The release of acetylcholine and of catecholamine from the cat's adrenal gland.

The cat's adrenal gland was perfused in situ with Krebs solution containing eserine; the amount of acetylcholine and of catecholamine released was measured. Splanchnic nerve stimulation (5 Hz for 2 min) increased the release of acetylcholine and catecholamine; the molar ratio of evoked release of catecholamine to acetylcholine was 122 +/- 8. It is suggested that this amplification is achieved because a chromaffin cell granule contains more mediator than does the acetylcholine quantum that releases it. The release per impulse of catecholamine during splanchnic nerve stimulation at 30 Hz was less than that released by stimulation at 1 or 5 Hz. This depression is attributed to a presynaptic failure, because the release of acetylcholine was similarly frequency dependent. The release of catecholamine was linearly related to the release of acetylcholine over the range tested, indicating that the input-output relationship at the splanchnic-adrenal medullary junction is linear. During continuous stimulation of the splanchnic nerve (5 Hz), catecholamine release declined to a level that was 32 +/- 2% of the initial output. This fatigue is attributed primarily to a postsynaptic depression, because the release of acetylcholine was maintained at 71 +/- 6% of its initial level. The presence of eserine in the perfusate was necessary for the release of acetylcholine to be detected, but in the presence of eserine catecholamine release was 90 +/- 10% that in the drug's absence. It is concluded that released acetylcholine is hydrolysed at some distance from its site of release and action. Glands perfused with raised K+ released acetylcholine and catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological dissection of receptor-associated and voltage-sensitive ionic channels involved in catecholamine release.

The experiments were designed to quantify pharmacologically the degree of participation of channels associated with the nicotinic cholinoceptor compared with voltage-sensitive channels during the evoked release of [3H]noradrenaline from prelabelled 3-7-day old cultured bovine adrenal chromaffin cells. To achieve this purpose we studied (a) the release of [3H]noradrenaline evoked by secretagogues known to trigger the secretory response through activation of receptor-associated channels (acetylcholine, nicotine), voltage-sensitive Na+ (veratridine) and Ca2+ (high [K+] ) channels or direct, channel-independent promotion of Ca2+ entry (ionomycin); and (b) the selective blockade of some of those responses using ionic manipulations (Na+ deprivation, high Mg2+) or drugs known to block the activity of receptor-operated channels (imipramine, cocaine), voltage-dependent Na+ (tetrodotoxin) or Ca2+ (nitrendipine) channels. Inhibition by nitrendipine, a potent Ca2+ antagonist, of the secretory responses to both nicotine and high [K+] indicates a preferential Ca2+ entry through voltage-sensitive channels during the secretory process. Blockade by cocaine and imipramine of the release of [3H]noradrenaline evoked by acetylcholine and nicotine, without alteration of the responses to high [K+], veratridine or ionomycin, speaks in favor of a selective inactivation of the nicotinic receptor-associated channel. Since Na+ deprivation abolished [3H]noradrenaline release produced by nicotine, it seems that Na+ entry through the receptor-linked ionophore might be a primary event in the initiation of the secretory process; the fact that tetrodotoxin did not affect the release favors this view. However, veratridine induced a tetrodotoxin-sensitive secretory response, suggesting the presence of voltage-sensitive Na+ channels which might physiologically be used to propagate action potentials through gap junctions between adjacent chromaffin cells, only in the intact gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of desensitization of nicotinic receptors of the cat adrenal medulla by concanavalin A.

1 The possibility of concanavalin A (Con A) blocking the development of desensitization of nicotinic receptors of the cat adrenal gland has been investigated. 2 During perfusion of the adrenal gland with Krebs-bicarbonate solution containing acetylcholine (ACh), the rate of catecholamine (CA) secretion was very high in the first 2 min; thereafter, as perfusion with ACh was continued the output fell, to reach about 20% of the initial value in 10 minutes. When the adrenal gland was pretreated with Con A, the subsequent desensitization of release during continued infusion of ACh was prevented. 3 When the adrenal gland was perfused with high K+ solution, there was always a large initial secretion of CA, and as perfusion with high K+ continued the output fell, to reach about 15% of the initial rate in 10 minutes. Con A did not affect the rate of CA secretion induced by high K+. 4 It is tentatively suggested that Con A blocks the desensitization of CA secretion evoked by ACh by interaction with the glycoprotein moiety of the nicotinic receptor of adrenal chromaffin cells.

Effect of beta-haloalkylamines and ephedrine on noradrenaline release from the intact spleen of the cat.

1. The effects of beta-haloalkylamines, phenoxybenzamine (PBZ), N-alpha-naphthyl-methyl-N-ethyl-beta-bromoethylamine (SY28), N-cyclohexyl-methyl-N-ethyl-beta-chloroethylamine (GD131) and ephedrine on noradrenaline (NA) output following nerve stimulation were studied in the intact spleen of the cat. Postganglionic sympathetic nerves were stimulated at frequencies of 10 and 30 Hz for a total of 210 stimuli.2. PBZ and SY28 (10 mg/kg) increased the transmitter output at 10 Hz by nearly 5-10 times.3. Administration of GD131 (10 mg/kg) had no effect on output at either frequency. Phentolamine (3 mg/kg) given after GD131 increased NA output at 10 Hz.4. Ephedrine (0.5 mg/kg) caused no changes in catecholamine output, while higher doses of ephedrine (10-20 mg/kg) increased the amount released at 10 Hz.5. Perfusion of the spleen with Krebs-bicarbonate solution containing either SY28, GD131 or ephedrine (10 mug/ml) increased the recovery of infused NA by 80-90%.6. It is concluded that presynaptic as well as postsynaptic events determine the overflow of NA following stimulation of sympathetic nerves of the spleen.

Release of noradrenaline and dopamine by nerve stimulation in the cat spleen perfused with 3 H-dopamine.

1. Recovery of (3)H-dopamine and (3)H-L-dopa infused intra-arterially at a constant rate of 20 ng/min into the cat spleen perfused with Krebs bicarbonate solution was 52.6+/-3.8% and 90.5+/-3.1% respectively. Recovery of (3)H-dopamine in spleens treated with alpha-methyl-p-tyrosine was 67.7+/-6.7% and was not significantly different from untreated spleens.2. Release of (3)H-dopamine by nerve stimulation after an infusion of (3)H-dopamine resembled release of endogenous noradrenaline. Release of (3)H-L-dopa and (3)H-L-tyrosine after their infusion did not occur in any consistent manner by nerve stimulation.3. Preferential release of (3)H-noradrenaline formed from (3)H-dopamine was not observed during continuous nerve stimulation. Specific activity of released (3)H-noradrenaline remained constant during any single stimulation period with or without (3)H-dopamine infusion. Treating the cats with alpha-methyl-p-tyrosine did not change the time course of (3)H-noradrenaline release.4. Output of noradrenaline expressed as a percentage of the first minute output in both normal and alpha-methyl-p-tyrosine treated spleens was not significantly different at various times during continuous nerve stimulation.5. Specific activity of released noradrenaline formed from (3)H-dopamine was always greater than the specific activity of the spleen in normal spleens and spleens treated with alpha-methyl-p-tyrosine.6. It is concluded that newly synthesized or infused noradrenaline initially mixes with a more rapidly turning pool and only slowly with the entire tissue store. During continuous nerve stimulation there is no further preferential release of newly synthesized noradrenaline. Released noradrenaline truly represents the state of the releasable pool and will vary with the latter.

Effect of flow-stop on noradrenaline release from normal spleens and spleens treated with cocaine, phentolamine or phenoxybenzamine.

1. Cat spleens were perfused with Krebs-bicarbonate solution, using a constant-flow pump at a rate of about 7 ml/min at 33-35 degrees C. Noradrenaline (NA) overflow by nerve stimulation at 10 Hz for 20 s was determined with or without flow-stop before and after treatment with cocaine, phentolamine or phenoxybenzamine. In order to determine the effect of flow-stop on overflow, the arterial and the venous flows were occluded by clamping the inflow and outflow tubes during the period of stimulation plus 30, 60 or 120 seconds.2. Without flow-stop, NA output was 0.93+/-0.25 ng/stimulus, which was significantly increased after cocaine (123+/-6.6%), phentolamine (415+/-93%) and phenoxybenzamine (578+/-107%). Phentolamine and phenoxybenzamine were much more effective than cocaine in enhancing overflow.3. Before treatment with drugs, flow-stops of 30, 60 and 120 s reduced NA outputs to 70+/-6.6, 27.5+/-2 and 7%, respectively, of the control outputs without flow-stop. None of the drugs significantly influenced the percentage reductions in NA outputs during a 30 s flow-stop. However, the percentage outputs after cocaine or phenoxybenzamine treatment during a 60 s flow-stop significantly increased to 45+/-2.5% and 57+/-6%, respectively, as compared to the percentage output of 27.5+/-2% from untreated spleens during a corresponding flow-stop period. During flow-stop, there was no appreciable metabolism of the released transmitter.4. Diffusion of the released transmitter from the site of liberation plays only a minor role in the removal of the released NA.5. It is suggested that the NA released by nerve stimulation acts on the presynaptic alpha sites to inhibit its own release by a negative feedback mechanism. Adrenoceptor blocking agents enhance the NA overflow from spleen because they remove this autoinhibition by blocking the presynaptic alpha sites.

Release of noradrenaline from the cat spleen by sodium deprivation.

1. The endogenous noradrenaline content of cat spleen slices was markedly reduced when the slices were incubated at 37 degrees C in a medium in which sodium was replaced by sucrose, lithium, choline or potassium. Depletion of tissue noradrenaline was accounted for by its release into the incubating medium. At an external sodium concentration of 20 mM, about 50% depletion was obtained in 2 hours.2. The enhanced release induced by sodium deprivation occurred in the absence of calcium, with or without ethyleneglycol-bis (beta-aminoethyl ether) N,N' tetraacetic acid. Manganese potentiated release, while magnesium was without effect.3. Ouabain caused a dose-dependent release of noradrenaline which was partially calcium-dependent. Removal of potassium from the incubation medium caused some release, which was potentiated in 25 mM sodium Krebs solution or by ouabain.4. At 4 degrees C, the release did not occur in sodium-free medium.5. Dinitrophenol did not affect the loss of noradrenaline caused by sodium withdrawal. Iodoacetic acid and N-ethylmaleimide caused a time-dependent depletion of noradrenaline. Tetracaine caused release and partly opposed the release caused by sodium deprivation. Tetrodotoxin had no effect. Guanethidine, but not phenoxybenzamine, released noradrenaline and potentiated the release induced by sodium withdrawal.6. The rate of release of (3)H-noradrenaline from reserpine-treated spleen slices was not altered by sodium withdrawal.7. Uptake-retention of (3)H-noradrenaline in slices depleted of their endogenous noradrenaline content by sodium deprivation was about 60% of the control slices. This was effectively blocked by cocaine. Release of (3)H-noradrenaline evoked by high potassium from both control and treated slices was calcium-dependent.8. It is suggested that sodium-potassium-activated ATPase maintains the integrity of the axonal membrane, and any procedure which depresses the activity of the enzyme or the sodium-potassium pump would cause transmitter release by causing temporary disturbance in the membrane. Evidence is presented to suggest that vesicles depleted of their endogenous noradrenaline content by sodium deprivation are re-used for the storage and release of transmitter.

Effect of guanidine on release of noradrenaline from the perfused spleen of the cat.

1 Guanidine increased noradrenaline (NA) output at 5 Hz by 3 to 6 fold, and doubled it at 30 Hz. Onset of maximum activity was slow, and reversal was also slow. Output of NA induced by potassium, sodium deprivation, or tyramine was not affected. 2 NA output was doubled at low concentrations (1 to 2 mM) of guanidine, but maximal effect was obtained at 4 mM. At 10 mM, spontaneous release was occasionally observed. 3 The effect of guanidine on NA release was related to the external calcium concentration. Outputs which previously have been shown to be insignificant at 5 Hz in 0.25 and 0.75 mM calcium-Krebs solution were markedly enhanced by guanidine. Guanidine enhanced release at all calcium concentrations up to 7.5 mM, but maximum output was obtained at 2.5 mM. 4 Guanidine had no effect on the recovery of intra-arterially infused NA. 5 The effects of guanidine and tetraethyl-ammonium (TEA) on NA release at 5 Hz were additive. 6 Guanidine reversed the inhibition of NA release by guanethidine during nerve stimulation at 5 and 10 Hz, and the NA output increased nearly 2.5 fold after repeated stimulation of the nerves. Guanidine was less effective in reversing the inhibitory effects of guanethidine on NA release at 30 Hz. 7 Guanidine did not affect release of catecholamines (CA) from the perfused cat adrenal gland by splanchnic nerve stimulation. 8 It is suggested that guanidine enhances NA release partly by increasing the influx of calcium into the neurone during an action potential, and also by interfering with intracellular binding of calcium.

Reversal of guanethidine blockade of sympathetic nerve terminals by tetraethylammonium and 4-aminopyridine.

1 The effect of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on the inhibitory effect of guanethidine on noradrenaline (NA) release was investigated in the perfused spleen of the cat. 2 Guanethidine blocked the release of NA evoked by nerve stimulation. TEA and 4-AP readily reversed this inhibitory effect, and the NA output was nearly doubled after repeated stimulation of the nerves. On subsequent perfusion with Krebs solution without TEA or 4-AP, the inhibitory effect of guanethidine reappeared. 3 The reversal of guanethidine blockade of sympathetic nerves by TEA and 4-AP is discussed.

Release of noradrenaline from cat spleen slices by potassium.

1 When cat spleen slices were exposed to a potassium-enriched (140 mM) Krebs solution, 367 +/- 31 ng g-1 5 min-1 of noradrenaline (NA) was released into the bathing medium. 2 Phenylephrine and clonidine (10(-7) to 10(-3) M) did not significantly modify the potassium-evoked NA release; acetylcholine decreased it in a dose-dependent manner. 3 Phenoxybenzamine increased NA release by 50% but phentolamine did not alter it; high concentrations of this drug greatly decreased NA release. Cocaine increased the NA release by about 30%. 4 It is suggested that the failure of sympathomimetic amines to depress, and of alpha-adrenoceptor blocking agents to enhance the release of NA by high potassium concentrations may be related to prolonged depolarization of the nerve terminals, which may desensitize presynaptic alpha-receptors. The fact that the same drugs are able to modify NA release during electrical nerve stimulation may be ascribed to the much shorter periods of depolarization occurring under these conditions.

Effect of 6-hydroxydopamine on bovine adrenal chromaffin cells in culture.

The effect of 6-hydroxydopamine on the catecholamine content and cell morphology of bovine adrenal chromaffin cells in culture was investigated. 6-Hydroxydopamine markedly reduced the catecholamine content of the cultured chromaffin cells after 6 and 24 h exposure. The effect was dose-related, with half-maximal depletion occurring at 2.4 X 10(-5) M. Cells exposed to 6-hydroxydopamine for 3 h and then to normal medium without the drug for 3 h more showed the same degree of toxicity as cells exposed to the drug for the entire 6 h. Ascorbate at high concentrations also exhibited toxicity toward chromaffin cells between 6 and 24 h of exposure. 6-Hydroxydopamine produced marked changes in cell morphology. At 1 h the cells appeared normal, at 3 h the processes were markedly shortened, and at 6 h they were completely retracted. On exposure for 24 h there were gross morphological changes and most cells were detached and free-floating in the medium. The toxicity of 6-hydroxydopamine in bovine adrenal chromaffin cells is discussed.

Ion dependence of the release of noradrenaline by tetraethylammonium and 4-aminopyridine from cat splenic slices.

Cat splenic slices prelabelled with [3H]-noradrenaline were incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, and the spontaneous total 3H release into different incubation media monitored. In normal Krebs bicarbonate solution, the spontaneous tritium fractional release amounted to 3.7% of the tissue radioactivity content per 5 min collection period. Tetraethylammonium (TEA) increased spontaneous transmitter release in a concentration-dependent manner; the release was maximal at 30 mM and was 3.5 times the basal release. 4-Aminopyridine (4-AP) also enhanced the spontaneous release of tritium. The response increased linearly with 4-AP concentration (1-10 mM). With 10 mM 4-AP, the release was as much as 6 times the basal transmitter release. Guanidine was much less potent than either TEA or 4-AP. The secretory response to TEA or 4-AP was little affected by changes in external Ca2+, Mg2+, Na+, Cl-, H2PO4- or by tetrodotoxin. However, transmitter release evoked by TEA or 4-AP strongly depended upon the concentration of HCO3- of the incubation solution; in fact, the secretory response varied almost linearly between 1 and 25 mM HCO3-. The mechanisms underlying these effects are probably related to the well-known ability of TEA and 4-AP to block K+ conductance that would cause depolarization of the splenic sympathetic nerve terminals. The HCO3- requirements for the secretory response are probably related to the ability of CO2/HCO3- solutions to mobilize and release Ca2+ from intracellular organelles.

Effect of muscarine on release of catecholamines from the perfused adrenal gland of the cat.

1 The secretory effect of muscarine was studied in the perfused adrenal gland of the cat. During perfusion of the adrenal gland with Krebs-bicarbonate solution containing muscarine 480 microM, the rate of catecholamine (CA) secretion was 2.02 +/- 0.43 micrograms/2 min in the first 2 min; thereafter, CA output declined only moderately, to reach about 70% of the initial value after 10 min. Secretory responses to brief infusions of muscarine remained reproducible for at least the first 3 infusions. 2 When the adrenal gland was perfused with muscarine (480 microM), infusions of high K+, nicotine, or veratridine produced their usual responses. A 100 fold lower dose of muscarine also failed to modify these responses. 3 During perfusion with high K+, muscarine evoked a secretory response that was only slightly smaller than the response to muscarine alone. 4 It is concluded that muscarine and nicotine activate CA secretion in the cat adrenal gland by independent mechanisms and that the muscarinic response, unlike the nicotinic response, is not readily desensitized.

Orthograde and retrograde axonal transport of calmodulin in a cat noradrenergic neurone.

Subcellular distribution studies of calmodulin in cat sympathetic ganglia demonstrated that about 90% of the protein remained in the 27,000 g supernatant, suggesting that it is a cytosolic protein. Only 4.5% was recovered in the microsomal fraction pellet. The inferior mesenteric ganglia contained 93.3 +/- 3 ng calmodulin per ganglion, and segments of unligated cat hypogastric nerves had 6.53 +/- 0.32 ng per 5 mm segment. When the nerve was ligated in the middle and left in the cat for 1-6 days, substantial amounts of calmodulin accumulated in segments of nerve immediately proximal (P1) and distal (D1) to the ligature. The amounts found in P1 amounted to 15.3, 20, 30.4 and 39.4 ng calmodulin per 5 mm segment 1, 2, 3 and 6 days after ligation, respectively. The average rate of transport was 5.5 mm per day, which corresponds to a slow component b of axonal transport (SCb). The accumulation of calmodulin in D1 was also increased with the time of ligation. After 1, 2, 3 and 6 days, the amounts of the protein found in D1 were 14.4, 17.7, 19 and 21 ng per 5 mm segment, respectively. The calculated mean rate for the retrograde transport was 3.9 mm per day. Decentralization of the inferior mesenteric ganglia did not affect the rate of accumulation of calmodulin or the basal amounts found in ganglia and nerves. Local injection inhibited the orthograde, but not the retrograde axonal transport of the protein. It is concluded that calmodulin undergoes a process of slow orthograde axonal transport probably incorporated into the axoplasmic matrix of a network of actin microfilaments. The protein is also transported in a retrograde manner.

Modification of potassium-evoked release of noradrenaline by various ions and agents.

1 Release of noradrenaline (NA) from isolated spleen slices of the cat by high K(+) and tetraethylammonium (TEA) was investigated. Studies were conducted with spleen slices whose tissue stores were prelabelled with [(3)H]-noradrenaline ([(3)H]-NA).2 Release by high K(+) was related to the K(+) concentration of the incubation medium. Release of [(3)H]-NA by 28.5 mM K(+) was only barely detectable over the background, while 70 mM K(+) enhanced release to more than 600% of the background output. Tetrodotoxin (TTX) did not block responses to 28.5 or 35 mM K(+).3 Background release was not modified by 1 or 3 mM TEA, but 10 and 30 mM TEA enhanced the release of [(3)H]-NA by about 50% and 150%, respectively, over the background level. Neither TTX nor hexamethonium (C(6)) blocked the TEA response. Release by TEA was also not blocked in Ca(2+)-free medium or in Ca(2+)-free medium containing up to 3 mM EGTA. Release by TEA was blocked in Ca(2+)-free medium containing 5 mM EGTA, and by La(3+) or Mn(2+).4 The response to 35 mM K(+) was not modified by 1 or 3 mM TEA; 10 mM TEA had an additive effect; and 30 mM TEA with 35 mM K(+) produced a response which was greater than the simple sum of responses to 35 mM K(+) and 30 mM TEA. At 45 mM K(+), 3 and 10 mM TEA potentiated the response, and at 30 mM K(+) only 1 mM TEA showed potentiation. TTX did not alter the response to high K(+) plus TEA.5 When TEA (30 mM) was added during prolonged incubation with 140 mM K(+), the response was only slightly enhanced. This suggests that a large part of the secretory response to TEA is mediated through mobilization of Ca(2+) activated by depolarization.6 Phenoxybenzamine (3.3 muM) potentiated responses to 35 and 140 mM K(+) by about 50%, and TTX did not influence this potentiation. Acetylcholine (ACh) blocked responses to 28.5 and 35 mM K(+), and 1 mM TEA antagonized this ACh blockade.7 In the perfused adrenal gland of the cat, the secretory response to TEA was related to its concentration. The response was not diminished by low Na(+), TTX, or C(6), but was markedly attenuated when TEA was applied 10 min after the start of perfusion with high K(+).

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.

Release of endogenous noradrenaline from ligated cat hypogastric nerve by veratridine.

The effect of veratridine on in vitro release of noradrenaline (NA) from ligated cat hypogastric nerve was investigated. After in vivo ligation for 24-48 h, large amounts of NA and dopamine-beta-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 degrees 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.

Release of noradrenaline from the ligated cat hypogastric nerve.

After in vivo ligation for 24 of the cat hypogastric nerve, large amounts of noradrenaline (NA) and dopamine beta-hydroxylase (DBH) accumulated in the nerve segment immediately proximal to the ligature (P 1). In vitro incubation of 24-h-ligated nerves ((segments P1 and P2) in oxygenated Krebs solution at 37 degrees 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.

Effect of tetraethylammonium and barium on the release of noradrenaline from the perfused cat spleen by nerve stimulation and potassium.

The effect of tetraethylammonium (TEA) and barium on release of noradrenaline (NA) from the cat spleen by nerve stimulation or potassium was investigated. 2. In spleens perfused with normal Krebs solution, the NA output at 5 Hz was barely detectable, and the output at 30 Hz was about 5-fold greater than the output at 5 Hz. 3. TEA (1 mM) or barium (2.5 mM) increased NA output at 5 Hz by 5-fold, but did not enhance it at 30 Hz. A maximal effect of TEA was obtained at about 1-3 mM. Enhancement of NA release by TEA was readily reversible. Output of NA induced by high potassium was not affected by TEA or barium. 4. The effect of TEA on release was related to the external calcium concentration. Insignificant outputs obtained at 5 Hz in 0.1 and 0.5 mM calcium-Krebs solutions were markedly increased by TEA, and were 2- and 5-fold greater than the control output at 5 Hz in normal Krebs solution containing 2.5 mM calcium. TEA enhanced release at all calcium concentrations up to 5 mM, but maximum output was still obtained at 2.5 mM. 5. Increasing the potassium concentrations of normal Krebs solution to 10, 15 and 20 mM depressed NA outputs at 5 Hz by 50, 55 and 75% respectively, TEA (1 mM) partially antagonized the inhibitory effect of potassium on release, and in zero potassium-Krebs solution it increased output by about 50% over that obtained in normal Krebs solution. 6. The ratio of NA outputs at 30 and 5 Hz during perfusion with Krebs solution containing TEA was about 0.6, and it approached the normal value as the calcium concentration of the perfusion medium was reduced. TEA facilitated release even at 30 Hz in low-calcium solutions. 7. It is suggested that the enhancement of NA release by TEA and barium is due to the greater influx of calcium ions into the sympathetic nerves during the course of an action potential.

Effect of calcium on the relationship between frequency of stimulation and release of noradrenaline from the perfused spleen of the cat.

The sympathetic nerves of the cat spleen were stimulated electrically (20 v, 1 msec, 7 sec) with increasing frequencies (1-30 Hz) during perfusion of the spleen with normal or low-calcium Krebs solution. Noradrenaline (NA) output per stimulus was measured. In spleens perfused with normal Krebs solution, the NA output per stimulus at 5Hz was barely detectable, while the output at 30 Hz was about 5-fold greater than at 5 Hz. Reduction of the calcium concentration in the Krebs solution to 0.2-0.5 mM, while reducing outputs at both frequencies, resulted in an output at 30 Hz that was nearly 8 to 10 times that at 5 Hz. In spleens pretreated with phenoxybenzamine (PBZ) the NA output was increased at all frequencies, and the maximal output occurred at 5 Hz, whereas the output at 30 Hz was only one-third of the maximal output. Reduction of the calcium concentration of the perfusion solution to 0.5 mM, while reducing outputs from PBZ-treated spleens, increased the output at 30 Hz by about 5-fold over the output at 5 Hz. The muscarinic inhibition of stimulation-evoked release of NA by acetylcholine was much more pronounced at low than at high frequency of stimulation. The increase in the release of NA per stimulus with increase in the frequency of sympathetic nerve stimulation is explained on the assumption that the specific entry of calcium ions required for transmitter release is also frequency-dependent.