Christian Legros

Reduced availability of voltage-gated sodium channels by depolarization or blockade by tetrodotoxin boosts burst firing and catecholamine release in mouse chromaffin cells

Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast‐inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca2+‐dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast‐inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst‐firing patterns that give rise to elevated Ca2+‐influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels.

GENOMIC ORGANIZATION OF THE KTX2 GENE, ENCODING A 'SHORT' SCORPION TOXIN ACTIVE ON K+ CHANNELS

A single intron of 87 bp, close to the region encoding the C‐terminal part of the signal peptide, was found in the gene of the ‘short’ scorpion toxin kaliotoxin 2 of Androctonus australis acting on various types of K+ channels. Its A+T content was particularly high (up to 86%). By walking and ligation‐mediated PCR, the promoter sequences of the kaliotoxin 2 gene of Androctonus australis were studied. The transcription unit of the gene is 390 bp long. Consensus sequences were identified. The genes of ‘short’ scorpion toxins active on K+ channels are organized similarly to those of the ‘long’ scorpion toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.

Roles of connexins and pannexins in (neuro)endocrine physiology

To ensure appropriate secretion in response to demand, (neuro)endocrine tissues liberate massive quantities of hormones, which act to coordinate and synchronize biological signals in distant secretory and nonsecretory cell populations. Intercellular communication plays a central role in this control. With regard to molecular identity, junctional cell–cell communication is supported by connexin-based gap junctions. In addition, connexin hemichannels, the structural precursors of gap junctions, as well as pannexin channels have recently emerged as possible modulators of the secretory process. This review focuses on the expression of connexins and pannexins in various (neuro)endocrine tissues, including the adrenal cortex and medulla, the anterior pituitary, the endocrine hypothalamus and the pineal, thyroid and parathyroid glands. Upon a physiological or pathological stimulus, junctional intercellular coupling can be acutely modulated or persistently remodeled, thus offering multiple regulatory possibilities. The functional roles of gap junction-mediated intercellular communication in endocrine physiology as well as the involvement of connexin/pannexin-related hemichannels are also discussed.

Molecular biology of scorpion toxins active on potassium channels

Peptidyl scorpion toxins are known to block diverse types of K+ channels with high affinity and, thus, can be used as powerful tools to study the physiological role of the ionic selectivity, and the architecture of the pore-region of this class of channels. Yet, diversity among K+ channels is large and there has been a profusion of research for new selective ligands in order to elucidate their mechanisms of action and pharmacology significance. Scorpion toxins active on K+ channels are short polypeptides of about 30 to 40 amino acid residues, cross-linked by three or four disulfide bridges. They display a high degree of primary sequence homologies. 1H Nuclear Magnetic Resonance (NMR) analysis has demonstrated that these toxins are composed of an α-helix and a two-stranded antiparallel β-sheet, linked by two disulfide bridges. This structural motif is also found in the insect defensins. A 370 bp cDNA encoding the Kaliotoxin 2 (KTX2) precursor (a 37 amino acid residues peptide purified from the North African scorpion Androctonus australis and acting as a high affinity blocker of K+ channels) was obtained by PCR amplification and the organization of the KTX2 precursor depicted. This precursor is composed of a signal peptide followed by the mature toxin. The transcriptional unit and the promotor region of the gene encoding KTX2 was then amplified from the genomic DNA of Androctonus australis and its sequence determined. A single intron of 87 bp, located close to the region encoding the C-terminal part of the signal peptide, was found. Its A+T content was particularly high (up to 86%). The transcription unit of the gene was 390 bp long. Regulatory consensus sequences were identified. The genes of scorpion ‘short’ toxins active on K+ channels are organized similarly to those of the scorpion ‘long’ toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.

Monitoring the Secretory Behavior of the Rat Adrenal Medulla by High-Performance Liquid Chromatography-Based Catecholamine Assay from Slice Supernatants

Catecholamine (CA) secretion from the adrenal medullary tissue is a key step of the adaptive response triggered by an organism to cope with stress. Whereas molecular and cellular secretory processes have been extensively studied at the single chromaffin cell level, data available for the whole gland level are much scarcer. We tackled this issue in rat by developing an easy to implement experimental strategy combining the adrenal acute slice supernatant collection with a high-performance liquid chromatography-based epinephrine and norepinephrine (NE) assay. This technique affords a convenient method for measuring basal and stimulated CA release from single acute slices, allowing thus to individually address the secretory function of the left and right glands. Our data point that the two glands are equally competent to secrete epinephrine and NE, exhibiting an equivalent epinephrine:NE ratio, both at rest and in response to a cholinergic stimulation. Nicotine is, however, more efficient than acetylcholine to evoke NE release. A pharmacological challenge with hexamethonium, an α3-containing nicotinic acetylcholine receptor antagonist, disclosed that epinephrine- and NE-secreting chromaffin cells distinctly expressed α3 nicotinic receptors, with a dominant contribution in NE cells. As such, beyond the novelty of CA assays from acute slice supernatants, our study contributes at refining the secretory behavior of the rat adrenal medullary tissue, and opens new perspectives for monitoring the release of other hormones and transmitters, especially those involved in the stress response.