Free Ringer Solution Research Articles

Simulation of the electroretinographic slow PIII component by injection of K+ -free Ringer's solution in the frog retina

The light-induced hyperpolarization of the photoreceptor results in a decrease in extracellular potassium ion concentration, [K+]. A decrease in [K+] in the photoreceptor layer was caused by injecting a small amount of K+-free Ringer's solution in the isolated frog retina, and the distribution of the field potential as induced by K+-free Ringer's solution was mapped. The depth profile of the response resembled that of the slow PIII component of the electroretinogram. These results support the notion that the slow PIII is generated by the Müller cells as they hyperpolarize in response to a light-induced decrease in [K+] in the photoreceptor layer.

Rod phototransduction modulated by bicarbonate in the frog retina: roles of carbonic anhydrase and bicarbonate exchange.

1. Effects on rod phototransduction following manipulation of retinal CO2-HCO3- and H+ fluxes were studied in dark-adapted retinas of the frog and the tiger salamander. 2. Rod photoresponses to brief flashes of light were recorded from the isolated sensory retina as electroretinogram mass receptor potentials and from isolated rods by the suction-pipette technique. The experimental treatments were: (1) varying [CO2] + [HCO3-] in the perfusion fluid: (2) applying acetazolamide (AAA), which inhibits the enzyme carbonic anhydrase (CA); and (3) applying 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) which blocks exchange mechanisms transporting HCO3- across cell membranes. 3. The concentration of the internal transmitter of the rods, cyclic GMP, was biochemically determined from the rod outer segment layer of retinas that had been incubated in the same solutions as were used for perfusion in the electrophysiological experiments. 4. The introduction of 6 mM-sodium bicarbonate to replace half the buffer of a nominally CO2-HCO3(-)-free (12 mM-phosphate or HEPES, [Na+] constant) Ringer solution doubled the cyclic GMP concentration in the rod outer segment layer and increased the saturating response amplitude and the relative sensitivity of rods in the intact retina. 5. The introduction of 0.5 mM-AAA into bicarbonate-containing Ringer solution accelerated the growth of saturated responses and sensitivity. Incubation of the retina in AAA-bicarbonate Ringer solution elevated the concentration of cyclic GMP ninefold compared with the phosphate control. 6. No effects of switching to bicarbonate-AAA Ringer solution were observed in the photocurrent of isolated rods drawn into suction pipettes with only the outer segment protruding into the perfusion fluid. The target of AAA is probably the CA-containing Müller cell. 7. The introduction of DIDS into the perfusate (at normal pH 7.5) set off a continuous decay of photoresponses which finally abolished light sensitivity completely. The decay proceeded regardless of whether bicarbonate and AAA were present or not. 8. Rods that had lost their photosensitivity in DIDS recovered almost fully when the pH of the DIDS perfusate was raised to 8.5. They also recovered when DIDS was washed out with bicarbonate Ringer solution at constant pH (7.5). 9. It is proposed that all our treatments ultimately modulate the intracellular pH of the rods which is determined by the relative rates of H+ leakage and HCO3- transport into the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Active potassium absorption in rat distal colon.

1. Active potassium (K+) absorption in rat distal colon was investigated by measuring mucosal-to-serosal (JK, ms) and serosal-to-mucosal (JK, sm) 42K+ fluxes (mu equiv h-1 cm-2) across isolated stripped mucosa under short-circuit conditions in normal and dietary Na-depleted animals. As previously demonstrated, removal of Na+ from both mucosal and serosal solutions bathing the normal colon slightly increased net K+ absorption as a result of inhibition of JK, sm without affecting JK, ms, while in the Na-depleted group net K+ secretion (-0.54 +/- 0.11) was converted to a marked net K+ absorption (1.68 +/- 0.30, P less than 0.001). 2. In both groups of animals in Na(+)-free Ringer solution, JK, ms exhibited saturable and linear components, while JK, sm was a linear function of [K+]. Estimated affinity constants (mM) for saturable net K+ absorption were similar in normal (0.52 +/- 0.12) and Na-depleted (0.67 +/- 0.11) animals; however, there was a greater than 3-fold increase in the saturable flux (Jmax) from 0.54 +/- 0.04 in the normal colon to 1.78 +/- 0.08 mu equiv h-1 cm-2 in Na-depleted animals. 3. Mucosal orthovanadate (100 microM) inhibited JK, ms in both normal (control, 0.66 +/- 0.05 vs. orthovanadate, 0.36 +/- 0.03 mu equiv h-1 cm-2, P less than 0.001) and Na-depleted animals (control 1.20 +/- 0.13 vs. orthovanadate 0.77 +/- 0.07 mu equiv h-1 cm-2, P less than 0.01) without affecting JK, sm or the short-circuit current. In the Na-depleted group mucosal omeprazole or SCH28080 (100 microM), inhibitors of gastric K(+)-H(+)-ATPase, insignificantly or slightly reduced (by 10%) JK, ms respectively; in contrast, mucosal ouabain (1 mM) markedly inhibited JK, ms (control, 1.61 +/- 0.16 vs. ouabain, 0.83 +/- 0.98 mu equiv h-1 cm-2, P less than 0.001). 4. Mucosal Na+ appeared to be a competitor of K+ uptake across the apical membrane. 5. These results indicate that dietary Na-depletion increases electroneutral K+ absorption by increasing its transport capacity and suggest that the mechanism of this active K+ absorption process may involve an apical K(+)-ATPase with properties that are unlike the gastric K(+)-H(+)-ATPase but similar, in part, to Na(+)-K(+)-ATPase.

Dependence of spontaneous release at frog junctions on synaptic strength, external calcium and terminal length.

1. The calcium dependence of spontaneous transmitter release from nerve terminals of different lengths was examined at neuromuscular junctions in frog muscle. Miniature endplate potential (MEPP) frequency was positively correlated with the endplate potential (EPP) quantal content and was dependent on external Ca2+. The higher the resting MEPP frequency in a 0.25 mM-Ca2+ Ringer solution, the greater the dependence on external Ca2+. MEPP frequency in all terminals dropped to approximately the same low level in a Ca2(+)-free Ringer solution containing EGTA. This suggests that terminals with higher release levels have a larger Ca2+ influx at rest. 2. Several tests were done to try to characterize the mode of Ca2+ entry into resting terminals. omega-Conotoxin (omega-CgTx) blocked evoked release and reduced MEPP frequency, but not as effectively as zero Ca2(+)-EGTA Ringer solution. Some component of Ca2+ influx thus appears to enter through channels insensitive to omega-CgTx. Tetrodotoxin (TTX) did not affect MEPP frequency, indicating that the Ca2+ did not enter through TTX-sensitive Na+ channels that might be opening spontaneously at rest. Hyperpolarization of the terminal by reducing the K+ in the Ringer solution caused no consistent differences in MEPP frequency, suggesting that the Ca2+ influx is relatively insensitive to small changes in membrane potential around the resting level. Strong buffering of the Ringer solution with citrate, to overwhelm any differences in Ca2+ buffering within different junctional clefts, had no significant effect on the MEPP frequency. 3. Evidence that the Na(+)-Ca2+ exchanger helps set the internal Ca2+ level was obtained. Reduction of the Na+ concentration in the Ringer solution caused increases in MEPP frequency ranging from 6 to 440%. However, these changes were not correlated with resting MEPP frequency, hence differences in MEPP frequency probably are not the result of differences in Na(+)-Ca2+ exchanger function in terminals having a uniform Ca2+ leak. 4. Although MEPP frequency was generally correlated with quantal content, in subsets of junctions grouped according to their similar quantal contents, there was a positive correlation between MEPP frequency and terminal length. 5. In zero Ca2(+)-EGTA Ringer solution, the low residual MEPP frequency is independent of terminal length, even when MPP frequency is sharply increased by tetanic stimulation.

Different actions of a short-acting barbiturate on sodium and potassium conductances in invertebrate and vertebrate neurons

In this study, the effects of methohexital are compared on the voltage-gated sodium (Na +) and potassium ion (K +) conductances of Retzius cells in the leech Macrobdella and of dorsal root cells of the chick in culture. Under current-clamp conditions methohexital prolonged the Na +-dependent action potential of neurons in the leech. This prolongation occurred in the absence of changes in resting membrane potential or the maximum rate of depolarization of the spike. The prolonged action potentials were identical to those recorded in the same neurons in the absence of outward currents [i.e. in Ca 2+-free Ringer's solution containing Mn 2+, tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP)]. They consisted of an initial spike, followed by a plateau lasting several hundreds of milliseconds. Both components of the action potential were Na + -dependent and resistant to tetrodotoxin (TTX), while the plateau was selectively blocked by saxitoxin (STX), suggesting that it originated from the flow of Na + through a conductance different from that underlying the spike potential (Johansen and Kleinhaus, 1987). Similarly, the plateau of the action potential prolonged by methohexital, described in this study was abolished by 50 μM saxitoxin. These results suggest that the action of the drug resulted from a block of repolarizing K +-conductances. This was confirmed by voltage-clamp experiments which showed that methohexital (100–1000μM) reduced both I K and I A in the Retzius cell, essential mimicking the combined effects of TEA and 4-AP (Johansen and Kleinhaus, 1986b). In contrast, in dorsal root cells, methohexital decreased the amplitude of Na + and K + currents equally. This modulation of ionic conductances by methohexital may be important for the sedative and anesthetic actions of the drug.

PH in principal cells of frog skin (Rana pipiens): dependence on extracellular Na+.

Measurements of intracellular pH (pHi) and of apical cell membrane potential (Va) were made in principal cells of frog skin (Rana pipiens) with double-barrel microelectrodes under open-circuit conditions. The tissues were pretreated with stilbenes (10(-3) M) and bathed in HCO3- -free NaCl Ringer solution that was buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.8). Substitution of extracellular Na+ on both sides of the epithelium with N-methyl-D-glucamine caused intracellular acidification by 0.27 pH units. Restoration of Na+ on the apical side alone or on both sides caused a pHi recovery of 0.24 and 0.28 pH units, respectively, whereas return of Na+ on the basolateral side caused no recovery. Recovery of pHi on restoration of Na+ to the apical side was prevented by 10(-5) M 5-(N-ethyl-N-isopropyl)-amiloride. In individual preparations there was no correlation between pHi recovery due to return of apical Na+ and changes in Va. The average change in pHi was several times greater than the one expected from voltage clamp-induced changes in Va at constant extracellular Na+. The results suggest the presence of a Na+-H+ exchange on the apical side of principal cells. Such a process could be part of a negative feedback mechanism for regulation of Na+ entry via apical Na+ channels into principal cells.

Differential effects of ADH on sodium, chloride, potassium, calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron.

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na+, Cl-, K+, Ca2+ and Mg2+ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (JNa+, JCl-, JK+, JCa2+, JMg2+) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PDte) and transepithelial resistance (Rte) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO3- containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO3- free Ringer solutions. In cTAL segments, AVP (10(-10) mol.l-1) significantly increased JMg2+ and JCa2+ from 0.39 +/- 0.08 to 0.58 +/- 0.10 and from 0.86 +/- 0.13 to 1.19 +/- 0.15 pmol.min-1 mm-1 respectively. Neither JNa+ nor JCl-, (JNa+: 213 +/- 30 versus 221 +/- 28 pmol.min-1 mm-1, JCl-: 206 +/- 30 versus 220 +/- 23 pmol.min-1 mm-1) nor PDte (13.4 +/- 1.3 mV versus 14.1 +/- 1.9 mV) or Rte (24.6 +/- 6.5 omega cm2 versus 22.6 +/- 6.4 omega cm2) were significantly changed by AVP. No significant effect of AVP on net K+ transport was observed. In mTAL segments, Mg2+ and Ca2+ net transports were close to zero and AVP (10(-10) mol.l-1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone, JNa+ increased from 107 +/- 33 to 148 +/- 30 and JCl- from 121 +/- 33 to 165 +/- 32 pmol.min-1 mm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of calcium current in bull-frog atrial myocytes.

1. A single-microelectrode technique has been used to study the voltage dependence and the kinetics of inactivation and reactivation of a tetrodotoxin-resistant inward current (ICa) in single cells from bull-frog atrium. 2. In most cases the kinetics of both inactivation and reactivation can be well described as a single-exponential process. 3. Several different observations indicate that inactivation of ICa in these cells is controlled by both voltage-dependent and current-dependent processes, as has been demonstrated previously in heart (Kass & Sanguinetti, 1984; Lee, Marban & Tsien, 1985) and in other tissues (Hagiwara & Byerly, 1981; Tsien, 1983; Eckert & Chad, 1984). 4. Evidence in favour of a voltage-dependent inactivation mechanism included: (a) In paired-pulse measurements of steady-state inactivation ('f infinity') a 'conventional' steady-state f infinity vs. membrane potential (Vm) relationship was obtained in the range of membrane potentials from -60 to 0 mV. (b) Increasing [Ca2+]o from 2.5 to 7.5 mM, which resulted in a 2-3-fold increase in ICa, did not produce any significant increase in the amount of inactivation. (c) Using a 'gapped' double-pulse protocol non-monotonic U-shaped inactivation relationships were obtained, i.e. positive to approximately +20 mV some removal of inactivation occurred. However, f never approached a value near 1.00 at very depolarized potentials; it reached a maximum between 0.5 and 0.6. (d) In constant [Ca2+]o and at fixed Vm, the kinetics of ICa inactivation were independent of peak size of ICa. This was demonstrated by: (i) varying the holding potential (-90 to -30 mV), (ii) using paired-pulse 'recovery' protocols, and (iii) partial block by La3+ (1-10 microM) and Cd2+ (0.1 mM). (e) Influx of Ca2+ ions was not an obligatory prerequisite for development of inactivation. In all ionic conditions (Ca2+, Sr2+, Ba2+, Na+-free and Ca2+-free Ringer solutions) currents displayed inactivation phenomena, although the extent and kinetics of inactivation were dependent upon ionic conditions. Outward currents recorded above the reversal potential for ICa exhibited time- and voltage-dependent inactivation, and could be inactivated by brief depolarizing pre-pulses that produced no net inward current flow. Evidence against a possible role of the electrogenic Na+-Ca2+ exchanger in producing inactivation of these outward currents was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Ion transfer characteristics of the calcium current in bull-frog atrial myocytes.

1. Voltage clamp studies on single cells from bull-frog atrium have been carried out to study the ion transfer characteristics of the calcium current, ICa. In agreement with the preliminary results of Hume & Giles (1983), a TTX-resistant, 'second transient inward current' was recorded consistently. Its average peak size at 0 mV in 2.5 mM [Ca2+]o Ringer solution was approximately -200 pA, and it was blocked by Cd2+ and La3+ but not by tetrodotoxin (TTX, 3 x 10(-6) M). 2. The peak size of this current increases by approximately 4 times when [Ca2+]o is raised from 1.25 to 7.5 mM, indicating that Ca2+ is a major charge carrier. 3. A well-defined reversal potential, Erev, for ICa can be recorded in normal Ringer solution and also when Ba2+ or Sr2+ serve as the charge carriers. When [Ca2+]o is changed the shifts in Erev follow the predictions of a Nernstian Ca2+ electrode. However, all Erev values are well below those predicted from the thermodynamic Nernstian ECa values (see Campbell, Giles, Hume, Noble & Shibata, 1988a). 4. The Ca2+ current exhibits voltage-dependent inactivation, whether the direction of net current flow is inward or outward; however, the rate of inactivation is affected by the species of cation carrying the current. Inactivation is reduced substantially in Ba2+ Ringer solution. 5. Magnesium (5 mM) is not a significant carrier or blocker of ICa in normal [Ca2+]o Ringer solution; however, 5 mM [Mg2+]o can block the current carried by either Sr2+ or Ba2+. In the absence of Mg2+, equimolar substitutions of Sr2+ or Ba2+ for Ca2+ result in larger currents than those carried by Ca2+ in the normal Ringer solution. 6. Sodium appears not to be a significant charge carrier in the presence of normal [Ca2+]o. However, after free [Ca2+]o has been reduced to extremely low levels (less than 10(-6) M) Na+ can carry a significant fraction of 'ICa'. Thus, it appears that the high selectivity of ICa for Ca2+ ions depends upon the presence of Ca2+. 7. 'Slow tails' are frequently recorded after repolarizing clamp steps back to the holding potential. These 'slow tails' are prominent in normal [Na+]o, [Ca2+]o and [Sr2+]o Ringer solution; however, they are markedly reduced in [Ba2+]o, in Na+-free and Ca2+-free Ringer solutions. Experimental and theoretical work suggests these slow tails may be generated by an electrogenic Na+-Ca2+ exchanger (see Campbell, Giles, Robinson & Shibata, 1988b).(ABSTRACT TRUNCATED AT 400 WORDS)

Ionic conductances of cultured principal cell epithelium of renal collecting duct.

The ionic conductive properties were studied of epithelia of collecting duct principal cells which had been grown in primary tissue culture from renal cortex/capsule explants. When pretreated with aldosterone (10(-6) mol/l) and bathed on either surface with isotonic HCO3(-)-free Ringer's solution, the transepithelial voltage, Vte, varied between -21 and -72 mV (apical surface negative) while the transepithelial resistance, Rte, ranged from 0.4 to 1.5 k omega cm2. By 10:1 step-changes in Na+ concentration the apical cell membrane was shown to have a high conductivity for sodium, inhibitable by amiloride, 10(-6) mol/l. However, contrary to observations in natural collecting duct under control conditions, amiloride never reversed the polarity of Vte even at 10(-4) mol/l. Both the apical and the basolateral cell membranes were conductive for potassium and both conductivities were inhibitable by Ba2+ (5 mmol/l). 10:1 reduction of apical Cl- concentration strongly hyperpolarized Vte with a monophasic time course suggesting the presence of a paracellular shunt conductance for Cl-. In addition there may be a small Cl- conductance present in the apical cell membrane since apical application of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB) at 10(-7) mol/l produced a minute but significant hyperpolarization. On the other hand, 10:1 reduction of basolateral Cl- concentration caused a biphasic change in Vte (initial depolarization, followed by repolarization) which indicates the presence of a large Cl- conductance in the basolateral cell membrane. The latter was not inhibitable by 10(-7) mol/l NPPAB. Higher concentrations of this and of an other Cl-channel blocker produced non-specific effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Na +/H + exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pH 1) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4′,5′-dimethylfluorescein. Steady-state pH 1 in nominally bicarbonate free Ringer's solution averaged 6.87 ± 0.02 (mean ± S.E., n = 53). After intracellular acidification induced by the NH 4Cl-prepulse technique, there was a sodium-dependent pH 1 recovery towards the normal steady-state pH 1. The initial pH 1 recovery rate was a saturable function of extracellular sodium concentration with an apparent K m for external sodium of about 25 mM and a V max of about 0.28 pH units/min. Virtually no pH 1 recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 ± 0.05 pH units (mean ± S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pH 1 recovery after NH 4Cl prepulse by amiloride with an IC 50 of about 15 μM. Amiloride in a concentration of 1 mM almost completely abolished pH 1 recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 ± 0.03 pH units (mean ± S.E., n = 7) within 5 min. These findings suggest that a Na +/H + exchange is present in SIRC rabbit corneal epithelial cells. Na +/H + exchange seems to be the major process involved in pH 1 recovery in SIRC cells after an intracellular acid load. Na +/H + exchange also plays a role in the maintenance of steady-state pH 1.

Effect of alpha-latrotoxin on the frog neuromuscular junction at low temperature.

1. alpha-Latrotoxin (alpha-LTx) was applied to frog cutaneous pectoris muscles bathed at 1-3 degrees C in either Ringer solution, Ca2+-free Ringer solution with 1 mM-EGTA and 4 mM-Mg2+ or Ringer solution plus 4 mM-Mg2+, and its effects on miniature end-plate potential (MEPP) frequency, nerve terminal ultrastructure and uptake of horseradish peroxidase (HRP) were studied. 2. Large concentrations (2 micrograms/ml) of alpha-LTx increased MEPP rates to levels above 100/s at all junctions, but the time course of the increases depended upon the divalent cation content of the bathing solution. However, similar numbers of MEPPs (0.3-0.7 x 10(6] were recorded at all junctions during 2 h of secretion. 3. Nerve terminals exposed to alpha-LTx for 2 h lost 60-75% of their synaptic vesicles and were swollen; their presynaptic membranes were deeply infolded and they often contained many large vesicular structures. Terminals in Ringer solution retained the largest number of synaptic vesicles; terminals in Ringer solution plus Mg2+ swelled the least and contained the largest number of coated vesicles. The average number of synaptic vesicles lost was approximately equal to the average number of MEPPs recorded. 4. Few vesicles became loaded with HRP when this extracellular tracer was present in the bathing solution and the muscles were fixed near the peak of secretion. 5. When the terminals were warmed to 20 degrees C, those in the Ca2+-free solution with Mg2+ secreted additional quanta and lost almost all their residual vesicles; those in Ringer solution without Mg2+ secreted few additional quanta and retained most of their residual vesicles. 6. These results suggest that recycling was blocked at these terminals and that for each quantum secreted a vesicle became permanently incorporated into the axolemma.

Modulation of calcium channels of twitch skeletal muscle fibres of the frog by adrenaline and cyclic adenosine monophosphate.

1. Modulation of fast and slow Ca2+ channels of frog skeletal muscle by adrenaline (10(-6) to 10(-5) M) and cyclic AMP was investigated using intracellular voltage recordings in intact fibres and a voltage-clamp technique in cut fibres. 2. In tetraethylammonium (TEA), Cl(-)-free Ringer solution, adrenaline increased the maximum rate of rise of Ca2+ spikes by 85% and in a similar solution, peak slow Ca2+ current (ICa,s) by 51%. 3. Application of cyclic AMP to the cut ends of fibres, produced a relative increase of ICa,s of ca. 24%. The effect was maintained for ca. 2 h. 4. Changes in the time course of ICa,s were produced by adrenaline and cyclic AMP: the limiting values of time-to-peak current measured as a function of membrane potential were lower (ca. 41% in adrenaline and ca. 34% in cyclic AMP) than those found in control experiments. Also, ICa,s decayed faster in the presence of adrenaline or cyclic AMP. These changes can be explained by exhaustion of Ca2+ in the lumen of transverse tubular system and do not require the assumption of kinetic variations. 5. Fast Ca2+ currents (ICa,f) which could not be blocked by nifedipine were also recorded. Cyclic AMP greatly increased the amplitude of ICa,f but had no obvious effects on ICa,f kinetics. 6. Application of catalytic subunit of cyclic AMP-dependent protein kinase by diffusion or by pressure injection also increased the amplitude of ICa,s and ICa,f. Pressure injection brought about modifications in the time course of ICa,s that cannot be explained by depletion of Ca2+. 7. Mechanical experiments were performed on single fibres. Nominally Ca2+-free solutions prevented the development and the maintenance of positive inotropic effect of adrenaline on twitch tension. Development of twitch potentiation was dependent upon the frequency of stimulation. Adrenaline was practically ineffective if no stimulation was applied. 8. It is concluded that both populations of Ca2+ channels are modulated by adrenergic stimulation probably via cyclic AMP, and that twitch potentiation may be mediated by a Ca2+ entry through Ca2+ channels.

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder.

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.

Na+-H+ and Cl(-)-OH-(HCO3-) exchange in gastric glands.

The pH-sensitive, fluorescent, cytoplasmic-trapped dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to measure intracellular (pHi) and pH electrode to measure extracellular pH (pHo) in suspensions of gastric glands isolated from rabbit stomachs. The fluorescence of BCECF-loaded glands was calibrated in terms of pHi by equilibrating pHo and pHi using ionophores or digitonin and titrating pHo to different values. An APPENDIX is included that covers details of dye calibration and interpretation of fluorescence signals. Glands incubated in NaCl Ringer solution had pHi 7.11. Na+-free Ringer solution caused pHi to decrease reversibly to 6.80. Na+-dependent alkalinization of pHi followed a similar time course to the acidification of pHo. These changes were blocked by 1 mM amiloride. When gland cells were acidified (using two different techniques) realkalinization was completely Na+ dependent but was independent of the presence of Cl-; also, neither high extracellular K+ concentration ([K+]o) nor high [K+]o plus 10(-5) M valinomycin affected the rates of Na+-dependent alkalinization. A neutral Na+-H+ exchanger was implicated. Glands also exhibited Cl(-)-dependent changes of pHi that were blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (2 X 10(-4) M). A Cl(-)-OH-(HCO3-) exchanger was indicated. Other studies showed that intracellular buffering capacity was approximately 45 mM (pH-1) and that the apparent proton conductance of gland cell membranes was small.

Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase.

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.

Optical measurements of intracellular pH in single LLC-PK1 cells: demonstration of Cl-HCO3 exchange.

An optical method was used to continuously monitor intracellular pH (pHi) in single cultured LLC-PK1 cells. Rapidly growing or quiescent cells, attached to coverslips, were loaded with the pH-sensitive dye 4',5'-dimethyl-5(and -6)-carboxyfluorescein by exposing them to the dye's permeant precursor. pHi was calculated from the intracellular absorbance spectrum of the dye in a single cell. For cells incubated in HCO3(-)-free Ringer's solution, pHi recovered exponentially from acid loads applied by NH+4 prepulsing. Because the recovery was Na+-dependent and amiloride-sensitive, it was probably caused by Na-H exchange at the plasma membrane. In HCO3- Ringer's solution, external Cl- removal caused pHi to reversibly increase by approximately equal to 0.3. This pHi increase was substantially reduced by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by conducting the Cl- removal in the nominal absence of HCO3-. Reducing [HCO3-]o from 25 to 5 mM at constant pCO2 (lowering pHo from 7.4 to 6.7) caused pHi to reversibly fall by approximately equal to 0.2. This pHi change was greatly diminished by DIDS, by removal of extracellular Cl-, or by performing the same pHo shift in the nominal absence of HCO3-. The pHi changes induced by altering [Cl-]o or pHo were not inhibited by Na+ removal. Our data indicate that LLC-PK1 cells possess a Na-independent Cl-HCO3 exchanger and that this transporter may be as important as the Na-H exchanger in determining pHi.

Effect of bovine heart atrial natriuretic and diuretic fractions on sodium transport in isolated toad skin.

The effects of two bovine heart atrial fractions (AF I and AF II) were investigated in toad skin. When added to the inner (serosal) solution, AF I caused a maximal reversible depression of the transepithelial potential and short-circuit current of about 60% and AF II induced maximal decline of these parameters of only 35%. When added to the outer (mucosal) solution, the effect of both fractions was often only partially reversible. Isaacson's amiloride test showed that AF I decreased the numerical estimates of all the components in the electrical equivalent circuit. The net Na+ flux decreased due to a fall in Na+ flux in the active direction. Incubation with Ca2+-free Ringer's solution decreased and Ca2+ ionophore A 23187 increased the effect of AF II. Partial replacement of sodium by choline in the outer solution slightly reduced the effect and substitution of normal Ringer's solution with sulfate or isethionate Ringer's solution in the outer solution significantly enhanced the effect of the AF. These results indicate that bovine heart atrial natriuretic fractions decrease the sodium potential (ENa), sodium conductance (GNa) and passive conductance (Gsh). The fall in net Na+ transport measured with sodium tracer fluxes confirms the decrease in ENa and GNa.

Na+-sensitive component of 3-O-methylglucose uptake in frog skeletal muscle.

A Na+-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na+ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na+ removed. At a 3-O-MG concentration of 2 mM, the Na+-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na+-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na+-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na+-sensitive component was at a 3-O-MG concentration of about 13 mM, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5 mM), a potent inhibitor specific for Na+-coupled glucose transport, reduced the uptake in a solution containing Na+ to the level in Na+-free Ringer's solution. Glucose of concentrations higher than 20 mM suppressed 3-O-MG uptake to a level slightly lower than that in Na+-free Ringer's solution. These observations indicate that there are Na+-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na +, K +-ATPase and electrogenic Na +, K +-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na +,K +-pump and the Na +,K +-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K +-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K H +), induced by the re-introduction of potassium, was used as an index of electrogenic Na +,K +-pumping. The K H + was blocked by 1 μM ouabain. Na +,K +-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na +,K +-ATPase (IC 50 = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K H + of the frog ganglion. CMA inhibited Na +,K +-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC 50 = 4.5 μM) than did ouabain (IC 50 = 10 μM). In contrast, electrogenic Na +,K +-pumping (i.e. the K H +) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na +,K +-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na +,K +-pump in situ.