of secretion leads Na,K-ATPase to be recruited from cytoplasmic pools and inserted into basal-lateral plasma membranes, we surveyed the subcellular distributions of Na,K-ATPase in resting and stimulated fragments of rat exorbital lacrimal gland. After a two-dimensional separation procedure based on differential sedimentation and density gradient centrifugation, we defined six density windows, which differ from one another in their contents of biochemical markers. The membranes equilibrating in window I could be identified as a sample of basal-lateral membranes; in resting preparations these membranes contained Na,K-ATPase enriched 16.6-fold with respect to the initial homogenates. Windows II through VI contained various cytoplasmic membrane populations; these accounted for roughly 80% of the total recovered Na,K-ATPase activity. Thirty-minute stimulation with 10 microM carbachol caused a 1.4-fold increase (P less than 0.05) in the total Na,K-ATPase content of window I; this increase could be largely accounted for by a 1.7-fold decrease in the total Na,K-ATPase content of density window V. Acid phosphatase activity also redistributed following stimulation, but it was recruited from a different source, and it was inserted into membranes equilibrating in windows II and III as well as into the membranes of window I.

Acid Phosphatase↗
PubMed1988

Ca2+ - and cAMP-induced protein phosphorylation in lacrimal gland basolateral membranes.

Basolateral plasma membranes play an integral role in coupling of stimulus to secretion of fluid and protein from the lacrimal gland. To determine if basolateral plasma membranes contain Ca2+- or adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent protein kinases, which could phosphorylate specific proteins important for secretion, a purified preparation of basolateral plasma membranes was prepared from rat exorbital lacrimal glands by differential and density gradient centrifugation. Phosphorylation of basolateral plasma membrane proteins was studied in the presence of [gamma-32P]ATP and was analyzed by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Increasing the Ca2+ concentration in the presence of calmodulin stimulated phosphorylation of a 52,000-Mr peptide with a maximal increase in phosphorylation obtained at 3 and 66 microM free Ca2+. The phenothiazines trifluoperazine and promethazine inhibited phosphorylation of this 52,000-Mr peptide; 50% inhibition was obtained at 15 and 95 microM, respectively. Increasing the cAMP level from 0 to 10 microM stimulated phosphorylation of another peptide of 91,000 Mr. This effect could be reproduced by guanosine 3', 5'-cyclic monophosphate, but only at 100 microM. The cAMP concentration causing 50% of maximal phosphorylation was 0.3 microM. We conclude that lacrimal gland basolateral plasma membranes contain Ca2+/calmodulin- and cAMP-dependent protein kinases and protein substrates.

Adenosine Triphosphate↗
PubMed1988

Cl(-)-HCO3- antiport in rat lacrimal gland.

With the use of analytical subcellular fractionation and tracer uptake methods we have demonstrated the presence of a Cl(-)-HCO3- antiport mechanism in the rat exorbital lacrimal gland. We find that outwardly directed gradients of HCO3- and of 35Cl- accelerated the flux of 36Cl- into isolated membrane vesicles. Because vesicle membrane potentials were clamped to 0 mV with K+-valinomycin, the observed anion gradient-dependent acceleration of Cl- influx could not be attributed to conductive fluxes. The antiporter had an apparent K0.5 for Cl- between 6 and 10 mM. It was sensitive to the stilbene derivatives 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). It was also sensitive to the loop diuretic furosemide, which has frequently been used in tests for NaKCl2 symporter activity. Other anions inhibited anion gradient-driven Cl- influx in the sequence SCN- greater than NO3- greater than Cl- greater than HCO3- greater than SO2-4. The density distribution of Cl(-)-HCO3- antiport activity indicated that approximately 80% of the transporter was associated with intracellular membranes, suggesting the presence of cytoplasmic pools of functional antiporters. Because several studies have already shown the presence of Na+-H+ antiporter activity in lacrimal acinar cell basolateral membranes, a cellular model for lacrimal acinar electrolyte secretion is proposed in which a parallel array of Cl- -HCO3- and Na+-H+ antiporters mediates the Na+-dependent accumulation of Cl- against its electrochemical potential gradient.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Laterobasal membranes from intestinal epithelial cells: isolation free of intracellular membrane contaminants.

A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8 mM CaCl2 concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.

Animals↗
PubMed1987

Na+/H+ antiporter in lacrimal acinar cell basal-lateral membranes.

The first step in the formation of lacrimal gland fluid is believed to depend on transport systems which couple a flux of Cl- ions to the passive influx of Na+ ions across the acinar cell basal-lateral plasma membrane. The transport systems which mediate these fluxes have not yet been characterized, but a review of previous studies (Parod and Putney, Am J Physiol 239:G106, 1980) raises the possibility that Na+/H+ antiporters might represent a major pathway for Na+ influx. This conclusion is of interest, because antiporter mediated Na+ fluxes can, potentially, drive net Cl- fluxes. We have now examined a sample of basal-lateral membrane vesicles from rat exorbital lacrimal gland to verify the presence of a Na+/H+ antiporter activity. Imposition of an outward H+ gradient caused a 4.4-fold increase in the 22Na influx rate, while imposition of an outward Na+ gradient accelerated H+ uptake as determined by changes in acridine orange absorbance. All transport experiments were done in the presence of valinomycin and symmetrical K+ concentrations, eliminating the possibility of conductive Na+ or H+ fluxes driven by diffusion potentials. The pH gradient dependent Na+ influx was completely inhibited by 1 mM amiloride, indicating that it was mediated by a Na+/H+ antiporter similar to those described in other tissues. Comparison of the density distributions of Na+/H+ antiport and standard membrane marker enzyme activities confirmed that the antiporter was primarily localized to the basal-lateral membranes.

Animals↗
PubMed1986

Subcellular distribution of ATP-dependent calcium transport in rat duodenal epithelium.

Subcellular fractionation studies were performed to delineate plasma membrane and intracellular membrane populations which might be involved in intracellular Ca2+-homeostasis of rat small intestinal epithelial cells. After a low-speed supernatant fraction had been suspended in 5% sorbitol and subjected to equilibrium centrifugation in a zonal rotor, the Golgi and endoplasmic reticulum markers, galactosyltransferase and NADPH-cytochrome -c reductase, were concentrated in a density region designated Window II. The basal-lateral membrane marker (Na+-K+)-ATPase was concentrated in a higher-density region designated Window III. ATP-dependent Ca2+ transport was equally distributed between the two windows. Several membrane populations could be resolved from each window with good recovery of Ca2+-transport activity by a second density gradient centrifugation step. Second density gradient fractions were subjected to counter-current partitioning in an aqueous polymer two-phase system. Basal-lateral membranes, characterized by an 11-fold enrichment of (Na+-K+)-ATPase, contained ATP-dependent Ca2+-transport activity with Vmax = 3.7 nmol/mg per min and Km = 0.5 microM. A major Golgi-derived population exhibited Ca2+-transport activity with Vmax and Km values similar to those of the basal-lateral membranes. One membrane population, presumed to have been derived from the endoplasmic reticulum, contained Ca2+-transport activity with Vmax = 4 nmol/mg per min and Km = 0.5 microM. In addition to demonstrating that ATP-dependent Ca2+-transport activity has a complex distribution within enterocytes, this study raises the possibility that the basolateral plasma membranes might account for a relatively minor portion of the cell's Ca2+-pumping ability.

Adenosine Triphosphate↗
PubMed1986

Mapping subcellular distribution of Na+-K+-ATPase in rat parotid gland.

Recent subcellular fractionation studies have raised the possibility that Na+-K+-ATPase might be present in both the apical and the basal-lateral membranes of exocrine gland acinar cells. Analytical fractionation and immunofluorescence microscopy studies of rat parotid glands were performed to confirm this interpretation. The distributions of biochemical markers after analyses based on differential sedimentation, equilibrium density-gradient centrifugation, and partitioning in an aqueous polymer two-phase system defined a total of 15 physically and biochemically distinct membrane populations. Among these populations, it was possible to select one (designated population i) with the characteristics expected of acinar cell basal-lateral plasma membranes. It contained Na+-K+-ATPase enriched 33-fold, and gamma-glutamyl transpeptidase enriched 23-fold with respect to the initial homogenate. A second population (designated population c) had the characteristics expected of acinar cell apical plasma membranes; it contained Na+-K+-ATPase enriched 28-fold, and gamma-glutamyl transpeptidase enriched 53-fold with respect to the initial homogenate. Although the identification of population c remains provisional, immunofluorescence studies verified that Na+-K+-ATPase is present in both the apical and the basal-lateral acinar cell plasma membranes. In view of these results, it is likely that the apical Na+-K+-ATPase would participate in series with basal-lateral sodium- and chloride-entry pathways in driving the secretory electrolyte fluxes.

Animals↗
PubMed1986

Apical and basal-lateral Na/K-ATPase in rat lacrimal gland acinar cells.

The distribution of Na/K-ATPase in rat exorbital lacrimal gland was studied using immunofluorescent localization of an antibody raised against rat kidney Na/K-ATPase. In cryostat sections, intralobular ducts were strongly immunoreactive and acinar cells showed localization on both apical and basal-lateral surfaces. Acinar cells also had strong intracellular reactivity in apical regions. These results are discussed in light of current models of exocrine gland electrolyte secretion.

Animals↗
PubMed1985

Complex subcellular distributions of enzymatic markers in intestinal epithelial cells.

Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constituent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent of a priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer two-phase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated with Golgi, endoplasmic reticulum, and brush-border membranes at specific activities two- to seven-fold that of the initial homogenate.

Acid Phosphatase↗
PubMed1984

A map of membrane populations isolated from rat exorbital gland.

We combined separation procedures based on three independent physical properties, sedimentation coefficient, density, and partitioning in an aqueous polymer two-phase system, to generate a three-dimensional subcellular fractionation of rat exorbital lacrimal gland. The distributions of protein and five enzymatic markers define a total of 13 physically and biochemically distinct membrane populations. These include epithelial cell apical membranes, purified 330-fold with respect to the initial homogenate; basal-lateral membranes, purified 80-fold; mitochondria, purified 19-fold; and a major endoplasmic reticulum population, purified 22-fold. Also apparent is a major Golgi population, which is extensively overlapped by other membrane populations; two populations that can be visualized as forming transitions between the endoplasmic reticulum and Golgi membranes; and several populations with unknown subcellular origins. Most of the markers have complex distributions among the isolated membrane populations; this complexity is consistent with current concepts of the synthesis and recycling of membrane constituents and the regulation of cytosolic electrolyte activities.

Animals↗
PubMed1984

Na+/H+ antiporter in membrane populations resolved from a renal brush border vesicle preparation.

A conventional brush border membrane preparation, obtained by divalent cation precipitation of homogenates of rabbit renal cortex, was analyzed by countercurrent distribution in an aqueous dextran:polyethylene glycol two-phase system. The resulting fractions were assayed for the presence of the Na+/H+ antiporter and for a variety of biochemical marker enzymes. This analysis revealed four physically distinct membrane populations (A-D). Population A consisted of two subpopulations, A' and A", which were enriched an average of 49-fold in maltase; they were also highly enriched in alkaline phosphatase, leucine aminopeptidase, and Na+/H+ antiporter. On the basis of their marker contents, populations A' and A" appear to represent highly purified, functional brush border membrane vesicles. Population B was enriched twofold in NADPH-cytochrome c reductase and population C was enriched 12-fold in galactosyltransferase. Populations B and C accounted for 25% of the protein in the starting material and appear to reflect contamination of the brush border membrane preparation by subpopulations of endoplasmic reticulum and Golgi fragments. Population D was enriched in Na+/H+ antiporter, alkaline phosphatase, leucine aminopeptidase, Na-K-ATPase, and acid phosphatase but not maltase, NADPH-cytochrome c reductase, galactosyltransferase, or succinate dehydrogenase. Its identity is unclear, and it might consist of a multiplicity of populations from different origins.

Animals↗