Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.     

Effects of pro-opiomelanocortin fragments on release of catecholamines from adrenal chromaffin cells

The effects of pro-opiomelanocortin (POMC) peptide fragments on the basal and agonist-induced release of catecholamines (CAs) from monolayer cultures of purified bovine adrenal chromaffin cells were tested. None of the 5 peptides tested, i.e. ß-MSH, ACTH_1–39, γ-MSH_1–13. γ₃-MSH and N-terminal POMC fragment, had any effect on basal CA release. However, ß-MSH (10^?5 M), γ-MSH_1–13 (10^?6–10^?5 M), γ₃-MSH (10^?5 M) and N-terminal POMC fragment (10^?5 M) inhibited the nicotine-induced release of CAs from the chromaffin cells. The possible physiological significance of this inhibitory neuromodulation is discussed.     

An electrophysiological investigation of the characteristics and function of GABAA receptors on bovine adrenomedullary chromaffin cells

The characteristics and function of -aminobutyric acid_A (GABA_A) receptors expressed on bovine chromaffin cells in culture have been investigated using patch-clamp techniques. In voltage-clamped whole-cells, locally applied GABA (100 M) evoked a transmembrane chloride current which demonstrated outward rectification. The amplitude of such currents was reversibly suppressed by the GABA_A receptor antagonists bicuculline, picrotoxin and RU5135, and enhanced by the general anaesthetic propanidid. Glycine (100 M) and baclofen (100 M) were ineffective as agonists. In support of a physiological role for GABA in the adrenal medulla, the co-existence of GABA_A and nicotinic acetylcholine (ACh) receptors was demonstrated on whole cells and outside-out membrane patches. Ionophoretically applied GABA reduced the amplitude of depolarization and action potential discharge occurring in response to locally applied ACh (100 M), but had no effect upon the underlying ACh-induced current. In addition, an excitatory action of GABA was demonstrated by recording action potential waveforms in cell-attached patches. The results are discussed in the context of a GABA-ergic regulation of catecholamine secretion.     

The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells

The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [(3)H]noradrenaline ([(3)H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca(2+)-evoked [(3)H]NA release but increased basal release (that occurring in the absence of Ca(2+)). Preincubation with antibodies against PLCgamma1 or PLCbeta3 (but not PLCdelta1, delta2, beta1 and beta2) also inhibited [(3)H]NA release evoked by Ca(2+) and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCgamma1 (but not PLCbeta3) antibodies inhibited the ability of Ca(2+) to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCgamma1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.     

Quantal size of catecholamine release from rat chromaffin cells is regulated by tonic activity of protein kinase A

To address the roles of tonic activity of protein kinase A (PKA) in the regulation of exocytosis, we examined the effects of selective PKA inhibitors on catecholamine (CA) release from rat chromaffin cells in the absence of PKA activators. Myristoylated protein kinase inhibitor (myr-PKI) reduced the total amount of CA released from a single cell, whereas H-89 did not affect the total amount and frequency of CA release. Amperometric recording revealed that myr-PKI had only a small effect on the number of amperometric spikes per cell, but preferentially inhibited exocytotic events of large quantal size. The change in quantal size distribution was not associated with a reduction of cellular CA content, quantified by using HPLC. These results suggest that the tonic activity of PKA near the cell membrane regulates neurotransmitter release by modulating quantal size.     

Properties of the nicotinic-receptor-activated current in adrenal chromaffin cells of the guinea-pig

Properties of acetylcholine(ACh)- and nicotine-induced currents were studied in the guinea-pig chromaffin cell, using the whole-cell and cell-attached versions of the patch-clamp technique. Bath application of ACh or nicotine, but not muscarine, produced an inward current associated with an increase in current noise at a membrane potential of –70 mV. Low concentrations of both agonists produced a sustained inward current whereas high concentrations produced a transient, then a sustained inward current. Nicotine was about twice as potent as ACh in inducing the inward current. Hexamethonium (6 M) inhibited the ACh-induced current but not in a competitive manner. By contrast, atropine (6 M) inhibited the ACh-induced current more strongly with increasing concentrations of ACh. The nicotinic-receptor-activated current (nicotinic current) showed inward rectification and, when Cs⁺ was used instead of K⁺ in the pipette solution, the polarity of the current changed at around –5 mV and a negative slope occurred between +10 mV and +30 mV. The nicotinic channel had a unit conductance of 33 pS. During the initial 20–30 min of whole-cell voltage-clamp recording, the time course of the agonist-induced desensitization was markedly facilitated. Addition of 5 mM MgATP in the pipette solution at least partly prevented this facilitation of the desensitization. The frequency of activation of the nicotinic receptor and the extracellular Ca²⁺ were not primary factors in the acceleration of rate of desensitization.     

Transplantation of cultured fetal human adrenal chromaffin cells to rat brain

Adrenal chromaffin cells, obtained from a therapeutically aborted human fetus of about 11 weeks gestation, were cultured for 3 weeks in vitro and then transplanted to the striatum of rats. Transplanted cells became established through strands of tissue growing into the host striatum. No signs of inflammation or rejection were observed up to the time of sacrifice one month post-transplantation. Histofluorescence examination of the implanted areas showed many clusters of cells having an intensely positive catecholamine fluorescence with some of the cells developing conspicuous processes. This study, showing survival of cultured human adrenal chromaffin cells transplanted into rat brain tissue, might indicate the feasibility of using cultured human material for future human neuronal transplantation studies as a therapeutic measure.     

Pertussis toxin attenuates clonidine inhibition of catecholamine release in adrenal chromaffin cells

Characteristics of the inhibitory action of clonidine on catecholamine release in bovine adrenal chromaffin cells were investigated. Clonidine at 3 x 10(-5) M inhibited acetylcholine (ACh)-evoked release by about 50%, but not catecholamine release evoked by high K+. Another alpha 2-agonist alpha-methyladrenaline was ineffective at inhibiting ACh-evoked release. The inhibition by clonidine of ACh-evoked release was not reversed by alpha 2-antagonists. Treatment of these cells with pertussis toxin reversed the inhibitory effect of clonidine, while it did not affect the inhibitory action of hexamethonium and of nifedipine. Therefore, clonidine inhibition of catecholamine release in these cells seems not to be mediated by the alpha 2-adrenoceptor, but might be mediated by a specific receptor for clonidine.     

Lack of effect of opioid compounds on angiotensin II responses of bovine adrenal medullary cells

Angiotensin II (10 nM) increased basal adrenaline and noradrenaline secretion from cultured bovine adrenal chromaffin cells by 2.5- to 3-fold and 4- to 6-fold, respectively, and stimulated basal accumulation of inositol phosphates more than 2-fold. Etorphine and diprenorphine in the range 10⁻⁹ to 10⁻⁵ M had no effect on the catecholamine secretion induced by angiotensin II, and, at 10⁻⁸ and 10⁻⁵ M, had no effect on angiotensin II-induced inositol phosphate accumulation. The functions of adrenal medullary opioid receptors remain to be determined.     

Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca²⁺-activated K⁺ current [I _K(Ca)] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K⁺ current [I _K(ATP)] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for I _K(ATP) and I _K(Ca) was 50 and 29.5%, respectively, whereas at F19, I _K(ATP) is reduced to 14%, and I _K(Ca) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of K_ATP and K_Ca channels.     

Palytoxin: a potent stimulator of catecholamine release from cultured bovine adrenal chromaffin cells.

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine release from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-9) M induced catecholamine release dose-dependently. About 40-50% of the total cellular catecholamine was released during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced catecholamine release was dependent on both extracellular Na+ and Ca2+, and was inhibited by organic and inorganic Ca2+ channel blockers, but not by tetrodotoxin. PTX-induced increase in 45Ca2+ influx into the cells, which was associated with catecholamine release, was also inhibited by these Ca2+ channel blockers. These results indicated that PTX-induced catecholamine release was mediated by activation of Na(+)-dependent, tetrodotoxin (TTX) insensitive voltage-dependent Ca2+ channels.     

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At –70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.     

Calcium channel subtypes in porcine adrenal chromaffin cells

 The effects of nifedipine, ω-conotoxin GVIA (ω-CgTx) and ω-agatoxin IVA (ω-AgTx) on Ca²⁺ currents, a 60-mM-K⁺-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and catecholamine secretion were examined to clarify the subtypes of Ca²⁺ channels in cultured adrenal chromaffin cells from the pig. Nifedipine, ω-CgTx, and ω-AgTx inhibited Ca²⁺ currents in a dose-dependent manner, suggesting the presence of L-, N- and P-type Ca²⁺ channels. The maximal doses of nifedipine (10 μM), ω-CgTx (1 μM), and ω-AgTx (0.1 μM) inhibited Ca²⁺ currents to 85%, 22%, and 94% of control currents, respectively. The inhibitory effects of these three blockers were observed in the same cell, indicating that at least three subtypes of Ca²⁺ channels are present in porcine chromaffin cells. The increase in [Ca²⁺]_i and catecholamine secretion induced by 60 mM K⁺ were inhibited equally by nifedipine (10 μM) and ω-CgTx (1 μM), but not by ω-AgTx (0.1 μM). These results suggest that L-, N- and P-type Ca²⁺ channels are present in porcine adrenal chromaffin cells, and that the major pathways of Ca²⁺ entry evoked by a high concentration of K⁺ are L- and N-type Ca²⁺ channels. Received: 6 September 1996 / Received after revision: 3 February 1997 / Accepted: 18 February 1997     

Swelling-induced catecholamine secretion recorded from single chromaffin cells

We have studied osmotically induced catecholamine secretion from bovine adrenal chromaffin cells by combining patch-clamp measurements, electrochemical detection of secretion, and Fura-2 measurements of intracellular free calcium concentration ([Ca²⁺]_i). We find that osmotically induced catecholamine release is exocytotic and calcium dependent. Furthermore, we demonstrate that cell swelling is coupled to such secretion via a volume-activated current, carrying predominantly chloride, which causes a plateau depolarization of the cell membrane potential and thus promotes voltage-activated calcium influx. Therefore, cell volume changes may modulate the secretory activity.     

Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca²⁺ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca²⁺ current (I _Ca) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p0.05). Analysis of voltage-dependent steady-state activation (m ) curves revealed a 0.70±0.27 charge increase in the activation-gate valency (z _m) following 8-Br-cAMP perfusion. Similar responses were observed when Ba²⁺ was the charge carrier, where z _m was increased by 1.33±0.34 charges (n=8). The membrane potential for half activation (V _1/2) was also significantly shifted 6 mV more negative for I _Ba (mean, n=8). The time course for I _Ba (and I _Ca) activation was well described by second-order m ² kinetics. The derived time constant for activation (_m) was voltage-dependent, and the _m/V relation shifted negatively after 8-Br-cAMP treatment. Ca²⁺ channel gating rates were derived from the (_m) and m ² values according to a Hodgkin-Huxley type m ² activation process. The forward rate ( _m) for channel activation was increased by 8-Br-cAMP at membrane potentials 0 mV, and the backward rate (_m) decreased at potentials +10 mV. Time-dependent inactivation of I _Ca consisted of a slowly decaying component (_h 300 ms) and a non-inactivating steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.     

Cytoplasmic organelles determine complexity and specificity of calcium signalling in adrenal chromaffin cells

Complex and coordinated fluctuations of intracellular free Ca2+ concentration ([Ca2+]c) regulate secretion of adrenaline from chromaffin cells. The physiologically relevant intracellular Ca2+ signals occur either as localized microdomains of high Ca2+ concentrations or as propagating Ca2+ waves, which give rise to global Ca2+ elevations. Intracellular organelles, the endoplasmic reticulum (ER), mitochondria and nuclear envelope, are endowed with powerful Ca2+ transport systems. Calcium uptake and Ca2+ release from these organelles determine the spatial and temporal parameters of Ca2+ signalling events. Furthermore, the ER and mitochondria form close relations with the sites of plasmalemmal Ca2+ entry, creating 'Ca2+ signalling triads' which act as elementary operational units, which regulate exocytosis. Ca2+ ions accumulating in the ER and mitochondria integrate exocytotic activity with energy production and protein synthesis.     

Barium-evoked release of catecholamines from digitonin-permeabilized adrenal medullary cells

We investigated the release of catecholamines by barium (Ba) in digitonin-permeabilized bovine adrenal medullary cells, to find out whether Ba acts directly or through the mobilization of endogenous calcium (Ca). We also tried to ascertain whether the release of catecholamines from the permeabilized cells occurs in the same manner as from the non-permeabilized control cells. In the permeabilized cells, a rise in either free Ca or Ba caused a saturable release of catecholamines. The maximal release of catecholamines caused by Ba was greater than that caused by Ca, suggesting that Ba directly triggered the release process. Release of catecholamines by Ba was accompanied by the release of dopamine beta-hydroxylase, both in the permeabilized and control cells, showing that Ba causes an exocytotic release of catecholamines. The concentration of Ba which was required for the release of catecholamines in the permeabilized cells agreed with that required in control cells. We conclude that Ba can substitute Ca in triggering the exocytotic release of catecholamines from the adrenal medullary cells. Permeabilized cells provide accurate information on the direct action of Ba in these cells.     

Calcium channel current facilitation in porcine adrenal chromaffin cells

 The mechanisms of depolarizing-prepulse-induced facilitation of Ca²⁺ channel current were investigated in a study of porcine chromaffin cells. The Ba²⁺ current evoked by a pulse to 0 mV was increased by a strong depolarizing prepulse (conditioning pulse), termed ”facilitation”. This facilitation increased with an increase in either the duration or the voltage of the conditioning pulse, and decreased with an increase in the interpulse interval. For example, the Ba²⁺ current was increased to 1.14 times the control (facilitation ratio) by a 150-ms conditioning pulse to +100 mV followed by a 10-ms interpulse interval. Forskolin, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-bromo-cAMP) and Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) did not affect the facilitation of the Ba²⁺ current, suggesting that a cAMP-dependent mechanism is not involved. Intracellular guanosine 5′-O-(3-thiotriphosphate) (GTPγS) decreased the Ba²⁺ current to 0.59 times the control and GDPβS increased it to 1.19. However, neither GTPγS nor guanosine 5′-O-(2-thiodiphosphate) (GDPβS) changed the amplitude of the Ba²⁺ current that was facilitated by the conditioning pulse. Thus, GTPγS increased the facilitation ratio to 2.05 and GDPβS decreased it to 1.05. Furthermore, the facilitation of the Ba²⁺ current was abolished by ω-conotoxin GVIA but not by either ω-agatoxin IVA or nifedipine. These results suggest that, in porcine chromaffin cells, there is a ω-conotoxin GVIA-sensitive N-type Ca²⁺ channel that is under the inhibitory control of a G protein, which can be relieved by a conditioning pulse. Received: 25 September 1997 / Received after revision: 14 November 1997 / Accepted: 16 December 1997