Localization of the Na-K pump in turtle retina.

The kinetics of ouabain binding to Na-K pump and the distribution of pump sites were examined in the retina of Pseudemys scripta elegans. Binding to retinal slices followed bimolecular kinetics characterized by a KD of 1.5 X 10(-6) M and a maximum binding capacity of 11.2 X 10(-8) mol g-1 of protein. Quantitative autoradiography of slices revealed a high concentration of bound ouabain in the inner segment, outer plexiform, inner plexiform and optic nerve layers, and correspondingly, a low level of binding in layers containing cell bodies. In the few instances that outer segments remained attached to cones, little or no binding to outer segments was observed. The membrane density of inner segment binding sites was measured by combining autoradiographic measurements of pump site concentration with stereological measurements of membrane concentration. The densities were 6.3 and 3.7 X 10(3) sites micron-2 of cone-ellipsoid and cone-fin cell membrane, respectively. The density of Müller cell microvilli was measured similarly but in enzymatically isolated cells and found to be 600 sites micron-2 of membrane. Measurements of Measurements of cone-ellipsoid pump site density in enzymatically isolated cones were not different from measurements in slices. Calculation of Na-K pump site turnover number for the cone inner segment from pump site densities and published dark current measurements yielded a value of 30 Hz.

Quantitative localization of Na-K pump sites in the frog sacculus.

The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [3H]ouabain autoradiography. Excised tissue was incubated for 10-30 min (23 degrees C) in micromolar concentrations of high specific activity [3H]ouabain (14-70 muCi ml-1, 5-15 Ci mmol-1), washed for 30 min (4 degrees C), then rapidly frozen (-175 degrees C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K+ (50 mM) in the incubation and (2) low specific activity [3H]ouabain (1 mM, 0.013-0.025 Ci mmol-1) indicated negligible nonspecific binding of the [3H]ouabain. Measurable levels of specific [3H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [3H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [3H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals. Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites micron-3 X 10(3), means +/- S.E.M.): saccule macula, 1.9 +/- 0.2; saccule perimacula, 1.1 +/- 0.1; saccule wall, 2.3 +/- 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (Sv). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and stereological data. The following values were obtained (sites micron-2 +/- 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites micron-2 +/- 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.

Quantitative localization of Na-K pump site in frog inner ear dark cells.

The Na-K pump site distribution within the dark cells of the frog inner ear was examined with quantitative freeze-dry [3H]ouabain autoradiography. Control experiments revealed that ouabain binding was specific. Na-K pumps were located in the basolateral dark cell plasma membrane and were distributed nonuniformly across the epithelial apical-basal axis. The highest concentration (sites per volume) was found over the basal region and the lowest over the apical region. The average pump site concentration for dark cells from four animals was 25 X 10(3) sites/micron3. Stereological analysis of conventionally fixed tissue revealed that the plasma membrane area per volume (Sv) was also nonuniform across the apical-basal axis with the highest surface to volume ratio in the basal region. The average Sv for two dark cell regions was 5.5 micron2/micron3. Combination of autoradiography and stereology revealed that the pump site density (sites per membrane area) was independent of position along the apical-basal axis and was equal to 4500 +/- 25%, a value close to the upper limit as determined by the diameter of the isolated Na-K-ATPase.

[3H]ouabain autoradiography of frog retina.

The kinetics and distribution of ouabain binding in retinas of Rana pipiens were examined quantitatively by scintillation counting and freeze-dry autoradiography. The time-course of binding at several concentrations was consistent with a bimolecular reaction. Estimated equilibrium binding levels gave a Michaelis-Menton relationship with a Km = 8.3 x 10(-8) M and a maximum binding level (Bmax) = 4.4 x 10(-8) mol/g protein. The distribution of binding sites measured autoradiographically varied considerably between layers. The photoreceptor, inner plexiform, and optic nerve fiber layers exhibited the heaviest binding. Within the photoreceptor layer, binding was nonuniform. Binding in the outer segment decreased distally, averaging approximately 4% of that in the proximal receptor layers (Bmax = 4.6 x 10(-6) M). The origin of the outer segment activity is uncertain at light microscope resolution, as it may be a result of inner segment calyceal processes. Binding within the proximal receptor layers was also nonuniform. Several peaks were observed, with those at the inner segment and synaptic layers being especially noticeable. Assuming an absence of glial cell binding in the proximal receptor layers, we calculated there to be 13 x 10(6) ouabain or Na+,K+ pump sites per rod receptor. Limited measurements suggest a Bmax of approximately 8 x 10(-6) M for the inner plexiform layer.

Transport of purine nucleotides and nucleosides by in vitro rabbit ileum.

The kinetics of [14C]ATP and [14C]adenosine entry into rabbit ileum were measured using 1-min mucosal exposures. ATP influx was the sum of a linear (P = 1.02 X 10(-3) cm/min) and nonlinear component (Jm = 4.07 nmol/min cm2, Km = 0.31 mM). Adenosine yielded quantitatively similar results. The kinetics of [gamma-32P]ATP entry were only linear (P = 0.55 X 10(-3) cm/min). These data indicate that at least 85% of the terminal phosphate of ATP is hydrolyzed during transport. Inhibitory measurements indicate that ring-labeled ATP and adenosine compete for a common entry path; however, adenosine does not inhibit the saturable component of [14C]ATP to the degree expected of a simple carrier-mediated system. Adenosine (5 mM) produced no inhibition of [gamma-32P]ATP (0.1-1.0 mM) entry. Measurement of hydrolysis during incubation indicates a negligible fraction of uptake results from labeled adenosine released into the bulk solution. Hydrolysis of ATP followed by a preferred uptake of the adenosine released at the membrane surface probably accounts for most of the observed transport. [14C]ATP entry depended neither on [Na+], [Mg2+], nor [Ca2+] of the bulk solution. Structural requirements for the saturable pathway were also investigated. Autoradiography of [3H]ATP confirmed that the labeled material entered the epithelial cells and that uptake was nonuniform among cells.

Teleost chloride cell. II. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments.

The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport.

High-resolution autoradiography of 3H-ouabain binding in salt transporting epithelia.

The distribution of salt transporting sites in epithelia is a problem of special interest to electrolyte metabolism. Autoradiography of freeze-dried plastic embedded tissue was used to localize 3H-ouabain in rabbit small intestine and renal medulla and gills of killifish. Resolution approaching theoretical limits for tritium at the light microscope level was achieved. The results of several tests are presented which demonstrate the resolution of the method and the specificity of the observed ouabain binding patterns. A large uptake of ouabain was found along the basolateral membranes of the enterocyte, thick ascending tubules and chloride cells (fish gill).

Mercurial perturbation of brush border membrane permeability in rabbit ileum.

The sulfhydryl reagents Hg++ and p-chloromercuribenzene sulfonate (PCMBS) at millimolar concentrations reduced the mucosal entry of sugars and amino acids to 80-90% of control levels within several minutes. Based on 50% levels of inhibition, Hg++ proved to be 20 and 10 times as potent as PCMBS in blocking sugar and amino acid transport, respectively; both systems were equally sensitive to Hg++. Concomitant measurements of 203Hg-PCMBS demonstrated a progressive tissue uptake, which, unlike inhibition, did not saturate with increasing times of exposure, thus suggesting appreciable epithelial entry with prolonged exposures (less than 30 min at 1 mM). At similar dose levels, no significant change in mucosal Na+ entry was detected. Inhibition was not reversed by 30-min washes in cholinesalt solutions; however, 10-min exposures to dithiothreitol [10 mM] reversed Hg++ and PCMBS inhibition by 40 and 100%, respectively. Alanine and galactose influx kinetics measured at concentrations of 0-100 mM exhibited a linear or diffusional entry component in addition to the usual saturable component for both control and Hg++-treated ileum. The presence of a diffusional term in the flux equation resulted in two sets of parameters giving nearly equal fits to these measurements. It was shown that this ambiguity could be resolved by determining the change in diffusional entry with Hg++ treatment. A 20-min exposure to 0.5 mM Hg++ caused an increase from 0.050 and 0.045 to 0.064 and 0.070 cm/hr in the coefficient of diffusional entry for alanine and galactose, respectively. On the basis of this increase, it is argued that Hg++ causes a decrease in Jmax and little change in Km for both transport mechanisms. This analysis has a general bearing on kinetic measurements of transport in which passive fluxes are comparable to those mediated by specific pathways. The alanine results are consistent with bimolecular reactions between mercurial and two membrane inhibitory sites, each producing approximately 40% reduction in membrane translocation rate. The estimated reaction rate constants were 5.0 and 0.4 mM min.

Radioautographic localization of sodium pump sites in rabbit intestine.

Direct demonstration of the cellular location of sodium pumping constitutes a key problem in the solution of intestinal sodium absorption. Utilizing silicone-impregnated epoxy sections of freeze-dried, osmium-fixed tissue, ouabain-(3)H and inulin-(3)H light microscope radioautographs have been produced which show that: lateral but not brush border membranes of rabbit small intestine bind ouabain-(3)H (high specific activity) with an affinity so great that a subsequent washing in ouabain-free medium has little effect on binding; lateral membrane binding is not apparent with low specific activity ouabain-(3)H, and inulin-(3)H and ouabain-(3)H (low specific activity) in the cores of the villi do not equilibrate with the intercellular spaces. Preliminary tracer measurements of ouabain-(3)H and inulin-(14)C spaces also agree with these findings As ouabain is a specific inhibitor of active sodium transport, these observations provide direct support for the view that lateral membrane pumping of sodium into the intercellular spaces causes, through osmotic forces on water, a flow of fluid out of these spaces into the interstitium.

Quantitative radioautography of sugar transport in intestinal biopsies from normal humans and a patient with glucose-galactose malabsorption.

Both galactose accumulation and phlorizin binding by columnar epithelial cells have been investigated in vitro with a recently developed technique for high-resolution, plastic-section radioautography which is particularly suited to small quantities of biopsy tissue. Grain density analysis of the radioautographs provides definitive support for the view that the cellular mechanisms underlying glucose-galactose absorption in laboratory animals are fully applicable to the small intestine of man. Even the number of sugar carriers at the microvillar membrane appears similar and the major quantitative difference, lower affinity for phlorizin in man, correlates with the finding that phlorizin is also a less potent inhibitor of uphill, galactose transport at the microvilli. In addition, radioautographs of biopsies taken 2 yr apart from a patient with glucose-galactose malabsorption provide evidence that the cellular defect in this inborn error of transport is a persistent reduction in the number of functioning sugar carriers at the microvillar membrane.

High-resolution radioautography of phlorizin-3H in rings of hamster intestine.

Quantitative light microscope radioautographs of galactose-(3)H and phlorizin-(3)H were prepared from freeze-dried plastic-embedded hamster small intestine incubated in vitro. The usual uphill epithelial cell accumulation of galactose accompanied by a somewhat smaller lamina propria accumulation was observed in control tissue incubated 3 min in 1 mM galactose-(3)H. The addition of 5 x 10(-4)M phlorizin to the medium blocked uphill accumulation, but did not prevent galactose equilibration with the epithelial cells. The galactose content of the lamina propria was considerably less than the galactose content of the epithelial cell. Varying the phlorizin-(3)H content of the medium from 0.6 to 60 microM revealed a brush border binding of phlorizin which followed a Langmuir adsorption isotherm with a half-saturation constant of 13 microM and a maximum binding of 84 micromoles of phlorizin/liter of microvilli or 2.6 x 10(6) sites/epithelial cell. The phlorizin content of the epithelial cell compartment, excluding microvilli, never exceeded 10% that of the medium after 20 min of incubation. These findings directly support the view that phlorizin is a nontransported inhibitor which binds glucose-galactose carriers at the surface of epithelial cell microvilli.

High-resolution radioautography of galactose-3H accumulation in rings of hamster intestine.

Radioautography of water-soluble substances has posed a major technical problem for the past decade. Utilizing silicone-impregnated plastic sections of frozen-dried tissue, a quantitative method was developed for studying distribution of (3)H-labeled galactose, mannitol, and phlorizin. The content of a 2-micro band may be measured with an accuracy of +/-20% by light microscopy; radioautographs may also be prepared for the electron microscope. Results with intestinal tissue incubated 1-10 min in vitro and, then, frozen rapidly indicate that the first step in galactose absorption is uphill transport into the brush border of the columnar epithelium. Correction of galactose content for the mannitol space in the brush border suggests that the sugar pump is located at the surface of the microvilli. Further evidence for the surface locus of the glucose-galactose pump was obtained with phlorizin (next paper, reference 40). The galactose content of columnar cell cytoplasm always equalled that of microvilli and no transcellular diffusion gradient could be detected; during the first minutes of incubation, however, a gradient did exist between nucleoplasm and cytoplasm. Downhill exit of galactose from columnar cells may have proceeded either directly across basal membranes to adjacent lamina propria or indirectly via open intercellular spaces. Lastly, even in the absence of muscularis, the connective tissue of the lamina propria constituted enough of a diffusion barrier so that it served as a secondary accumulating compartment for galactose under present in vitro conditions.