Comparisons of the effects of ryanodine on catecholamine secretion evoked by caffeine and acetylcholine in perfused adrenal glands of the guinea-pig.

1. The effect of ryanodine on catecholamine secretion induced by caffeine and muscarinic receptor activation was investigated in perfused adrenal glands of the guinea-pig. 2. Caffeine (40 mM) caused only a small increase in catecholamine secretion during perfusion with standard Locke solution. Caffeine-induced catecholamine secretion was markedly enhanced after removal of CaCl2 together with replacement of NaCl with sucrose. 3. In the absence of CaCl2 and NaCl, 50 microM ryanodine had no effect on the resting catecholamine secretion. Caffeine (40 mM) administered 15 min after treatment with ryanodine caused an increase in catecholamine secretion similar to that prior to application of ryanodine, but failed to have any effect thereafter. Combined application of ryanodine and caffeine also prevented catecholamine secretion induced by caffeine applied subsequently. 4. Catecholamine secretion induced by 100 microM acetylcholine (ACh) was only partially inhibited after treatment with ryanodine plus caffeine under Ca(2+)-free, Na(+)-deficient conditions. 5. Preferential influence of ryanodine on the response to caffeine was also confirmed in catecholamine secretion evoked by paired stimuli with caffeine and ACh alternately, during perfusion with either Ca(2+)-free Locke or sucrose-substituted solutions. 6. These results indicate that caffeine increases catecholamine secretion by mobilizing Ca2+ from intracellular Ca2+ stores through ryanodine-sensitive mechanisms in guinea-pig adrenal chromaffin cells. Ca2+ stores sensitive to caffeine and muscarinic receptor activation may not overlap entirely.     

Effect of 2,4,6-trinitrophenol on catecholamine secretion from perfused bovine adrenal glands

2,4,6-Trinitrophenol (PA) evoked a prolonged catecholamine (CA) secretion from perfused bovine adrenal glands. The PA-evoked CA secretion was concentration-dependent, required the presence of extracellular calcium and resulted from a direct action of PA on the chromaffin cells. Furthermore, PA reduced Mg2+-ATPase activities in the plasma membrane-rich microsome and granule-rich fraction from the adrenal medulla. These results indicate that PA evokes CA secretion through the actions on both the chromaffin cell membranes and granule membranes.     

Inhibitory effects of caffeine on secretagogue-induced catecholamine secretion from adrenal chromaffin cells of the guinea-pig.

1. The inhibitory action of caffeine on catecholamine secretion induced by secretagogues was investigated in perfused adrenal glands and dispersed chromaffin cells of the guinea-pig. 2. Caffeine (10 mM) caused a reversible inhibition of catecholamine secretion evoked by acetylcholine (ACh, 50 microM), KCl (56 mM, high K+) and veratridine (100 microM) and that induced by muscarinic receptor activation in the absence of extracellular Ca2+ in perfused adrenal glands. 3. In dispersed chromaffin cells, caffeine caused a dose-dependent inhibition of the secretory responses to 100 microM ACh and veratridine. Forskolin (30 microM), dibutyryl cyclic AMP (1 mM) and 8-bromo cyclic AMP (1 mM) did not mimic the action of caffeine. 4. In the voltage-clamp, whole-cell recording mode (at a holding potential of -60 mV or -70 mV), ACh (100 microM) evoked an inward current, and depolarizing pulses elicited inward Na+, Ca2+ and outward K+ currents. All these responses were partially inhibited by caffeine (20 mM). 5. ACh rapidly increased the intracellular concentration of Ca2+ ([Ca2+]i) in fura-2-loaded cells in either the presence or the absence of external Ca2+, though its magnitude was decreased by about 50% in Ca(2+)-free conditions. Caffeine (20 mM) inhibited these ACh-induced increases in [Ca2+]i. 6. In permeabilized chromaffin cells, caffeine (20 mM) caused an inhibition of catecholamine secretion evoked by Ca2+ (10 microM). 7. These results suggest that caffeine inhibits evoked catecholamine secretion through mechanisms such as the blockade of voltage-dependent Na+ and Ca2+ currents and ACh receptor current, and reduction of the release of intracellularly stored Ca2+ and/or Ca(2+)-sensitivity of the secretory apparatus.     

Antagonism of aminoglutethimide and adrenocorticotropic hormone (ACTH) studied on slices of adrenal glands of the guinea-pig

Aminoglutethimide at concentrations from 0.1 to 5 nM is able to inhibit the cortisol release elicited by adrenocorticotropic hormone (ACTH) (from 2.5 to 50 ng/ml) in guinea-pig adrenal cortex slices. The antagonism is a non-competitive one (in a Lineweaver-Burk plot), whereas other drugs (morphine, endorphin, indomethacin, etc.) inhibit ACTH competitively. This is in agreement with the known mechanism of action of aminoglutethimide, which inhibits the synthesis of cortisol by blocking reactions of enzymes such as aromatase and desmolase. From the data one can calculate the dissociation constant (Km) of ACTH with its receptor(s) to be 0.27 pg/ml and the inhibiting constant (Kl) of aminoglutethimide to be 49.78 x 10(-10) M. The maximal response of ACTH was 52.9 ng/ml.     

Influence of cytisine on catecholamine release in isolated perfused rat adrenal glands

The aim of the present study was to determine the characteristics of cytisine on the secretion of catecholamines (CA) in isolated perfused rat adrenal glands, and to clarify its mechanism of action. The release of CA evoked by the continuous infusion of cytisine (1.5 x 10(-5) M) was time-dependently reduced from 15 min following the initiation of cytisine infusion. Furthermore, upon the repeated injection of cytisine (5 x 10(-5) M), at 30 min intervals into an adrenal vein, the secretion of CA was rapidly decreased following the second injection. Tachyphylaxis to the release of CA was observed by the repeated administration of cytisine. The cytisine-induced secretion of CA was markedly inhibited by pretreatment with chlorisondamine, nicardipine, TMB-8, and the perfusion of Ca2+-free Krebs solution, while it was not affected by pirenzepine or diphenhydramine. Moreover, the secretion of CA evoked by ACh was time-dependently inhibited by the prior perfusion of cytisine (5 x 10(-6) M). Taken together, these experimental data suggest that cytisine causes secretion of catecholamines from the perfused rat adrenal glands in a calcium-dependent fashion through the activation of neuronal nicotinic ACh receptors located in adrenomedullary chromaffin cells. It also seems that the cytisine-evoked release of catecholamine is not relevant to the activation of cholinergic M1-muscarinic or histaminergic receptors.     

Gintonin facilitates catecholamine secretion from the perfused adrenal medulla

The present study was designed to investigate the characteristics of gintonin, one of components isolated from Korean Ginseng on secretion of catecholamines (CA) from the isolated perfused model of rat adrenal gland and to clarify its mechanism of action. Gintonin (1 to 30 µg/ml), perfused into an adrenal vein, markedly increased the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. The gintonin-evoked CA secretion was greatly inhibited in the presence of chlorisondamine (1 µM, an autonomic ganglionic bloker), pirenzepine (2 µM, a muscarinic M₁ receptor antagonist), Ki14625 (10 µM, an LPA_1/3 receptor antagonist), amiloride (1 mM, an inhibitor of Na⁺/Ca²⁺ exchanger), a nicardipine (1 µM, a voltage-dependent Ca²⁺ channel blocker), TMB-8 (1 µM, an intracellular Ca²⁺ antagonist), and perfusion of Ca²⁺-free Krebs solution with 5mM EGTA (a Ca²⁺chelater), while was not affected by sodium nitroprusside (100 µM, a nitrosovasodialtor). Interestingly, LPA (0.3~3 µM, an LPA receptor agonist) also dose-dependently enhanced the CA secretion from the adrenal medulla, but this facilitatory effect of LPA was greatly inhibited in the presence of Ki 14625 (10 µM). Moreover, acetylcholine (AC)-evoked CA secretion was greatly potentiated during the perfusion of gintonin (3 µg/ml). Taken together, these results demonstrate the first evidence that gintonin increases the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. This facilitatory effect of gintonin seems to be associated with activation of LPA- and cholinergic-receptors, which are relevant to the cytoplasmic Ca²⁺ increase by stimulation of the Ca²⁺ influx as well as by the inhibition of Ca²⁺ uptake into the cytoplasmic Ca²⁺ stores, without the increased nitric oxide (NO). Based on these results, it is thought that gintonin, one of ginseng components, can elevate the CA secretion from adrenal medulla by regulating the Ca²⁺ mobilization for exocytosis, suggesting facilitation of cardiovascular system. Also, these findings show that gintonin might be at least one of ginseng-induced hypertensive components.     

Facilitation of stimulation-evoked catecholamine release by somatostatin in dog perfused adrenal glands

Summary The effect of somatostatin on the catecholamine (CA) release from the chromaffin cells was determined on the isolated dog adrenals perfused with 1.8 mM Ca²⁺ containing fluid except indicated. The extremely low concentrations of somatostatin (0.18 nM – 18 nM) were found to stimulate acetylcholine (ACh, 5 M)-evoked CA release with the maximum response (by 114% above control) at 1.8 nM but the relatively high concentrations (61, 610 nM) caused an inhibition. Somatostatin (6.1 nM) also facilitated excess K⁺ (15 mM)-induced CA release but failed to enhance the release evoked by a Ca²⁺ ionophore, A23187 (50 M) and the release by caffeine (50 mM) under the condition of Ca²⁺-free. Somatostatin by itself did not affect significantly on the basal release of CA. Elevation of Ca²⁺ concentrations from 1.8 mM to 5 mM in the perfusion fluid reduced the stimulatory and inhibitory effect of somatostatin. It is possible that somatostatin enhances CA release by facilitating the influx of Ca²⁺ via the potential-sensitive permeability channel when chromaffin cells are depolarized. The present results provide the first demonstration that somatostatin stimulate the release of CA from the adrenal gland suggesting that somatostatin may function as a facilitatory modulator of the response to ACh at chromaffin cells.     

Role of capsaicin-sensitive neurons in catecholamine secretion from rat adrenal glands

Sensory fibres innervate the adrenal medulla but their function is not known. In the present study, we have used the sensory neurotoxin capsaicin to evaluate the effect of capsaicin sensitive sensory fibres on catecholamine (CA) secretion from isolated perfused rat adrenal glands. CA secretion in response to 1 and 10 min electrical field stimulation of adrenal nerve terminals was significantly attenuated in the adrenal glands of adult rats pretreated as neonates with capsaicin and was frequency dependent, being more pronounced at the higher frequencies of stimulation (5 to 30 Hz) than at the low (0.3, 1 Hz) frequencies. Perfusion of control rat adrenal glands with capsaicin did not evoke CA secretion, but did increase CA secretion in response to perfusion with nicotine. Perfusion with capsaicin for 30 min (but not for 4 min) reduced the CA secretory response to subsequent nerve stimulation. The results suggest that capsaicin sensitive sensory neurons innervating the adrenal medulla are involved in the regulation of adrenal CA secretion evoked by electrical stimulation of adrenal nerve terminals.     

Inhibitory mechanism of pinacidil on catecholamine secretion from the rat perfused adrenal gland evoked by cholinergic stimulation and membrane depolarization

1. The present study attempted to investigate the effect of potassium channel openers on secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from rat isolated perfused adrenal gland. 2. The perfusion of pinacidil (30-300 microM) into an adrenal vein for 20 min produced dose-dependent inhibition of CA secretion evoked by acetylcholine (ACh; 5.32 mM), high K+ (56 mM), 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 100 microM for 2 min), 3-(m-chloro-phenyl-carbamoyl-oxy)-2-butynyl trimethyl ammonium chloride (McN-A-343; 100 microM for 2 min), cyclopiazonic acid (CPA; 10 microM for 4 min) and methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyri dine-5-carboxylate (Bay-K-8644; 10 microM for 4 min). 3. In the presence of minoxidil (100 microM), which is also known to be a potassium channel activator, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and CPA were also significantly depressed. 4. In adrenal glands preloaded with pinacidil (100 microM) in the presence of glibenclamide (GB; 1 microM), a specific blocker of ATP-regulated potassium channels, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and CPA were restored to a considerable extent of the control release as compared with that of pinacidil only. 5. These results suggest that pinacidil causes marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, as well as by membrane depolarization, indicating that this effect may be mediated by inhibiting influx of extracellular calcium and release of intracellular calcium in the rat adrenomedullary chromaffin cells. Furthermore, these findings suggest that these potassium channel opener-sensitive membrane potassium channels also play a modulatory role in regulating CA secretion.     

Exocytotic release of catecholamine from perfused adrenal gland of guinea-pig induced by veratridine.

1 Experiments were carried out on perfused adrenal glands of guinea-pig to determine whether veratridine caused the exocytotic release of catecholamine by comparing its effect with that of splanchnic nerve stimulation and secretagogues such as acetylcholine and excess K+. 2 Veratridine (100 microM) and excess K+ (56 mM) caused secretion of catecholamine and dopamine-beta-hydroxylase (DBH) activity in the venous effluents in the presence of atropine (30 microM) and hexamethonium (2 mM). Splanchnic nerve stimulation in the presence or absence of physostigmine (100 nM) and infusion of acetylcholine in the presence of physostigmine had the same effect. In all the responses, the release of DBH tended to last for a longer period than that of catecholamine. 3 The ratio of catecholamine to DBH activity appearing in the venous effluents was approximately 9, regardless of the method of stimulation. This value was close to the ratio of catecholamines to the 'soluble' DBH activity found in the chromaffin granules. 4 All the types of stimulation used caused a proportional release of adenine nucleotides and catecholamines in the effluents. The adenine nucleotides were mainly adenosine 5'-phosphate. 5 The ratio of catecholamine to adenine nucleotides was approximately 11, regardless of the method of stimulation. 6 It is suggested that the release of catecholamine induced by veratridine occurs by exocytosis in adrenal glands of guinea-pig.     

Substance P modulates the time course of nicotinic but not muscarinic catecholamine secretion from perfused adrenal glands of rat.

1. Substance P (SP) and acetylcholine (ACh) are contained within the splanchnic nerve terminals in the adrenal gland and can be released in response to stress. In the rat, the release of aCh brings about secretion of catecholamines (CA) by acting on nicotinic and muscarinic receptors on the adrenal chromaffin cells. 2. In the present study, we have used a rat isolated adrenal gland preparation to investigate the effects of SP, perfused at different concentrations, on CA secretion evoked by 10(-5) M nicotine and 10(-4) M muscarine. 3. In the first 10 min stimulation period (S1), in the absence of SP, nicotine (10(-5) M) evoked substantial and equal secretion of noradrenaline (NA) and adrenaline (Ad). In a second 10 min stimulation period (S2), carried out 18 min after S1, the nicotinic response was desensitized. In contrast, the muscarinic response, which preferentially evoked Ad secretion in S1 (Ad/NA: 8.7/1), was well maintained in S2. 4. SP present in S1 had no effect on desensitization of the subsequent nicotinic response in S2. 5. At low concentrations (10(-7)-10(-10) M), SP changed the time course of nicotine-induced CA secretion during S1 by enhancing CA secretion in the first 4 min and inhibiting CA secretion thereafter. The maximal effect occurred at 10(-9) M SP. 6. At a higher concentration (10(-5) M), SP inhibited total nicotinic CA secretion throughout S1 and produced a biphasic secretion of CA (depressed in the presence of SP and enhanced after wash out of SP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory action of hydralazine on catecholamine secretion from cultured bovine adrenal chromaffin cells

Hydralazine caused a concentration-dependent inhibition of the secretion of catecholamines induced by carbamylcholine or high K+ from cultured bovine adrenal chromaffin cells, and also caused the significant inhibition of radioactive calcium uptake induced by carbamylcholine into the cells. However, hydralazine failed to inhibit the secretion of catecholamines evoked by the calcium-ionophore, A23187. The inhibitory action of hydralazine on catecholamine secretion induced by carbamylcholine was not affected by increasing the concentration of calcium ion in the reaction mixture. These observations therefore seem to indicate that the inhibitory action of hydralazine is not due to either the blocking of receptors for carbamylcholine or the disruption of the secretory machinery, and suggest that the drug may cause the inhibition of catecholamine secretion through its blocking action on calcium influx into the cells.     

Short term effect of steroids on catecholamine secretion from bovine adrenal medulla chromaffin cells

Bovine chromaffin cells were used to examine neuronal modulation, as their function is similar to sympathetic post-ganglionic neurons. The effect of steroids on evoked catecholamine secretion from primary culture of bovine adrenal medullary cells was investigated. A wide range of progestins, androgens and estrogens was found to have a significant effect on catecholamine secretion induced by the natural neurotransmitter acetylcholine (ACh). The androgens (especially androstandione and androsterone), as a class were the most effective in inhibition of stimulated secretion, while the estrogens had little, to no, effect. Among all steroids tested, progesterone had the most significant effect, other progestins were less potent. Progesterone inhibited catecholamine secretion evoked by ACh, nicotine and oxotremorine-M in a dose-dependent manner with similar IC₅₀ values in the μM range. It also blocked the secretion evoked by high potassium concentration (59 nM) or veratradine (100 μM), but no effect was seen on the secretion evoked by the calcium ionophore A-23187 (10 μM). Progesterone inhibition of ACh or oxotremorine-M stimulation was immediate and sustained. These results suggest that progesterone and other steroids might have a membrane effect probably acting through blockade of calcium influx necessary for the secretory response.     

Voltage-independent catecholamine release mediated by the activation of muscarinic receptors in guinea-pig adrenal glands.

1. The differences between the mechanisms of muscarinic and nicotinic receptor-mediated catecholamine secretion with respect to their dependence on voltage changes and extracellular Ca were examined using perfused adrenal glands of the guinea-pig. 2. Acetylcholine (ACh, 10(-6) to 10(-3) M) caused a dose-dependent increase in catecholamine secretion. The ED50 value for ACh was 7 x 10(-5) M. In the presence of atropine (10(-5) M), the dose-response curve for ACh was shifted to the right. Hexamethonium (5 x 10(-4) M) preferentially reduced the responses to higher concentrations of ACh (greater than 10(-5) M). Pilocarpine (5 x 10(-4) M) and nicotine (3 x 10(-5) M) also stimulated catecholamine release. 3. During perfusion with isotonic KCl solution, ACh and pilocarpine, but not nicotine, evoked catecholamine secretion. These responses were abolished by atropine (10(-6) M). Pilocarpine-stimulated catecholamine secretion was enhanced during perfusion with isotonic KCl solution. Under these conditions, hexamethonium (10(-3) M) significantly augmented ACh-evoked catecholamine release. 4. During perfusion with either Ca-free isotonic KCl or Ca-free Locke solution, ACh and pilocarpine caused a partial increase in catecholamine secretion whereas nicotine and high K solution (56 mM) did not. The responses to ACh and pilocarpine were completely inhibited by atropine but not by hexamethonium. 5. When guinea-pig adrenal glands were perfused with isotonic KCl solution containing 2.2 mM Ca which was subsequently removed and replaced with EGTA, ACh-induced catecholamine secretion was similar in magnitude to that observed during perfusion with Locke solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Influence of TMB-8 on secretion of catecholamines from the perfused rat adrenal glands

An attempt was made to investigate the effect of TMB-8 [3,4,5-trimethoxybenzoate-8 (N,N-diethylamino) octyl ester], which is known to be an inhibitor of intracellular Ca²⁺ release, on catecholamines (CA) secretion evoked by Ach, excess K⁺, DMPP, McN-A-343 and caffeine from the isolated perfused rat adrenal glands and to clearify its mechanism of action. The pretreatment with a low dose of TMB-8 (10 μM) for 20 min led to marked inhibition in CA secretion evoked by Ach (5.32 mM), excess K⁺ (56 mM), DMPP (100 μM), McN-A-343 (100 μM) and BAY-K 8644 (10^?5M). Caffeine-induced CA secretion was similar to that of control only during the first periods (0–3 min) but thereafter marked inhibition in CA secretion evoked by caffeine was observed during the rest periods up to 30 min. The increased moderate concentration of TMB-8 (30 μM) caused the result similar to that of 10 μM TMB-8. However, in adrenal glands preloaded with a high dose of TMB-8 (100 μM), CA releases evoked by Ach, excess K⁺, DMPP, McN-A-343 and caffeine were almost completely blocked by the drug. These experimental data demonstrate that TMB-8 may inhibit cholinergic receptor-mediated and also depolarization-dependent CA secretion, suggesting that these TMB-8 effects seem to be mediated through inhibiting influx of extracellular calcium into the rat adrenal medullary chromaffin cells as well as reducing the release of calcium from intracellular sources.     

Studies on secretion of catecholamine evoked by caffeine from the isolated perfused rat adrenal gland

The influence of caffeine on secretion of catecholamines (CA) was examined in the isolated perfused rat adrenal gland. Caffeine (0.3 mM) perfused into an adrenal vein of the gland produced a marked increase in secretion of CA. This secretory effect of CA evoked by perfusion of caffeine for one minute was considerably prolonged, lasting for more than 90 minutes. The tachyphylaxis to releasing effect of CA induced by caffeine was observed by repeated perfusion of this drug. The caffeine-evoked CA secretion was markedly inhibited by pretreatment with ouabain, trifluoperazine, TMB-8 and perfusion with calcium-free Krebs solution containing 5 mM EGTA, but was not affected by perfusion of calcium-free Krebs solution without other addition. CA secretion evoked by caffeine was not reduced significantly by pretreatment with chlorisondamine but after the first collection of perfusate for 3 min was clearly inhibited. Interestingly, the caffeine-evoked CA secretion was considerably potentiated by pretreatment with atropine or pirenzepine, but after the first collection for 3 min it was markedly decreased. These experimental results suggest that caffeine causes a marked increase in secretion of CA from the isolated perfused rat adrenal gland by an extracellular calcium-independent exocytotic mechanism. The secretory effect of caffeine may be mainly due to mobilization of calcium from an intracellular calcium pool in the rat chromaffin cells and partly due to stimulation of both muscarinic and nicotinic receptors.     

Effect of picrotoxin on adrenal catecholamine secretion

The effect of picrotoxin (PT) on catecholamine (CA) secretion was investigated in perfused bovine adrenal glands. A low dose of PT (3 microM) enhanced the CA secretion evoked by a 15-min exposure to 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic agonist; 0.1 mM), but a higher dose (0.3 mM) of PT inhibited the DMPP-evoked CA secretion. The rate of decline of secretory response to the prolonged DMPP stimulation was also accelerated by a higher dose (0.1 mM) of PT. In the dose-response curves for DMPP-evoked CA secretion, the inhibitory action of PT (0.3-1 mM) was more prominent at high doses than at low doses of DMPP. The inhibition pattern was similar to the pattern of a barbiturates blockade. In separate experiments, PT (0.1 mM) augmented calcium (10 mM)- and high potassium (56 mM)-evoked secretory responses. Spontaneous CA secretion was unaffected by PT at the concentrations indicated above. These results indicate that a low dose of PT potentiates, but higher doses inhibit, the adrenal CA secretion by a nicotinic agonist and that the inhibitory effect of PT resembles that of barbiturates.