Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon

Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na⁺ or K⁺ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na⁺ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K⁺ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f _c=15.6±0.91 Hz and a mean plateau valueS _o=(7.04±2.94)×10^–20 A² sec/cm². The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K⁺ entry across the apical membrane. Elimination of the K⁺ gradient by bathing the colon on both sides with K⁺ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs⁺ or tetraethylammonium (TEA) and by serosal addition of Ba²⁺. The one-sided action of these K⁺ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K⁺ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na⁺ transport pathway.     

Intracellular binding of spin-labeled amiloride: an alternative explanation for amiloride's effects at high concentration

Amiloride, an important inhibitor of Na+ transport and Na+/H+ exchange, has been used in nontransporting tissues to investigate the relationship between ionic fluxes or intracellular pH change and proliferative or synthetic events. Reports that amiloride is permeant and had direct effects on intracellular processes have led us to investigate the possibility that amiloride binds intracellularly to nuclei, mitochondria, and to purified nucleic acids. Using a nitroxide spin-labeled derivative of amiloride (ASp) and electron paramagnetic resonance (EPR) spectroscopy, we have demonstrated that nuclei and mitochondria isolated from trout liver bind significant amounts of ASp especially at the high amiloride concentrations (approximately mM) commonly used to inhibit proliferative events. While the chemical component responsible for ASp binding in these organelles was not identified, native DNA binds significant amounts of ASp whereas single stranded DNA and RNA bind much less. When these observations are taken together with reports of amiloride's direct action on cellular processes, they support the possibility that some of the effects attributed to inhibition of a transport event are caused by amiloride directly.     

Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

The H+ Pump in Frog Skin (Rana esculenta): Identification and Localization of a V-ATPase

We here report on studies on the frog skin epithelium to identify the nature of its excretory H⁺ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A₁ (10 μm) blocked H⁺ excretion (69 ± 8% inhibition) and therefore Na⁺ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na₂SO₄ solution, ``low-Na<sup>+</sup> conditions' under which H<sup>+</sup> and Na<sup>+</sup> fluxes are coupled 1:1. The electrogenic outward H<sup>+</sup> current measured in absence of Na<sup>+</sup> transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A<sub>1</sub> or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na<sup>+</sup> transport (short-circuit current), which develops with apical solutions containing 115 mm Na<sup>+</sup> (``high-Na⁺ conditions'), demonstrating a specific action on H⁺ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H⁺ excretion is mediated by a V-ATPase being the electrogenic H⁺ pump in frog skin. Received: 21 May 1996/Revised: 24 December 1996     

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

External Ni<Superscript>2+</Superscript> and ENaC in A6 Cells: Na<Superscript>+</Superscript> Current Stimulation by Competition at a Binding Site for Amiloride and Na<Superscript>+</Superscript>

In cultured A6 monolayers from distal Xenopus kidney, external Ni²⁺ stimulated active Na⁺ uptake via the epithelial Na⁺ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni²⁺ effect. Na⁺ current noise analysis was performed using the neutral Na⁺-channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni²⁺ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni²⁺ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni²⁺ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na⁺—known to compete with amiloride and so producing the self-inhibition phenomenon—and Ni²⁺ exerted mutually exclusive analogous effects on amiloride kinetics. Na⁺ current kinetics revealed that Ni²⁺ prevents ENaC to be downregulated by self-inhibition. Co²⁺ behaved similarly to Ni²⁺, whereas Zn²⁺ did not. Attempts to disclose the chemical nature of the site reacting with Ni²⁺ suggested cysteine but not histidine as reaction partner.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Day–night specific binding of 2-[125I]Iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species

Some of melatonin’s (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[¹²⁵I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K _d) and maximal binding densities (B _max) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K _d values in the tissues in the picomolar range indicated the high affinity. K _d and B _max values were tissue- and species-dependent. The GTP analogue [Guanosine 5′-O-(3-thiotriphosphate)] treatment significantly reduced the B _max value, indicating that the 2-[¹²⁵I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K _d and B _max values were observed. These are the first studies of the presence of 2-[¹²⁵I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

The effect of poly-l-lysine,amiloride and methyl-l-lysine on gill ion transport and permeability in the rainbow trout

Summary The action of poly-l-lysine (PLL) on Na and Cl transport across freshwater fish gills was studied. Low concentrations (10^–6M) were added to the external medium for brief periods (1–5 min), then removed. During the next 20 min there was a rapid net loss of Na (117±17 Equiv[100 g]^–1 hr^–1) and Cl (129±17 Equiv[100 g]^–1 hr^–1). Both values are an order of magnitude larger than unidirectional effluxes in control fish. The efflux of both ions decreased to control values within 60 min after application and removal of PLL. In contrast, unidirectional influxes (J _in ^Cl andJ _in ^Na ) were inhibited by about 40% and showed no sign of returning to the original rates for 3 hr. Thus, PLL has two independent actions, causing a large increase in gill permeability which is reversible within an hour and a partial inhibition of influx which showed no sign of reversing for 2–3 hr. When PLL was applied for a longer period (60 min, the results were qualitatively similar but the permeability change was larger and persisted longer. These effects were compared with those of the small organic amines, amiloride and methyl-l-lysine. The latter inhibitedJ _in ^Na , but there was no other similarity to PLL. Neither affected sodium efflux, nor did they have any effect on Cl movements, in or out, across the gill. Inhibition ofJ _in ^Na , was rapidly and completely reversible, amd amiloride was shown to act by competing with Na for an entry site.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Phosphorylated intermediate of the ouabain-insensitive,Na-stimulated ATPase in rat kidney cortex and rainbow trout gills

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na⁺-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg²⁺ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg²⁺-dependent, Na⁺-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH₂OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na⁺ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1–E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na·E1∼P to Na·E2-P.     

Acid–base regulation in isolated gill cells of the goldfish (Carassius auratus)

Mechanisms of acid release and intracellular pH (pH_i) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pH_i was determined using the fluorescent probe 2,7-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na⁺ channel and Na⁺/H⁺ exchanger (NHE) inhibitor, had no effect on pH_i, but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH₄Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na⁺. Under steady-state conditions, 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid (DIDS), a HCO₃^–-transport inhibitor, caused a slow acidification of pH_i, and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A₁, an inhibitor of V-ATPase, had no effect on steady-state pH_i and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na⁺-dependent HCO₃^– transport is the dominant mechanism besides an NHE and V-ATPase to control pH_i in goldfish gill cells.Communicated by G. Heldmaier     

On the cross-reactivity of amiloride and 2,4,6 Triaminopyrimidine (TAP) for the cellular entry and tight junctional cation permeation pathways in epithelia

Summary 2,4,6 Triaminopyrimidine (TAP) has been previously shown to inhibit the passive tight junctional cation permeation pathway in various leaky epithelia. Amiloride has been shown to be an effective inhibitor of the cation cellular entry pathway in tight epithelia. In this paper we demonstrate that TAP and amiloride at appropriate concentrations are able to block either of these epithelial cation permeation pathways. TAP was found to block the Na entry pathway in frog skin with the following characteristics: it (1) inhibits from the external solution only, (2) is completely reversible, (3) increases the transepithelial resistance, (4) is active in the monoprotonated form, (5) is noncompetitive with Na, (6) displays saturation kinetics which obey a simple kinetic model (K _I=1×10^–3 m), (7) is independent of external calcium, (8) is dependent on external buffering capacity, and (9) is competitive with amiloride. Amiloride inhibition of the junctional permeation in gallbladder had the following characteristics: it (1) increases the transepithelial resistance, (2) decreases cation conductance without affecting the anion conductance, (3) displays saturation kinetics which obey a simple kinetic model (K _I=1×10^–3 m), and (4) possesses inhibitory activity in both its protonated and unprotonated form. These results not only indicate that a similar inhibitory site may exist in both of these cation permeation pathways, but also provide information on the chemical nature and possible location of these inhibitory sites.