Antonio G. García

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: the recording of basal levels and fluctuations of catecholamine release with good reliability; 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; 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; 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.

Relative sensitivities of chromaffin cell calcium channels to organic and inorganic calcium antagonists

K-evoked 45Ca uptake into, and catecholamine release from cat adrenomedullary tissues were potently inhibited by the dihydropyridine (DHP) Ca antagonist (+)-PN 200-110 (IC50 = 0.8 nM). Verapamil and diltiazem were 2000-fold less potent and flunarizine behaved as the least potent blocker (IC50 = 2980 nM); other DHP had a full range of potencies between (+)-PN 200-110 and verapamil. The order of potencies for inorganic antagonists was Zn greater than Cd greater than La greater than Ni greater than Co greater than Mn greater than Mg. Since a great controversy exists on the sensitivity of Ca channels to various antagonists, this comprehensive study will facilitate the selection of appropriate Ca antagonists to answer fundamental questions concerning chromaffin and neural Ca channels.

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 60 s) increased total catecholamine secretion from perfused cat adrenal glands; this respnse 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 μm) equally inhibited [3H]‐noradrenaline release evoked by nicotine or acetylcholine from cultured bovine adrenal chromaffin cells; clonidine (10 μm) 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 α‐or β‐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.

Effects of Bay K 8644 on cat adrenal catecholamine secretory responses to A23187 or ouabain

1 Calcium ionophore A23187 increases the rate of spontaneous catecholamine release from cat adrenal glands perfused at 37°C with oxygenated Krebs bicarbonate solution, in a time‐ and Caconcentration‐dependent manner. The secretory profile obtained with the ionophore was not modified in the presence of the Ca channel activator Bay K 8644. 2 Ouabain also enhanced the rate of spontaneous catecholamine outputs in a time‐ and concentration‐dependent manner. The threshold ouabain concentration capable of producing a clear, yet delayed secretory response was 10−6 m. Increasing ouabain concentrations up to 10−6 m enhanced catecholamine release and shortened the time to peak release. 3 The dihydropyridine Ca channel activator Bay K 8644 (10−6 m) markedly potentiated the secretory effects of all ouabain concentrations used (10−7‐10−4 m). However, the most impressive potentiations were seen at 10−5 m ouabain; while at this concentration ouabain alone released 2.6 ±0.07 μg catecholamines per 30 min, in the presence of Bay K 8644 the release was 73.4 ± 5.7 μg per 30 min. Conversely, at a fixed ouabain concentration (10−5 m), the potentiation was also dependent on the Bay K 8644 concentration (10−8‐10−5 m). 4 Although K deprivation inhibits Na pumping as does ouabain, Bay K 8644 did not modify the rate of catecholamine release evoked by K removal from the perfusion medium. 5 Potassium deletion, nimodipine or high Mg all reversed the fully developed secretory response evoked by ouabain plus Bay K 8644. 6 In glands depolarized by continuous perfusion with high K solutions, once the secretory response was inactivated, the introduction of ouabain caused an enhancement of the catecholamine secretory rate. This increase was dependent on the extracellular Na concentration and was not affected by Bay K 8644. In the presence of 6 mm Na the secretory effects of Bay K 8644 plus ouabain were abolished. 7 These results are compatible with the following conclusions: (i) Bay K 8644 potentiates only those catecholamine secretory responses that are known to be mediated through the activation of voltage‐sensitive Ca channels; the drug does not seem to affect secretory responses by acting on the membrane Na/Ca exchange system or at some intracellular Ca‐dependent component of the secretory machinery of Ca buffering systems. (ii) It is likely that ouabain enhances the rates of adrenal catecholamine release by a dual mechanism: chromaffin cell depolarization and activation of a membrane Na/Ca exchange system.

Dihydropyridine chirality at the chromaffin cell calcium channel

The racemic mixture of the dihydropyridine PN200-110 (Sandoz) inhibits K+-evoked catecholamine release from cat adrenal glands with an IC50 of 4.1 nM; IC50s for (+)- and (-)-PN200-110 were 0.84 and 45.8 nM, respectively. While the (+)-enantiomer of the dihydropyridine Sandoz 202-791 potentiated secretion (EC50 = 100 nM), the (-)-enantiomer behaved as a potent inhibitor (IC50 = 10 nM). Since K+-evoked 45Ca-uptake was also potently inhibited by (+)-PN200-110, it seems that the chromaffin cell dihydropyridine receptor is associated to the voltage-dependent Ca-channel and that it exhibits an exquisite stereoselectivity.

Inactivation of potassium-evoked adrenomedullary catecholamine release in the presence of calcium, strontium or BAY-K-8644

The rate of catecholamine release from cat adrenal glands perfused with Krebs solution containing 59 mM K declined exponentially during the first few minutes of depolarization. The rate of decline was considerably slower when Ca was substituted by Sr. The late addition of Ca, Sr or the Ca‐channel activator BAY‐K‐8644 evoked a revival of secretion when catecholamine release was inactivated by prior K depolarization; the revival of secretion was independent of the depolarization time. These data demonstrate that inactivation of catecholamine release is specifically dependent on Ca; the modulatory role of Ca on secretion seems to be exerted at a step distal to the transmembraneous Ca channel.

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

1 Cat splenic slices prelabelled with [3H]‐noradrenaline were incubated in oxygenated Krebs‐bicarbonate solution at 37°C, and the spontaneous total 3H release into different incubation media monitored. In normal Krebs bicarbonate solution, the spontaneous tritium fractional release amounted to 3.7% of the tissue radiactivity content per 5 min collection period. 2 Tetraethylammonium (TEA) increased spontaneous transmitter release in a concentration‐dependent manner; the release was maximal at 30 mm and was 3.5 times the basal release. 3 4‐Aminopyridine (4‐AP) also enhanced the spontaneous release of tritium. The response increased linearly with 4‐AP concentration (1–10 mm). With 10 mm 4‐AP, the release was as much as 6 times the basal transmitter release. Guanidine was much less potent than either TEA or 4‐AP. 4 The secretory response to TEA or 4‐AP was little affected by changes in external Ca2+, Mg2+, Na+, Cl−, H2PO4− or by tetrodotoxin. 5 However, transmitter release evoked by TEA or 4‐AP strongly depended upon the concentration of HCO3− of the incubation solution; in fact, the secretory response varied almost linearly between 1 and 25 mm HCO3−. 6 The mechanisms underlying these effects are probably related to the well‐known ability of TEA and 4‐AP to block K+ conductance that would cause depolarization of the splenic sympathetic nerve terminals. The HCO3− requirements for the secretory response are probably related to the ability of CO2/HCO3− solutions to mobilize and release Ca2+ from intracellular organelles.

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

1 Subcellular distribution studies of calmodulin in cat sympathetic ganglia demonstrated that about 90% of the protein remained in the 27,000 g supernatant, suggesting that it is a cytosolic protein. Only 4.5% was recovered in the microsomal fraction pellet. 2 The inferior mesenteric ganglia contained 93.3 ± 3 ng calmodulin per ganglion, and segments of unligated cat hypogastric nerves had 6.53 ± 0.32 ng per 5 mm segment. 3 When the nerve was ligated in the middle and left in the cat for 1–6 days, substantial amounts of calmodulin accumulated in segments of nerve immediately proximal (P1) and distal (D1) to the ligature. The amounts found in P1 amounted to 15.3, 20, 30.4 and 39.4 ng calmodulin per 5 mm segment 1, 2, 3 and 6 days after ligation, respectively. The average rate of transport was 5.5 mm per day, which corresponds to a slow component b of axonal transport (SCb). 4 The accumulation of calmodulin in D1 was also increased with the time of ligation. After 1, 2, 3 and 6 days, the amounts of the protein found in D1 were 14.4,17.7,19 and 21 ng per 5 mm segment, respectively. The calculated mean rate for the retrograde transport was 3.9 mm per day. 5 Decentralization of the inferior mesenteric ganglia did not affect the rate of accumulation of calmodulin or the basal amounts found in ganglia and nerves. Local injection inhibited the orthograde, but not the retrograde axonal transport of the protein. 6 It is concluded that calmodulin undergoes a process of slow orthograde axonal transport probably incorporated into the axoplasmic matrix of a network of actin microfilaments. The protein is also transported in a retrograde manner.

Pharmacological Properties of the Chromaffin Cell Calcium Channel

In 1961, Douglas and Rubin21 concluded that “the role of acetylcholine as a transmitter at the adrenal medulla is to cause some brief change in medullary cells which allows extracellular Ca2+ to penetrate them and trigger the catecholamine ejection process.” The formation accumulated since then can be summarized in the diagram of the sequence of events taking place during the secretory cycle depicted in Fig. 1.