Protein kinase C is necessary for recovery from the thyrotropin-releasing hormone-induced r-ERG current reduction in GH3 rat anterior pituitary cells

Abstract

The biochemical cascade linking activation of phospholipase C-coupled thyrotropin-releasing hormone (TRH) receptors to rat ERG (r-ERG) channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH3 cells and pharmacological inhibitors. To check the recent suggestion that Rho kinase is involved in the TRH-induced r-ERG current suppression, the hormonal effects were studied in cells pretreated with the Rho kinase inhibitors Y-27632 and HA-1077. The TRH-induced r-ERG inhibition was not significantly modified in the presence of the inhibitors. Surprisingly, the hormonal effects became irreversible in the presence of HA-1077 but not in the presence of the more potent Rho kinase inhibitor Y-27632. Further experiments indicated that the effect of HA-1077 correlated with its ability to inhibit protein kinase C (PKC). The hormonal effects also became irreversible in cells in which PKC activity was selectively impaired with GF109203X, Gö6976 or long-term incubation with phorbol esters. Furthermore, the reversal of the effects of TRH, but not its ability to suppress r-ERG currents, was blocked if diacylglycerol generation was prevented by blocking phospholipase C activity with U-73122. Our results suggest that a pathway involving an as yet unidentified protein kinase is the main cause of r-ERG inhibition in perforated-patch clamped GH3 cells. Furthermore, they demonstrate that although not necessary to trigger the ERG current reductions induced by TRH, an intracellular signal cascade involving phosphatidylinositol-4,5-bisphosphate hydrolysis by phospholipase C, activation of an alpha/betaII conventional PKC and one or more dephosphorylation steps catalysed by protein phosphatase 2A, mediates recovery of ERG currents following TRH withdrawal.

Figure 1. E-4031-insensitive currents are not a main target for TRH in perforated-patch clamped GH₃ cells studied in high-K⁺, low-Ca²⁺ extracellular solutions

Current recordings from two representative cells showing high and low proportions of E-4031-insensitive inward current components are shown in A and B. The E-4031 sensitivity of currents recovered following TRH-induced inhibition after a prolonged period of TRH washout is illustrated in C. Currents were recorded in response to the voltage protocol shown in A. A and B, upper panel, currents under control conditions, after blockade of r-ERG channels with 5 μm E-4031, and 3 min after adding 100 nm TRH in the continuous presence of the inhibitor. Lower panel, E-4031-sensitive and TRH-sensitive currents obtained by subtracting the traces in the presence of E-4031 or TRH + E-4031 from control currents. C, current traces in the upper panel correspond to those recorded before adding TRH (Control), after 2 min of TRH treatment, following a 15 min period of washout with hormone-free medium, and in the presence of 5 μm E-4031. E-4031-sensitive currents obtained by subtracting the trace in the presence of E-4031 from those without inhibitor are shown in the lower panel. Zero current level is indicated with dashed lines on the non-subtracted current records.

Figure 2. TRH-induced r-ERG current suppression is not significantly modified by ROCK inhibitors

The time course of relative r-ERG current reduction by TRH and E-4031 is shown for three different cells without any previous treatment (A), following preincubation for 1 h with 20 μm Y-27632 (B), or after 1.5 h with HA-1077 (C) at 37 °C. For further details on drug treatments see Methods. Pulse protocols as described in Fig. 1 were used. Current estimations were performed from total inward charge during the hyperpolarization steps at −100 mV as described in Methods. Averaged charge values before any addition and those corresponding to the minimum following addition of E-4031 were considered as 0 and 100 %, respectively. Filled circles correspond to the current traces shown above. The first one or two data points following addition of TRH, when total inward currents became transiently enhanced by activation of Ca²⁺-dependent K⁺ channels due to massive liberation of Ca²⁺ from intracellular stores have been deleted for clarity. Perfusion of 100 nm TRH and addition of 5 μm E-4031 to the recording chamber are indicated. Values for the data in the absence and presence of TRH are indicated by horizontal dashed lines. D, comparison of TRH-induced reduction of E-4031-sensitive r-ERG currents for control cells and cells treated for 1–3 h at 37 °C with Y-27632 or HA-1077. Current reductions were estimated from total inward charge measurements as indicated above. n.s., not significant. Numbers in parentheses indicate the number of experiments.

Figure 3. Effect of ROCK inhibitors on reversal of the TRH-induced r-ERG current reductions

The time course of variations in inward current magnitude is shown for three different cells without any previous treatment (A) or following treatment for 1–3 h at 37 °C with 20 μm HA-1077 (B) or Y-27632 (C). Filled circles correspond to the current traces shown above. Pulse protocols as described in Fig. 1 were used. Current estimations were performed from total inward charge during the hyperpolarization steps at −100 mV. The magnitude of the TRH-sensitive current component was estimated by subtracting the minimum current level reached in the presence of TRH from every data point. Averaged values for the data before adding TRH are indicated by horizontal dashed lines. D, comparison of the magnitude of current recovery upon TRH washout in the absence or presence of ROCK inhibitors. Data are expressed as percentage of TRH-inhibited current recovered after 15 min of hormone washout. The values were derived from the data shown in A–C. Data from cells preincubated with 2 μm HA-1077 are also shown for comparison. ***P = 0.0001, *P = 0.05. Numbers in parentheses indicate the number of experiments.

Figure 4. Effect of PKC inhibition on reversal of the TRH-induced r-ERG current reductions

A, time course of variations in inward current magnitude in response to the addition of 1 μm GF109203X (GF) and the subsequent response to a short application of 100 nm TRH in the continuous presence of the inhibitor. B, time course of the TRH effect in a PKC-downregulated cell following a long-term incubation for 24 h with 1 μm PMA. C, comparison of the magnitude of current recovery in control and PKC-inhibited cells. Pulse protocols and data evaluation were performed as described in Fig. 3. ***P = 0.0001, **P = 0.01.

Figure 5. Effect of U-73122 on the Ca²⁺ responses of GH₃ cells

A and B, time course of variation in [Ca²⁺]_i in two representative cells and their response to TRH addition in the absence (A) or the presence (B) of 10 μm U-73122. Measurement of [Ca²⁺]_i in Fura-2-loaded cells was performed as described in Methods. Addition of U-73122 to the recording chamber and the period of perfusion with 100 nm TRH are indicated. Expanded fragments of the [Ca²⁺]_i recordings during perfusion of TRH are shown in the insets. Note the contribution of two precedent spontaneous Ca²⁺ oscillations to the otherwise quite small Ca²⁺ elevation induced by TRH entry in B. Inset calibration bars correspond to 100 nm Ca²⁺ and 10 s. C, averaged recordings of [Ca²⁺]_i showing the initial TRH-induced Ca²⁺ transients in the absence (Control) or presence of U-73122. Data traces synchronized to the moment of TRH addition were averaged point by point and averaged values ±s.e.m. are shown. Averaged [Ca²⁺]_i values in the first point of the recordings have been subtracted from the data. Numbers in parentheses indicate the number of cells from which averaged data were obtained.

Figure 6. Effect of PLC inhibition by U-73122 on the TRH effects

A, TRH-induced r-ERG current suppression is not modified by U-73122. The time course of relative r-ERG current reduction by TRH and E-4031 following a 5 min incubation with 10 μm U-73122 is shown in the middle panel. Filled circles correspond to the current traces shown above. The start of perfusion with U-73122 and the duration of the TRH treatment are marked. Introduction of 5 μm E-4031 into the recording chamber and the averaged magnitude of the current reduction at the end of the 5 min treatment with U-73122 before adding TRH are also indicated on the graph. A comparison of TRH-induced reduction of E-4031-sensitive r-ERG currents for control cells and cells treated with U-73122 is shown in the lower panel. B, the r-ERG current reductions caused by TRH became irreversible in the presence of U-73122. The time course of variations in inward current magnitude in response to an addition of 10 μm U-73122 and the subsequent response to a short application of 100 nm TRH in the continuous presence of the inhibitor are shown in the middle panel. Filled circles correspond to the current traces shown above. A comparison of the magnitude of current recovery upon TRH washout in the absence and presence of U-73122 is shown in the lower panel. Pulse protocols and data evaluation were performed as described in Figs 2 and 3. ***P = 0.0001. Numbers in parentheses indicate the number of experiments.

Figure 7. Effect of wortmannin on the TRH-induced current reductions

Data from a cell preincubated for 2 h with 10 μm wortmannin at 37 °C are shown. The inhibitor was also maintained in the chamber at the same concentration during the full time course of the experiment. Filled circles correspond to the current traces shown above. Similar results were obtained in five of the eight cells treated with wortmannin.

Figure 8. Effect of the PKC inhibitor Gö6976 on the TRH effects

The time course of relative r-ERG current reduction by TRH and E-4031 following a 5 min treatment with 0.5 μm Gö6976 is shown in the upper right panel. Filled circles correspond to the current traces shown on the left. Note the almost complete absence of reversal of the TRH-induced inhibition after more than 20 min of hormone washout. A comparison of the TRH-induced reductions of the E-4031-sensitive r-ERG currents for control cells and cells treated with Gö6976 is shown in the lower left panel. Averaged values of the magnitude of current recovery upon TRH washout in the absence or the presence of Gö6976 is shown in the lower right panel. ***P = 0.0001.

Figure 9. Schematic representation of the biochemical pathways controlling hormone secretion in GH₃ cells (A) and the steps blocked by different pharmacological agents as shown in this report (B)

The first phase (marked 1 on the scheme) corresponds to the transient release of stored Ca²⁺ into the cytosol after production of IP₃ in response to PIP₂ hydrolysis catalysed by PLC, with concomitant hyperpolarization due to activation of Ca²⁺-dependent K⁺ channels. The second phase (marked 2) is the result of the increase in Ca²⁺-dependent action potential frequency due to reductions in r-ERG conductance, leading to an increase in intracellular Ca²⁺ oscillations and manifested in a cell population as a plateau of increased Ca²⁺ and secretion. Participation of a protein kinase-catalysed step is marked PK on the scheme. The minus sign indicates development of the TRH-induced r-ERG current inhibition. The plus sign stands for reversal of the inhibitory effect. The proposed mechanism for reversal of the hormonal inhibition by means of DAG elevation, activation of an α or βII subtype of cPKC, and one or more dephosphorylation steps catalysed by PP_2A is depicted in the centre of the cell. Whether phosphorylation and/or phosphate removal are exerted on the channel itself or on a regulatory component associated with it remains to be established. The alternative possibilities that PKC activation either increases PP_2A activity or acts by inactivating the kinase involved in r-ERG inhibition are also illustrated. For more explanation see text. TRH, thyrotropin-releasing hormone; R, TRH receptor; q, G protein of the G_q/11 type; PLC, phospholipase C; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP₃, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; cPKC α/β, conventional protein kinase C of the α or β type; PP_2A, protein phosphatase 2A; PRL, prolactin; OKA, okadaic acid; VDCC, voltage-dependent calcium channel; K_Ca, calcium-dependent potassium channel.