Luis Gandía

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.

Q‐ and L‐type Ca2+ channels dominate the control of secretion in bovine chromaffin cells

Potassium‐stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K+ in the presence of 2 mM Ca2+ for 10 s, applied at 5‐min intervals) was inhibited by the dihydropyridine furnidipine (3 μM) by 50%. ω‐Conotoxin MVIIC (CTx‐MVIIC, 3 μM) also reduced the secretory response by about half. Combined CTx‐MVIIC plus furnidipine blocked 100% catecholamine release. 45Ca2+ uptake and cytosolic Ca2+ concentrations ([Ca2+]i) in K+‐depolarized cells were partially blocked by furnidipine or CTx‐MVIIC, and completely inhibited by both agents. The whole cell current through Ca2+ channels carried by Ba2+ (I Ba) was partially blocked by CTx‐MVIIC. Although ω‐conotoxin GVIA (CTx‐GVIA, 1 μM) and ω‐agatoxin IVA (Aga‐IVA, 0.2 μM) partially inhibited 45Ca2+ entry, I Ba and the increase in [Ca2+]i, the combination of both toxins did not affect the K+‐evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q‐like Ca2+ channel which has a prominent role in controlling exocytosis. They also suggest that Q‐ and L‐type Ca2+ channels, but not N‐ or P‐types are localized near exocytotic active sites in the plasmalemma.

Sphingosine Facilitates SNARE Complex Assembly and Activates Synaptic Vesicle Exocytosis

Summary Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

Neuroprotectant minocycline depresses glutamatergic neurotransmission and Ca2+ signalling in hippocampal neurons

The mechanism of the neuroprotective action of the tetracycline antibiotic minocycline against various neuron insults is controversial. In an attempt to clarify this mechanism, we have studied here its effects on various electrophysiological parameters, Ca2+ signalling, and glutamate release, in primary cultures of rat hippocampal neurons, and in synaptosomes. Spontaneous excitatory postsynaptic currents and action potential firing were drastically decreased by minocycline at concentrations known to afford neuroprotection. The drug also blocked whole‐cell inward Na+ currents (INa) by 20%, and the whole‐cell Ca2+ current (ICa) by about 30%. Minocycline inhibited glutamate‐evoked elevation of the cytosolic Ca2+ concentration ([Ca2+]c) by nearly 40%, and K+‐evoked glutamate release from synaptosomes by 63%. Minocycline also depressed the frequency and amplitude of spontaneous excitatory postsynaptic currents, but did not affect the whole‐cell inward current elicited by γ‐aminobutyric acid or glutamate. This pharmacological profile suggests that the neuroprotective effects of minocycline might be associated with the mitigation of neuronal excitability, glutamate release, and Ca2+ overloading.

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of −80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (IBa) snowed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 μM and 10 μM respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 μM) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of IBa was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin ω-conotoxin GVIA (GVIA; 1 μM) blocked 31% of IBa; blockade was irreversible upon removal of the toxin from the extracellular medium, ω-Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of IBa. ω-Conotoxin MVIIC (MVIIC; 5 μM) produced a 36% blockade of IBa; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 μM), GVIA (1 μM), IVA (100 nM) and MVIIC (3 μM) produced partial inhibition of IBa, which were additive. Our data suggest that the whole cell IBa in rat chromaffin cells exhibits at least four components. About 50% of IBa is carried by L-type Ca2+ channels, 30% by N-type Ca2+channels and 15% by P-type Ca2+ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca2+channels account for 45% of the current, N-type carry 35% and L-type Ca2+ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.

ω‐Agatoxin‐IVA‐sensitive calcium channels in bovine chromaffin cells

A large component of the whole‐cell currents through Ca2+ channels in bovine adrenomedullary chromaffin cells has been shown to be insensitive to both L‐type and N‐type Ca2+ channel Mockers, suggesting the existence of a third type of Ca2+ channel. In the present paper, ω‐agatoxin‐IVA (AgTx), a selective blocker of P‐type Ca2+ channels in mammalian neurons, has been used to investigate the presence of this subtype of Ca2+ channel in bovine chromaffin cells. Barium currents (I Ba) through Ca2+ channels were recorded in whole‐cell patch‐clamped bovine chromaffin cells. I Ba was blocked by AgTx in a dose‐dependent and irreversible manner. At the maximal concentration used (1 μM), AgTx inhibited T Ba by 49.5 ± 3%. Such a blockade was also present when bovine chromaffin cells were pretreated with 10 μM fumidipine, a novel 1,4‐dihydropyridine L‐type channel blocker, and after treatment with 1 μM of the N‐type channel blocker, ω‐conotoxin GVIA (CgTx). A combination of these three types of Ca2+ channel blockers suppressed the macroscopic Ba2+ currents by 88%. We conclude that bovine chromaffin cells, in addition to N‐ and L‐type Ca2+ channels, possess a P‐like component in their whole‐cell currents through the Ca2+ channels.

Neuroprotection afforded by nicotine against oxygen and glucose deprivation in hippocampal slices is lost in α7 nicotinic receptor knockout mice

Although alpha7-receptors are considered the main target for neuroprotection, other receptor subtypes (alpha4beta2 or alpha3beta4) have also been implicated. Hence, we have used alpha7-transgenic mice, to study the hypothesis that alpha7-receptors play a dominant role in mediating neuroprotection in an in vitro model of ischemia. We have used rat and mouse hippocampal slices to establish the model of nicotinic neuroprotection against oxygen and glucose deprivation (OGD). Neuronal damage caused by OGD during 1 h plus 3 h re-oxygenation, was quantified by measuring lactate dehydrogenase (LDH) release from hippocampal slices. In rat hippocampal slices, OGD increased over twofold basal LDH release. Such increase was reduced when treated with 10-100 microM nicotine; maximal protection afforded by nicotine amounted to 46%. This neuroprotection was antagonized by the non-selective nicotinic receptor for acetylcholine (nAChR) blocker mecamylamine (10 microM). In hippocampal slices from wild-type control mice, nicotine (100 microM) decreased by 54.4% LDH release evoked by OGD plus re-oxygenation. In contrast, nicotine failed to exert neuroprotection in alpha7 knockout mice. This finding reinforces the view that the hippocampal neuroprotective effects of nicotine are predominantly linked to alpha7 receptors.

A physiological view of the central and peripheral mechanisms that regulate the release of catecholamines at the adrenal medulla

Here we review the tight neural control of the differential secretion into the circulation, of the adrenal medullary hormones adrenaline and noradrenaline. One or the other catecholamines are differentially released on various stress conditions. This is specifically controlled by central nervous system nuclei at the cortex, hypothalamus and spinal cord. Different firing patterns of splanchnic nerves and nicotinic or muscarinic receptors cause the selective release of noradrenaline or adrenaline, to adapt the body to the ‘fight or flight’ reaction, or during severe hypoglycaemia, haemorrhage, cold, acute myocardial infarction or other severe stressful conflicts. Endogenously acetylcholine (ACh) released at the splanchnic nerve‐chromaffin cell synapse, acting on muscarinic and nicotinic receptors, causes membrane depolarization and action potentials (AP) in chromaffin cells. These changes vary with the animal species, the cell preparation (intact bisected adrenal, adrenal slices, or isolated fresh or cultured cells) or the recording technique (intracellular microelectrodes, patch‐clamp, perforated‐patch, cell‐attached). Conflicting results leave many open questions concerning the actions of ACh on chromaffin cell excitability. The use of adrenal slices and field electrical stimulation will surely provide new insights into these mechanisms. Chromaffin cells have been thoroughly used as models to study the relationship between Ca2+ entry, cytosolic Ca2+ signals, exocytosis and endocytosis, using patch‐clamp and amperometric techniques. Cells have been stimulated with single depolarizing pulses (DPs), DP trains and with simulated AP waveforms. These approaches have provided useful information but we have no data on APs generated by pulsatile secretory quanta of ACh, trying to mimic the intermittent and repetitive splanchnic nerve discharge of the neurotransmitter. We present some recent experiments using ultrashort ACh pulses (25 ms), that cause non‐desensitizing repetitive APs with each ACh pulse, at low ACh concentrations (30 μm). Ultrashort pulses of a high ACh concentration (1000 μm) causes a single AP followed by a prolonged depolarization. It could be interesting trying to correlate these ‘patterns of splanchnic nerve discharge’ with Ca2+ signals and exocytosis. This, together with the use of adrenal slices and transmural electrical stimulation of splanchnic nerves will provide new physiologically sound data on the regulation of adrenal medullary secretion.

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 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) 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 μM; 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, howevever, 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.

Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations

This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of ω-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of −80 mV and depolarising pulses to 0 mV. Mean peak IBa was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, ω-conotoxin MVIIC (3 μM) inhibited IBa by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of ω-agatoxin IVA (20 nM) inhibited IBa by 9%; 2 μM inhibited IBa by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 μM) and ω-conotoxin GVIA (1 μM), 2 μM ω-agatoxin IVA inhibited the remaining current (about 40–45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/gw-conotoxin GVIA/high ω-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of IBa is blocked by micromolar concentrations of ω-agatoxin IVA and ω-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by ω-conotoxin MVIIC, the blockade by high concentrations of ω-agatoxin IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca2+ channels. An R-type residual current is not present in these cells.