Naunyn-Schmiedeberg’s Arch Pharmacol (2000) 361 : 120–126 Digital Object Identifier (DOI) 10.1007/s002109900183

O R I G I N A L A RT I C L E

T. Meyer · M. Oles · L. Pott

Ca2+ entry but not Ca2+ release is necessary for desensitization of ETA receptors in airway epithelial cells

Received: 7 September 1999 / Accepted: 2 November 1999 / Published online: 8 December 1999 © Springer-Verlag 1999

Abstract Ca2+ transients evoked by endothelin-1 (ET-1) were measured in single cells of a human tracheal epithelial cell line using the fluorescent Ca2+ indicator fura-2. In line with a previous study, a single exposure to ET-1 (10 nM) for 10–20 s resulted in a long-lasting desensitization to a subsequent challenge by the peptide, without affecting sensitivity to agonists for other Ca2+-mobilizing receptors such as P2y or H1, respectively. In the absence of extracellular Ca2+ ET-1 elicited a Ca2+ signal of comparable amplitude as in the presence of extracellular Ca2+ but of shorter duration. Exposure to ET-1 in the absence of Ca2+ caused significantly less desensitization. Inhibition of the Ca2+ entry component of the Ca2+ transient by means of SK&F 96365, an inhibitor of Ca2+ entry, had effects comparable to Ca2+ removal. The Ca2+ transient was shortened but not significantly reduced in amplitude, and desensitization was reduced in the presence of the compound. These data demonstrate that desensitization of ETA receptors (ETAR) is promoted by transmembrane Ca2+ entry but not by Ca2+ release. Key words Epithelial cell · Airway · Endothelin-1 · Desensitization · Ca2+ signalling · Fura

Introduction Endothelins have first been described in 1988 as most potent vasoconstrictors produced by vascular endothelial cells (Yanagisawa et al. 1988). Since then actions of endothelins – predominantly the ET-1 isoform – have been demonstrated in a number of organ systems and cell types (McMillen and Sumpio 1995). Apart from the vascular endothelium, one of the richest sources for ET-1 seems to

T. Meyer · M. Oles · L. Pott (✉) Institut für Physiologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany e-mail: [email protected], Fax: +49-234-3214449

be the airway epithelium (Black et al. 1989). In apparent analogy to the system of the vascular endothelial layer, airway epithelial cells not only produce endothelin(s) but also express high-affinity functional endothelin receptors (Ninomiya et al. 1995). Neither their physiological role nor the specific properties of ET-1-induced signalling in airway epithelial cells presently are understood. As the various actions of endothelins in different cell types seem to be mediated – at least partly – by intracellular Ca2+ mobilization (e.g. Pollock et al. 1995 for review), more insight into the specific properties and underlying mechanisms of endothelin-induced Ca2+ signals will help to understanding cell-specific functions of this class of signalling peptides and their receptors. An unusual property of signalling via ETAR which impedes to study actions of ET-peptides in a systematic manner is the limited reproducibility of responses. One example is the rise in blood pressure, which lasts for several hours following a single injection of endothelin. On the cellular level we have shown previously that a single exposure of airway epithelial cells for as short as 10–20 s caused a reduction or a complete loss of sensitivity to a subsequent exposure. Complete recovery from this desensitization required more than 20 h (Oles et al. 1997). It is likely to reflect a rapid endocytotic internalization of the surface receptors (Chun et al. 1995). In analogy to Ca2+ signals linked to other receptor/agonist systems in a variety of cells, Ca2+ transients in airway epithelial cells are composed of a component which is independent of extracellular Ca2+ and a plateau-like component which is absent in Ca2+-free solution. This component is assumed to reflect inward Ca2+ current through a pathway which shares similarity with the store depletion-operated Ca2+ entry typical of many types of non-excitable cells. In the present study using measurement of ET-1evoked Ca2+ transients we showed that desensitization of ETAR requires a rise in [Ca2+]i by Ca2+ entry, whereas a Ca2+ transient of comparable amplitude caused exclusively by Ca2+ release from intracellular stores failed to promote desensitization.

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Materials and methods Cell culture. Experiments were performed on an epithelial cell line (passage number 19–32) of human origin, immortalized by transfection with the HPV-18 E6 and E7 genes (Yankaskas et al. 1993). The clone was kindly provided by Dr. J.R. Yankaskas (Chapel Hill, N.C., USA). Cultures grown in tissue culture flasks (35 mm2; Falcon, Dreieich, Germany) to confluency were trypsinized and five little droplets (20 µl) of cell suspension (about 3×105 cells/ml), spaced by about 10 mm, were placed on the bottom of a 35-mm tissue culture dish, where they formed confluent islets of 1–2 mm in diameter within 2 days. In order to prevent contamination by microsuperfusion with ET-1-containing solution, each measurement or sequence of measurements, respectively, was performed on an individual islet. Cultures were used between days 4 and 11 after trypsinization and re-plating. No time-dependent differences in the behaviour to be described were found within this period of time. At earlier times sensitivity to ET-1 was variable and in some cultures completely absent. Culture medium. The culture medium was a serum-free DMEM/Nut Mix F12 (Gibco BRL, Dreieich, Germany) supplemented according to the original publication (Yankaskas et al. 1993) with ITS (insulin, transferrin, selenium supplement; 10 µg/ml; BoehringerMannheim, Germany), hydrocortisone (1 µM; Sigma, Deisenhofen, Germany), pituitary extract (3.7 µg/ml; Sigma), EGF (25 ng/ml; Sigma), cholera toxin (10 ng/ml; Sigma), kanamycin (0.5 mg/ml; Sigma), gentamycin (0.5 mg/ml; Sigma). This medium is not well defined and affects various signal traduction pathways in an unpredictable way. As, however, this cell line has been characterized in a number of previous studies using this particular medium, which is necessary for maintanance of its phenotype, we refrained from analysing putative effects of any of the constituents on properties of receptor-linked Ca2+ signalling. Chemicals. Other chemicals were from the following sources: fura2/AM, Molecular Probes Europe (Leiden, Netherlands); ET-1, Calbiochem (Bad Soden, Germany); SK&F 96365 was obtained from Biomol (Hamburg, Germany), ATP and histamine from Sigma (Steinheim, Germany). Single cell fura-2 measurements. Prior to an experiment medium was removed and replaced by the standard experimental solution containing fura-2/AM (7 µM). The dishes were placed in the incubator at 37°C for 35 min. Thereafter the cultures were washed with excess of fura-2/AM-free solution and placed in the incubator for another 60–120 min in order to allow for hydrolysis of the ester. The culture dish was placed on the temperature-controlled (35°C) stage of the microscope and was perfused at 250 µl/min with standard solution at 35°C. Standard solution had the following composition (in mM): NaCl 140; KCl 5.4; CaCl2 1.8; MgCl2 1.0; Hepes/ NaOH 10.0, pH adjusted to 7.4; glucose 5.0. Ca2+-free solution was made without CaCl2; EGTA was added to a final concentration of 2 mM. Dual-wavelength measurements of fura-2 fluorescence were performed using a setup based on a Zeiss Axiovert microscope to which a custom-made dual-wavelength excitation device without moving optical components was adapted (Bals et al. 1990). The microscope was equipped with a long-working-distance objective (Achroplan 40, 0.6 corr.; Zeiss, Cologne, Germany) which allowed for sufficient intensity also at 340 nm excitation wavelength from cells growing on conventional tissue-culture plastics rather than glass-coverslips. Fluorescence was recorded by means of a photomultiplier tube (Hamamatsu R4632; Munich, Germany) from a selected field of a group of confluent cells limited to a single cell using a variable diaphragm. Single cells or groups of a few cells usually did not or very poorly respond to ET-1. The wavelength for excitation of the dye was altered between 340 nm and 380 nm usually at 200 s–1. Emission was measured at 512 nm. Photomultiplier signals were background-corrected and the ratio (f340/f380) was calculated. Fluorescence signals and the ratio were stored on the hard

disc of a computer. Offline analysis of the signals was performed by means of custom-made software. Application and washout of agonists to a group (islet) of cells was performed by means of a solenoid switch-operated superfusion device. The tip diameter of the common outlet tube was 0.5 mm. The half-time of exchange between two solutions achieved by this local perfusion device was previously determined as less than 200 ms and thus was not limiting to the time course of the response to an agonist.

Results As shown in Fig. 1, in line with our previous study (Oles et al. 1997), a brief challenge (10–20 s) of a single epithelial cell by 10 nM ET-1 resulted in a Ca2+ transient which lasted for several minutes. The width at 90% recovery under standard conditions was always >6 min and was independent of the duration of exposure to ET-1. The amplitude was comparable to that of Ca2+ signals evoked by a saturating concentration of either histamine (100 µM) or the purinergic agonist ATP (100 µM). A second exposure to the peptide following a washing period of totally 300 s resulted in a transient of much smaller amplitude, whereas a third application of ET-1 failed to evoke a measurable change in [Ca2+]i. This sequence of measurements was bracketed by brief exposures to a saturating concentration of either histamine (100 µM) or ATP (100 µM) which yielded identical fura-2 signals. Whereas almost every cell tested responded to ATP or His with a sizable Ca2+ signal, the amplitude of the ET-1-induced signal ranged from virtually 0% to 120% of the signal evoked by histamine. Only data from cells were included in this study in which the ET-1-induced signal (10 nM) was ≥80% of the signal induced by 100 µM histamine (65% of cells studied). In a fraction of cells there was a gradual decay of the fura-2 ratio (rundown). In those cases a linear extrapolation was made between the first and the last reference signal induced by histamine or ATP, respectively. The homologous nature of the loss in sensitivity to ET-1 demonstrates that it does not reflect a desensitization on the post-receptor level such as the Ca2+ signalling ma-

Fig. 1 Homologous desensitization of Ca2+ transients induced by ET-1. Histamine (His; 100 µM) and ET-1 (ET; 10 nM) were applied as indicated. The arrowheads correspond to washing periods of 250 s in duration

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Fig. 2 Comparison of Ca2+ transients elicited by ET-1 in the absence of Ca2+ in the bathing solution (Ca2+-free plus 2 mM EGTA) and at 2 mM Ca2+. The signal labelled Ca2+-free was recorded first. Exposure to ET-1 was followed by 5-min superfusion with Ca2+-free solution and 2-min standard (2 mM Ca2+) solution

chinery, but reflects a genuine property of the ETAR. It has been shown previously that recovery from the type of desensitization studied here is a slow process with a halftime of around 15 h in terms of recovery of the size of the fluorescence ratio signal. Like Ca2+ signals induced via activation of various 2+ Ca -mobilizing receptors in different types of cells, the ET-1-induced Ca2+ transient is composed of a component caused by release from internal stores and a component due to transmembrane entry, most likely via a pathway reflecting capacitative (store depletion-activated) Ca2+ entry. Figure 2 illustrates that a transient evoked by ET-1 in the absence of extracellular Ca2+ has a comparable amplitude as in the presence of Ca2+ but is much shorter in duration; the width of the signal at 90% recovery was 96±39 s (n=56) in Ca2+-free solution vs. 382±133 s at 2 mM [Ca2+]o (n=86). In this experiment the signal was first recorded in Ca2+-free condition, this was followed by a 3min wash in Ca2+-free solution. Thereafter the cell was superfused with Ca2+-containing solution for another 3 min before the next challenge by ET-1. The amplitudes of both signals were identical, i.e. the first exposure to ET-1 – in the absence of Ca2+ – did not desensitize the ETAR. Desensitization did not occur when the interval between the exposure to ET-1 in the absence of Ca2+ and re-admission of Ca2+ was at least 250 s, as illustrated in Fig. 3. The ET-1-induced transients in Fig. 3A were separated by a total of 530 s. This time interval consisted of a period ranging between 300, 100 or 50 s, respectively, in Ca2+-free solution plus a period of 200, 400 or 450 s, respectively, of superfusion with 2 mM Ca2+. In each run 30 s prior to exposing the cell again to ET-1,

Fig. 3A–C Ca2+-free solution outlasting exposure to ET-1 is necessary to prevent desensitization. A Recording of Ca2+ signals from one representative cell. ATP (at 2 mM Ca2+) was used at a saturating concentration of 100 µM. ET-1 concentration was 10 nM. Duration of exposure to agonist was 10 s. The filled arrowheads denote interruptions of 500 s in duration consisting of variable Ca2+-free and Ca2+-containing periods (see text). The time indicated represents the duration of superfusion with Ca2+-free solution. B Summarized data (mean values) from a total of four cells. The amplitude is expressed as fraction of the signal evoked by the first exposure to ET-1. C Sample recording of Ca2+ reloading after eliciting a Ca2+ transient in the absence of extracellular Ca2+

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Fig. 4 Repetitive activation of Ca2+ transients in the absence of extracellular Ca2+. The filled arrowheads indicate a gap of 500 s in duration consisting of a Ca2+-free period of 300 s and a period in standard (2 mM Ca2+) solution of 200 s

Ca2+-containig solution was replaced by Ca2+-free solution. Whereas the long (300 s) Ca2+-free period resulted in a signal of identical amplitude as the first (reference) signal, the subsequent transients were decreased as the Ca2+free period became shorter. This does not reflect a difference in loading of the intracellular Ca2+ stores, since the period for loading was longer for the smaller (more desensitized) signals. The summarized data from four experiments of this type in Fig. 3B demonstrate that for t≥250 s in Ca2+-free solution complete recovery was observed. During the Ca2+ re-loading period (i.e. upon changing from Ca2+-free to Ca2+-containing solution) there was a transient rise in [Ca2+]i, most likely reflecting store depletion-operated Ca2+ entry. An example is illustrated in Fig. 3C. For long-term experiments as in Fig. 3A signal recording was discontinued during the Ca2+-free and Ca2+loading periods in order to minimize bleaching and photodynamic damage. The period subsequent to ET-1 exposure during which desensitization could be induced by switching to Ca2+containing solution might be related to the slow dissociation rate of the peptide from its receptor. It is also conceivable and not contradictory to this hypothesis that this time window is determined by activity of the Ca2+ entry mechanism, which contributes to the Ca2+ transient for several hundred seconds after an exposure to ET-1 of only 10 s in duration as shown previously (Oles et al. 1997; see also Fig. 3C). Using a protocol of Ca2+-free and Ca2+-reloading periods, multiple Ca2+ transients with little or no desensitization could be recorded from an individual cell. A representative example is illustrated in Fig. 4. Ca2+-free and Ca2+-reloading periods were 300 s and 200 s, respectively. Taken together these data demonstrate that agonist-induced desensitization of the ETAR depends on the presence of Ca2+ in the extracellular solution. This could either reflect a requirement for extracellular Ca2+ as such, or alternatively it could mean that Ca2+ entry is required for desensitization of this receptor. To distinguish between these two hypotheses, we used a compound (SK&F

Fig. 5 Repetitive activation of transients in the presence of SK&F 96365 (15 µM). The arrowheads indicate gaps of 500 s in duration composed of a Ca2+-free period (300 s) and a reloading period (2 mM Ca2+) of 250 s

96365) which has been shown to inhibit Ca2+ entry (Merritt et al. 1990; Iwamuro et al. 1998), though with a specificity which at present is not well defined . As shown in Fig. 5, in the continuous presence of this compound (15 µM), ET-1-induced Ca2+ signals have a width at 90% recovery in the order of 100 s (107±24 s; n=8 cells) comparable to signals obtained in Ca2+-free solution, i.e. they are lacking the much longer lasting entry-component. The compound did neither affect the amplitude of Ca2+ signals evoked by histamine or ATP nor the amplitude of the ET1induced transient in the presence of Ca2+ normalized to the histamine-evoked signal (data not shown). Repetitive exposures to ET-1 yielded signals of almost identical amplitude. Taking into account the rundown of the histamine-induced signal in this particular experiment, there was virtually no desensitization. Thus, blocking Ca2+ entry results in removal of agonist-induced long-term desensitization of ETAR. Both Ca2+-free extracellular solution and the entry blocker SK&F 96365 resulted in a shortening of the ET-1-induced Ca2+ signal without significantly affecting its amplitude. The summarized data illustrating the effects of Ca2+ removal and SKF 96365 on the amplitude of signals evoked by a second and third exposure to ET-1 are shown in Fig. 6. As the duration of the ET-1-induced Ca2+ transient is substantially shortened by Ca2+ removal as well as by SK&F 96365, it cannot be excluded that a long-lasting rise in [Ca2+]i as such results in a reduction in responsiveness of the ETAR-linked signalling pathway, irrespective of its source. Figure 7 shows a representative experiment (n=5) to test for this hypothesis. After eliciting the standard reference signal using 100 µM histamine, ET-1 was applied for 20 s in the absence of Ca2+. This was followed by a long-lasting (100 s) exposure to histamine, which yielded a corresponding long-lasting rise in [Ca2+]i. Taking into account a moderate rundown of the signal evoked

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Fig. 6 Summarized data illustrating removal of desensitization by Ca2+-free solution or SK&F 96365 in the presence of Ca2+. Responses to second and third exposures to 10 nM ET-1 were normalized to the amplitude of response to the first exposure. Experimental protocols were as in Figs. 4 and 5

Fig. 7 A long-lasting Ca2+ transient induced by histamine does not affect sensitivity to ET-1. Agonists were superfused as indicated by the horizontal bars. Open arrowheads correspond to gaps of 200 s in the presence of Ca2+ (2 mM). The filled arrowhead denotes a period of 250 s Ca2+-free plus 250 s 2 mM Ca2+

by histamine, the amplitude of a transient evoked by 10 nM ET-1 in the presence of Ca2+ subsequent to the longlasting pulse of histamine was not affected. In line with the data described above, the second ET-1-induced signal was reduced by about 80%, confirming that desensitization of ETAR was operating normally in this cell. Because of the irreversible nature of actions of ET-1 it has been difficult, if not impossible, to measure a quantitative concentration-response relation in cellular experimental models. A single exposure to the peptide usually results in a substantial loss in sensitivity and, if no particular precautions are taken, contamination to an unknown degree of other cells in the same culture dish. As the absolute amplitude of ET-1-induced transients is variable in individual cells, also with regard to an internal standard, such as a transient evoked by a conventional Ca2+-mobilizing agonist, also measurements of concentration-re-

Fig. 8A–B ET-1 concentration-response curve. A Sample recordings from one individual cell. The traces were separated by gaps of 500 s in duration (250 s Ca2+-free; 250 s 2 mM Ca2+). ET was superfused in the absence of Ca2+ as shown in previous figures. B Summarized data. Signals were normalized to the amplitude of the transient elicited by 10 nM ET-1 in each cell. Data are mean values ± SD from 9–11 cells except for the highest concentration (50 nM) which represents the average of two measurements

sponse curves using single exposures per cell is hampered. By means of the experimental protocol described above it was possible for the first time to obtain functional concentration-response data for this peptide. Figure 8A shows sample recordings from one cell of Ca2+ transients induced in the absence of extracellular Ca2+ by various concentrations of ET-1 and, for standardization, by 10 µM ATP. Summarized data from 9–11 cells per concentration have been plotted in Fig. 8B. The curve was fitted using simple saturation kinetics with an EC50 of 1.06 nM and a slope factor of 0.94. Although the EC50 obtained in a functional study does not necessarily correspond to the dissociation constant of the agonist-receptor interaction

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(Stephenson 1956), the value in the low nanomolar range is in line with the high affinity of ETA receptors for ET-1 determined in radioligand binding assays in various cell types (e.g. Kobayashi et al. 1994).

Discussion In many systems studied, endothelin receptors are coupled to the phospholipase C pathway, resulting in Ca2+ transients composed of release from InsP3-sensitive intracellular stores and Ca2+ entry, most likely reflecting the capacitative (store depletion-activated) entry pathway (e.g. Xuan et al. 1994; Yang et al. 1995), although evidence has been provided that different entry pathways might become activated by ET-1 in parallel (Iwamuro et al. 1999). In comparison with conventional receptors converging on the PLC-InsP3 signalling cascade, ET-1-induced Ca2+ transients are characterized by their much longer duration and by a lack in reproducibility in an acute experiment. The latter has been demonstrated to reflect a novel type of long-term desensitization. (Oles et al. 1997). It should be noted that the term ‘desensitization’ has not been coined to a distinct mechanism but is normally used as a generic term to refer to any process whereby receptors become refractory or less responsive to a given stimulus (DebBurman and Hosey 1995). Whereas the knowledge about mechanisms contributing to desensitization is rather detailed for the prototypic β2-adrenergic receptor (see Krupnick and Benovic 1998; Pitcher et al. 1998 for recent reviews), much less is known about other GPCRs. There is no doubt that other mechanisms of regulating GPCRs exist than those described for the β2AR (cf. Bünemann et al. 1999), but also that one type of GPCR can undergo desensitization by more than one mechanism. Phosphorylation of ETAR by overexpressed receptor kinases (GRK2, -5, -6) concomitant with various degrees of desensitization has been demonstrated in a heterologous expression system (Freedman et al. 1997) using an inositol phosphate assay as readout. If this represents a genuine mechanism operating in a differentiated system or merely reflects overexpression of the kinases, however, remained open. More recently it has been demonstrated that cardiac atrial ETAR in their native environment can be desensitized via PKC as well as PKA, depending on the animal species (rat vs. guinea pig), despite complete identity of putative phosphorylation sites between these two species (Ono et al. 1998). Thus Chun et al. (1995) have demonstrated that ETA receptors, heterologously expressed in CHO cells, undergo a rapid agonist-dependent internalization in caveolae-like structures, with the intact agonist remaining bound to the receptor for several hours. These authors suggested the interesting hypothesis that the internalized complexes might continue to act as signalling elements and thus could represent a novel mode of long-term signal transduction via a heptahelical receptor.Rapid endocytosis of ETAR concomitant with a loss in agonist sensitivity may also occur upon binding of an antagonist (BQ123), i.e. in the absence of downstream signalling (Bhowmick

et al. 1998). This can be excluded to be relevant for the system under study, since exposure to the antagonist BQ-788 at a concentration which completely blocked ET-1induced Ca2+ signalling did not attenuate a subsequently recorded Ca2+ transient evoked by ET-1 (Oles et al. 1997). Moreover, the key observation of the present study, namely that receptor occupancy and Ca2+ entry, i.e. transduction of the receptor signal, are conditional for desensitization of ETAR signalling, is not compatible with the notion that an agonist/antagonist-induced conformational change of the receptor is sufficient for its desensitization. Whatever the reactions are that lead from binding of ET-1 to the ETAR to the long-lasting loss in responsiveness, an early step requires a rise in intracellular Ca2+ concentration by transmembrane entry which cannot be mimicked by Ca2+ release from internal stores. Neither the finding alone that in the absence of extracellular Ca2+ or in the presence of SK&F 96365 desensitization is reduced or completely abolished in some cells is an unequivocal proof that Ca2+ entry is conditional for desensitization of the ETAR. The effect of Ca2+ removal could simply represent an effect via an external Ca2+ or divalent cation binding site or a faster dissociation of ET-1 from its receptor in the presence of Ca2+. This would, however, be difficult to reconcile with the effect of SK&F 96365. This compound was initially described as an inhibitor of receptor-activated Ca2+ entry with low selectivity for a distinct mechanism (Merritt et al. 1990; Iwamuro et al. 1998). The similarity of the effects of Ca2+ removal and SK&F 96365 on the shape of the Ca2+ transient and on desensitization strongly support the notion that Ca2+ entry is conditional for desensitization. In the same cell line as studied here, evidence has been provided that the Ca2+ entry mechanism activated by ET-1 reflects a store depletion-activated pathway, although it cannot be excluded that other Ca2+ entry pathways operate in parallel, as has been demonstrated recently in a smooth muscle cell line (Iwamuro et al. 1998). The lack of an effect of Ca2+ release on desensitization, despite the fact that the amplitude reaches about the same level as signals composed of both, an entry and a release component, uncovers the limitations of measuring global Ca2+ signals. Moreover, using a Ca2+-chelating compound for measuring Ca2+ concentrations has effects on basal Ca2+ concentration as well as Ca2+ transients due to entry and/or release. Although a small concentration of fura was used in the present study, resulting in a low level of loading, it is conceivable that the difference between entry and release on ETAR desensitization is absent or less pronounced in a cell in situ. There are numerous examples that Ca2+ signals in various types of cells are non-homogeneous and that subcellular inhomogeneities can be of functional relevance, e.g. by localized activation of a membrane current (e.g. Zhuge et al. 1998). This applies to both Ca2+ release from InsP3-sensitive and insensitive stores as well as to Ca2+ entry. Ca2+ entry through individual Ca2+-conducting ion channels is supposed to create submembrane Ca2+ domains at the inner mouth of the channel whose amplitude, spatial spreading, and lifetime

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apart from the size of the current depend on cytosolic Ca2+-buffering properties (Augustine and Neher 1992). Much higher free Ca2+ concentrations can be reached in such microdomains as compared to the global (average) Ca2+ concentration measured by means of a dye. Assuming that a Ca2+-sensing molecule involved in initiating the process which finally results in endocytosis of the ETAR is co-localized with the entry channel, a selective dependence of desensitization on Ca2+ entry could be explained. Calmodulin, the classic ubiquitous Ca2+ sensor protein, has been shown to represent the Ca2+ sensor for endocytotic vesicle recycling in chromaffin cells (Artalejo et al. 1996). In a series of experiments two calmodulin antagonists (W-7, calmidazolium) tested had no effect on ET-1induced Ca2+ transients or their desensitization properties, respectively (data not shown). Further work is required to identify the Ca2+ sensor involved in desensitization of ETA-R.

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