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Endocrinology Vol. 139, No. 12 4801-4810
Copyright © 1998 by The Endocrine Society

ARTICLES

Inhibition of Voltage-Dependent Calcium Channels by Prostaglandin E2 in Rat Melanotrophs1

Keiko Tanaka, Izumi Shibuya, Narutoshi Kabashima, Yoichi Ueta and Hiroshi Yamashita

Department of Physiology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan

Address all correspondence and requests for reprints to: Izumi Shibuya, Ph.D., Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, 807 Japan. E-mail: shibuya{at}med.uoeh-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of -80 to 0 mV, with an ED50 of 68 nM. The maximum inhibition of Ba2+ currents by 1 µM PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nM) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 µM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents.

These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PGs ARE KNOWN to influence hormone release from endocrine cells in the anterior and intermediate lobes and nerve terminals of the posterior lobe of the pituitary gland. PGE2 seems to be the most potent in modulating secretion of pituitary hormones of several prostanoids investigated so far, such as PGD2, PGF2a, and PGI2 (1, 2). It has been shown that PGE2 inhibits release of ACTH and ß- endorphin (1, 2) and enhances release of gonadotropins, GH, vasopressin, and oxytocin (3, 4, 5). It has been shown that the sites of actions of PGE2 are exerted either at the hypothalamus (4, 5, 6) or directly on endocrine cells or nerve endings in the pituitary gland (1, 2, 3). Moreover, several lines of evidence suggest that PGE2 is synthesized locally in the hypothalamus and in the pituitary gland (1, 2, 3, 5).

Melanotrophs of the pituitary pars intermedia exhibit spontaneous secretion of various biologically active peptides derived from POMC, such as {alpha}-MSH and ß-endorphin, at a high rate. Measurements of cytosolic Ca2+ concentrations ([Ca2+]i) with the fluorescent Ca2+ indicator, fura-2, and the rate of Mn2+ quenching of fura-2 in rat melanotrophs revealed spontaneous Ca2+ entry through the plasma membrane (7, 8). It has been suggested that such spontaneous Ca2+ entry accounts for the spontaneous secretion (9). Melanotroph secretion is under inhibitory control by neurotransmitters released by hypothalamic neurons directly innervating melanotrophs, dopamine, and {delta}-amino butric acid (GABA) (10, 11). A study with the Mn2+ quenching technique also revealed that these secretoinhibitory transmitters, acting through dopamine D2 and GABAB receptors, arrest spontaneous Ca2+ entry (7). It has been reported that melanotroph secretion is inhibited by PGE2 and that indomethacin, an inhibitor of PG synthesis, enhanced spontaneous secretion of ß-endorphin from explants of the hypothalamo-pituitary gland complex (2). Moreover, [Ca2+]i, recorded from dissociated rat melanotrophs, was reversibly suppressed by PGE2 (12). These results suggest that PGE2 synthesized endogenously inhibits spontaneous melanotroph secretion in a tonic manner; however, the cellular mechanism of inhibition by PGE2 is not clear.

It has been reported that rat melanotrophs possess several distinct subtypes of voltage-dependent Ca2+ channels and that such channels are inhibited by the secretoinhibitory transmitters (13, 14, 15, 16, 17). Moreover, expression of messenger RNAs (mRNAs) for POMC and voltage-dependent Ca2+ channels has been shown to be down-regulated by dopamine D2 receptors (18, 19). These results indicate that inhibition of voltage-dependent Ca2+ channels is one of the key mechanisms by which melanotroph function is regulated. To date, there is no report on the effects of PGE2 on voltage-dependent Ca2+ channels of melanotrophs, but a few studies conducted in other types of cells have reported the PGE2 modulates voltage-dependent Ca2+ channels (20, 21).

The aim of the present study was to examine whether voltage-dependent Ca2+ channels are modulated by PGE2 in melanotrophs and, if so, through what mechanisms they are modulated. For this purpose, we used acutely dissociated rat melanotrophs and studied the effects of PGE2 and EP receptor ligands on voltage-dependent Ba2+ currents of these cells, by the whole-cell patch-clamp technique.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell dissociation
Rat melanotrophs were prepared using a method described previously (22). In brief, male Wistar rats of 6–8 weeks (150–200 g) were used. The pituitary neurointermediate lobe was carefully isolated from the anterior lobe and transferred to a HEPES-buffered solution (HBS), the composition of which was (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH adjusted to 7.4 with NaOH), supplemented with 0.1% BSA. The lobes were incubated in HBS, containing 0.2% trypsin (type III; Sigma Chemical Co., St. Louis, MO), at 37 C for 20 min with continuous shaking (100 cycles/min). The lobes were then incubated in Ca2+-free HBS containing 0.1% collagenase (type I; Sigma Chemical Co.) for 15 min and triturated with glass pipettes. Isolated cells thus obtained were washed twice with enzyme-free HBS and maintained at room temperature (23 C) for at least 4 h, until used. In experiments with pertussis toxin (PTX), melanotrophs were maintained in primary culture for 18 h in humidified air containing 5% CO2 at 37 C. The culture medium was DMEM, with the addition of 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Patch-clamp recordings
Melanotrophs were plated in a culture dish (35-mm diameter); and perfusion of cells with standard HBS (without BSA) was begun about 10 min later, when the cells had become attached to the bottom of culture dish. The arrangements for perfusing cells and recording membrane currents have been described in detail previously (22). The inner pipette solution used in the recording electrodes contained (mM); 140 CsCl, 10 EGTA, 2 CaCl2, 1 MgCl2, 2 Mg-ATP, 0.3 GTP, and 10 HEPES (pH adjusted to 7.2 with Tris). After making a high-resistance seal, the perfusion solution was switched from a standard HBS to a Ba2+-containing solution, the composition of which was (mM) 10 BaCl2, 140 TEA-Cl, 10 HEPES, 10 glucose and 5 KCl (pH adjusted to 7.4 with Tris). Voltage-dependent Ba2+ currents were elicited by voltage steps from a holding potential of -80 mV to a test potential of 0 mV unless otherwise noted. Leak currents and capacitative transients were canceled by off-line subtraction of Cd2+ (200 µM)-insensitive currents. The sampling rate was 10 kHz. Membrane currents were recorded with a patch-clamp amplifier (AxoPatch 200A; Axon Instruments Inc., Foster City, CA) and were digitized using Pclamp software (version 6.0.2; Axon Instruments Inc). Analysis of the data were done using Axograph software (version 3.5.5; Axon Instruments Inc.). All electrophysiological measurements were made at room temperature.

Statistics
Results are expressed as the mean ± SE and n represents the number of experiments. Statistical differences (P < 0.05) were determined by Wilcoxon signed-rank test or by Mann-Whitney’s U test.

Drugs
SC-51322 and butaprost were generously provided by Ono Pharmaceutical Co. (Osaka, Japan). PGE2 and 17-phenyl-trinor-PGE2 (17PT-PGE2) were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), misoprostol was from Cascade Biochem Limited (Berkshire, UK), sulprostone was from Cayman Chemical Company (Ann Arbor, MI), PTX was from List Biological Laboratories (Campbell, CA), all the peptide toxin Ca2+-channel blockers were from Peptide Institute (Osaka, Japan), and other chemicals (which were of analytical grade) were from Nacalai Tesque (Kyoto, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PGE2 on voltage-dependent Ba2+ currents
Voltage-dependent Ba2+ currents were measured from 121 cells dissociated from the neurointermediate lobe of pituitary glands obtained from 22 rats. PGE2 (100 nM or 1 µM) inhibited Ba2+ currents elicited by a voltage step from a holding potential (Vh) of -80 to 0 mV, in 110 out of 121 cells examined (90.9%). Representative time courses of inhibition of Ba2+ currents induced by 100 nM dopamine and 1 µM PGE2, measured at 3–8 and 40–45 msec after the start of the depolarizing test potential (IBa3–8 and IBa40–45), are shown in Fig. 1AGo. PGE2 inhibited Ba2+ currents with a clear kinetic slowing of the currents, as well as with steady-state inhibition (Fig. 1BGo). The time course of the kinetic slowing of Ba2+ currents can be seen as a reduction in the ratio between IBa3–8 and IBa40–45 shown in Fig. 1AGo. The patterns of kinetic slowing and steady-state inhibition produced by PGE2 were similar to those produced by dopamine: the ratio of the maximum inhibition of IBa3–8 against the maximum inhibition of IBa40–45 reached 1.62 ± 0.14 and 1.71 ± 0.12 during application of PGE2 and dopamine, respectively (Fig. 1CGo). On the other hand, recovery from inhibition by PGE2 was considerably slower than that by dopamine (Fig. 1CGo). Because larger inhibition was observed when currents were measured between 3 and 8 ms, IBa3–8 was used for further analysis, unless otherwise noted.



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  		Figure 1. Inhibition of voltage-dependent Ba2+ currents by PGE2 and dopamine. A, A representative time course of inhibition of Ba2+ currents (elicited by voltage steps to 0 mV, from a holding potential, Vh, of -80 mV) by PGE2 (1 µM) and dopamine (DA; 100 nM). Open and closed circles, The amplitudes of Ba2+ currents measured between 3–8 and 40–45 msec after the start of depolarizing voltage commands, respectively (IBa3–8 and IBa40–45). In the lower panel, the ratio of IBa3–8 against IBa40–45 is plotted. B, Representative Ba2+-current traces recorded before and during 100 nM dopamine or 1 µM PGE2 application (indicated by a-d in A). C, The ratio of percent inhibition of IBa3–8 against that of IBa40–45 (open bars) and the recovery time after removal of the ligands (closed bars). The recovery time was calculated as the time difference between the time when the drug was removed and the time when Ba2+ currents returned to control levels.

 
The PGE2-induced inhibition of Ba2+ currents was concentration-dependent (Fig. 2Go). PGE2, at concentrations ranging from 10 nM to 1 µM, significantly inhibited Ba2+ currents; and nearly maximal inhibition was observed at 1 µM (Fig. 2BGo). Little or no inhibition was observed at 100 pM. The EC50 of the PGE2-induced inhibition was estimated to be 68 nM, from the concentration-response curve (Fig. 2BGo). The maximal inhibition of Ba2+ currents by PGE2 was similar to that by dopamine at the maximally effective concentration (100 nM) (Fig. 2BGo). When PGE2 (1 µM) was added twice to the same cells, inhibition of Ba2+ currents induced by the second PGE2 application was 92.2 ± 6.1% (n = 5) of that induced by the first application. The reduction of inhibition was 2.8 ± 2.0% of the first response and was not significant. This value was used in later analysis as a control value to examine a significant difference on inhibition of each subtype of Ba2+ currents induced by two repeated applications of PGE2.



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  		Figure 2. The concentration-response relation of PGE2-induced inhibition of Ba2+ currents. A, A representative time course of concentration-dependent inhibition of IBa3–8 by increasing concentrations of PGE2; B, the concentration-response curve of PGE2-induced inhibition of IBa3–8. The curve was calculated by the least-square method. The ED50 was estimated to be 68 nM. The Hill coefficient obtained from the curve was 0.61, suggesting that negative cooperativity may exist in the PGE2-induced inhibition of Ba2+ currents. The data are shown as the mean ± SE of the values obtained from five to seven experiments. Closed triangle, Inhibition of IBa3–8 by 100 nM dopamine (n = 12); asterisks, significant inhibition (P < 0.05).

 
Current-voltage relations of Ba2+ currents measured before, during, and after PGE2 (1 µM) application are shown in Fig. 3AGo. PGE2 significantly inhibited Ba2+ currents elicited by the test potentials, ranging from -30 to 40 mV (n = 7), which was similar to dopamine-induced inhibition of Ba2+ currents (n = 4, an example is shown in Fig. 3CGo).



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  		Figure 3. The current-voltage relation of Ba2+ currents measured before, during, and after PGE2 application. A, Representative traces of Ba2+ currents in response to voltage steps to -60 to 50 mV, from Vh of -80 mV before, during, and after application of 1 µM PGE2; B, the current-voltage relation of IBa3–8 and the effects of 1 µM PGE2 obtained from the traces shown in A; C, the current-voltage relation of IBa3–8 in response to 100 nM dopamine obtained from the same cell as A and B.

 
Effects of EP agonists and antagonist on Ba2+ currents
When tested at 1 µM, the EP1/EP3 agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition of Ba2+ currents by PGE2; whereas the selective EP2 agonist, butaprost, had little effect (Fig. 4AGo). The effects of the EP agonists on Ba2+ currents, obtained from 5 cells by randomizing the order of application, are summarized in Fig. 4CGo. Misoprostol and sulprostone produced a long-lasting inhibition of Ba2+ currents (just as PGE2), whereas 17PT-PGE2 caused more rapidly reversible inhibition: the mean recovery time of 17PT-PGE2-induced inhibition obtained from 12 cells was 45.8 ± 2.6 sec. Inhibition of Ba2+ currents by 1 µM PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322 (23). SC-51322, at 1 µM, reversibly reduced the PGE2-induced inhibition of Ba2+ currents by 23% (Fig. 4Go, A and B).



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  		Figure 4. Analysis of receptor subtypes involved in the inhibition of Ba2+ currents by PGE2. A, Representative time courses of inhibition of IBa3–8 by PGE2 (1 µM) and other EP agonists and effect of the EP1 antagonist, SC-51322, on the PGE2-induced inhibition. All the drugs were tested at 1 µM, and the traces shown in a–d were obtained from different cells. Sulprostone (Sulp) and 17PT-PGE2 (EP1/EP3 agonists), and misoprostol (Miso) (EP2/EP3 agonist) caused inhibition of Ba2+ currents, whereas butaprost (Buta) (EP2 agonist) showed little effect. B, Summary data of the effect of 1 µM SC-51322 on the inhibition of IBa3–8 by 1 µM PGE2. The data are shown as the mean ± SE of the values obtained from six experiments. Asterisk, Statistical significance against 1 µM PGE2 (control; P < 0.05). C, Summary data for the effects of PGE2 and other EP agonists on IBa3–8. The data are shown as the mean ± SE of the values obtained from five experiments. Asterisks, Significant inhibition (P < 0.05).

 
Effects of a prepulse on inhibition of Ba2+ currents by PGE2
The magnitude of the inhibition of Ba2+ currents by PGE2 (1 µM) was greatly reduced by a depolarizing prepulse, from -80 to +80 mV, for 50 msec, similar to the inhibition of Ba2+ currents by dopamine (100 nM) (Fig. 5Go). The prepulse removed 64.7 ± 7.0 and 61.9 ± 5.4% of the total inhibition of IBa3–8 induced by dopamine (n = 7) and PGE2 (n = 13), respectively, but caused moderate relief of the inhibition of IBa40–45 (34.7 ± 9.1 and 21.2 ± 4.2% of the total inhibition, respectively) (Fig. 5CGo).



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  		Figure 5. Effects of a depolarizing prepulse on inhibition of Ba2+ currents induced by PGE2 or dopamine. A, A representative time course of inhibition of IBa3–8 induced by 1 µM PGE2 or 100 nM dopamine and the effects of a depolarizing prepulse (to 80 mV, for 50 msec, preceding the test pulse with an interval of 5 ms) on the inhibition. Closed circles, IBa3–8 recorded with the prepulse. B, Superimposed traces of Ba2+ currents obtained before and during PGE2 (1 µM) or dopamine (100 nM) application with or without the prepulse. C, Summary data for the total inhibition of IBa3–8 and Iba40–45 induced by PGE2 (n = 13) or dopamine (n = 7) and the voltage-insensitive inhibition persisted when the prepulse was applied (closed bars).

 
Effects of pretreatment with PTX on inhibition of Ba2+ currents by PGE2
When cells were pretreated with PTX during primary culture for 18 h, the inhibition of Ba2+ currents by PGE2 (1 µM) was nearly abolished (Fig. 6Go). Ba2+ currents, measured from cells maintained in primary culture for 18 h without PTX pretreatment, were still susceptible to the inhibition by PGE2. There was no significant difference in the amplitude of Ba2+ current between the PTX-pretreated cells and the control cells.



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  		Figure 6. Effects of pretreatment with PTX on the inhibition of Ba2+ currents by PGE2. A, Representative traces of Ba2+ currents before and during 1 µM PGE2 application obtained from melanotrophs pretreated with PTX (300 ng/ml, 37 C, 18 h); B, summary data for the Ba2+ current amplitude and the inhibition of IBa3–8 by PGE2 obtained from cells pretreated with PTX (n = 7) and from cells maintained in primary culture without PTX (n = 8). Asterisk, Significance against control (P < 0.05).

 
Effects of blockers of Ca2+ channels on inhibition by PGE2
The subtypes of Ca2+ channels inhibited by PGE2 were examined by blocking each type of current with selective inhibitors, 10 µM nicardipine (L-type), 1 µM {omega}-conotoxin GVIA (N-type), and 20 nM {omega}-agatoxin IVA plus 1 µM {omega}-conotoxin MVIIC (P/Q-type). Nicardpine and {omega}-agatoxin IVA/{omega}-conotoxin MVIIC produced consistent inhibition in all 16 cells examined, whereas {omega}-conotoxin GVIA was without clear effects in 8 cells but caused marked inhibition ranging from 3–24% of the total current in 8 other cells. In total, each of the Ca2+-channel blockers produced significant inhibition, and the amplitude of the residual component, after application of all the blockers, was also significant (Fig. 7Go, A and B). Inhibition by {omega}-conotoxin GVIA and {omega}-agatoxin IVA/{omega}-conotoxin MVIIC was irreversible, but that by nicardipine was partially reversible. The amplitude of currents remaining after application of the Ca2+-channel blockers was not reduced by increasing the concentration of {omega}-conotoxin GVIA to 3 µM (n = 4), or that of {omega}-agatoxin IVA to 100 nM (n = 4). Fractional components of Ba2+ currents of N-, P/Q-, L-, and R-types (calculated from 16 experiments) were 4.2 ± 1.6, 48.1 ± 4.9, 30.5 ± 5.0, and 17.2 ± 3.1%, respectively, of the total currents; and inhibition of Ba2+ currents of each type by PGE2 (1 µM), as a fraction of the total currents, were 2.2 ± 0.9, 17.3 ± 2.4, 10.1 ± 1.5, and 5.5 ± 1.3%, respectively. Only the inhibition of Ba2+ currents of P/Q- and L-types was significant (P < 0.05), when compared with the reduction of Ba2+ current inhibition observed in two repeated applications of PGE2 (1 µM).



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  		Figure 7. Analysis of Ca2+-channel subtypes that are susceptible to the inhibition by PGE2. A, A representative time course of IBa3–8 in response to PGE2 after blockade of N-, P/Q-, and L-type channels. Each type of Ca2+ channel was blocked by the selective blockers for N, P/Q, and L types [{omega}-conotoxin GVIA (1 µM), {omega}-agatoxin IVA (20 nM) plus {omega}-conotoxin MVIIC (1 µM), and nicardipine (10 µM), respectively]. B, Fractional components of N-, P/Q-, L-, and R(remaining)-type Ca2+-channel subtypes (open bars) and inhibition of each type of currents by PGE2, expressed as a fraction of the total currents (closed bars). Each component was calculated by subtracting Ba2+ currents recorded just before and after drug application. The data are shown as the mean ± SE of the values obtained from 16 experiments. C, Representative traces of total, P/Q-type, and L-type currents with or without PGE2 (1 µM). P/Q- and L-type currents were obtained by subtracting Ba2+ currents recorded before and after blockade of each currents. D, Summary data for the ratio of IBa3–8 against IBa40–45 of P/Q- and L-type currents (open bars), and the ratio of percent inhibition of IBa3–8 against that of IBa40–45 by 1 µM PGE2 for P/Q- and L-currents (closed bars; calculated by subtracting Ba2+ current traces before and after blockade of each current, as in C). Note that P/Q-type currents (n = 14) showed kinetic slowing, and that L-type currents (n = 14) showed steady-state inhibition, in response to PGE2. Asterisks, Significance against the value calculated from total currents (P < 0.05).

 
There was some difference between inhibition of the two major components, P/Q- and L-type currents: inhibition of P/Q-type currents was mainly kinetic slowing and that of L-type currents was mainly steady-state inhibition (Fig. 7CGo). This was further confirmed by calculating the ratio of inhibition of IBa3–8 against that of IBa40–45 induced by PGE2 (Fig. 7DGo), where the ratio was significantly larger for P/Q-type but significantly smaller for L-type, compared with the ratio for the total currents. Moreover, the majority of the inhibition of P/Q-type currents by PGE2 was removed by the depolarizing prepulse, whereas that of the L-type current inhibition was insensitive to the prepulse (Fig. 8Go, A and B). The prepulse-sensitive components in the PGE2-induced inhibition of P/Q-, and L-type currents were 87.3 ± 7.8 (n = 3) and 33.2 ± 10.8% (n = 7), respectively, of the total inhibition of each current.



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  		Figure 8. Effects of a depolarizing prepulse on PGE2-induced inhibition of P/Q- and L-type currents. A, Representative traces of Ba2+ currents before and during PGE2 (1 µM) application obtained with or without a depolarizing prepulse (to 80 mV, for 50 msec). Total, P/Q-type, and L-type currents obtained from a single cell are shown. P/Q-type and L-type currents were obtained by subtraction of Ba2+ currents recorded before and after blockade of each current. B, Summary data for the effects of a prepulse on PGE2-induced inhibition of the total, P/Q,- and L-type IBa3–8. Closed bars, Voltage-insensitive inhibition persisted when the prepulse was applied. The data are shown as the mean ± SE of the values obtained from three (P/Q-type) and 7 (L-type) experiments. Note that inhibition of P/Q-type currents was almost entirely removed by the prepulse, whereas that of L-type currents was largely unaffected.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that PGE2 produced reversible inhibition of voltage-dependent Ba2+ currents in the majority of rat melanotrophs examined. Moreover, the maximum inhibition by PGE2 was similar to that observed with dopamine, the classic secretoinhibitory transmitter for melanotrophs. Such potent inhibitory actions may be one of the mechanisms by which PGE2 suppresses spontaneous secretion and basal [Ca2+]i in rat melanotrophs (2, 12).

Comparison between responses to PGE2 and dopamine
The inhibitory effects of dopamine and PGE2 were similar in several aspects, including the magnitude of inhibition, the pattern of inhibition, and the prepulse sensitivity, indicating that activation of receptors of the two ligands evoke similar cellular mechanisms to inhibit voltage-dependent Ca2+ channels of melanotrophs. One difference between the inhibition by PGE2 and that by dopamine was that the time course of recovery from inhibition was significantly slower in PGE2-mediated inhibition. The difference could be accounted for by a difference in the dissociation rates of PGE2 and dopamine from their receptors, because an EP1/EP3 agonist, 17PT-PGE2, produced rapidly reversible inhibition, just as dopamine. It has been shown that neuropeptide Y, which is known to dissociate from its receptor 10–100 times more slowly than dopamine, showed more persistent inhibitory effects on peptide secretion than dopamine in melanotrophs of Xenopus laevis (24). It remains to be studied whether PGE2 causes persistent inhibition of peptide secretion or cytosolic Ca2+ behavior in rat melanotrophs.

The property of PG receptors in rat melanotrophs
Four subclasses of PGE receptors, EP1-EP4, have been cloned to date, and diverse cellular actions are known to be mediated by these receptors (25, 26). The present results with EP agonists and antagonist indicate that EP1 and EP3 receptors are present in rat melanotrophs and mediate inhibition of voltage-dependent Ca2+ channels. Of the two EP receptors, however, EP3 receptors seem to be the major EP receptor subtype, because the selective EP1 antagonist SC-51322 blocked the total inhibition by PGE2 by only 23%. In general, cellular functions mediated through EP3 receptors have been the best characterized among the four EP receptors and have been shown to be coupled to PTX-sensitive G proteins in various types of cells, whereas the signal transduction of EP1 receptors is poorly understood (26). The present results, showing that pretreatment with PTX potently reduced the magnitude of the PGE2-induced inhibition of Ba2+ currents, are in good agreement with PTX-sensitive cellular responses reported for EP3 receptors in other types of cells (25, 26). Several reports have revealed that PGE2 modulates Ca2+ channels: PGE2 inhibits voltage-dependent Ca2+ channels in sympathetic neurons (20) and enhances Ca2+ currents in dorsal root ganglion neurons (21). In these reports, however, the PG receptor subtype responsible for the modulation of Ca2+ channels has not been studied. Although EP receptors are known to play critical roles in various cellular functions, the receptor subtypes and the signal transduction involved in such functions are still unclear. This seems to be caused by the fact that only a few selective pharmacological tools for EP receptors have been available. The present study is the first report for EP receptor subtypes mediating modulation of voltage-dependent Ca2+ channels.

The mechanism of the PGE2-induced inhibition of Ca2+ channels
The inhibition of Ca2+ channels by PGE2 consisted of two distinct patterns of inhibition, namely, steady-state inhibition and kinetic slowing of the currents. The two patterns of inhibition closely resemble patterns of inhibition of voltage-dependent Ca2+ channels of rat melanotrophs observed with dopamine or serotonin (5HT) (17, 27). It has been reported that PTX-sensitive G protein-mediated inhibition of voltage-dependent Ca2+ channels shows characteristic kinetic slowing of currents in various types of neuronal or endocrine cells (28). Such G protein-mediated inhibition can be partially or entirely removed by applying a depolarizing prepulse (28), and was thus termed voltage-sensitive inhibition of Ca2+ channels. The mechanism underlying the phenomenon is believed to be mediated by membrane-delimited interaction between voltage-dependent Ca2+ channels and the ß{gamma}-subunits of heterotrimeric G proteins, because overexpression of ß{gamma}-subunits mimicked receptor-mediated inhibition with evident kinetic slowing, and the inhibition by Gß{gamma}-subunits could also be removed by a depolarizing prepulse (29). The present results, that the majority of the PGE2-induced kinetic slowing of Ba2+ currents were removed by a depolarizing prepulse, suggest that PGE2 exerts inhibitory actions mainly through the same mechanism. On the other hand, PGE2 consistently caused steady-state inhibition of Ba2+ currents that were relatively insensitive to a prepulse. Although little is known of voltage-insensitive inhibition of Ca2+ channels, it was ascribed to mechanisms involving protein kinases that are downstream of the G protein activation (30). Such inhibitory mechanisms of Ca2+ channels could account for the prepulse-insensitive inhibition of Ca2+ channels induced by PGE2 in melanotrophs.

The subtypes of voltage-dependent Ca2+ channels of melanotrophs
In the present study, four distinct subtypes (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were identified in rat melanotrophs by the use of selective inhibitors of Ca2+ channels. We did not separate P- and Q-type currents, because of uncertainty in separating the two types of currents (31, 32). In voltage-dependent Ba2+ currents of rat melanotrophs, P/Q-type currents were the largest and carried approximately half of the total Ba2+ currents, and L-type currents carried approximately one third of the total currents. We found that half of melanotrophs examined possessed Ba2+ currents sensitive to blockade by {omega}-conotoxin GVIA, a selective blocker of N-type currents. Although N-type currents have been shown to play a major role in Ca2+ oscillations in melanotrophs of Xenopus laevis, by the use of {omega}-conotoxin GVIA (33), the existence of N-type channels in rat melanotrophs has been controversial: earlier studies without using a selective channel blocker reported a large fraction of N-type channels, but this was based on electrophysiological properties of the currents, such as voltage-dependent inactivation and the lack of susceptibility to dihydropyridines (14, 16). A more recent study, performed by using {omega}-conotoxin GVIA, reported that N-type currents are absent in rat melanotrophs (17). The discrepancy between this and our results could be caused by the use of primary culture in the former results, because Ca2+ channels of rat melanotrophs are reported to undergo substantial changes during primary culture (34, 35). On the other hand, Beatty et al. (36), in their measurements of [Ca2+]i, observed Ca2+ influx through {omega}-conotoxin GVIA-sensitive channels in melanotrophs obtained from rats of postnatal days 1 and 12 but not in melanotrophs of day 42 rats. Thus, it is also possible that the number of N-type channels, expressed in melanotrophs decreases during the period of postnatal development, and the age of rats used in the present study were close to the threshold period. These possibilities should be examined by measuring N-type currents in melanotrophs maintained in long-term culture or in those obtained from newborn rats. A significant portion of Ba2+ currents remained after the blockers for N-, P/Q-, and L-type currents were added, indicating that the currents are carried by R-type channels. It seems unlikely that this is caused by incomplete blockade of N-, P/Q-, or L-type currents, because we used a supramaximal concentration of nicardipine, and, moreover, increasing concentrations of N- and P/Q-type channel blockers did not cause further inhibition of Ba2+ currents.

The Ca2+ channel subtypes susceptible to inhibition by PGE2
The present results revealed that among the high-threshold Ca2+ channels, only L- and P/Q- type Ca2+ channels receive significant inhibitory influence by PGE2. This is in good agreement with a selective block of L- and Q-type currents by 5HT, observed in rat melanotrophs (17). Although receptors coupled to PTX-sensitive G proteins are known to inhibit certain types of Ca2+ channels and the type varies between preparations, the most common targets of the modulation by receptor ligands seems to be N- and P/Q-type Ca2+ channels in most neuronal preparations (37, 38, 39). This is consistent with results obtained from cells expressing subunits of cloned neuronal Ca2+ channels, showing that Ca2+ currents in cells expressing {alpha}1A (P/Q-type) or {alpha}1B (N-type) were inhibited by neurotransmitters, whereas those in cells expressing {alpha}1C (L-type) or {alpha}1E (R-type) were unresponsive (40, 41). In endocrine cells, however, L-type channels play a major role in stimulus-secretion coupling and are indeed the major target of inhibition by transmitters or hormones (42, 43, 44, 45). Several classes of dihydropyridine-sensitive Ca2+ channels have been cloned to date (28), and such diverse molecules might explain the difference between the properties of L-type channels in neurons and endocrine cells. It should be noted that rat melanotrophs express mRNA for the {alpha}1D subunit of Ca2+ channels, which are also known to be dihydropyridine sensitive, and the selective D2 agonist, bromocriptine, decreased {alpha}1D mRNA levels (46).

The mode of inhibition of P/Q- and L-type Ca2+ channels was different, in that the former was mainly kinetic slowing and was potently removed by a prepulse, whereas the latter was mainly steady-state inhibition and was relatively resistant to a prepulse. Such a difference suggests that there may be two distinct mechanisms for PGE2-mediated inhibition of Ca2+ channels of rat melanotrophs: P/Q type Ca2+ channels receive mainly voltage-sensitive inhibition, whereas L-type Ca2+ channels receive mainly voltage-insensitive inhibition. The mode of P/Q-type inhibition observed in the present study closely resembles the pattern of inhibition of N- or P/Q-type Ca2+ channels observed in neuronal preparations (37, 38), suggesting that P/Q-type channels are the major target of inhibition by Gß{gamma} in melanotrophs. Although the mechanism of inhibition of L-type Ca2+ channels is unclear, this seems to be particularly important for secretoinhibitory actions of dopamine and other ligands, because spontaneous and high-K+-evoked melanotroph secretion and basal [Ca2+]i of melanotrophs was potently suppressed by dihydropyridines (47, 48). Because the pars intermedia consists of a virtually homogeneous population of melanotrophs, it may provide a good preparation for study of the precise cellular mechanism of the voltage-insensitive inhibition of Ca2+ channels.

Physiological or pathophysiological significance of inhibition of melanotrophs by PGE2
PGE2 is synthesized by various types of cells, including immune cells, in physiological and/or pathophysiological conditions. Moreover, the pituitary gland has been shown to be a site of synthesis of PGs, including PGE2 (2, 49). It has been reported that in preparations containing both the hypothalamus and the neurointermediate lobe of the pituitary gland, melanotroph secretion was suppressed by PGE2 (but not by PGD2, PGF2a, or PGI2) and potentiated by indomethacin, an inhibitor of PG synthesis (2). The results indicate that PGE2 may serve as an endogenous inhibitory factor regulating melanotroph secretion. On the other hand, cytokines are known to stimulate production of various PGs. This seems to be of particular importance because {alpha}MSH inhibits various actions mediated by cytokine receptors (50). The cascade from cytokines to PGE2, {alpha}MSH, and to cytokine receptors could form a positive feedback loop for cytokine actions when circulating cytokine and PGE2 concentrations increase. Although the function of the peptides released by melanotrophs in mammals, in contrast with that in amphibians, is largely unknown, such a cascade may be a candidate for the physiological and/or pathophysiological significance of mammalian pituitary pars intermedia.


   Acknowledgments
 
The authors are grateful to Ono Pharmaceutical Co. Ltd. for providing us SC-51322 and butaprost and to Dr. Kongsamut (Hoechst Marrion Roussel, Bridgewater, NJ) for the critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Grants-in-Aid 09470020 (to I.S.) and 08457022 (to H.Y.) from the Ministry of Education, Science and culture, Japan, and Research Grant 1173 (to K.T.) from the Japanese Society of the Promotion of Science (to K.T.). Back

Received March 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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