Perfused Cat Adrenal Glands Research Articles

Contribution of SK and BK channels in the control of catecholamine release by electrical stimulation of the cat adrenal gland.

1. Transmural electrical stimulation (10 Hz, 1 ms, 40 V for 10 s) of cat adrenal glands perfused at room temperature with Krebs-Hepes solution produced catecholamine secretory responses which were reproducible when stimulations were applied at 5 min intervals. Such responses were inhibited about 20% by atropine (1 microM) and 80% by hexamethonium (30 microM). Apamin (100 nM) increased the secretory response 2.5-fold in the presence of atropine and 8-fold in the presence of hexamethonium. 2. Potentiation by apamin of secretory responses evoked by 100-pulse trains was similar at 5, 10 and 20 Hz (about 2-fold). When glands were continuously stimulated at 3 Hz, apamin increased 4-fold the initial secretion plateau. Continuous stimulation at a higher frequency (20 Hz) produced a sharp secretory peak followed by a small, sustained plateau; apamin did not alter this plateau. Apamin also enhanced the secretory responses obtained with sustained stimulation with acetylcholine (10 or 200 microM). 3. Secretion peaks induced by brief acetylcholine pulses (10 microM for 10 s) applied to isolated and superfused cat adrenal chromaffin cells were enhanced more than 3-fold by 100 nM apamin. Charybdotoxin (10 nM) did not enhance these secretory peaks. 4. In perfused cat adrenal glands, charybdotoxin (10 nM) affected neither the secretion evoked by trains of electrical stimulation applied at different frequencies nor the secretion evoked by acetylcholine pulses. 5. In 0.5 mM [Ca2+]o, apamin enhanced 3-fold the secretion evoked by electrical stimulation trains of 100 pulses (10 Hz, 10 s) and almost 6-fold the acetylcholine (10 microM for 10 s)-induced secretion. In 5 mM Ca2+, apamin enhanced the secretory responses to electrical stimulation and acetylcholine 2- and 10-fold, respectively. Charybdotoxin enhanced 2.5-fold the secretory response to electrical stimulation in 0.5 mM Ca2+, although this effect was not statistically significant. A synergistic interaction between the two toxins on catecholamine release induced by electrical stimulation was observed at low but not at high [Ca2+]o. 6. Simultaneous release of acetylcholine and catecholamines upon electrical stimulation was achieved in glands in which the endogenous acetylcholine stores in the splanchnic nerve terminals had been prelabelled by perfusion with [3H]choline. While apamin enhanced more than 2-fold the postsynaptic release of catecholamines, the presynaptic release of acetylcholine remained unaffected. 7. The results are compatible with the hypothesis that, under physiological conditions, Ca(2+)-activated SK channels present in chromaffin cells control the firing patterns of action potentials induced by the acetylcholine released from splanchnic nerves during stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of -70 mV, induced whole-cell currents through Ca2+ channels (ICa) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 microM) reduced the peak current by 47% and the late current by 80%. omega-Conotoxin GVIA (CgTx, 1 microM) reduced the peak ICa by 42% and the late ICa by 55%. Pulses (10 s duration) with 70 mM K+/2.5 mM Ca2+ solution (70 K+/2.5 Ca2+), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca2+ concentration ([Ca2+]i) from 0.1 to 2.21 microM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K+/2.5 Ca2+ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca2+]o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca2+ channels in cat chromaffin cells. It seems, however, that though extracellular Ca2+ entry through both channel types leads to similar increments of averaged [Ca2+]i, the control of catecholamine release is dominated only by Ca2+ entering through L-type Ca2+ channels. This supports the idea of a preferential segregation of L-type Ca2+ channels to localized "hot spots" in the plasmalemma of chromaffin cells where exocytosis occurs.

Modulation by L-type Ca2+ channels and apamin-sensitive K+ channels of muscarinic responses in cat chromaffin cells

In the perfused cat adrenal gland stimulated with the muscarinic agonist methacholine chloride (100 microM for 3 min), two components were detected in the catecholamine secretory response: 1) an early phasic component that peaked at 300 ng/5 s catecholamine release and 2) a tonic component whose peak was transient and declined to a plateau of about 140 ng/5 s. Apamin (0.1 microM) increased the phasic component to 1,200 ng/5 s and the tonic component to approximately 350 ng/5 s. In single fura 2-loaded cat adrenal chromaffin cells, the cytosolic Ca2+ concentration ([Ca2+]i) also followed a biphasic pattern after stimulation with methacholine. Depletion of extracellular Ca2+ reduced the phasic [Ca2+]i peak by > 50% and the phasic secretory peak by approximately 90%; both the tonic components of [Ca2+]i and secretion were abolished. Depletion of intracellular Ca2+ pools decreased the phasic and tonic components of [Ca2+]i and secretion with respect to control values; however, the phasic components diminished more than the tonic components of [Ca2+]i and secretion. Although 3 microM furnidipine (a dihydropyridine L-type Ca2+ channel blocker) inhibited the phasic component of [Ca2+]i and secretion, its effects were more pronounced on the tonic component. omega-Conotoxin GVIA (1 microM, an N-type Ca2+ channel blocker) did not affect the [Ca2+]i or the methacholine secretory responses. The secretion peak seems to depend on both extracellular free Ca2+ (Cao2+) entry through L-type Ca2+ channels as well as on the mobilization of Ca2+ from intracellular stores; the plateau depends only on Cao2+ entry through L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Two components in the adrenal nicotinic secretory response revealed by cobalt ramps

Prolonged stimulation with nicotine (50 μM) enhanced the secretion of catecholamines from perfused cat adrenal glands. The profile of secretion consisted of a quick activation phase to peak of 7.68 μg/min followed by a second inactivation phase which exhibited a t 1 2 of 3.75 min. Sustained stimulation with a solution enriched in K + (59 mM) also evoked a transient secretory response, with a peak release of 8.62 μg/2 min and a t 1 2 for inactivation of 4.8 min. Co 2+ (10 mM) blocked the nicotinic response by 58% and the K +-evoked secretory response by over 96%. In the presence of Co 2+; (5 mM), continuous perfusion with nicotine produced a transient but large initial secretory response; the gradual decrease of the extracellular Co 2+ concentration, [Co 2+] 0, as a continuous ramp allowed the development of a second component of secretion which inactivated later on. When the glands were continuously stimulated with 59 mM K + in the presence of Co 2+, the first component of secretion was missing: the second component appeared as [Co 2+] 0 decreases as a ramp. In similar experiments performed in low-Na + solution (10 mM Na +), only the first secretion component evoked by nicotine was observed. This finding suggests that the second component of secretion depends on Na + entry through the nicotinic receptor, on the ensuing cell depolarization and on Ca 2+ entry through voltage-dependent Ca 2+ channels. These data are compatible with the idea that the nicotinic receptor-mediated catecholamine secretory response in the cat adrenal chromaffin cell has two components: the first seem to be triggered by Ca 2+ entry through the nicotinic receptor channels, and the second by Ca 2+ entry through voltage-dependent Ca 2+ channels. The data also suggest that Co 2+ ions protect against the Ca 2+-dependent desensitization of nicotinic receptors.

Electrically-evoked catecholamine release from cat adrenals: Role of cholinergic receptors

Catecholamine (CA) release from perfused cat adrenal glands was continuously monitored using an on-line system coupled to an electrochemical detector. This highly sensitive procedure allowed the detection of small changes in the rate of secretion, even using short trains of electrical stimulation or brief acetylcholine (ACh) pulses. CA release was linear with increasing strength of ACh, transmural or splanchnic nerve stimulation. By using specific blockers, the contribution of nicotinic or muscarinic receptors to the overall secretory response to various stimuli could be established. That nicotinic receptors play a major role in mediating the secretory response to all stimuli is shown by the clear inhibition of the response with mecamylamine (10 μM). In contrast, atropine (1 μM) halved secretion evoked by ACh or nerve stimulation but had little effect on the response to trains of transmural electrical stimulation. When transmural electrical stimulation was applied continuously (instead of in trains), increasing the frequency in a step-wise manner, a bell-shaped curve was obtained; secretion reached a peak at 8 Hz and then declined sharply at 16 and 32 Hz. With this stimulation pattern, atropine decreased by 50% the secretion response at the higher frequencies (4–32 Hz). Very few studies are available which define the role of receptors and ionic channels in mediating electrically-evoked CA release. These stimulation patterns have not been used previously and are likely to mimic more closely than those used in earlier studies the physiological firing pattern of splanchnic nerves innnervating adrenomedullary cells.

Alamethicin-evoked catecholamine release from cat adrenal glands

Alamethicin enhances the rate of catecholamine output from perfused cat adrenal glands in a concentration-dependent manner. At 37 ° C, catecholamine released went from 4.29±0.25 to 20.51±0.63 ug/stimulus at ionophore concentrations ranging from 20 to 100 ug/ml. Secretion was abolished at 22 ° C or in the absence of extracellular Ca. The time-course of secretion (quick activation followed by a decline) evoked by alamethicin considerably differs from the catecholamine release pattern seen with A23187, X537A or ionomycin, which evoke a slowly developing, non-inactivating secretory response. In fact, its transient secretion pattern resembles that of nicotinic or high-K stimulation of cat adrenal glands, thus suggesting that alamethicin might form Ca permeable artificial channels in chromaffin cell plasma membranes.

Secretory and radioligand binding studies on muscarinic receptors in bovine and feline chromaffin cells.

1. Muscarinic agonists enhanced catecholamine release from perfused cat adrenal glands with the following relative order of potencies: methacholine greater than oxotremorine greater than McN-A-343 greater than pilocarpine greater than bethanechol greater than muscarine. Because a continuous online electrochemical detection system was used to monitor catecholamine release, this sequence could be obtained at concentrations much lower (1-10 microM) and during much shorter stimulation times (3-30 s) than in previous reports. 2. All muscarinic agonists used secreted adrenaline preferentially over noradrenaline. Methacholine evoked a sustained, non-desensitizing response in the cat adrenal, which declined to basal levels of secretion immediately after Ca2+ removal: upon Ca2+ restoration secretion was restored to the previous plateau. 3. In addition to evoking a direct secretory response, low concentrations of methacholine, pilocarpine, bethanechol or muscarine clearly potentiated cat adrenal secretory responses evoked by pulses of nicotine (2 microM for 30 s) or high K+ (17.7 mM for 30 s). 4. [3H]Quinuclydinyl benzylate (QNB) specifically bound to cat adrenomedullary membranes with a saturating monophasic curve, suggesting a single binding site with a KD of 23 pM and a Bmax of 67 fmol (mg protein)-1. Preferential displacement by atropine over pirenzepine suggests that the binding site is associated to a M2-type muscarinoceptor. 5. Methacholine (3-300 microM) did not enhance the spontaneous catecholamine release from perfused bovine intact adrenal glands or superfused chromaffin cells. Neither did the drug affect secretion evoked by dimethylphenylpiperazinium (10 microM for 3 s) or K+ (35 mM for 3 s) from isolated superfused bovine adrenal chromaffin cells. 6. [3H]QNB bound to purified bovine adrenomedullary plasma membranes with a KD of 29 pM and a Bmax of 89 fmol (mg protein)-1. Displacement by pirenzepine suggests the presence of two binding sites (Hill coefficient = 0.64) with Ki1 of 39 nM and Ki2 of 2734 nM. 7. Because the ionophore A23187 enhanced K(+)-evoked secretion in both, bovine and cat adrenals, it seems that a similar cytosolic Ca2+ rise induced by muscarinic stimulation might constitute the underlying mechanism both to cause a secretory response per se as well as the potentiation of catecholamine release evoked by nicotinic or high K+ stimulation. However, it is unclear why the bovine behaves differently from the feline chromaffin cell as far as the muscarine-evoked effects are concerned.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

O-137 - Intracellular Na+ modulates catecholamine secretion induced by muscarinic receptor activation from perfused cat adrenal glands.

O-137 - Intracellular Na+ modulates catecholamine secretion induced by muscarinic receptor activation from perfused cat adrenal glands.

Sodium-dependent inhibition by PN200-110 enantiomers of nicotinic adrenal catecholamine release.

1. Dimethylphenylpiperazinium (DMPP) or high K concentrations evoke catecholamine release from perfused cat adrenal glands; in both cases the secretory response was significantly enhanced in the absence of Na. Tetrodotoxin did not modify the nicotinic secretory response. 2. The (+)- and (-)-enantiomers of the dihydropyridine Ca channel blocker PN200-110 show a high degree of stereoselectivity in the inhibition of catecholamine secretion evoked by high K or by DMPP in the presence of Na, the (+)-enantiomer being 57 and 80 times more potent, respectively, than the (-)-enantiomer. Both, noradrenaline and adrenaline release were equally depressed by PN200-110. 3. The IC50 values for (+)- and (-)-PN200-110 for blockade of the secretory response induced by K or DMPP in the presence of Na are in the same range. In the absence of Na, (-)-PN200-110 did not affect DMPP-evoked secretion; however, the (+)-enantiomer partially inhibited it. 4. The results suggest that the physiological catecholamine release from chromaffin cells is preceded by Na entry through the nicotinic receptor-associated ionophore; this causes cell depolarization, opening of voltage-dependent, dihydropyridine-sensitive Ca channels and Ca entry into the cell. In the absence of Na, additional Ca influx through an alternative pathway (the nicotinic cholinoceptor ionophore?) might also activate secretion.

Intracellular Ca 2+ antagonist TMB-8 blocks catecholamine secretion evoked by caffeine and acetylcholine from perfused cat adrenal glands in the absence of extracellular Ca 2+

Unlike acetylcholine, caffeine was much more effective in releasing catecholamine in the absence of extracellular Ca 2+ than in its presence in perfused cat adrenal glands. The intracellular Ca 2+ antagonist, TMB-8 (10 −4 M), inhibited reversibly the catecholamine secretion evoked by caffeine (40 mM) and that induced by acetylcholine (10 −4 M) in the presence of hexamethonium (10 −3 M) during perfusion with Ca 2+-free Locke solution containing EGTA (10 −5 M). These results support our view that muscarinic receptor activation causes catecholamine secretion by mobilizing Ca 2+ from an intracellular pool just as caffeine does.

Secretion from adrenaline- and noradrenaline-storing adrenomedullary cells is regulated by a common dihydropyridine-sensitive calcium channel

Dihydropiridines (+)-PN200-110 and (±)-Bay-K-8644 inhibit or potentiate, respectively, catecholamine release evoked by DMPP- or K-stimulation of perfused cat adrenal glands. Since both, secretion of adrenaline and noradrenaline are equally affected, and these two drugs specifically act on voltage-dependent chromaffin Ca channels, it seems that secretion of each amine from their respective cell is regulated by the same type of channel.

Voltage-dependence of nitrendipine provides direct evidence for dihydropyridine receptor coupling to calcium channels in intact cat adrenals

Nitrendipine behaves as a powerful inhibitor of catecholamine release in perfused cat adrenal glands stimulated with pulses of Ca (0.5 mM) in high K (118 mM). Its blocking ability is considerably enhanced if, before Ca stimulation, the adrenomedullary tissue is impregnated with nitrendipine in depolarizing (IC50 = 0.99 nM) as opposed to hyperpolarizing (IC50 = 214 nM) conditions. 3H-nitrendipine binding to adrenomedullary tissue was considerably enhanced in depolarization; blockade of 45Ca uptake into and catecholamine secretion from the same glands was also enhanced in depolarized tissues. This study shows for the first time the a good correlation between these three parameters in the same intact preparation, providing direct evidence for a functional coupling between dihydropyridine receptors, Ca channels and secretion.

Differential release of enkephalin and enkephalin-containing peptides from perfused cat adrenal glands.

We have compared the enkephalin-like material derived from proenkephalin released from perfused cat adrenal glands stimulated with pilocarpine (5 X 10(-4)M) and nicotine (5 X 10(-6) M). In addition, two doses of acetylcholine (10(-5) and 10(-4) M) and 50 mM K+ were tested. Free Met-enkephalin immunoreactivity and total Met-enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin-containing fragments, were coreleased with catecholamines. Free Met-enkephalin immunoreactivity represented 13% of total immunoreactivity for nicotinic stimulation, 46% for pilocarpine, 33% for 10(-5) M acetylcholine, 22% for 10(-4) M acetylcholine, and 16% for 50 mM K+. Analysis of the perfusate by gel filtration showed that 80% of the total Met-enkephalin immunoreactivity whose release was induced by pilocarpine was eluted in fractions corresponding to fragments of low molecular weight, whereas these fractions accounted only for 10% of the total Met-enkephalin immunoreactivity whose release was induced by nicotine. HPLC analysis of low-molecular-weight peptide fractions revealed that Met-enkephalin, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-Arg-Phe represented 69% of total Met-enkephalin immunoreactivity whose release was induced by pilocarpine. These results indicate that selective activation of muscarinic receptors is followed by release of low-molecular-weight material, whereas nicotine application also yielded high-molecular-weight peptides. Furthermore, increasing the acetylcholine concentration from 10(-5) to 10(-4) M and using 50 mM K+ increased proportionally the high-molecular-weight peptide secretion. Results are discussed in relation to the existence of a heterogeneous population of granules either in the same cell or in different cells, containing proenkephalin-derived peptides. (ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of adrenomedullary catecholamine release by propranolol isomers and clonidine involving mechanisms unrelated to adrenoceptors.

1 Transmural electrical stimulation (10 Hz, 40 V, 1 ms for 60s) increased total catecholamine secretion from perfused cat adrenal glands; this response was enhanced by neostigmine and inhibited by mecamylamine, suggesting that release of acetylcholine from splanchnic nerve terminals was stimulating nicotinic receptors and enhancing catecholamine secretion. 2 Isoprenaline, (-)-propranolol and (+)-propranolol (10(-7)-10(-5)M) inhibited the electrically-evoked secretory response by 40-70%; similar reductions were obtained with clonidine and yohimbine. Neither, (+)-propranolol nor (-)-propranolol inhibited K-evoked secretion from cat adrenals; in contrast, nimodipine potently inhibited it (IC50 = 24 nM). 3 Either, racemic propranolol or the (+)- or (-)-isomers (1-10 microM) equally inhibited [3H]-noradrenaline release evoked by nicotine or acetylcholine from cultured bovine adrenal chromaffin cells; clonidine (10 microM) inhibited secretion by 50% and yohimbine or isoprenaline did not affect it. 4 The results indicate that adrenomedullary catecholamine release evoked by splanchnic nerve stimulation is not modulated by alpha- or beta-adrenoceptors and suggest that propranolol may inhibit secretion by blocking ion fluxes through the acetylcholine receptor ionophore. Clonidine may inhibit secretion by this same mechanism, and/or by interfering with some intracellular event in the secretory mechanism.

Continuous monitoring of catecholamine release from perfused cat adrenals

Catecholamine release from perfused cat adrenal glands has been continuously monitored by on-line connection of the perfusion fluid emanating from the gland to an electrochemical detector. This method allowed: (1) the recording of basal levels and fluctuations of catecholamine release with good reliability; (2) the study of the release of catecholamines in response to even submicromolar concentrations of nicotine or acetylcholine and 3 mM increments of the K + concentration, perfused in pulses of a few seconds, and to determine the effects of drugs and ionic manipulations on such secretory responses; (3) the analysis of the ‘physiological’ secretory response evoked by electrical stimulation of the splanchnic nerves, allowing the study of presynaptic as well as postsynaptic components of the effects of drugs and ions on chemical neurotransmission at the splanchnic-chromaffin cell synapse; (4) the continuous monitoring of the kinetics of the secretory process during sustained depolarization with several secretagogues and its correlation with the kinetics of activation and inactivation of potential sensitive Ca channels. In addition, this method avoids the time-consuming procedures of collecting samples and assaying them individually through tedious fluorimetric or radioenzymatic techniques.

A dopaminergic receptor modulates catecholamine release from the cat adrenal gland.

Nicotine evokes the release of catecholamines from perfused cat adrenal glands in a concentration-dependent manner, the median effective concentration for nicotine being 5 microM. Two 2 min pulses of 5 microM-nicotine, 40 min apart (S1 and S2) gave net catecholamine outputs of 7.64 and 3.55 micrograms/8 min, respectively. The ratio S2/S1 in control glands was 0.5. Increasing concentrations of apomorphine (1-10 microM) markedly inhibited catecholamine release during the second nicotine pulse (S2). At 1 microM-apomorphine, the release during S2 was significantly reduced to 16% of S1; with 10 microM-apomorphine, the secretory response was reduced further to only 3% of S1, the ratio S2/S1 being 0.03. The presence of haloperidol, sulpiride or picobenzide (each 0.5 microM) during S2, completely reversed the inhibition of catecholamine release produced by apomorphine. Haloperidol itself increased the nicotinic secretory response during S2; so, while the ratio S2/S1 was 0.5 in control conditions, this ratio increased significantly to 0.95 if haloperidol (0.5 microM) was present during S2, suggesting that the presence of this dopaminergic antagonist removed a negative feed-back mechanism that inhibits nicotine-evoked catecholamine release. If present during S2, dopamine (1 microM) also markedly inhibited catecholamine release evoked by nicotine; this inhibition was again reversed by 0.5 microM-haloperidol. Neither the opiate antagonist naloxone nor the alpha-adrenoceptor blocking agent phentolamine (at concentrations of 0.5-5 microM) affected the inhibition by apomorphine of the secretory response to nicotine. These data strongly suggest that the cat adrenal medulla chromaffin cell membrane contains a dopaminergic receptor which modulates the catecholamine secretory process triggered by stimulation of the nicotinic cholinoceptor. The fact that dopamine is released in measurable amounts, together with adrenaline and noradrenaline, from perfused cat adrenal glands in response to nicotinic stimulation (V. Ceña, unpublished results), favours a role for this dopaminergic receptor in modulating catecholamine release from the chromaffin cell.

Co-release of enkephalins and precursors with catecholamines from the perfused cat adrenal gland in situ.

We have compared the nature of the enkephalin-like material derived from proenkephalin present in the intact cat adrenal gland with the material co-released with catecholamines from the perfused adrenal in response to splanchnic nerve stimulation and to perfusions with solutions containing acetylcholine (ACh) or high potassium chloride (KCl). In cat adrenals most of the enkephalin-like material was in the form of large enkephalin-containing peptides. Free (met)enkephalin immunoreactivity represented only 25% of the total (met)enkephalin immunoreactivity as determined by enzymatic digestion of large enkephalin-containing fragments. Electrical stimulation (15 Hz) of the splanchnic nerve or perfusion of the gland with ACh (0.1 mM) or KCl (50 mM), applied for 10 min, induced an immediate release of free (met)enkephalin immunoreactivity, (met)enkephalyl-arg-phe immunoreactivity, and of large (met)enkephalin-containing peptides. The release by all three modes of stimulation followed a pattern that paralleled the output of catecholamines. A rapid fatigue of all secretory processes developed during the stimulation periods, similar to that observed for catecholamines. During splanchnic nerve stimulation, each nanomole of catecholamine output was accompanied by the output of 0.4 pmol free (met)enkephalin immunoreactivity, of 1.1 pmol total (met)enkephalin immunoreactivity and of 0.1 pmol (met)enkephalyl-arg-phe immunoreactivity. Analysis of the perfusate by high-pressure liquid chromatography revealed that (met)enkephalin, (met)enkephalyl-arg-phe and (met)enkephalyl-arg-gly-leu were released in molar ratios of 4 to 1 to 1 which is similar to the ratio found in the precursor, proenkephalin. The ratio of total (met)enkephalin immunoreactivity to free (met)enkephalin immunoreactivity in the perfusate was the same (approximately 2.7) during two successive periods of splanchnic nerve stimulation separated by 10 min. When release was evoked by increasing the K+ concentration to 50 mM-KCl, this ratio was increased more than twofold compared with that obtained by electrical stimulation of the splanchnic nerve. Analysis of the perfusate by gel filtration showed that, during splanchnic nerve stimulation, 47% of the total (met)enkephalin immunoreactivity eluted in fractions containing fragments of low molecular weight. When KCl was used as stimulus only 12% of total (met)enkephalin immunoreactivity eluted in these fractions. The results indicate that the nature of the released peptides depends on the type of stimulus used to evoke release.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of hypertonic solutions on catecholamine release from cat adrenal glands

The effects of hypertonic solutions on the spontaneous and evoked catecholamine release from isolated, perfused cat adrenal glands were studied. Hypertonic solutions enhanced three times the spontaneous release of the amines, but markedly inhibited the release evoked by nicotine 15μM for 2 min or high K +(17.7 mM for 2 min). Using sucrose as osmoticant, the secretory response to high K + was decreased to 53 and 15% of controls at 344 and 416 mosM, respectively At 512 mosM sucrose inhibited poorly the release of catecholamines evoked by nicotine, but reduced it by 90% at 1000 mosM; sodium chloride behaved similarly to sucrose. A rise in the osmolarity of only 7.5% with choline chloride produed a complete inhibition of the K +-evoked response. These effects were not seen using isotonic choline chloride; on the contrary, isotonic choline chloride enhanced the K + response, probably by stimulating nicotinic receptors. Since atropine did not modify this effect, it seems that secretion of catecholamines evoked by choline is mediated by nicotinic receptors. While the inhibitory effects of sucrose and NaCl were completely reversed when the tonicity of the perfusion medium was restored to its normal value (320 mosM), the effects of choline seemed to be long lasting and were reversed only partially. It is worth noting that the inhibitory effects of hyperosmotic solutions developed very fast. It is concluded that an increase in the tonicity of the medium bathing the chromaffin cell depresses profoundly the exocytotic catecholamine release response to nicotine and high K + these experiments suggest that the existence of an osmotic gradient across the plasma and or vesicular membranes might be a prerequisite for fusion of both membranes and or the subsequent extracellular discharge of chromaffin granule contents to the cell exterior.