Pedro Michelena

Separation and culture of living adrenaline- and noradrenaline-containing cells from bovine adrenal medullae

Separation of viable adrenaline-containing from noradrenaline-containing chromaffin cells in large amounts has been achieved. The procedure involves collagenase digestion of bovine adrenomedullary tissue, isolation of cells through gentle filtration, separation of chromaffin from nonchromaffin cells on discontinuous gradients of the radiopaque contrast Renografin, and separation of adrenaline-enriched from noradrenaline-enriched fractions after centrifugation on self-generated Percoll gradients. Collection of 1-ml Percoll fractions gave two clear-cut catecholamine peaks. The denser peak was enriched in adrenaline and phenylethanolamine-N-methyltransferase (PNMT), suggesting that over 90% of cells were adrenergic. The lighter peak was preferentially enriched in noradrenaline but not in PNMT. With this information, we could collect by gentle aspiration two main fraction layers of larger volumes; one at the bottom of the Percoll gradient, which contained essentially adrenaline-storing cells and the other at the top of the gradient, enriched in noradrenaline cells. Those cells could be maintained viable for at least 1 week in primary monolayer cultures, as shown by neutral red staining and trypan blue exclusion. This method will allow the identification of chemical components, receptors, or ionic channels present in one specific type of cell, to determine their relevance to the regulation of the differential secretion of specific materials present in one but not in the other cell type and to ascertain whether the released materials from one cell type affect the functions of the other.

The mechanism of calcium channel facilitation in bovine chromaffin cells

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage‐clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole‐cell Ba2+ currents (IBa). 2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3.18 +/‐ 0.12 mM (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11.2 +/‐ 0.97 and 15.2 +/‐ 2 mM, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7‐fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2+ channel facilitation, persisted in the presence of 3 microM nifedipine. 4. Intracellular dialysis of GDP‐beta‐S (0.5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml‐1 for 18‐24 h) prevented the reduction in peak current caused by a 1:100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 microM) or suramin (100 microM) and PPADS (100 microM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (< 10 kDa) considerably reduced the activity of SVL. 6. Stopping the flow of the external solution (10 mM Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow‐stop conditions in the presence or absence of 3 microM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole‐cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G‐protein‐mediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non‐L‐type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridine‐sensitive Ca2+ channels are not tenable.

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

Abstract Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 µM nifedipine (L-type channels), 21% sensitive to 1 µM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 µM ω-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 µM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.

Activation of adrenal medullary L-arginine: nitric oxide pathway by stimuli which induce the release of catecholamines

The activation of the L-arginine: nitric oxide (NO) pathway in the cat adrenal medulla by different stimuli which induce the release of catecholamines was studied. Stimuli that evoke catecholamine release, such as electrical stimulation of splanchnic nerves (50 V, 5 Hz, 1 ms), methacholine (100 microM), dimethyl-4-phenylpiperazinium iodide (DMPP; 10 microM), high K+ (35 mM) and alamethicin (15 micrograms ml-1) also caused a rise in cyclic GMP in the perfused cat adrenal medulla. NG-nitro-L-arginine methyl ester (L-NAME; 1 mM) abolished the rise in cyclic GMP induced by these stimuli without affecting the catecholamine release. Bovine adrenal medulla cytosol contained an NO synthase which was L-arginine- and Ca(2+)-dependent. In conclusion cat and bovine adrenal medulla stimulated with a variety of secretagogues synthesize NO from L-arginine to activate the soluble guanylate cyclase. The present data do not rule out a role for cyclic GMP in the regulation of catecholamine secretion; however, it seems more plausible that cyclic GMP may play a role in controlling local blood flow and thus the access of the released catecholamines to the systemic circulation during stressful conflicts.

Drastic facilitation by α-latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i

1 Latrotoxin (LTX, 1–3 nm) caused a gradual increase of the supontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs–Hepes solution containing 2.5 mm Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mm) and to acetylcholine (ACh, 30 μm). 2 The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs–Hepes deprived of CaCl2) induced secretory responses that gradually reached 400–800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mm K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100–200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]0 or in electroporated cells. 3 Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole‐cell Ca2+ currents or basal [Ca2+]j. In fact, LTX attenuated the K+‐ or ACh‐evoked increases in 45Ca2+ uptake and [Ca2+]1. 4 It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+1 tranMents, produced by the Na+‐Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na+‐dependent action potentials.

R56865 inhibits catecholamine release from bovine chromaffin cells by blocking calcium channels

1 The effects of R56865 (a new class of cardioprotective agent which prevents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine release, whole‐cell current through Ca2+ channels (IBa) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin cells. 2 R56865 caused a time‐ and concentration‐dependent blockade of catecholamine release from superfused cells stimulated intermittently with 5 s pulses of 59 mm K+. After 5 min superfusion, a 3 μm concentration inhibited secretion by 20%; the blockade increased gradually with perfusion time, to reach 85% after 40 min. The IC50 to block secretion after 5 min periods of exposure to increasing concentrations of R56865 was around 3.1 μm. The blocking effects of R56865 were reversible after 5–15 min wash out. In high Ca2+ solution (10 mm Ca2+), the degree of blockade of secretion diminished by 20% in comparison with 1 mm Ca2+. 3 In electroporated cells, R56865 (10 μm) did not modify the secretory response induced by the application of 10 μm free Ca2+. 4 R56865 blocked the peak IBa current in a concentration‐ and time‐dependent manner; its IC50 was very similar to that obtained for secretion (3 μm). The compound not only reduced the size of the peak current but also promoted its inactivation; when the effects of R56865 were measured at the most inactivated part of the current, its IC50 was 1 μm. Both the inactivation and the reduction of the peak currents were reversible upon washing out the drug. 5 In fura‐2‐loaded single chromaffin cells the basal [Ca2+]i of around 100 nm was elevated to a peak of 1.5 μm by the application of a 5 s pulse of 59 mm K+. R56865 (10 μm) did not affect the basal [Ca2+]i but blocked by 90% the K+‐evoked increase. This effect was fully reversible after 5–10 min of wash out. 6 The results are compatible with the idea that R56865 blocks Ca2+ entry into K+‐depolarized chromaffin cells by promoting the inactivation of voltage‐dependent Ca2+ channels; this would lead to the limitation of the rise in [Ca2+]i and of the release of catecholamines. The restriction of catecholamine release may favour indirectly the known direct beneficial cardioprotective actions of R56865.

Effects of Ca2+ channel antagonist subtypes on mitochondrial Ca2+ transport

This study was carried out to define the effects of various Ca2+ channel modulatory drugs on mitochondrial Ca2+ movements. Bovine adrenal medulla mitochondria took up Ca2+ at an initial rate of 6.8 nmol mg protein-1 5 s-1, with a Km of 15 microM and a Bmax of 30 nmol mg protein-1. At 30 microM, neither verapamil, diltiazem, nitrendipine nor Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] affected the initial rate of Ca2+ uptake. Ca(2+)-loaded mitochondria retained their Ca2+ contents in the presence of ruthenium red for at least 30 min. Cinnarizine and flunarizine, but not verapamil, diltiazem, isradipine, Bay K 8644 or nitrendipine, caused a fast and dramatic Na(+)-independent Ca2+ loss. Other Ca2+ channel antagonists assayed such as penfluridol, R56865 [N-[1-(4-(4-fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2- benzothiazolamine], lidoflazine, R87926 [(+)-(S)-4-(2-benzothiazolyl-methylamino)-alpha-[(3,4-difluorophenoxy ) methyl] 1 piperidine] and sabeluzole, also had a mitochondrial Ca2+ depleting effect which seemed to be directly related to their octanol/water partition coefficient. The Na(+)-dependent Ca2+ efflux from mitochondria was completely inhibited by diltiazem and greatly blocked by nitrendipine. Isradipine caused a moderate blockade and Bay K 8644 and verapamil had no effect. All these data open the possibility of developing novel Ca2+ channel antagonists having selective actions on plasmalemmal Ca2+ channels, and others with additional and different effects on mitochondrial Ca2+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)

A preferential pole for exocytosis in cultured chromaffin cells revealed by confocal microscopy

Histological studies suggest that adrenal medulla chromaffin cells in situ are polarised, but functional evidence is lacking. We present here the first demonstration for polarisation of exocytosis in isolated, spherical, bovine chromaffin cells. Cells were stimulated with 70 mM K+ to cause a marked enhancement of catecholamine release, monitored amperometrically. FM1‐43 and dopamine β‐hydroxylase antibodies provided fluorescence confocal pictures that were 2–3‐fold more intense in the bottom of the cells, as compared to equatorial or apex planes. This suggests that the solid phase to which the cell attaches serves as a ‘trophic’ signal for the polarisation of its secretory apparatus.

PF9404C, a new slow NO donor with beta receptor blocking properties

PF9404C is the S‐S diesteroisomer of a novel blocker of beta adrenergic receptors with vasodilatory properties. It causes a concentration‐dependent relaxation of rat aorta helical strips pre‐contracted with 10−6 M noradrenaline (NA; IC50 33 nM). It was equipotent to nitroglycerin (NTG; IC50 49 nM), but much more potent than isosorbide dinitrate (ISD; IC50 15,000 nM). Oxyhaemoglobin (10 μM) shifted to the right the concentration‐response curve for the relaxation induced by PF9404C (IC50 530 nM) or NTG (IC50 61 nM). Either methylene blue (MB) or ODQ (1 μM each) largely prevented the vasorelaxing responses to increasing concentrations of PF9404C or NTG. In rat aorta smooth muscle cells, PF9404C increased the formation of cyclic GMP from 3 pmol mg−1 protein in basal conditions, to 53 pmol mg−1 protein in 10 μM PF9404C. Neither metoprolol nor carvedilol enhanced cyclic GMP. In the electrically driven guinea‐pig left atrium, PF9404C blocked the inotropic effects of isoprenaline in a concentration‐dependent manner. Its IC50 (30 nM) was similar to that for S‐propranolol (22.4 nM) and lower than the IC50s for metoprolol (120 nM) and atenolol (192 nM). The beta‐adrenergic ligand (−)‐[3H]‐CGP12177 (0.2 nM) was displaced from its binding to rat brain membranes with Ki of 7 nM, 17 nM, 170 nM and 1.2 μM respectively for PF9404C, S‐(−)propranolol, metoprolol, and atenolol. The data are consistent with the idea that the S‐S diesteroisomer PF9404C, is a potent vasorelaxing agent, as well as a blocker of cardiac beta adrenergic receptors. The mechanism of its vasorelaxing effects involves the slow generation of NO. This molecule can, therefore, exhibit antihypertensive and cardioprotective actions through a double mechanism, NO donation and beta blockade.

Effects of tyramine and calcium on the kinetics of secretion in intact and electroporated chromaffin cells superfused at high speed

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K+ glutamate-based solution containing 50 nM free Ca2+ and 2 mM adenosine 5′-triphosphate, dipotassium salt (K2ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca2+ concentrations ([Ca2+]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca2+] (3–100 μM), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200–400 vesicles per cell. Pretreatment of cells with tyramine (10 μM for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca2+] in the range of 0.1–3 μM, the activation rate of secretion increased from 2.3 to 35.3 nA · s−1, reached a plateau between 3–30 μM and rose again from 100 to 1000 μM [Ca2+] to a maximum of 91.9 nA · s−1. In contrast, total secretion first increased (0.1–1 μM Ca2+), then plateaud (1–100 μM Ca2+) and subsequently decreased (100–1000 μM Ca2+). At 30 and 1000 μM extracellular [Ca2+] or [Ca2+]o, the activation rates of secretion from intact cells depolarised with 70 mM K+were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 μM Ca2+) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 μA · s at 1000 μM Ca2+) than in electroporated cells (42.4 μA,s at 1 μM Ca2+). Separation of the time-courses of changes in intracellular [Ca2+] or [Ca2+]i and secretion in intact chromaffin cells depolarised with 70 mM K+was demonstrated at different [Ca2+]o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca2+]i. In contrast, the decline of secretion was faster than the decline of the peak [Ca2+]i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca2+ by the rates of opening and closing of voltage-dependent Ca2+ channels of the L-and Q-subtypes, which control the local [Ca2+]i near to exocytotic sites.