Isolation and identification of plasma membrane populations.
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Acid Phosphatase↗
Biomedical subjects
A K Mircheff
Publications and source records attributed to A K Mircheff.
Complex subcellular distribution of sodium-dependent amino acid transport systems in kidney cortex and LLC-PK1/Cl4 cells.
To characterize the amino acid transport system in basolateral membranes and to test for possible intracellular loci of amino acid transport activity, we surveyed the distribution of L-alanine transport activity in rabbit proximal tubular cells and LLC-PK1/Cl4 cells. A three-dimensional separation procedure based on differential sedimentation, density gradient centrifugation, and counter-current distribution resolved 21 physically and biochemically distinct membrane populations from rabbit cortex. Inhibition of L-alanine transport by phenylalanine and N-(methylamino)isobutyric acid was used to delineate parallel amino acid transport pathways. Population n was identified as brush border membranes by virtue of its 16-fold maltase enrichment; 94% of its Na(+)-dependent alanine transport activity was mediated by systems previously shown to be characteristic of brush border membranes. Two populations, c' and c", which accounted for 25% of the total Na,K-ATPase activity, were identified as basalateral membranes on the basis of Na,K-ATPase cumulative enrichment factors of 15 and 21; 82% of the total alanine transport in these populations was mediated by a Na(+)-independent system similar to the classical system L. Na,K-ATPase, Na(+)-independent and Na(+)-dependent alanine transport activities were associated with intracellular membrane populations as well as with the plasma membranes. The major intracellular locus of Na,K-ATPase activity, population i accounted for roughly 31% of the Na,K-ATPase, maximally enriched ninefold; it contained 29% of the total system L transport activity. Population l, which was identified as endoplasmic reticulum because it was the major locus of membrane-bound NADPH cytochrome c reductase activity, contained 44% of the total system A transport. Three distinct Golgi-derived populations, m', m", and o, accounted for 39% of the total system A transport. A survey of the amino acid transport systems in LLC-PK1/Cl4 cells showed that the majority of system A-mediated amino acid transport was present in membranes of intracellular and possibly apical origin. The presence of large intracellular pools of amino acid transport activities might reflect newly synthesized transport proteins, ongoing membrane recycling or, perhaps, intracellular reserves available for rapid recruitment to the plasma membrane.
Alanine↗
Autoimmunity of the lacrimal gland.
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Antigen Presentation↗
Traffic of major histocompatibility complex class II molecules in rabbit lacrimal gland acinar cells.
PURPOSE: It has been suggested that lacrimal gland acinar cells, which have been induced to express major histocompatibility complex class II (MHC II) molecules, might initiate local autoimmunity by using mechanisms similar to those operating in the specialized antigen-presenting cells to process and present autoantigens. Surface-labeling experiments indicate that constituents of the acinar cell plasma membrane participate in a rapid recycling traffic. The authors have surveyed the subcellular distribution of MHC II molecules and have evaluated their participation in the traffic between plasma membranes and intracellular compartments. METHODS: Acinar cells were isolated from rabbit lacrimal glands and maintained for two nights in a serum-free, hormone-supplemented culture medium containing 10 microM carbachol. MHC II molecules were detected with a monoclonal antibody (MAB 2C4), biotinylated goat-antimouse IgG (BGAM), and avidin-ferritin (AvFe) or streptavidin-gold (SAvAu) conjugates. RESULTS: Postembedding labeling with MAB 2C4, BGAM, and AvFe revealed MHC II molecules at the surface membranes, in cytoplasmic vesicles, and in secretory vesicles. When cells were chilled to 4 degrees C and subjected to preembedding labeling with MAB 2C4, BGAM, and AvFe, surface MHC II molecules were specifically labeled. Labeled complexes were rapidly internalized upon warming to 37 degrees C. Postembedding labeling with MAB 2C4, BGAM, and SAvAu revealed additional intracellular MHC II molecules, with a distribution overlapping that of the MHC II molecules labeled during the preembedding procedure. When cells were cultured overnight in the presence of MAB 2C4 and subjected to postembedding labeling with BGAM and AvFe, label was detectable in small vesicles and in secretory vesicles. However, the extent of labeling appeared less than obtained with postembedding labeling with MAB 2C4, BGAM, and AvFe. Preembedding labeling of cells that had been incubated overnight with MAB 2C4 indicated that the cells continued to express MHC II molecules at their surface membranes, and rapid internalization of label upon warming to 37 degrees C confirmed that MHC II molecule traffic continued in the presence of MAB 2C4. Postembedding labeling with MAB 2C4, BGAM, and SAvAu indicated the continued presence of a large intracellular pool of MHC II molecules. CONCLUSIONS: MHC II molecules in lacrimal acinar cells are present in large intracellular and small surface pools. They move rapidly between these two pools, but further work will be required to determine whether the MHC II molecule traffic represents recycling or turnover and whether recycling pools and sequestered pools coexist. The presently available data make it reasonable to propose that the traffic of MHC II molecules to plasma membranes provides a mechanism by which acinar cells display intracellularly generated autoantigens to potentially reactive helper T lymphocytes.
Animals↗
Rabbit lacrimal acinar cells in primary culture: morphology and acute responses to cholinergic stimulation.
PURPOSE: The rabbit lacrimal gland yields large numbers of viable acinar cells that, when exposed to carbachol, respond with accelerated protein release, fluid phase endocytosis (Lucifer yellow uptake), and Na/H antiport activation. The current study was undertaken to determine whether such cells exhibit similar responses after having been maintained in primary culture. METHODS: Cells were isolated from 2-kg, juvenile male New Zealand White rabbits and maintained in a supplemented DMEM/Ham's F-12 medium for up to 72 hours. RESULTS: Electron microscopy showed the organization of freshly isolated cells to be highly polarized, with secretory vesicles at one pole and nucleus at the other; vesicles were heterogeneous in size and in the electron density of their contents. The cells remained polarized after overnight culture, but the secretory vesicle population was more homogeneous in size and content, and the cells tended to aggregate. After 72 hours, roughly half the cells retained good morphology and cytoplasmic polarization, but the vesicles were enlarged and their contents less electron dense. Cells that had been maintained overnight responded to the addition of 10 microM carbachol with a 32.2% +/- 15.5% (n = 12, P < 0.04) increase in the total amount protein released during a standard 20-minute incubation. This represented a mean 125% increase in the temperature-dependent component of protein release. The protein secretory response was decreased to 14.6% +/- 6.1% (n = 3, P < 0.07) for cells that had been maintained for 72 hours. In the same samples, carbachol increased fluid phase endocytosis by 38.3% +/- 8.1% (P < 0.01) and 70.9% +/- 13.4% (P < 0.025), respectively. The protein secretory response was partially, and the endocytic response fully blocked by 1 mM atropine. CONCLUSIONS: This model could be useful as a simplified system in which to study regulation of acinar cell function over days, rather than hours, as is required in fresh tissue models.
Alkalies↗
Surface and intracellular pools of Na,K-ATPase catalytic and immuno-activities in rat exorbital lacrimal gland.
The subcellular distribution of Na,K-ATPase in rat lacrimal gland acinar cells was surveyed by subcellar fractionation followed by determination of two Na,K-ATPase catalytic activities, K(+)-dependent p-nitrophenylphosphate hydrolysis and ouabain-sensitive ATP hydrolysis, by Western blotting of isolated membrane fractions, and by analysis of lacrimal tissue with immunocytochemical methods at the light and EM levels. Both catalytic activities distributed in parallel after centrifugation on sorbitol gradients. They were associated with membrane samples that appeared to be derived from: (1) acinar cell basal-lateral membranes; (2) the Golgi complex; and (3) endocytic compartments. beta 1-subunit immunoreactivity closely paralleled catalytic activity. The alpha 1-subunit immunoreactivity distribution suggested the presence of alpha 1-subunits in alpha beta complexes and excess alpha 1 subunits which were not assembled with beta subunits and not catalytically active. In the putative basal-lateral membrane and endocytic samples, alpha 1-reactivity was associated primarily with a 100-kDa band, while in the Golgi samples it was associated primarily with 40 and 60-kDa bands. beta 1-reactivity was also heterogeneous, with reactivity in basal-lateral membrane and putative endocytic samples associated with a broad band of 50-54 kDa, and reactivity in Golgi samples associated with discrete bands of 50, 52, and 54 kDa. Staining with anti-holoenzyme and anti-alpha 1-subunit antibodies yielded strong indirect immunofluorescence signals both in plasma membrane and in intracellular regions of acinar cells. beta 1-like immunoreactivity was concentrated in cytoplasmic regions of acinar cells. Immunogold electron microscopy revealed positive staining with anti-holoenzyme in Golgi membranes of acinar cells and in basal-lateral membranes of duct cells. These data support the hypothesis that lacrimal acinar cells contain substantial cytoplasmic pools of Na,K-ATPase and that there is a location-dependent heterogeneity which is not detected by immunocytochemical methods.
Adenosine Triphosphate↗
Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini.
Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.
Amiloride↗
Sex-dependent parameters related to electrolyte, water and glycoprotein secretion in rabbit lacrimal glands.
The lacrimal glands of males and females of various species differ with respect to several morphological, biochemical and functional characteristics. The purpose of this study was to determine the effect of sexual maturation on Na+,K(+)-ATPase, muscarinic cholinergic receptors and beta-adrenergic receptors, which are closely related to the secretion of electrolytes and fluid by the gland, and on other membrane-associated enzymes, specifically galactosyltransferase and alkaline and acid phosphatase. Soluble and total membrane fractions were obtained from lacrimal glands of prepubertal (1.0 kg), pubertal (2 kg), and mature (4 kg) of New Zealand white rabbits of both sexes. Prepubertal and pubertal rabbits exhibited no sex differences in the total amount of lacrimal gland protein or in any of the enzymes or receptors, with the exception of galactosyltransferase and alkaline phosphatase. Galactosyltransferase had higher total and specific activities in prepubertal and pubertal males, and alkaline phosphatase had higher specific activity in prepubertal males. As animals matured, total protein and activities of the enzymes increased, and several quantitative differences between males and females became apparent. Samples from mature females contained significantly less DNA and membrane and total protein. Specific activities of Na+,K(+)-ATPase, cholinergic receptors, galactosyltransferase, and acid and alkaline phosphatase were 40% to 80% greater (p < 0.05) in mature females. Total and specific activity for beta-adrenergic receptors, on the other hand, were higher in the male rabbits. These findings suggest that sex hormones play a role in regulating the levels of expression of a number of enzymes and receptors, including several which are clearly involved in lacrimal secretory functions.
Aging↗
Plasma membrane internalization and recycling in rabbit lacrimal acinar cells.
PURPOSE: The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells. METHODS: Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive groups were biotinylated at 4 degrees C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin was then labeled with avidin-horseradish peroxidase complex for light microscopy, with avidin-lucifer yellow conjugate for fluorescence microscopy, and quantitative fluorometry, and with avidin-ferritin conjugate for electron microscopy. RESULTS: At 4 degrees C labels remained at the surfaces of intact cells. Surface avidin-lucifer yellow decreased markedly, giving way to punctate cytoplasmic labeling, on warming to 37 degrees C. Electron microscopy of cells warmed after labeling with avidin-ferritin revealed ferritin in smooth vesicles underlying the plasma membranes, in vesicles adjacent to Golgi membranes, and in multivesicular bodies. Incubation at 37 degrees C before chilling and labeling with avidin-lucifer yellow decreased the cells' capacity to bind avidin-lucifer yellow by 95%, with t0.5 < 0.5 min. If cells were then incubated with avidin-lucifer yellow at 37 degrees C, they took up the marker with a time course that indicated that 60% of the initial biotin either recycled back to the plasma membrane or remained in intracellular compartments that could be reached by endocytosed extracellular fluid. Internalized biotin communicated with extracellular avidin-lucifer yellow with a t0.5 of 2 min, and this process was accelerated by carbachol at concentrations of 10 mumol/l and 1 mmol/l. CONCLUSIONS: Acinar cell plasma membrane constituents participate in an ongoing, secretagogue-modulated recycling traffic between small surface-expressed pools and 10- to 20-fold larger intracellular pools.
Animals↗
Synthesis and translocation of Na(+)-K(+)-ATPase alpha- and beta-subunits to plasma membrane in MDCK cells.
Synthesis and translocation of Na(+)-K(+)-ATPase alpha-catalytic and beta-glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into beta-subunit was equal to that incorporated into alpha-subunit after 15 min of labeling. Because the ratio of total methionines in alpha- vs. beta-subunit is approximately 5:1, these results suggest that beta-subunit is synthesized in molar excess over alpha-subunit. Half of the newly synthesized beta-subunit, likely unassembled units, were degraded by 60 min after labeling, while alpha-subunits were stable through 120 min after synthesis, suggesting alpha may be limiting for alpha beta-assembly. By 120 min the ratio of counts incorporated into alpha vs. beta approached 5, which is predicted by a 1:1 ratio of alpha to beta. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature beta (beta i) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature beta (beta m) became detectable after 30 min, and conversion of beta i to beta m was 90% complete at 120 min. A peak of labeled alpha-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled beta m-subunit in this sample, suggesting movement as alpha beta-heterodimers.
Animals↗
Prolactin localization, binding, and effects on peroxidase release in rat exorbital lacrimal gland.
Prolactin immunoreactivity has been detected in human tears and in lacrimal glands, and it has been suggested that this hormone might be a modulator of lacrimal secretion as well as a component of lacrimal gland fluid. The present study was designed to confirm the immunocytochemical localization of prolactin in the rat lacrimal gland, to determine the source of the prolactin, and to evaluate the acute effects of prolactin on lacrimal secretory function. We have confirmed that prolactin-like immunoreactivity is present in secretory vesicles of acinar cells of male and female Sprague-Dawley rats. Prolactin message was present at detectable levels in RNA extracts of lacrimal glands from males, indicating that at least a component of the prolactin-like immunoreactivity was the product of synthesis within the lacrimal glands. Crude membrane fractions from acini isolated from males bound 43.1 +/- 3.2 femtomoles prolactin/mg protein (mean +/- standard error of the mean; n = 6), which was significantly (P less than 0.01) more than comparable fractions from females (15.4 +/- 2.4 fmoles/mg protein, n = 6). Preincubating membranes at 65 degrees for 20 min to release endogenous ligands increased prolactin binding to 84.8 +/- 20.8 fmoles/mg protein for males and 63.8 +/- 17.4 fmoles/mg protein for females (P greater than 0.1), suggesting that, on average, similar numbers of receptors are expressed in acinar cells of male and female rats but a larger fraction of the receptors is occupied by endogenous prolactin-like peptides in females. Because prolactin binding triggers prolactin receptor internalization in various cell types, we propose that the prolactin-like immunoreactivity in lacrimal acinar cells of females has been accumulated from the circulation, while the immunoreactivity seen in males results, at least in part, from de novo synthesis. Ovine prolactin at concentrations of 10-20 ng/ml inhibited carbachol-induced peroxidase release by 19.6% +/- 6.9% (n = 8, P less than 0.02) but failed to alter peroxidase release in the absence of carbachol. These observations suggest that prolactin might function as an endocrine, paracrine, or autocrine modulator in the lacrimal gland.
Animals↗
Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands.
The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.
Animals↗
pH-sensitive anion exchanger in rat lacrimal acinar cells.
Basolateral membranes from rat lacrimal acinar cells contain Na(+)-H+ and Cl(-)-HCO3- antiport activities [Invest. Ophthalmol. Visual Sci. 28: 1726-1729, 1989; Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G367-G373, 1988]. This study evaluated factors involved in coupling ion fluxes through these antiporters. 22Na+ flux into acini isolated from rat exorbital glands was 94 +/- 6 nmol.mg-1.min-1, and it was accelerated threefold by 10(-5) M carbachol; neither resting nor stimulated influx was affected by bumetanide. It is, therefore, likely that a portion of the carbachol-dependent Na+ influx is mediated by Na(+)-H+ antiporters. 36Cl- flux into Cl(-)-loaded, unstimulated acini was 275 +/- 21 nmol.mg-1.min-1; Cl- flux into HCO3(-)-loaded acini was 204 +/- 2; Cl- flux into acini loaded with both Cl- and HCO3- was 253 +/- 32; and influx in the absence of exchangeable intracellular anions was 176 +/- 13. Therefore, Cl(-)-Cl- self-exchange represented the major component of anion exchanger-mediated Cl- flux into resting cells. As pHi was increased above 7.2 by potassium-nigericin pH clamping, Cl- fluxes into Cl(-)- and HCO3(-)-containing acini, but not into Cl(-)-depleted acini, were significantly accelerated. SITS completely abolished the pHi-activated increment of Cl(-)-Cl- exchange. Carbachol increased Cl- unidirectional flux into Cl(-)-loaded cells by 25% (P less than 0.1), apparently as a result of Na(+)-H+ antiporter-mediated cytoplasmic alkalinization.(ABSTRACT TRUNCATED AT 250 WORDS)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Secretagogue-induced redistributions of Na,K-ATPase in rat lacrimal acini.
Lacrimal acinar cells secrete macromolecular products in an approximately isotonic, sodium chloride (NaCl)-rich fluid. The mechanisms of macromolecular product secretion depend in part on a recycling traffic of membrane constituents between the Golgi complex and the apical plasma membrane. In contrast, the acinar cell's mechanisms for secreting Na+ and Cl- depend largely on the fluxes of these ions through transporters expressed in the apical and basal-lateral membranes. In addition to accelerating the recycling of secretory vesicle membrane constituents, the cholinergic agonist carbachol also triggers a net redistribution of sodium potassium adenosine triphosphatase (Na,K-ATPase) ion pumps between Golgi-associated pools and the basal-lateral plasma membranes (Yiu SC, et al: J Membrane Biol 102:185, 1988). In the present study, acinar preparations from rat lacrimal glands were stimulated with either carbachol, epinephrine, or isoproterenol. All three agonist stimulated release of the secretory protein lactoperoxidase, but only carbachol significantly accelerated Na+ undirectional influx. Subcellular fractionation analyses of resting and stimulated preparations indicated that carbachol caused a significant translocation of Na,K-ATPase activity from a Golgi-associated compartment to the basal-lateral plasma membranes. Neither adrenergic agonist significantly increased the basal-lateral membrane Na,K-ATPase activity, but each triggered a distinct pattern of redistributions of Na,K-ATPase and the Golgi membrane marker, galactosyltransferase. The carbachol-induced augmentation of basal-lateral membrane Na,K-ATPase activity represents a mechanism by which the cell might compensate for increased Na+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Class II antigen expression by lacrimal epithelial cells. An updated working hypothesis for antigen presentation by epithelial cells.
It has been suggested that aberrant expression of Class II histocompatibility antigens (HLA) is involved in T cell activation and leads to autoimmunity. Although Class II antigen expression was found in various nonlymphoid tissues, including salivary glands, its expression on lacrimal epithelial cells has not been reported. In this study, 12 cadaver lacrimal glands were analyzed for HLA-DR and for the numbers and distributions of T suppressor cells (Ts), T helper cells (Th), B cells, and macrophages. None of these cases exhibited the high numbers of inflammatory cells, tissue damage, and fibrosis characteristic of Sjogren's syndrome. The HLA-DR-positive epithelial cells were detected in ten cases; they represented from less than 1% to more than 70% of the epithelial cells. In these ten positive cases, there were greater numbers of T cells per millimeter squared (229 +/- 94 [mean +/- the standard error of the mean]) than in the two HLA-DR-negative cases (37 +/- 1 [mean +/- range]). Three lacrimal gland specimens tested were negative for immunoglobulin (Ig) G-bearing B cells, and two of the three specimens tested had IgA-bearing cells. Acinar cells were isolated from rat and rabbit lacrimal glands and cultured overnight in serum-free media supplemented with several potential mediators of Class II antigen expression: interferon-gamma, carbachol, or isoproterenol. Freshly isolated cells did not express Class II antigens at detectable levels, but in most experiments, they began to express the antigen even in the absence of putative mediators.(ABSTRACT TRUNCATED AT 250 WORDS)
Aged↗
Isolation of enterocyte membranes.
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Animals↗
Subcellular distribution of Na+/H+ antiport activity in rat renal cortex.
Phase partitioning analyses of a brush border membrane preparation obtained with a divalent cation precipitation procedure (Am J Physiol 246:F853-F858, 1984) confirmed that Na+/H+ antiport activity was localized primarily to the brush border membrane of the rabbit proximal tubular epithelial cell. This analysis also indicated that antiport activity was associated with membrane populations that appeared to be derived from cytoplasmic structures. However, since the starting point of the analysis was a partially-purified brush border sample rather than a total membrane sample, it was not possible to discern the magnitude of the potential cytoplasmic pool of antiport activity. We have now used a three dimensional analytical fractionation procedure, based on differential centrifugation, equilibrium density gradient centrifugation, and partitioning in an aqueous polymer 2-phase system, to survey the subcellular distribution of Na/H antiport activity in rat kidney cortex. Roughly 53% of the recovered antiport activity could be assigned to a population of brush border membrane vesicles characterized by a 15-fold enrichment of maltase. An additional 26% of the recovered activity could be assigned to a group of three membrane populations whose biochemical characteristics appeared equally consistent with origins in distinct microdomains of the brush border membrane and with origins in microdomains of the Golgi complex involved in the assembly or recycling of brush border membrane constituents. Therefore, depending on the identities of membranes which contained the secondary pool of Na+/H+ antiport activity, no more than one-third of the total recoverable Na+/H+ antiport activity could be assigned to cytoplasmic membranes of the proximal tubular epithelium.
Animals↗
Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment epithelium.
We have attempted to isolate samples of apical and basal-lateral plasma membranes from cultured fetal human RPE. Cells from confluent, dome-forming cultures were disrupted with a Dounce apparatus. Nuclei and melanin granules were sedimented by centrifugation at 2600 g for 10 min. The supernates were layered over gradients of 17.5-65% sorbitol and centrifuged at 122,000 g for 5 hr. Fractions were grouped into "density windows" on the basis of their biochemical marker contents. Na,K-ATPase and alkaline phosphatase overlapped but did not precisely parallel one another, suggesting associations with two partially separated membrane populations; in density window I, alkaline phosphatase was enriched 4.3-fold, and Na,K-ATPase was enriched 1.7-fold, whereas in window II the corresponding enrichment factors were 7.7 and 6.7. These markers were well resolved from a mitochondrial marker, but they were overlapped by endoplasmic reticulum and Golgi markers. Additional density gradient centrifugations, performed after samples had been suspended in 55% sorbitol, further separated alkaline phosphatase- and Na,K-ATPase-containing membranes from endoplasmic reticulum and Golgi membranes, yielding alkaline phosphatase and Na,K-ATPase cumulative enrichment factors of 6.8 and 2.5 for the sample from window I and 9.3 and 10.9 for the sample from window II. Subsequent phase partitioning analysis of the sample from window I further enriched an alkaline-phosphatase-rich membrane population, which is believed to represent the RPE basal-lateral membranes. The sample from density window II contained two membrane populations, both enriched in Na,K-ATPase, alkaline phosphatase, and galactosyltransferase, and both of which appear to be derived from the apical plasma membrane. SDS-PAGE and Western blotting confirmed a correlation between Na,K-ATPase catalytic activity and Na,K-ATPase alpha subunit immunoreactivity.
Alkaline Phosphatase↗