Submucosal glands are the predominant site of CFTR expression in the human bronchus.

We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.

Transport ATPase cytochemistry: ultrastructural localization of potassium-dependent and potassium-independent phosphatase activities in rat kidney cortex.

A cytochemical method for the light and electron microscope localization of the K- and Mg-dependent phosphatase component of the Na-K-ATPase complex was applied to rat kidney cortex, utilizing p-nitrophenylphosphate (NPP) as substrate. Localization of K-N-ATPase activity in kidneys fixed by perfusion with 1% paraformaldehyde -0.25% glutaraldehyde demonstrated that distal tubules are the major cortical site for this sodium transport enzyme. Cortical collecting tubules were moderately reactive, whereas activity in proximal tubules was resolved only after short fixation times and long incubations. In all cases, K-NPPase activity was restricted to the cytoplasmic side of the basolateral plasma membranes, which are characterized in these neplron segments by elaborate folding of the cell surface. Although the rat K-NPPase appeared almost completely insensitive to ouabain with this cytochemical medium, parallel studies with the more glycoside-sensitive rabbit kidney indicated that K-NPPase activity in these nephron segments is sensitive to this inhibitor. In addition to K-NPPase, nonspecific alkaline phosphatase also hydrolyzed NPP. The latter could be differentiated cytochemically from the specific phosphatase, since alkaline phosphatase was K-independent, insensitive to ouabain, and specifically inhibited by cysteine. Unlike K-NPPPase, alkaline phosphatase was localized primarily to the extracellular side of the microvillar border of proximal tubules. A small amount of cysteine-sensitive activity was resolved along peritubular surfaces of proximal tubules. Distal tubules were unreactive. In comparative studies, Mg-ATPase activity was localized along the extracellular side of the luminal and basolateral surfaces of proximal and distal tubules and the basolateral membranes of collecting tubules.

Characterization of muscarinic acetylcholine receptors in the avian salt gland.

Electrolyte and fluid secretion by the avian salt gland is regulated by activation of muscarinic acetylcholine receptors (R). In this study, these receptors were characterized and quantitated in homogenates of salt gland from domestic ducks adapted to conditions of low (freshwater, FW) and high (saltwater, SW) salt stress using the cholinergic antagonist [3H]-quinuclidinyl benzilate (QNB). Specific binding of the antagonist to receptors in both FW- and SW-adapted glands reveals a single population of high affinity binding sites (KdFW = 40.1 +/- 3.0 pM; KdSW = 35.1 +/- 2.1 pM). Binding is saturable; RLmaxFW = 1.73 +/- 0.10 fmol/micrograms DNA; RLmaxSW = 4.16 +/- 0.31 fmol/micrograms DNA (where L is [3H]QNB and RL the high affinity complex). Calculated average cellular receptor populations of 5,800 sites/cell in FW-adapted glands and 14,100 sites/cell in SW-adapted glands demonstrate that upward regulation of acetylcholine receptors in the secretory epithelium follows chronic salt stress. The receptor exhibits typical pharmacological specificities for muscarinic cholinergic antagonists (QNB, atropine, scopolamine) and agonists (oxotremorine, methacholine, carbachol). In addition, the loop diuretic furosemide, which interferes with ion transport processes in the salt gland, competitively inhibits [3H]QNB binding. Preliminary studies of furosemide effects on [3H]QNB binding to rat exorbital lacrimal gland membranes showed a similar inhibition, although the diuretic had no effect on antagonist binding to rat brain or atrial receptors.

Ultrastructural localization of Na+,K+-ATPase in rat and rabbit kidney medulla.

Na+,K+-ATPase was localized at the ultrastructural level in rat and rabbit kidney medulla. The cytochemical method for the K+-dependent phosphatase component of the enzyme, using p-nitrophenylphosphate (NPP) as substrate, was employed to demonstrate the distribution of Na+, K+-ATPase in tissue-chopped sections from kidneys perfusion-fixed with 1% paraformaldehyde-0.25% glutaraldehyde. In other outer medulla of rat kidney, ascending thick limbs (MATL) were sites of intense K+-dependent NPPase (K+-NPPase) activity, whereas descending thick limbs and collecting tubules were barely reactive. Although descending thin limbs (DTL) of short loop nephrons were unstained, DTL from long loop nephrons in outer medulla were sites of moderate K+-NPPase activity. In rat inner medulla, DTL and ascending thin limbs (ATL) were unreactive for K+-NPPase. In rabbit medulla, only MATL were sites of significant K+-NPPase activity. The specificity of the cytochemical localization of Na+,K+-ATPase at reactive sites in rat and rabbit kidney medulla was demonstrated by K+-dependence of reaction product deposition, localization of reaction product (precipitated phosphate hydrolyzed from NPP) to the cytoplasmic side of basolateral plasma membranes, insensitivity of the reaction to inhibitors of nonspecific alkaline phosphatase, and, in the glycoside-sensitive rabbit kidney, substantial inhibition of staining by ouabain. The observed pattern of distribution of the sodium transport enzyme in kidney medulla is particularly relevant to current models for urine concentration. The presence of substantial Na+,K+-ATPase in MATL is consistent with the putative role of this segment as the driving force for the countercurrent multiplication system in the outer medulla. The absence of significant activity in inner medullary ATL and DTL, however, implies that interstitial solute accumulation in this region probably occurs by passive processes. The localization of significant Na+,K+-ATPase in outer medullary DTL of long loop nephrons in the rat suggests that solute addition in this segment may occur in part by an active salt secretory mechanism that could ultimately contribute to the generation of inner medullary interstitial hypertonicity and urine concentration.

The development of surface specialization in the secretory epithelium of the avian salt gland in response to osmotic stress.

Cell surface specialization, a characteristic common to most ion-transporting epithelia, was studied in the salt (nasal) gland of the domestic duck in relation to osmotic stress. Three days after hatching, experimental ducklings were given 1% NaCl to drink for 12 hr and freshwater for the remainder of each day. Control ducklings were maintained exclusively on freshwater. The fine structure of the secretory epithelium was examined on various days of the regimen. The nasal gland epithelium of the secretory lobule is composed of several types of cells. Peripheral cells, lying at the blind ends of the branched secretory tubules, are similar in both control and experimental animals at all stages of glandular development. These generative cells contain few mitochondria and have nearly smooth cell surfaces. Partially specialized secretory cells predominate in the secretory tubules of control animals and appear as transitional cells in the tubular epithelium of salt-stressed animals. These cells contain few mitochondria and bear short folds along their lateral cell surfaces. Fully specialized cells dominate the secretory epithelium of osmotically stressed ducklings. The lateral and basal surfaces of these cells are deeply folded, forming complex intra- and extracellular compartments. This vast increase in absorptive surface area is paralleled by an increase in the number of mitochondria that pack the basal compartments. The development of this fully specialized cell is correlated with the marked increase in (Na(+)-K(+))-ATPase activity in the glands of osmotically stressed birds.

Basolateral plasma membrane localiztion of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland.

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 muM ouabain (containing 5 muCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.

Structural diversity of occluding junctions in the low-resistance chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus).

The structural features of the chloride-secreting opercular epithelium of seawater-adapted killifish (Fundulus heteroclitus) were examined by thin-section and freeze-fracture electron microscopy, with particular emphasis on the morphological appearance of occluding junctions. This epithelium is a flat sheet consisting predominantly of groups of mitochondriarich chloride cells with their apices associated to form apical crypts. These multicellular groups are interspersed in an otherwise continuous pavement cell epithelial lining. The epithelium may be mounted in Ussing-type chambers, which allow ready access to mucosal and serosal solutions and measurement of electrocal properties. The mean short-circuit current, potential difference (mucosal-side negative), and DC resistance for 19 opercular epithelia were, respectively, 120.0 +/- 18.2 microA/cm2, 12.3 +/- 1.7 mV, and 132.5 +/- 26.4 omega cm2. Short-circuit current, a direct measure of Cl- transport, was inhibited by ouabain (5 micron) when introduced on the serosal side, but not when applied to the mucosal side alone. Autoradiographic analysis of [3H]-ouabain-binding sites demonstrated that Na+,K+-ATPase was localized exclusively to basolateral membranes of chloride cells; pavement cells were unlabeled. Occluding junctions between adjacent chloride cells were remarkably shallow (20-25 nm), consisting of two parallel and juxtaposed junctional strands. Junctional interactions between pavement cells or between pavement cells and chloride cells were considerably more elaborate, extending 0.3-0.5 micron in depth and consisting of five or more interlocking junctional strands. Chloride cells at the lateral margins of crypts make simple junctional contacts with neighboring chloride cells and extensive junctions with contiguous pavement cells. Accordingly, in this heterogeneous epithelium, only junctions between Na+,K+-ATPase-rich chloride cells are shallow. Apical crypts may serve, therefore, as focal areas of high cation conductivity across the junctional route. This view is consistent with the electrical data showing that transmural resistance across the opercular eptihelium is low, and with recent studies demonstrating that transepithelial Na+ fluxes are passive. The simplicity of these junctions parallels that described recently for secretory cells of avian salt gland (Riddle and Ernst, 1979, J. Membr. Biol., 45:21-35) and elasmobranch rectal gland (Ernst et al., 1979, J. Cell Biol., 83:(2, Pt. 2):83 a[Abstr.]) and lends morphological support to the concept that paracellular ion permeation plays a central role in ouabain-sensitive transepithelial NaCl secretion.

Teleost chloride cell. I. Response of pupfish Cyprinodon variegatus gill Na,K-ATPase and chloride cell fine structure to various high salinity environments.

Certain euryhaline teleosts can tolerate media of very high salinity, i.e. greater than that of seawater itself. The osmotic gradient across the integument of these fish is very high and the key to their survival appears to be the enhanced ability of the gill to excrete excess NaCl. These fish provide an opportunity to study morphological and biochemical aspects of transepithelial salt secretion under conditions of vastly different transport rates. Since the cellular site of gill salt excretion is believed to be the "chloride cell" of the branchial epithelium and since the enzyme Na,K-ATPase has been implicated in salt transport in this and other secretory tissues, we have focused our attention on the differences in chloride cell structure and gill ATPase activity in the variegated pupfish Cyprinodon variegatus adapted to half-strength seawater (50% SW), seawater (100% SW), or double-stregth seawater (200% SW). The Na,K-ATPase activity in gill homogenates was 1.6 times greater in 100% SW. When 50% SW gills were compared to 100% SW gills, differences in chloride cell morphology were minimal. However, chloride cells from 200% SW displayed a marked hypertrophy and a striking increase in basal-lateral cell surface area. These results suggest that there are correlations among higher levels of osmotic stress, basal-lateral extensions of the cell surface, and the activity of the enzyme Na,K-ATPase.

Localization of Na+-pump sites in frog skin.

The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.

Overexpression of Rab3D enhances regulated amylase secretion from pancreatic acini of transgenic mice.

Rab3D, a member of the ras-related GTP-binding protein Rab family, is localized to secretory granules of various exocrine tissues such as acinar cells of the pancreas, chief cells of the stomach, and parotid and lacrimal secretory cells. To elucidate the function of Rab3D in exocytosis, we have generated transgenic mice that over-express Rab3D specifically in pancreatic acinar cells. Hemagglutinin-tagged Rab3D was localized to zymogen granules by immunohistochemistry, and was shown to be present on zymogen granule membranes by Western blotting; both results are similar to previous studies of endogenous Rab3D. Secretion measurements in isolated acinar preparations showed that overexpression of Rab3D enhanced amylase release. Amylase secretion from intact acini of transgenic mice 5 min after 10 pM cholecystokinin octapeptide (CCK) stimulation was enhanced by 160% of control. In streptolysin-O-permeabilized acini of transgenic mice, amylase secretion induced by 100 microM GTP-gamma-S was enhanced by 150%, and 10 microM Ca2+-stimulated amylase secretion was augmented by 206% of that of the control. To further elucidate Rab3D involvement in stimulus-secretion coupling, we examined the effect of CCK on the rate of GTP binding to Rab3D. Stimulation of permeabilized acini with 10 pM CCK increased the incorporation of radiolabeled GTP into HA-tagged Rab3D. These results indicate that overexpression of Rab3D enhances secretagogue-stimulated amylase secretion through both calcium and GTP pathways. We conclude that Rab3D protein on zymogen granules plays a stimulatory role in regulated amylase secretion from pancreatic acini.

Localization of sodium pump sites in frog urinary bladder.

[3H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and autoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na+ transport was completely reversible in toad bladder whereas, in frog bladder, [3H]ouabain was tightly bound and Na+ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [3H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of certain (Na+ -K+)-ATPase ligands(K+, ATP) to levels known to reduce ouabain binding. High K+ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [3H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na+ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significantly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.

Muscarinic receptor-stimulated Ca2+ signaling and inositol lipid metabolism in avian salt gland cells.

Activation of muscarinic cholinergic receptors was studied by measuring agonist-stimulated inositol lipid turnover and changes in [Ca2+]i in dissociated salt gland secretory cells. Carbachol stimulation of quin2-loaded cells results in a sustained 4-fold increase in [Ca2+]i, while incorporation of [32P]Pi into phosphatidylinositol (PI) and phosphatidate are similarly increased. [3H]Inositol phosphates, measured in the presence of Li+, increased 13-fold. The stimulated increment in [Ca2+]i required extracellular Ca2+, whereas [3H]inositol phosphate accumulation was independent of external Ca2+. Dose-response curves for carbachol-induced increments in [Ca2+]i, PI labeling, and labeled inositol phosphate release are similar, with EC50 values of 6, 4.5 and 8 microM, respectively. Dissociation constants for atropine vs. the quin2 and phospholipid responses are 0.59 +/- 0.3 nM and 0.48 +/- 0.28 nM, respectively. These cells thus provide a model system for the study of non-exocytotic secretion as a consequence of stimulated inositol lipid turnover.

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland.

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.

Differentiation of two distinct K conductances in the basolateral membrane of turtle colon.

The K conductance of the basolateral membrane of turtle colon was measured in amphotericin-treated cell layers under a variety of ionic conditions. Changing the composition of the bathing solutions changed not only the magnitude but also the physical properties of the basolateral K conductance. The results are consistent with the notion that altered ionic environments can lead to changes in the relative abundance of two different populations of K channels in the basolateral membrane, which can be differentiated on the basis of pharmacological specificity, ion selectivity, and tracer kinetics. In the following article (Germann, W. J., S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:253-274), we present evidence consistent with the hypothesis that one of these conductances was due to the same channels that give rise to the normal resting basolateral K conductance of the transporting cells, while the other was associated with experimental maneuvers that led to extreme swelling of the epithelial cells.

Resting and osmotically induced basolateral K conductances in turtle colon.

Two types of K conductance can be distinguished in the basolateral membranes of polyene-treated colonic epithelial cells (see Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:237-251). The significance of these two types of K conductance was investigated by measuring the properties of the basolateral membrane under conditions that we presumed would lead to marked swelling of the epithelial cells. We compared the basolateral conductance under these conditions of osmotic stress with those observed under other conditions where changes in cell volume would be expected to be less dramatic. In the presence of a permeant salt (KCl) or nonelectrolyte (urea), amphotericin-treated colonic cell layers exhibited a quinidine-sensitive conductance. Light microscopy revealed that these conditions were also associated with pronounced swelling of the epithelial cells. Incubation of tissues in solutions containing the organic anion benzene sulfonate led to the activation of the quinidine-sensitive gK and was also associated with dramatic cell swelling. In contrast, tissues incubated with an impermeant salt (K-gluconate) or nonelectrolyte (sucrose) did not exhibit a quinidine-sensitive basolateral conductance in the presence of the polyene. Although such conditions were also associated with changes in cell volume, they did not lead to the extreme cell swelling detected under conditions that activated the quinidine-sensitive gK. The quinidine-sensitive basolateral conductance that was activated under conditions of osmotic stress was also highly selective for K over Rb, in contrast to the behavior of normal Na transport by the tissue, which was supported equally well by K or Rb and was relatively insensitive to quinidine. The results are consistent with the notion that the basolateral K conductance measured in the amphotericin-treated epithelium bathed by mucosal K-gluconate solutions or in the presence of sucrose was due to the same channels that are responsible for the basolateral K conductance under conditions of normal transport. Conditions of extreme osmotic stress, however, which led to pronounced swelling of the epithelial cells, were associated with the activation of a new conductance, which was highly selective for K over Rb and was blocked by quinidine or lidocaine.

Muscarinic stimulation of phospholipid turnover in dissociated avian salt gland cells.

Addition of carbamylcholine to 32P-prelabeled dissociated avian salt gland cells resulted in increased turnover of phosphatidic acid, phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, which could be prevented by the inclusion of atropine. Carbamylcholine had no discernable effect on protein phosphorylation, measured either in the total preparation or in subcellular fractions. It is concluded that for the avian salt gland, no obligatory link is indicated between protein phosphorylation and either phospholipid turnover or salt secretion.

Autoradiographic localization of tritiated ouabain-sensitive sodium pump sites in ion transporting epithelia.

The freeze-dry autoradiographic method devised originally by Stirling (J Cell Biol 53:704, 1972) to localize Na+ pump sites with (3H)ouabain is reviewed. Biochemical, physiological, and autoradiographic data are discussed which establish that ouabain binding to intact tissue conforms to rigid criteria for high Na+ pump specificity. Among these are that glycoside binding exhibits saturation kinetics, ligand dependence, and close correlation with degrees of inhibition of Na+-K+-ATPase and Na+ transport. Moreover, localization of Na+ pump sites by this technique shows a cell and membrane specificity which mirrors that obtained by cytochemical and immunocytochemical methods. In addition to resolving cell-specific patterns of localization in heterogeneous tissues, the demonstration of Na+-K+-ATPase by these techniques indicates that Na+ pumps are distributed uniformly along plasmalemmal surfaces and are restricted to the basolateral interface in reabsorptive and secretory epithelia despite the opposing polarity of net transepithelial electrolyte transport.

Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus.

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.

Purification of mouse brain (Na+ + K+)-ATPase catalytic unit, characterization of antiserum, and immunocytochemical localization in cerebellum, choroid plexus, and kidney.

The denatured catalytic polypeptide of mouse brain (Na+ + K+)-adenosine triphosphatase(ATPase) was separated from microsomal membranes on polyacrylamide gels and used as an immunogen. The antiserum, characterized by immunoblots, recognizes the polypeptide corresponding to the catalytic unit in various fractions of mouse brain and cross-reacts with the catalytic unit from lamb kidney, duck salt gland, and electroplax. The same polypeptide in brain and salt gland is recognized by antiserum raised against purified lamb kidney enzyme. Light microscopy was performed with the peroxidase-conjugated second antibody method. In mouse cerebellum, immunochemical staining outlines Purkinje cell and granule cell perikarya. Intense activity is associated with regions of high synaptic content including the pericellular basket meshes and preaxonal regions of Purkinje cells and the glomeruli in the granular layer. In the molecular layer, the neuropil is diffusely reactive with distinct vertically oriented processes evident. White matter exhibits light stain deposition. Choroid plexus presents abundant reaction product only at ependymal apical surfaces, while the ependymal lining of the fourth ventricle displays little or no immunoreactivity. Specificity of the antiserum was demonstrated further in mouse kidney where staining conforms to the well-characterized localization of the enzyme along basolateral surfaces of cortical and medullary tubules. The biochemical and immunocytochemical data show the efficacy of generating antisera to brain (Na+ + K+)-ATPase using catalytic polypeptide as an immunogen.

Histamine H1 receptor-mediated Ca2+ signaling in cultured bovine corneal endothelial cells.

The corneal endothelium pumps ions and water from the stroma to the aqueous humor, maintaining corneal transparency. This report investigates the possibility that cultured corneal endothelial cells express neurohormonal Ca2+ signaling pathways employed by other epithelia to regulate transport or other cellular functions. Agonist-stimulated changes in intracellular calcium ([Ca2+]i) in single bovine corneal endothelial cells (BCEC) derived from confluent cultures were measured by microspectrofluorimetry using the Ca(2+)-sensitive probe, fura 2. Mean resting [Ca2+]i in BCEC was 46 +/- 2 nM (n = 124). The muscarinic cholinergic agonist, carbachol, did not mobilize Ca2+, whereas histamine induced a rapid increase in [Ca2+]i to initial peak levels of 549 +/- 22 nM (n = 46) at maximally stimulating doses. The initial rise in [Ca2+]i in response to histamine was dose dependent, with a minimum effective dose of 50 nM, EC50 = 0.84 mumol/l, and a maximum effective dose of 10 mumol/l. [Ca2+]i decreased from the initial peak, but then stabilized to form an agonist-dependent sustained elevation or abruptly fell back to baseline to begin oscillatory fluctuations. The initial peak was insensitive to removal of extracellular calcium (Ca2+o), whereas subsequent elevations in [Ca2+]i or sustained [Ca2+]i oscillations required Ca2+o. The amplitude of the oscillations in [Ca2+]i increased with an increase in [histamine]. However, frequency was independent of [histamine] (mean = 0.62 spikes min-1 +/- 0.06, n = 33). Histamine-induced Ca2+ mobilization was inhibited by the H1 receptor antagonist triprolidine, but was unaffected by ranitidine (H2 antagonist) or thioperamide (H3 antagonist).(ABSTRACT TRUNCATED AT 250 WORDS)