María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells.

María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.

The Adrenal Medulla Modulates Mechanical Allodynia in a Rat Model of Neuropathic Pain.

We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not affect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chromaffin cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerve.

Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain.

We have tested the hypothesis that neuropathic pain acting as a stressor drives functional plasticity in the sympathoadrenal system. The relation between neuropathic pain and adrenal medulla function was studied with behavioral, immunohistochemical and electrophysiological techniques in rats subjected to chronic constriction injury of the sciatic nerve. In slices of the adrenal gland from neuropathic animals, we have evidenced increased cholinergic innervation and spontaneous synaptic activity at the splanchnic nerve⁻chromaffin cell junction. Likewise, adrenomedullary chromaffin cells displayed enlarged acetylcholine-evoked currents with greater sensitivity to α-conotoxin RgIA, a selective blocker of α9 subunit-containing nicotinic acetylcholine receptors, as well as increased exocytosis triggered by voltage-activated Ca2+ entry. Altogether, these adaptations are expected to facilitate catecholamine output into the bloodstream. Last, but most intriguing, functional and immunohistochemical data indicate that P2X3 and P2X7 purinergic receptors and transient receptor potential vanilloid-1 (TRPV1) channels are overexpressed in chromaffin cells from neuropathic animals. These latter observations are reminiscent of molecular changes characteristic of peripheral sensitization of nociceptors following the lesion of a peripheral nerve, and suggest that similar phenomena can occur in other tissues, potentially contributing to behavioral manifestations of neuropathic pain.

Alpha2-adrenoceptors in adrenomedullary chromaffin cells: functional role and pathophysiological implications.

Chromaffin cells from the adrenal medulla participate in stress responses by releasing catecholamines into the bloodstream. Main control of adrenal catecholamine secretion is exerted both neurally (by the splanchnic nerve fibers) and humorally (by corticosteroids, circulating noradrenaline, etc.). It should be noted, however, that secretory products themselves (catecholamines, ATP, opioids, ascorbic acid, chromogranins) could also influence the secretory response in an autocrine/paracrine manner. This form of control is activity-dependent and can be either inhibitory or excitatory. Among the inhibitory influences, it stands out the one mediated by α2-adrenergic autoreceptors activated by released catecholamines. α2-adrenoceptors are G protein-coupled receptors capable to inhibit exocytotic secretion through a direct interaction of Gßγ subunits with voltage-gated Ca2+ channels. Interestingly, upon intense and/or prolonged stimulation, α2-adrenergic receptors become desensitized by the intervention of G protein-coupled receptor kinase 2 (GRK2). In several experimental models of heart failure, there has been reported the up-regulation of GRK2 and the loss of functioning of inhibitory α2-adrenoceptors resulting in enhanced release of adrenomedullary catecholamines. Given the importance of circulating catecholamines in the pathophysiology of heart failure, the recovery of α2-adrenergic modulation of the secretory response from chromaffin cells appears as a novel strategy for a better control of the patients with this cardiac disease.

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells.

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca(2+) during action potentials. Indeed, Ca(2+)-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K(+) currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca(2+)) Ca(2+) concentrations. Despite their low open probability under these conditions (V(50) of +146.8 mV), BK channels were responsible for more than 25% of the total K(+) efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K(+) channels. The other main source of fast voltage-dependent K(+) efflux at such a low Ca(2+) was a transient K(+) (I(A)-type) current activating with V (50) = -14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca(2+). Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca(2+) concentration, and suggest that they could do so also in the presence of extracellular Ca(2+), before Ca(2+) entering the cell facilitates their activity.

Subcellular distribution and early signalling events of P2X7 receptors from mouse cerebellar granule neurons.

The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 µM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 µM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.