Free Ringer Research Articles

Potassium-dependent chloride and water transport across the seawater eel intestine

Simultaneous measurements of net ion and water fluxes and transepithelial potential difference (PD) were made in the stripped intestine of the seawater eel, and it was examined whether Cl- was driven following electrochemical gradient for Na+ across the brush border membrane of the epithelium or not. When mucosal Na+ was completely replaced with K+, while the serosa was being bathed with normal Ringer's solution, net Cl- and water fluxes were maintained as high as those in normal Ringer's solution. After serosal Na+ was completely replaced with choline+ while the mucosa was being bathed with Na+-free KCl Ringer's solution, 80% of the original Cl- and water fluxes still persisted, indicating significant Na+-independent Cl- and water transport. These results are against a hypothesis that Cl- is driven by electrochemical gradient of Na+ across the brush border membrane. The Na+-independent Cl- and water fluxes were a saturable function of mucosal K+ concentration, suggesting K+-dependent Cl- and water transport. A possible mechanism of Cl- transport is discussed in relation to K+ transport. On the other hand, a good correlation was observed between the net Cl- and water fluxes. This suggests that water transport depends on Cl- transport system; NaCl and/or KCl cotransport.

Extracellular chloride replacement by isethionate induces abnormal spontaneous release of transmitter at the frog neuromuscular junction.

1 Replacement of chloride by isethionate in Ringer solution bathing frog skeletal muscle fibres induces, after a delay of about 30 min, marked mechanical activity which was blocked by tubocurarine. This effect is reversed by washing out the isethionate. 2 Miniature end plate potentials (m.e.p.ps) and giant potentials (potentials greater than or equal to 2 X modal value) were recorded intracellularly in normal Ringer and isethionate Ringer solution. 3 The frequency of m.e.p.ps was unaltered by isethionate. The proportion of giant potentials increased from 3% in normal Ringer to 24.5% in isethionate Ringer after 90 min. This effect is usually reversible if the exposure to isethionate does not exceed 2 h. 4 The giant potentials were large enough to initiate trains of action potentials and still occurred in the presence of tetrodotoxin or Ca2+-free Ringer. Isethionate produced no change in the tau D of miniature endplate currents. 5 Chloride replacement by propionate produced no change in the proportion of giant potentials. 6 It is suggested that the isethionate anion can induce giant potentials and the possible mechanism of action is discussed.

The role of the electrogenic sodium pump in the glutamate afterhyperpolarization of frog spinal cord.

Drug responses of isolated hemisected frog spinal cords were examined by means of the sucrose-gap technique. The glutamate-induced depolarizations (glu-d) of motoneurones (recorded from ventral roots), and primary afferents (recorded from dorsal roots), were followed by an afterhyperpolarization (glu-a.h.). The depolarization induced by DL-homocysteic acid (DLH) was only occasionally followed by an afterhyperpolarization (DLH-a.h.). The glu-a.h. on both roots persisted in the presence of tetrodotoxin (TTX, 0.1-1 microM), or Ringer solution containing 10 mM-Mg2+; 0.1 mM-Ca2+ or 2 mM-Mn2+; 0.2 mM-Ca2+. This indicated that the response was neither due to the release of endogenous neurally active substances nor to the activation of a Ca2+-sensitive K+ conductance. The glu-a.h. was reduced or blocked by K+-free Ringer solution, 3-acetylstrophanthin (3-Ac-Str; 1 microM) or Li+ ions, and was therefore attributed to the activity of the electrogenic Na+ pump. The duration of depolarization induced by glu or DLH was increased in the presence of K+-free Ringer solution, 1 microM 3-Ac-Str or Li ions. It is therefore suggested that the electrogenic Na+ pump may play a role in limiting the duration of depolarization induced by the action of excitatory amino acids. The re-admission of K+ ions to preparations which had been incubated in K+-free Ringer solution produced a transient hyperpolarization (K-a.h.) of the membrane potential of ventral roots which is also attributable to the activation of the electrogenic Na+ pump. Both the K-a.h. and the glu-a.h. were enhanced in Ca2+-free Ringer solution. It is therefore suggested that the Ca2+ ions may modulate the activity of the electrogenic pump in central nervous tissue.

Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange.

Using pH-sensitive microelectrodes to measure intracellular pH (pHi) in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum, we have found that when cells are acid-loaded by pretreatment with NH+4 in a nominally HCO3--free Ringer, pHi spontaneously recovers with an exponential time course. This pHi recovery, which is indicative of active (i.e., uphill) transport, is blocked by removal of Na+ from both the luminal and basolateral (i.e., bath) solutions. Re-addition of Na+ to either the lumen or the bath results in a full pHi recovery, but at a lower-than-normal rate; the maximal rate is achieved only with Na+ in both solutions. The diuretic amiloride reversibly inhibits the pHi recovery when present on either the luminal or basolateral sides, and has its maximal effect when present in both solutions. The pHi recovery is insensitive to stilbene derivatives and to Cl- removal. A transient rise of intracellular Na+ activity accompanies the pHi recovery; there is no change of intracellular Cl- activity. These data suggest that these proximal tubule cells have Na-H exchangers in both the luminal and basolateral membranes.

Electrogenesis of the slow inhibitory postsynaptic potential in bullfrog sympathetic ganglia.

The ionic mechanisms of the slow surface positive (P)-potential and the slow inhibitory postsynaptic potential (IPSP), an intracellularly recorded P-potential in sympathetic ganglia, were analysed by means of sucrose-gap, intracellular microelectrode techniques, and voltage clamp technique. Both the P-potential and the slow IPSP consist of two different potential components, namely the ouabain-sensitive and the ouabain-insensitive components. The ouabain-sensitive component was enhanced by a moderate conditioning hyperpolarization. This component was most reasonably explained as a potential change generated by an activation of the electrogenic Na+ pump. The ouabain-insensitive potential component of the P-potential and the slow IPSP decreased in the amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of ouabain-insensitive component of slow IPSP and slow inhibitory postsynaptic current (IPSC) was close to the EK. The amplitude of ouabain-insensitive component of P-potential and slow IPSP was markedly decreased by an elevation of external K+ concentration. The reversal potential of ouabain-insensitive component shifted to a more positive potential level in high K+ Ringer's solution. On the other hand, it was augmented in K+-free Ringer's solution. A reduction of the membrane resistance was observed during the generation of the slow IPSP, when the membrane potential of ganglion cells was held at a membrane potential level more negative than -60 mV. The slow IPSC recorded by voltage-clamp method was associated with an increase in membrane conductance. It was concluded that the ouabain-insensitive component was generated by an activation of K+ conductance.

The contribution of the electrogenic sodium-potassium pump to the electrical activity of toad rods.

1. The membrane potential of rods in the isolated toad retina was recorded while changing the ionic composition of the extracellular medium.2. Caesium (Cs(+)) at a concentration of 1 mM was sufficient to completely block the sag from the peak to the plateau in the bright-flash voltage response.3. In the presence of 10 mM-Cs(+) the bright-flash response increased in amplitude to about 90 mV, thus reaching an absolute membrane potential of between -110 and -135 mV. These responses consisted of an initial fast component of about 35 mV followed by a much slower component which could be as large as 50 mV.4. At the peak of the initial fast component the rod membrane conformed closely to the behaviour of a K(+) electrode with a P(Na)/P(K) ratio of 0.023. On average the amplitude of the slow component was about 35 mV in the presence of 2.6 mM-K(+) and was reduced to about 25 mV in a K(+)-free Ringer.5. Addition of 100 muM-strophanthidin to the perfusate induced several reversible changes in the electrical activity of rods. The dark resting membrane potential depolarized by about 5 mV and the kinetics of the voltage response to dim flashes of light slowed down. The voltage sensitivity initially increased by about 30%, but the peak of the response to a bright flash of light was reduced by about 13 mV.6. In rods treated with 10 mM-Cs(+) the slow component present in the bright flash response was abolished by strophanthidin with an apparent K(m) of 3 muM.7. The amplitude of the slow component decreased with a time lag of about 2 min when external Na(+) was reduced. A previous exposure of the retina to a Na(+)-free Ringer solution for at least 3 min modified the voltage photoresponse in a way similar to that observed in the presence of 100 muM-strophanthidin.8. When external Ca(2+) concentration ([Ca(2+)](o)) was increased from 2 to 5 mM the slow component decreased by about 30%. When [Ca(2+)](o) was reduced the slow component increased. A twofold increase was observed when [Ca(2+)](o) was lower than 10(-4) M.9. It is suggested that the slow component of the voltage response in the presence of external Cs(+) is caused by an electrogenic current driven by the Na(+)-K(+) transport system, during a voltage-dependent block of external Cs(+) of some K(+) channels.

Action potential repolarization may involve a transient, Ca2+-sensitive outward current in a vertebrate neurone.

Repolarization of the action potential in squid axon1 and several types of neurones2-4 involves a voltage-activated potassium (K+) current. Voltage clamp analysis has demonstrated that this current has rapid activation kinetics1,3-5. In several neuronal types, the same technique has also revealed a slowly activated K+ current that is calcium (Ca2+)-sensitive3,5-10. This slow Ca2+-activated K+ current is the major current underlying the late, slower portion of the after-hyperpolarization following an action potential11-14. In several muscle types, fast, transient Ca2+-dependent K+ currents have been described15-17 which may contribute to repolarization of the action potential. Rapidly activating, Ca2+-dependent K+ currents have been observed in sympathetic neurones of the bullfrog and it has been suggested that they contribute to action potential repolarization of those neurones8,9,18. We have studied the membrane currents in bullfrog sympathetic neurones using voltage clamp methods and report here a transient outward current that appears to be composed of two separate currents. One of those currents is a transient, Ca2+-sensitive outward current as indicated by a significant reduction of the current by treatments that reduce or block Ca2+ entry (Mn2+, Cd2+, Co2+, Mg2+ or Ca2+-free Ringer). Such treatments also decreased the rate of action potential repolarization. The results suggest that this current is involved in repolarization of the action potential and consequently may regulate Ca2+ entry into the neurone during spike activity.

Ammonium action on post-synaptic inhibition in crayfish neurones: implications for the mechanism of chloride extrusion.

1. The reversal potential of the Cl(-)-dependent, inhibitory post-synaptic potential (E(i.p.s.p.)) was measured in the isolated crayfish stretch receptor neurone using two intracellular micro-electrodes. The difference between E(i.p.s.p.) and the resting membrane potential (E(m)), the i.p.s.p. driving force, was reversibly decreased by addition of NH(3)/NH(4) (+), and the mechanism of this decrease was investigated.2. The NH(3)/NH(4) (+)-induced decrease in i.p.s.p. driving force was dose-dependent with an onset at about 0.2 mM. E(i.p.s.p.) always remained more negative than E(m) or, when the neurone was spontaneously firing, the threshold potential. E(m) and resting membrane resistance (R(m)) also decreased in a dose-dependent fashion. Synaptic conductance (g(s)) increased with low doses, but decreased on application of 20 mM-NH(3)/NH(4) (+). All the effects were fully reversible on return to normal Ringer solution.3. Intracellular acidification (substitution of 50% Cl(-) by acetate compared with isethionate) considerably reduced the i.p.s.p. driving force. Simultaneous application of NH(3)/NH(4) (+) and acetate-substituted Ringer solution caused a similar decrease in the driving force to application of the same concentration of NH(3)/NH(4) (+) under normal conditions. Increasing the extracellular pH at which a given concentration of NH(3)/NH(4) (+) was applied caused a smaller decline in the i.p.s.p. driving force. These results suggest that intracellular acidification decreases the i.p.s.p. driving force and that the NH(3)/NH(4) (+)-induced decline is caused by an action of the ammonium ion.4. Elevation of extracellular K(+) (K(+) (0)) decreased the i.p.s.p. driving force, E(m) and R(m), and increased g(s). Reduction of K(+) (0) had the converse effects on all parameters.5. Application of Rb(+) or Cs(+) mimicked the effects of NH(3)/NH(4) (+). Substitution of K(+) (0) by Rb(+), Cs(+) or NH(3)/NH(4) (+) opposed or even reversed the increase in i.p.s.p. driving force when Na(+) was used as the substitute. The effectiveness of the various cations in decreasing the driving force was in the following order: Rb(+) > NH(4) (+) > K(+) > Cs(+).6. Inhibition of the Na pump by ouabain or K(+)-free Ringer solution caused a gradual reduction in the i.p.s.p. driving force. Since the driving force also decreased when the Na(+) gradient probably was increased (elevated K(+) (0)), this suggests a dependence on the K(+) gradient rather than the Na(+) gradient or the Na pump itself.7. Frusemide (6 x 10(-4) M) reversibly decreased the i.p.s.p. driving force and E(m), and increased g(s). R(m) was not significantly affected. Application of frusemide in the presence of 5 mM-Rb(+) and vice versa, caused a further reduction in the driving force. The recovery of the driving force on removal of either agent was slowed by the presence of the other.8. Application of 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS; 10(-4) M) caused spontaneous firing and reduced E(i.p.s.p.) to the threshold potential. R(m) and g(s) increased. The effects were slowly reversible on removal of the drug.9. It is proposed that the i.p.s.p. driving force is maintained by a K(+)-Cl(-) co-transport mechanism, driven by the K(+) gradient. The K(+) site exhibits the binding selectivity: Rb(+) > NH(4) (+) > K(+) > Cs(+) and the mechanism is inhibited partially by frusemide and completely by DIDS.

Divalent cations and fast axonal transport in chemically desheathed (triton X-treated) frog sciatic nerve

We have studied the ability of divalent cations to restore to normal axonal transport (AXT) which was inhibited by deprivation of Ca 2+ and/or Mg 2+ ions. The epi- and perineurium of the frog sciatic nerve were damaged by a 30-s wash in Triton X-100 containing frog Ringer's. This treatment did not affect either AXT or nerve levels of Ca 2+ and Mg 2+, but made the ions more easily extractable with a Ca 2+- and Mg 2+-free Ringer's solution (CMFR). Inhibition of AXT was achieved by incubating Triton X-100-treated nerves in CMFR + EGTA for 5 h, followed by an additional incubation for 12 h in CMFR or Ringer's devoid of only Ca 2+ (CFR). These treatments reduced Ca 2+ and Mg 2+ contents by 77% and 38% respectively. Addition of Ca 2+ (1.1 mM) during the 12-h period stimulated AXT, measured as accumulation of 3H-labelled components in front of a ligature, several fold. Mg 2+ could not substitute for Ca 2+ but potentiated the stimulating effect of Ca 2+. Addition of other divalent cations did not affect AXT (Sr 2+ and Ba 2+) or potentiated the inhibition caused by Ca 2+-deprived medium (Mn 2+ and Co 2+). ATP and creatine phosphate contents were similar in nerves incubated in Ca 2+-deprived medium and in Ca 2+-containing Ringer's. Thus, inhibition of AXT in the former situation was not due to a decreased availability of high energy phosphates. Two calcium antagonists, D-600 and nifedipin, which are potent smooth muscle relaxants, effectively blocked AXT. The present ressults suggest that Ca 2+ is specifically required to maintain AXT and that an anlogy exists between Ca 2+ regulation during smooth muscle contraction and AXT.

Reversal of copper-induced inhibition of vasopressin responsiveness by reducing agents

Copper inhibits the hydro-osmotic response to vasopressin in the urinary bladder of Bufo marinus at a site proximal to cyclic AMP production. This effect is not reversed by washing in Cu 2+-free Ringer's solution but is overcome by serosal addition of reducing agents, suggesting that vasopressin responsiveness in this tissue is modulated by either the redox potential or the sulfhydryl content of the serosal membrane.

Chloride extrusion in the isolated perfused teleost gill

Chloride extrusion is examined in the isolated perfused gill of the pinfish,Lagodon rhomboides. In both sea water and Ringer's baths, the Cl efflux from the isolated gill is 45% that of the intact animal. The transepithelial electrical potential (TEP) across the isolated gill in sea water is equal to that in vivo, in Ringer's the gill TEP is slightly less than in vivo. Cl efflux is linearly dependent upon afferent flow of the perfusate. Furosemide, added to the perfusate inhibits 57% of the Cl efflux in gills bathed bilaterally by Ringer's. Ouabain causes a marked vasoconstriction and increase in afferent pressure. Removal of Na from the perfusate produces an inhibition of the Cl efflux that is not potential mediated. Net extrusion of Cl is inhibited in isolated gills bathed bilaterally by sodium free Ringer's.

Involvement of extracellular calcium in gastric stimulation.

We have tested whether external Ca2+ is required for either initiation or maintenance of secretory parameters, including membrane elaboration of oxyntic cells, in frog gastric mucosa. Ca2+ was removed from in vitro mucosal preparations [by washing repeatedly in Ca2+-free Ringer solution and adding 0.1 mM ethylene glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid to the serosal solution] either before (i.e., resting tissues) or after addition of stimulants. Electrophysiology [transepithelial potential difference (PD) and resistance], morphology (morphometric analysis of transmission electron micrographs), and transport (H+ secretion) were monitored. La3+ (1 mM) was added to the mucosal solution to help maintain resistance and PD. La3+ decreased tissue shunt conductance during Ca2+-free conditions, as evidenced by a decreased mucosal-serosal flux of 22Na+, presumably by preserving tight-junction integrity. Secretion was elicited by histamine alone or in combination with dibutyryl cAMP and isobutylmethylxanthine (a phosphodiesterase inhibitor). External Ca2+ is not required for the initiation of H+ secretion or the accompanying morphological changes when the combined stimulants are used, whereas H+ secretion and the morphological change showed some Ca2+ dependency when histamine alone was used. Thus, histamine-elicited secretion seems to be more sensitive to Ca2+ removal than that brought about by the combined stimulants. Long-term effects of Ca2+-free solutions on resistance, PD, and H+ secretion can largely be explained by disruptive effects on tight junctions.

Effects of secretagogues on the K + permeability of mucosal and serosal borders of rabbit colonic mucosa

(1) K + efflux rates from the mucosal and serosal surfaces of sheets of rabbit colonic mucosa have been determined by measuring net K + loss into K +-free Ringer solution bathing each side of the tissue. (2) Initially, there is a high rate of K + loss from the tissue, this falls to a lower steady-state rate after 20 min. Loss of K + from the tissue into the serosal bath is 6–8-fold faster than loss to the mucosal bath. (3) A number of intestinal secretagogues, e.g. theophylline, cyclic AMP, carbachol, ionophore A23187, as well as the laxative bisacodyl, raise the K + efflux rate across the mucosal border by 200–300%. In the case of K + efflux induced by carbachol the effect is shown to be dependent on raised levels of intracellular Ca 2+. Ca 2+-calmodulin complex does not appear to be be involved in activation of K + efflux across the mucosal border. (4) Amiloride does not block mucosal K + efflux, but tetraethyl-ammonium does inhibit K + efflux across the mucosal border, induced by either bisacodyl or raised intracellular Ca 2+. (5) The results suggest that laxatives may increase the rate of K + secretion into the colonic lumen by raising the K + permeability of the mucosal border.

The ionic mechanism of intracellular pH regulation in crayfish neurones.

1. Intracellular pH (pHi) regulation in crayfish neurones was studied using pH-, Na+-, and Cl- sensitive micro-electrodes. Neuronal pH regulation has previously been studied only in molluscs. 2. The average resting pHi of crayfish neurones was 7.12 +/- 0.09, which is 1 pH unit more alkaline than that predicted were H+ ions distributed in equilibrium with the membrane potential. 3. When the cytoplasm was acidified (by NH4Cl loading, CO2 application, or HCl injection), pHi recovered towards its resting value. 4. Removal of Na+ from the external solution inhibited pHi recovery from an acid load by more than 90%. pHi recovery resumed immediately when external Na+ was reintroduced. 5. The resting intracellular Na+ concentration ([Na+]i) of crayfish neurones was 15-25 mM. During pHi recovery from an acid load, [Na+]i increased by 10-50 mM. 6. Reducing the external HCO3(-) concentration from 5 mM to 0 mM slowed pHi recovery by an average of about 45%. This slowing was appreciable even in cells in which Na+ removal almost totally blocked pHi recovery. 7. The resting intracellular Cl- concentration ([Cl-]i) was 30-40 mM, indicating that these cells actively accumulate Cl-. During pHi recovery from an acid load, [Cl-]i decreased by 3-5 mM. 8. In the presence of the anion exchange inhibitor SITS (4-acetamide-4'-isothiocyanostilbene-2,2'-disulphonic acid), pHi recovery was slowed to the rate which was normally seen in HCO3(-)-free Ringer solution. SITS abolished the dependence of pHi recovery on the external HCO3(-) concentration. 9. It is concluded that pHi regulation in crayfish neurones involves two separate mechanisms: a Na+-dependent, HCO3(-)-independent acid extrusion process, and a Cl---HCO3(-) exchange which is probably also Na+-dependent.

Ultrastructural dynamics of exocytosis in the ovulation-neurohormone producing caudo-dorsal cells of the freshwater snail Lymnaea stagnalis (L.).

The ultrastructural dynamics of exocytosis in the ovulation-stimulating neurosecretory Caudo-Dorsal Cells (CDC) of the freshwater snail L. stagnalis were studied after incubation of cerebral ganglia in Ringer's solutions with different concentrations of K+ and Ca2+. Detection of exocytosis was facilitated by the use of the tannic acid-glutaraldehyde fixation method (TAGO-method). In control Ringer (low K+) the frequency of exocytosis was rather low. Exocytosis mainly occurred as "terminal" exocytosis (TE); "intracellular" (ICE) and, particularly, "multiple" exocytosis (ME) took place infrequently. Incubation in high K+-containing Ringer strongly increased exocytotic activity. Compared to the controls the total frequency of exocytosis was 50 X as high, whereas TE, ICE and ME occurred 6 X, 47 X, and more than 300 X as frequently, respectively. In high K+/Ca2+-free Ringer the total frequency of exocytosis was only 2 X as high as in control Ringer. It is concluded that TE, ICE, and ME are normal, Ca2+-dependent exocytotic phenomena. The significance of their dynamics in response to K+-stimulation is discussed. The extremely high frequency of exocytosis, as well as the presence of "unaltered granule contents in transit", is explained by assuming that an exocytotic event in the CDC lasts rather long, viz. some minutes. The results may reflect the physiological mechanism by which the CDC release their ovulation hormone. The possible involvement of "clear" and "large" electron lucent vesicles in membrane reuptake after exocytosis is considered.

Effects of ouabain on chloride movements across the seawater eel intestine

Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K+ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na+−K+ pump by ouabain, K+-free or Na+-free Ringer solution increased the diffusion potential for Cl−. Inhibition of Cl− transport and increment in Cl− permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl− transport as well as Cl− permeability is dependent on Na+−K+ pump activity. A possible mechanism of dependence of Cl− transport on the Na+−K+ pump is discussed in relation to the increment in Cl− permeability.

Effect of glucagon on ion transport in mouse intestine.

Effects of glucagon (GN) on short-circuited mouse intestine were studied. GN (30 microgram . ml-1), added to the serosa of intestine mounted in an Ussing chamber and bathed in glucose-free Ringer, induced significant increases of 43% in serosal-to-mucosal Cl- flux (Js leads to m Cl), 315% in net Cl- secretion (Jnet Cl), 85% in net residual flux (J net R), 61% in short-circuit current (Isc), and 44% in open-circuit potential difference (PD). The mucosal-to-serosal Cl- flux and both unidirectional Na+ fluxes (Jm leads to s Na and Js leads to m Na) were unchanged. In a glucose Ringer bathing medium, GN exhibited no significant effects on ion fluxes and electrical parameters. To eliminate the possibility that observed GN-induced changes in PD and Isc were partially due to changes in membrane surface charge, the effects of GN in Cl- -free Ringer were studied. Under these conditions, GN had no effect on electrical parameters. Furthermore, GN elicited no effect on cAMP levels in either the presence or absence of glucose. These findings suggest that 1) the effect of GN on Jnet Cl is masked in the presence of glucose, 2) GN-induced increases in Isc and PD are a reflection of the increase in Jnet Cl and are neither due to changes in membrane surface charge nor to an increase in net Na+ flux, and 3) GN-induced secretory diarrhea is in part due to changes in electrolyte transport.

Two modes of phosphate transport by turtle urinary bladder.

The effects of changes in pH and addition of CO2/HCO3- on transepithelial phosphate transport were studied in turtle urinary bladder. Net mucosa-to-serosa flux of phosphate (JP) was determined as the difference between unidirectional 32P fluxes in the absence of transepithelial electrochemical gradients. With 5 mM phosphate in HCO3--free Ringer at pH 8.4, JP was 21.8 +/- 7.4 nmol . 8 cm-2 . h-1. There was a slight increase in JP with isohydric addition of 10 mM HCO3-. Addition of 5% CO2, which reduced pH to 7.3, did not affect JP. At pH 8.4, JP was not affected by ouabain or dinitrophenol and increased progressively as phosphate concentration was raised between 0.5 and 10 mM. At pH 6.2 in the absence of exogenous CO2 and HCO3-, JP was undectable. With 2.5 mM HCO3- and 5% CO2 at pH 6.5, JP was 61.3 +/- 16.0 and decreased to 30.6 +/- 1.6 nmol . 8 cm-2 . h-1 when pH was raised to 7.2 by increasing HCO3- to 10 mM. At pH 6.5 JP was inhibited by both ouabain and dinitrophenol. These results suggest that at acidic pH, JP results from the tranport of H2PO4-. The transport of H2PO4- is CO2 dependent and inhibited by ouabain and dinitrophenol. In contrast, at alkaline pH, JP results from the transport of HPO4(2-), which is neither CO2 dependent nor inhibited by ouabain or dinitrophenol.

THE ACTION OF CHLORPHENESIN CARBAMATE ON THE FROG SPINAL CORD

Studies were carried out to elucidate the mechanism of action of chlorphenesin carbamate (CPC) and to compare the effect of the drug with that of mephenesin on the isolated bullfrog spinal cord. Ventral and dorsal root potentials were recorded by means of the sucrose-gap method. CPC caused marked hyperpolarizations and depressed spontaneous activities in both of the primary afferent terminals (PAT) and motoneurons (MN). These hyperpolarizations were observed even in high-Mg2+ and Ca2+-free Ringer’s solution, suggesting that CPC has direct actions on PAT and MN. Various reflex potentials (dorsal and ventral root potentials elicited by stimulating dorsal and ventral root, respectively) tended to be depressed by CPC as well as by mephenesin. Excitatory amino acids (L-aspartic acid and L-glutamic acid) caused marked depolarizations in PAT and MN, and increased the firing rate in MN. CPC did not modify the depolarization but abolished the motoneuron firing induced by these amino acids. However, mephenesin reduced both the depolarization and the motoneuron firing. The dorsal and ventral root potentials evoked by tetanic stimulation (40 Hz) of the dorsal root were depressed by the drugs. These results indicate that CPC has an apparent depressing action on the spinal neuron, and this action may be ascribed to the slight hyperpolarization and/or the prolongation of refractory period.

Effects of some divalent cations on synaptic transmission in frog spinal neurones.

1. Synaptic transmission between dorsal root afferents and motoneurones was studied in the isolated and hemisected spinal cord of frogs, using intracellular and extracellular recording techniques, and ionic substitutions of divalent cations in the bathing fluid. 2. Delayed components of excitatory post-synaptic potentials (e.p.s.p.s) evoked in motoneurones by dorsal root supramaximal stimuli, as well as the Ca2+-dependent slow after-hyperpolarization which follows antidromic spikes, were reversibly blocked by superfusing the cords with 'Ca2+-free' media containing Co2+ (4 mM) or Mg2+ (6-10 mM). However, short latency e.p.s.p.s persisted in these media for more than 8 hr. 3. The minimum synaptic delay of the Co2+ and Mg2+, resistant e.p.s.p.s, measured from the peak negativity of the extracellularly recorded presynaptic spike to the onset of the e.p.s.p., was 0.3 msec at 10 +/- 1 degrees C. 4. The Co2+, Mg2+-resistant e.p.s.p.s were graded, and could be elicited by stimulation of segmental or adjacent roots. Those evoked by each of two adjacent roots showed linear summation when the roots were stimulated simultaneously. 5. The Co2+, Mg2+-resistant e.p.s.p.s decreased in amplitude at stimulating frequencies between 10 and 100 Hz, and with paired stimuli at intervals shorter than 20-40 msec. These reductions in amplitude were paralleled by decreases in amplitude of the presynaptic population spike. 6. Solutions free of divalent ions, containing EGTA (2 mM) abolished the Co2+, Mg2+-resistant e.p.s.p.s. They remained blocked for a variable time after returning to Ca2+-free Ringer containing Mg2+ (8 mM). Their continued abolition at this stage is probably not due to changes in electrical properties of motoneuronal membranes. Eventually, the Mg2+-resistant e.p.s.p.s started recovering in the Ca2+-free Ringer containing Mg2+. The time of onset of this recovery depended on the duration of exposure to EGTA. 7. Sr2+ (2-11 mM), although less effective than Ca2+, restored the composite e.p.s.p.s evoked by dorsal root supramaximal stimuli, as well as the Ca2+-dependent slow after-hyperpolarization of the motoneurone. The composite e.p.s.p.s could not be restored with Ba2+ (2-10 mM). 8. The results suggest that the Co2+, Mg2+-resistant e.p.s.p is generated by electrical coupling between some afferent fibres (probably primary afferents) and motoneurones. The after-effects of EGTA treatments probably reflect uncoupling of electrotonic junctions. In contrast, the delayed components of the composite e.p.s.p.s are generated through chemical synapses whose divalent cation requirement is similar to that of the neuromuscular junction.