Temperature dependence of chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells

1. The temperature dependence of the steady-state self-exchange of chloride between human red cells and a plasma-like electrolyte medium has been studied by measuring the rate of (36)Cl(-) efflux from radioactively labelled cells. Between 0 and 10 degrees C the rate increased by a factor of eight corresponding to an Arrhenius activation energy of 33 kcal/mole.2. The rate of chloride exchange decreased significantly in experiments where 95% of the chloride ions in cells and medium were replaced by other monovalent anions of a lyotropic series. The rate of chloride self-exchange was increasingly reduced by bromide, bicarbonate, nitrate, iodide, thiocyanate, and salicylate. The latter aromatic anion was by far the most potent inhibitor, reducing the rate of chloride self-exchange to 0.2% of the value found in a chloride medium.3. The temperature sensitivity of the chloride self-exchange was not affected significantly by the anionic inhibitors. The Arrhenius activation energies of chloride exchange were between 30 and 40 kcal/mole in the presence of the six inhibitory anions mentioned above.4. The rate of self-exchange of bromide, thiocyanate, and iodide between human red cells and media was determined after washing and labelling cells in media containing 120 mM bromide, thiocyanate, or iodide respectively. The rate of self-exchange of the three anions were 12, 3, and 0.4% of the rate of chloride self-exchange found in the chloride medium.5. The Arrhenius activation energies of the self-exchange of bromide, iodide, and thiocyanate were all between 29 and 37 kcal/mole, the same magnitude as found for the self-exchange of chloride.6. Although approximately 40% of the intracellular iodide and salicylate ions appeared to be adsorbed to intracellular proteins, the rate of tracer anion efflux followed first order kinetics until at least 98% of the intracellular anions had been exchanged.7. The self-exchange of salicylate across the human red cell membrane occurred by a different mechanism than the one utilized by the inorganic monovalent anions. The activation energy of salicylate exchange (13.2 kcal/mole) was significantly lower than that of inorganic anion exchange. Salicylate exchange increased with decreasing pH in contrast to the exchange of chloride, which decreases when pH is lowered.     

The effect of pH on the 36Cl efflux from frog skeletal muscle

1. Frog sartorii and toe muscles were loaded in a 100 mM-K 216 mM-Cl solution at pH 7·4 labelled with either ³⁶Cl, or ⁴²K, or with both isotopes. The efflux of the isotopes into inactive solution of the same composition at pH 5·0 and 9·8 was followed.2. The efflux of ³⁶Cl into acid solution was extremely slow so that the cellular fraction could easily be distinguished. On raising the pH the efflux of ³⁶Cl from sartorii increased 4·5 ± 0·43 (mean ± S.E.) times; from toe muscles 6·7 ± 0·69 (mean ± S.E.) times. The effect was fully reversible.3. The efflux of ⁴²K from sartorii was only slightly increased by alkaline solutions. When the effluxes of ³⁶Cl and ⁴²K were measured simultaneously the increment in chloride efflux was much greater than the increment in ⁴²K efflux.4. Toe muscles depolarized by high potassium solution showed an approximately twofold increase in ⁴²K efflux on transfer from acid to alkaline solution. This effect was independent of the presence of chloride ions.5. The results confirm the identification of chloride as the ion species principally concerned in the pH sensitivity of the membrane conductance.     

The pH sensitivity of the chloride conductance of frog skeletal muscle

1. The effect of changes in the pH of the extracellular solution on the membrane conductance of frog sartorius and toe muscle fibres was measured with intracellular micro-electrodes.2. In Ringer solution the membrane conductance was found to be highly sensitive to changes in pH between 5.0 and 9.8. In alkaline solution the conductance rose; in acid solution it fell.3. After replacement of chloride by the relatively impermeant methylsulphate ion the membrane conductance showed little change when pH was altered. It is concluded that chloride is the ion species principally concerned in the pH sensitivity of the resting membrane conductance.4. The relation between pH and the chloride conductance was sigmoid, with the steepest part of the curve lying in the region of neutrality.5. The membrane conductance of muscles equilibrated in a 100 mM-K 216 mM-Cl solution was also sensitive to changes of extracellular pH. As in Ringer solution, the membrane conductance rose in alkaline and fell in acid solutions in a sigmoid fashion.6. Sartorius muscles in isotonic potassium methylsulphate solution showed no change in membrane conductance at different pH values.7. In chloride-free solution a fall in pH tended to cause depolarization; a rise in pH had the opposite effect.8. In Ringer solution the initial effect of a rise in pH was usually a transient depolarization. The indication is that the intracellular concentration of chloride ions may be slightly in excess of that which corresponds to the resting potential. The long-term effects of changes in pH on the membrane potential in Ringer solution were in the same direction as in the absence of chloride.9. The transient potential changes produced on addition and withdrawal of chloride ions were found to be larger in alkaline solutions than in acid solutions. This is further evidence for a higher chloride permeability in alkaline solutions.     

Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle.

1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (a(i) (Cl)) of frog sartorius fibres at 25 degrees C.2. The internal pH (pH(i)) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass).3. In normal bicarbonate-free solution (containing 2.5 mM potassium), the average chloride equilibrium potential, E(Cl) (calculated from a(i) (Cl) and the measured chloride activity of the external solution (a(o) (Cl)) was 87.7 +/- 1.7 mV (mean +/- S.E.; n = 16) in fibres where the average membrane potential, E(m), was 88.3 +/- 1.5 mV (mean +/- S.E.; n = 16). In experiments where a(i) (Cl) was varied between about 1 and 10 mM (which corresponds to values of E(m) between about -105 and -50 mV) E(Cl) was within 1-3 mV of E(m) at equilibrium. These measurements of a(i) (Cl) were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of a(i) (Cl) below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode.4. In solutions containing 15 mM bicarbonate and gassed with 5% CO(2), pH(i) was 6.9, corresponding to an internal bicarbonate concentration of 7.6 mM. E(Cl) measured in this solution was some 4 mV positive to E(m). Most of the difference between E(Cl) and E(m) could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of a(Cl).5. If bicarbonate/CO(2) in the external solution was replaced by HEPES/pure O(2) at constant pH, then pH(i) rose from 6.88 +/- 0.02 (mean +/- S.E.) to 7.05 +/- 0.02. A change in external pH of 1 unit caused pH(i) to change by about 0.02 unit and the intracellular buffering power was calculated to be about 35.6. In solution made hypertonic by the addition of sucrose, E(m) changed little or depolarized and E(Cl) and E(m) remained close. In contrast, in solution made hypertonic by the addition of solid sodium chloride (high-chloride solution) E(Cl) became negative to E(m). Conversely in low chloride solution E(Cl) became positive to E(m).7. When the chloride permeability (P(Cl)) was reduced by the use of acid solution, E(Cl) moved positive to E(m) indicating an accumulation of internal chloride. When P(Cl) was increased again by returning to more alkaline solution, E(m) depolarized to E(Cl).8. The results are consistent with the existence of a small, active movement of chloride, the effects of which are normally obscured by large passive movements of chloride when P(Cl) is large.     

Effect of some monovalent anions on chloride and sulphate permeability of human red cells

1. The permeability of human red cells to (36)Cl(-) and to [(35)S]SO(4) (2-) was studied in the presence of various monovalent anions.2. A maximum decrease of anion permeability was found in a study of the steady-state exchange of (36)Cl in a medium containing 120 mM salicylate. The exchange had a half-time of 3 hr at 0 degrees C, a reduction of normal chloride permeability by a factor of 10(5). The activation energy of chloride exchange decreased from a value of 45 to 22 kcal/mole in the interval between 0 and 10 degrees C. Simultaneous determination of the permeability to potassium and chloride proved that salicylate induced a reversal of the normal selectivity of red cells at 0 degrees C (permeability coefficient P(K) of 3.5 x 10(-9) cm/sec to be compared with a P(Cl) of 2 x 10(-9) cm/sec).3. In contradistinction to the slow movement of (36)Cl, the exchange of [(14)C]salicylate was completed within 4 min, when red cells were suspended at 0 degrees C in the salicylate medium.4. A study of the sulphate permeability at 38 degrees C showed that the rate of steady-state exchange decreased, when chloride was replaced by lyotropic anions other than bromide. The sequence of the permeability decrease was: Cl(-) = Br(-) < I(-) < NO(3) < SCN(-) < salicylate, the same sequence which previously has been shown to increase the permeability to sodium and potassium. The activation energies of sulphate exchange were 32 kcal/mole (chloride medium), and 38 kcal/mole (thiocyanate medium).5. Sufficient data were obtained during the study to demonstrate that when equilibrium has been obtained, there is a good agreement between the values of (36)Cl (cell water)/(36)Cl (extracellular water) and of {[(35)S]SO(4) (cell water)/[(35)S]SO(4) (extracellular water)}((1/2)).6. It is concluded that the anion-induced changes of permeability are due to binding of anions to fixed cationic charges in the red cell membrane.     

Ion fluxes during the inhibitory junction potential in the guinea-pig taenia coli.

1. Contribution of different ions to the inhibitory junction potential (i.j.p.) in the guinea-pig taenia coli was studied by measuring the 42K, 24Na and 36Cl fluxes, the membrane resistance and the influence of various external ion concentrations. 2. The membrane resistance, as measured by the electrotonic potential, decreased transiently during the i.j.p. A maximal reduction of the electrotonic potential of about 50% was found at the top of the i.j.p. 3. The i.j.p. amplitude could be reduced by raising the external potassium concentration. Extrapolation of the relationship observed shows that the inhibitory response would be abolished at 115 mM potassium. Similar experiments were made in chloride-free medium, chloride being replaced by isethionate. Amplitude and time course of the response were not different in chloride containing Locke solution and chloride-free medium. 4. The half-times of 42K, 24Na and 36Cl effluxes during rest were 29, 10 and 9 min respectively. The 42K-efflux from the preparation was markedly increased to about three times the resting efflux during field stimulation. In low-chloride solution a similar effect on 42K-efflux was observed during field stimulation. Only a slight increase in the chloride efflux was observed but the sodium efflux was not affected during field stimulation. 5. From the results presented it is concluded that the inhibitory junction potential is caused by a selective increase in potassium permeability of the smooth-muscle cell membrane.     

Effect of divalent cations on AMPA-evoked extracellular alkaline shifts in rat hippocampal slices.

The generation of activity-evoked extracellular alkaline shifts has been linked to the presence of external Ca(2+) or Ba(2+). We further investigated this dependence using pH- and Ca(2+)-selective microelectrodes in the CA1 area of juvenile, rat hippocampal slices. In HEPES-buffered media, alkaline transients evoked by pressure ejection of RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) averaged approximately 0.07 unit pH and were calculated to arise from the equivalent net addition of approximately 1 mM strong base to the interstitial space. These alkaline responses were correlated with a mean decrease in [Ca(2+)](o) of approximately 300 microM. The alkalinizations were abolished reversibly in zero-Ca(2+) media, becoming indiscernible at a [Ca(2+)](o) of 117+/-29 microM. Addition of as little as 30-50 microM Ba(2+) caused the reappearance of an alkaline response. In approximately one-fourth of slices, a persistent alkaline shift of approximately 0.03 unit pH was observed in zero-Ca(2+) saline containing EGTA. In HEPES media, addition of 300 microM Cd(2+), 100 microM Ni(2+), or 100 microM nimodipine inhibited the alkaline shifts by roughly one-half, one-third, and one-third, respectively, whereas Cd(+) and Ni(2+) in combination fully blocked the response. In bicarbonate media, by contrast, Cd(+) and Ni(2+) blocked only two-thirds of the response. In the presence of bicarbonate, Ni(2+) caused an unexpected enhancement of the alkalinization by approximately 150%. However, when the extracellular carbonic anhydrase was blocked by benzolamide, addition of Ni(2+) reduced the alkaline shift. These results suggested that Ni(2+) partially inhibited the interstitial carbonic anhydrase and thereby increased the alkaline responses. These data indicate that an activity-dependent alkaline shift is largely dependent on the entry of Ca(2+) or Ba(2+) via voltage-gated calcium channels. However, sizable alkaline transients still can be generated with little or no external presence of these ions. Implications for the mechanism of the activity-dependent alkaline shift are discussed.     

Purification and characterization of a protease produced by Bacillus megaterium RRM2: application in detergent and dehairing industries

An alkaline serine protease produced by Bacillus megaterium RRM2 isolated from the red alga, Kappaphycus alvarezii (Doty) Doty ex Silva was studied for the first time and the same analyzed for the production of protease in the present study. Identification of the bacterium was done on the basis of both biochemical analysis and by 16S rDNA sequence analysis. The extracellular protease obtained from B. megaterium RRM2 was purified by a three-step process involving ammonium sulphate precipitation, gel filtration (Sephadex G100) and Q-Sepharose column chromatography. The purity was found to be 30.6-fold with a specific activity of 3591.5 U/mg protein with a molecular weight of 27 kDa. The metal ions Ca(2+), Mg(2+), K(+) and Na(+) marginally enhanced the activity of the purified enzyme while Hg(2+), Cu(2+), Fe(2+), CO(2+) and Zn(2+), had reduced the activity. The enzyme was found to be active in the pH range of 9.0-10.0 and remained active up to 60 °C. Phenyl Methyl Sulfonyl Fluoride (PMSF) inhibited the enzyme activity, thus, confirming that this enzyme is an alkaline serine protease. Likewise, DTT also inhibited the enzyme thus confirming the disulfide nature of the enzyme. The enzyme exhibited a high degree of tolerance to Sodium Dodecyl Sulphate (SDS). The partially purified protease when used as an additive in the commercial detergents was found to be a suitable source for washing clothes especially those stained with blood. Further, it showed good dehairing activity within a short duration in goat skin without affecting its collagen component.     

Surface potential reflected in both gating and permeation mechanisms of sodium and calcium channels of the tunicate egg cell membrane

1. Threshold changes of Na and Ca currents due to various polyvalent cations (stabilizing cations) or H(+) ions were studied in the egg cell membrane of a tunicate, Halocynthia roretzi, by using the voltage-clamp technique.2. With an increase in [Ca](o) or a decrease in pH in the external solution, the current-voltage (I-V) relations for the peak of the Na and Ca currents shifted along the voltage axis in the positive direction. These voltage shifts in the I-V relations, measured at a potential of V((1/2)) where inward current attains its half-maximum, were shown to be identical to shifts in voltage-dependence of the time courses of Na and Ca currents, and also identical to shifts in the inactivation curves of Na current along the voltage axis.3. The shifts in V((1/2)) produced by various polyvalent cations or H(+) ions were analysed by the Gouy-Chapman equation for the diffuse double layer, by assuming that a change in V((1/2)) directly corresponds to a change in the surface double layer potential.4. The V((1/2))-divalent cation concentration relations of Na current were exactly described by the predictions of the theory with a constant value of the surface charge density of 1e(-)/(9 A)(2). The weak stabilizing effects of Mg(2+), Sr(2+) and Ba(2+) were quite similar to each other and were explained in terms of a ;screening' effect. Other divalent cations, such as Ca(2+), Mn(2+) and Ni(2+), showed various different stabilizing effects which were explained in terms of a ;binding' effect. The binding constants (K(1)'s) for Ca(2+), Mn(2+) and Ni(2+) were 0.21, 0.45 and 0.94 M(-1), respectively.5. H(+) ions showed a powerful stabilizing effect upon the Na current with a K(H) of 6 x 10(4)M(-1). This value indicates that the acidic sites around Na channels have a pK(a) of 4.78. La(3+) ions also acted as a strong stabilizer upon the Na current with a K(La) of 15 M(-1). For both H(+) and La(3+), the V((1/2))-concentration relations were also exactly described by the Gouy-Chapman equation with the same charge density of 1e(-)/(9 A)(2) as estimated by varying divalent cations.6. The stabilizing effect of permeant cations such as Ca(2+), Sr(2+) and Ba(2+) on Ca channel currents was analysed. The effect of lowering pH was also studied. It was found that the surface charge density of 1e(-)/(9 A)(2) estimated by Na current is also applicable to the explanation for the V((1/2))-divalent cation concentration or - pH relationships. The estimated binding constants for H(+), Ca(2+) and Sr(2+) were 1.2x10(5), 0.58 and 0.035 M(-1), respectively. Ba(2+) does not bind to charged sites near to the Ca channels.7. It was noticed that a considerable reduction in the conductances of Na and Ca currents occurred in parallel with a stabilizing effect. This reduction was ascribed to a decrease in the concentration of permeant cations at the external surface of the cell membrane, as predicted by the theory of the diffuse double layer. The Goldman, Hodgkin-Katz equation for ionic currents was applied to explain the conductance suppression.8. The conductance suppressions of Na and Ca channel currents due to Ca(2+), Sr(2+) and Ba(2+) were found to be apparent ones, only reflecting decreases in the surface concentration of permeant cations without any changes in the permeability. After correction for the apparent suppression, the real permeability ratio among Ca(2+), Sr(2+) and Ba(2+) for Ca channels was determined as 1.00, 0.56 and 0.21 respectively.9. The conductance suppression of Na current by lowering pH was explained in terms of a real suppression or blocking which is superimposed on the apparent suppression. Considering the surface [Na](o), the plot of P(Na) against the surface pH yielded a blocking curve of Na channel by H(+) ions, which implies that two H(+) ions are necessary to block each Na channel. For Ca channels no real blockage was observed in acidic pH.10. It was concluded from the present experiment that there exists a surface potential capable of affecting both gating and permeation mechanisms of ionic channels in this tunicate egg cell membrane.     

Chloride self-exchange in toad skeletal muscle in vivo and in vitro

The exchange of cellular Cl with 36Cl has been measured in saline-perfused hindlimb muscles of the pithed toad and compared with cellular Cl exchange in isolated muscles incubated in the vitro either in toad Ringer solution or in toad plasma. In the perfused hindlimb, the rate of 36Cl efflux from muscle cells [17.0 +/- 0.9 pmol Cl.(cm2 plasma membrane.s)-1] was only 40% as fast as that of the 36Cl influx. The discrepancy between Cl influx and efflux was accompanied by a cellular accumulation of Cl against the electrochemical gradient for this anion. Concurrently, the cells took up Na in amounts at least equal to the accumulated Cl. During this accumulation of Na and Cl, the mean resting potential remained constant at a value of -89.2 +/- 1.9 (SE) mV. Na and Cl were taken up by the muscle cells of the perfused hindlimb without a concomitant decrease in cellular K content; i.e., without evidence of inhibition of the Na-K pump. The rate constant for cellular 36Cl efflux from isolated toad muscles preincubated for 3 h in vitro in toad Ringer solution was about five times faster than that of muscles in the perfused hindlimb and similar in magnitude to published values for Cl fluxes in frog muscle. Cellular Cl efflux from muscles briefly preincubated in vitro for 15 min instead of 3 h was significantly slower than after prolonged preincubation. In vitro incubation of isolated toad muscles in toad plasma slowed the cellular 36Cl efflux to values approaching those measured in the perfused hindlimb, without comparably depressing the 36Cl influx. It is suggested that the uptake of NaCl by the cells of perfused hindlimb muscle may proceed by an electroneutral inward cotransport of Na and Cl on the same carrier.     

Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112

Thermoalkalophilic Enterobacter sp. MTCC 5112 was isolated from a sediment sample collected from the Mandovi estuary on the west coast of India. This culture produced extracellular xylanase. The xylanase enzyme was isolated by ammonium sulfate (80%) fractionation and purified to homogeneity using size exclusion and ion exchange chromatography. The molecular mass of the xylanase was approximately 43 kDa. The optimal pH of the xylanase activity was 9, and at room temperature it showed 100% stability at pH 7, 8 and 9 for 3 h. The optimal temperature for the enzyme activity was 100 degrees C at pH 9.0. At 80 degrees C and pH 9, 90% of the enzyme activity was retained after 40 min. At 70 and 60 degrees C, the enzyme retained 64% and 85% of its activity after 18 h, respectively, while at 50 degrees C and pH 9 the enzyme remained stable for days. For xylan, the enzyme gave a K(m) value of 3.3 mg ml(-1) and a V(max) value of 5,000 micromol min(-1) mg(-1) when the reaction was carried out at 100 degrees C and pH 9. In the presence of metal ions such as Co(2+), Zn(2+), Fe(2+), Cu(2+), Mg(2+) and Ca(2+) the activity of the enzyme increased, whereas strong inhibition of enzyme activity was observed in the presence of Hg(2+) and EDTA. To the best of our knowledge, this is the first report on the production of xylanase by this bacterium.     

Isolation, characterization of heavy metal resistant strain of Pseudomonas aeruginosa isolated from polluted sites in Assiut city, Egypt

Sixty six isolates of Pseudomonas spp. were isolated from wastewater of El-Malah canal located in Assiut, Egypt and were checked for their heavy metal tolerance. One isolate has tested for its multiple metal resistances and found to be plasmid mediated with molecular weight 27 Kb for nickel and lead. It was identified as Pseudomonas aeruginosa ASU 6a. Its minimal inhibitory concentration (MIC) for Cu(2+), Co(2+), Ni(2+), Zn(2+), Cr(3+), Cd(2+)and Pb(2+) were 6.3, 5.9, 6.8, 9.2, 5.8, 4.4, and 3.1 mM, respectively. Growth kinetics and the maximum adsorption capacities were determined under Ni(2+) and Pb(2+) stress. The latter heavy metals induced potassium efflux and were used as indicator for plasma membrane permeabilization.     

The distribution of chloride ions in the smooth muscle cells of the guinea-pig's taenia coli

1. The intracellular Cl concentration of taenia coli cells, determined by an analytical procedure and by an extrapolation procedure, has a value between 60 and 73 m-mole/l. cell water.2. This concentration is too high to be explained by a passive distribution. The discrepancy could be due to a binding of Cl in the intracellular or extracellular compartment or to an active uptake of Cl by the cells.3. Determination of the activity coefficient for Cl in homogenates of smooth muscle did not support the hypothesis of binding of Cl ions.4. The efflux of (36)Cl from taenia coli cells was not affected by foreign anions. After 1 hr exposure to a Cl-free solution, the tissues contained less than 1 m-mole of Cl/kg wet wt., even if Cl had been replaced by a slowly penetrating anion. Because the intracellular cation concentration remained constant, it has to be assumed that new anionic groups can be formed in the cells.5. The intracellular Cl concentration decreases during exposure to ouabain or to K-free solution. The uptake seems therefore to be linked to the uptake of K through the Na pump.6. Exposure to K-free solution increases the K permeability of the membrane. Under the same experimental conditions the Cl permeability of the membrane increases as long as K is leaking out of the cells.7. The anions in the external solution exert an important influence on the K permeability of the membrane. NO(3) and I cause a small increase of the permeability and large anions such as benzenesulphonate, propionate or pyroglutamate cause a pronounced decrease of this permeability.     

Evidence of nucleotidyl phosphatase activity associated with core protein sigma A of avian reovirus S1133

Both avian reovirus core protein sigma A purified from virus-infected cell extracts and the purified bacterially expressed protein sigma A (e sigma A) were characterized for their nucleoside triphosphate (NTP) hydrolysis activity by thin-layer chromotography. Protein sigma A from both preparations has a nonspecific nucleotidyl phosphatase activity that hydrolyzes four types of NTP to their corresponding nucleoside di- and monophosphates and free phosphate. The divalent cation requirement for this activity of e sigma A was further examined by the addition of Mn(2+), Mg(2+), Ca(2+), and Zn(2+) ions. NTP hydrolysis by e sigma A was maximal when Mn(2+), Mg(2+), or Ca(2+) concentrations were 5, 4, or 1 mM, respectively. Addition of Mn(2+) or Mg(2+) stimulated the reactions up to 4- or 3-fold, respectively, higher than Ca(2+) (2.2-fold). However, Zn(2+) ion inhibited this activity of e sigma A. The results suggest that nucleotidyl phosphatase activity of e sigma A is absolutely dependent on the divalent cations Mn(2+), Mg(2+), or Ca(2+), but not Zn(2+). Similar results were obtained from the analysis of divalent cation requirements for the protein sigma A nucleotidyl phosphatase activity. Optimal pH for nucleotidyl phosphatase activity of protein sigma A from both preparations was determined using reaction mixtures buffered at different pH. The results show that the optimal activities of both proteins were similar and were achieved between pH 7.5 and 8.5.     

Extracellular pH changes and accompanying cation shifts during ouabain-induced spreading depression

Interstitial ionic shifts that accompany ouabain-induced spreading depression (SD) were studied in rat hippocampal and cortical slices in the presence and absence of extracellular Ca(2+). A double-barreled ion-selective microelectrode specific for H(+), K(+), Na(+), or Ca(2+) was placed in the CA1 stratum radiatum or midcortical layer. Superfusion of 100 microM ouabain caused a rapid, negative, interstitial voltage shift (2-10 mV) after 3-5 min. The negativity was accompanied by a rapid alkaline transient followed by prolonged acidosis. In media containing 3 mM Ca(2+), the alkalosis induced by ouabain averaged 0.07 +/- 0.01 unit pH. In media with no added Ca(2+) and 2 mM EGTA, the alkaline shift was not significantly different (0.09 +/- 0.02 unit pH). The alkaline transient was unaffected by inhibiting Na(+)-H(+) exchange with ethylisopropylamiloride (EIPA) or by blocking endoplasmic reticulum Ca(2+) uptake with thapsigargin or cyclopiazonic acid. Alkaline transients were also observed in Ca(2+)-free media when SD was induced by microinjecting high K(+). The late acidification accompanying ouabain-induced SD was significantly reduced in Ca(2+)-free media and in solutions containing EIPA. The ouabain-induced SD was associated with a rapid but relatively modest increase in [K(+)](o). In the presence of 3 mM external Ca(2+), the mean peak elevation of [K(+)](o) was 12 +/- 0.62 mM. In Ca(2+)-free media, the elevation of [K(+)](o) had a more gradual onset and reached a significantly larger peak value, which averaged 22 +/- 1.1 mM. The decrease in [Na(+)](o) that accompanied ouabain-induced SD was somewhat greater. The [Na(+)](o) decreased by averages of 40 +/- 7 and 33 +/- 3 mM in Ca(2+) and Ca(2+)-free media, respectively. In media containing 1.2 mM Ca(2+), ouabain-induced SD was associated with a substantial decrease in [Ca(2+)](o) that averaged 0.73 +/- 0. 07 mM. These data demonstrate that in comparison with conventional SD, ouabain-induced SD exhibits ion shifts that are qualitatively similar but quantitatively diminished. The presence of external Ca(2+) can modulate the phenomenon but is irrelevant to the generation of the SD and its accompanying alkaline pH transient. Significance of these results is discussed in reference to the propagation of SD and the generation of interstitial pH changes.     

Analysis of the effluxes of sodium, potassium and chloride ions from smooth muscle in normal and hypertonic solutions

1. Efflux curves of (24)Na, (42)K and (36)Cl from the guinea-pig taenia coli were obtained in normal Krebs solution, and in hypertonic Krebs solution in which the osmolarity had been doubled by the addition of sucrose.2. The efflux curves were complex, and in order to get average curves each was analysed as the sum of three exponential terms, and average curves were constructed from the means of the constants found.3. In order to estimate the membrane permeability to the ions, it was necessary to make assumptions as to the distribution of ions in the tissue. Several models have been examined and the predictions of the models with respect to the membrane properties were compared with the data obtained by using electrophysiological methods by other workers.4. It was found that reasonable predictions of membrane properties could only be made using models in which the majority of the rapidly exchanging sodium is considered to be extracellular. This amount of sodium is more than can be accounted for as freely dissolved in the extracellular water.5. A possible interpretation of the ion exchange and distribution would be to suppose that some proportion of the three ions is contained in an extracellular compartment not available to the normal extracellular markers, and limited in its exchange by the rate of diffusion in the extracellular phase, and that the truly intracellular fractions of the tissue ions do not exchange with the external solution in a simple exponential manner, but in a manner described by an aggregate of exponential terms due to inherent variation in the permeabilities of the individual cell membranes.6. There is no evidence for any change in the membrane permeabilities to sodium and potassium in hypertonic solution, but there is evidence for a decrease in chloride permeability in this solution.     

Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb

1. Experiments were done on exteriorized foetal lambs of 123-144 days gestation to measure bidirectional ion fluxes through the pulmonary epithelium and to compare them with those predicted from the sum of the measured forces determining passive flux according to the Ussing flux-ratio equation. Fluxes of Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Cl(-), Br(-) and I(-) were measured and permeability constants obtained by following concentrations of their labelled isotopes in lung liquid and plasma after injection into either. Activity ratios were obtained from chemical measurements or tracer distributions, electrical potential differences by placing KCl-agar bridges, connected to calomel half-cells, in blood and lung liquid. Lung liquid volume and secretion rate were measured by adding an impermeant tracer (inulin or [(125)I]albumin) to lung liquid.2. The permeability sequence of the pulmonary epithelium for alkali metals was, Na(+) > K(+) > Rb(+) > Li(+) > Cs(+) and that for halides I(-) approximately Br(-) > Cl(-). Permeabilities to alkaline earths were lower than for the other ions, no definite sequence being established.3. There was an electrical potential difference of -1 to -10 mV (mean -4.3 mV) between lung liquid and plasma (lung liquid negative). Plasma/lung liquid chemical activity ratios were less than unity for the halides (Cl(-), Br(-), I(-)), and for K(+) and Rb(+), whereas the ratio of one-way fluxes (plasma --> lung liquid)/(lung liquid --> plasma) was in each case greater than unity. From the difference between the measured flux ratios and those predicted from the forces determining passive flux, it was concluded that the halides, K(+) and Rb(+) were actively transported from plasma to lung liquid, Cl(-) being quantitatively the most important. Na(+) and Ca(2+) appeared to move passively down a gradient of electrochemical potential.4. When alveolar liquid [HCO(3) (-)] was artificially raised, a net flux of HCO(3) (-) from lung liquid against a gradient of electrochemical activity was observed, suggesting active transport of that ion out of lung liquid.5. The addition of KCN to lung liquid stopped the secretion of liquid and absorption took place.     

Studies of radiocalcium efflux in single barnacle muscle fibres: effects of procaine and external divalent cations

1. (45)Ca efflux from single barnacle muscle fibres loaded with radio-calcium by microinjection was studied.2. The (45)Ca washout curve consisted of three exponential phases with half-times of 4.8, 12.6 and 111.1 min.3. Removal of external Ca(2+) reduced (45)Ca efflux by 65%. The (45)Ca efflux recovered upon restoring external Ca(2+), the magnitude of the recovery being dependent upon the external Ca(2+) concentration. 10 mM procaine was found to reduce the magnitude of the recovery.4. Removal of external Mg(2+) resulted in a 38% increase in (45)Ca efflux.5. External application of procaine at pH 7.8 caused a dose-dependent inhibition of (45)Ca efflux. The magnitude of the inhibition was reduced in the presence of low external Ca(2+) concentrations. 10 mM procaine at pH 9.3 caused a biphasic effect: inhibition was followed by stimulation.6. Microinjection of 0.5 M procaine caused only inhibition of (45)Ca efflux, whereas microinjection of 1.5 M procaine caused stimulation followed by inhibition. These effects were observed at an external pH of 7.8 and 9.3.7. Injection of 100 mM-EGTA abolished the stimulatory but not the inhibitory effect produced by procaine injection.8. These results are interpreted as indicating that a major fraction of the (45)Ca efflux involves Ca-Ca exchange which is inhibited by the charged form of procaine in a non-competitive manner at the external surface of the muscle fibre. The stimulatory action is attributed to release by procaine of Ca(2+) from internal binding sites.