Barbara Rosati

Glucose- and arginine-induced insulin secretion by human pancreatic {beta}-cells: the role of HERG K+ channels in firing and release

The human ether‐a‐go‐go‐related genes (herg) are expressed in tissues other than heart and brain where the HERG K+ channels are known to regulate the repolarization of the heart action potential and the neuronal spike‐frequency accommodation. We provide evidence that herg1 transcripts are present in human pancreatic islets that were used to study both insulin secretion and electrical activity with radioimmunoassay and single cell perforated patch‐clamp techniques, respectively. Glucose‐ and arginine‐induced islets insulin secretion data suggested a net increase of release under perfusion with antiarrhythmic drugs known to selectively block HERG channels. Indeed we could routinely isolate a K+ current that was recognized as biophysically and pharmacologically similar to the HERG current. An analysis of the glucose‐ and arginine‐induced electrical activity (several applications during 30 min) in terms of firing frequency and putative insulin release was done in control and in the presence of selective blockers of HERG channels: the firing frequency and the release increased by 32% and 77%, respectively. It is concluded that HERG channels have a crucial role in regulating insulin secretion and firing of human β‐cells. This raises the possibility that some genetically characterized hyperinsulinemic diseases of unknown origin might involve mutations in the HERG channels.—Rosati, B., Marchetti, P., Crociani, O., Lecchi, M., Lupi, R., Arcangeli, A., Olivotto, M., Wanke, E. Glucose‐ and arginine‐in‐duced insulin secretion by human pancreatic β‐cells: the role of HERG K+ channels in firing and release. The FASEB J. 14, 2601–2610 (2000)

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether‐a‐go‐go‐related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long‐QT syndrome and Drosophila ‘seizure’ mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50 = 16±1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure‐function relationships and novel pharmacological tools with potential therapeutic efficacy.—Gurrola, G. B., Rosati, B., Rocchetti, M., Pimienta, G., Zaza, A., Arcangeli, A., Olivotto, M., Possani, L. D., Wanke, E. A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom. FASEB J. 13, 953–962 (1999)

HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an up-regulation of alpha(v)beta(3) integrin in the preosteoclastic leukemia cell line FLG 29.1

Integrin receptors have been demonstrated to mediate either “inside-to-out” and “outside-to-in” signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (VREST) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K+ channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/αvβ3 integrin and calcitonin receptor. An early activation of HERG current (IHERG), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the β1 integrin subunit activation, through the involvement of a pertussis-toxin sensitive Gi protein, and appears to be a determinant signal for the up-regulation of αvβ3 integrin, as well as for the increased expression of calcitonin receptor.

ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?

The ether‐a‐go‐go‐related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike‐frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single‐cell level by means of perforated patch‐clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N‐methyltransferase and ERG channels suggested expression of these channels in epinephrine‐ but not in norepinephrine‐containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long‐QT running heart.

Isolation of a Long-Lasting eag-Related Gene-Type K+ Current in MMQ Lactotrophs and Its Accommodating Role during Slow Firing and Prolactin Release

Native rat lactotrophs express thyrotrophin-releasing hormone-dependent K+ currents consisting of fast and slow deactivating components that are both sensitive to the class III anti-arrhythmic drugs that block the eag-related gene (ERG) K+ current (IERG). Here we describe in MMQ prolactin-releasing pituitary cells the isolation of the slowly deactivating long-lasting component (IERGS), which, unlike the fast component (IERGF), is insensitive to verapamil 2 μm but sensitive to a novel scorpion toxin (ErgTx-2) that hardly affects IERGF. The time constants of IERGS activation, deactivation, and recovery from inactivation are more than one order of magnitude greater than in IERGF, and the voltage-dependent inactivation is left-shifted by ∼25 mV. The very slow MMQ firing frequency (∼0.2 Hz) investigated in perforated patch is increased approximately four times by anti-arrhythmic agents, by ErgTx-2, and by the abrupt IERGSdeactivation. Prolactin secretion in the presence of anti-arrhythmics is three- to fourfold higher in comparison with controls. We provide evidence from IERGS andIERGF simulations in a firing model cell to indicate that only IERGS has an accommodating role during the experimentally observed very slow firing. Thus, we suggest that IERGS potently modulates both firing and prolactin release in lactotroph cells.

Modulation of HERG Current and herg Gene Expression during Retinoic Acid Treatment of Human Neuroblastoma Cells: Potentiating Effects of BDNF

The modulation of herg gene and HERG currents (I(HERG)) was studied in SH-SY5Y neuroblastoma (NB) cells treated with all-trans-retinoic acid (RA) in the absence or presence of the neurotrophin brain-derived neurotrophic factor (BDNF). Both treatments produced a strong increase in the percentage of cells differentiated along the neuronal pathway, with an orientation to a cholinergic phenotype, while a minority of cells displayed a glial phenotype particularly evident after long-term exposure to the inducers. Differentiation of NB cells was accompanied by an increase in herg gene transcription, which attained its maximum after 6 days of treatment with RA and was not further increased by BDNF. This effect evidently reflected on HERG currents: In fact, RA produced an increase in HERG current density which was strongly potentiated by BDNF. Moreover, RA treatment affected the biophysical properties of I(HERG), inducing an increase in the deactivation time constant and a left shift of the activation curve. These effects were not substantially affected by BDNF. This modulation of I(HERG) influenced the value of the resting potential (V(REST)), which resulted significantly hyperpolarized in (RA with or without BDNF)-treated cells. Interestingly, these effects were absent in the glial population, which prevailed in cultures after long-term exposure to the inducers. On the whole, we demonstrate that besides expressing IRK currents, NB cells display another strategy to hyperpolarize their V(REST), based on the appropriate modulation of HERG currents. Different from what happens in normal neuroblast development, the latter are never lost by cancer cells despite the progression of these cells along the neuronal differentiative pathway, raising intriguing questions about the role of HERG currents in tumour behavior.

erg gene(s) expression during development of the nervous and muscular system of quail embryos

The expression pattern of K(+) currents is the principal regulator of electrical activity during development of the nervous and muscular system. We report here a study showing the expression pattern of HERG K(+) currents-encoding (erg) genes in various nervous and muscular tissues at different stages of quail embryo development.

Excitable properties in astrocytes derived from human embryonic CNS stem cells

Although it is widely believed that astrocytes lack excitability in adult tissue, primitive action potential‐like responses have been elicited from holding potentials negative to −80u2003mV, in cultured and injury‐induced gliotic rodent astrocytes and in human glia under pathological conditions such as glioblastomas and temporal lobe epilepsy. The present study was designed to investigate the properties of astrocytes (identified by immunoreactivity for glial fibrillary acidic protein) derived from multipotent human embryonic CNS stem cells and cultured for 12–25u2003days in differentiating conditions. We describe here for the first time that brief (1u2003ms) current pulses elicit spikes from a resting potential (VREST) of ≈u200a−37u2003mV and, more interestingly, that spontaneous firing can be occasionally recorded in human astrocytes. A voltage‐clamp study revealed that in these cells: (i) the half‐inactivation of the tetrodotoxin (TTX)‐sensitive Na+ channels is around VREST; (ii) the delayed rectifier K+ current is very small; (iii) the ever‐present transient outward A‐type K+ channels are paradoxically capable of inhibiting the action potentials elicited from a negative membrane potential (−55 to −60u2003mV); and (iv) inwardly rectifying currents are not present. The responses predicted from a simulation model are in agreement with the experiments. As suggested by recent studies, the decrease of Na+ channel expression and the changes of the electrophysiological properties during the postnatal maturation of the CNS seem to exclude the possibility that astrocytes may play an excitable role in adult tissue. Our data show that excitability and firing should be considered an intrinsic attribute of human astrocytes during CNS development. This is likely to have physiological importance because the role of astrocytes during development is different from the [K+]o‐buffering role played in adult CNS, namely the glutamate release and/or the guiding of migrating neurons.

Inhibition of depolarization-induced [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by some second-generation H1 receptor antagonists through blockade of store-operated Ca2+ channels (SOCs)

In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration ([K(+)](e)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high [K(+)](e)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM [K(+)](e)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated [K(+)](e). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.