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.     

Calmodulin antagonists inhibit dihydropyridine calcium channel activator (BAY-K-8644) induced cGMP synthesis in pituitary tumor cells

The dihydropyridine calcium channel activator, BAY-K-8644, stimulates cGMP formation in ACTH-secreting mouse AtT-20 clonal corticotrophs. The recent report that calmodulin antagonists could inhibit dihydropyridine binding in several tissues suggested that these agents might also affect the cyclic nucleotide response to BAY-K-8644. In fact, TMB-8, trifluoperazine, and melittin, described as in vitro antagonists of calmodulin-dependent enzyme activities, all inhibited BAY-K-8644 induced cGMP synthesis in a concentration-dependent manner. The antagonists had no effect on cGMP formation stimulated by sodium nitroprusside or sodium azide. The calcium channel antagonist, nifedipine, did not stimulate cGMP formation nor did it alter the effect of BAY-K-8644 on accumulation of the nucleotide; one explanation thus is that the cyclase involved in cGMP formation is coupled to a low affinity binding site for BAY-K-8644, which is less accessible to other dihydropyridines. The relation of cyclic GMP formation to the function of the calcium channel in AtT-20 cells remains unknown.     

Angiotensin II increases cytosolic calcium and stimulates catecholamine release in cultured bovine adrenomedullary cells

In bovine adrenomedullary cells in primary culture, angiotensin II (AII) elicited virtually immediate, dose-related increments in cytosolic calcium [( Ca++]i) measured by the Quin 2 technique and stimulated approximately proportional secretion of norepinephrine, epinephrine, and dopamine measured by liquid chromatography with electrochemical detection. Peak responses of [Ca++]i to AII were similar to peak responses to nicotine or KCl. Pre-treatment with verapamil or washing the cells in calcium-free medium attenuated the stimulatory effect of AII on [Ca++]i. Pre-treatment with nicotine, which temporarily inactivates cholinergic receptor-activated calcium channels, did not affect [Ca++]i responses to AII. The results indicate functional effects of AII on cultured chromaffin cells. The mechanism of cellular activation by AII appears to include increases in [Ca++]i due to opening of membrane calcium channels which may be unrelated to cholinergic receptor-operated calcium channels.     

Opposing actions of the enantiomers of BAY-K-8644 on calcium currents and ACTH secretion in clonal pituitary corticotrophs

BAY-K-8644 in low concentrations is known to stimulate, and in higher concentrations, to depress calcium-dependent ACTH secretion from mouse clonal (tumor) pituitary corticotrophs, AtT-20/D16-16 (AtT-20). In the present study, voltage-dependent inward calcium currents in these cells were potentiated by low concentrations of this compound and depressed by higher concentrations consistent with its actions on ACTH secretion. A similar relationship was demonstrated for a different but related compound, CGP 28,392. Each of BAY-K-8644's enantiomers, BAY-R(-)5417 and BAY-R(+)4407, had opposing effects upon these inward calcium currents and ACTH secretion. The (+)isomer antagonized both inward calcium currents and ACTH secretion. In contrast, the (-)enantiomer was responsible for the stimulatory effects of BAY-K-8644. Nevertheless, some antagonistic properties were noted with high concentrations of this latter enantiomer. The stimulation of ACTH secretion in AtT-20 cells by low concentrations of BAY-K-8644 can be attributed to a potentiation of voltage-activated calcium currents by one of its enantiomers, BAY-R-(-)5417. In contrast, the depression of secretion that occurs at higher concentrations is likely to be the result of the reduction of these currents by the other enantiomer (BAY-R(+)4407).     

Alteration by methadone of catecholamine uptake and release in isolated rat adrenomedullary storage vesicles

Incubation of isolated rat adrenomedullary storage vesicles with methadone produced inhibition of ³H-epinephrine uptake and promotion of release of endogenous catecholamines. Neither effect was seen using morphine, nor could morphine antagonize methadone-induced catecholamine release, suggesting that these actions are not mediated by opiate receptors. Inhibition of uptake by methadone appeared to contain a competitive component with a lower Ki for methadone compared to the Km for ³H-epinephrine. Despite competitive inhibition by methadone, the maximal uptake capacity (analogous to Vmax) as determined by double-reciprocal plots, was increased by the drug, probably as a result of greater availability of intravesicular storage sites because of the drug-induced of release endogenous catecholamines. Agents which enhance or block catecholamine transport into vehicles had no effect on the catecholamine release by methadone, indicating that the latter is separable from the action on uptake. These alterations of catecholamine uptake and release may play a role in the effects of methadone on the adrenal medulla $\text{in vivo}$.     

Benzodiazepine inhibition of the stimulation-evoked catecholamine release from bovine adrenomedullary cells in culture

Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 μ M) inhibited CA release evoked by acetylcholine (ACh), excess K⁺ and veratridine but not by A23187 or caffeine in Ca²⁺ -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-β-carboline-3-carboxylate (βCCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K⁺ was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked ⁴⁵Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca²⁺ influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed.     

Effects of calcium and bay K-8644 on calcium currents in adrenal medullary chromaffin cells

Summary The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca²⁺ currents, recorded in the presence of either 5.2 or 2.6mm Ca²⁺ exhibited a single, noninactivating component. To analyze the effects of Ca²⁺ and Bay K-8644 on the kinetics of the Ca²⁺ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca²⁺] (2.5mm) the midpoint of the steady-state Ca²⁺-channel activation curve lay at –6.9 mV. Increasing the [Ca²⁺] to 5.2mm shifted the midpoint by –4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2mm Ca²⁺) and 9.2 mV (for 2.5mm Ca²⁺) induced ane-fold change in the activation of the current. Increasing [Ca²⁺]₀ from 2.5 to 5.2mm induced a marked increase in the rate constant for turning on the Ca²⁺ permeability. Conductances were estimated from the slope of the linear part of the currentvoltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5mm Ca²⁺, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 m increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 m induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 m) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from –6.9 to –13 mV. At the midpoint potential of –13 mV, a change in potential of 6.9 mV caused ane-fold change in Ca²⁺ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca²⁺ permeability.     

Neuropeptide Y regulates catecholamine release evoked by interleukin-1beta in mouse chromaffin cells

Activation of the hypothalamic-pituitary-adrenal gland (HPA) axis can modulate the immune system. Cytokines and neuropeptide Y (NPY) are potent regulators of the HPA axis and are both produced by the adrenal medulla. The cytokine interleukin-1beta (IL-1beta) belongs to the interleukin-1 family along with interleukin-1alpha and the interleukin receptor antagonist (IL-1ra). The aim of the present study was to determine the interaction between NPY and IL-1beta in catecholamine (norepinephrine, NE and epinephrine, EP) release from mouse chromaffin cells in culture. We found that IL-1beta increased the constitutive release of NPY, NE and EP from mouse chromaffin cells. This IL-1beta stimulatory effect was blocked by IL-1ra. The immunoneutralization of NPY and the use of the NPY Y(1) receptor antagonist (BIBP 3226) inhibited the stimulatory effect of IL-1beta on catecholamine release from these cells. The present work shows that IL-1beta induces catecholamine release, and in turn this peptide will induce an additional increase in catecholamine release acting through the Y(1) receptor. This work suggests that NPY is involved in the regulatory loop between the immune and the adrenal system in some pathophysiological conditions where plasmatic IL-1beta increases, like in sepsis, rheumatoid arthritis, stress or hypertension.     

Effects of mastoparan on catecholamine release from chromaffin cells

Release of catecholamines from bovine adrenal chromaffin cells exposed to mastoparan, a wasp venom peptide which activates GTP-binding proteins and phospholipase A2, was evaluated. Release of catecholamines was dependent on mastoparan concentration and time of exposure. This release was, however, independent of extracellular calcium and accompanied by release of the cytoplasmic marker lactate dehydrogenase. Mastoparan also inhibited catecholamine secretion evoked by nicotine, but the peptide had little or no effect on release induced by other secretagogues. These findings suggest that in chromaffin cells mastoparan is not a secretagogue but rather causes cell lysis and blocks nicotinic receptor function.     

Calcium channel modulation by the dihydropyridine derivative Bay K 8644 in mammalian visceral smooth muscle cells

1. Modulation of Ca channels by the dihydropyridine Ca agonist Bay K 8644 in guinea-pig taenia coli smooth muscle cells was investigated using the patch clamp technique. 2. Single Ca channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 50 mM Ba. Bath application of the drug markedly increased the opening probability of Ca channels. 3. The effect was found to be due to an increase in the mean opening times of Ca channels. Due to this increase, the mean current reconstructed by averaging individual current trace responses was markedly increased in the presence of Bay K 8644.     

Exhaustion of calcium does not terminate evoked neurotransmitter release

Theories are considered which assume that termination of evoked release is caused by the exhaustion of intracellular Ca. It is shown that such theories predict, contrary to experiment, that total release is an unsaturated function of intracellular Ca whose duration depends strongly on extracellular Ca. These and other findings lead to the conclusion that termination must be due to the fast change of another parameter (not intracellular Ca).     

Modulation by beta-adrenoceptors and angiotensin II receptors of splanchnic nerve evoked catecholamine release from the adrenal medulla.

In the present study, we have evaluated the effect of both facilitatory beta 2-adrenoceptor and angiotensin II receptor on the release of adrenal catecholamines induced by electrical stimulation of the splanchnic nerve in anaesthetized and vagotomized dog. In these experiments, individual or combined treatments with the beta 2-adrenoceptor antagonist ICI 118551 (0.3 mg/kg i.v.), the converting enzyme inhibitor captopril (2 mg/kg i.v.), or the angiotensin II receptor antagonist saralasin (2 micrograms.kg-1.min-1 i.v.) were found to significantly decrease the release of adrenal catecholamines during splanchnic nerve stimulation (5-V pulses of 2 ms duration for 3 min at 1 Hz) whatever the order of administration of the drugs. On the other hand, the infusion of angiotensin II (20 ng.kg-1.min-1) was shown to potentiate the release of adrenal catecholamines in response to electrical stimulation, and this effect was totally blocked by treatment with saralasin (4 micrograms.kg-1.min-1 i.v.). This facilitating angiotensin mechanism differed from beta-adrenoceptor facilitating mechanism, since following beta-blockade with ICI 118551, angiotensin II infusion still significantly potentiated the release of catecholamines during splanchnic nerve stimulation. These observations thus suggest that both facilitating beta 2-adrenoceptors and angiotensin II receptors can independently modulate the release of adrenal catecholamines.     

Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.     

Effects of imidazole compounds on catecholamine release in adrenal chromaffin cells

1. Effects of imidazole compounds and guanabenz on the stimulus-evoked release of catecholamine (CA) were studied in cultured bovine adrenal chromaffin cells. 2. Clonidine, oxymetazoline, phentolamine, chlorpheniramine, and guanabenz inhibited acetylcholine (ACh)-evoked CA release in a dose-dependent manner, but not high K(+)-evoked release. 3. The inhibition by these compounds was not antagonized by nonimidazole and nonguanidine alpha 2-antagonists (yohimbine and phenoxybenzamine) but was significantly antagonized by tolazoline (imidazole alpha 2-antagonist) and cimetidine (imidazole H2-antagonist). Moreover, tolazoline by itself augmented the ACh-evoked, but not the high K(+)-evoked, CA release. 4. Although chlorpheniramine and cimetidine are antagonists for H1 and H2 histaminergic receptors, the site of action for these compounds in our results seemed to differ from the histamine receptors. 5. These results suggest that the inhibitory action of imidazole compounds and guanabenz on ACh-evoked CA release in adrenal chromaffin cells is mediated through an imidazole receptor. Adrenal chromaffin cells may contain an endogenous clonidine-displacing substance (CDS) which has been found in adrenal gland and brain as an endogenous ligand for imidazole receptors. Thus, CDS may have a regulatory role in the stimulus-secretion coupling in these cells.     

Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels.

A new 1,4-dihydropyridine photoaffinity ligand, [3H]diazipine, has been assessed by binding and photolabeling, and compared with a currently used [3H]azidopine. [3H]Diazipine reversibly binds to skeletal muscle Ca2+ channels with a similar affinity to [3H]azidopine, but [3H]diazipine labels the channel two times more efficiently and no release of the incorported amount is observed after dithiothreitol treatment.     

Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

The effects of calcium and strontium on the quantal content of nerve-evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2-0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca(2+)](o) was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca(2+)](o). Hence, extracellular calcium affected mainly the late, 'asynchronous', portion of phasic release. The average quantal content grew exponentially from 0.09 +/- 0.01 to 1.04 +/- 0.07 with the increase in [Ca(2+)](o) without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2-1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) (25 micromol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 micromol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms.