Activation of cell membrane potassium conductance by mercury in cultured renal epithelioid (MDCK) cells

To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)-cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1 microM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by approximately 0.2 microM. Higher concentrations of mercury ions (greater than 10 microM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 microM, 1 microM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 microM, but not 1 microM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin- or pertussis toxin-sensitive G-proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.     

Cellular mechanisms of ATP-induced hyperpolarization in renal epitheloid MDCK-cells

Previous studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)-cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoyl phorbol 13-acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5-trisphosphate (IP3), inositol 1,3,4,5-tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the cell membrane.     

Effect of BHT 920 on calcium-activated K+ channels in renal epithelioid MDCK cells.

In Madin Darby canine kidney (MDCK) cells, epinephrine has been shown to increase intracellular calcium, activate calcium-dependent K+ channels and hyperpolarize the cell membrane. The present study has been performed to test for the possible involvement of alpha 2-adrenergic receptors. To this end, the effects of alpha 2-adrenoceptor agonist BHT 920 have been studied on cell membrane potential, ion channel activity and intracellular calcium: Similar to epinephrine, BHT 920 hyperpolarizes the cell membrane, increases intracellular calcium and activates inwardly rectifying K+ channels (single channel slope conductances 30-80 pS). Half-maximal hyperpolarization is achieved at concentrations between 10 and 100 nmol/l. The hyperpolarizing effect of BHT 920 is abolished in the presence of alpha 2-adrenoceptor antagonist yohimbine (100 nmol/l) but not in the presence of alpha 1-adrenoceptor antagonist prazosin (100 nmol/l). At extracellular calcium activity below 100 nmol/l BHT 920 still leads to a transient hyperpolarization of the cell membrane but, in contrast to epinephrine, is unable to significantly increase intracellular calcium or significantly activate the calcium-sensitive K+ channels. The observations indicate that stimulation of alpha 2-receptors participates in the epinephrine-induced increase of intracellular calcium, channel activation and hyperpolarization.     

Effects of extracellular nucleotides on electrical properties of subconfluent Madin Darby canine kidney cells

ATP and ADP but not AMP lead to sustained hyperpolarization of Madin Darby canine kidney (MDCK) cells. The present study has been performed to test for an influence of other nucleotides on the potential difference across the cell membrane (PD) in subconfluent MDCK cells. PD has been continuously monitored with conventional microelectrodes during rapid exchange of extracellular fluid. Application of 1 mumol/1 UTP leads to a rapid (less than 2 s) hyperpolarization of the cell membrane by -17.0 +/- 0.4 mV (from -50.1 +/- 0.6 mV), a reduction of cell membrane resistance and an increase of the sensitivity of PD to alterations of extracellular potassium. The concentration needed for half maximal effect of UTP is approximately equal to 0.2 mumol/1. ITP is similarly effective, whereas UDP, GTP and GDP are less effective. Up to 1 mmol/1 UMP, GMP, TTP or CTP do not significantly alter PD. In calcium-free extracellular fluid the hyperpolarizing effect of UTP is blunted (-11.6 +/- 2.3 mV) and only transient. In conclusion, UTP similar to purine triphosphates hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium, which is apparently recruited from both intra- and extracellular sources.     

Ion channels in Madin-Darby canine kidney cells.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.     

Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP.     

Cellular mechanisms of bradykinin-induced hyperpolarization in renal epitheloid MDCK-cells.

Previous studies have demonstrated that bradykinin hyperpolarizes the cell membrane of subconfluent MDCK cells by increase of the potassium conductance. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of bradykinin on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate diester (TPA). In untreated cells, bradykinin leads to a transient increase of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, increase of Cai, activation of potassium channels and hyperpolarization of the cell membrane. The effects of bradykinin on PD and Cai are still present in the absence of extracellular calcium. In cells pretreated with pertussis toxin the effect of bradykinin on inositol trisphosphate formation is almost abolished but bradykinin still leads to a transient increase of Cai and PD in the presence and absence of extracellular calcium. In cells pretreated with TPA the bradykinin-induced increase of inositol trisphosphate formation is blunted, the bradykinin-induced increase of Cai abolished, but the bradykinin-induced hyperpolarization still present. The observations indicate that bradykinin increases Cai in part by phorbol ester and pertussis toxin sensitive activation of phospholipase C. In addition, bradykinin is capable of enhancing Cai by utilizing pertussis toxin insensitive mechanisms. Furthermore, bradykinin is able to transiently enhance the potassium conductance without a general increase of intracellular calcium.     

Role of Hyperpolarization Attained by Linoleic Acid in Chick Myoblast Fusion

Our previous report has suggested that hyperpolarization generated by reciprocal activation of calcium-activated potassium (K(Ca)) channels and stretch-activated channels induces calcium influx that triggers myoblast fusion. Here we show that linoleic acid is involved in the process of generating hyperpolarization in cultured chick myoblasts and hence in promotion of the cell fusion. Linoleic acid dramatically hyperpolarized the membrane potential from -14 +/- 3 to -58 +/- 5 mV within 10 min. This effect was partially blocked by 1 mM tetraethylammonium (TEA) or 30 nM charybdotoxin, a selective K(Ca) channel inhibitor, and completely abolished by 10 mM TEA. Single-channel recordings revealed that linoleic acid activates TEA-resistant potassium channels as well as K(Ca) channels. Furthermore, linoleic acid induced calcium influx from extracellular solution, and this effect was partially blocked by 1 mM TEA and completely prevented at 10 mM, similar to the effect of TEA on linoleic acid-mediated hyperpolarization. Since the valinomycin-mediated hyperpolarization promoted calcium influx, hyperpolarization itself appears capable of inducing calcium influx. In addition, gadolinium prevented the valinomycin-mediated increase in intracellular calcium level under hypotonic conditions, revealing the involvement of stretch-activated channels in calcium influx. Furthermore, linoleic acid stimulated myoblast fusion, and this stimulatory effect could completely be prevented by 10 mM TEA. These results suggest that linoleic acid induces hyperpolarization of membrane potential by activation of potassium channels, which induces calcium influx through stretch-activated channels, and thereby triggers myoblast fusion.     

Effect of trifluoperazine on renal epithelioid Madin-Darby canine kidney cells

Following exposure to a number of hormones, the cell membrane in Madin-Darby Canine Kidney (MDCK) cells is hyperpolarized by increase of intracellular calcium activity. The present study has been performed to elucidate the possible role of calmodulin in the regulation of intracellular calcium activity and cell membrane potential. To this end trifluoperazine has been added during continuous recording of cell membrane potential or intracellular calcium. Trifluoperazine leads to a transient increase of intracellular calcium as well as a sustained hyperpolarization of the cell membrane by activation of calcium sensitive K+ channels. Half-maximal effects are observed between 1 and 10 mumol/L trifluoperazine. A further calmodulin antagonist, chlorpromazine, (50 mumol/L), similarly hyperpolarizes the cell membrane. The effects of trifluoperazine are virtually abolished in the absence of extracellular calcium. Pretreatment of the cells with either pertussis toxin or phorbol-ester TPA does not interfere with the hyperpolarizing effect of trifluoperazine. In conclusion, calmodulin is apparently involved in the regulation of calcium transfer across the cell membrane but not in the stimulation of K+ channels by intracellular calcium.     

Pertussis toxin-dependent and -independent hormonal effects on cultured renal epithelioid cells

The present study has been performed to test for the involvement of pertussis toxin-sensitive GTP-binding proteins (G-proteins) in the cellular transduction of hormone-induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin-dependent and -independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Electrophysiology of phagocytic membranes. III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques. The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is -90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero. Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore. The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10(-5) M. It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Changes in the neuronal membrane potential of the mollusk Planorbarius corneus under the action of cardiac glycosides

In experiments on isolated neurones from the gastropod mollusc P. corneus, strophantin and digoxin in low concentrations produce slow hyperpolarization, in higher ones--depolarization; at concentrations about 1 mM, hyperpolarization was more evident. In all cases, the decrease in membrane resistance was observed. Presumably, membrane permeability for potassium ions increases. During application of the drugs in concentrations 10-100 microM, hyperpolarization may be masked by depolarization due to block of Na,K-pump. Higher concentrations, increasing potassium permeability of the membrane, may result in substitution of depolarization by hyperpolarization.     

Electrophysiology of phagocytic membranes: III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques.The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is ?90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero.Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore.The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10^?5 M.It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used.

The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.     

Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea.

Acetylcholine has long been thought to be the neurotransmitter of the cochlear efferent system in mammals although the evidence is largely indirect. By using whole-cell recordings from isolated outer hair cells, we show that acetylcholine activates a large rapidly desensitizing outward potassium current. This corresponds to hyperpolarization of the membrane potential from rest. The half maximal dose for acetylcholine was 13.5 microM with a cooperativity of 2. The response was not due to a conventional muscarinic action of acetylcholine for it was not blocked by 0.1 microM atropine and muscarinic antagonists but it could be blocked by 0.1 microM curare, suggesting that it shared many properties of a nicotinic receptor. It was, however, inhibited by 10 microM strychnine. The potassium current activated by acetylcholine required external calcium and was characterized by a significant delay at room temperature. This points to the involvement of a second messenger system, possibly calcium itself.     

Intracellular calcium and extra-retinal photoreception in Aplysia giant neurons

The early or “instantaneous” current-voltage relationship for the light-activated potassium current in Aplysia giant neurons was linear during the first second of illumination. However, the light current was greatly reduced or abolished by prolonged hyperpolarization. It was also greatly reduced by the injection of calcium EGTA buffers having calcium activities of 5.6 × 10^?8 M and simulated by injecting buffers with calcium activities of 2.8–5.6 × 10^?7 M. Removal of calcium from the extracellular fluid had no effect. Both the light-and calcium-activated outward potassium currents were reduced by tetra-ethylammonium (TEA) ions. The light current was not affected by substituting rubidium for potassium nor by substituting either lithium or Tris for sodium. The calcium-activated potassium current persisted when the neuron was cooled to 5°C. However, the light response could no longer be elicited. Light hyperpolarizes Aplysia neurons probably by increasing intracellular calcium activity two-to six-fold which activates a membrane potassium conductance. Calcium levels appear to be restored within the cell and are energy dependent. The light-activated release of calcium is inhibited by cooling. The body wall of Aplysia transmits enough visible or 500-nm light to hyperpolarize some Aplysia giant neurons under ambient conditions. These neurons may be involved in the extraretinal light entrainment that occurs in Aplysia.     

Governance of arteriolar oscillation by ryanodine receptors

To investigate the role of ryanodine receptors in glomerular arterioles, experiments were performed using an isolated perfused hydronephrotic kidney model. In the first series of studies, BAYK-8644 (300 nM), a calcium agonist, constricted afferent (19.6 +/- 0.6 to 17.6 +/- 0.5 microm, n = 6, P < 0.01) but not efferent arterioles. Furthermore, BAYK-8644 elicited afferent arteriolar oscillatory movements. Subsequent administration of nifedipine (1 microM) inhibited both afferent arteriolar oscillation and constriction by BAYK-8644 (to 19.4 +/- 0.5 microm). In the second group, although BAYK-8644 constricted afferent arterioles treated with 1 microM of thapsigargin (19.7 +/- 0.6 to 16.8 +/- 0.6 microm, n = 5, P < 0.05), it failed to induce rhythmic contraction. Removal of extracellular calcium with EGTA (2 mM) reversed BAYK-8644-induced afferent arteriolar constriction (to 20.0 +/- 0.5 microm). In the third series of investigations, ryanodine (10 microM) but not 2-aminoethoxyphenyl borate (100 microM) abolished afferent arteriolar vasomotion by BAYK-8644. In the fourth series of experiments, in the presence of caffeine (1 mM), the stronger activation of voltage-dependent calcium channels by higher potassium media resulted in greater afferent arteriolar constriction and faster oscillation. Our results indicate that L-type calcium channels are rich in preglomerular but not postglomerular microvessels. Furthermore, the present findings suggest that either prolonged calcium influx through voltage-dependent calcium channels (BAYK-8644) or sensitized ryanodine receptors (caffeine) is required to trigger periodic calcium release through ryanodine receptors in afferent arterioles.     

Cloning and functional expression of a plant voltage-dependent chloride channel.

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.     

Intestinal zinc uptake in freshwater rainbow trout: evidence for apical pathways associated with potassium efflux and modified by calcium

Understanding the mechanisms of intestinal zinc uptake in fish is of considerable interest from both nutritional and toxicological perspectives. In this study, properties of zinc transport across the apical membrane of freshwater rainbow trout intestinal epithelia were examined using right-side-out brush border membrane vesicles (BBMV's). Extravesicular calcium was found to have complex actions on zinc uptake. At a low zinc concentration of 1 microM, calcium (0.1-2 mM) significantly stimulated zinc uptake. In contrast, calcium inhibited zinc uptake at higher zinc levels (100 microM). Lanthanum and cadmium in the external medium did not block zinc uptake, suggesting that interactions between zinc and calcium were not exerted at a calcium channel. Copper also failed to exercise any inhibitory action. Zinc association with the BBMV's was enhanced by an outward potassium gradient. This stimulatory effect was only present at a zinc concentration of 100 microM. The potassium channel blocker, tetraethylammonium chloride inhibited zinc uptake at this relatively high zinc concentration, suggesting the presence of a low affinity zinc uptake pathway linked to potassium efflux. The present study provides evidence that the mechanism of intestinal zinc uptake in rainbow trout is pharmacologically very different from that of the piscine gill and the mammalian intestine.