Microvillar Region Research Articles

Intestinal brush border formation requires a TMIGD1-based intermicrovillar adhesion complex.

Intestinal epithelial cells absorb nutrients through the brush border, composed of dense arrays of highly ordered microvilli at their apical membranes. A protocadherin-based intermicrovillar adhesion complex localized at microvilli tips mediates microvilli packing and organization. Here, we identified a second adhesion complex localized at the proximal base region of microvilli. This complex contained the immunoglobulin superfamily member TMIGD1, which directly interacted with the microvillar scaffolding proteins EBP50 and E3KARP. Complex formation with EBP50 required the activation of EBP50 by the actin-binding protein ezrin and was enhanced by the dephosphorylation of Ser162 in the PDZ2 domain of EBP50 by the phosphatase PP1α. Binding of the EBP50-ezrin complex to TMIGD1 enhanced the dynamic turnover of EBP50 at microvilli. Enterocyte-specific inactivation of Tmigd1 in mice resulted in microvillar blebbing, loss of intermicrovillar adhesion, and perturbed brush border formation. Thus, we identified a second adhesion complex in microvilli and propose a mechanism that promotes microvillar formation and dynamics.

Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains.

Cordon Bleu (Cobl) is a WH2-containing protein believed to act as an actin nucleator. We show that it has a very specific localization in epithelial cells at the basal region of microvilli, a localization unlikely to be involved in actin nucleation. The protein is localized by a central region between the N-terminal COBL domain and the three C-terminal WH2 domains. Ectopic expression of Cobl shortens apical microvilli, and this requires functional WH2 domains. Proteomic studies reveal that the COBL domain binds several BAR-containing proteins, including SNX9, PACSIN 2/syndapin 2, and ASAP1. ASAP1 is recruited to the base of microvilli by binding the COBL domain through its SH3. We propose that Cobl is localized to the basal region of microvilli both to participate in length regulation and to recruit BAR proteins that associate with the curved membrane found at the microvillar base.

Increased expression of ERp57 in rat oocytes during meiotic maturation is associated with sperm–egg fusion

Oocyte meiotic maturation is a developmental transition that starts during germinal-vesicle breakdown and ends at the arrest in metaphase of meiosis II. This transition is associated with changes to both the proteins that are synthesized and the abundance/distribution of post-translational modifications that are crucial for subsequent fertilization and embryogenesis. Here, we isolated and cultured rat oocytes in vitro during both metaphase of meiosis I (MI) and meiosis II (MII) stages, respectively, and then compared their proteomic profiles by high-resolution, two-dimensional gel electrophoresis (2DE) followed by mass spectrometry. We found that the expression of five proteins was up-regulated while six proteins were down-regulated when comparing MI to MII oocytes. The expression of ERp57, an endoplasmic reticulum chaperone, underwent a dramatic increase between MI and MII oocytes, and became concentrated in a dome-shaped area of the cell surface within the microvillar region. A similar profile was observed during spermatogenesis, and sperm ERp57 eventually localized to the head and flagellum surfaces, finally ending in the equatorial region of acrosome-reacted sperm. Given the localization pattern, we tested and found that a polyclonal antiserum created against recombinant rat ERp57 significantly inhibited spermatozoa from penetrating zona pellucida-free oocytes without affecting either sperm motility or the acrosome reaction. These results indicate that ERp57 expression on oocytes, and possibly sperm, plays an important physiological role during sperm-egg fusion.

SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition

Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.

Biochemical activity and multiple locations of particulate guanylate cyclase in Rhyacophila dorsalis acutidens (Insecta: Trichoptera) provide insights into the cGMP signalling pathway in Malpighian tubules

In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs).However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system.

Metabolic fate of monovalent and multivalent antibodies of Heymann nephritis following formation of surface immune complexes on glomerular epithelial cells.

Heymann nephritis of the rat has several similarities to human idiopathic membranous glomerulo-nephropathy, and therefore serves as an animal model for study of the human disease. The disease process in the rat is initiated by autoantibodies forming in situ immune deposits on the surface of the glomerular epithelial cell in the lamina rara externa region of the glomerular basement membrane. It is now understood that multispecific antibodies (like anti-gp600) form the nephritogenic deposits which cause proteinuria, while MoAb (like anti-gp330, anti-90 kD and anti-gp70) form only transient deposits which do not give rise to proteinuria. The reasons for differences between pathologic effects of mono- and multispecific antibodies are not known. Following characterization of putative antigens of Heymann nephritis by immunofluorescence, immunogold and immunoblot techniques, we have investigated the metabolic handling of a multispecific (anti-600), monospecific antibody (anti-gp70) and a MoAb (anti-gp330) by the cultured glomerular epithelial cell to gain an insight into the mechanism of nephritogenicity of multispecific antibody. Anti-gp600 reacted to multiple antigens in the 330-kD and 70-kD regions; anti-gp70 reacted to only the 70-kD region. Ultrastructurally, all three types of antigens were present on the plasma membrane, concentrated in the microvillar region. The cells were incubated with the antibodies, and clearance of the antibodies from the cells was evaluated. Following binding, all three antibodies were internalized by the cells. However, it was found that monoclonal anti-gp330 MoAb was cleared most rapidly from the cell (t1/2 5 min), followed by anti-gp70 (t1/2 30 min). Anti-gp600 was cleared at four times slower rate than anti-gp70 (t1/2 2h) by the cell. While anti-gp330 MoAb and monospecific anti-gp70 antibody were expelled from the cell in 25-32% digested form, the multispecific anti-gp600 was digested to the extent of only 5-8%. No immune complexes were detected in the medium with any of the three antibodies. The shed label was in intact IgG form. It is concluded that multispecific antibodies of Heymann nephritis are nephritogenic because of their slower clearance and digestion and therefore, higher accretion rate on the surface of the glomerular epithelial cell. The differential handling of the antibodies by the glomerular epithelial cell offers an explanation for the in vivo differences in the nephritogenicity of these antibodies observed earlier.

The SOCS Box Protein STOPS Is Required for Phototransduction through Its Effects on Phospholipase C

Phosphoinositide-specific phospholipase C (PLC) isozymes play roles in a diversity of processes including Drosophila phototransduction. In fly photoreceptor cells, the PLCbeta encoded by norpA is critical for activation of TRP channels. Here, we describe a PLCbeta regulator, STOPS, which encodes a SOCS box protein. Mutation of stops resulted in a reduced concentration of NORPA and a defect in stopping signaling following cessation of the light stimulus. NORPA has been proposed to have dual roles as a PLC- and GTPase-activating protein (GAP). We found that the slow termination resulting from expressing low levels of wild-type NORPA was suppressed by addition of normal amounts of an altered NORPA, which had wild-type GAP activity, but no PLC activity. STOPS is the first protein identified that specifically regulates PLCbeta protein concentration. Moreover, this work demonstrates that a PLCbeta derivative that does not promote TRP channel activation, still contributes to signaling in vivo.

EPI64 regulates microvillar subdomains and structure

EPI64 is a TBC domain–containing protein that binds the PDZ domains of EBP50, which binds ezrin, a major actin-binding protein of microvilli. High-resolution light microscopy revealed that ezrin and EBP50 localize exclusively to the membrane-surrounded region of microvilli, whereas EPI64 localizes to variable regions in the structures. Overexpressing EPI64 results in its and EBP50's relocalization to the base of microvilli, including to the actin rootlet devoid of ezrin or plasma membrane. Uncoupling EPI64's binding to EBP50, expression of any construct mislocalizing its TBC domain, or knock down of EBP50 results in loss of microvilli. The TBC domain of EPI64 binds directly to Arf6-GTP. Overexpressing the TBC domain increases Arf6-GTP levels, and expressing dominant-active Arf6 results in microvillar loss. These data reveal that microvilli have distinct cytoskeletal subdomains and that EPI64 regulates microvillar structure.

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 +/- 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 +/- 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP(3)R(3))(,) alpha-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP(3)R(3), PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP(3)R(3), alpha-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of approximately 3 mum-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10-12 mum into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm–egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme- like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm–egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant ( P ≤ 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm–oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 μM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm–egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

Does membrane trafficking play a role in regulating the sodium/hydrogen exchanger isoform 3 in the proximal tubule?

The proximal tubule sodium/hydrogen exchanger continuously reabsorbs the bulk of the filtered sodium, controlling salt delivery to the distal nephron which is critical for tubuloglomerular feedback autoregulation and for fine control of salt excretion in the distal nephron. This review focuses on recent studies of the mechanisms of regulation of sodium transport in the proximal tubule, and addresses whether results from studies in proximal tubule cell lines are applicable to the proximal tubule in situ. Recent in-vivo studies provided evidence that sodium/hydrogen exchanger isoform 3 can move into and out of the apical microvilli accompanied by parallel changes in renal sodium transport: the exchanger is retracted from the microvilli in response to hypertension, parathyroid hormone or dopamine treatment and moved into the microvilli in response to sympathetic nervous system stimulation, puromycin aminonucleoside induced nephritic syndrome, and insulin treatment. Studies in cultured opossum kidney proximal tubule cells provided evidence for clathrin coated vesicle mediated, dynamin dependent, cytoskeleton dependent internalization of sodium/hydrogen exchanger isoform 3 from the surface to an endosomal pool in response to dopamine or parathyroid hormone. In the intact proximal tubule there is evidence for a two-step internalization process: (1) from villi to the intermicrovillar cleft region and (2) to a higher density membrane pool that may be either below the microvilli or deep in intermicrovillar clefts. Recent studies have described a significant inactive pool of the exchanger in the intermicrovillar region in vivo that may serve as a storage and recruitable pool. The molecular mechanisms responsible for increasing or decreasing sodium transport in the proximal tubule appear to include redistribution of sodium/hydrogen exchanger isoform 3 to or from the microvillar region. Detailed studies in cultured proximal tubule cell lines provide evidence for endocytosis and exocytosis of the exchanger dependent on cytoskeleton and clathrin coated vesicles. In vivo, the apical membrane is differentiated into discrete villar and intermicrovillar membrane domains and the intermicrovillar domain, not observed in cultured cells, may serve as a recruitable storage pool for sodium/hydrogen exchanger isoform 3.

Effects of perturbation of cell polarity on molecular markers of sperm-egg binding sites on mouse eggs.

The mouse germinal vesicle (GV)-intact oocyte is a symmetric cell, with the GV centrally localized and with components of the plasma membrane and cortex symmetrically distributed around the periphery of the oocyte. During oocyte maturation, two distinct regions of the egg plasma membrane and cortex develop: the amicrovillar region overlying the meiotic spindle and the microvillar region. The development of this polarity is significant, since sperm bind to and fuse with the microvillar region. We are interested in the development of egg polarity and have characterized the localizations of several markers for egg polarity in normal metaphase II eggs and GV-intact oocytes. The asymmetric distributions of these markers (including actin, cortical granules, binding sites for the sperm proteins fertilin alpha and fertilin beta, and two different beta(1) integrin epitopes) develop during oocyte maturation in vitro, and this polarity can be perturbed by treatments that disrupt the actin microfilaments or microtubules. In addition, immunoelectron microscopy reveals that binding sites for recombinant fertilin beta are specifically localized to the microvillar region, suggesting that the binding sites for this sperm ligand are either specifically localized or activated in this region. These results indicate that structural remodeling of the mouse egg plasma membrane is accompanied by molecular remodeling, resulting in the localization or activation of specific molecules in subdomains of the plasma membrane.

The Removal of the Sperm Perinuclear Theca and Its Association with the Bovine Oocyte Surface during Fertilization

The perinuclear theca (PT) is a unique cytoskeletal structure whose anterior part is intercalated between the inner acrosomal membrane and the nuclear envelope of the mammalian sperm head and is important for spermiogenesis and stabilization of sperm structures (Oko and Maravei,Biol. Reprod.50, 1000–1014, 1994; Oko and Maravei,Microsc. Res. Tech.32, 520–532, 1995). Using immunofluorescence labeling of inseminated bovine oocytes and serial sectioning–ultrastructural analysis, we demonstrate that the PT is removed from the sperm nucleus following the loss of the sperm plasma membrane and the interaction of oocyte cortex with the PT. These events precede the development of the male pronucleus. The removal of the PT involves the elongated oocyte microvilli, rich in actin microfilaments, since it can be blocked by the microfilament-disrupting drug cytochalasin B. Reduction of disulfide bonds, which is a major factor supporting the disassembly of the sperm nucleus and accessory structures during mammalian fertilization, seems to exert little effect on the PTin vitro,as evidenced by the treatment of isolated bull sperm with the disulfide bond-reducing agent dithiothreitol.In vivo,intact bull sperm microinjected into mature oocytes do not undergo disassembly of the PT. Consequently, the decondensation of the sperm nucleus does not occur. These data suggest that the binding of the PT to the oocyte microvillar region and its removal from the sperm nucleus constitute an early step in mammalian fertilization, which is required for the conversion of the sperm nucleus into a male pronucleus.

Characterization of the Binding of Recombinant Mouse Sperm Fertilin α Subunit to Mouse Eggs: Evidence for Function as a Cell Adhesion Molecule in Sperm-Egg Binding

Fertilin (previously known as PH-30) is a sperm protein that is a candidate molecule for mediating the binding and fusion of the sperm and egg plasma membranes. Fertilin is a heterodimer, with a β subunit that has a region of homology to the disintegrin family of integrin ligands and an α subunit that has a region of homology to viral fusion peptides. It has been hypothesized that fertilin β and α subunits mediate the interactions between sperm and egg plasma membranes, namely, binding and fusion, respectively. To address this hypothesis and to examine specifically the role of fertilin α in fertilization, we have expressed the predicted extracellular domain of mouse fertilin α as a bacterial fusion protein with maltose-binding protein. This fusion protein (hereafter referred to as recombinant fertilin α-EC) binds to the microvillar region ofzona pellucida(ZP)-free eggs, the region of the membrane to which sperm bind. This binding is reduced in the absence of divalent cations and is supported by Ca2+, Mg2+, or Mn2+. Eggs that have been treated with chymotrypsin bind less recombinant fertilin α-EC than do untreated eggs, suggesting that a chymotrypsin-sensitive binding site for recombinant fertilin α-EC is present on egg surfaces. Binding to eggs is also affected by the method used to remove the ZP. Finally, recombinant fertilin α-EC inhibits the binding of sperm to eggs duringin vitrofertilization of ZP-free eggs. These data are the first evidence to suggest that fertilin α can function as a cell adhesion molecule during fertilization, mediating the binding of sperm and egg plasma membranes.

Structural characterization of muscles and epithelial sheaths of the oviduct of Stomoxys calcitrans (Diptera: Muscidae).

Fine structure of both muscle and epithelial cells in the oviduct of Stomoxys calcitrans (L.) was characterized. Each tubular section of the oviduct consisted of an inner epithelial sheath enveloped by an outer network of muscle fibers that showed noticeable variation in cross-sectional thickness. Some regions consisted of a single cellular layer, whereas others were composed of two or more layers of cells. Moreover, a wide variation in muscle fiber orientation was observed, with some cells appearing circular and others appearing nearly longitudinal. The myofibrils of oviduct muscles were separated into sarcomeres of irregular alignment. The classical A (1 microns in length) and I bands of myofilaments were evident, but the Z disk consisted of discontinuous rows of dense bodies. In partially contracted muscle cells, some myofilaments of the A band passed between the dense bodies of the Z band. T-system tubules were evident and formed dyads with sarcoplasmic reticulum. The structural integrity of the oviduct was maintained by connective tissue strands between muscles and the epithelial sheath. Many of these strands consisted of basal lamina. Epithelial cells that line the inner sheath of the oviduct were cuboidal. In transection, these cells revealed a prominent apical microvillar region with large numbers of mitochondria and an extensive infolding of the basal plasma membrane.

Human taste cells express the G protein α-gustducin and neuron-specific enolase

Expression of the α-subunit of the taste-specific G protein α-gustducin and the glycolytic enzyme neuron-specific enolase (NSE) was investigated immunohistochemically in human circumvallate and foliate taste papillae. Immunofluorescence for α-gustducin was observed in taste cells of both types of papillae and exhibited two patterns of immunofluorescence, plasmalemmal and cytosolic. The plasmalemmal pattern showed intense immunofluorescence localized to the apical region, and was exhibited by most immunoreactive taste cells. In contrast, the cytosolic pattern, observed in one or two immunoreactive cells in a taste bud per section, showed immunofluorescence distributed throughout the cytoplasm. A subpopulation of α-gustducin-immunoreactive taste receptor cells, most of which exhibited the cytosolic pattern, also expressed NSE. Optical sectioning, using confocal laser scanning microscopy, demonstrated the highest level of expression of α-gustducin in the apical microvillar region of the taste cells in close apposition to the taste pore. These studies indicate conservation of epitopes of α-gustducin in humans and rats, and suggest that this G protein is associated with taste transduction in both rats and humans. The patterns of expression of α-gustducin, and coexpression with NSE, may correlate with specialized subtypes or developmental stages of taste receptor cells.

Ultrastructure of the life-cycle stages of Goussia janae (Apicomplexa, Eimeriidae), with X-ray microanalysis of accompanying precipitates

The ultrastructural features of the life-cycle stages of Goussia janae from the intestinal epithelium of the dace Leuciscus leuciscus and chub L. cephalus are described. All merogonial, gamogonial, and early sporogonial stages were localized in the microvillar region in an intracellular and extracytoplasmic position, covered by closely apposed enterocyte and parasitophorous vacuole membranes. Two types of location in the host cell were observed: (i) a more frequent "monopodial" type with a single zone of attachment to the host-cell cytoplasm, and (ii) a "spider-like" type with several isolated zones of attachment. Merozoites were formed by either ecto- or endo-merogony. Microgamonts produced elongated biflagellate microgametes at their periphery. The oocyst wall, produced exclusively by the parasite, was formed at the end of the intracellular phase of the life cycle. Exogenous sporulation resulted in the formation of elongated sporocysts with a thin sporocyst wall bearing a longitudinal suture accompanied by a narrow membranaceous veil. In the cytoplasm and cytoplasmic and parasitophorous vacuoles of the parasite, fine, dense precipitates were present. X-ray microanalysis of these precipitates from osmicated and non-osmicated samples revealed high levels of Ca and P, indicating the possible presence of hydroxyapatite.

Expression of intestinal alkaline phosphatase in human organs.

Human intestinal alkaline phosphatase was immunohistochemically identified and localized in the pancreas, liver and kidney by use of a monoclonal antibody specific for intestinal alkaline phosphatase isozyme and by amplified biotin-streptavidin staining. In all the examined organs, the intestinal isozyme was found to be localized in the epithelial cells of ducts: bile ducts in the liver, distal convoluted tubules and collecting tubules in the kidney and ducts in the secretory epithelium in the pancreas. In the liver the antibody also stained some sinus-lining cells. In all the examined organs the endothelial cells of the capillaries and some vessels were stained. By use of immunoelectron microscopy, intestinal alkaline phosphatase was, as expected, found to be localized to the microvillar region of the small intestine. The isozyme was abundantly expressed in the apical area of the microvilli and in membrane remnants in the fuzzy coat. Capillaries and vessels in the submucosa were also stained, as well as small vesicles in the endothelial cells. The present investigation demonstrates the expression and localization of the intestinal alkaline phosphatase in several organs, though previously believed to be expressed only in the intestine.

Characterization of ischemia-induced loss of epithelial polarity

Total renal ischemia for various time intervals (0-50 min) resulted in the rapid and duration-dependent redistribution of polarized membrane lipids and proteins in renal proximal tubule cells. Following only 15 min of ischemia, apical membrane enrichment of NaK-ATPase, normally a basolateral membrane (BLM) enzyme, had increased (1.6 +/- 0.6 vs. 2.9 +/- 1.2, P less than 0.01). In vivo histochemical localization of NaK-ATPase showed reaction product throughout the apical microvillar region. PTH-stimulatable adenylate cyclase, another BLM protein, was also found in ischemic but not control apical membrane fractions. One dimensional SDS-PAGE showed four bands, present in control BLM and ischemic apical membranes, which could not be found in control apical membrane fractions. Immunohistochemical localization of leucine aminopeptidase (LAP) showed the enzyme was limited to the apical domain in control cells. Following ischemic injury (50 min), LAP staining could be seen within the cell and along the BLM. Following 24 hr of reperfusion, the BLM distribution of LAP was further enhanced. With cellular recovery from ischemic injury (5 days), LAP was again only visualized in the apical membrane. Duration-dependent alterations in apical and BLM lipids were also observed. Apical sphingomyelin and phosphatidylserine and the cholesterol-to-phospholipid ratio decreased rapidly while apical phosphatidylcholine and phosphatidylinositol increased. Taken together, these results indicate renal ischemia causes rapid duration-dependent reversible loss of surface membrane polarity in proximal tubule cells.

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland.

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.