An inhibitor of T lymphocyte blastogenesis derived from the unfertilized ova of Shad (Alosa sapidissima).

Unfertilized ova from Shad, a North Atlantic herring, contains a cytostatic inhibitor of T lymphocyte blastogenesis. The inhibitor has an estimated molecular weight of 10,000-30,000 Da, is heat stable, non dialyzable, and resistant to protease digestion and periodate oxidation. Although the inhibitor functions at an early metabolic event in T lymphocyte mitogenesis, it does not appear to interfere with thymidine transport, antagonize lectin binding to lymphocyte surface receptors, or interfere with the function of an essential serum component in the cell culture media.

A tumor growth inhibitory factor and a tumor growth promoting factor isolated from unfertilized ova of shad (Alosa sapidissima).

In the present study, a cytostatic tumor growth inhibitory peptide and a tumor growth promoting peptide with molecular weights of 20,000-30,000 Da have been identified in the supernatant fraction of unfertilized ova from Shad. The factors can be separated by gel chromatography, thus indicating that the factors are individual molecules. Both of the factors are nondialyzable, heat stable, and resistant to trypsin digestion and periodate oxidation.

Sympathetic neurons extend neurites in a culture medium containing cyanide and dinitrophenol but not iodoacetate.

A culture medium circulated through the rat heart and supplemented with insulin, transferrin and nerve growth factor leads to a massive proliferation of neurite outgrowth from neurons of peripheral sympathetic ganglia of the chick embryo. Addition of 1 mM cyanide or 50 microM dinitrophenol to such medium for 2 days had no adverse effect on the neurite outgrowth and ATP content of these neurons. However, 0.5 microM iodoacetate lowered ATP content 65% without affecting the number of surviving neurons up to 2 days. Only when ATP content was reduced to 80% by 2.5 microM iodoacetate was the number of surviving neurons significantly reduced (30%). It is concluded that the glycolytic pathway is the major route of ATP synthesis in embryonic sympathetic neurons maintained in culture, and only a small fraction of ATP is utilized for the survival and neurite extension.

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(+).

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.

Ionomycin stimulates secretion of catecholamines from cat adrenal gland and spleen.

Ionomycin, a polyether antibiotic, stimulated the secretion of catecholamines and dopamine beta-hydroxylase from perfused adrenal glands and [3H]norepinephrine ([3H]NE) from spleens of the cat. Release was calcium dependent, and strontium or barium did not substitute for calcium. Ionomycin failed to release [3H]NE from reserpinized spleens. High magnesium did not interfere in the ionomycin response, but lanthanum and manganese blocked it. Ionomycin response that was pH dependent was not affected by potassium depolarization. The secretory response to ionomycin was enhanced when both glycolysis and oxidative metabolism were inhibited. It is concluded that ionomycin introduces calcium into the chromaffin cells and adrenergic nerve terminals to cause the secretory response and that a rise in intracellular calcium may be an adequate stimulus for secretion.

Adrenergic nerve-blocking activity of a new guanidine derivative.

The effects of 4-7-exo-methylene-hexahydroisoindoline-ethyl guanidine hemisulfate (no. 865-123) on norepinephrine release were investigated in the perfused spleen on the cat. No. 865-123 irreversibly blocked the release of norepinephrine evoked by nerve stimulation. Tetraethylammonium, 4-amino-pyridine and guanidine readily reversed this inhibitory effect, and the norepinephrine output was nearly tripled after repeated stimulation of the nerves. On subsequent perfusion with Krebs' solution without any drugs, the inhibitory effect of no. 865-123 partially reappeared. Perfusion pressure responses followed the same pattern as release except during the final perfusion period with Krebs' solution. It is suggested that tetraethylammonium, 4-aminopyridine and guanidine allow greater than normal amounts of calcium to accumulate inside the adrenergic nerve terminals during an action potential to reverse no. 865-123 blockade of norepinephrine release.

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.

Action of nicotine on sympathetic nerve terminals.

Interaction between 4-aminophrydine (4-AP) and nicotine on sympathetic nerve terminals was studied in the isolated cat spleen slices, labeled with [3H]norepinephrine ([3H]NE). Incubation of slices for 5 min at 37 degrees C in low (50 microM) and high (2 mM) concentrations of nicotine released 0.8 +/- 0.08 and 2.73 +/- 0.39% of tissue [3H]NE. Tetrodotoxin (TTX) blocked the response to low nicotine but not to high nicotine. Low nicotine did not release [3H]NE in the absence of calcium. Response to high nicotine which persisted in calcium-free solution was blocked by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetra-acetic acid. 4-AP (1 mM) not only enhanced the response to low nicotine but it effectively antagonized the suppressant effects of TTX and calcium-free solution on release induced by low nitotine. Restoration of release by 4-AP from TTX-blocked preparations occurred in the absence of calcium in the perfusion medium, but lanthanum (1 mM) blocked it. Restoration of release from spleen slices incubated in calcium-free Krebs' solution by 4-AP was blocked by lanthanum and prolonged incubation in calcium-free ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraaectic acid solution. It is concluded that at lower doses nicotine, by acting on nicotinic receptors, depolarizes the sympathetic nerve terminals to set off propagated action potentials which are responsible for NE release, and that 4-AP restores nicotine response in the presence of TTX or in the absence of calcium by mobilizing calcium both from extracellular and intracellular sources.

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.

Release of catecholamines from perfused cat adrenal gland by veratridine.

Experiments were undertaken to verify the existence of fast sodium channels in the adrenal chromaffin cell membrane and to assess their contribution to the physiological release of catecholamines. We have used veratridine to activate sodium channels. Veratridine causes secretion of catecholamines from perfused cat adrenal gland. Secretory response to veratridine is calcium dependent and abolished by tetrodotoxin. Secretion of catecholamines by acetylcholine is only partially blocked by tetrodotoxin. It is concluded that the adrenal chromaffin cell membrane contains fast sodium channels directly comparable to those of impulse-propagating neurons, but they do not appear to be essential in the secretory response to acetylcholine or splanchnic nerve stimulation.

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.

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.

Effect of 4-aminopyridine on release of noradrenaline from the perfused cat spleen by nerve stimulation.

1. 4-aminopyridine (4-AP, 1 mM) increased noradrenaline (NA) output from the perfused cat spleen at 5 Hz by about fivefold. Enhancement of NA release by 4-AP was reversible. Output of NA induced by potassium was not affected. 2. NA output was doubled at low concentrations (0.1--0.3 mM) of 4-AP, but maximal effect was obtained at 1--3 mM. At 10 mM, it induced spontaneous release of NA which was insensitive to calcium. 3. Insignificant outputs obtained at 5 Hz in 0.1 and 0.3 mM calcium-Krebs solution were markedly enhanced by 4-AP. 4-AP enhanced release at all calcium concentrations up to 5 mM, but maximum output was obtained at 2.5 mM. 4. 4-AP at pH 8.5 was more effective in enhancing NA release than at pH 7.4. 5. 4-AP increased the recovery of intra-arterially infused NA from the control 26 to 47%. 6. 4-AP did not affect release of catecholamines (CA) from the perfused cat adrenal gland by acetylcholine (ACh). 7. It is suggested that 4-AP inactivates potassium current in sympathetic nerves and prolongs the duration of the action potential, thereby allowing a greater influx of calcium ions into the neurone to enhance release of NA.

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.