0013-7227/91/1284-2015$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 4 Printed in U.S.A.

Reevaluation of the Electrophysiological Actions of Thyrotropin-Releasing Hormone in a Rat Pituitary Cell Line (GH3) STEVEN M. SIMASKO Department of Physiology, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214

ABSTRACT. The electrophysiological actions of TRH were examined in the clonal pituitary cell line GH3 with the use of the perforated patch variation of the standard whole cell patchclamp technique. The action of TRH on spontaneously spiking cells was to cause a brief hyperpolarization (first phase action), followed by a period during which action potential behavior was significantly modified (second phase action). The modifications during second phase action included a reduction in the slope of the up-stroke, a reduced peak potential, an increase in duration, and a depolarizing shift of the after-hyperpolarization. The modification of voltage- and calcium-dependent conductances that underlie these changes were investigated in voltage clamp experiments. During first phase action TRH was found to increase calcium-dependent potassium current. During second

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RH IS a tripeptide that is released by the hypothalamus and regulates the secretion of both TSH and PRL from the pituitary (1). It is known that TRH stimulates secretion from pituitary cells via activation of the phosphoinositide turnover pathway (2). Activation of this pathway produces a bifurcating signal that involves first the release of intracellular stores of Ca2+ through the production of inositol trisphosphate and then activation of protein kinase-C (PKC) through the production of diacylglycerol (3). In clonal GH3 pituitary cells this bifurcating signal system results in a biphasic secretion response: first, a burst of secretion, followed by a slower rate of secretion (4-6). When the actions of TRH on intracellular Ca2+ levels are examined, a biphasic pattern also emerges: first, a rapid increase in intracellular Ca2+ due to the release of intracellular stores, followed by an elevated plateau phase that is dependent on extracellular Ca2+ and is prevented by agents that block voltage-dependent calcium channels (5, 7). It has also been shown that removal of extracellular Ca2+ or the addition of agents that block voltageReceived September 24, 1990. Address all correspondence and requests for reprints to: Dr. Steven M. Simasko, Department of Physiology, 124 Sherman Hall, State University of New York, Buffalo, New York 14214.

phase action TRH was found to significantly reduce the L-type calcium current (35%), with no alteration in the T-type calcium current. The second phase action of TRH on calcium-dependent potassium conductance was complex. First, a decrease was observed. This was followed by an increase that did not become fully manifest until after TRH was washed from the cell. TRH caused no change in voltage-dependent potassium current. These results indicate that the second phase action of TRH on action potential behavior in GH3 cells is mediated by a reduction in Ltype calcium current and alterations in the behavior of calciumdependent potassium currents, but not through changes in voltage-dependent potassium currents. (Endocrinology 128: 20152026, 1991)

dependent calcium channels reduces the secretory response produced by TRH (4-6). Since voltage-dependent calcium channels appear to be a conduit for the influx of extracellular Ca2+, which, in turn, is important to the secretory response, much attention has focused on the electrophysiological actions of TRH. GH3 cells spontaneously fire calcium action potentials (8). When TRH is applied to these cells, there is first a hyperpolarization that causes cessation of action potential activity, followed by a period of depolarization, during which there is an increase in action potential frequency (9). It has also been observed that TRH produces a biphasic effect on membrane conductance; first, there is an increase that corresponds to the hyperpolarization (first phase), followed by a decrease that corresponds to the period of slight depolarization (second phase) (10). It has been shown with simultaneous patch-clamp electrophysiological and calcium-dye measurements that the hyperpolarization is due to activation of calcium-dependent potassium channels produced by the release of intracellular stores of Ca2+ (11). The increased firing of Ca2+ action potentials during second phase action is thought to lead to an increase in the influx of Ca2+ though voltagedependent calcium channels that is important to the secretory response (9). It has been suggested that the 2015

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ELECTROPHYSIOLOGICAL ACTIONS OF TRH

second phase of TRH action on membrane electrophysiological events is due to a TRH-induced decrease in voltage-dependent potassium current (12). The second phase of the electrophysiological action of TRH is mimicked by activators of PKC (13, 14). This latter observation suggests that activation of PKC should decrease the voltage-dependent potassium current in GH3 cells. However, I have not been able to observe such an action in standard whole cell patch-clamp experiments. One possibility for the failure to observe this predicted effect of PKC activators is that essential intracellular components may have been lost due to the intracellular dialysis that occurs with standard whole cell patch-clamp methods (15). To circumvent the potential artifacts introduced by standard whole cell methods I have used the perforated-patch modification of the standard whole cell patch-clamp configuration (16). In the perforated patch, electrical access to the interior of the cell is achieved through the use of the pore-forming agent nystatin. Whereas in the standard whole cell configuration I have found inconsistent responses and frequently a total lack of response to the application of TRH, in the perforated patch configuration, cells consistently responded to the application of hormone. Using the perforated patch method in current clamp experiments I have found that TRH produces a biphasic response similar to that previously described. However, results of voltage clamp experiments suggest that the actions of TRH that underlie the second phase actions on membrane potential are significantly different from those previously described (12). Calcium- and voltage-dependent potassium currents were examined with a double pulse protocol that permits separation of these two current components. Using this pulse protocol in perforated patch experiments I have found that TRH produces no change in voltage-dependent potassium currents. However, TRH has a complicated effect on the calcium-dependent potassium current. First, there is an increase consistent with a release of intracellular stores of Ca2+. This is followed by a decrease in this current. Finally, TRH produces a late increase in this current that becomes fully manifest only after TRH is washed from the cell. The decrease in the calcium-dependent potassium current is at least partially caused by a reduced Ca2+ influx due to a TRH-induced decrease in voltage-dependent calcium current.

Endo • 1991 Voll28«No4

supplemented with 15% horse serum and 2.5% fetal calf serum. Flasks were kept in a 95% air-5% CO2 humidified atmosphere at 36.5 C. Growth medium was replaced twice a week, and the cells were split (1:3) into subcultures once a week. At each pass some cells were plated onto glass coverslips for electrophysiological measurements. Electrophysiological measurements were made 6-10 days after plating of the subcultures. Cells frozen in 10% dimethylsulfoxide-medium plus serum were periodically thawed (every 10-12 weeks) and introduced as the stock line. All tissue culture medium and serum were obtained from Gibco (Grand Island, NY). Electrophysiological measurements A coverslip on which cells were growing was rinsed with bath buffer (see below) and then placed in a Plexiglass chamber (volume, 0.4 ml) mounted on a microscope stage (Nikon Diaphot, Nikon, Inc., Garden City, NJ). Bath buffer was continuously applied to the chamber (1 ml/min) by a peristaltic pump and removed by an adjustable aspirator. Switching bath solutions was performed with a manual valve on the inflow line suspended near the recording chamber. The characteristics of the bath exchange can be seen in Fig. 1, where approximately 20 sec after switching the valve, TRH reached the cell. Electrophysiological recordings were made from isolated cells similar in size to the majority of the cells on the coverslip and with a minimum of extracellular debris on their surface. Patch electrodes (1-2 Mfi resistance), pulled from borosilicate glass (TW150-6, World Precision Instruments, New Haven, CT), were coated to within 30 /im of the tip with Sylgard (DowCorning Corp., Midland, MI) and then fire polished on a microforge. The patch electrode was connected to the headstage of a List Electronics EPC-7 patch-clamp amplifier (Medical Systems Corp., Greenvale, NY). In current clamp experiments the voltage signal from the patch-clamp amplifier was stored on VCR tape with use of a digital data recorder (VR-10, Instrutech Corp., Elmont, NY). In voltage clamp experiments the voltage signal from the patch-clamp amplifier was filtered at 3 kHz with an eight-pole Bessel filter, and digitally stored on an IBM AT computer equipped with a Labmaster TM-40-

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Materials and Methods Cell culture

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GH3 cells from pass 19, obtained from American Type Culture Collection (Rockville, MD), were grown as previously described (18). Briefly, a stock line was maintained by growing cells in 50-ml plastic culture flasks in 5 ml Ham's F-10 medium

FIG. 1. Effect of TRH on action potential behavior. The inflow line was changed to one containing 1.0 ixU TRH at the arrow labeled on and returned to control at the arrow labeled off. Action potentials from areas indicted by labeled bars are shown at greater resolution in the bottom tracings.

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ELECTROPHYSIOLOGICAL ACTIONS OF TRH PGH A-to-D board (Scientific Solutions, Inc., Solon, OH). The FASTLAB software package from INDEC Systems (Sunnyvale, CA) was used to control the operation of the computer. For the perforated patch modification of the whole cell recording technique (16), a nystatin stock solution (50 mg/ml) was made in dimethylsulfoxide before addition to the pipette solution (10 n\ stock to 3 ml pipette solution). Successful perforation of the patch (access resistance,

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TIME (sec) remains accelerated even after TRH is washed from the cells. Effects of TRH on voltage-dependent calcium current The results illustrated in Figs. 2-5 indicate the importance of Ca2+ influx through voltage-dependent calcium channels in the current response to the second voltage pulse of the double pulse protocol. Since TRH induces changes in the current response to the second voltage pulse, it is possible that these changes could be accounted for by alterations in voltage-dependent calcium current. This possibility was examined in experiments performed as illustrated in Fig. 8. Calcium currents were measured as described in Materials and Methods. A period of control values were obtained, and then 1.0 ixM TRH was applied to the cell. As can be seen in Fig. 8, TRH

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produced a large decrease in the sustained current (Ltype), with little effect on the transient current (T-type). The average (±SE) of the decrease in L-type calcium current from five cells was 35 ± 9% (P < 0.02, by paired t test). The change in T-type current was not significant (+2 ± 3%). This effect of TRH on L-type current was somewhat reversible (Fig. 8).

Discussion In this study the actions of TRH on membrane electrophysiological properties of GH3 cells were studied with the perforate patch variation of a standard whole cell patch-clamp technique. While the observations of the actions of TRH on action potential behavior are consistent with what has previously been observed (9, 12, 14),

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ELECTROPHYSIOLOGICAL ACTIONS OF TRH

20+

A transient D sustained 100

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TIME (sec) FIG. 8. Effect of TRH on calcium currents (traces above graph). The top trace indicates voltage protocol (holding, —84 mV; test, 0 mV). Control in the graph is the average current response from three or four current traces immediately before TRH application. Sustained current values (squares) were obtained just before the end of the test pulse. Transient current values (triangles) were obtained by subtracting sustained current from peak current. The bar on the graph indicates the approximate time of TRH exposure (first dot before bar indicates exactly when inflow line was changed to one containing 1.0 /*M TRH). Solid symbols on graph correspond to current traces above graph. This experiment was repeated on four additional cells with similar results (see text for summary).

the actions of TRH on potassium and calcium currents that underlie this membrane potential behavior indicate the need for a new interpretation of the mechanisms that underlie the second phase action of TRH. However, before the effects of TRH can be appreciated, the nature of the currents measured in voltage clamp experiments needs to be addressed. Justification for separating voltage-dependent calcium currents into L-type and T-type can be found elsewhere (17, 19). On the other hand, the use of a double pulse protocol to separate voltage-dependent potassium currents from calcium-dependent potassium currents is a unique aspect of this study. Characterization of potassium currents Because the first pulse of the double pulse protocol is to a potential where little Ca2+ would enter the cell, the current in response to the this pulse of the protocol is mostly voltage-dependent potassium current, although some contamination with calcium-dependent current is apparent. Several lines of evidence indicate that the current response to the second pulse is that due to

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calcium activation of calcium-dependent potassium current. It is unlikely that the current in response to the second pulse is from recovery of the transient voltagedependent potassium current seen in the first pulse. First, the transient potassium current requires tens of seconds to recover from inactivation (21), and at most, the interspike interval is 0.25 sec (except see Fig. 5). Second, the peak of the current response to the second pulse is frequently greater than that to the first pulse. If incomplete recovery of the transient potassium current occurred during the abbreviated recovery period, the current response to the second pulse should always be less than that to the first pulse. Third, the inactivation rate of the current in response to the first pulse is substantially different from that in response to the second pulse. Whereas these observations demonstrate that the current in response to the second voltage pulse is not transient voltage-dependent potassium current, the calcium dependence of the current response to the second pulse was shown by several different experiments. First, when the voltage of the interpulse period is reduced to the holding potential, a voltage at which voltage-dependent calcium current would rapidly deactivate, resulting in no Ca2+ influx, there is no additional current in response to the second pulse. Furthermore, when bath Ca2+ was replaced with Mg2+, it was found that there was no increase in current in response to the second voltage pulse. Finally, when the interpulse period was increased, thus allowing more Ca2+ to enter the cell, the initial current in response to the second pulse was elevated. A second aspect of the current in response to the second voltage pulse is that it decays or grows with time depending on the particular circumstance. Since the open probability of calcium-dependent potassium channels under a particular set of circumstances (voltage and free Ca2+ concentration on the cytoplasmic surface) is constant over minutes of recording (23), and the voltage is clamped, the decay or growth of this current must reflect changes in the Ca2+ concentration at the cytoplasmic surface. This concentration of Ca2+ would be dependent on the influx of Ca2+ into the compartment adjacent to the cytoplasmic surface of the plasma membrane and efflux of Ca2+ out of this compartment. The fact that the shape of this current (either when decaying or growing) is not exponential indicates that the changes in Ca2+ concentration in this compartment must be due to the sum result of many processes (diffusion away from the plasma membrane, cytosolic calcium buffering capacity, reuptake into intracellular stores, and extrusion of Ca2+ across the plasma membrane by the Ca2+-ATPase and the Na-Ca exchanger). The results presented in this study do not establish the relative significance of calcium buffering and these influx and efflux pathways. The observations that the decay of this current is unique to

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ELECTROPHYSIOLOGICAL ACTIONS OF TRH

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each cell would suggest that the within a particular cell the relative importance of these various calcium regulatory mechanisms may be different. For example, just as some cells express a large voltage-dependent calcium current whereas some express a small current, some cells may express more Ca2+-ATPase than others. The observations that the shape of the current in response to the second pulse is dependent on voltage suggests that at least one of the influx or efflux mechanisms must be voltage dependent. The most likely mechanism for the voltage dependence of the shape of this current is continued Ca2+ influx through voltage-dependent calcium channels during the duration of the second pulse. Thus, the more positive the pulse, the less Ca2+ that enters, and the faster the decay. Effects of TRH As previously observed (9, 11, 12, 14), the first phase of TRH action is to cause a hyperpolarization of the membrane that lasts only a brief period of time (18 sec). Previously, this has been suggested to be due to release of intracellular Ca2+, which, in turn, activates calciumdependent potassium channels (11, 12, 26). Results obtained in this study are consistent with this interpretation. Immediately after the application of TRH (i.e. during the hyperpolarization), the current in response to the first pulse of the double pulse protocol is elevated. The observation that the elevated outward current is not simply a scaling of the currents expressed in the control trace suggests that a new current component has been recruited. The most likely candidate is calcium-dependent potassium current. The observation that this current is elevated on the first pulse suggests that the source of calcium to activate this current cannot be influx through voltage-dependent calcium channels. Thus, either the calcium that is activating these potassium channels enters through a nonvoltage-dependent mechanism, or it is due to the release of intracellular calcium. The latter interpretation is consistent with previous observations that TRH causes a release of intracellular stores of Ca2+ (5, 7). Detailed examination of action potential behavior during the second phase of TRH action indicates that TRH primarily influences action potential behavior by decreasing the slope of the up-stroke, reducing the peak depolarization, and increasing action potential duration. All of these observations are consistent with a reduction of voltage-dependent calcium current. In a study submitted for publication (24) the effect of increasing bath Ca2+ from 2-10 mM was examined. When bath Ca2+ was increased, thus increasing the calcium current, the upstroke of action potentials became steeper, the peak was more depolarized, and the duration was shortened. In addition, TRH caused the AHP to move in a depolarized

Endo-1991 Voll28-No4

direction. Such an effect of TRH implies a more complicated mechanism of action than just reducing calcium current, since this parameter was not significantly changed when bath Ca2+ concentration was altered (24). Examination of the actions of TRH in voltage clamp experiments confirmed the prediction that TRH would decrease the voltage-dependent calcium current. It was found that this decrease was exclusively in the L-type current. It is likely that this TRH-induced decrease is mediated by activation of PKC, since it has previously been shown that direct activators of PKC reduce voltagedependent calcium current in GH3 cells (25). The finding that TRH decreases the L-type calcium current is at odds with a previous report that TRH has no effect on this current (12). The most reasonable explanation for the failure of the previous investigators to observe this action of TRH is that their measurements were made with standard whole cell methods and that an intracellular component essential for the expression of this action of TRH was lost during the intracellular dialysis that occurs in the standard whole cell technique (15). During the second phase of TRH actions, TRH was found to produce complicated alterations in the behavior of the calcium-dependent potassium current without altering the behavior of voltage-dependent potassium currents. The lack of alteration in voltage-dependent current is contrary to what has previously been reported (12). One possibility for the observed effect in the previous study was that the measurement of voltage-dependent current was measured at a voltage at which significant Ca2+ would enter the cell. Although these investigators selected cells in which the voltage-dependent potassium current dominated the calcium-dependent current, a decrease in calcium-dependent current could have confounded their measurement. Another difference between this study and the previous study was that in the present study the perforated patch technique, a much less intrusive configuration was used. The observation that TRH does not alter voltage-dependent potassium current indicates that this current component is probably not regulated by PKC and explains why I have been unable to detect an effect of phorbol esters on this current (data not shown). One minute after TRH application (i.e. a point in time after the initial hyperpolarization), a decrease in the initial amplitude of the calcium-dependent potassium current and an acceleration of the rate of decay was observed. Both of these observations may be accounted for by the observation that TRH decreases the voltagedependent calcium current. However, the potential contribution of a TRH-induced increase in the activity of a Ca2+ efflux mechanism cannot be discounted (22). A second observation was that the initial magnitude of the calcium-dependent potassium current began to increase

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ELECTROPHYSIOLOGICAL ACTIONS OF TRH

before TRH was washed from the cell and continued to increase to a plateau level after TRH was washed from the cell. This increase in the initial magnitude of the calcium-dependent current occurred even though the voltage-dependent calcium current was suppressed. One possibility is that TRH activates other, nonvoltage-dependent pathways of elevating intracellular Ca2+, such as a receptor-operated channel. Activation of such a pathway would lead to an increase in the background Ca2+ level to which the Ca2+ that enters during the interpulse period would be added, thus resulting in Ca2+ levels at the beginning of the second pulse that are actually elevated over those found in control conditions. If this interpretation was correct, one would also expect the calcium-dependent potassium current during the first voltage pulse (i.e. the sustained current value) to be elevated. This was not observed to be the case. Since the results are not consistent with an elevated level of Ca2+ as the mechanism for the increase in calcium-dependent potassium current, one is forced to conclude that TRH recruits more calcium-dependent potassium channels, increases single channel conductance, or increases the sensitivity of these channels to Ca2+. These possibilities are currently under investigation. Another aspect to this increase in calcium-dependent potassium current is that it appears to increase most dramatically after TRH has been washed from the cell. This late increase can be explained by the reversal of the block of voltage-dependent calcium current. The fact that this elevated current continues for several minutes after TRH removal indicates that the process must reverse slowly, if at all. The increase in the calcium-dependent potassium current may underlie the change in the AHP produced by TRH. It has previously been observed that activation of calcium-dependent potassium current is important to repolarization of the action potential (24, 26). If the increase in this current produced by TRH is mediated on BK potassium channels (voltage and calcium dependent) (27), it may result in repolarization of the action potential at lower concentrations of intracellular Ca2+. If it is assumed that SK potassium channels (voltage insensitive but calcium dependent) (27) underlie the AHP (28), then this lower Ca2+ would result in less stimulation of this current at the negative potentials, which, in turn, would reduce the observed AHP. This same effect may also account for the increase in firing rate previously reported (9, 12, 14) and observed in this study in cells that had spontaneous firing rates slower than 1/sec. If repolarization of the action potential occurs at lower concentrations of Ca2+ immediately adjacent to the cytoplasmic surface of the plasma membrane, it would require less time for the cell to clear this Ca2+ between action potentials. It is likely that this residual Ca2+ contributes to prolongation of the interspike interval by

2025

activation of calcium-dependent potassium channels. Validation of these assumptions and testing of these proposed mechanisms require further experimentation. An issue that arises from the results presented in this study is how does TRH produce an increase in intracellular Ca2+ during the second phase of action that is sensitive to removal of extracellular Ca2+ and blockers of voltage-dependent calcium channels (5, 7) when TRH actually decreases the amplitude of the voltage-dependent calcium current. One possibility is that because of spatial and temporal considerations of Ca2+ entry and removal, under the new circumstances of ion current behavior produced by TRH, more net Ca2+ enters the cell. For example, more net Ca2+ may enter when the calcium current is small and action potentials are relatively long, to a more hyperpolarized potential, and more frequent than when the calcium current is large and action potentials are relatively brief, more depolarized, and less frequent. On the other hand, it could be that the increase in intracellular Ca2+ during the second phase of TRH action is due to an action of TRH at some other step in the regulatory process than Ca2+ influx through voltage-dependent calcium channels. For example, the net change in Ca2+ influx through voltage-dependent calcium channels may be insignificant relative to an alteration in an efflux mechanism (extrusion across the plasma membrane by the Ca2+-ATPase or the Na-Ca exchanger), another influx mechanism, or changes in intracellular buffering or sequestration. Resolution of these possibilities can only be achieved through a more thorough understanding of action potential behavior, the role of Ca2+ in controlling action potential behavior, and the factors that regulate the movement of Ca2+ into and out of the compartment immediately under the plasma membrane. In conclusion, in this investigation the perforated patch technique was used to study the electrophysiological actions of TRH. With this technique it was found that TRH caused a brief hyperpolarization of the membrane potential, followed by a more prolonged period during which action potentials exhibited a reduced slope of up-stroke, a reduced peak depolarization, a prolonged duration, and a reduced AHP. In relatively slow firing cells the interspike interval was also reduced. These actions of TRH on action potential behavior are in part explained by a reduction in the voltage-dependent calcium current and a change in the calcium-dependent potassium current. In contrast to what has previously been reported (12), TRH was not found to have any action on the voltage-dependent potassium current. References 1. Goodman HM 1988 Basic Medical Endocrinology. Raven Press, New York, p 37

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ELECTROPHYSIOLOGICAL ACTIONS OF TRH

2. Gershengorn MC 1986 Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol 48:515-526 3. Berridge MJ 1987 Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56:159-193 4. Aizawa TA, Hinkle PM 1985 Thyrotropin-releasing hormone rapidly stimulates biphasic secretion of prolactin and growth hormone in GH4Ci rat pituitary tumor cells. Endocrinology 116:73-82 5. Albert PR, Tashjian Jr AH 1985 Dual actions of phorbol esters on cytosolic free Ca2+ concentration and reconstitution with ionomycin of acute thyrotropin-releasing hormone responses. J Biol Chem 260:8746-8759 6. Martin TFJ, Kowalchyk JA 1984 Evidence for the role of calcium and diacylglycerol as dual second messengers in thyrotropin-releasing hormone action: involvement of Ca2+. Endocrinology 115:1527-1536 7. Gershengorn MC, Thaw C 1985 Thyrotropin-releasing hormone (TRH) stimulates biphasic elevation of cytoplasmic free calcium in GH3 cells. Further evidence that TRH mobilizes cellular and extracellular Ca2+. Endocrinology 116:591-596 8. Kidokoro Y 1975 Spontaneous calcium action potentials in a clonal pituitary cell line and their relationship to prolactin secretion. Nature 258:741-742 9. Ozawa S, Kimura N 1979 Membrane potential changes caused by thyrotropin-releasing hormone in the clonal GH3 cell and their relationship to secretion of pituitary hormone. Proc Natl Acad Sci USA 76:6017-6020 10. Ozawa S 1981 Biphasic effect of thyrotropin-releasing hormone on membrane K+ permeability in rat clonal pituitary cells. Brain Res 209:240-244 11. Mollard P, Vacher P, Dufy B, Winger BP, Schlegal W 1988 Thyrotropin-releasing hormone-induced rise in cytosolic calcium and activation of outward K+ current monitored simultaneously in individual GH3B6 pituitary cells. J Biol Chem 263:19570-19576 12. Dubinsky JM, Oxford GS 1985 Dual modulation of K channels by thyrotropin-releasing hormone in clonal pituitary cells. Proc Natl Acad Sci USA 82:4282-4286 13. Osteberg BC, Sand O, Bjoro T, Haug E 1986 The phorbol ester TPA induces hormone release and electrical activity in clonal rat pituitary cells. Acta Physiol Scand 126:517-524 14. Dufy B, Jaken S, Barker JL 1987 Intracellular Ca2+-dependent protein kinase C activation mimics delayed effects of thyrotropin-

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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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releasing hormone on clonal pituitary cell excitability. Endocrinology 121:793-802 Dufy B, MacDermott AM, Barker JL 1986 Rundown of GH3 cell K+ conductance response to TRH following patch recording can be obviated with GH3 cell extract. Biochem Biophys Res Commun 137:388-396 Horn R, Marty A 1988 Muscarinic activation of ionic currents measured by a new whole cell recording method. J Gen Physiol 92:145-160 Matteson DR, Armstrong CM 1986 Properties of two types of calcium channels in clonal pituitary cells. J Gen Physiol 87:161182 Tashjian Jr AH 1979 Clonal strains of hormone-producing pituitary cells. Methods Enzymol 57:527-535 Simasko SM, Weiland GA, Oswald RE 1988 Pharmacological characterization of two calcium currents in GH3 cells. Am J Physiol 254:E328-E336 Zar JH 1974 Biostatistical Analysis. Prentice-Hall, Englewood Cliffs Oxford GS, Wagoner PK 1989 The inactivating K+ current in GH3 pituitary cells and its modification by chemical reagents. J Physiol 410:587-612 Drummond AH 1985 Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells. Nature 316:752-755 Pallotta BS, Hepler JR, Oglesby SA, Harden TK 1987 A comparison of calcium-activated potassium channel currents in cell-attached and excised patches. J Gen Physiol 89:985-997 Simasko SM 1989 Action potential duration in a rat pituitary cell line (GH3) is dependent on intracellular calcium accumulation. Soc. Neurosci Abstr 15:1143 Marchetti C, Brown AM 1988 Protein kinase activator l-oleoyl-2acetyl-sn-glycerol inhibits two types of calcium current in GH3 cells. Am J Physiol 254:C206-C210 Ritchie AK 1987 Thyrotropin-releasing hormone stimulates a calcium-activated potassium current in a rat anterior pituitary cell line. J Physiol 385:611-625 Lang DG, Ritchie AK 1987 Large and small conductance calciumactivated potassium channels in the GH3 anterior pituitary cell line. Pfluegers Arch 410:614-622 Blatz AL, Magleby KL 1987 Calcium-activated potassium channels. Trends Neurosci 10:463-467

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Reevaluation of the electrophysiological actions of thyrotropin-releasing hormone in a rat pituitary cell line (GH3).

The electrophysiological actions of TRH were examined in the clonal pituitary cell line GH3 with the use of the perforated patch variation of the stan...
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