Increase In Catecholamine Output Research Articles

An investigation of the effects of angiotensin on the release of neurohumoral transmitters at motor, adrenergic and cholinergic nerve terminals.

Abstract On the sciatic nerve-gastrocnemius-soleus muscle preparation of the cat, angiotensin (1–5 μg/kg, i.v.) potentiated the twitch response to maximal and submaximal stimulation of the sciatic nerve and produced partial reversal of an incomplete tubocurarine blockade. These actions could not be explained in terms of increased acetylcholine release since they were not seen in isolated motor nerve-striated muscle preparations and were probably secondary to the cardiovascular actions of angiotensin. Blockade of conduction in the postganglionic cholinergic nerves in the guinea-pig isolated ileum preparation by cooling or anoxia antagonized the response of this tissue to angiotensin. These procedures left the response to exogenous acetylcholine unchanged though they removed the cholinergic component of the response to angiotensin which is known to be present in this tissue. No evidence of increase in catecholamine output could be found in preparations of guinea-pig and rabbit vasa deferentia or rabbit duodenum responding to submaximal stimulation of their adrenergic nerves. It is concluded that angiotensin has no direct action on the stores of neurohumoral transmitter at motor, postganglionic cholinergic or postganglionic adrenergic nerves and that its known acetylcholine releasing action in the isolated ileum results from stimulation of the ganglia.

Functional role of local angiotensin-converting enzyme (ACE) in adrenal catecholamine secretion in vivo

The aim of the present study was to investigate whether exogenous angiotensin I (AngI) is locally converted to angiotensin II (AngII), which in turn results in an increase in the adrenal catecholamine (CA) secretion in the adrenal gland in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by an HPLC-electrochemical method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngI (0.0062 to 6.2 microg, 0.0096 to 9.6 microM) to the left adrenal gland resulted in significant increases in CA output in a dose-dependent manner. Following administration of 0.62 microg (0.96 microM) of AngI, adrenal epinephrine and norepinephrine outputs increased from 20.8+/-13.6 to 250.9+/-96.4 ng x min(-1) x g(-1) (p<0.05, n = 5) and from 2.8+/-1.7 to 29.6+/-11.1 ng x min(-1) x g(-1) (p<0.05, n = 5), respectively. From the same left adrenal gland, the output of AngII increased from -0.02+/-0.04 to 26.39+/-11.38 ng x min(-1) x g(-1) (p<0.05, n = 5), while plasma concentrations of AngII in aortic blood remained unchanged. In dogs receiving captopril (12.5 microg, 0.5 mM) 10 min prior to AngI, the net amounts of CA and AngII secreted during the first 3 min after AngI were diminished by about 80% (p<0.05, n = 5) compared with those obtained from the control group. There was a close correlation (r2 = 0.91, n = 6) between the net increases in AngII and CA outputs induced by AngI. The results indicate that the local angiotensin converting enzyme is functionally involved in regional AngII formation in the canine adrenal gland in vivo. The study suggests that AngII thus generated may play a role in the local regulation of adrenal CA secretion.

Effects of adrenomedullin and PAMP on adrenal catecholamine release in dogs

We examined the effects of proadrenomedullin-derived peptides on the release of adrenal catecholamines in response to cholinergic stimuli in pentobarbital sodium-anesthetized dogs. Drugs were administered into the adrenal gland through the phrenicoabdominal artery. Splanchnic nerve stimulation (1, 2, and 3 Hz) and ACh injection (0.75, 1.5, and 3 microgram) produced frequency- or dose-dependent increases in adrenal catecholamine output. These responses were unaffected by infusion of adrenomedullin (1, 3, and 10 ng. kg-1. min-1) or its selective antagonist adrenomedullin-(22-52) (5, 15, and 50 ng. kg-1. min-1). Proadrenomedullin NH2-terminal 20 peptide (PAMP; 5, 15, and 50 ng. kg-1. min-1) suppressed both the splanchnic nerve stimulation- and ACh-induced increases in catecholamine output in a dose-dependent manner. PAMP also suppressed the catecholamine release responses to the nicotinic agonist 1, 1-dimethyl-4-phenylpiperazinium (0.5, 1, and 2 microgram) and to muscarine (0.5, 1, and 2 microgram), although the muscarine-induced response was relatively resistant to PAMP. These results suggest that PAMP, but not adrenomedullin, can act as an inhibitory regulator of adrenal catecholamine release in vivo.

Modulation of adrenal catecholamine release by PACAP in vivo.

The aim of the present study was to investigate whether pituitary adenylate cyclase-activating polypeptide-(1-27) (PACAP27) can modulate the adrenal catecholamine (CA) secretion induced by splanchnic nerve stimulation (SNS) and by exogenous acetylcholine (ACh) in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were quantified by a high-performance liquid chromatography coupled with electrochemical detection. Adrenal venous blood flow was measured by gravimetry. Local infusion of PACAP27 (0.5, 5, and 50 ng) to the left adrenal gland via the adrenolumbar artery resulted in an increase in CA output, reaching a significant level at the highest dose tested. Either direct SNS (2 Hz) or local infusion of ACh (0.5 microgram) to the left adrenal gland produced significant increases in CA output to an extent similar to that obtained with 50 ng of PACAP27 alone. In the presence of PACAP27 (50 ng), CA responses to either SNS or exogenous ACh were significantly potentiated by approximately four- and sixfold, respectively, compared with those obtained in response to each stimulus alone. However, the enhanced CA responses to ACh were not significantly different from those to SNS. The results indicate that the increase in adrenal CA secretion, induced by either direct SNS or exogenous ACh, is synergistically enhanced by PACAP27. The study suggests that the enhanced CA secretion may result from the activation of a PACAP-mediated facilitatory mechanism(s) localized presumably at the postsynaptic level in the canine adrenal medulla in vivo, although the possible involvement of presynaptic mechanisms cannot completely be ruled out in the present study.

Functional role of local angiotensin-converting enzyme (ACE) in adrenal catecholamine secretion in vivo

The aim of the present study was to investigate whether exogenous angiotensin I (AngI) is locally converted to angiotensin II (AngII), which in turn results in an increase in the adrenal catecholamine (CA) secretion in the adrenal gland in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by an HPLC-electrochemical method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngI (0.0062 to 6.2 µg, 0.0096 to 9.6 µM) to the left adrenal gland resulted in significant increases in CA output in a dose-dependent manner. Following administration of 0.62 µg (0.96 µM) of AngI, adrenal epinephrine and norepinephrine outputs increased from 20.8 ± 13.6 to 250.9 ± 96.4 ng·min-1·g-1 (p < 0.05, n = 5) and from 2.8 ± 1.7 to 29.6 ± 11.1 ng·min-1·g-1 (p < 0.05, n = 5), respectively. From the same left adrenal gland, the output of AngII increased from -0.02 ± 0.04 to 26.39 ± 11.38 ng·min-1·g-1 (p < 0.05, n = 5), while plasma concentrations of AngII in aortic blood remained unchanged. In dogs receiving captopril (12.5 µg, 0.5 mM) 10 min prior to AngI, the net amounts of CA and AngII secreted during the first 3 min after AngI were diminished by about 80% (p < 0.05, n = 5) compared with those obtained from the control group. There was a close correlation (r2 = 0.91, n = 6) between the net increases in AngII and CA outputs induced by AngI. The results indicate that the local angiotensin converting enzyme is functionally involved in regional AngII formation in the canine adrenal gland in vivo. The study suggests that AngII thus generated may play a role in the local regulation of adrenal CA secretion.Key words: angiotensin I, angiotensin II, captopril, adrenal gland, anesthetized dog.

Role of nitric oxide in adrenal catecholamine secretion in anesthetized dogs.

We examined the role of nitric oxide (NO) in adrenal catecholamine secretion in response to splanchnic nerve stimulation (SNS) and exogenous acetylcholine (ACh) in anesthetized dogs. The NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME), NO donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e (NOC 7), and ACh were administered intra-arterially into the adrenal gland. The increases in catecholamine output induced by ACh (0.75-3 microgram) were enhanced by L-NAME (0.1-1 mg/min) and inhibited by NOC 7 (0.2-2 microgram/min). Inhibition by NOC 7 (2 microgram/min) was observed during treatment with L-NAME (1 mg/min). The increases in catecholamine output induced by SNS (1-2 Hz) were inhibited by L-NAME and by NOC 7. No inhibitory effect of NOC 7 was observed during treatment with L-NAME. These results suggest that NO may play an inhibitory role in the regulation of adrenal catecholamine secretion in response to exogenous ACh.

The role of BK Ca channels in the nitric oxide-mediated regulation of adrenal catecholamine secretion

We examined whether high conductance Ca 2+-activated K + (BK Ca) channels are involved in the modulatory action of nitric oxide (NO) on the secretion of adrenal catecholamines in response to splanchnic nerve stimulation and acetylcholine in anesthetized dogs. The NO donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)- N-methyl-1-propanamine (NOC 7), the BK Ca channel blocker charybdotoxin and acetylcholine were administered intraarterially (i.a.) into the adrenal gland. NOC 7 infusion (2 μg min −1) inhibited increases in catecholamine output induced by splanchnic nerve stimulation (1–3 Hz) and acetylcholine (0.75–3 μg). Charybdotoxin infusion (100 ng min −1) did not affect increases in catecholamine output induced by splanchnic nerve stimulation and acetylcholine. Charybdotoxin blocked the NOC 7-induced inhibition of increases in catecholamine output induced by splanchnic nerve stimulation but not by acetylcholine. These results suggest that NO may inhibit the secretion of adrenal catecholamines induced by splanchnic nerve stimulation through activation of BK Ca channels.

Role of K+ channels in adrenal catecholamine secretion in anesthetized dogs.

We examined the role of K+ channels in the secretion of adrenal catecholamine (CA) in response to splanchnic nerve stimulation (SNS), acetylcholine (ACh), 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and muscarine in anesthetized dogs. K+ channel blockers and the cholinergic agonists were infused and injected, respectively, into the adrenal gland. The voltage-dependent K+ channel (KA type) blocker mast cell degranulating (MCD) peptide infusion (10-100 ng/min) enhanced increases in CA output induced by SNS (1-3 Hz), but it did not affect increases in CA output induced by ACh (0.75-3 micrograms), DMPP (0.1-0.4 microgram), or muscarine (0.5-2 micrograms). The small-conductance Ca(2+)-activated K+ (SKCa) channel blocker scyllatoxin infusion (10-100 ng/min) enhanced the ACh-, DMPP-, and muscarine-induced increases in CA output, but it did not affect the SNS-induced increases in CA output. These results suggest that KA channels may play an inhibitory role in the regulation of adrenal CA secretion in response to SNS and that SKCa channels may play the same role in the secretion in response to exogenously applied cholinergic agonists.

Apamin-sensitive SK Ca channels modulate adrenal catecholamine release in anesthetized dogs

We investigated the role of high conductance (BK Ca) and small conductance Ca 2+-activated K + (SK Ca) channels in adrenal catecholamine release in response to splanchnic nerve stimulation, acetylcholine, the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and muscarine in anesthetized dogs. The selective SK Ca channel blocker apamin and the selective BK Ca channel blocker charybdotoxin were infused into the adrenal gland through the phrenicoabdominal artery, and the cholinergic agonists were injected into the same artery. Splanchnic nerve stimulation (1, 2, 3 and 10 Hz), acetylcholine (0.75, 1.5 and 3 μg), DMPP (0.1, 0.2 and 0.4 μg) and muscarine (0.5, 1 and 2 μg) produced frequency- or dose-dependent increases in catecholamine output as measured in adrenal venous blood. Apamin infusion (1, 3 and 10 ng/min) enhanced the acetylcholine-, DMPP- and muscarine-induced increases in catecholamine output in a dose-dependent manner, but it did not affect the splanchnic nerve stimulation-induced catecholamine response. Charybdotoxin infusion (10, 30 and 100 ng/min) did not affect the increases in catecholamine output induced by the agonists and splanchnic nerve stimulation. Neither apamin nor charybdotoxin affected basal catecholamine output. These results suggest that apamin-sensitive SK Ca channels located in adrenal medullary cells may play an inhibitory role in the regulation of adrenal catecholamine release mediated by extrasynaptic nicotinic and muscarinic receptors.

Role of ET(A) and ET(B) receptors in endothelin-1-induced adrenal catecholamine secretion in vivo.

The present study was designed to test whether endothelin (ET) A and/or B receptors in the adrenal medulla are functionally involved in ET-1-induced catecholamine (CA) release in anesthetized dogs. ET-1 was locally infused into the gland via the left adrenolumbar artery. Plasma CA in adrenal venous and aortic blood was determined by a high-performance liquid chromatography method. In the control group, the local infusion of ET-1 (0.5 microg, 0.4 microM) resulted in a significant increase in CA output. In the presence of a low dose of BQ-123 (5 microg, 16.4 microM), the ET-1-induced CA response was significantly attenuated by approximately 80%. With a high dose of BQ-123 (50 microg, 164 microM), the CA response was further blocked by approximately 95%. This inhibition was significantly greater than that obtained with the low dose of BQ-123. By contrast, a low dose of BQ-788 (5 microg, 15.1 microM) did not significantly affect the CA response. With a high dose of BQ-788 (50 microg, 151 microM), the CA response was only partially inhibited by approximately 70%. The results indicate that BQ-123 significantly inhibited ET-1-induced adrenal CA release in a dose-dependent manner. With the low doses, the CA response was markedly inhibited by BQ-123 but remained unchanged in the presence of BQ-788. Moreover, the high dose of BQ-123 virtually abolished the CA response, whereas BQ-788 failed to do so within the dose range tested. The present study suggests that the ET(A) receptor may play a predominant role in mediating the ET-1-induced CA secretion in the canine adrenal gland in vivo, although the possible involvement of the ET(B) receptor could not completely be excluded under the present experimental conditions.

Implication of L-type Ca2+ channels in noncholinergic adrenal catecholamine secretion by endothelin-1 in vivo.

The aim of the present study was to investigate if either dihydropyridine-sensitive L-type Ca2+ channels or cholinergic receptor-mediated mechanisms are implicated in endothelin-1 (ET)-induced adrenal catecholamine (CA) secretion in anesthetized dogs. ET was locally administered to the left adrenal gland via the left adrenolumbar artery. Plasma CA concentrations in adrenal venous and aortic blood were determined by a high-performance liquid chromatography method. In the control group, local infusion (1 min, 0.5 ml/min) of ET (the fixed total dose of 0.5 microgram given to the gland or approximately 0.0197 microgram/kg of body weight) resulted in a sharp increase in the basal CA output, followed by a rapid decline, and a relatively slow secondary response lasted over a period of 15-30 min. In the second group treated with nifedipine (5 micrograms or approximately 0.207 microgram/kg) similarly administered 10 min before ET infusion, the ET-induced first steep increase in CA output was significantly attenuated by approximately 75% (P < 0.05, n = 6). In dogs similarly receiving either pentolinium (1 mg or approximately 0.041 mg/kg) or atropine (0.5 mg or approximately 0.018 mg/kg), the ET-induced CA response remained unchanged. The results indicate that ET-induced adrenal CA release was largely mediated by the activation of dihydropyridine-sensitive L-type Ca2+ channels. Furthermore, neither nicotinic nor muscarinic receptors were functionally implicated in the CA response to ET. The study suggests the existence of noncholinergic mechanisms involved in the secretory action of ET on the adrenal medulla in the dog in vivo.

Inhibition by ω-conotoxin GVIA of adrenal catecholamine release in response to endogenous and exogenous acetylcholine

Effects of the N-type voltage-dependent Ca 2+ channel (VDCC) blocker, ω-conotoxin GVIA, and the L-type VDCC blockers, nifedipine and verapamil, on adrenal catecholamine release were examined in anesthetized dogs. These blockers were infused into the adrenal gland through the phrenicoabdominal artery. Splanchnic nerve stimulation at 1 and 3 Hz produced frequency-dependent increases in epinephrine and norepinephrine output determined from adrenal venous blood. Infusion of ω-conotoxin GVIA (0.4 μg/min) significantly inhibited the splanchnic nerve stimulation-evoked increases in epinephrine and norepinephrine output. Furthermore, increases in epinephrine and norepinephrine output induced by intraarterial injection of acetylcholine (3 μg) into the adrenal gland also were inhibited by ω-conotoxin GVIA (0.4 μg/min). Further inhibition of splanchnic nerve stimulation- or exogenous acetylcholine-induced increases in catecholamine output was observed even after the cessation of ω-conotoxin GVIA infusion. Neither nifedipine (1 μg/min) nor verapamil (10 μg/min) affected the splanchnic nerve stimulation-evoked increases in catecholamine output, whereas they inhibited the oxogenous acetylcholine-evoked catecholamine release. These results suggest that N-type VDCCs located in adrenal medullary cells may contribute to the release of adrenal catecholamines in response to endogenous and exogenous acetylcholine in the dog.

Effects of protein kinase C activator and inhibitor on adrenal catecholamine release in response to splanchnic nerve stimulation in anesthetized dogs.

Effects of protein kinase C (PKC) activator and inhibitors on adrenal catecholamine release were examined in anesthetized dogs. Output of epinephrine (EPI) and norepinephrine (NE) was determined from adrenal venous blood by high-performance liquid chromatography (HPLC) with electrochemical detection. All drugs were infused intraarterially (i.a.) into the adrenal gland through the phrenic abdominal artery. Infusion of the PKC activator phorbol-12,13-dibutyrate (PDB 0.1 micrograms/min) increased EPI and NE output during basal state and enhanced increases in catecholamine output induced by splanchnic nerve stimulation (SNS 1 and 3 Hz). These effects of PDB were abolished by the PKC inhibitor staurosporine (SSP 0.3 microgram/min), when both drugs were infused simultaneously. Infusion of SSP (0.1, 0.3, and 1 micrograms/min) caused a dose-dependent inhibition of the SNS-induced increases in EPI and NE output. SNS-induced increases in catecholamine output were also inhibited by another PKC inhibitor polymyxin B (PMB 0.1, 0.3, and 1 micrograms/min) and by the phospholipase C (PLC) inhibitor neomycin (NM 0.3, 1, and 3 mg/min). SSP, PMB, and NM did not affect basal output of EPI and NE. These results suggest that activation of PKC promotes release of adrenal catecholamines and provide indirect evidence that activation of PKC and PLC may be involved in SNS-induced release of catecholamines from dog adrenal gland.

Effects of nifedipine and Bay-K-8644 on the release of catecholamines from the dog adrenal gland in response to splanchnic nerve stimulation.

1. The effects of nifedipine and Bay-K-8644 on the release of adrenal catecholamines were examined in anaesthetized dogs. 2. Splanchnic nerve stimulation (SNS) at 1 and 3 Hz produced frequency-dependent increases in adrenaline (ADR) and noradrenaline (NA) output determined from adrenal venous blood. 3. Neither nifedipine (10 and 30 micrograms/kg, i.v.) nor Bay-K-8644 (10 and 30 micrograms/kg, i.v.) modified the SNS-induced increases in catecholamine output. Basal catecholamine output tended to be increased and decreased by nifedipine and Bay-K-8644, respectively. 4. Nifedipine produced significant decreases in arterial pressure and renal blood flow rate. Bay-K-8644 produced a significant increase in arterial pressure associated with a decrease in renal blood flow rate. 5. These results suggest that dihydropyridine-sensitive calcium channels do not play a major role in adrenal catecholamine release evoked by SNS.

Facilitatory Role of the Renin-Angiotensin System in Controlling Adrenal Catecholamine Release in Hemorrhaged Dogs

Effects of the renin-angiotension system (RAS) on adrenal catecholamine release in response to hemorrhagic hypotension and splanchnic nerve stimulation (SNS) were studied in pentobarbital-anesthetized dogs. In hemorrhage experiments, mean blood pressure (MBP) was maintained at 50 mm Hg for 60 min by bleeding the arterial blood into a pressurized bottle. In the renal intact group (control), epinephrine (EPI) and norepinephrine (NE) output from the adrenal gland increased markedly during hemorrhagic hypotension: from 45 +/- 13 and 4.7 +/- 0.9 to 1,167 +/- 202 and 169 +/- 30 ng/min at 60 min after onset of hemorrhage, respectively. The increases in catecholamine output during hemorrhagic hypotension in the renal-intact group pretreated with captopril (1 mg/kg intravenously, i.v.) and in the renal-ligated group were significantly smaller than those in the control group. The increases in catecholamine output in the renal-ligated group infused with angiotensin II (AngII 10 ng/kg/min i.v.) were comparable to those in the control group. In SNS experiments, AngII infusion (10 ng/kg/min i.v.) enhanced increases in catecholamine output induced by 3 Hz SNS significantly. Captopril (1 mg/kg i.v.) did not affect the SNS-induced increases in catecholamine output. These results suggest that the renal RAS facilitates reflex release of adrenal catecholamines during hemorrhagic hypotension, at least in part, by acting directly on the release process of catecholamines from dog adrenal gland.

Evidence against a humoral control mechanism in adrenal catecholamine secretion during insulin-induced hypoglycemia.

The present study tested the hypothesis that a humoral control mechanism is involved in the enhanced adrenal catecholamine secretion during insulin-induced hypoglycemia. The experiments were carried out in anesthetized dogs in which neuronal and humoral components were simultaneously determined by measuring catecholamine output from the right innervated and the left acutely denervated adrenal gland, respectively. Different levels of hypoglycemia were induced by intravenous injection of insulin with doses of 0.075 (n = 6), 0.150 (n = 6), and 0.300 IU/kg (n = 6) in three separate groups of dogs. Catecholamine output in the right innervated gland increased dose dependently (P less than 0.05), reaching a maximum level 45 min after insulin administration. By contrast, catecholamine output from the left denervated adrenal gland remained unchanged at all doses tested. In sham-denervated animals (n = 7), catecholamine output from the left adrenal gland increased to a magnitude similar to that observed in the right innervated gland after insulin administration. Plasma glucose concentration significantly decreased in a dose-dependent manner, reaching a nadir 30 min after insulin administration. Maximum decreases in plasma glucose concentration could be strongly correlated with maximum increases in catecholamine output from the right innervated adrenal gland (r = -0.66, n = 18, P = 0.011), but not with those from the left denervated gland (r = -0.32, n = 18, P = 0.455). The present results do not support the functional existence of a humoral mechanism permitting the release of adrenal catecholamines during insulin-induced hypoglycemia.

Catecholamine secretion from bullfrog adrenals in response to osmotic changes

1. 1. High K +-induced catecholamine (CA) secretion from bullfrog adrenals was suppressed in hypertonic solutions made by adding sucrose or NaCl and enhanced in solutions made hypotonic with reduced [NaCl] 0. 2. 2. When the preparation was transferred from a solution made hypertonic with sucrose or NaCl to a normal saline solution, CA secretion was increased transitorily. 3. 3. The magnitude of the transitory response depended on the duration of soaking in the hypertonic solution. 4. 4. Higher tonicities produced larger transitory responses, irrespective of whether the solute added was sucrose or NaCl. 5. 5. When the preparation was passed from normal saline to a hypotonic solution made by lowering [NaCl] 0, a similar increase in CA output was observed. 6. 6. The transitory effect of the osmotic gradient was similarly observed in low Ca 2+, Mg 2+ EGTA-containing solutions and in solutions whose sole divalent cation was Mg 2+.

FURTHER EVIDENCE FOR THE INVOLVEMENT OF Na+CHANNELS IN THE RELEASE OF ADRENAL CATECHOLAMINE: THE EFFECT OF SCORPION VENOM AND GRAYANOTOXIN I

1 The effects of venom from the scorpion, Leiurus quinquestriatus, and grayanotoxin I on catecholamine secretion were studied in the perfused adrenal glands of guinea-pig. 2 Scorpion venom (0.1 to 10 micrograms/ml) caused a dose-dependent increase in catecholamine output. The response to the venom was partially inhibited by atropine (0.5 mM) plus hexamethonium (1mM). The dose-response curve was shifted to the right in the presence of these blocking agents. 3 Grayanotoxin I (0.1 to 0.5 mM) caused a dose-dependent increase in catecholamine output which was significantly reduced by atropine (0.5 mM) plus hexamethonium (1 mM). However, when grayanotoxin I (0.1 mM) was applied together with scorpion venom (0.1 micrograms/ml, a concentration which alone, was almost ineffective) the maximum catecholamine output was reached even in the presence of atropine plus hexamethonium. 4 Tetrodotoxin (0.1 or 0.2 microM) reversibly inhibited the secretory response induced by scorpion venom (10 micrograms/ml) and grayanotoxin I (0.1 mM) plus scorpion venom (0.1 micrograms/ml). 5 Scorpion venom and grayanotoxin I plus scorpion venus did not cause catecholamine secretion in the absence of extracellular Na+ or Ca2+ ions. However, the secretory response was restored by reintroduction of Na+ or Ca2+ ions. 6 It is suggested that both scorpion venom and grayanotoxin I activate Na+ channels on the chromaffin cell and result in catecholamine secretion.

Morphological characteristics and stimulus-secretion coupling in bovine adrenal chromaffin cell cultures

Chromaffin cells isolated from adult bovine adrenal medullae were plated on collagen-coated dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 8–16 h in culture, the chromaffin cells had started to develop one, two or, more rarely, three neurites. The neurites grew at a rate of7.25 ± 0.07μm/day showing a linear correlation between process length and age of the culture. The neurites displayed one or more varicosity-like structures. Some neurites made long lasting contacts with other cell bodies and processes. Cytochemical tests indicated positive catecholamine fluorescence in boti cell bodies and processes. Fluorometric determinations indicated that by day 7 the catecholamine content was75.7 ± 1% of that found on day 1 and that noradrenaline and adrenaline represented24.6 ±1% and75.3 ± 1% of the total amine content respectively. Catecholamines were released from these cultured chromaffin cells in response to stimulation by either 56 m m KCl or acetylcholine. The stimulation-induced release of amines was Ca 2+-dependent and was blocked by increasing the extracellular c oncentration of Mg 2+. Substitution of (a) extracellular Na 2+ by either choline or sucrose or (b) Ca 2+ by Ba 2+ produced a sharp increase in catecholamine output. Acetylcholine produced a doserelated increase in catecholamine release; half maximal release was produced by3.5 × 10 −5 m acetylcholine and maximal release by 10 −4 m acetylcholine. The cholinergic receptor of cultured chromaffin cells appears to be nicotinic since catecholamine release was stimulated by nicotine and carbamylcholine but not by pilocarpine. Acetylcholine-evoked catecholamine release was blocked by d-tubocurarine (ID 50 = 5.5 × 10 −7 m), and by hexamethonium (ID 50 = 8 × 10 −6 m). However, the acetylcholine-evoked amine release was not blocked by α-bungarotoxin at a concentration (1.25 × 10 −7 m) which blocked frog rectus abdominus responses to acetylcholine (10 −6–10 −3 m). Tetrodotoxin (5 × 10 −6 m) produced47 ± 2% inhibition of the secretory response to acetylcholine (10 −4 m) but it did not modify the amine release in response to 56 m m KCl. Adult bovine chromaffin cells in culture display some of the morphological and functional characteristics of sympathetic neurones. Consequently, the bovine chromaffin cell in culture appears to be a promising model for studies on development and secretion.

The effect of veratridine on the release of catecholamines from the perfused adrenal gland.

1. Experiments on perfused adrenal glands of guinea-pigs were carried out to study the catecholamine output induced by veratridine in the presence of hexamethonium and atropine. 2. Veratridine (10 micrometer to 200 micrometer) caused a dose-dependent increase in catecholamine output. 3. The addition of veratridine to the perfusion medium for a period of 3 min caused an increase in catecholamine output which reached a maximum 5 min to 10 min after withdrawal of the drug. The catecholamine output then gradually declined and reached near resting values within 30 minutes. It was never sustained for a longer period, even when veratridine was infused for 1 hour. 4. Veratridine failed to increase the catecholamine output in the absence of extracellular Ca2+. However, the addition of Ca2+ after an infusion of veratridine (100 micrometer) in the absence of Ca2+ caused an increase in the catecholamine output which was proportional to the concentration of Ca2+ (0.55 mM to 8.8 mM) used. 5. Veratridine did not increase the catecholamine output in the absence of extracellular Na+ ions, NaCl being replaced by equimolar choline chloride or LiCl. Veratridine also failed to evoke catecholamine output in a Na+-free solution in which Na+ was replaced by sucrose; this was the case even in the presence of a high concentration of Ca2+ (8.8 mM). 6. Tetrodotoxin (0.1 micrometer) and excess Mg2+ (20 mM) reversibly inhibited the catecholamine output induced by veratridine. 7. Ouabain (10 micrometer) significantly potentiated the veratridine-induced catecholamine output. 8. It is suggested that Na+-dependent Ca2+ influx as well as voltage-dependent Ca2+ influx mechanisms may be involved in the catecholamine output induced by veratridine.