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.

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.

Elasmobranch rectal gland cell: autoradiographic localization of [3H]ouabain-sensitive Na, K-ATPase in rectal gland of dogfish, Squalus acanthias.

Specific binding of radiolabeled inhibitor was employed to localize the Na-pump sites (Na,K-ATPase) in rectal gland epithelium, a NaCl-secreting osmoregulatory tissue which is particularly rich in pump sites. Slices of gland tissue from spiny dogfish were incubated in suitable [3H]ouabain-containing media and then prepared for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative freeze-dry autoradiography at the light microscope level. Gross freezing or drying artifacts were excluded by comparison with additional aldehyde-fixed slices. Characterization experiments demonstrated high-affinity binding which correlated with Na,K-ATPase inhibition and half-saturated at approximately 5 microM [3H]ouabain. At this concentration, the normal half-loading time was approximately 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed approximately 85% of the bound [3H]ouabain to be localized within a 1-micrometer wide boundary region where the highly infolded basal-lateral cell membrane are closest to the mitochondria. These results establish that most of the enormous Na,K-ATPase activity associated with rectal gland epithelium is in the basal-lateral cell membrane facing interstitial fluid and not in the luminal membrane facing secreted fluid. Moreover, distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.

DDT: disrupted osmoregulatory events in the intestine of the eel Anguilla rostrata adapted to seawater.

The drinking of seawater and absorption of water along with sodium across the intestinal epithelium are well-known osmoregulatory events in marine teleosts. The insecticide DDT impairs fluid absorption in intestinal sacs from eels adapted to seawater. Furthermore, this functional impairment has an enzymatic basis; DDT also inhibits the (Na(+) and K(+)) activated, Mg(2+)-dependent adenosine triphosphatase in homogenates of the intestinal mucosa. Thus, the extreme sensitivity of teleosts to organochlorine pollutants may involve the disruption of osmoregulatory transport mechanisms.

Ingestion of crude oil: sublethal effects in herring gull chicks.

A single small oral dose of Kuwait or South Louisiana crude oil caused cessation of growth, osmoregulatory impairment, and hypertrophy of hepatic, adrenal, and nasal gland tissue in herring gull chicks living in a simulated marine environment. These findings suggest that ingesting crude oil causes multiple sublethal effects that might impair a bird's ability to survive at sea.

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.

Killifish opercular skin: a flat epithelium with a high density of chloride cells.

In teleosts the head region, particularly the gills, plays the key role in osmoregulatory NaCl transport, presumably by mechanisms located in the chloride cell. As interest has focused on specific mechanisms of chloride cell function, two classical preparations, the intact fish and the isolated, perfused gill, have continued to serve as the only available model systems. However, both of these preparations have severe limitations, e.g., as they are not flat sheets, they cannot be studied readily under the ideal thermodynamic conditions achieved with the short-circuit current technique. The present sutdy describes the histology and ultrastructure of a particular area of skin in the head region of both killifish (Fundulus heteroclitus) and sea raven (Hemitripterus americanus). This skin lies on the inside of the operculum and possesses a flat epithelium containing chloride cells. In the present study the identity of the chloride cell in this epithelium was definitively established with the electron microscope. Although the opercular epithelium from the marine sea raven contains few chloride cells, that from the euryhaline killifish adapted to pond water, 100%-, and 200% artificial seawater is predominately chloride cells. Significantly, the teleost gill has never been reported to contain more than 10% chloride cells. Thus the opercular skin of the killifish can serve as a model to study the adaptive role of chloride cells in euryhaline teleosts. A separate electrophysiological study has deminstrated that the short-circuit current technique can be applied to this skin.

Pathways of cycloleucine transport in killifish small intestine.

By using blocking concentrations of competitors, i.e., concentrations of other neutral amino acids that cause maximal inhibition, cycloleucine transport into slices or everted sacs of killifish (fundulus heteroclitus) small intestine could be partitioned into three pathways. One is apparently not mediated, a second is inhibited by all neutral amino acids tested (component 1), and a third, is inhibited by alpha-aminocarboxylic acids (component 2), but not by beta-alanine or taurine. Both mediated pathways were Na dependent, and each yielded a linear double reciprocal plot of initial slice uptake vs. cycloleucine concentration. Apparent Kt and Vmax values for component 1 were 0.03 mM and 33 pmol/mg tissue per 3 min, respectively; corresponding values for component 2 were 0.12 mM and 28 pmol/mg tissue per 3 min. Additional experiments with an intestinal brush border membrane vesicle preparation indicate that these mediated components reflect true differences in carrier specificity rather than the differential effects of inhibitors on metabolism or on the Na gradient that drives cycloleucine transport.

p-Aminohippuric acid transport into brush border vesicles isolated from flounder kidney.

p-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: 1) unlabeled PAH inhibited the uptake of [3H]PAH; 2)[3H]PAH efflux from the vesicles was stimulated in the presence of unlabeled PAH in the extravesicular medium; and 3) PAH influx was inhibited by 2,4-dinitrophenol (DNP) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS). D-Glucose plus a sodium gradient stimulated PAH uptake, as did a K2SO4 gradient plus valinomycin, suggesting that PAH is transported as an anion. In contrast, PAH uptake into a membrane fraction containing mainly basal-lateral plasma membranes exhibited a larger inhibition by probenecid but a smaller inhibition by unlabeled PAH and SITS. Thus, carrier-mediated transfer of PAH driven by the electrochemical potential difference for PAH is demonstrated in the brush border membrane of the flounder kidney.

Renal excretion of chlorphenol red and related organic acids in the intact flounder Pseudopleuronectes americanus.

Renal clearance experiments were performed on unanesthetized winter flounder from which bladder urine was collected continuously and caudal vein blood was sampled periodically; renal tissue was also obtained terminally for comparison of test organic acid content in vivo and after incubation in vitro. Urine flow rates and inulin U/P (urine to plasma concentration) ratios were relatively constant for a given fish and averaged 1.0 ml/hr X kg fish and 2.6, respectively. In contrast, U/P ratios for all three test acids cycled from minima of near 100 to maxima of over 1000 roughly every 24 hr when plasma concentrations of unbound acid were below 1 micron; correction of plasma protein binding was required in the case of chlorphenol red, but not PAH or Diodrast. Both in vivo and in vitro the organic acid content of renal tissue was intermediate between plasma and urine concentrations. These results demonstrate that kidneys of intact flounder exhibit the remarkable concentrative capacity for exogenous organic acids previously observed with isolated tubules and suggest that the tubular urine concentration is established in two steps by cell transport first at the peritubular and second at the luminal membrane. The anterior kidney position and the magnitude of maximal PAH and Diodrast clearances, about 1000 ml/hr X kg flounder, are consistent with most of the cardiac output returning to the heart through the renal portal circulation; a regulatory shunt bypassing the peritubular capillaries is proposed to explain cycling of organic acid clearances to minimal values.

Excretion of phenol red by the Necturus kidney.

Phenol red (phenolsulfonphthalein, PSP) is thought to be secreted by proximal kidney tubules in all vertebrates. The present study examined PSP transport by the kidney of the salamander, Necturus maculosus. In Necturus kidneys perfused with oxygenated Ringer solution, the PSP/creatinine clearance ratio was unity. Perfusion with 1 mM octanoate converted net p-aminohippurate (PAH) reabsorption to net secretion, but had no effect on PSP. In seven urethan-anesthetized Necturi, the PSP/inulin clearance ratio averaged 0.85 +/- 0.21 (SD), not significantly different from unity. Thin slices from Necturus kidneys incubated in vitro for 2 h failed to accumulate PSP; slice-to-medium (S/M) concentration ratios averaged 0.8 +/- 0.2 (n = 6). With frog kidney slices, (S/M)PSP was 9.6 +/- 1.4 (n = 6). Necturus kidney slices accumulated PAH ((S/M)PAH = 4.1 +/- 0.7) (n = 6), but uptake was not inhibited by 1 mM PSP. We conclude that Necturus kidney tubules transport PAH, but do not transport PSP. These results are consistent with the hypothesis that the organic acid secretory system in most animals involves several carriers.

Ion transport across the isolated intestinal mucosa of the winter flounder, Pseudopleuronectes americanus. I. Functional and structural properties of cellular and paracellular pathways for Na and Cl.

The isolated intestinal mucosa of the flounder, Pseudopleuronectes americanus, when bathed in a 20 mM HCO3-Ringer's solution bubbled with 1% CO2 in O2, generated a serosa-negative PD and, when short-circuited, absorbed Cl at almost 3 times the rate of Na. Reducing HCO3 to 5 mM decreased the net Cl flux by more than 60%. The following results suggest that, despite the PD, Na and Cl transport processes are nonelectrically coupled: replacing all Na with choline abolished both the PD and net Cl flux; replacing all Cl with SO4 and mannitol abolished the PD and the net Na flux; and adding ouabain (to 0.5 mM) abolished the PD and the net Cl flux. Nearly all of the unidirectional serosa-to-mucosa Cl flux (JClsm) seemed to be paracellular since it varied with PD and Cl concentration in a manner consistent with simple diffusion. JClsm was only about one-fourth of JNasm, suggesting that the paracellular pathway is highly cation-selective. The data can be explained by the following model: (i) Na and Cl uptake across the brush border are coupled 1 : 1; Na is pumped into the lateral space and Cl follows passively, elevating the salt concentration there; (ii) the tight junction is permeable to Na but relatively impermeable to Cl; and (iii) resistance to Na diffusion is greater in the lateral space (considered in its entirety) than in the tight junction. If these assumptions are correct, the serosa-negative transmural PD is due mainly to a salt diffusion potential across the tight junction and, under short-circuit condition, most of the Na pumped into the lateral space diffuses back into the luminal solution, whereas most of the Cl enters the serosal solution. Morphological features of the epithelium support this interpretation: the cells are unusually long (60 micrometer); there is little distension of the apical 12 micrometer of the lateral space during active fluid absorption; and distension distal to this region is intermittently constricted by desmosomes.

Renal handling of the polar DDT metabolite DDA (2,2-bis[p-chlorophenyl] acetic acid) by marine fish.

The renal handling of 2,2-bis(p-chlorophenyl) acetic acid (DDA) was examined in the isolated tubules of the winter flounder (Pseudopleuronectes americanus) in vitro in conjunction with clearance studies in the flounder and in the aglomerular goosefish (Lophius americanus). In vitro, both uptake studies and autoradiography showed extensive energy-dependent accumulation within the cytoplasm of tubular cells and the tubular lumen. The uptake was strongly inhibited by p-aminohippurate and chlorophenol red. A second component of uptake was insensitive to metabolic inhibitors or organic acids and represented tissue binding. In vivo, both species showed net secretion which was inhibited by probenecid. Comparison of DDT and DDA distribution and excretion emphasized the importance of the greater water solubility of DDA and of its secretory transport, since DDA was excreted at over 200 times the rate of DDT. Liver, kidney, and bile also showed elevated DDA tissue-to-plasma ratios. Thus, the organic acid system mediates the accumulation and excretion of DDA in these fish.

DDE feeding and plasma osmoregulation in ducks, guillemots, and puffins.

To assess the possibility that organochlorine pesticide disruption of osmoregulation is responsible for recent large kills of young seabirds, we have studied the effects of DDE feeding (10-250 ppm) on plasma osmoregulation and nasal gland function in the following species: mallared and white Pekin ducks (both Anas platyrhynchos), black guillemot (Cepphus grylle), and common (Fratercula arctica). Other investigators have recently reported that dietary DDE (10-1,000 ppm) inhibits nasal gland secretion in freshwater-maintained mallards; our initial experiments with white Pekins showed no such inhibition during either freshwater or seawater maintainance. Moreover, DDE had minimal effects on plasma electrolyte levels and total nasal gland Na-K-ATPase activities in all species studied. Liver DDE levels in experimental ducks and guillemots were comparable to those reported for seabirds found dead after kills; levels in starved experimental puffins were much higher. Thus DDE at environmental levels does not affect osmoregulation or nasal gland Na-K-ATPase either in ducks or in two species of oceanic birds.

Na-K-ATPase localization in teleost urinary bladder by [3H]ouabain autoradiography.

Previous studies on the urinary bladder of the seawater-acclimated winter flounder (pseudopleuronectes americanus) demonstrated that active Na and Cl transport were ouabain sensitive. This suggested a relationship between the Na pump and Na-K-ATP-ase. The specific binding of [H]ouabain to Na-K-ATPase provides a means of localizing the site of active Na transport. In isolated bladders, a positive linear correlation (r= 0.89) was found between the active Na transport rate and the Na-K-ATPase activity. Ouabain binding by the bladder surface appeared to be saturable and relatively specific, e.g., was reduced by a high K concentration. When only the mucosal side of the bladder was exposed to 5 muM ouabain, both inhibitory effects and binding were small and are explained by finite permeability of the bladder to ouabain. In contrast, binding and inhibitory effects from the serosal side were much greater. Autoradiographs demonstrated that [3H]ouabain was bound only to the serosal side of the epithelial cells. Ultrastructural examination revealed that the area of ouabain binding coincided with the basal and lateral plasma membranes.