Activation of Na+,K+,Cl- cotransport in squid giant axon by extracellular ions: evidence for ordered binding.

Activation of the influx mode of the Na+,K+,Cl- cotransporter (NKCC) by extracellular Na+, K+ and Cl- was studied using the internally dialyzed squid giant axon. Cooperative interactions among the three transported ions were assessed using ion activation of NKCC-mediated 36Cl influx under two sets of experimental conditions. The first, or control condition, used high, non-limiting concentrations of two of the cotransported ions (the co-ions) while activating cotransport with the third ion. Under this non-limiting co-ion condition the calculated Vmax of the cotransporter was between 57 and 60 pmol/cm2/s. The apparent activation (KApp, or half-saturation) constants were: K+, 9 mM; Na+, 52 mM; and Cl-, 146 mM. The second condition used limiting co-ion concentration conditions. In this case, activation by each ion was determined when one of the other two co-ions was present at or near its apparent half-saturation concentration as determined above. Under these limiting conditions, the KApp values for all three co-ions were significantly increased regardless of which co-ion was present at a limiting concentration. The effects on the apparent Vmax were more complicated. When K+ was the limiting co-ion, there was little effect on the Vmax for Na+ or Cl- activation. In contrast, limiting concentrations of Na+ or Cl- both resulted in a large reduction of the apparent Vmax when activating with the other two co-ions. These results are consistent with an ordered binding mechanism for the NKCC in which K+ binds before Na+ or Cl-. Physiological implications for these results are discussed.

Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection.

We examined the effects of human cytomegalovirus (HCMV) infection on the Na+-K+-Cl- cotransporter (NKCC) in a human fibroblast cell line. Using the Cl--sensitive dye MQAE, we showed that the mock-infected MRC-5 cells express a functional NKCC. 1) Intracellular Cl- concentration ([Cl-]i) was significantly reduced from 53.4 +/- 3.4 mM to 35.1 +/- 3.6 mM following bumetanide treatment. 2) Net Cl- efflux caused by replacement of external Cl- with gluconate was bumetanide sensitive. 3) In Cl--depleted mock-infected cells, the Cl- reuptake rate (in HCO-3-free media) was reduced in the absence of external Na+ and by treatment with bumetanide. After HCMV infection, we found that although [Cl-]i increased progressively [24 h postexposure (PE), 65.2 +/- 4.5 mM; 72 h PE, 80.4 +/- 5.0 mM], the bumetanide and Na+ sensitivities of [Cl-]i and net Cl- uptake and loss were reduced by 24 h PE and abolished by 72 h PE. Western blots using the NKCC-specific monoclonal antibody T4 showed an approximately ninefold decrease in the amount of NKCC protein after 72 h of infection. Thus HCMV infection resulted in the abolition of NKCC function coincident with the severe reduction in the amount of NKCC protein expressed.

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts.

The effects of human cytomegalovirus (HCMV) infection on Cl-/HCO-3 exchanger activity in human lung fibroblasts (MRC-5 cells) were studied using fluorescent, ion-sensitive dyes. The intracellular pH (pHi) of mock- and HCMV-infected cells bathed in a solution containing 5% CO2-25 mM HCO-3 were nearly the same. However, replacement of external Cl- with gluconate caused an H2DIDS-inhibitable (100 microM) increase in the pHi of HCMV-infected cells but not in mock-infected cells. Continuous exposure to hyperosmotic external media containing CO2/HCO-3 caused the pHi of both cell types to increase. The pHi remained elevated in mock-infected cells. However, in HCMV-infected cells, the pHi peaked and then recovered toward control values. This pHi recovery phase was completely blocked by 100 microM H2DIDS. In the presence of CO2/HCO-3, there was an H2DIDS-sensitive component of net Cl- efflux (external Cl- was substituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl- (in the nominal absence of CO2/HCO-3), the H2DIDS-sensitive net Cl- efflux was much greater from HCMV- than from mock-infected cells. In mock-infected cells, H2DIDS-sensitive, net Cl- efflux decreased as pHi increased, whereas for HCMV-infected cells, efflux increased as pHi increased. All these results are consistent with an HCMV-induced enhancement of Cl-/HCO-3 exchanger activity.

Human cytomegalovirus infection enhances osmotic stimulation of Na+/H+ exchange in human fibroblasts.

Infection with human cytomegalovirus (HCMV) causes an enlargement (cytomegaly) of human fibroblasts (MRC-5). As a first step toward determining whether solute uptake, mediated in part by Na+/H+ exchange, is responsible for the development of cytomegaly, we studied the effects of HCMV infection on intracellular pH (pHi) regulation (nominal CO2/ HCO3- concn = 0) by comparing cytomegalic cells with mock-infected cells. Seventy-two hours after HCMV infection of MRC-5 cells we observed the following changes relative to mock-infected cells: resting pHi is 0.1-0.2 pH unit more alkaline; the intrinsic buffering power of the cytoplasm was reduced by approximately 40-50%; acid-loading H(+)-equivalent fluxes were reduced; and there were alterations of Na+/H+ exchanger (NHE) properties, including an alkaline shift of the pHi dependence of activity, a reduction of the apparent affinity for extracellular Na+, and an increase of the apparent maximum velocity and a large increase in stimulation by a hyperosmotic challenge. These results indicate that HCMV infection exerts a profound effect on functional properties of the NHE, on acid-loading mechanisms, and on intrinsic cellular buffering power. These effects are consistent with a role for the NHE in the development of cytomegaly.

Elevated [Cl-]i, and [Na+]i inhibit Na+, K+, Cl- cotransport by different mechanisms in squid giant axons.

Bumetanide-sensitive (BS) unidirectional fluxes of (36)Cl- or (22)Na+ were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-]i or [Na+]i resulted in a concentration-dependent reduction of the BS influx of both (36)Cl- and (22)Na+. Raising [Cl-]i above 200 mM completely blocked BS influxes. However, raising [Na+]i to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-]i values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-]i levels > 125 mM resulted in a [Cl-]i-dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl may interact with an intracellular site (or sites), which does not mediate Cl transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.

Effects of okadaic acid and intracellular Cl- on Na(+)-K(+)-Cl- cotransport.

The Na(+)-K(+)-Cl- cotransporter of the squid giant axon requires ATP and is inhibited by intracellular Cl- (Cli-) in a concentration-dependent manner ([Cl-]i > or = 200 mM completely inhibits the cotransporter). In the present study we address the question of whether inhibition of cotransport by Cli- is due to a Cl(i-)-induced increase of protein phosphatase activity. Intracellular dialysis was used to apply the phosphatase inhibitor okadaic acid (OKA) under conditions of [Cl-]i at 0, 150, or 300 mM during measurement of cotransporter-mediated unidirectional Cl- influx into axons. At 0 mM [Cl-]i, the application of 250 nM OKA had no effect on the cotransport-mediated Cl- influx when axons were dialyzed with the normal intracellular ATP concentration ([ATP]i = 4 mM). Reduction of [ATP] to 50 microM resulted in a significant decrease of the bumetanide-sensitive CL- influx, which could be partially reversed by OKA treatment. Similarly, in ATP-limited axons with [Cl-]i at 150 mM, cotransporter influx was partially stimulated by treatment with OKA. However, axons dialyzed with 300 mM [Cl-]i ([ATP]i = 50 microM) had no measurable cotransport influx, nor was subsequent treatment with OKA able to induce a cotransport-mediated Cl- influx. We conclude that the inhibition of cotransport caused by Cli- is not the result of an increase in the OKA-sensitive protein phosphatase activity.

Human cytomegalovirus infection increases the number of ouabain-binding sites in human fibroblasts.

Infection of permissive human embryo fibroblasts (MRC-5) with human cytomegalovirus (HCMV) increased the number of copies of the Na+,K(+)-ATPase (the Na+ pump) in the plasma membrane, measured as ouabain-binding sites. The increase was preceded by cell enlargement by about 24 hr, becoming significant between 48 and 72 hr after infection. Reduction in Na+ or Cl- concentration in the culture media immediately after infection partially prevented the increase in the number of ouabain-binding sites. The effect was reversible upon restoring Cl- or Na+ to the incubation medium, but withdrawal of either ion at 24 or 48 hr PE failed to prevent the increase in the number of binding sites. These results suggest that the processes that resulted in the increase of copies of the Na+,K(+)-ATPase required both Na+ and Cl- during the first 24 hr PE. Amiloride and ethylisopropylamiloride, two inhibitors of Na+ transport mechanisms of the plasma membrane have been previously shown to reduce the amount of virus yields and to prevent the onset of cytomegaly (Fons et al., 1991, Proc. Soc. Exp. Biol. Med. 196, 89-96). We show here that these agents partially block the increase in ouabain-binding sites caused by HCMV infection.

Binding of bumetanide to microsomes from optic ganglia of the squid, Loligo pealei.

Saturable high-affinity binding of [3H] bumetanide [dissociation constant (KD) = 80 nM] was measured in microsomal membranes prepared from squid optic ganglia. Under control conditions, the maximal specific binding of labeled bumetanide (Bmax) was approximately 6-7 pmol/mg protein. Binding had a higher relative affinity for bumetanide than for furosemide and depended on the presence of Cl- and K+, but not Na+, in the incubation media. In the case of K+, [3H]bumetanide binding was half-saturated at [K+] = 100 mM. The Cl- effect was biphasic. At [Cl-] between 0 and 150 mM, [3H]bumetanide binding increased with increasing [Cl-]. However, when [Cl-] was increased above 150 mM, [3H]bumetanide binding was progressively reduced. ATP acted as a nonessential activator [mean affinity constant (K0.5) approximately 1 microM] of the ion-dependent [3H]bumetanide binding by increasing the apparent binding capacity. The activation by ATP did not require Mg2+. Other adenosine analogues also stimulated the binding of bumetanide.

Osmotic stimulation of Na(+)-K(+)-Cl- cotransport in squid giant axon is [Cl-]i dependent.

The effects of increasing extracellular osmolality on unidirectional Cl- fluxes through the Na(+)-K(+)-Cl- cotransporter were studied in internally dialyzed squid giant axons. Hyperosmotic seawater stimulated bumetanide-sensitive Cl-influx at 150 mM intracellular Cl- concentration ([Cl-]i), whereas Cl- efflux was unaffected under comparable ionic conditions. Stimulation of bumetanide-sensitive Cl- influx was proportional to the increase in extracellular osmolality. Bumetanide-sensitive Cl- influx began to increase after a latency of approximately 20 min after a stepwise increase of extracellular osmolality and continued to increase for at least 70 min. The increased bumetanide-sensitive Cl- influx measured after 65 min of exposure to hyperosmotic external fluid was a function of the intracellular Cl- concentration; stimulation by hyperosmotic external fluids was observed at physiological [Cl-]i levels (greater than 100 mM) but not at lower [Cl-]i levels. Under both normo- and hyperosmotic conditions, intracellular Cl- inhibited Na(+)-K(+)-Cl- cotransport influx in a concentration-dependent manner. However, in hyperosmotic seawater, the dose dependence of inhibition by intracellular Cl- was shifted to higher [Cl-]i values. Therefore, we conclude that hyperosmotic extracellular fluids stimulate influx via the Na(+)-K(+)-Cl- cotransport by resetting the relation between [Cl-]i and transport activity.

A plasma membrane-enriched preparation from giant barnacle muscle fibers.

We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2'-disulfo-4,4'-diisothiocyano)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.

Vanadate and fluoride effects on Na+-K+-Cl- cotransport in squid giant axon.

The effects of vanadate and fluoride on the Na+-K+-Cl- cotransporter of the squid giant axon were assessed. In axons not treated with these agents, intracellular dialysis with ATP-depleting fluids caused bumetanide-inhibitable 36Cl influx to fall with a half time of approximately 16 min. In the presence of either 40 microM vanadate or 5 mM fluoride, the decay of bumetanide-inhibitable 36Cl influx was significantly slowed; half time for vanadate-treated axons is 45 min and for fluoride-treated axons is 37 min. These agents are not exerting their effects on Na+-K+-Cl- cotransport by influencing the rate of ATP depletion of the axon, since they had no effect on the ATP hydrolysis rate of an optic ganglia homogenate. We therefore suggest that these data support the hypothesis that Na+-K+-Cl- cotransport in squid axons is regulated by a phosphorylation-dephosphorylation mechanism and that vanadate and fluoride reduce the rate of dephosphorylation by inhibiting a protein phosphatase.

Effects of pH changes on sodium pump fluxes in squid giant axon.

The effects of independently varying intracellular and extracellular pH on sodium pump fluxes were studied in the squid giant axon. By means of intracellular dialysis, we found that changes of intracellular pH (pHi), but not of extracellular pH, affected ouabain-sensitive Na+ efflux and K+ influx over the pH range of 6.0-8.6. Both fluxes were maximum at a pHi of 7.2-7.4. Variations away from this optimal pHi in either the acidic or alkaline direction resulted in a graded inhibition of both ouabain-sensitive fluxes. The kinetic basis for the inhibitory effect of acidic pHi was examined by comparing the kinetic parameters of activation of ouabain-sensitive sodium efflux by intracellular Na+ (Na+i) and extracellular K+ (K+o) at normal pHi with those at acidic pHi. We found that the inhibitory effect of intracellular acidity results from a reversible decrease in maximum velocity (Vmax), without an effect on the activation parameters for Na+i (K1/2 Na+i) or K+o (K1/2 K+o).

Coupled Na/K/Cl efflux. "Reverse" unidirectional fluxes in squid giant axons.

Studies of unidirectional Cl-, Na+, and K+ effluxes were performed on isolated, internally dialyzed squid giant axons. The studies were designed to determine whether the coupled Na/K/Cl co-transporter previously identified as mediating influxes (Russell. 1983. Journal of General Physiology. 81:909-925) could also mediate the reverse fluxes (effluxes). We found that 10 microM bumetanide blocked 7-8 pmol/cm2 X s of Cl- efflux from axons containing ATP, Na+, and K+. However, if any one of these solutes was removed from the internal dialysis fluid, Cl- efflux was reduced by 7-8 pmol/cm2 X s and the remainder was insensitive to bumetanide. About 5 pmol/cm2 X s of Na+ efflux was inhibited by 10 microM bumetanide in the continuous presence of 10(-5) M ouabain and 10(-7) M tetrodotoxin if Cl-, K+, and ATP were all present in the internal dialysis fluid. However, the omission of Cl- or K+ or ATP reduced the Na+ efflux, leaving it bumetanide insensitive. K+ efflux had to be studied under voltage-clamp conditions with the membrane potential held at -90 mV because the dominant pathway for K+ efflux (the delayed rectifier) has a high degree of voltage sensitivity. Under this voltage-clamped condition, 1.8 pmol/cm2 X s of K+ efflux could be inhibited by 10 microM bumetanide. All of these results are consistent with a tightly coupled Na/K/Cl co-transporting efflux mechanism. Furthermore, the requirements for cis-side co-ions and intracellular ATP are exactly like those previously described for the coupled Na/K/Cl influx process. We propose that the same transporter mediates both influx and efflux, hence demonstrating "reversibility," a necessary property for an ion-gradient-driven transport process.

Calcium transport mechanisms in dog red blood cells studied from measurements of initial flux rates.

Ca2+ efflux from dog red blood cells loaded with Ca2+ using the A23187 ionophore could be separated into two main components: (1) Mg- and ATP-dependent (active transport) and (2) dependent on external Na (K1/2 around 15 mM); at 80 microM internal free Ca the relative magnitudes of these fluxes were 70% and 30% respectively. The Na-dependent Ca2+ efflux had the following additional properties: (i) it was partially inhibited by ATP depletion or preincubation with vanadate, but it was not affected by Mg2+ depletion; (ii) it failed to be stimulated by external monovalent cations other than Na: (iii) it was stimulated by reduction in the internal Na+ concentration. Both active and Na-dependent Ca2+ efflux remained unchanged in hypotonic solutions or in solutions with alkaline pH (8.5). In cells containing ATP and Mg2+, external Ca2+ inhibited Ca2+ efflux (K1/2 around 1 mM); on the other hand, in Mg-free dog red cells external Ca2+ stimulated Ca2+ efflux (K1/2 about 30 microM). In Mg-depleted red cells incubated in the absence of external Na2+, Ca2+ influx as a function of external Ca2+ followed a monotonically saturable function (K1/2 around 20 microM): addition of Na resulted in (i) inhibition of Ca2+ influx and (ii) a sigmoid relationship between flux and external Ca2+. Intracellular Ca2+ stimulated the external Na-dependent Ca2+ efflux along a sigmoid curve (K1/2 around 30 microM); on the other hand the Ca pump had a biphasic response to internal Ca2+: stimulation at low internal Ca2+ (K1/2 between 1 and 10 microM), followed by a decline at internal Ca2+ concentrations higher than 50 microM.