Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

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     

Effect of amiloride,ouabain and Ba++ on the nonsteady-state Na−K pump flux and short-circuit current in isolated frog skin epithelia

Summary Effect of amiloride, ouabain, and Ba⁺⁺ on the nonsteady-state Na–K pump flux and short-circuit current in isolated frog skin epithelia.The active Na⁺ transport across isolated frog skin occurs in two steps: passive diffusion across the apical membrane of the cells followed by an active extrusion from the cells via the Na⁺–K⁺ pump at the basolateral membrane. In isolated epithelia with a very small Na⁺ efflux, the appearing Na⁺-flux in the basolateral solution is equal to the rate of the pump, whereas the short-circuit current (SCC) is equal to the active transepithelial Na⁺ transport. It was found that blocking the passive diffusion of Na⁺ across the apical membrane (addition of amiloride) resulted in an instantaneous inhibition of the SCC (the transepithelial Na⁺ transport, whereas the appearing flux (the rate of the Na⁺–K⁺ pump) decreased with a halftime of 1.9 min. Addition of the Na⁺–K⁺ pump inhibitor ouabain (0.1mm) resulted in a faster and bigger inhibition of the appearing flux than of the SCC. Thus, by simultaneous measurement of the SCC and the appearing Na⁺ flux one can elucidate whether an inhibitor exerts its effect by inhibiting the pump or by decreasing the passive permeability. Addition of the K⁺ channel inhibitor Ba⁺⁺, in a concentration which gave maximum inhibition of the SCC, had no effect on the appearing flux (the rate of the Na–K pump) in the first 2 min, although the inhibition of the SCC was already at its maximum.It is argued that in the short period, where the Ba⁺⁺-induced inhibition of SCC is at its maximum and the appearing flux in unchanged, the decrease in the SCC (SCC) is equal to the net K⁺ flux via the Na⁺–K⁺ pump, and the coupling ratio () of the Na⁺–K⁺ pump can be calculated from the following equation =SCC _t=0/SCC where SCC _t=0 is the steady-state SCC before the addition of Ba⁺⁺.     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin

Summary A modified cytochemical technique with 5-adenylylimidodiphosphate as substrate, was used to examine the distribution of adenylate cyclase in cells comprising the transepithelial Na⁺ transport pathway in isolated frog skin epithelium. Particular attention was paid to the effects of fixation on the activity and localization of adenylate cyclase. Fixation in glutaraldehyde alone or in combination with paraformaldehyde reduced the amount of reaction product, while better results were obtained using unfixed tissues. Optimum results were obtained following stimulation of adenylate cyclase with forskolin and in the presence of specific metabolic inhibitors. Adenylate cyclase was localized in the basolateral membranes of the principal cells which constitute a functional syncytium for Na⁺ transport and was absent from the apical membranes of the outermost granulosum cells. This distribution is consistent with the transepithelial Na⁺ transport model and defines the functional morphology of the cells involved in Na⁺ transport across frog skin. The results are compatible with the process of Na⁺ re-absorption across other epithelial cells, verifying that frog skin is a convenient model-tissue to study Na⁺ transport mechanisms. Adenylate cyclase was also found in membranes of the mitochondria-rich cells, a minor and parallel Na⁺ transporting pathway.     

Basolateral K+ Conductance Establishes Driving Force for Cation Absorption by Outer Sulcus Epithelial Cells

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of −90.4 ± 0.7 mV (n= 78), close to the Nernst potential for K⁺ (−95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K⁺ concentration with a K⁺/Na⁺ permeability ratio of 36. The most frequently observed K⁺ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10⁻⁵ to 10⁻³ m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 μm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K⁺ conductance, in concert with the Na⁺,K⁺-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.     

Effect of ouabain,amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frog skin (Rana temporaria)

Summary When tracer Na⁺ is added to the solution bathing the apical side of isolated epithelia the observed transepithelial tracer influx increases with time until a steady state is reached. The build-up of the tracer flux follows a single exponential course. The halftime for this build-up under control conditions was 0.92 ±0.06 min, and in the presence of ouabain 4.51±0.7 min. It is shown that the calculated Na⁺-transport pool is located in the cells. The Na⁺-transport pool under control conditions was 35.6 ±3.4 nmol/cm², which corresponds to an intracellular Na⁺ concentration of 7.9mm. Activation of the active Na⁺ transport by addition of antidiuretic hormone resulted in a highly significant increase in the Na⁺ transport pool, and inhibition of the transcellular Na⁺ transport with amiloride resulted in a decrease in the Na⁺-transport pool.Furthermore, the active Na⁺ transport increased along anS-shaped curve with increasing intracellular Na⁺ concentration (Na⁺-transport pool). The Na⁺ pump was found to be half saturated at an intracellular Na⁺ concentration of 12.5mm.     

Regulation of Epithelial Na+ Permeability by Protein Kinase C is Tissue Specific

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na⁺ transport across frog skin epithelium by increasing the apical Na⁺ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na⁺ transport (measured as amiloride-sensitive short-circuit current, I _SC, across skins from Rana temporaria and Bufo marinus, and inhibited I _SC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of I _SC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na⁺ channels is organ, and not species specific. The stimulation of Na⁺ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected in frog skin. Received: 19 January 1996/Revised: 10 April 1996     

Ca2+ dependency of Na+ transport by rabbit renal brush border membrane

Summary Intracellular Ca²⁺ has been suggested to play an important role in the regulation of epithelial Na⁺ transport. Previous studies showed that preincubation of toad urinary bladder, a tight epithelium, in Ca²⁺-free medium enhanced Na⁺ uptake by the subsequently isolated apical membrane vesicles, suggesting the downregulation of Na⁺ entry across the apical membrane by intracellular Ca²⁺. In the present study, we have examined the effect of Ca²⁺-free preincubation on apical membrane Na⁺ transport in a leaky epithelium, i.e., brush border membrane (BBM) of rabbit renal proximal tubule. In contrast to toad urinary bladder, it was found that BBM vesicles derived from proximal tubules incubated in 1mm Ca²⁺ medium exhibited higher Na⁺ uptake than those derived from proximal tubules incubated in Ca²⁺-free EGTA medium. Such effect of Ca²⁺ in the preincubation medium was temperature dependent and could not be replaced by another divalent cation, Ba²⁺ (1mm). Ca²⁺ in the preincubation medium did not affect Na⁺-dependent BBM glucose uptake, and its effect on BBM Na⁺ uptake was pH gradient dependent and amiloride (10^–3 m) sensitive, suggesting the involvement of Na⁺/H⁺ antiport system. Addition of verapamil (10^–4 m) to 1mm Ca²⁺ preincubation medium abolished while ionomycin (10^–6 m) potentiated the effect of Ca²⁺ to increase BBM Na⁺ uptake, suggesting that the effect of Ca²⁺ in the preincubation medium is likely to be mediated by Ca²⁺-dependent cellular pathways and not due to a direct effect of extracellular Ca²⁺ on BBM. Neither the proximal tubule content of cAMP nor the inhibitory effect of 8, bromo-cAMP (0.1mm) on BBM Na⁺ uptake was affected by the presence of Ca²⁺ in the preincubation medium, suggesting that Ca²⁺ in the preincubation medium did not increase BBM Na⁺ uptake by removing the inhibitory effect of cAMP. Addition of calmodulin inhibitor, trifluoperazine (10^–4 m) to 1mm Ca²⁺ preincubation medium did not prevent the increase in BBM Na⁺ uptake. The effect of Ca²⁺ was, however, abolished when protein kinase C in the proximal tubule was downregulated by prolonged (24 hr) incubation with phorbol 12-myristate 13-acetate (10^–6 m). In summary, these results show the Ca²⁺ dependency of Na⁺ transport by renal BBM, possibly through stimulation of Na⁺/H⁺ exchanger by protein kinase C.     

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.     

Single-file diffusion through K+ channels in frog skin epithelium

Summary The ratio between the unidirectional fluxes of K⁺ across the frog skin with K-permeable outer membranes was determined in the absence of Na⁺ in the apical solutions. The experiments were performed under presteady-state conditions to be able to separate the flux ratio for K⁺ through the cells from contributions to the fluxes through extracellular leaks. The cellular flux ratio deviated strongly from the value calculated from the flux ratio for electrodiffusion. The experiments can be explained if the passive K transport through the epithelial cells proceeds through specific channels by single-file diffusion with a flux ratio exponent of about 2.5.     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Extracellular Ca2+ controls outward rectification by apical cation channels in toad urinary bladder: Patch-clamp and whole-bladder studies

Summary Outward rectifying. cation channels were observed in the epithelial cells of the urinary bladder of the toad.Bufo marinus. As studied in isolated cells using the patch-clamp technique, the channel has an average conductance of 24 and 157 pS for pipette potentials between 0 and +60 mV and –60 to –100 mV, respectively, when the major cation in both bath and pipette solutions is K⁺. The conductance of the cannel decreasen with increasing dehydration energy of the permeant monovalent cation in the oder Rb⁺=K⁺>Na⁺>Li⁺. Reversal potentials near zero under biionic conditions imply that the permeabilities for all four of these cations are smiliar. The channel is sensitive to quinidine sulfate but not to amiloride. It shares several pharmacological and biophysical properties with an outwardly-rectifying, vasopressin-sensitive pical K⁺ conductive pathway described previously for the toad urinary bladder. We demonstrate, in both single-channel and whole-bladder studies, that the outward rectification is a consequence of interaction of the chanel with extracellular divalent cations, particularly Ca²⁺, which blocks inward but not outward current. Various divalent cations impart different degrees of outward rectification to the conductive pathway. Concentrations of Mg²⁺ and Ca²⁺ required for halfmaximal effect are 3×10^–4 and 10^–4 m, resopectively. For Co²⁺ the values are 10^–6 m at +50 mV and a 10^–4 m at +200 mV. The mechanism of blockade by divalent cations is not established, but does not seem to involve a voltage-dependent interaction in which the blocker penetrates the transmembrane electric field. In the absence of divalent cations in the mucosal solution, the magnitudes of inward current carried by Rb⁺, K⁺, Na⁺ and Li⁺ through the apical K⁺ pathway at any transepithelial voltage, are in the same order as in the single-channel studies. We propose that the cation channel observed by us in isolated epithelial cells is the single-channel correlate of the vasopressin-sensitive apical K⁺ conductive pathway in the toad urinary bladder and is also related to the oxytocin- and divalent cation-sensitive apical condictivity observed in frog skin and urinary bladder.     

Identification of apical membranes from tight epithelia using spin-labeled amiloride and electron paramagnetic resonance spectroscopy

Summary Apical cell membranes from Na⁺-transporting epithelia were identified in centrifugal fractions prepared from homogenates of rainbow trout kidney, gill and frog skin using a spinlabeled, nitroxide derivative of amiloride and electron paramagnetic resonance spectroscopy. Spin-labeled amiloride (ASp) is a potent inhibitor of Na⁺ transport. Frog skin shortcircuit current was inhibited by 50% in the presence of 7×10^–8 m ASp, whereas 4×10^–7 m amiloride was required to obtain the same effect. ASp is a suitable probe for the amiloride binding site based on analytical criteria: Unbound ASp produces an EPR signal linear with concentration and detectable at micromolar concentrations. Estimates of ASp binding can usually be made on less than 100 g of membrane protein. While ASp binds nonspecifically to many materials, amiloride- or benzamil-displaceable binding occurred only in trout gill and kidney, and in frog skin, but not in trout skeletal muscle. ASp binds to membrane fractions produced by differential centrifugation of trout gill, kidney and frog skin. In trout gill and kidney, 81% and 91%, respectively, of the amiloride-displaceable ASp binding is found in the 10,000 xg fraction. All of the ASp binding in frog skin is found in the 10,000 xg fraction. These data indicate that spin-labeled amiloride is a useful probe for the identification of the amiloride binding site, and electron paramagnetic resonance spectroscopy will allow the amiloride binding site to be used as a molecular marker for apical membranes.     

An L-type Calcium Channel in Renal Epithelial Cells

A voltage-activated Ca⁺⁺ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl₂ solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba⁺⁺ and Ca⁺⁺ is highly selective over Na⁺ and K⁺ (P_Ca(Ba):P_Na(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca⁺⁺ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca⁺⁺ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 1996     

Calcium entry in rabbit corneal epithelial cells: Evidence for a nonvoltage dependent pathway

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca⁺⁺ channels, Na⁺/Ca⁺⁺ exchange, and nonvoltage-dependent Ca⁺⁺-permeable channels. Whole cell inward currents carrying either Ca⁺⁺ or Ba⁺⁺ were not detected using voltage clamp techniques. We also used imaging technology and the Ca⁺⁺-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca⁺⁺ concentration ([Ca]_i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 m), or Ni⁺⁺ (40 m), a blocker of many voltage-dependent calcium channels, did not affect [Ca⁺⁺]_i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca⁺⁺]_i. These results are inconsistent with the presence of voltage gated Ca⁺⁺ channels. Nonvoltage gated Ca⁺⁺ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K⁺ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn⁺⁺ and bath perfusion with 5 mm Ni⁺⁺ decreased [Ca⁺⁺]_i suggesting that the Ca⁺⁺ conductance was blocked. These results are most consistent with a nonvoltage gated Ca⁺⁺ influx pathway. Finally, replacing extracellular Na⁺ with Li⁺ resulted in an increase in [Ca⁺⁺]_i if the cells were first Na⁺-loaded using the Na⁺ ionophore monensin and ouabain, a Na⁺-K⁺-ATPase inhibitor. These results suggest that Na⁺/Ca⁺⁺ exchange may also regulate [Ca⁺⁺] in this cell type.The authors are grateful to Chris Bartling for expert technical assistance with the imaging experiments, Helen Hendrickson for cell preparation, and Jonathon Monck for helpful discussions regarding imaging technology. This work was supported by National Institutes of Health grants EYO3282, EYO6005, DK08677, and an unrestricted award from Research to Prevent Blindness.     

Diacylglycerols stimulate short-circuit current across frog skin by increasing apical Na+ permeability

Summary The phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates baseline Na⁺ transport across frog skin epithelium and partially inhibits the natriferic response to vasopressin. The effects are produced largely or solely when TPA is added to the mucosal surface of the tissue. Although TPA activates protein kinase C, it has other effects, as well. Thus, the biochemical basis for the effects and the ionic events involved have been unclear. Furthermore, the physiologic implications have been obscure because of the sidedness of TPA's actions.We now report that two synthetic diacylglycerols (DAG) replicate the stimulatory and inhibitory effects of TPA on frog skin. DAG is the physiologic activator of PKC. In this tissue, it produces half-maximal stimulation at a concentration of 19 M. In contrast to TPA, DAG is about equally effective from either tissue surface.In a series of eight experiments, DAG was found to depolarize the apical membrane. Diacylglycerol also increases the paracellular conductance of frog skins bathed with mucosal Cl^– Ringer's solution. The latter effect can be minimized by replacing NO ₃ ^– for Cl^– in the mucosal solution. Under these conditions, combined intracellular and transepithelial measurements indicated that DAG increased both the apical Na⁺ permeability and intracellular Na⁺ concentration. These results are qualitatively similar to the effects of cyclic 3, 5-AMP on this tissue, suggesting that activation of PKC by DAG causes phosphorylation of the same or nearby gating sites phosphorylated by cAMP.We propose that apical Na⁺ entry is regulated in part by activation of PKC, and that insulin may be a physiologic trigger of this activation.     

Colchicine effect on the permeability of the whole epithelium and of isolated cells of frog skin

The effect of 2×10^–5 M colchicine on epithelial cells isolated from frog skin was investigated. Three hours of treatment with colchicine did not change either Na⁺ and K⁺ content of isolated cells or nonelectrolyte permeability. When ADH (50 mU/ml) was added, thiourea uptake values became greater than without the hormone; the same values were found in the cells previously treated with colchicine. Na⁺ transepithelial transport, measured by means of short-circuit current, was inhibited by the antimitotic agent both under control conditions and after ADH stimulation. These results support the view that colchicine does not directly affect ADH action on membrane permeability, but influences some mechanism that controls ADH action on transepithelial transport. Intercellular junctions appear to be the location of such a mechanism.