Gastric H+/K(+)-ATPase: targeting signals in the regulation of physiologic function.

The H+/K(+)-ATPase of gastric parietal cells catalyzes acid secretion in the stomach. Regulation of this pump's function involves its targeting to an intracellular storage compartment, which fuses with the apical plasma membrane in response to secretagogue stimulation. The H+/K(+)-ATPase must also be returned to this intracellular storage compartment to inactivate acid secretion. Recent research suggests that a number of targeting signals and protein-protein interactions participate in governing this process.

An ion-transporting ATPase encodes multiple apical localization signals.

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.

A transmembrane segment determines the steady-state localization of an ion-transporting adenosine triphosphatase.

The H,K-adenosine triphosphatase (ATPase) of gastric parietal cells is targeted to a regulated membrane compartment that fuses with the apical plasma membrane in response to secretagogue stimulation. Previous work has demonstrated that the alpha subunit of the H, K-ATPase encodes localization information responsible for this pump's apical distribution, whereas the beta subunit carries the signal responsible for the cessation of acid secretion through the retrieval of the pump from the surface to the regulated intracellular compartment. By analyzing the sorting behaviors of a number of chimeric pumps composed of complementary portions of the H, K-ATPase alpha subunit and the highly homologous Na,K-ATPase alpha subunit, we have identified a portion of the gastric H,K-ATPase, which is sufficient to redirect the normally basolateral Na,K-ATPase to the apical surface in transfected epithelial cells. This motif resides within the fourth of the H,K-ATPase alpha subunit's ten predicted transmembrane domains. Although interactions with glycosphingolipid-rich membrane domains have been proposed to play an important role in the targeting of several apical membrane proteins, the apically located chimeras are not found in detergent-insoluble complexes, which are typically enriched in glycosphingolipids. Furthermore, a chimera incorporating the Na, K-ATPase alpha subunit fourth transmembrane domain is apically targeted when both of its flanking sequences derive from H,K-ATPase sequence. These results provide the identification of a defined apical localization signal in a polytopic membrane transport protein, and suggest that this signal functions through conformational interactions between the fourth transmembrane spanning segment and its surrounding sequence domains.

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

The NH(2)-terminus of norepinephrine transporter contains a basolateral localization signal for epithelial cells.

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT-NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH(2)-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH(2)-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH(2)-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.

The cell biology of ion pumps: sorting and regulation.

The physiologic function of an ion pump is determined, in part, by its subcellular localization and by the cellular mechanisms that modulate its activity. The Na,K-ATPase and the gastric H,K-ATPase are two closely related members of the P-type family of ion transporting ATPases. Despite their homology, these pumps are sorted to different domains in polarized epithelial cells and their enzymatic activities are subject to distinct regulatory pathways. The molecular signals responsible for these properties have begun to be elucidated. It appears that a complex array of inter- and intra-molecular interactions govern these proteins' trafficking, distribution and catalytic capacity.

Immunocytochemical localization of plasmalemmal proteins in semi-thin sections of epithelial monolayers.

We describe a technique for analysis by light microscopic immunocytochemistry of the distribution of plasmalemmal proteins in polarized epithelial cells. For this purpose, Madin Darby Canine Kidney (MDCK) cells were grown to confluency on Cytodex beads, the beads were fixed with formaldehyde, and semi-thin (0.5 micron) sections were cut at liquid nitrogen temperature on an ultracryomicrotome. The distribution of the basolaterally distributed plasmalemmal protein, Na,K-ATPase, was assessed by indirect immunofluorescence using a monospecific polyclonal antibody directed against the alpha-subunit of the Na pump. Such preparations enable epithelial monolayers to be evaluated in cross-section, thus permitting unambiguous topological assessment of apical and basolateral membrane proteins. Thus, the spatial uncertainties encountered in en face examination of membrane protein distribution in epithelia grown on solid supports are largely obviated. In addition, we describe a technique for removal of the bead matrix, which markedly reduces nonspecific background staining and improves access of reagents to the basal cell surface, thus permitting localization of basal lamina components.

The dimensions of the triangle of Koch in children.

We measured the dimensions of Koch's triangle in children with normal intracardiac anatomy to determine the relation between the size of the triangle of Koch and patient age, weight, height, and body surface area. We found that the dimensions of Koch's triangle varies significantly and directly with patient age and body habitus in this pediatric population.

Regulation of myocardial glucose uptake and transport during ischemia and energetic stress.

Myocardial glucose utilization increases in response to the energetic stress imposed on the heart by exercise, pressure overload, and myocardial ischemia. Recruitment of glucose transport proteins is the cellular mechanism by which the heart increases glucose transport for subsequent metabolism. Moderate regional ischemia leads to the translocation of both glucose transporters, GLUT4 and GLUT1, to the sarcolemma in vivo. Myocardial ischemia also stimulates 5'-adenosine monophosphate-activated protein kinase, which may be a fuel gauge in the heart and other tissues signaling the need to turn on energy-generating metabolic pathways. Pharmacologic stimulation of this kinase increases cardiac glucose uptake and transporter translocation, suggesting that it may play an important role in augmenting glucose entry in the setting of ischemic or energetic stress. Thus, recent work has provided insight into the cellular and molecular mechanisms responsible for glucose uptake during energetic stress, which may lead to new approaches to the treatment of patients with coronary artery disease.

Nongastric H+,K+-ATPase: cell biologic and functional properties.

Several members of the H+,K+-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H+,K+-ATPase as well as a number of nongastric H+,K+-ATPase isoforms. Physiological studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. Although much has been learned about the pattern of H+,K+-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties of renal H+,K+-ATPases both in vitro and in situ. Our analysis of ion fluxes driven by a nongastric H+,K+-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H+,K+-ATPase isoforms may make diverse contributions to renal cation transport. We find that the activities of renal H+,K+-ATPases in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H+,K+-ATPase beta-subunit. Transgenic mice expressing a version of this protein in which the signal has been disabled show constitutively active renal K resorption. The identities of the H+,K+-ATPase isoforms that are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established.

The effect of premolar extractions on the soft-tissue profile in adult African American females.

The present study was designed to evaluate the effect of four first premolar extractions on the soft tissue profile in African American patients. Pretreatment and posttreatment cephalograms of 28 adult female patients were assessed. The data were subjected to ANOVA, and correlation coefficients were performed between the significantly different dental and soft tissue variables. Variables that showed correlation at r value of greater than 0.6 were subjected to a stepwise multiple regression. The results of the study indicate that retraction of the lower lip correlates with retraction of both maxillary and mandibular anterior teeth. A ratio of 1.2:1 [corrected] was obtained between mandibular incisor retraction and retraction of the lower lip. The relationship between the upper lip and retraction of maxillary incisors was not significant. A ratio of 1.75:1 [corrected] was attained between maxillary incisor retraction and upper lip change. The upper lip correlated most strongly with lower lip retraction. Mandibular incisor angulation was the only hard-tissue variable that could be used as a predictor in a regression model to explain lip response to orthodontic therapy. Changes in the maxillary complex were more difficult to predict because of the complex nature of the soft-tissue integument and the details of muscle tension and soft-tissue tone that were lost by conversion of a three-dimensional structure into a roentgenographic cephalogram. A significant profile change did occur following the extraction of four first premolars and subsequent orthodontic therapy.

Sex differences in cerebral blood flow following chorioamnionitis in healthy term infants.

OBJECTIVE: Sex is an important determinant of neonatal outcomes and may have a significant role in the physiologic response to maternal chorioamnionitis. Our goal was to determine cerebral blood flow (CBF) parameters by sex and subsequent neurodevelopment in healthy term infants exposed to chorioamnionitis. STUDY DESIGN: CBF by Doppler ultrasound in anterior and middle cerebral (ACA, MCA) and basilar arteries were analyzed for time-averaged maximum velocity (TAMX) and corrected resistive index in 52 term control and chorioamnionitis-exposed infants between 24 and 72 h after birth. Placental pathology confirmed histologic evidence of chorioamnionitis (HC). Bayley Scales of Infant Development-III were administered at 12 months. RESULT: HC male infants had significantly greater TAMX in the MCA and lower mean MCA and ACA resistance than HC females. Abnormal CBF correlated negatively with neurodevelopmental outcome. CONCLUSION: CBF is altered in term infants with histologically confirmed chorioamnionitis compared with control infants with sex-specific differences.

Ion pump sorting in polarized renal epithelial cells.

The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

Ion pumps in epithelial cells: sorting, stabilization, and polarity.

The P-type family of ion-transporting ATPases includes all of the isoforms of Na(+)-K(+)-adenosinetriphosphatase (ATPase), the plasma membrane and organellar Ca(2+)-ATPases, gastric H(+)-K(+)-ATPase, and the recently characterized nongastric H(+)-K(+)-ATPases, among others. In epithelial cells, members of this pump family generate the cation gradients responsible for vectorial fluid and solute transport. To carry out this function, each P-type ATPase must be restricted to a specific membrane domain. Newly synthesized ion pumps must be sorted to their appropriate destinations and retained there after their delivery. Recently, progress has been made toward understanding the signals and targeting pathways that epithelial cells employ to generate anisotropic pump distributions. The mechanisms involved in generating these pump distributions may serve as well to regulate pump function.