Andrea Marcantoni

Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin

The mechanisms underlying the cardiac activities of synthetic growth hormone secretagogues (GHS) are still unclear. The natural ligand of the GHS receptors, i.e. ghrelin, classically binds the GHS receptor and exerts endocrine actions in acylated forms only; its cardiovascular actions still need to be investigated further. In order to clarify these aspects, we studied the effects of either the synthetic peptidyl GHS hexarelin (1 microM), or the natural ghrelin (50 nM) and the endogenous ghrelin derivatives des-Gln14-ghrelin (1-100 nM) and des-octanoyl ghrelin (50 nM), on the tension developed by guinea pig papillary muscle and on L-type Ca2+ current (ICa) of isolated ventricular cells. The binding of these molecules to ventricular cell membrane homogenates was also studied. We observed that all peptides reduced the tension developed at low frequencies (60-120 beats/min) in a dose-dependent manner. No alteration in cardiac contractility was induced by des-Gln14-ghrelin or des-octanoylated ghrelin when the endocardial endothelium had been removed or after cyclooxygenase blockade. Pretreatment with tyramine (2 microM) had no effect on the inotropic response induced by des-Gln(14)-ghrelin. No significant effect on I(Ca) of isolated ventricular cells was observed in the presence of des-Gln14-ghrelin (100 nM). The order of potency on the tension of papillary muscle was: des-octanoyl ghrelin > ghrelin = des-Gln14-ghrelin > hexarelin. This gradient of potency was consistent with the binding experiments performed on ventricular membranes where either acylated or unacylated ghrelin forms, and hexarelin, recognized a common high-affinity binding site. In conclusion, ghrelin, des-Gln14-ghrelin and des-octanoyl ghrelin, show similar negative inotropic effect on papillary muscle; as des-octanoyl ghrelin is peculiarly devoid of any GH-releasing activity, the cardiotropic action of these molecules is independent of GH release. The binding studies and the experiments performed both on the isolated cells and on papillary muscle after endothelium removal or cyclooxygenase blockade indicate that the cardiotropic action of natural and synthetic ghrelin analogues reflects the interaction with a novel GHS receptor (peculiarly common for ghrelin and des-octanoyl ghrelin), leading to release of cyclooxygenase metabolites from endothelial cells, as indicated by direct measurement of prostacyclin metabolite 6-keto-PGF(1alpha).

Loss of Cav1.3 Channels Reveals the Critical Role of L-Type and BK Channel Coupling in Pacemaking Mouse Adrenal Chromaffin Cells

We studied wild-type (WT) and Cav1.3−/− mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca2+ channel (LTCC) and BK channels that underlie the pacemaker current controlling spontaneous firing. Most WT-MCCs (80%) were spontaneously active (1.5 Hz) and highly sensitive to nifedipine and BayK-8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methyl ester). Nifedipine blocked the firing, whereas BayK-8644 increased threefold the firing rate. The two dihydropyridines and the BK channel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BK channels. WT-MCCs expressed equal fractions of functionally active Cav1.2 and Cav1.3 channels. Cav1.3 channel deficiency decreased the number of normally firing MCCs (30%; 2.0 Hz), suggesting a critical role of these channels on firing, which derived from their slow inactivation rate, sizeable activation at subthreshold potentials, and close coupling to fast inactivating BK channels as determined by using EGTA and BAPTA Ca2+ buffering. By means of the action potential clamp, in TTX-treated WT-MCCs, we found that the interpulse pacemaker current was always net inward and dominated by LTCCs. Fast inactivating and non-inactivating BK currents sustained mainly the afterhyperpolarization of the short APs (2–3 ms) and only partially the pacemaker current during the long interspike (300–500 ms). Deletion of Cav1.3 channels reduced drastically the inward Ca2+ current and the corresponding Ca2+-activated BK current during spikes. Our data highlight the role of Cav1.3, and to a minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs and open new interesting features about their role in the control of firing and catecholamine secretion at rest and during sustained stimulations matching acute stress.

Chronic hypoxia up‐regulates α1H T‐type channels and low‐threshold catecholamine secretion in rat chromaffin cells

α1H T‐type channels recruited by β1‐adrenergic stimulation in rat chromaffin cells (RCCs) are coupled to fast exocytosis with the same Ca2+ dependence of high‐threshold Ca2+ channels. Here we show that RCCs exposed to chronic hypoxia (CH) for 12–18 h in 3% O2 express comparable densities of functional T‐type channels that depolarize the resting cells and contribute to low‐voltage exocytosis. Following chronic hypoxia, most RCCs exhibited T‐type Ca2+ channels already available at −50 mV with the same gating, pharmacological and molecular features as the α1H isoform. Chronic hypoxia had no effects on cell size and high‐threshold Ca2+ current density and was mimicked by overnight incubation with the iron‐chelating agent desferrioxamine (DFX), suggesting the involvement of hypoxia‐inducible factors (HIFs). T‐type channel recruitment occurred independently of PKA activation and the presence of extracellular Ca2+. Hypoxia‐recruited T‐type channels were partially open at rest (T‐type ‘window‐current’) and contributed to raising the resting potential to more positive values. Their block by 50 μm Ni2+ caused a 5–8 mV hyperpolarization. The secretory response associated with T‐type channels could be detected following mild cell depolarizations, either by capacitance increases induced by step depolarizations or by amperometric current spikes induced by increased [KCl]. In the latter case, exocytotic bursts could be evoked even with 2–4 mm KCl and spike frequency was drastically reduced by 50 μm Ni2+. Chronic hypoxia did not alter the shape of spikes, suggesting that hypoxia‐recruited T‐type channels increase the number of secreted vesicles at low voltages, without altering the mechanism of catecholamine release and the quantal content of released molecules.

Cav1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca2+ channels (LTCCs, Cav1) open readily during membrane depolarization and allow Ca2+ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca2+-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Cav1.2 and Cav1.3), Cav1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Cav1.3−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Cav1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Cav1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Cav1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.

Nanocrystalline diamond microelectrode arrays fabricated on sapphire technology for high-time resolution of quantal catecholamine secretion from chromaffin cells.

The quantal release of oxidizable molecules can be successfully monitored by means of polarized carbon fiber microelectrodes (CFEs) positioned in close proximity to the cell membrane. To partially overcome certain CFE limitations, mainly related to their low spatial resolution and lack of optical transparency, we developed a planar boron-doped nanocrystalline diamond (NCD) prototype, grown on a transparent sapphire wafer. Responsiveness to applied catecholamines as well as the electrochemical and optical properties of the NCD-based device were first characterized by cyclic voltammetry and optical transmittance measurements. By stimulating chromaffin cells positioned on the device with external KCl, well-resolved quantal exocytotic events could be detected either from one NCD microelectrode, or simultaneously from an array of four microelectrodes, indicating that the chip is able to monitor secretory events (amperometric spikes) from a number of isolated chromaffin cells. Spikes detected by the planar NCD device had comparable amplitudes, kinetics and vesicle diameter distributions as those measured by conventional CFEs from the same chromaffin cell.

Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes.

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST‐BODIPY‐Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X‐Rhod‐1. Immunofluorescence experiments showed the presence of connexin‐43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage‐dependent calcium signaling in cocultured MSCs. MSCs showed a time‐dependent increase of labeling of ST‐BODIPY‐Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non‐fluorescent DHP, Nifedipine, completely abolished ST‐BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization‐induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage‐dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte‐like phenotype. J. Cell. Biochem. 100: 86–99, 2007.

Calcium channel types contributing to chromaffin cell excitability, exocytosis and endocytosis

Voltage gated Ca(2+) channels are effective voltage sensors of plasma membrane which convert cell depolarizations into Ca(2+) signaling. The chromaffin cells of the adrenal medulla utilize a large number of Ca(2+) channel types to drive the Ca(2+)-dependent release of catecholamines into blood circulation, during normal or stress-induced conditions. Some of the Ca(2+) channels expressed in chromaffin cells (L, N, P/Q, R and T), however, do not control only vesicle fusion and catecholamine release. They also subserve a variety of key activities which are vital for the physiological and pathological functioning of the cell, like: (i) shaping the action potentials of electrical oscillations driven either spontaneously or by ACh stimulation, (ii) controlling the action potential frequency of tonic or bursts firing, (iii) regulating the compensatory and excess endocytosis following robust exocytosis and (iv) driving the remodeling of Ca(2+) signaling which occurs during stressors stimulation. Here, we will briefly review the well-established properties of voltage-gated Ca(2+) channels accumulated over the past three decades focusing on the most recent discoveries on the role that L- (Cav1.2, Cav1.3) and T-type (Cav3.2) channels play in the control of excitability, exocytosis and endocytosis of chromaffin cells in normal and stress-mimicking conditions.

A diamond-based biosensor for the recording of neuronal activity.

We have developed a device for recording the extracellular electrical activity of cultured neuronal networks based on a hydrogen terminated (H-terminated) conductive diamond. GT1-7 cells, a neuronal cell line showing spontaneous action potentials firing, could maintain their functional properties for days in culture when plated on the H-terminated diamond surface. The recorded extracellular electrical activity appeared in the form of well-resolved bursts of fast and slow biphasic signals with a mean duration of about 8ms for the fast and 60ms for the slow events. The time courses of these signals were in good agreement with those recorded by means of conventional microelectrode array (MEAs) and with the negative derivative of the action potentials intracellularly recorded with the patch clamp technique from single cells. Thus, although hydrophobic in nature, the conductive H-terminated diamond surface is able to reveal the spontaneous electrical activity of neurons mainly by capacitative coupling to the cell membrane. Having previously shown that the optical properties of H-terminated diamond allow to record cellular activity by means of fluorescent probes (Ariano, P., Baldelli, P., Carbone, E., Giardino, A., Lo Giudice, A., Lovisolo, D., Manfredotti, C., Novara, M., Sternschulte, H., Vittone, E., 2005. Diam. Relat. Mater. 14, 669-674), we now provide evidence for the feasibility of using diamond-based cellular biosensors for multiparametrical recordings of electrical activity from living cells.

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO−) in the modulation of calcium current (ICa) in guinea pig ventricular myocytes with the whole‐cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half‐life of ONOO− at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O2−). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 μM) or SNAP (300 μM) increased basal ICa (50.3 ± 4.6%, n = 7 and 46.2 ± 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 μM) also stimulated basal ICa (44.6 ± 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 μM), although separately ineffective on ICa, enhanced the current if applied together (33.5 ± 0.7%, n = 7). The simultaneous donor of O2− and NO, SIN‐1 (500 μM), also stimulated basal ICa (22.8 ± 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 μM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 μM), ICa was still increased by SIN‐1 (32.0 ± 6.1%, n = 4 and 30.0 ± 5.4%, n = 8). Both Manganese(III)tetrakis(4‐benzoic acid) porphyrin chloride (MnTBAP, 100 μM) a ONOO− scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN‐1 on ICa (respectively −0.6 ± 4.1%, n = 4 and 3.6 ± 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN‐1, while it was enhanced by blocking the NO–cGMP pathway with the NO synthase inhibitor L‐NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP. J. Cell. Physiol. 197: 284–296, 2003© 2003 Wiley‐Liss, Inc.

Calcium channels in chromaffin cells: focus on L and T types

Voltage‐gated Ca2+ channels (Cav) are highly expressed in the adrenal chromaffin cells of mammalian species. Besides shaping action potential waveforms, they are directly involved in the excitation–secretion coupling underlying catecholamine release and, possibly, control other Ca2+‐dependent events that originate near the membrane. These functions are shared by a number of Cav channel types (L, N, P/Q, R and T) which have different structure–function characteristics and whose degree of expression changes remarkably among mammalian species. Understanding precisely the functioning of each voltage‐gated Ca2+ channels is a crucial task that helps clarifying the Ca2+‐dependent mechanisms controlling exocytosis during physiological and pathological conditions. In this paper, we focus on classical and new roles that L‐ and T‐type channels play in the control of chromaffin cell excitability and neurotransmitter release. Interestingly, L‐type channels are shown to be implicated in the spontaneous autorhythmicity of chromaffin cells, while T‐type channels, which are absent in adult chromaffin cells, are coupled with secretion and can be recruited following long‐term β‐adrenergic stimulation or chronic hypoxia. This suggests that like other cells, adrenal chromaffin cells undergo effective remodelling of membrane ion channels and cell functioning during prolonged stress conditions.