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Endocrinology Vol. 138, No. 8 3141-3153
Copyright © 1997 by The Endocrine Society

ARTICLES

Endothelin Activates Large-Conductance K+ Channels in Rat Lactotrophs: Reversal by Long-Term Exposure to Dopamine Agonist1

Béla Kanyicska, Marc E. Freeman and Stuart E. Dryer

Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, Florida 32306

Address all correspondence and requests for reprints to: Marc E. Freeman, Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4075. E-mail: freeman{at}neuro.fsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ETA receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment.

The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 µM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BKCa) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 µM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 µM dopamine and 20 nM ET-1 caused activation of BKCa channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (±)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BKCa channels.

These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BKCa channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ETA receptor to BKCa channels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL, a protein hormone secreted by lactotrophs in the anterior pituitary gland, plays an important role in the regulation of development, reproduction, lactation, and adaptation to a changing environment (1). The secretion of PRL is regulated primarily by persistent inhibitory signals emanating from the central nervous system (2). The release of dopamine from neurons of the tuberoinfundibular and tuberohypophyseal pathways is thought to be the principal factor responsible for the tonic inhibition of PRL secretion (3). It has been suggested, however, that endothelin-like peptides (ETs) may play a role in the regulation of PRL secretion. For instance, ETs and their messenger RNAs are expressed in the pituitary and in the hypothalamic magnocellular nuclei (4, 5, 6, 7). Moreover, ET receptors are found in the anterior lobe of the pituitary gland (8), and ETs are potent inhibitors of PRL secretion from cultured pituitary cells (9, 10, 11). The pharmacological profile of ET-like peptides on PRL secretion (12) is consistent with the observation that the ETA receptor is the predominant subtype expressed in the pituitary gland (8). The inhibitory effects of ETs and dopamine on PRL secretion are mediated by pertussis toxin (PTX)-sensitive G proteins (11, 13). Therefore, it has been proposed that endothelins (ETs) may act in concert with dopamine to regulate PRL secretion in vivo (14).

Endothelins, like angiotensin II, are members of the structurally dissimilar but functionally related vasoactive peptide goup (15). Because angiotensin II is well established as a stimulator of PRL secretion from lactotrophs (1), the discovery of the inhibitory effect of ETs on PRL secretion was unexpected (9, 10). Moreover, previous studies have suggested that the signal transduction pathways activated via ET receptors include activation of L-type Ca2+-channels and an increase in phosphatidyl-inositol turnover leading to release of Ca2+ from intracellular storage pools and activation of certain protein kinases (16, 17). Paradoxically, these events would tend to promote PRL secretion (18). However, it has been previously shown that activation of K+ channels plays an important role in the inhibition of PRL secretion produced by dopamine (19, 20, 21, 22, 23, 24) or somatostatin (25). Therefore it seemed possible that ET-1 inhibits PRL secretion by activating K+ channels.

In a previous study of interactions between dopamine and ET-1 in vitro, we found that long term (>48 h) exposure to dopamine or the D2 agonist PPHT converted the action of ET-1 on PRL secretion from inhibitory to stimulatory (14). In the present study, using the cell-attached and inside-out patch recording configurations on cultured rat lactotrophs, we present evidence that ET-1 causes long-term modulation of large-conductance Ca2+- dependent K+ channels (BKCa channels).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell preparation
Random cycling female rats (Sprague-Dawley, Charles River, Raleigh, NC) were used as pituitary gland donors. The animals were killed by decapitation and cells from the anterior pituitary gland were dispersed using collagenase-hyaluronidase as described previously (26, 27). The enzymatically dissociated cells were enriched in lactotrophs by differential sedimentation on a discontinuous Percoll gradient (28).The cells that formed a layer between 50 and 60% Percoll were plated onto poly-L-lysine coated glass coverslips (0.25 mg/ml in distilled water) and placed in six-well tissue culture plates containing 2 ml DMEM supplemented with 10% FBS and antibiotics (60 mg/liter sodium-penicillin, 100 mg/liter streptomycin sulfate, and 2.5 mg/liter amphotericin-B). All media components were obtained from Life Technologies (Gaithersburg, MD). Cells were maintained in vitro for 2–4 days before use in electrophysiological experiments.

The enrichment in lactotrophs was evaluated by immunocytochemical staining for PRL. Briefly, cells cultured as above were fixed in freshly prepared B5-fixative (6 g HgCl2 and 1.25 g sodium acetate dissolved in 90 ml distilled water and mixed with 10 ml 37% formaldehyde) for 10 min followed by 95% ethanol for 1 min. After several rinses in PBS, cells were incubated overnight with rabbit antirat PRL antiserum (NIDDK S-9, diluted to 1:7000 in 0.4% Triton X-100 containing PBS). Following incubation with biotinylated goat antirabbit IgG (1:600, Vector Laboratories, Burlingame, CA), the PRL-like immunoreactivity was visualized by the avidin-biotin peroxidase method using the Vector Elite kit (Vector Laboratories). The results showed that 70–75% of the cultured cells expressed PRL-like immunoreactivity and could therefore be classified as lactotrophs.

Electrophysiology
Coverslips containing lactotrophs were mounted in a recording chamber (500 µl volume) and perifused with various external salines at 2 ml/min. Cell-attached patch recordings were made using standard methods (29). Briefly, recording electrodes were pulled in two stages from borosilicate glass (1.5 mm diameter, Drummond Scientific, Broomall, PA), coated with Sylgard 184 resin (Dow Corning, Midland, MI) and fire-polished. In many experiments, bath and recording pipettes contained a normal external saline consisting of (mM): 145 NaCl, 5.4 KCl, 0.8 MgCl2, 5.4 CaCl2, 10 D-glucose, 13 HEPES/NaOH (pH 7.4) at room temperature (22 C). In these experiments, the cell-attached patch was depolarized by 60 mV, close to 0 mV assuming a resting potential of -60 mV. With this recording configuration, and in normal external saline, the patch potential is free to fluctuate as the intracellular potential is not controlled. Therefore, in several experiments lactotrophs were perifused with a high K+ solution consisting of (mM): 140 KCl; 8 MgCl2; 0.1CaCl2; 10 D-glucose; 10 HEPES/KOH, pH 7.4. Under those conditions, cell membrane potential is clamped to 0 mV and the potential across the patch can be set by the potential applied to the recording pipette, which could be filled with either normal or high K+ salines, depending on the desired ionic gradient. To buffer intracellular free Ca2+, cells were incubated in the presence of 50 µM BAPTA-AM (Molecular Probes, Eugene, OR) for 60 min in Ca2+-free extracellular solution (30). ET-1 or dopamine were dissolved in bath salines and applied by perifusing the entire chamber from separate gravity-fed reservoirs controlled by valves. The dead time of the perifusion system was approximately 45 sec. For inside-out patches, cells were perifused with a saline that contained (mM): 150 KCl, 0.8 MgCl2, 10 EGTA, 10 HEPES, pH 7.4 (Ca2+-free saline) or in a similar saline that contained 10 mM EGTA and 0.2 mM CaCl2, pH 7.4 (free Ca2+ of 0.1 µM). Recording pipettes were filled with normal external salines.

All recordings were made using an Axon Instruments Axopatch 1D amplifier, and data were filtered at 2 kHz and stored on magnetic videotape for off-line analysis using pclamp software (version 6.03, Axon Instruments, Foster City, CA). Throughout this paper, currents flowing out of the patch membrane (and into the recording electrode) are shown as upward deflections of the traces. Dopamine was obtained from Calbiochem (La Jolla, CA), PPHT from Research Biochemicals International (Natick, MA), and BQ-610 from Penninsula (Belmont, CA). Pertussis toxin, and all other chemicals were purchased from Sigma (St. Louis, MO).

Data analyses
Data stored on magnetic videotape were digitized off-line (25 kHz) and saved on removable hard disks (Iomega Corporation, Denver, CO). Before analysis of channel activity, an all-point histogram of current amplitudes was created to determine the half-amplitude threshold. Using a half-threshold crossing procedure that ignored transitions of less than 50 µsec, an idealized record of the original data was created and used for kinetic analysis. The probability of a channel being open [P (open)] was used as a parameter to assess the level of channel activity. The P (open) values were estimated in 6 sec segments over a varying length of continuous data and plotted as a function of time. Mean P (open) values were compared using Student’s unpaired t test (Stat-View, Abacus Concept, Berkeley, CA). Open-time histograms were constructed from patches in which there was only one main amplitude class even at the highest rate of channel activation, suggesting the presence of a single functional channel in the patch. Histograms were fitted with exponential curves using the Levenburg-Marquardt and maximum likelihood nonlinear least squares routines implemented in pclamp software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ET-1 activates large-conductance calcium-dependent potassium channels (BKCa channels) on rat lactotrophs
Bath application of 20 nM ET-1 caused a robust and persistent activation of large-conductance K+ channels in 26 out of 37 cell-attached patches tested. In these experiments, patches were depolarized by 60 mV from the resting potential in normal external saline. With this recording configuration, bath-applied agonists do not have access to the external face of the patch membrane. Figure 1Go illustrates the effect of 20 nM ET-1 on large-conductance K+ channels in rat lactotrophs perifused with normal external saline. In this patch, the channels were quiescent before ET-1 application. Robust channel activation occurred after 8-min exposure to ET-1 (Fig. 1Go). It should be noted, however, that there is considerable cell-to-cell variation in the latency of the response to ET-1 (4–20 min, mean 10.5 ± 4.8 min, n = 19). A similar latency was observed when 20 nM ET-1 was present only in the recording electrode (not shown). The stimulatory effect of ET-1 on these channels often started with an intermittent, burst-like activity followed by sustained tonic activity. In general, the effect of ET-1 was long lasting and could be maintained for as long as ET-1 was present. Complete recovery usually required at least 10 min washout of ET-1. This time course is similar to that of ET-1 effects on PRL secretion from lactotrophs maintained under comparable experimental conditions (14).



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  		Figure 1. Endothelin (ET-1) evokes sustained activation of K+ channels in a cell-attached patch on rat lactotrophs. The recording electrode and bath contained normal extracellular saline and the patch was depolarized 60 mV from the resting membrane potential. ET-1 (20 nM) was applied by bath perifusion. With this recording configuration, ET-1 is excluded from the patch membrane. Traces show representative channel activity observed at the times indicated to the left. Times are relative to the start of ET-1 application (0 min). ET-1 was washed out of the bath after 10 min. Current level of the closed state is indicated by a solid line to the right of each trace. Note slow onset and recovery of ET-induced stimulation of channel activity.

 
With physiological ionic gradients (5.4 mM KCl in the patch pipette), the slope conductance of ET-evoked unitary currents was approximately 95 pS. (Fig. 2Go). Under these ionic conditions, the ET-induced unitary currents could not be reversed, but instead gradually vanished with patch hyperpolarization. The extrapolated reversal potential was -77 mV, close to the calculated EK of -86 mV. When the patch pipette contained 150 mM KCl, unitary currents reversed at 0 mV, which is equal to the predicted EK under these conditions. Under these conditions, the unitary slope conductance at calculated patch potentials of +80 to -80 mV was 165 pS, and there was little rectification (Fig. 2Go). The ET-evoked large-conductance K+ channels invariably increased their activity with patch depolarization reaching half-maximal activity around +30 mV (Fig. 3Go). Before ET-1 application, large-conductance K+ channel openings were infrequent and could not be increased substantially by depolarization up to +40 mV (Fig. 3Go). Note that the bath saline contained high K+ in these experiments (Figs. 2Go and 3Go). These characteristics suggested that the ET-activated channels of rat lactotrophs are large-conductance Ca2+-dependent K+ channels (BKCa channels), similar or identical to the channels described by Lang and Ritchie (31) on GH3 cells (a lactotroph-derived pituitary cell line).



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  		Figure 2. Current-voltage relationship of ET-activated K+ channels in cell-attached patches. Representative unitary currents are shown from two different cell-attached patches recorded at various holding potentials. The electrodes were filled with either 5.4 mM KCl (top left) or 150 mM KCl (top right) containing saline. In both cases, the cells were perifused with saline containing 150 mM KCl, 0.1 mM CaCl2, 8 mM MgCl2, 10 mM HEPES and 20 nM ET-1. Current level of closed state is shown to the left of each trace by a solid line, while the calculated patch potentials (assuming a resting membrane potential of 0 mV) are indicated at the right. I-V curves constructed from these patches are shown in the bottom panel for recordings made with pipettes containing either 5.4 mM KCl (open circles) or 150 mM KCl (filled circles). Data points are fitted with linear regression lines with slopes of 95 and 165 pS, respectively. With physiological K+ gadient, the extrapolated reversal potential was -77 mV, and with the symmetrical K+ the reversal potential was close to 0 mV.

 


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  		Figure 3. ET-1 increases voltage-sensitivity of BK channels in cell-attached patches. Electrodes were filled with normal external saline while the cells were perifused with 20 nM ET-1 in the high K+ saline described in Fig. 2Go. Patch was held at -60 mV and depolarized in 20 mV steps before and after application of 20 nM ET-1. Individual traces shown (left) show channel activation observed after ET-1 application. The short bars at the beginning of each trace denote current level of the closed state, whereas the calculated patch membrane potentials are indicated at the right (assuming a resting potential of 0 mV). The probability of a channel being open [P (open)] before and after ET-1 application was assessed in 2-sec segments over 2–3 min of recordings at each holding potential and the mean P (open) values were plotted against the calculated patch membrane potential (left). Very little channel activity was observed at any membrane potential before ET-1 application. The level of channel activity observed after ET-1 application was voltage-dependent and increased with depolarizing membrane potential. The sigmoid curve was fitted by nonlinear regression analysis (Graphpad, San Diego, CA). The channel activity reached its half maximal level when the patch was depolarized by 30.6 ± 1.1 mV from the resting potential, assumed to be close to 0 mV under these recording conditions.

 
To address the Ca2+-dependency of the ET-activated channels, three different experimental approaches were used. First, 20 nM ET-1 was applied through the external bath saline which contained 150 mM KCl to clamp the cell membrane potential, and thus the patch potential, at close to 0 mV. In cell-attached patch configuration, the recording electrode contained 5.4 mM KCl and was held at 0 mV. Robust activation of large conductance channels occurred about 10 min after ET-1 application. Upon excision of the patch into Ca2+-free saline containing 150 mM KCl and 10 mM EGTA, the channels became quiescent. A subsequent bath application of saline containing 0.1 µM Ca2+ restored the activity of these channels in the inside-out patch with a unitary current amplitude similar to that observed before patch excision (Fig. 4AGo). Therefore, at least some Ca2+ is required for the continued gating of ET-activated channels. In another series of experiments (Fig. 4BGo), the lactotrophs were preincubated for 60 min with 50 µM of the cell permeable Ca2+-chelator BAPTA-AM (30). No BKCa channel activity was observed in 16 patches tested, even in the continuous presence of ET-1 in the bath for 30–50 min. In five of these totally quiescent patches, the presence of functional BKCa channels could be demonstrated by excising inside-out patches into Ca2+-rich saline (Fig. 4BGo). Note that the effects of BAPTA could reflect in part a requirement for Ca2+ in ET-mediated signal transduction such as activation of phospholipase C, protein kinase C, or phospholipase A. In another set of experiments, we noted that ET-1 failed to activate BKCa channels in any of 11 cell-attached patches when the recording pipettes were filled with Ca2+-free salines, even when the bath salines contained 5.4 mM Ca2+ (data not shown). These data suggest that localized Ca2+ influx is necessary for the initiation of ET-1 activation of BKCa channels.



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  		Figure 4. Endothelin-1 activates Ca2+-dependent K+ channels. A, The recording electrode contained normal external saline and was held at 0 mV. The bath saline contained 150 mM KCl. BKCa channels became active approximately 10 min after 20 nM ET-1 was applied through the bath (top traces). Current level of the closed state is indicated to the right of each trace. The bath solution was changed to Ca2+-free saline containing 150 mM KCl and 10 mM EGTA before attaining an inside-out patch configuration. Patch excision into Ca2+-free saline caused an immediate cessation of BKCa channel activity (middle traces). A subsequent bath application of saline containing 0.1 µM free Ca2+ caused activation of BKCa channels with a unitary conductance similar to that observed before patch excision. B, The cells were preincubated for 60 min with the cell-permeant Ca2+-chelator BAPTA-AM and then perifused with high K+ extracellular saline. The recording electrode was filled with normal saline containing 20 nM ET-1 and the patch potential was held at 0 mV. In cell-attached configuration, no large conductance channel activity was detected (upper traces). After excising the patch inside-out into Ca2+-containing medium (0.1 mM), two large conductance channels immediately became active (middle traces). The Ca2+-dependency of these channels where again demonstrated by replacing the bath solution with a Ca2+-free medium (lower traces).

 
To characterize further the ET-activated BKCa channels, we tested their sensitivity to neurotoxins having varying specificity in inhibiting different K+ channels. The toxins were dissolved in the pipette solution while ET-1 was applied in the bath perifused with normal extracellular saline (5–7 patches in each case, data not shown). The general K+-channel blocker 20 mM tetraethylammonium (32) in the pipette blocked the ET-induced channel activation. When the pipette solution contained 1 µM apamin, an inhibitor of small conductance K+ channels (33) or 5 mM 4-aminopyridine, an inhibitor of voltage-activated K+ channels (32), 20 nM ET-1 caused an activation of large conductance K+ channels comparable to that observed when the recording electrodes were filled with normal saline alone. The lack of effect of apamin and 4-aminopyridine on ET-activated channels supports our previous categorization of these channels as BKCa-type. Surprisingly, bath application of 20 nM ET-1 was able to cause full activation of BKCa channels in cell-attached patches in the presence of the BKCa channel inhibitor charybdotoxin (32, 34, 35) or iberiotoxin (36), even at toxin concentrations of 200 nM. These results suggest that the ET-activated K+-channels in lactotrophs are similar to the Type II BKCa channels of rat brain, which are also resistant to charybdotoxin (37).

Dopamine and ET-1 activate identical BKCa channels
As with ET-1, application of 10 µM dopamine also caused activation of BKCa channels. In most cases (12 of 17 patches), dopamine and ET-1 were able to activate indistinguishable K+ channels in the same patches. However, the effects of dopamine occurred with a somewhat shorter latency (2–10 min, mean time to peak was 5.4 ± 2.6 min) and recovered more rapidly. In a few cases, the cell-attached patch contained only one functional BKCa channel and lasted long enough to test both agonists sequentially. Two representative recordings are presented in Fig. 5Go. The probability of the channel being open [P (open)] was assessed in 6-sec segments throughout the entire recording. When the bath contained normal saline, dopamine caused a modest increase in overall channel activity, while the removal of dopamine precipitated a robust activation of the channel (Fig. 5AGo). This latter phenomenon correlates well with the recent finding that removal of dopamine results in a transient increase in intracellular free Ca2+ in lactotrophs (38), consistent with the Ca2+-dependent nature of the ET-and/or dopamine-activated channel. In the recording shown in Fig. 5AGo, application of 20 nM ET evoked a high level of channel activity after a delay of 8 min. Following removal of ET-1, BKCa channel activity remained elevated for about 80 min before activity returned to baseline levels. Subsequent application of ET-1 was still capable of increasing BKCa channel activity, indicating that the mechanism involved in ET-induced channel activation retained its functional integrity during this exceptionally long recording (Fig 5AGo). These experiments were repeated with extracellular saline containing high K+ (150 mM) to clamp the cell membrane potential near 0 mV. We tested these conditions because they would eliminate most of the ambiguity concerning the actual patch potential in the cell-attached configuration and because they could provide recordings more suitable for kinetic analysis. When the cells were perifused with high K+ saline, dopamine and ET-1 were still capable of activating BKCa channels in 11 out of 17 patches. It is notable, however, that under these conditions, dopamine withdrawal did not cause an additional increase in the activity of the BKCa channel (Fig. 5BGo).



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  		Figure 5. Time course of dopamine (DA) and ET-induced activation of BKCa channels. The cells were perifused either in normal saline (A) or high K+ saline (B). In both cases, the patches appear to contain only one functional BKCa channel. Agonists (10 µM DA or 20 nM ET-1) were applied by bath perifusion. Recording electrodes contained normal external saline and patches were depolarized by 60 mV (A) and 0 mV (B), respectively. The probability of a channel being open [P (open)] was assessed in 6-sec segments throughout the entire recording period and plotted as a function of time. Both agonists unequivocally increased the level of channel activity although their effects differed in terms of duration and intensity. In addition, with normal external saline in the bath, withdrawal of DA from the perifusing medium precipitated a strong activation of the BKCa channel (A). The longevity of this particular patch allowed application of a second ET-1 challenge following the complete recovery from previous challenge. The second application of ET-1 resulted in an increase in channel activity, indicating that the cell preserved its functional integrity throughout the entire length of the recording. On panel C, two traces from original recordings of A and B are presented at higher time resolution. Using cell attached configuration with normal external saline in the bath, the large and long lasting outward currents displayed characteristic relaxations (a). However, when the bath contained 150 mM KCl (which effectively clamped the membrane potential near to zero), no relaxations were observed (b).

 
Using the cell-attached configuration with physiological ionic gradients, pronounced inward relaxations were apparent in individual unitary currents (Fig. 5CGo, trace a). A similar phenomenon was noted by Fenwick et al. (39) in chromaffin cells. Their equivalent circuit analysis suggested that the opening of a single BKCa channel is sufficient to produce significant membrane hyperpolarization, resulting in decrease in the driving force for K+ across the patch membrane (39). That a similar process occurs in lactotrophs is strongly supported by the fact that we never observed relaxations in unitary currents in cell-attached patches in the presence of 150 mM external K+, conditions that should effectively clamp the membrane potential to 0 mV. (Fig. 5CGo, trace b). The BKCa channels activated by dopamine or ET-1 had similar kinetic properties. The open-time distribution in the presence of either agonist could be fitted best as the sum of two exponential components with time constants of 0.21 and 8.11 msec for ET-1 (Fig. 6Go) and 0.18 and 7.91 msec for dopamine (histogram not shown), with the longer time constant having greater weight. The open-time distributions of spontaneous BKCa channel events (determined before application of agonist) also required two time constants for an adequate fit (0.15 and 1.84 msec), but under those conditions the shorter time constant had greater weight (Fig. 6Go). Thus, while both dopamine and ET-1 increased the open probability of BKCa channels (Fig. 5Go, A and B), they also shifted the distribution of open times in favor of longer open events.



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  		Figure 6. Kinetic properties of BKCa channels activated by ET-1. Data were taken from the same patch shown in Fig. 5BGo. Representative traces are shown to illustrate the low level of channel activity before ET-1 application (upper left), and the increased activity with characteristically long open events induced by 20 nM ET-1 (lower left). Open-time histograms (right) were constructed from 2 min of continuous data and were fitted as the sum of two exponential components (solid line) with proportions (p1 and p2) and time constants ({tau}1 and {tau}2) as indicated.

 
The effects of ET-1 on BKCa channels are mediated by ETA receptors via pertussis toxin-sensitive G proteins
When lactotrophs were treated for 5 min with 200 nM of the specific ETA receptor antagonist BQ-610 (40) before exposure to ET-1, application of 20 nM ET-1 (in the continued presence of 200 nM BQ-610) failed to activate BKCa channels in any of 12 cell-attached patches tested. In five cases, it was possible to apply 20 nM ET-1 twice, first in the presence of BQ-610, and later after the antagonist had been removed by washing for 35 min. Endothelin-1 did not activate BKCa channels in the presence of BQ-610 but caused persistent activation once the antagonist had been removed (Fig. 7AGo).



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  		Figure 7. Effects of the ETA receptor antagonist BQ-610 on BKCa channel activation evoked by ET-1. The probability of a channel being open is shown over the course of a recording (A). The times of drug applications were indicated by the horizontal lines placed above the diagram. The presence of the competitive ETA receptor antagonist BQ610 applied at 200 nM concentration before and during application of 20 nM ET-1 prevented activation of BKCa channels. Following washout of the antagonist, reapplication of 20 nM ET-1 resulted in a significant increase in channel activity (A). B, combined data from five different patches treated as shown in A. Mean P (open) was calculated from 30 min of data starting at the beginning of ET-1 application (B). Asterisks denote significant difference (P < 0.05) between groups of ET-1+BQ610 vs. ET-1 alone. Representative traces used to construct panel A are shown in panel C. The cell-attached patch was depolarized by 60 mV from the resting membrane potential. Current level of the closed state is indicated to the right of the traces. These traces were taken approximately 12 min after the switch to ET-containing saline, in the presence of antagonist (BQ610+ET-1), and after the antagonist has been washed out (ET-1). In the presence of antagonist, there was no increase in BKCa channel activity. When the antagonist had been washed out completely, reapplication of 20 nM ET-1 led to a robust increase in BKCa channel activity. Note long open times and the relaxations in the outward currents, which are characteristic for this recording condition (cell-attached patch in normal saline). The effect of ET-1 was reversed after about 30 min washing in normal saline.

 
In another series of recordings, control and pertussis toxin-treated cells from the same dispersion were observed alternately. After cell-attached patch formation, channel activities were recorded in control saline for about 10 min followed by challenge with ET-1 for 20 min. After pertussis toxin treatment, no response was observed in nine patches, whereas a short-lived slight increase in BKCa channel activity was observed in three patches. The probability of a channel being open was calculated before and during the exposure to ET-1 and the net changes in P(open) by ET-1 were calculated and compared between control and pertussis toxin-treated groups. Only those patches that showed BKCa-like events and lasted for at least 30 min were considered. The effects of 20 nM ET-1 on BKCa channels were markedly reduced or eliminated when the cells were pretreated for 12 h with 200 ng/ml pertussis toxin (Fig. 8Go).



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  		Figure 8. The effect of ET-1 on BKCa channels is mediated by a pertussis toxin- (PTX)sensitive G protein. In a series of recordings, control and PTX-treated cells (200 ng/ml for 16–24 h) were used alternately in cell-attached configuration. The bath was perifused with high-K+ external saline and the patch was held at 0 mV. After patch formation, basal channel activity was recorded for 10 min followed by the challenge with 20 nM ET-1 for 20 min. Representative traces are presented for control (A) and for PTX-treated cells (B), obtained before (saline) and during ET-1 treatment (ET-1). Mean P (open) values were estimated as in Fig. 7Go. Only those patches which displayed large-conductance channel activity and lasted at least for 30 min were considered for this analysis. In untreated cells (n = 5, panel B), ET-induced a significant increase in channel activity (*, P < 0.05) while this effect of ET-1 was diminished by PTX (200 ng/ml) pretreatment (n = 9, D).

 
Long-term exposure to a dopamine agonist alters the response of pituitary cells to ET-1
In recordings made from pituitary cells that had been cultured for 48 h in the presence of 500 nM PPHT, a selective D2 receptor agonist (41), we observed a markedly higher level of basal activity of BKCa channels (9 of 12 patches). When 20 nM ET-1 was applied to these PPHT-treated cells, basal BKCa channel activity was inhibited in 6 out of 9 patches. ET-1 had no effect on the remaining 3 patches. The inhibitory effect of ET-1 on BKCa channels was reversed completely after a 20–30 min washing (Fig. 9Go). It should be emphasized that we observed ET-induced inhibition of BKCa channel activity only in lactotrophs that were exposed to dopamine agonist for at least 48 h. ET-1 still caused activation of BKCa channels in lactotrophs that were treated with PPHT for a shorter period of time (18–24 h, five patches, data not shown). This is consistent with our previous studies showing that the conversion of the effect of ET-1 on PRL secretion from inhibitory to stimulatory requires at least 48 h exposure to a D2 dopamine agonist (14).



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  		Figure 9. ET-1 inhibits spontaneously active BKCa channels in rat lactotrophs treated for 48 h with the selective D2 agonist PPHT. Traces presented in A and B are from two different cell-attached patches depolarized by 60 mV from the resting membrane potential. The recording chamber and the pipette contained normal extracellular saline. Current level of the closed state is indicated to the right of the traces by a solid line. Note that the relatively high level of spontaneous BKCa channel activity in PPHT treated cells was inhibited by bath application of 20 nM ET-1 at 0 min. ET-1 was washed out after 10 min exposure. The inhibitory effect of ET-1 was fully reversible. To display the time course of the effect of ET-1 on BKCa channel activities, P (open) was assessed using data from patch B and plotted as a function of time (C). Followed application of 20 nM ET-1 in the perifusion medium, the P (open) value dropped gradually from 0.427 ± 0.085 (calculated over 0.2 to 4.3 min) to 0.072 ± 0.012 (over 10.8 to 22.1 min). The robust ET-induced suppression of BKCa channel activity (-83%) was reversible and required about 15-min washout to recover fully (C).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In untreated lactotrophs, bath application of ET-1 caused a robust activation of BKCa channels in cell-attached patches. When lactotrophs were cultured in the presence of a D2 dopamine agonist for at least 2 days, the elevated basal BKCa channel activity was inhibited by the same application of ET-1. The effect of ET-1 on BKCa channels, i.e. its time course, its sensitivity to pertussis toxin or ETA receptor antagonist, as well as its characteristic conversion from stimulatory to inhibitory by long term exposure to D2 agonist, all correlate closely with the effect of ET-1 on PRL secretion (11, 12, 14).

The intracellular events leading to the ET-evoked modulation of BKCa channels and change of PRL secretion are poorly understood. Nevertheless, a general mechanistic feature of ET-induced BKCa channel activation is suggested by the present data. The fact that activation of BKCa channels occurred in cell-attached patches where ET-1 was excluded from the patch membrane suggests that activation of ETA receptors gives rise to one or more mobile second messengers. The nature of this messenger cannot be determined from the present experiments. One possibility is that ET-1 mobilizes a pool of Ca2+ that is colocalized with the BKCa channels but does not have access to active PRL release zones in the lactotrophs. The gating of ET-activated BKCa channels is dependent on intracellular free Ca2+. Moreover, we have noted that if the recording pipette is filled with Ca2+-free solution, bath application of ET-1 was unable to evoke persistent BKCa channel activation, even if the bath contained 5.4 mM CaCl2. It has been shown that BKCa channels often form a functional complex with voltage-sensitive Ca2+-channels (42, 43). Therefore, it is possible that the modulation of BKCa channels by ET-1 requires the activation of a subpopulation of voltage-sensitive Ca2+-channels that are functionally coupled to BKCa channels. Because in naïve lactotrophs the predominant effect of ET-1 on PRL secretion is inhibitory, it seems likely that this local increase in free Ca2+ is spatially segregated from the lactotroph’s PRL releasing zones.

Many of our recordings were made when the patch membrane was held at 0 mV. Under these conditions, the noninactivating voltage-sensitive Ca2+-channels are probably active continuously. However, without ET-1 or DA application, the level of BKCa channel activity remained low, and spontaneous openings were rare and short lived. Thus, although local Ca2+ influx is necessary to initiate the effects of ET-1, an increase in Ca2+ by itself is not sufficient to produce long term stimulation of BKCa channels. It is likely that other modulatory events contribute to the stimulatory effects of ET-1, possibly by influencing the voltage- and/or Ca2+-sensitivity of the BKCa channels. For instance, previous studies suggested that BKCa channels can be modulated by protein kinases and phosphatases (37, 44, 45). It should be added that activation of BKCa channels by somatostatin in GH3 cells requires activation of an okadaic acid-sensitive protein phosphatase (25, 46). Moreover, the deduced amino acid sequences of the recently cloned BKCa channels contain multiple consensus phosphorylation sites for protein kinase A, calmodulin-activated protein kinase II, and protein kinase C (47, 48, 49). Finally, we have previously observed that pharmacological blockade of certain protein kinases and phosphatases prevents the long lasting inhibitory effects of ET-1 on PRL secretion, while these treatments seemed to facilitate the stimulatory effect of ET-1 on PRL secretion (50). Therefore, it is possible that ET-1 induced modulation of BKCa channels is achieved by specific alterations of the phosphorylation state of these channels. We are currently studying the role of protein phosphorylation-dephosphorylation cascades in the ET-induced signaling mechanisms in normal lactotrophs.

In the present study, there was considerable similarity between ET-1 and dopamine-induced activation of BKCa channels on lactotrophs. The action of both agonists required pertussis toxin-sensitive G proteins (24) and the channels, after activation by either agonist, showed similar open-time distributions. However, the effects of dopamine and ET-1 differed in their latency to onset and time required for complete recovery. Several studies have already indicated that the same K+ channel can be coupled to more than one receptor, either by the same or by different subsets of G proteins (51). Therefore, it is possible that dopamine and ET-1 initiate separate transduction pathways that converge to cause activation of the same population of BKCa channels.

Previous studies on the Ca2+- and voltage-sensitivity of BKCa channels indicated that gating of these channels is reduced at or around the resting membrane potential (52). Similar results were obtained here. Nevertheless BKCa channels may still play an important role in the regulation of basal hormone secretion. For example, lactotrophs (20, 53) and GH3 cells (54, 55) often fire action potentials spontaneously. These action potentials are driven by an increased membrane conductance for Ca2+ ions, and the spike frequency and duration is directly proportional to hormone secretion (54). An increase in the activity of BKCa channels, by decreasing the duration of action potentials and the associated Ca2+ influx, could contribute to setting the rate of basal PRL secretion. It is also possible that ET-1 modifies the voltage- and/or Ca2+-sensitivity of BKCa channels in lactotrophs sufficiently for them to exhibit some activity at or around the resting membrane potential. This could result in hyperpolarization and deactivation of voltage-sensitive Ca2+ channels. This mechanism provides an additional basis for the inhibitory action of ET-1 on basal PRL secretion (14) as well as modulation of lactotroph responses to other agents (50).

Lactotrophs and PRL secreting cell lines of pituitary origin express several types of K+ channels (51, 56, 57). Previous studies have examined the role of these channels in modulation of PRL secretion (19, 21, 22, 58). For example, Oxford and co-workers (20, 22) have shown that dopamine or D2 agonists cause rapid activation of a 50 pS K+ channel via pertussis toxin sensitive G proteins. Additional studies on excised patches suggested that the activation of those channels was membrane delimited and did not require any soluble cytoplasmic mediator (20, 22, 23, 59). The ET-1 and dopamine-activated K+ channels described here are clearly different from those described above, as their gating is Ca2+-dependent. The kinetic and pharmacological properties of these channels resemble the type II large-conductance Ca2+-activated K+ channels isolated from rat brain (37, 60) and the BKCa channels in the nerve terminals of rat neurohypophysis (61) that are resistant to blockade by charybdotoxin.

We have previously reported that the effects of ET-1 on PRL secretion depend upon the prior history of the cells (14). Thus, 48-h (or longer) exposure to dopamine or D2 agonists causes a reduced basal secretion of PRL and converts the effects of ET-1 from inhibitory to stimulatory. The temporal pattern of ET-induced stimulation of PRL secretion after chronic dopamine treatment is comprised of two distinct phases: a large but transient increase, followed by a sustained modest elevation. The stimulatory effects of ET-1, unmasked by long-term dopamine treatment, were also mediated by ETA receptors. In the present study, we have observed an increase in the basal activity of BKCa channels in cells exposed to the D2 agonist PPHT for 48 h. Application of ET-1 under these conditions caused a sustained inhibition of BKCa channel activity. Therefore, it appears that chronic exposure to dopamine altered the intracellular signaling pathways coupling ETA receptors to the BKCa channels. The possible mechanisms whereby chronic activation of D2 dopamine receptors could reverse ET’s actions on lactotrophs have been discussed in detail elsewhere (14). The inhibitory effects of ET-1 on BKCa channels described above can account for the sustained increase in basal PRL secretion observed in lactotrophs chronically exposed to dopamine, but it seems unlikely that they are responsible for the much larger transient stimulation of secretion (14). Indeed, there is no reason to believe that modulation of BKCa channels is the only action produced by ET-1 in lactotrophs. However, since the persistent activation of D2 receptors closely resembles the in vivo environment of lactotrophs, we believe that further studies on this model will help to elucidate the physiological role of ETs and dopamine in the regulation of PRL secretion.

In summary, we have shown that ET-1 at a physiologically relevant concentration can profoundly influence BKCa channels in rat lactotrophs. Changes in gating of these channels may influence the rate of PRL secretion and the responsiveness of lactotrophs to PRL secretagogues. Indeed, the previously described changes in PRL secretion brought about by ET-1 are mirrored by its effects on BKCa channel activity under a variety of conditions. Structural elements of BKCa channels and their modulatory subunits indicate that they can be targeted by many signaling pathways (49, 62). This suggests that modulation of BKCa channels may be part of the overall mechanism by which lactotrophs integrate different chemical signals elicited by extracellular messengers such as ET-1 and dopamine.


   Acknowledgments
 
The authors thank The National Pituitary Agency and Dr. Albert Parlow for the rat PRL antiserum as well as Theresa D’Souza for her contribution to the single-channel recordings.


   Footnotes
 
1 Supported by NIH Grants HD-11669 and DK-43200 (to M.E.F.) and NS-32748 (to S.E.D.) and AFOSR Grant F49620 (to S.E.D.) Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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