Involvement of the Na+/Ca2+ exchanger in ouabain-induced inotropy and arrhythmogenesis in guinea-pig myocardium as revealed by SEA0400

Involvement of the Na+/Ca2+ exchanger in ouabain-induced inotropy and arrhythmogenesis was examined with a specific inhibitor, SEA0400. In right ventricular papillary muscle isolated from guinea-pig ventricle, 1 microM SEA0400, which specifically inhibits the Na+/Ca2+ exchanger by 80%, reduced the ouabain (1 microM)-induced positive inotropy by 40%, but had no effect on the inotropy induced by 100 microM isobutyl methylxantine. SEA0400 significantly inhibited the contracture induced by low Na+ solution. In HEK293 cells expressing the Na+/Ca2+ exchanger, 1 microM ouabain induced an increase in intracellular Ca2+, which was inhibited by SEA0400. The arrhythmic contractions induced by 3 microM ouabain were significantly reduced by SEA0400. These results provide pharmacological evidence that the Na+/Ca2+ exchanger is involved in ouabain-induced inotropy and arrhythmogenesis.     

Effect of Na+/Ca2+ exchange inhibitor, KB-R7943 on ouabain-induced arrhythmias in guinea-pigs.

1. We investigated protective effects of KB-R7943, a Na+/Ca2+ exchange (NCX) inhibitor, on ouabain-induced tonotropy and arrhythmias in isolated whole atria and ouabain-induced changes in electrocardiogram (ECG) in the guinea-pig. 2. KB-R7943 (10 and 30 microM) suppressed the tonotropic effect of ouabain, and prolonged the onset time of extra-systole induced by ouabain in isolated atria. 3. The intravenous injection of KB-R7943 (1 and 3 mg kg-1) significantly increased the doses of ouabain required to induce ventricular premature beats (VPB), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA) in anaesthetized guinea-pigs. 4. Lidocaine (Na+channel inhibitor) and R56865 (Na+ and Ca2+ overload inhibitor) also suppressed the ouabain-induced tonotropic effect and extra-systole in isolated atria, but Hoe-694 (Na+/H+ exchange inhibitor) or diltiazem (Ca2+ channel inhibitor) did not affect them. 5. Lidocaine also increased the doses of ouabain required to induce VPB, VT, VF and CA in anaesthetized guinea-pigs. 6. From these results, we conclude that KB-R7943 suppresses ouabain-induced arrhythmias through inhibition of the reverse-mode NCX.     

Dual mechanism of ouabain action on noradrenaline output from guinea-pig vas deferens

Isolated guinea-pig vas deferens was used to test the effects of varying the concentration of extracellular Na+, Ca2+ and Mg2+ and the influence of tetanic transmural stimulation and of tetrodotoxin on the ouabain-induced increase in noradrenaline output. The total amount of noradrenaline released by ouabain increased with increasing concentration of either Na+ (from 35 to 143 mM) or Ca2+ (from 0.3 to 2.5 mM). The rise in the ouabain-induced increase of noradrenaline output was retarded by increasing the concentration of either Ca2+ from 0.3 to 5 mM or Mg2+ from 1.2 to 20 mM. Tetanic transmural stimulation facilitated the occurrence of ouabain-induced noradrenaline output, while tetrodotoxin (5 X 10(-7) g/ml) greatly inhibited it. These results suggest that ouabain causes an increase in noradrenaline output through at least two different mechanisms, a tetrodotoxin-sensitive action potential and Na+-dependent Ca2+ influx resulting from the inhibition of the Na+-K+ pump.     

Effects of ouabain on muscle tension and intracellular Ca2+ level in guinea-pig aorta.

The effects of ouabain on muscle tension and the intracellular Ca2+ level ([Ca2+]i) were examined in guinea-pig aorta loaded with fura-2. Ouabain caused a gradual and sustained increase in both [Ca2+]i and muscle tension. There was a positive correlation between these two parameters. In Ca(2+)-free solution, ouabain did not affect either [Ca2+]i or muscle tension, suggesting that the ouabain-induced increase in [Ca2+]i was not due to Ca2+ release from storage sites. The ouabain-induced increase in [Ca2+]i and muscle tension was inhibited by Ni2+, which inhibits the Na+/Ca2+ exchanger, but not by verapamil. Furthermore, anionic and cationic amphiphiles were used as modulators of the Na+/Ca2+ exchanger. Sodium dodecyl sulfate accelerated the responses to ouabain, whereas dodecyltrimethylammonium bromide inhibited them. These results suggest that in the guinea-pig aorta, ouabain induces contraction by increasing the Ca2+ influx through the Na+/Ca2+ exchanger on the plasma membrane, but not through verapamil-sensitive Ca2+ channels.     

Analysis of ouabain-induced contractions in isolated coronary arteries

Contractile responses to ouabain in helical strips of dog and monkey coronary arteries were investigated. Ouabain (5 X 10(-8) to 5 X 10(-6) M) caused a dose-related contraction in dog and monkey arteries; the response of monkey coronary arteries was significantly greater. In dog coronary arteries, contractile responses to high concentrations of ouabain were potentiated by treatment with propranolol. In the arteries contracted with ouabain, the addition of phentolamine caused a relaxation. Contractile responses of dog coronary arteries to ouabain were markedly suppressed by exposure to Ca2+-free media or by treatment with verapamil. Reduction of external concentration of K+ or lowering the temperature of bathing media did not selectively influence the ouabain-induced contraction. These results suggest that ouabain-induced contractions of dog coronary arteries are associated mainly with an increase in the Ca2+-influx, which does not result from an inhibition of the Na+, K+-activated ATPase nor from an activation of alpha adrenoceptors by noradrenaline released from adrenergic nerves. Ouabain in high concentrations seems to liberate noradrenaline from adrenergic nerves, which preferentially activates beta adrenoceptors in dog coronary artery.     

The role of Na+ in muscarinic receptor-mediated catecholamine secretion in the absence of extracellular Ca2+ in cat perfused adrenal glands.

1. The role of Na+ in muscarinic receptor-mediated catecholamine secretion, which is independent of extracellular Ca2+, was investigated by observing the effect of veratridine and ouabain in perfused adrenal glands of the cat. 2. Veratridine (10(-4) M) markedly enhanced catecholamine secretion evoked by acetylcholine (ACh, 10(-4) M) during perfusion with Ca2(+)-free Locke solution containing hexamethonium (10(-3) M). The enhancement tended to be larger for noradrenaline secretion than for adrenaline secretion. Qualitatively the same result was obtained in the response to pilocarpine (5 x 10(-4) M). 3. Ouabain (10(-4) M) also enhanced ACh- and pilocarpine-induced catecholamine secretions, especially noradrenaline secretion in the absence of extracellular Ca2+. 4. Tetrodotoxin (10(-6) M) blocked the enhancing effect of veratridine on ACh-induced catecholamine secretion, but not that of ouabain in the absence of extracellular Ca2+. 5. When NaCl was replaced with sucrose, there was no secretory response to ACh regardless of the presence or absence of veratridine or ouabain. However, when ouabain, but not veratridine, was infused with Na+ before the replacement of NaCl, the response to ACh was substantially augmented. 6. These results indicate that Na+ is essential in the initiation of muscarinic receptor-mediated catecholamine secretion and its enhancement by veratridine and ouabain in the absence of extracellular Ca2+. Both drugs seem to increase the intracellular concentration of Na+ through different mechanisms and result in increases in the efficiency of Ca2+ mobilization from intracellular Ca2+ pools linked to muscarinic receptors.     

Release of noradrenaline from the ligated cat hypogastric nerve

After in vivo ligation for 24 h of the cat hypogastric nerve, large amounts of noradrenaline (NA) and dopamine β-hydroxylase (DBH) accumulated in the nerve segment immediately proximal to the ligature (P₁). In vitro incubation of 24-h-ligated nerves (segments P₁ and P₂) in oxygenated Krebs solution at 37% C in the presence of the ionophore X537A or high K⁺ concentrations caused a marked release of endogenously accumulated NA into the incubation medium. High-K⁺-evoked release was entirely dependent on extracellular Ca²⁺. Electrical nerve stimulation caused an 80% tissue NA loss, but the transmitter could not be found in the medium as intact NA. These results suggest that the in vivo ligated cat hypogastric nerve may serve as a useful model of adrenergic nerve terminals free of effector cells.     

Na+/Ca2+ exchange mediation in the ouabain-induced contraction in human placental vessels.

1. Ouabain induced concentration-dependent contractions in segments of human placental arteries and veins, which were practically abolished in a Ca(2+)-free medium and not modified by the calcium antagonist nifedipine or the calcium agonist Bay K 8644. 2. Ouabain (10(-4) M) elicited a time-dependent enhancement of the 45Ca2+ uptake, which remained equal in presence of nifedipine or Bay K 8644. 3. The Na+/Ca2+ exchange blocker amiloride reduced both the contractions and the 45Ca2+ uptake induced by ouabain, whereas the Na ionophore monensin produced a parallel shift to the left of the concentration-response curve to ouabain. 4. These results suggest that ouabain-induced contractions in these vessels are dependent on the extracellular Ca2+, which mainly enters into the cell through the Na+/Ca2+ exchange system.     

Noradrenaline release induced by ouabain and vanadate in cat cerebral and peripheral arteries

The effects of 10(-4) M ouabain and 10(-3) M vanadate (Na3VO4) on [3H]noradrenaline release from cat cerebral and femoral arteries was studied. Ouabain induced tritium secretion in cerebral arteries, but not in femoral ones, which was reduced by Ca suppression and potentiated by extracellular Na reduction to 11.9 mM. However, vanadate evoked tritium release from both kinds of vessels was unaffected under these experimental conditions. These data suggest: ouabain elicited secretion from adrenergic nerve endings is likely due to inhibition of the Na, K-ATPase and subsequent Ca influx through Na-Ca exchange, and vanadate action is mediated by another mechanism different to the Na pump blockade.     

Comparison of the effect of amiloride and its analogue dichlorobenzamil on cardiac chronotropic responses to ouabain in myocardial cell aggregates in culture

The purpose of this study was to compare the effects of amiloride, an inhibitor of Na(+)-H+ exchange, and its analog 3',4'-dichlorobenzamil, a more specific inhibitor of Na(+)-Ca2+ exchange on the response of cardiac myocytes to ouabain. Cardiac myocyte aggregates were prepared from myocytes obtained from 7-day-old chick embryo hearts. Ouabain at 10(-6) M produced a marked and significant (p less than 0.05) reduction in contractile frequency. Amiloride, at 10(-7) to 10(-5) M produced a definite, significant (p less than 0.05) and dose-dependent reduction in this effect of ouabain. In contrast, dichlorobenzamil, 10(-7) to 10(-6) M, significantly (p less than 0.05) accentuated this effect of ouabain. Thus, amiloride and its analog dichlorobenzamil have different effects on the cardiac responses to ouabain presumably because of the differences in the specificity of their inhibition of Na+/H+ and Na+/Ca2+ exchange. Thus to the extent that the effects of amiloride and dichlorobenzamil are mediated through, respectively Na(+)-H+ and Na(+)-Ca2+ exchange, these data suggest that ouabain-induced reduction in contractile frequency is mediated through Na(+)-H+ exchange while Na(+)-Ca2+ exchange acts to minimize this action of ouabain. Amiloride may be useful to oppose the negative chronotropic effect of ouabain while dichlorobenzamil accentuates this effect of ouabain.     

Effects of ouabain on isolated cerebral and femoral arteries of the cat: a functional and biochemical study

1. This study analyzes the mechanisms involved in the responses to ouabain in cat cerebral and femoral arteries and characterizes the electrogenic Na+ pump present in these vessels. The latter was accomplished by measurement of [3H]-ouabain binding to arterial membrane fractions, K+-elicited relaxation and ouabain-sensitive 86Rb+ uptake. 2. Ouabain induced transient contraction in cylindrical segments of cerebral arteries. This contraction was reduced by verapamil (3 X 10(-6) M) and Ca2+-removal from the medium but was not modified by phentolamine (3 X 10(-6) M) or pretreatment with reserpine. However, the contraction elicited by ouabain in femoral artery segments lasted longer, and was reduced by Ca2+-omission, phentolamine or reserpine, but remained unaffected by verapamil. 3. The immersion of the arteries in low-Na+ (25 mM) medium abolished the contraction caused by ouabain. 4. The exposure of the arteries to a K+-free medium induced a small transient increase in tension, and the subsequent application of K+ (7.5 mM) elicited a marked relaxation. This effect was greater in cerebral than in peripheral arteries, and was suppressed by ouabain (10(-4) M). 5. Scatchard analysis of the [3H]-ouabain binding to arterial membrane fractions suggested a single class of binding sites. The KD values for both kinds of arteries were of similar order, while the Bmax value was greater in cerebral than in femoral arteries. 6. Total and ouabain-sensitive 86Rb+ uptakes were greater in cerebral than in femoral vessels. 7. These results indicate that: (1) ouabain-induced contraction of cerebral arteries is due to a direct effect on vascular smooth muscle cells, while in femoral arteries it is due to noradrenaline release from adrenergic nerve terminals; and (2) the electrogenic Na+ pump activity is greater in cerebral than in peripheral arteries.     

Ouabain-induced increases in resting tone of human hyperplastic prostate following repeated noradrenaline and electrical field stimulation.

1. The effect of ouabain on contractions to repeated noradrenaline stimulation and electrical field stimulation of human hyperplastic prostate was examined. Ouabain (1 microM) did not induce contractile response per se but progressively increased the resting tone (i.e., the tone between one noradrenaline stimulation, or electrical field stimulation, and the following) of human hyperplastic prostate. 2. The increased tone by ouabain following repeated noradrenaline stimulations or electrical field stimulation was fully relaxed by the removal of external calcium, and recovered following restoration of calcium. 3. The effect of noradrenaline on NA+ uptake was measured. Noradrenaline (10 microM) significantly increased the rate of Na+ accumulation in the presence of ouabain (1 microM); this stimulatory effect was almost completely blocked by prazosin (0.1 microM) and ethylisopropylamiloride (100 microM). In contrast, tetrodotoxin (1 microM) had no effect on noradrenaline-stimulated Na+ transport in human hyperplastic prostate. 4. Intracellular Na+ loading by noradrenaline (10 microM) in the presence of ouabain (1 microM) significantly increased the transmembrane Ca2+ uptake as compared with the absence of ouabain; however, nifedipine (1 microM) was ineffective on Ca2+ uptake under this condition. 5. Transmembrane CA2+ efflux was stimulated by noradrenaline (10 microM) in human hyperplastic prostate; this effect was significantly decreased in the presence of ouabain (1 microM). 6. It is suggested that the increased tone of human hyperplastic prostate following repeated excitation in the presence of ouabain is due to increased Ca2+ entry and reduced efflux of Ca2+ through the Na+/Ca+ exchange system as a consequence of Na+ pump inhibition by ouabain.     

Influence of lidocaine on ouabain-induced inotropic response in rat atria

In this paper we demonstrated that lidocaine broadens the therapeutic range of ouabain action having a protective effect on ouabain-induced toxicity on rat atria. The lidocaine effect on therapeutic ouabain action was associated with the increase in the sensitivity of Na(+)-K(+)-ATPase related to a decreased in the equilibrium dissociation constant (K(d)) of high affinity binding sites. Lidocaine suppressed the ouabain-induced tonotropic effect and arrhythmias, decreasing the number of low affinity binding sites (B(max)) without changes in K(d). Blockade of Na(+)-Ca(2+) exchange with KB-R7943 or dual Na(+)-Ca(2+) channel with flunarizine, mimicked lidocaine effect increasing ouabain therapeutic action, extending its concentration range tolerated, delaying the onset of contracture. Lidocaine itself triggered negative inotropic response at high concentration. This effect was increased in the presence of flunarizine and verapamil but not by the inhibition of calcium/calmodulin with W-7. The mechanism underlying the lidocaine-induced negative inotropic response, appears to be different that underlying the positive inotropic effect on ouabain action. This study provides evidence that lidocaine can interact with the same or similar binding sites for ouabain in rat atrial tissue, providing a protective effect on ouabain-induced changes in contractility. The contribution of Na(+)-Ca(2+) exchange and/or Ca(2+) overload on lidocaine effect is discussed.     

Solubilization of an active opiate receptor from Bufo marinus

The effect of veratridine on in vitro release of noradrenaline (NA) from ligand cat hypogastric nerve was investigated. After in vivo ligation for 24–48 h, large amounts of NA and dopamine-β-hydroxylase (DBH) accumulated in the nerve segment immediately proximal to the ligature (P₁). In vitro incubation of ligated nerves (segments P₁ and P₂) in oxygenated Krebs solution at 37°C in the presence of veratridine caused a marked and dose-dependent release of endogenously accumulated NA into the incubation medium. The release continued to occur for a considerable time, even after washout of the drug. Veratridine-induced depletion of tissue NA was accounted for by its release, as NA, into the incubating medium. The secretory response to veratridine was readily blocked by tetrodotoxin (TTX). Veratridine-induced release was dependent on calcium and abolished by high magnesium. On the basis of the similarity between the NA secretory response to veratridine in this preparation and in adrenergically innervated organs, the results favour the view that the in vivo-ligated cat hypogastric nerve may serve as a useful model of adrenergic nerve terminals free of effector cells.     

The mode of action of caffeine on catecholamine release from perfused adrenal glands of cat.

1 Adrenaline and noradrenaline secretion induced by caffeine was investigated in the perfused cat adrenal glands. 2 Caffeine (10-80 mM) caused a dose-dependent increase in both adrenaline and noradrenaline secretion when applied for 1 min and 10 min after replacing Ca2+ with 10(-5)M EGTA in the perfusion solution. The ratio of adrenaline to noradrenaline was about 1:1. Mg2+ and/or Ca2+ inhibited the response to caffeine. 3 When caffeine (40 mM) was repeatedly applied in the absence of extracellular Ca2+, the secretory response almost disappeared but only at the second challenge with caffeine. However, the response was partially restored after readmission of Ca2+ (2.2 mM) and was augmented after the readmission of Ca2+ with ouabain (10(-5) M). 4 Caffeine-induced secretion of adrenaline and noradrenaline increased with the increase in the preloaded concentration of Ca2+ and attained a maximum at 16 mM Ca2+. 5 During perfusion with Ca2+-free Locke solution containing hexamethonium (10(-3)M), acetylcholine (10(-4)M) caused increases in both adrenaline and noradrenaline secretions with a ratio of about 1:2. The secretory responses were partially inhibited by preceding stimulation with exposure to caffeine (80 mM). 6 These results suggest that caffeine mobilizes Ca2+ from an intracellular storage site that may not be entirely the same as that linked to muscarinic receptors, and causes an increase in both adrenaline and noradrenaline secretion from cat adrenal chromaffin cells.     

Moguisteine: a novel peripheral non-narcotic antitussive drug.

1. Contributions of intra- and extracellular Ca2+ to noradrenaline (NA) release evoked by increasing intracellular Na+ concentrations (ouabain plus monensin) from adrenergic nerves of guinea-pig vas deferens were evaluated under conditions eliminating carrier-mediated NA release (with 100 microM cocaine). 2. Ouabain (100 microM) plus monensin (10 microM), unlike 100 mM KCl, produced a marked NA release which was unchanged by Ca(2+)-removal. 3. In normal solution but not in Ca(2+)-free solution, the release of NA evoked by ouabain plus monensin was reduced by adenosine, clonidine and neuropeptide Y, and by Ca(2+)-channel blockers such as omega-conotoxin GVIA and nifedipine. The release of NA was also decreased by cromakalim in a glibenclamide-sensitive fashion. 4. In contrast, in the absence but not in the presence of Ca2+, the drug-evoked NA release was inhibited by mitochondrial inhibitors (carbonylcyanide-m-chlorophenylhydrazone and oligomycin) and further by immobilizers of intracellular Ca2+ (TMB-8 and BAPTA-AM) and calmodulin antagonists (W-7 and trifluoperazine). 5. These findings suggest that the release of NA evoked by elevation of [Na+]i from adrenergic nerves in the presence and absence of Ca2+ involves, in part, exocytotic processes which are triggered by depolarization-induced Ca2+ influx and by utilization of Ca2+ from intracellular Ca2+ store sites such as mitochondria, respectively.     

NORADRENALINE RELEASE FROM THE RAT HEART DURING ANOXIA: EFFECTS OF CHANGES IN EXTRACELLULAR SODIUM CONCENTRATION AND INHIBITION OF SODIUM UPTAKE MECHANISMS

1. Anoxic perfusion of the isolated rat heart releases noradrenaline in the absence of sympathetic nerve fibre stimulation. 2. Anoxic noradrenaline release is enhanced by reducing the extracellular Na+ concentration, consistent with the proposal that such release occurs by carrier-mediated efflux. 3. Release is also enhanced by lignocaine but inhibited by amiloride and ethylisopropylamiloride, suggesting that sodium entry into adrenergic nerve terminals during anoxia occurs by Na+/H+ (and possibly Na+/Ca2+) exchange.     

Ouabain distinguishes between nicotinic and muscarinic receptor-mediated catecholamine secretions in perfused adrenal glands of cat.

1. The effect of ouabain on catecholamine (adrenaline and noradrenaline) secretion induced by agents acting on cholinoceptors was studied in perfused cat adrenal glands. Acetylcholine (ACh) (5 x 10(-7) to 10(-3) M), pilocarpine (10(-5) to 10(-3) M) and nicotine (10(-6) to 5 x 10(-5) M) caused dose-dependent increases in catecholamine secretion. Both ACh and nicotine released more noradrenaline than adrenaline and the reverse was the case for pilocarpine. 2. Ouabain (10(-5) M) enhanced catecholamine secretion induced by ACh (10(-5) M), pilocarpine (10(-3) M) and nicotine (3 x 10(-6) M) during perfusion with Locke solution. The ratio of adrenaline to noradrenaline was not affected by ouabain. 3. In the absence of extracellular Ca2+, ACh and pilocarpine, but not nicotine, still caused a small increase in catecholamine secretions, which were enhanced by treatment with ouabain (10(-5) M) plus Ca2+ (2.2 mM) for 25 min. The effect of ouabain was much more significant on noradrenaline secretion than on adrenaline secretion. The enhanced response was blocked by atropine (10(-6) M) but not by hexamethonium (5 x 10(-4) M). 4. Nifedipine (2 x 10(-6) M) inhibited the responses to pilocarpine and nicotine. The treatment with ouabain (10(-5) M) reversed only the response to pilocarpine and resulted in a significant increase in the proportion of noradrenaline released. 5. It is suggested that ouabain enhances evoked catecholamine secretions by facilitating Ca2+ entry through nicotinic receptor-linked Ca2+ channels and by increasing the intracellular Ca2+ pool linked to muscarinic receptors.     

Ameliorative effects of amiloride and pralidoxime in chronic constriction injury and vincristine induced painful neuropathy in rats

The present study was designed to investigate the ameliorative effects of clinically available drugs, with Na+/Ca2+ and Na+/H+ exchange inhibitory actions, in chronic constriction injury and vincristine induced painful neuropathy in rats. Sciatic nerve ligation and vincristine treatment (50 microg/kg for 10 days) was employed to induce neuropathy in rats. Paw pressure, von Frey hair, acetone drop, and tail heat immersion tests were performed to assess degree of mechano-hyperalgesia, mechano-allodynia, cold chemical allodynia and spinal thermal sensation respectively. Axonal degeneration of sciatic nerve was assessed histopathologically. The levels of thio-barbituric acid reactive species, reduced glutathione, and total calcium were determined to assess biochemical alterations. Amiloride (15 mg/kg i.p.), Na+/Ca2+ and Na+/H+ exchange inhibitor, and pralidoxime (20 mg/kg i.p.), Na+/Ca2+ exchange inhibitor, were administered for 10 consecutive days starting from the day of surgery or vincristine administration. Sciatic nerve ligation and vincristine treatment resulted in significant axonal degeneration, development of mechano-hyperalgesia, mechano-allodynia, cold chemical allodynia and spinal heat hyperalgesia and also resulted in rise in thio-barbituric acid reactive species, total calcium and decrease in reduced glutathione levels. Administration of amiloride and pralidoxime attenuated chronic constriction injury and vincristine induced axonal degeneration and reduction of nociceptive threshold along with reduction in calcium levels and oxidative stress. The observed anti-nociceptive effects of amiloride and pralidoxime may possibly be attributed to inhibition of Na+/Ca2+ and Na+/H+ exchangers with subsequent decrease in Ca2+ ions and oxidative stress.     

Potentiation of K+-evoked catecholamine release in the cat adrenal gland treated with ouabain.

1 A vigorous catecholamine secretory response was evoked by small increments (2-10 mM) of the extracellular concentration of K+ ([K+])o) in cat adrenal glands treated with ouabain (10(-4) M), and perfused with Krebs-bicarbonate solution at room temperature. 2 The secretory response depends on [K+]o; increments of [K+]o as small as 2 mM for 2 min evoked a clear secretory response; at 10-17.7 mM K+, the maximal secretory response was observed. In normal glands, not treated with ouabain, no increase of the rate of catecholamine output was observed by raising [K+]o up to 17.7 mM for 2 min. 3 The K+ secretory response was time-dependent, requiring at least 1 min to be initiated; on continued exposure to 10 mM [K+]o, the enhanced response remained for at least 1 h. 4 In low [Na+]o, the K+-secretory response was unchanged. However, in 0-Ca2+, high-Mg2+ solutions, or in the presence of D600, an organic Ca2+ antagonist, it was abolished. 5 The K+-induced secretory response was not altered in the presence of tetrodoxin or tetraethylammonium. 6 It is concluded that ouabain potentiated the catecholamine secretory response to raised [K+]o by increasing the amount of Ca2+ available to the secretory machinery through (a) mobilization of an enhanced pool of membrane-bound Ca2+, (b) activation of membrane Ca2+ inward current; or (c) decrease of intracellular Ca2+ buffering systems. The activation by ouabain of a membrane Na+-Ca2+ exchange system is not involved in this K+-secretory response. It is suggested that the plasma membrane ATPase enzyme system, by changing the affinity of its Ca2+ binding sites, might control the availability of this cation to the secretory machinery and, therefore, modulate catecholamine secretion in the adrenal gland.