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ERG K⁺ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?¹

FRANCESCA GULLO, EVA ALES^*, BARBARA ROSATI³, MARZIA LECCHI⁴, ALESSIO MASI, LEONARDO GUASTI, MARÍA F. CANO-ABAD^*, ANNAROSA ARCANGELI, MANUELA G. LOPEZ^* and ENZO WANKE²

Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy;
^* Instituto Teófilo Hernando. Departamento de Farmacología, Universidad Autónoma de Madrid, Arzobispo Morcillo 4, E-28029 Madrid, Spain; and
Dipartimento di Patologia e Oncologia Sperimentali, Università di Firenze, Viale Morgagni 50, I-50134 Firenze, Italy

²Correspondence: Dipartimento di Biotecnologie e Bioscienze, Università de Milano-Biocca, Piazza della Scienza, 2, 20126 Milano, Italy. E-mail: enzo.wanke{at}unimib.it

SPECIFIC AIMS

HERG potassium channels were discovered as regulators of the duration of the heart action potential. The slowly decreasing action potential (AP) plateau shifts these channels to a less inactivated state that leads to an increasing outward K⁺ current (I_Kr), and this sustains further repolarization until complete repolarization is attained. Several mutations discovered in this channel are the origin of one (LQT2) of the long QT syndromes. It is known that syncope and sudden death may be precipitated by acute arousal, and ß-blockers have become standard prophylactic therapy.

HERG channels may sustain a process of spike frequency adaptation during long trains of spikes because, at rest, an outward ERG current (I_ERG) develops that is sufficient to inhibit firing. Thus, ERG channels contribute to the control of burst duration in smooth muscle, carotid body, lactotrophs, and human ß-pancreatic cells. This suggests that HERG channels may play a crucial role in other tissues in which excitability is of primary significance for neurotransmitter and hormone release.

Since the heart is under the action of catecholaminergic neurotransmitters we investigated the possibility that chromaffin cells control the release of catecholamines by using ERG channels. Although it has been suggested that ERG mRNA is present in sympathetic ganglia, more recently it has been shown that adult sympathetic neurons are devoid of functional ERG channels.

PRINCIPAL FINDINGS

1. erg genes are expressed in rat chromaffin cells
RNAse Protection Assay experiments show the presence of erg1 transcripts while the other two genes (erg2, erg3) were undetectable by this method. Erg1 resulted to be expressed at the protein level in a highly glycosylated form as reported in the heart.

2. ERG channels sustain a K⁺ current sensitive to specific ERG channels blockers
In voltage-clamped chromaffin cells, we observed a K⁺ current showing the typical biophysical and pharmacological properties of ERG channels.

3. Blockers of ERG channels modify the excitability of single chromaffin cell
In single chromaffin cells we have determined the electrical properties by using the perforated patch-clamp technique (Fig. 1 ). We routinely performed two tests with the following drugs: 1) nicotine to check the classical chromaffin response (a strong firing increase frequently associated to depolarization), and 2) WAY (a highly specific blocker of ERG channels) to check for hyperexcitability due to the block of ERG K⁺ channels (in 43% of the tested cells, n=28). Using other blockers, we tested and compared the expression and functioning of other K⁺ channels such as K_(Ca) and K_(ATP), which were not affected by WAY.

View larger version (61K): [in this window] [in a new window]   Figure 1. Pharmacological identification of chromaffin cells and the properties of histamine- and WAY-induced responses. A–C) The perforated current-clamp recordings of the response of the same cell during the application of 10 µM nicotine (nic), 10 µM histamine (his), and 1 µM WAY (way). See the line below. D–E) In another cell the same experiment as in (A), but only the histamine and WAY application are shown. The indicated bars have the following values: (A) 10s, 10 mV; (B) 10s; (C) 20s; (D), 10s; (E) 20s, 10 mV. F) In another cell the response elicited by WAY (shown by the continuous line below the recording) was analyzed in terms of instantaneous spike frequency (open circles, see the y-axis on the left). The two upper and lower insets show the type of firing and the shape of a single AP at the appropriate time scale before WAY application and before washout, respectively. The brief (0.8 s) negative-going deflections of the membrane potential are responses to -1 pA current pulses. G) An example (same cell) of the dose-response experiments done with applications of different concentrations (1, 0.7, and 0.4 µgr/ml) of ErgTx. H) Dose-response relationship resulting from different experiments: The fractional firing frequency increase vs. [ErgTx] (number of experiments was 2, 3, 5, 6, 6, 2 at [ErgTx] of 0.1, 0.2, 0.4, 0.7, 1, and 2 µgr/ml).

4. Catecholamine release is increased by the ERG channels blockers
Single cells were used to measure simultaneously the cytosolic Ca²⁺ concentration and, by carbon fiber electrochemical detection, the catecholamine secretion (Fig. 2 ) after brief (5–10 s) applications of nicotine and high [K⁺]_o with and without an ERG channel blocker. When ERG channels were inhibited, the catecholamine release was increased (in 59% of the tested cells, n=32).

View larger version (21K): [in this window] [in a new window]   Figure 2. [Ca2+]c and catecholamine release simultaneously observed in a chromaffin cell during application of high [K+]o, WAY, and histamine. The upper panel shows a typical fluorometric tracing, corresponding to the [Ca2+]c, in a rat chromaffin cell stimulated for 5 s with 70 mM K+, for 1 min with 1 µM way 123,398 (WAY), and for 30 s with 10 µM histamine (His). The lower panel corresponds to simultaneous recording of the neurotransmitter release obtained with the carbon fiber technique and shows a clear correlation with the [Ca2+]c, signal. The latency for WAY’s action can be observed in the [Ca2+]c and amperometric tracings. The inset shows detailed secretory spikes, each corresponding to the release of catecholamines from a single granule, obtained in the presence of the ERG channel blocker. Application of WAY shows a slight increase in the basal amperometric signal by itself.

5. ERG channels are preferentially expressed in epinephrine-containing cells
To understand whether epinephrine-(E cells) or norepinephrine-containing (NE cells) (or both) were implicated in the inhibitory ERG-mediated mechanism, we adopted two strategies. First, during the patch clamp and carbon fiber experiments, we routinely checked chromaffin cells for a histamine response because it is known that E cells preferentially respond to this stimulus. In the perforated patch and carbon fiber experiments, 84% and 78% of the cells responded to histamine. These percentages agree with the fact that E cells account for 82% of all chromaffin cells in rat and that virtually all respond to histamine. These results substantially confirmed that our rat chromaffin cells behave like those studied earlier using other methods. Second, to correlate the electrophysiological and secretion data described above, we performed immunocytochemistry experiments to verify whether ERG channels were expressed randomly or confined to one of the two principal types of chromaffin cells (E or NE cells). We used antibodies against ERG channels and PNMT (an antigen present exclusively in E-containing cells). Rat chromaffin cells positive for ERG were also positive for PNMT staining. Conversely, cells negative for ERG were negative for PNMT staining. These results indicate that ERG channels are preferentially located in E but not in NE cells. For control purposes we performed double immunostaining in C2C12 cells, a murine myoblast line in which we detected neither ERG currents nor erg1 RNA (not shown). As expected, these cells showed negative immunostaing against PNMT and ERG antibodies.

CONCLUSION AND SIGNIFICANCE

Our results demonstrate that selective ERG channels blockers cause an increase in the firing frequency or a profound depolarization of chromaffin cells, and therefore lead to the considerable increase in catecholamine secretion that we demonstrate is derived mainly from E cells. This is the first demonstration that in addition to cardiac tissue ERG channels are expressed in the adrenal medulla, a tissue closely interconnected with the heart as a result of catecholaminergic activity. In ventricular myocytes and chromaffin cells, ERG channels respectively play the two different roles of action potential repolarization and firing frequency accommodation (or inhibition).

Our results show that ERG channels contribute efficiently to the physiological function of chromaffin cells in terms of excitability and epinephrine release. As epinephrine is one of the most important neurotransmitters controlling cardiac function and ERG channels expressed in cardiac myocytes are crucial in determining the repolarization of the action potential (and thus the QT interval), our results could have interesting and novel implications in the context of cardiac LQT syndromes (LQTS).

Studies of genotype–phenotype correlations have shown that specific triggers such as exercise, emotion, and sleep/rest are distributed differently, the first being more common in LQT1 patients, the second among LQT2 patients, and the third among those with LQT3. Syncope and sudden death have been described in association with loud noises, implicating adrenergic stimuli, such as the ringing of an alarm clock, and these provided the rationale for the ß-blocker anti-adrenergic therapy that has proved to have a potent protective effect against LQT1 and LQT2 syndromes.

All of these findings highlight the cooperative relationship between the catecholaminergic system and heart K⁺ channels in originating LQT1 and LQT2 syndromes; although this connection has been suggested and therapeutically used, no other research has suggested that the same gene may be involved in this tissue relationship.

We suggest that LQT2 patients untreated with ß-blockers, before being awakened by the noise of their alarm clocks, have a low heart rate and consequently their action potential duration is particularly long because of the loss-of-function of HERG channels. Being in deep sleep, the majority of their unstimulated chromaffin cells release small amounts of epinephrine. At the time of sudden awakening, the chromaffin cells greatly stimulated by the cholinergic input and successive massive secretion of epinephrine (not inhibited by ERG channel-sustained feedback) can reach the heart and prolong AP to the point of fibrillation and sudden death. If treated with ß-blockers, such patients can survive.

View larger version (45K): [in this window] [in a new window]   Figure 3. Diagram summarizing the results of the paper, known facts about the LQT2 syndrome (inset); arrows indicate discovered suggested links.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0200fje; to cite this article, use FASEB J. (December 17, 2002) 10.1096/fj.02-0200fje

³ Present address: Department of Physiology and Biophysics, SUNY at Stony Brook, Stony Brook, New York, USA

⁴ Present address: Department of Physiology, CMU, 1211 Geneva 4, Switzerland

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