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Endocrinology Vol. 142, No. 11 4785-4794
Copyright © 2001 by The Endocrine Society

ARTICLES

Ceramide Inhibits the Inwardly Rectifying Potassium Current in GH3 Lactotrophs

Sheng-Nan Wu, Yuk-Keung Lo, Benjamin Ing-Tiau Kuo and Hung-Ting Chiang

Departments of Medical Education and Research (S.-N.W.) and Internal Medicine (H.-T.C.) and Section of Neurology (Y.-K.L.), Kaohsiung Veterans General Hospital, Kaohsiung City, Taiwan; Institute of Biomedical Sciences, National Sun Yat-Sen University (S.-N.W.), Kaohsiung City, Taiwan; and National Yang-Ming University (Y.-K.L., I.-T.K., H.-T.C.), Taipei City, Taiwan

Address all correspondence and requests for reprints to: Sheng-Nan Wu, M.D., Ph.D., Department of Medical Education and Research, Kaohsiung Veterans General Hospital, No. 386, Ta-Chung First Road, Kaohsiung City, Taiwan, Republic of China. E-mail: snwu{at}isca.vghks.gov.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of ceramide on ion currents in rat pituitary GH3 cells were investigated. Hyperpolarization-elicited K+ currents present in GH3 cells were studied to determine the effect of ceramide and other related compounds on the inwardly rectifying K+ current (IK(IR)). Ceramide (C2-ceramide) suppressed the amplitude of IK(IR) in a concentration-dependent manner, with an IC50 value of 5 µM. Ceramide caused a rightward shift in the midpoint for the activation curve of IK(IR). Pretreatment with PD-98059 (30 µM) or U-0126 (30 µM) did not prevent ceramide-mediated inhibition of IK(IR). However, the magnitude of ceramide-induced inhibition of IK(IR) was attenuated in GH3 cells preincubated with dithiothreitol (10 µM). TNF{alpha} (100 ng/g) also suppressed IK(IR). In the inside-out configuration, application of ceramide (30 µM) to the bath slightly suppressed the activity of large conductance Ca2+-activated K+ channels. Under the current clamp mode, ceramide (10 µM) increased the firing of action potentials. Cells that exhibited an irregular firing pattern were converted to those displaying a regular firing pattern after application of ceramide (10 µM). Ceramide also suppressed IK(IR) in neuroblastoma IMR-32 cells. Therefore, ceramide can produce a depressant effect on IK(IR). The blockade of this current by ceramide may affect cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CERAMIDE, A PRODUCT of sphingomyelin turnover, is a lipid second messenger that is implicated in the regulation of several cellular responses to extracellular stimuli, including differentiation, growth suppression, cell senescence, and apoptosis (1). Ceramide (C2-ceramide) can be generated within the cell via the hydrolysis of sphingomyelin or de novo synthesis. It has been shown that an increase in ceramide levels within the cell would occur in response to several inducers of cellular stress (1, 2, 3, 4).

There are several lines of evidence showing that ceramide can regulate ion channels. For example, ceramide was found to block Ca2+-activated K+ (BKCa) channels in coronary smooth myocytes (5) and to suppress an inwardly rectifying K+ current in oligodendrocytes (6). In contrast, ceramide enhanced the delayed rectifier K+ current in cortical neurons (7). In cerebral vascular muscle, ceramide was also reported to induce contraction and increase intracellular Ca2+ (8). To date, however, limited information has been reported regarding the underlying mechanism of actions of ceramide on ion currents in pituitary lactotrophs, although ceramide was found to inhibit depolarization-evoked Ca2+ entry in pituitary cells (9, 10).

Pituitary GH3 lactotrophs, in addition to the presence of voltage-dependent K+ and Ca2+ currents, have been shown to exhibit an inwardly rectifying K+ current (IK(IR)). On the basis of biophysical and pharmacological properties, this current that was sensitive to the inhibition by E-4031, and TRH was previously identified as an erg (ether-à-go-go-related)-mediated K+ current (11, 12, 13, 14, 15). This current was also thought to be an important determinant of the resting membrane potential (13, 15). The inhibition of this current may produce an increase in the firing rate of action potentials (13, 15, 16) and lead to an increase in PRL secretion by lactotrophs (17).

Therefore, in the present study the electrophysiological effects of ceramide and other related compounds in GH3 cells were investigated. We sought to 1) determine whether ceramide (C2-ceramide) has any effect on the erg-like IK(IR) in GH3 cells; 2) compare the potency of other related compounds in blocking the amplitude of IK(IR); 3) examine the effect of ceramide on other types of ion currents, including voltage-dependent L-type Ca2+ currents and large conductance BKCa channels; and 4) ascertain whether ceramide can influence the membrane potential and the firing pattern of spontaneous action potentials in these cells. The present results indicate that the underlying ceramide-induced inhibition of IK(IR) in GH3 cells can significantly contribute to the change in membrane potential, thus affecting PRL secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The clonal strain GH3 cell line, originally derived from a rat anterior pituitary adenoma, was obtained from the Culture Collection and Research Center (CCRC-60015, Hsinchu, Taiwan) (13). Cells were cultured routinely in monolayer culture in 50-ml plastic culture flasks in a humidified environment of 5% CO2/95% air in 5 ml Ham’s F-12 nutrient medium (Life Technologies, Inc., Grand Island, NY). The media were supplemented with 15% heat-inactivated horse serum (vol/vol), 2.5% FCS (vol/vol), and 2 mM L-glutamine (Life Technologies, Inc.). Cells were subcultured once a week, and a new stock line was generated from frozen cells (frozen in 10% glycerol in medium plus serum) every 3 months. The experiments were performed after 5 or 6 d of subcultivation (60–80% confluence).

Stock cultures of human neuroblastoma IMR-32 cells were also obtained from the Culture Collection and Research Center (CCRC-60014). IMR-32 cells were maintained in Eagle’s MEM (Life Technologies, Inc.) supplemented with 2 mM L-glutamine and Earle’s balanced salt solution adjusted to contain 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 10% FBS (vol/vol).

Electrophysiological measurements
Immediately before each experiment, GH3 or IMR-32 cells were dissociated, and an aliquot of cell suspension was transferred to a recording chamber mounted on the stage of an inverted microscope (Diaphot 200, Nikon, Tokyo, Japan). Cells were bathed at room temperature (20-25 C) in normal Tyrode’s solution containing 1.8 mM CaCl2. Patch pipettes (3–5 M{Omega} in bathing solution) were prepared from Kimax capillary tubes (Vineland, NJ) using a two-step electrode puller (PP-83, Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83, Narishige). Membrane currents were recorded in the whole cell or inside-out mode of the patch-clamp technique with an RK-400 patch amplifier (Biologic, Claix, France) (13, 18). All potentials were corrected for liquid junction potential, a value that would develop at the tip of the pipette when the composition of pipette solution was different from that of the bath.

Data recording and analysis
The signals consisting of voltage and current tracings were displayed with digital storage oscilloscope (model 1602, Gould, Valley View, OH) and LCD projector (AV600, Delta, Taipei, Taiwan). The data were simultaneously recorded on a digital audio tape recorder (model ZA5ES, Sony, Tokyo, Japan). Current signals were low pass filtered at 1 kHz before digitization. A Digidata 1320A interface (Axon Instruments, Inc., Union City, CA) was used for the analog to digital/digital to analog conversion. To reduce electrical noise, this interface device was connected to a Pentium III-based portable computer (Slimnote VX3, Lemel, Taipei, Taiwan) through a USB port and was then controlled with the aid of the Clampex subroutine in the pCLAMP 8.02 software (Axon Instruments). Voltage-activated currents recorded during whole cell experiments were stored without leakage correction and analyzed subsequently using the Clampfit subroutine of pCLAMP (Axon Instruments) or the Origin 6.0 software (Microcal Software, Inc., Northampton, MA) to construct a current-voltage (I-V) relationship for ion currents.

To calculate percentage inhibition of ceramide on IK(IR), cells were bathed in a high K+, Ca2+-free solution, and each cell was hyperpolarized from -10 to -120 mV. Current amplitudes during the application of ceramide were compared with those measured after a subsequent application of E-4031 (10 µM). E-4031 is known to be a selective blocker of IK(IR) (13, 15). The concentration of ceramide required to inhibit 50% of current amplitude was fitted to a Hill equation: y = Emax/{1 + (IC50n/[C]n)}, where [C] is the concentration of ceramide, IC50 and n are the half-maximal concentration of ceramide required to inhibit IK(IR) (i.e. E-4031-sensitive current) and the Hill coefficient, respectively, and Emax is ceramide-induced maximal inhibition of IK(IR).

Unitary currents of BKCa channels were analyzed with Fetchan and Pstat subroutines in the pCLAMP software (Axon Instruments). Multi-Gaussian adjustments of the amplitude distributions between channels were used to determine unitary currents. The functional independence between channels was verified by comparing the observed stationary probabilities with the values calculated according to the binomial law. The number of active channels in the patch was counted at the end of each experiment through perfusion of a solution with 100 µM Ca2+ and then used to normalize opening probability at each potential.

The alteration in membrane potentials of GH3 cells was examined under the current clamp conditions. The frequency of spontaneous action potentials was characterized by transforming the oscillating signals from their time domain to their representation in the frequency domain with the aid of power spectral analysis. Spectral analysis was performed based on a discrete Fourier transform algorithm with the aid of Origin software (Microcal Software) (13). When spontaneous action potentials in GH3 exhibited a regular discharge pattern, a concentrated peak shown in the power spectrogram would correspond to the mean firing rate.

All values are reported as the mean ± SEM. The paired or unpaired t test and one-way ANOVA with the least significance difference method for multiple comparisons were used for the statistical evaluation of differences among the mean values. Differences between the values were considered significant at P < 0.05 or P < 0.01.

Drugs and solutions
PD-98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), U-0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene), and (Bu)2AMP were obtained from Tocris (Bristol, UK). C2-ceramide (N-acetylsphingosine), C2-dihydroceramide (N-acetylsphinganine), E-4031, and penitrem A were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). 4,4'-dithiodipyridine (DTDP), dithiothreitol, TRH, tetraethylammonium chloride, tetrodotoxin, IL-1ß and TNF{alpha} were purchased from Sigma (St. Louis, MO). Azimilide was a gift from Procter & Gamble (Cincinnati, OH). All other chemicals were commercially available and of reagent grade.

The composition of normal Tyrode’s solution was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES-NaOH buffer, pH 7.4. To record K+ currents, patch pipette was filled with the solution consisting of 140 mM KCl, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer, pH 7.2. To record the inwardly rectifying K+ current, high K+, Ca2+-free solution contained 130 mM KCl, 10 mM NaCl, 3 mM MgCl2, 6 mM glucose, and 10 mM HEPES-KOH, pH 7.4. To record Ca2+ current, KCl inside the pipette solution was replaced with equimolar CsCl, and the pH was adjusted to 7.2 with CsOH.

In the single channel recording, high K+-bathing solution contained 145 mM KCl, 0.53 mM MgCl2, and 5 mM HEPES-KOH, pH 7.4, and the pipette solution contained 145 mM KCl, 2 mM MgCl2, and 5 mM HEPES-KOH, pH 7.2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ceramide on hyperpolarization-activated currents in GH3 cells
The whole cell configuration of the patch-clamp technique was used to investigate the effect of ceramide on macroscopic ion currents. When GH3 cells were bathed in a high K+-Ca2+ free solution, a family of large inward current upon membrane hyperpolarization could be observed. Examples of ion currents elicited by the 1-sec long clamp pulses to various membrane potentials from a holding potential of -10 mV are shown in Fig. 1Go. Hyperpolarizing voltage pulses were found to induce an instantaneous current, followed by a voltage- and time-dependent activation of K+ inward current. These inward currents decayed at potentials below -50 mV, and the decay became faster with greater hyperpolarization (13, 15, 19).



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  		Figure 1. Inhibitory effect of ceramide on the I-V relationships of the hyperpolarization-evoked currents in rat pituitary GH3 cells. Cells were bathed in a high K+, Ca2+-free solution containing tetrodotoxin (1 µM) and CdCl2 (0.5 mM). A, Superimposed current traces obtained when a cell was held at the level of -10 mV, and various voltage pulses ranging from 0 to -120 mV in 20-mV increments were applied. Current traces shown in Aa are controls, and those in Ab were obtained 1 min after application of ceramide (30 µM). The right side of A shows the ceramide-sensitive inward current (i.e. a-b). B, Averaged I-V relationships for initial (circles) and steady state (squares) components of ion currents in the absence (upper part) and presence (lower part) of 30 µM ceramide. Each point represents the mean ± SEM (n = 8–12).

 
Within 1 min of exposing the cell to ceramide (30 µM), the amplitude of the hyperpolarization-elicited currents was significantly reduced. For example, when cells were hyperpolarized from -10 to -120 mV, the presence of ceramide (30 µM) significantly decreased the amplitude of peak inward currents from 3950 ± 182 to 1225 ± 102 pA (n = 9). This inhibitory effect was readily reversed on the removal of ceramide. However, there was no significant difference in current amplitudes taken at the end of the hyperpolarizing pulses from -10 to -120 mV (i.e. late sustained inward current) between the absence (1280 ± 62 pA; n = 9) and presence (1272 ± 59 pA; n = 8) of 30 µM ceramide. When the difference of current traces between the presence and absence of ceramide (30 µM) was taken, the net change in membrane currents, i.e. the ceramide-sensitive component, was obtained. As shown in Fig. 1AGo, these ceramide-sensitive inward currents exhibited a voltage dependence of activation and current decay similar to that of control currents. The presence of ceramide (30 µM) significantly decreased the slope of the linear fit of current amplitudes to the voltages between -120 and -60 mV from 37.5 ± 8.5 to 6.9 ± 2.8 nS (n = 8). These data indicate that an inwardly rectifying K+ current (IK(IR)) was present in GH3 cells (13, 15, 19, 20), and that the blocking effect of ceramide was exerted primarily on the component of membrane currents that is responsible for an inward rectification.

The relationship between the concentration of ceramide and the percent inhibition of IK(IR) is illustrated in Fig. 2Go. The current amplitudes of IK(IR) in the presence of ceramide was compared with those after a subsequent application of E-4031 (10 µM). Application of ceramide (0.3–100 µM) was found to suppress the amplitude of E-4031-sensitive currents in a concentration-dependent manner. The half-maximal concentration required for the inhibitory effect of ceramide on IK(IR) was 5 µM, and 100 µM ceramide nearly abolished current amplitude.



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  		Figure 2. Concentration-dependent inhibition of IK(IR) by ceramide in GH3 cells. A, Superimposed current traces obtained in the absence and presence of ceramide. Cells were bathed in a high K+, Ca2+-free solution, and hyperpolarizing pulses from -10 to -120 mV were applied. 1, Control; 2 and 3, obtained after addition of 3 and 10 µM ceramide, respectively; 4, after the addition of E-4031 (10 µM), but in the presence of ceramide (10 µM). B, Concentrationresponse relationship for ceramideinduced inhibition of IK(IR), i.e. E-4031-sensitive current. Each point represents the mean ± SEM (n = 6–9). The smooth line represents the best fit to the Hill equation. The IC50 values, maximally inhibited percentage of E-4031-sensitive current, and Hill coefficient were 5 µM, 99%, and 1.1, respectively.

 
Comparison between the effect of C2-ceramide and those of C2-dihydroceramide, E-4031, azimilide, 4-dithiodipyridine, and TRH
The effects of other related compounds, including C2dihydroceramide, E-4031, azimilide, 4-dithiodipyridine, and TRH, on the amplitude of IK(IR) in these cells were examined and compared. C2-Dihydroceramide is an inactive C2ceramide analog. Azimilide was reported to be a blocker of HERG channels (21). 4,4'-Dithiodipyridine is known to be a lipophilic sulfhydryl oxidizing agent. As depicted in Fig. 3Go, C2-dihydroceramide (10 µM) had little effect on the amplitude of IK(IR). However, like E-4031, azimilide (10 µM) significantly suppressed IK(IR) by approximately 90%. Similar to previous studies (11, 14), TRH (10 µM) could inhibit IK(IR) in these cells. Interestingly, 4,4'-dithiodipyridine (10 µM) also suppressed this current by about 45%. Therefore, the data presented here suggested that, unlike C2-dihydroceramide, ceramide can suppress IK(IR) in GH3 cells.



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  		Figure 3. Comparison between the effect of ceramide (C2-ceramide) and those of C2-dihydroceramide, E-4031, azimilide, DTDP, and TRH on the amplitude of IK(IR). Each cell was hyperpolarized from -10 to -120 mV with a duration of 1 sec. The peak amplitude of IK(IR) in the control was considered to be 1.0, and the relative amplitude of IK(IR) after application of each agent was plotted. The parentheses next to each bar indicate the number of cells examined. Values are the mean ± SEM.

 
Voltage dependence of ceramide-induced inhibition of IK(IR)
To further characterize the inhibitory effects of ceramide on IK(IR), we also examined the voltage dependence of the effect of ceramide on IK(IR). Figure 4Go shows the steady state activation curve of IK(IR) in the absence and presence of ceramide (10 µM). A two-step voltage protocol was applied (22, 23, 24). A 15-sec conditioning pulse to various membrane potentials preceded a test pulse (1 sec in duration) to -120 mV from a holding potential of -10 mV. The relationships between the membrane potentials and the normalized amplitudes of IK(IR) with or without the application of ceramide (10 µM) were plotted and fitted by the Boltzmann function using nonlinear regression analysis (25): I = Imax/{1 + exp[(V -a)/b]}, where Imax is the maximal activated IK(IR), V is the membrane potential in mV, a is the membrane potential for a half-maximal activation, and b is the slope factor of activation curve. In control, a = -28.7 ± 1.1 mV, and b = -9.8 ± 0.6 mV (n = 5), whereas in the presence of ceramide (10 µM), a = -18.6 ± 0.8 mV, and b = -9.6 ± 0.4 mV (n = 5). Thus, ceramide not only inhibited the maximal conductance of IK(IR), but also shifted the activation curve to depolarized potentials by approximately 10 mV. In contrast, no significant change in the slope (i.e. b value) of the curve was seen in the presence of ceramide. These results indicate that the voltage-dependent behavior of IK(IR) in GH3 cells is different in the presence of ceramide.



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  		Figure 4. Effect of ceramide on steady state activation curve of IK(IR). By use of a two-step protocol, the steady state activation parameters of IK(IR) were obtained in the absence and presence of ceramide (10 µM). The conditioning voltage pulses with a duration of 15 sec to various membrane potential between +10 and -70 mV in 10-mV increments were applied from a holding potential of -10 mV. After each conditioning pulse, a test pulse to -120 mV with a duration of 1 sec was applied to evoke IK(IR). The superimposed current traces obtained in the control are illustrated in A. The uppermost part in A indicates the voltage protocol. The lower part in A shows original current traces obtained in an expanded time scale, as indicated by an open arrow in the middle of A. The filled arrows indicate the zero current level. In B, the normalized amplitude of IK(IR) (I/Imax) was constructed against the conditioning potential, and the curves obtained in the absence and presence of ceramide (10 µM) were fitted by the Boltzmann function. Each point represents the mean ± SEM (n = 5–7).

 
Effect of ceramide on IK(IR) in cells preincubated with PD-98059 or U-0126
It was previously reported that the effects of ceramide on apoptotic changes in astrocytes or in airway smooth myocytes was related to the activity of the ERK or p42/p44 MAPK (p42/p44 MAPK) (26, 27). The effect of ceramide on IK(IR) was thus assessed in cells treated with PD-98059 (10 µM) or U-0126 (10 µM). PD-98059 can inhibit the ERK cascade and be an inhibitor of p44/p42 MAPK, whereas U-0126 can suppress the activity of p44/p42 MAPK (26, 28). However, in this study it was found that neither PD-98059 (10 µM) nor U-0126 (10 µM) significantly affected the ceramide-inhibited IK(IR) (Fig. 5Go). The magnitude of ceramide-inhibited IK(IR) remained unaltered in cells preincubated with PD-98059 or U-0126 cells. Thus, the inhibitory effect of ceramide on IK(IR) observed in GH3 cells did not seem to be mediated by the activation of ERKs or p42/p44 MAPKs.



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  		Figure 5. Effect of ceramide on the averaged I-V relations of IK(IR) in GH3 cells treated with PD-98059 (upper part) and U-0126 (lower part). GH3 cells were preincubated with PD-98059 (10 µM) or U-0126 (10 µM) for 5 h. Each cell was held at -10 mV, and various potentials ranging from 0 to -120 mV in 20-mV increments were applied. Each point represents the mean ± SEM (n = 5–8). Open symbols, Control; closed symbols, in the presence of ceramide (10 µM).

 
Effect of ceramide on IK(IR) in dithiothreitol-treated GH3 cells
It has recently been shown that ceramide could increase free oxygen radicals in smooth muscle cells and neutrophils (29, 30). 4,4'-Dithiopyridine was also noted to suppress IK(IR) in GH3 cells. Therefore, we examined whether changes in reactive oxygen species can affect ceramide-induced inhibition of IK(IR) in GH3 cells. Interestingly, we showed that ceramide-induced inhibition in the amplitude of IK(IR) was attenuated in GH3 cells preincubated with 10 µM dithiothreitol. Dithiothreitol is a sulfhydryl reducing agent. Furthermore, the subsequent application of 4,4'-dithiodipyridine greatly suppressed IK(IR) (Fig. 6Go). For example, when the dithiothreitol-treated cells were hyperpolarized from -10 to -120 mV, ceramide (30 µM) suppressed IK(IR) by only about 15%. In contrast, in control cells ceramide (30 µM) almost fully inhibited these currents. These results suggest that reducing and oxidizing agents have some effect on IK(IR) in these cells.



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  		Figure 6. Effect of ceramide on IK(IR) in GH3 cells preincubated with dithiothreitol. In these experiments GH3 cells were incubated with dithiothreitol (10 µM) for 5 h. Cells bathed in a high K+ Ca2+-free solution were held at -10 mV, and voltage pulses ranging from 0 to -140 mV in 20-mV increments were applied. A, Original current traces obtained for controls (upper part), in the presence (middle part) of ceramide (30 µM), and in the presence (lower part) of ceramide (30 µM) plus DTDP (30 µM). Open arrows indicate the zero current level. The uppermost part in A indicates the voltage protocol. Of note, in dithiothreitol-treated GH3 cells the presence of ceramide produced a slight reduction in IK(IR); however, the subsequent application of DTDP greatly suppressed IK(IR). B, Current density vs. membrane potential relationships of IK(IR) measured at the peak components of IK(IR) in dithiothreitol-treated GH3 cells. {circ}, Control; •, in the presence of ceramide (30 µM);. {blacksquare}, ceramide (30 µM) plus DTDP (30 µM). Each point represents the mean ± SEM (n = 5–8).

 
Effect of IL-1ß and TNF{alpha} on the amplitude of IK(IR)
Cytokines (e.g. IL-1ß or TNF{alpha}) are known to affect neuroendocrine secretory activities and increase the production of ceramide (31). Thus, it will be of importance to examine the effects of IL-1ß and TNF{alpha} on IK(IR) in these cells. The results showed that IL-1ß (100 ng/g) had no effect on the amplitude of IK(IR), whereas TNF{alpha} (100 ng/g) suppressed it (Fig. 7Go). TNF{alpha} (100 ng/g) significantly decreased IK(IR) from 1984 ± 210 pA (n = 7) to 1462 ± 118 pA (n = 5). Thus, similar to ceramide, TNF{alpha} can cause an inhibitory effect on IK(IR) in GH3 cells.



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  		Figure 7. Effect of IL-1ß and TNF{alpha} on the amplitude of IK(IR) in GH3 cells. Cells were bathed in a high K+, Ca2+ free solution. Each cell was hyperpolarized from -10 to -120 mV with a duration of 1 sec. A, Original current traces obtained in the absence and presence of IL-1ß (Aa) and TNF{alpha} (Ab). 1, Controls; 2, obtained 2 min after the addition of 100 ng/g IL-1ß (Aa) or 100 ng/g TNF{alpha} (Ab). B, Bar graph showing the effects of 100 ng/g IL-1ß and 100 ng/g TNF{alpha} on the amplitude of IK(IR). In parentheses above each bar are the number of cells examined. Values are the mean ± SEM. *, P < 0.05 vs. control.

 
Lack of effect of ceramide on voltage-dependent L-type Ca2+ currents (ICa,L)
The effect of ceramide on the amplitude of ICa,L was also assessed in this study. In these experiments cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2, and the recording pipettes were filled with a Cs+-containing solution. As shown in Fig. 8Go, the presence of ceramide (10 µM) had no effect on the amplitude of ICa,L. For example, when cells were depolarized from -50 to 0 mV, there was no significant change in the amplitude of ICa,L between the absence (135 ± 14 pA; n = 6) and presence (134 ± 12 pA; n = 6) of 10 µM ceramide. However, subsequent application of tetrandrine (5 µM) produced a significant reduction of ICa,L without altering the overall I-V relationships of ICa,L. Tetrandrine was reported to suppress ICa,L in GH3 cells (32). Thus, in GH3 cells ICa,L, unlike IK(IR), was relatively unaffected in the presence of ceramide.



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  		Figure 8. Effect of ceramide on ICa, L in GH3 cells. Cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2, 1 µM tetrodotoxin, and 10 mM tetraethylammonium chloride. The recording pipette was filled with a Cs+-containing solution. In A, original current traces were recorded when a cell was depolarized from -50 to 0 mV. a, Control; b, ceramide (10 µM); c, ceramide (10 µM) plus tetrandrine (5 µM). The filled arrow indicates the zero current level. In B, the I-V relationships of ICa, L were obtained for controls ({circ}), in the presence of 10 µM ceramide (•), and in the presence of 10 µM ceramide plus 5 µM tetrandrine ({blacksquare}). Tetra, Tetrandrine.

 
Inhibitory effect of ceramide on large conductance BKCa channels
The effect of ceramide on the activity of BKCa channels was further examined. In these experiments the single channel recordings with an inside-out mode were performed in a symmetrical K+ (145 mM) solution (13). Bath medium contained 0.5 µM Ca2+, and holding potential was continuously set at +60 mV. The probability of channel openings at the level of +60 mV in the control was 0.21 ± 0.03 (n = 7). After application of ceramide (10 µM) to the bath, no significant change in the activity of BKCa channels was found [0.21 ± 0.03 (n = 7) vs. 0.21 ± 0.02 (n = 6)]. However, when the excised membrane patches were intracellularly exposed to ceramide at a concentration of 30 µM, the channel activity was significantly decreased (Fig. 9Go). The addition of ceramide (30 µM) decreased the opening probability to 0.14 ± 0.02 (n = 8). However, there was no significant alteration in the single channel conductance of BKCa channels between the absence (193 ± 9 pS; n = 6) and presence (192 ± 8 pS; n = 5) of 30 µM ceramide. On the other hand, penitrem A (1 µM) greatly inhibited the channel activity (Fig. 9BGo). Penitrem A was considered to be a potent blocker of BKCa channels (13, 33). These results indicate that ceramide at a concentration of 30 µM did not change the single channel conductance of BKCa channels, but was capable of suppressing the channel activity that was sensitive to inhibition by penitrem A. By comparison, GH3 cells seem to be less responsive to ceramide than smooth muscle cells in suppressing the activity of BKCa channels (5).



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  		Figure 9. Effect of ceramide on the activity of BKCa channels in GH3 cells. A, Original current traces showing the activity of BKCa channels in the absence (upper part) and presence (lower part) of ceramide (30 µM). The inside-out configuration was performed, and bath medium contained 0.5 µM Ca2+. The holding potential was +60 mV. Ceramide (30 µM) was applied to the intracellular surface of the detached membrane patch. Upward deflections are due to the channel opening. B, Bar graph showing the effects of ceramide and penitrem A on BKCa channel activity. In parentheses in each bar are the number of cells examined. Values are the mean ± SEM. *, P < 0.05 vs. control.

 
Effect of ceramide on the firing of action potentials
To determine whether ceramide can cause any change in the membrane potential of GH3 cells, the experiments were conducted with a K+-containing pipette solution, and cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2. Figure 10Go illustrates the effect of ceramide on the firing of action potentials in GH3 cells. When ceramide (10 µM) was added to the bath, the membrane potential was significantly depolarized to -39 ± 6 mV (n = 6) from a control value of -47 ± 7 mV (n = 6). The firing rate of action potentials was increased to 0.98 ± 0.12 Hz from a control value of 0.22 ± 0.07 Hz (n = 7).



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  		Figure 10. Effect of ceramide on spontaneous action potentials in GH3 cells. Cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2. The experiments were performed under current clamp conditions. The left panel is the control, and the right panel was obtained 2 min after application of ceramide (10 µM). Ceramide (10 µM) caused membrane depolarization and induced an increase in the frequency of action potentials. Open arrows in each potential trace indicate the 0 mV potential. The lower part in each panel shows the spectral pattern of firing action potentials in the absence (left) and presence (right) of 10 µM ceramide. Of note, when the cell was exposed to ceramide (10 µM), the repetitive firing was converted from an irregular (left) to a regular (right) pattern.

 
Effect of ceramide on the discharge pattern of spontaneous action potentials in GH3 cells
The effect of ceramide on the firing pattern of action potentials was also investigated. To analyze the discharge pattern of spontaneous action potentials, power spectral analyses of the change in membrane potential that can convert the time domain to the frequency domain were performed (13, 34). The frequency of spontaneous action potentials in GH3 cells was variable, ranging between 0.08 and 0.35 Hz. As shown in Fig. 10Go, the majority of GH3 cells in the control were noted to exhibit the scattered power density in its spectrogram. These data indicate that there was an irregular pattern of the repetitive firing in control GH3 cells. Interestingly, when cells were exposed to ceramide (10 µM), the frequency-domain analysis showed that the discharge pattern of these GH3 cells exhibited a concentrated power density at approximately 1.0 Hz, with its subsequent harmonic components appearing at multiples of 1.0 Hz, e.g. 2.0 Hz. Similar results were found in seven different cells. Thus, the results indicate that exposure of GH3 cells to ceramide produced an increase in the firing rate that was accompanied by the conversion from an irregular to a regular discharge pattern.

Inhibitory effect of ceramide on IK(IR) in neuroblastoma IMR-32 cells
In the final series of studies we examined the effect of ceramide in neuroblastoma IMR-32 cells to determine whether ceramide can affect IK(IR) in other types of neuroendocrine cells. As shown in Fig. 11Go, when cells were bathed in a high K+-Ca2+-free solution, the hyperpolarization-activated currents can be observed in IMR-32 cells. These currents evoked by membrane hyperpolarization correspond to those described as inwardly rectifying K+ currents. The application of azimilide (10 µM) or E-4031 (10 µM) significantly suppressed these currents (data not shown). The application of ceramide (10 µM) resulted in a strong reduction of the hyperpolarization-elicited currents in IMR-32 cells (Fig. 11Go). During hyperpolarizing pulses, the remaining currents were not found to decay as observed in the absence of ceramide. The results indicate that ceramide can suppress the amplitude of IK(IR) present in neuroblastoma IMR-32 cells.



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  		Figure 11. Inhibitory effect of ceramide on the I-V relationships of hyperpolarization-evoked currents in human neuroblastoma IMR-32 cells. Cells were bathed in a high K+, Ca2+-free solution containing tetrodotoxin (1 µM) and CdCl2 (0.5 mM). A, Superimposed current traces obtained when a cell was held at the level of -10 mV and various voltage pulses ranging from 0 to -140 mV in 20-mV increments were applied. Current traces shown in upper part are controls, and those in the lower part were obtained 1 min after the addition of ceramide (10 µM). The uppermost part denotes the voltage protocol. Open arrows indicate the zero current level. B, Averaged I-V relationships for initial (circles) and steady state (squares) components of K+ inward currents in the control (open symbols; left) and during the exposure to 10 µM ceramide (filled symbols; right). Each point represents the mean ± SEM (n = 9–12).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that 1) ceramide can effectively suppress the inwardly rectifying K+ current IK(IR) in a concentration-dependent manner in pituitary GH3 cells; 2) ceramide caused a right shift in the midpoint for the activation curve of IK(IR); 3) ceramide slightly decreased the activity of BKCa channels; 4) ceramide increased the firing rate of action potentials; and 5) ceramide suppressed IK(IR) in neuroblastoma IMR-32 cells. The ceramide-induced inhibition of IK(IR) could result in a depolarization and affect the firing frequency and pattern of spontaneous action potentials in these cells.

The IC50 value of ceramide required for the inhibition of IK(IR) was 5 µM in the present study. This value is lower than that found to suppress Ca2+ channels (10), but is quite close to those required for the increase in intracellular Ca2+ transients, the induction of apoptotic change, or the stimulation of cAMP production (1, 8, 10, 35). Therefore, there might be a link between the actions of ceramide on neurons or neuroendocrine cells and its observed effects on ion channels, although further research is needed to find out whether ceramide can affect IK(IR) in a variety of cells or different types of erg-like current (14).

The present study showed that ceramide not only reduced the maximal conductance of IK(IR), but it produced a positive shift in the steady state activation curve as well. It is thus possible that the inhibitory effect of ceramide on IK(IR) was different at different potentials. However, this effect was due to the fact that the different amounts of inwardly rectifying K+ channels might be in an inactivated state at these different potentials, not to the fact that the effect of ceramide on the channel was different. It thus remains to be determined whether the sensitivity of IK(IR) to ceramide is influenced by changes in membrane potential.

It was reported that ceramide can enhance the GHRH-stimulated increase in intracellular cAMP in pituitary cells (10). However, we did not find that (Bu)2cAMP (100 µM), a cell-permeable analog of cAMP, had any effect on the amplitude of IK(IR) in GH3 or IMR-32 cells (data not shown). It is thus unlikely that the ceramide-induced decrease in IK(IR) is due to the increased level of intracellular cAMP. In GH3 cells preincubated with PD-98059 or U-0126, ceramide also effectively suppressed the amplitude of IK(IR). Thus, the inhibitory effect of ceramide on this current is not associated with activation of the ERK- or p42/p44 mitogen-activated protein kinases. Moreover, the present results seem to differ from the findings of Hida et al. (6), demonstrating that in cultured oligodendrocytes, ceramide suppressed inwardly rectifying K+ currents and that this inhibition was mediated via a Ras- and Raf-1 dependent pathway. This discrepancy is currently unclear, but it could be due to the differences in the types of inwardly rectifying K+ channels and/or cells examined.

It was found that DTDP can suppress IK(IR) in GH3 cells. In dithiothreitol-treated cells, the inhibitory effect of ceramide on IK(IR) was attenuated, and subsequent application of DTDP effectively suppressed IK(IR). These results suggest that the sulfhydryl oxidizing and reducing agents can produce an effect on IK(IR) in GH3 cells. It will thus be of interest to determine whether the effect of ceramide on IK(IR) is related to the production of reactive oxygen species caused by ceramide. Indeed, it was reported that a reactive oxygen species scavenger could reverse the effect of ceramide on pituitary adenylate cyclase activating polypeptide-induced production of cAMP (36). In isolated coronary arteries, the vasodilation in response to ceramide was also shown to be associated with an increase in superoxide production (30). Therefore, it is possible that the production of reactive oxygen species caused by ceramide is upstream of the inhibition of IK(IR).

Ceramide, released as a consequence of sphingomyelinase, is thought to play a role in fundamental processes such as cell proliferation, membrane receptor function, oncogenesis, and immune inflammatory responses (1, 6, 26). Pituitary lactotrophs can generate ceramide under certain conditions, leading us to hypothesize that ceramides may exert effects on pituitary function (3). TNF{alpha}-induced neuronal apoptosis was noted to be implicated in the ceramide-generating pathways (31). Interestingly, we found that, unlike IL-1ß, TNF{alpha}, suppressed IK(IR) in GH3 cells. Indeed, previous reports have shown that KIR channels might be a relevant target of neoplastic transformation and that the cell cycle clock may exert a direct influence on the activity of these cells (22). It will thus remain to be clarified to what extent the ceramide-mediated effects on cellular function are associated with its inhibitory effect on the inwardly rectifying K+ channels. Further study is also needed to determine whether the increased production of ceramide is implicated in TNF{alpha}-mediated inhibition of IK(IR).

The physiological importance of erg-mediated currents has been recognized in cardiac myocytes, neuroblastoma cells, and lactotrophs (12, 22, 37, 38). In fact, in this study we also found that IK(IR), which was sensitive to the inhibition by ceramide, was present in neuroblastoma IMR-32 cells. Furthermore, erg RNA has been recently detected in other tissues. It is thus possible that the erg-mediated K+ currents will be present in a variety of cells, including neurons and neuroendocrine cells (23). It will also be of importance to assess whether ceramide directly suppresses the rapidly activating component of delayed rectifier K+ current in cardiac myocytes, because this effect may lead to a prolongation of the QT interval in the electrocardiogram (37, 38).

The present study showed that ceramide decreased the opening probability of BKCa channels in GH3 cells. The result is consistent with a previous report in vascular smooth muscle cells (5), although the concentration of ceramide (i.e. 30 µM) used in our study was relatively higher. This decrease in K+ channel activity can depolarize cell membrane and activate voltage-gated Ca2+ or Na+ channels, thereby leading to an increase in cell excitability (38). Ceramide was previously reported to induce a rise in intracellular Ca2+ in vascular smooth myocytes (8). Therefore, blockade of both IK(IR) and BKCa channel caused by ceramide may synergistically act to affect the functional activity of these cells in vivo.

The results of the present study show a significant block of IK(IR) by ceramide in GH3 and IMR-32 cells. This effect is presumably not mediated by its effects on the activity of p44/p42 MAPKs. Our results imply that ceramide-induced effects on pituitary function could be partly, if not entirely, attributed to the blockade of the inwardly rectifying K+ channels.


   Acknowledgments
 
The authors thank Yen-Hua Hung and Hui-Fang Li for excellent technical assistance, Dr. Chung-Ren Jan for helpful discussion of this work, and Prof. Larry Low-Tone Ho for his continuous encouragement.


   Footnotes
 
This work was supported by grants from National Science Council (NSC-89-2320-B-075B-016), Kaohsiung Veterans General Hospital (VGHKS90-06, VGHKS90-73), and VTY Joint Research Program, Tsou’s Foundation (VTY89-P3-23), Taiwan, Republic of China.

Abbreviations: BKCa, Ca2+-activated K+; DTDP, 4,4'-dithiodipyridine; ICa,L, L-type Ca2+ currents; IK(IR), inwardly rectifying K+ current; I-V, current-voltage.

Received May 21, 2001.

Accepted for publication August 2, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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