Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Effect of external magnesium on intracellular free sodium: Na+ flux via Na+/Mg2+ antiport is masked by other Na+ transport systems in rat cardiac myocytes.

Mg2+ efflux from heart cells on a Na+/Mg2+ antiport has been postulated, but the Na+ flux component of the antiport has not been demonstrated. The study aimed to establish if the Na+ flux component could be measured by following changes in [Na+]i with SBFI during conditions known to reverse the antiport (5 mmol/L Mg2+(o), Na+(o)- & Ca2+(o)-free): and after minimising the activity of other Na+ transport pathways. Resting [Na+]i was 8 +/- 0.7 mmol/L (mean +/- S.E., n = 39 cells) in normal Tyrode's solution. [Na+]i decreased below the normal level in all cells (a decline of 4-5 mmol/L, n = 21) during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free). Controls using 1 mmol/L Mg2+(o) showed similar declines in [Na+]i, but the fall was greatest when Na+(o) was replaced by K+(o) (decline of 6 mmol/L) rather than the tetramethylammonium ion (TMA+). The rate of decrease in [Na+]i during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free) was slowed by 20 microM ouabain (n = 5) or by elevation of pHo to pH 9 (n = 7) so that [Na+]i remained close to the initial value. The decrease of [Na+]i was not affected by 10 microM imipramine (n = 15). These data suggest that the Na+ efflux component of the Na+/Mg2+ antiport is masked in Na+(o)- and Ca2+(o)-free conditions by other Na+(i) efflux pathways.     

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.     

Intracellular free magnesium and its regulation, studied in isolated ferret ventricular muscle with ion-selective microelectrodes

Intracellular free magnesium ([Mg2+]i) was measured in isolated ferret papillary muscles using ion-selective microelectrodes filled with the new magnesium sensor ETH 5214. This new sensor, unlike its predecessor ETH 1117, does not react to marked changes in K+, Na+ or pH. Reducing Ca2+ from 20 microM to around 10 nM also did not affect the response so these electrodes are ideally suited to study intracellular Mg2+ and its regulation. The mean value for the [Mg2+]i from thirty-two experiments (forty-two impalements) was 0.85 mM, confirming previous estimates from this laboratory. Intracellular Mg2+ is not passively distributed and the possibility that Mg2+ is transported out of the cell by a Na(+)-Mg2+ exchanger was investigated. An increase in [Mg2+]o caused an increase in [Mg2+]i, as did stepwise reduction in the [Na+]o. However, this increase in [Mg2+]i on Na+ reduction also occurred in Mg2(+)-free solution suggesting that the increase in [Mg2+]i was due to the increase in intracellular Ca2+ on Na+ reduction. Moreover, increasing [Na+]i by strophanthidin did not change the [Mg2+]i and on increasing [Mg2+]o there was no reduction in the [Na+]i. Blocking ATP production lead to small increases in the [Mg2+]i. These results are not consistent with a Na(+)-Mg2+ exchanger as being the main outward transport mechanism for Mg2+ in this tissue.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Fluid movements across rabbit ileum coupled to passive paracellular ion movements.

1. Theophylline (10 mM) and choleragen (1 x 10(-6) g ml.-1) abolish net fluid absorption by everted sacs of rabbit ileum. Triaminopyrimidine (20 mM) and ethacrynate (0.1 mM) prevent this inhibition of net fluid movement. Replacing Ringer Cl- with isethionate prevents the theophylline-dependent decrease in fluid absorption also. 2. Ouabain (0.1 mM) abolishes net fluid movements in both control and theophylline-treated tissue. 3. With ouabain present, hypertonic NaCl (200 mM) in the mucosal solution causes net fluid secretion (serosal-mucosal flux). With theophylline added to both the mucosal and serosal solution, net fluid absorption (mucosal-serosal flux) is observed (P less than 0.001). Triaminopyrimidine (20 mM), or ethacrynate (0.1 mM), or replacement of Ringer Na+ with choline, or Ringer Cl- with isethionate all prevent the theophylline-induced reversal of osmotic flow. 4. Theophylline increases passive net flux of Na+ and Cl- from mucosal solution containing hypertonic (200 mM) NaCl+ ouabain (0.1 mM) across sheets of ileum into serosal solution containing mannitol Ringer + ouabain. The increased passive Na+ flux is blocked by triaminopyrimidine and the increased Na+ and Cl- fluxes are blocked by ethacrynate (0.1 mM). 5. The suggested route of increased NaCl leakage is via the paracellular pathway as it is inhibited by triaminopyrimidine. The increase, itself, is a consequence of the increased passive permeability of the mucosal border to Cl-, induced by theophylline or choleragen. Water is apparently electro-osmotically coupled to the paracellular Na+ leakage (100 mole water mole-1 Na+), hence increased passive leakage reverses osmotic flow. In active tissue the lateral intercellular space contains hypertonic NaCl, and hence increased leakage of NaCl across the tight-junction in theophylline or choleragen-treated tissue gives rise to net fluid secretion.     

The effect of adenosine triphosphate on the transmural potential in rat small intestine

1. ATP either in the mucosal or serosal fluid caused a transient increase in the potential difference and short-circuit current across the wall of rat jejunum in vitro, the serosal solution becoming more positive.2. Similar responses were observed in the ileum and colon, and in in vivo preparations of small intestine.3. The response is relatively specific for ATP.4. The transient nature of the response is not due to rapid hydrolysis of extracellular ATP.5. High concentrations of K(+) in the mucosal medium, serosal ouabain or mucosal 2,4-dinitrophenol all inhibit the response without altering the time course.6. Significant inhibition was not observed in the presence of ADP or in Mg(2+)-free salines.7. The results are consistent with an intracellular action of ATP in the epithelium to stimulate net ion transport. The results do not demonstrate whether or not extracellular ATP can act as an energy donor for an electrogenic ion pump.8. Theophylline prolongs the time course of the response, and the involvement of the adenyl cyclase system cannot be excluded as an explanation for the findings.     

Myocardial cation contents during induction of calcium paradox

Myocardial cation contents were measured in isolated rat hearts perfused under various conditions. Reperfusion of Ca2+-deprived hearts produced marked increases in myocardial Ca2+ and Na+ and decreases in Mg2+ and K+ contents. These changes were dependent on the Ca2+ concentration and duration of perfusion during the periods of Ca2+ deprivation and reperfusion. The loss of Ca2+ and K+ contents normally seen after Ca2+-free exposure as well as the reperfusion-induced changes were prevented if the Ca2+-free medium contained low (35 mM) Na+ or was cooled to 21 degrees C. Reperfusion with normal Ca2+, low Na+ medium augmented the increase in myocardial Ca2+ content, while reducing K+ or Mg2+ or increasing Mg2+ in the reperfusion medium had no effect. Addition of verapamil, D600, or propranolol to the reperfusion solution did not alter the reperfusion-induced cation changes observed using control medium. These data suggest that during Ca2+ depletion, the mechanisms responsible for regulating calcium influx are either lost or inactivated, so that reperfusion-induced changes are governed solely by the driving force favoring calcium influx. The occurrence of Ca2+ overload under this condition has been implicated in the irreversible damage to myocardium and contractile failure.     

Aconitine-induced delayed afterdepolarization in frog atrium and guinea pig papillary muscles in the presence of low concentrations of Ca2+

Aconitine will induce arrhythmias after the fiber has been completely repolarized. This arrhythmia is generally facilitated in the presence of high Ca2+ solution, yet the aconitine-induced arrhythmia occurs even in the presence of low Ca2+ solutions. We studied aconitine-induced arrhythmia (particularly the amplitude of delayed afterdepolarization) in the frog atrium or guinea-pig papillary muscles in Ca2+-free solution, in the presence or absence of Ca2+ channel blocking agents. In Ca2+-free solution, aconitine (10(-5) g/ml) decreased the resting potential, overshoot, Vmax, and shortened the duration of the 90% action potential, before the onset of delayed afterdepolarization in frog atrial preparations. Tetrodotoxin (TTX) (2 X 10(-7) g/ml) blocked these aconitine-induced electrical changes. Verapamil (10(-6) g/ml) in nominally Ca2+-free solution blocked neither the generation of delayed afterdepolarization nor the triggered activity, while LaCl3 (0.5 mM) or TTX halted it. Delayed afterdepolarization appeared following the aconitine-induced transient increase in twitch tension. This transient increase in twitch tension was blocked by LaCl3 and TTX but not by verapamil. Delayed afterdepolarization in Ca2+-free solution demonstrated the voltage dependence of a U shape between -40 and -80 mV and was inhibited by low Na+ and high K+. Under the influence of aconitine in the guinea pig papillary muscle exposed to the Ca2+-free solution, depolarizing clamp pulses produced a transient inward current, and here the sigmoid time- and voltage-dependent characteristics were similar to those seen in the case of digitalis intoxication. These results suggest that intracellular Na+ loading plays an important role in the aconitine-induced delayed afterdepolarization and transient inward currents in low Ca2+ solution.     

Effect of Mg2+ on non-quantal acetylcholine release at the mouse neuromuscular junction

The effects of extracellular concentrations of Mg2+ on the non-quantal release of acetylcholine (ACh) from nerve terminals was studied by extra- and intracellular electrophysiological methods. Anticholinesterase-treated mouse diaphragms were used in vitro. In the presence of Ca2+, the non-quantal release was maximal in the absence of Mg2+ and was inhibited by 3 mmol/l Mg2+. The inhibitory effect of Mg2+ was antagonized by ouabain and was absent in Ca2+-free (EGTA) solutions. The non-quantal release of ACh was found to be more sensitive to inhibition by Mg2+ than the quantal one which was measured as the amplitude of miniature endplate currents.     

Na+-Ca2+ exchange induces low Na+contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca2+-ATPase

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.     

Bursting activity in leech Retzius neurons induced by low external chloride

We have investigated the bursting activity of Retzius neurons in the central nervous system of the leech Hirudo medicinalis as induced in Cl(-)-free saline by measuring membrane potential, membrane current and the intracellular calcium concentration ([Ca2+]i), using fura-2 or Oregon-Green488-Bapta-1. The Retzius neurons changed their low tonic firing to rhythmical bursting activity when the extracellular Cl- concentration ([Cl-]o) was lowered to 1 mM or less. In Cl(-)-free saline (Cl- exchanged by gluconate), bursting was accompanied by a rise in intracellular Ca2+ in both cell body and axon, which oscillated in synchrony with the bursts. The Ca2+ transients depended on the amplitude and duration of the depolarization underlying the burst, and were presumably due to Ca2+ influx through voltage-dependent Ca2+ channels. In Ca(2+)-free, EGTA-buffered saline or in the presence of Ca2+ channel blockers verapamil (1 mM) or diltiazem (500 microM) the depolarizations underlying the bursts in Cl(-)-free saline were enhanced in amplitude and duration. Bursting was not affected by depleting the intracellular Ca2+ stores with cyclopiazonic acid. The depolarization in Cl(-)- and Ca(2+)-free saline did not evoke intracellular Ca2+ changes. The burst-underlying membrane depolarization induced by Cl- removal was found to be due to a Na(+)-dependent persistent inward current and could be inhibited by saxitoxin (25-50 microM). The results suggest that a persistent Na+ current is generated in Cl(-)-free saline and induces the depolarization underlying rhythmic activity, and that presumably Ca(2+)-induced K+ currents modulate the bursting behaviour.     

Mg2+-malate co-transport, a mechanism for Na+-independent Mg2+ transport in neurons of the leech Hirudo medicinalis

Mg2+-extrusion from Mg2+-loaded neurons of the leech, Hirudo medicinalis, is mediated mainly by Na+/Mg2+ antiport. However, in a number of leech neurons, Mg2+ is extruded in the nominal absence of extracellular Na+, indicating the existence of an additional, Na+-independent Mg2+ transport mechanism. This mechanism was investigated using electrophysiological and microfluorimetrical techniques. The rate of Na+-independent Mg2+ extrusion from Mg2+-loaded leech neurons was found to be independent of extracellular Ca2+, K+, NO3-, HCO3-, SO4(2-), HPO4(2-), and of intra- and extracellular pH. Na+-independent Mg2+ extrusion was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), furosemide, ouabain, vanadate, iodoacetate, 4-amino-hippurate, or alpha-cyano-4-hydroxycinnamate and was not influenced by changes in the membrane potential in voltage-clamp experiments. Na+-independent Mg2+ extrusion was, however, inhibited by the application of 2 mM probenecid, a blocker of organic anion transporters, suggesting that Mg2+ might be co-transported with organic anions. Extracellularly, of all organic anions tested (malate, citrate, lactate, alpha-ketoglutarate, and 4-amino-hippurate) only high, but physiological, concentrations of malate (30 mM) had a significant inhibitory effect on Na+-independent Mg2+ extrusion. Intracellularly, iontophoretically injected malate, citrate, or fura-2, but not Cl-, alpha-ketoglutarate, glutamate, succinate, or urate, were stimulating Na+-independent Mg2+ extrusion from those neurons that initially did not extrude Mg2+ in Na+-free solutions. Our data indicate that Mg2+ is co-transported with organic anions, preferably with malate, the predominant extracellular anion in the leech. The proposed model implies that, under experimental conditions, malate drives Mg2+ extrusion, whereas under physiological conditions, malate is actively taken up, driven by Mg2+, so that malate can be metabolized.     

Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system.

1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+)-K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions.     

Na(+)/Ca(2+) exchanger in GABAergic presynaptic boutons of rat central neurons

Rat Meynert neurons were acutely isolated using a dissociation technique that maintains functional GABAergic presynaptic boutons. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded under voltage-clamp conditions using whole cell patch-clamp recordings. Using the frequency of mIPSCs as a measure of presynaptic terminal excitability, the existence of a Na(+)/Ca(2+) exchanger (NCX) in these GABAergic nerve terminals was clearly demonstrated. Both the frequency and the amplitude of mIPSCs were unaffected by replacement of extracellular Na(2+). However, in this Na(+)-free external solution, ouabain could now induce a transient increase of mIPSCs frequency, which was not inhibited by adding Cd(2+) or cyclopiazonic acid but was inhibited by removing external Ca(2+). This indicates that this transient potentiation was dependent on external Ca(2+), but that this Ca(2+) influx was not via voltage-dependent Ca(2+) channels. KB-R7943, an inhibitor of NCX, at a concentration of 3 x 10(-6) M, reduced this transient increase of mIPSCs frequency without affecting mIPSCs amplitude and the response to exogenous GABA. These results demonstrate the existence of NCX in these GABAergic nerve terminals. In zero external Na(+), ouabain causes an accumulation of intraterminal Na(+) and a resultant influx of Ca(2+) through the reversed mode operation of NCX. However, under more physiological conditions, NCX may also operate in a forward mode and serve to maintain low intracellular [Ca(2+)] in nerve terminals.     

Calcium-independent GABA release from striatal slices: the role of calcium channels

We have investigated the role of Ca2+ and Ca2+ channels in the modulation of GABA release. Brain slices prepared from rat striatum were preincubated with [3H]GABA, superfused with Krebs bicarbonate buffer, and exposed to electrical field stimulation (2 Hz for 3 min). Tritium efflux was measured as an index of GABA release. Both resting and evoked efflux were greatly accelerated by deleting Ca2+ from the medium and adding EGTA (1 mM). However, when the concentration of Mg2+ in the buffer was elevated to 10 mM, no effect of the Ca2(+)-deficiency was observed on resting release and its impact on evoked overflow was diminished. Moreover, addition of verapamil (10 microM), a Ca2+ channel blocking agent, reduced evoked overflow even in the absence of external Ca2+, while 4-aminopyridine (10 microM), a K+ channel inhibitor, enhanced GABA efflux in normal buffer but had no effect in the absence of Ca2+. Finally, we have shown previously that nipecotic acid, an inhibitor of high affinity GABA transport, increases GABA overflow in normal buffer, but blocks it in Ca2(+)-free buffer. Collectively, these results suggest that Ca2+ channels may play two roles in the regulation of depolarization-induced GABA release. Firstly, these channels permit a depolarization-induced influx of Ca2+ which then promotes GABA release. In addition, these channels influence GABA release through a mechanism that does not involve external Ca2+. Although the precise nature of this latter involvement is unclear, we propose that the Ca2+ channels serve to permit an influx of Na+, which in turn promotes Ca2(+)-independent release through an influence on the high affinity GABA transport system.     

The effect of sodium and calcium ions on the release of catecholamines from the adrenal medulla: sodium deprivation induces release by exocytosis in the absence of extracellular calcium

1. Perfusing bovine adrenal glands with Na(+)-free Locke solution for 15-40 min did not modify the increase in the release of catecholamines from glands stimulated by acetylcholine. However, after 80-100 min of perfusion with Na(+)-free solution, the response to acetylcholine stimulation was decreased or abolished.2. Immediately after switching the perfusion medium to Na(+)-free solution, there was a sharp increase (6-10 times over control values) in catecholamine output.3. Graded substitution of Na(+) in the perfusion fluid enhanced the output of catecholamines. This increase in the output of amines was linearly related to the logarithm of the extracellular Na(+) concentration.4. The release of catecholamines in the absence of Na(+) was not reduced by the presence of atropine and hexamethonium nor by the omission of Ca(2+) in the presence of EDTA or EGTA.5. Excess of Mg(2+) in the perfusion fluid reduced (10 mM-Mg(2+)) or blocked (20 mM-Mg(2+)) the increase in the output of catecholamines induced by Na(+) deprivation in the presence or absence of extracellular Ca(2+).6. Na(+) deprivation induced release of catecholamines during perfusion of the glands with depolarizing concentrations (56 mM) of K(+).7. In the presence or the absence of extracellular Ca(2+), the increase in the output of catecholamines induced by Na(+) deprivation was accompanied by an increase in the output of dopamine beta-hydroxylase, but not of lactate dehydrogenase. In addition, during perfusion with Ca(2+) free solution, Na(+) deprivation induced a parallel increase in both catecholamine and adenosine triphosphate outputs.8. The ratios of catecholamines to dopamine beta-hydroxylase and catecholamines to adenosine triphosphate determined in the perfusates collected from glands during perfusion with Na(+)-free medium were similar to those measured in the soluble contents of isolated chromaffin granules. These results provided biochemical evidence in favour of exocytosis as the mechanism of secretion during Na(+) deprivation.     

The kinetics of influx of calcium and strontium into rat intestine in vitro

1. The role of uptake across the brush border in the intestinal absorption of calcium has been studied by examining the kinetics of influx into slices of rat intestine in vitro. Both mucosal and serosal surfaces were exposed to the medium.2. The rate of influx was accurately defined by a two-component expression comprising a saturable (Michaelis-Menten) term and a second term linear with concentration. Influx across the mucosal surface of closed sacs was similar, and the saturable component for slice influx could be ascribed mainly to transport across the mucosal surface. The half-saturation constant for Ca was near 1 mM. This component was predominant at normal luminal concentrations of free Ca in the duodenum of young rats, but less so in jejunum and ileum and in older rats.3. The same kinetic expression applied to Sr influx, with a half-saturation constant of 2-3 mM, and possibly also to Ba with an even higher value.4. The saturable component of Ca influx was greatly reduced by 2,4: dinitrophenol (DNP); influx was also inhibited by iodoacetate, cyanide and at 0 degrees C. Inhibition commenced soon after exposure of the slices. A high concentration of DNP also caused an increase in the linear component of Ca influx.5. The kinetics of Ca influx across the mucosal surface agreed closely with the kinetics of steady-state absorption of Ca either across the whole mucosal epithelium in vivo or across the entire intestinal wall in vitro. This agreement supports the hypothesis that Ca entry across the brush border is the rate-limiting step in absorption; such a hypothesis would allow net Ca translocation while preserving a low intracellular concentration of ionic Ca in the mucosal epithelial cells.     

Role of Ca2+ in genesis of lower esophageal sphincter tone and other active contractions.

The effects of absent or low Ca2+ (0.5 mM), verapamil, nifedipine, Na nitroprusside, theophylline, La2+, and ethanol on basal active tension (tone), "off" contractions, and carbachol contractions were studied in opossum lower esophageal sphincter strips. Incubation in Ca2+-free Ringer (0.1 mM EGTA) abolished tone and contractions. Low Ca2+, verapamil, nifedipine, and theophylline depressed tone more rapidly than "off" contractions. Only verapamil and nifedipine depressed carbachol contractions. Na nitroprusside rapidly depressed tone but left contractions unchanged. La3+ at 1 X 10(-3) M behaved like Ca2+-free incubation but produced sustained contractions with muscle stimulation. Ethanol depressed "off" contractions more than tone and did not affect carbachol-induced contractions. These results suggest that tone probably results from inward leak of Ca2+, whereas "off" contractions depend on release of Ca2+ sequestered in the cell by a mechanism not immediately dependent on increased Ca2+ influx. Carbachol may increase Ca2+ influx as well as utilize sequestered Ca2+. Nifedipine and verapamil may act to block both resting and stimulated Ca2+ influx. Na nitroprusside may act by increasing Ca2+ efflux. Ethanol may act by decreasing the availability of sequestered Ca2+ or by inhibiting the function of a mediator responsible for "off" contractions.     

Unidirectional influx of phosphate across the mucosal membrane of rabbit small intestine

The influx of phosphate across the mucosal border of different regions of rabbit small intestine was investigated using the technique of Schultz et al., J. Gen. Physiol.50, 1241–1260 (1967). In the duodenum, the phosphate influx consisted of two components: 1. a saturable part, inhibited competitively by the presence of arsenate in the mucosal solution and strongly dependent on the mucosal Na concentration, and 2. a Na-independent part, linearly related to the mucosal phosphate concentration. In the jejunum and the ileum, the phosphate influx was a linear function of the mucosal phosphate concentration. In these regions arsenate had no effect on the influx, supporting the idea of a diffusional transport. HgCl₂ (0.5 mM) reduced the phosphate influx in the duodenum, at 140 mM Na, to the levels under Na-free conditions. The Na-independent influx was only slightly decreased by HgCl₂, suggesting that this agent affects mainly the Na-dependent phosphate influx. In the ileum HgCl₂ decreased the influx by about the same amount as under Na-free conditions in the duodenum. Thus, in the rabbit, the duodenum only appears to have a Na-dependent, carrier mediated phosphate transport mechanism at the mucosal membrane. In the jejunum and the ileum the phosphate uptake seems to be by simple diffusion.Presented in part at the Second Meeting of the European Intestinal Transport Group, Pont-à-Mousson, France, September 6–9, 1978. Abstract published in Gastroenterol. Clin. Biol.3, 173 (1979)