Nerve growth factor affects Ca2+ currents via the p75 receptor to enhance prolactin mRNA levels in GH3 rat pituitary cells

Abstract

In clonal pituitary GH(3) cells, spontaneous action potentials drive the opening of Ca(v)1 (L-type) channels, leading to Ca(2+) transients that are coupled to prolactin gene transcription. Nerve growth factor (NGF) has been shown to stimulate prolactin synthesis by GH(3) cells, but the underlying mechanisms are unknown. Here we studied whether NGF influences prolactin gene expression and Ca(2+) currents. By using RT-PCR, NGF (50 ng ml(-1)) was found to augment prolactin mRNA levels by approximately 80% when applied to GH(3) cells for 3 days. A parallel change in the prolactin content was detected by Western blotting. Both NGF-induced responses were mimicked by an agonist (Bay K 8644) and prevented by a blocker (nimodipine) of L-type channels. In whole-cell patch-clamp experiments, NGF enhanced the L-type Ca(2+) current by approximately 2-fold within 60 min. This effect reversed quickly upon growth factor withdrawal, but was maintained for days in the continued presence of NGF. In addition, chronic treatment (>or= 24 h) with NGF amplified the T-type current, which flows through Ca(v)3 channels and is thought to support pacemaking activity. Thus, NGF probably increases the amount of Ca(2+) that enters per action potential and may also induce a late increase in spike frequency. MC192, a specific antibody for the p75 neurotrophin receptor, but not tyrosine kinase inhibitors (K252a and lavendustin A), blocked the effects of NGF on Ca(2+) currents. Overall, the results indicate that NGF activates the p75 receptor to cause a prolonged increase in Ca(2+) influx through L-type channels, which in turn up-regulates the prolactin mRNA.

Figure 1. The effect of NGF on prolactin mRNA and protein levels is mimicked by Bay K 8644 and prevented by nimodipine

A, representative gel showing the effects of long-term exposure to NGF or DHP drugs on PCR signals for the prolactin and cyclophilin messages. Total RNA was extracted from sibling cultures of GH₃ cells that were grown in the absence (control) or presence of 50 ng ml⁻¹ NGF, 0.5 μm (−)-Bay K 8644 (BayK), 1 μm nimodipine (NIM), or NGF plus nimodipine (NGF + NIM) for 72 h. The cDNAs for cyclophilin and prolactin were then amplified in parallel from the RNA samples using optimal conditions for semiquantitative analysis. B, average intensity of the prolactin mRNA signal, normalized to the cyclophilin signal and expressed as a percentage of control values, in the different cell groups. The results (mean ± s.e.m) of three independent experiments are shown. Distinct letters denote significantly different (P < 0.05) means. C, example of Western blot designed to measure changes in the intracellular content of prolactin. Before protein extraction, cells were treated with NGF and DHP drugs as described in A. D, relative prolactin levels in the indicated cell groups. Data were derived from the densitometric analysis of three separate blots, with prolactin signals normalized to the corresponding β-actin levels.

Figure 2. EGF elevates the prolactin mRNA even in the presence of nimodipine

A, PCR signals for the prolactin and cyclophilin messages in a representative gel. Semi-quantitative RT-PCR was performed on total RNA from control GH₃ cells and cells that were treated with 0.15 nm EGF, 1 μm nimodipine (NIM), or EGF plus nimodipine (EGF + NIM) for 72 h. B, relative levels of the prolactin mRNA in these cells as determined from four separate experiments.

Figure 3. A 60-min exposure to NGF increases the activity of HVA Ca²⁺ channels

A, representative Ca²⁺ current recordings in a control cell and a cell that was exposed to NGF for 60 min at 37°C before being examined with NGF present in the bathing solution (1 h NGF). Step depolarizations of 10 ms were applied from a holding potential of −80 mV. Currents are shown for potentials between −50 and +30 mV in 20 mV increments, as indicated by the diagram in the left panel. Tail currents recorded on repolarization are also shown. B, dependence of peak Ca²⁺ current on the membrane potential (V_m) during the depolarizing steps in control and NGF-treated cells (n = 7 in each case). From −10 to +50 mV, the magnitude of the current was significantly larger in NGF-treated cells. C, plots of the initial amplitude of the tail current for HVA (fast tail) and T-type (slow tail) channels against V_m. Same cells and symbols as in B. Each data set was fitted with a Boltzmann function (continuous lines) to determine the maximal tail current (I_max), V_1/2 and k. Values for I_max are given in the text. The values for V_1/2 were 7 ± 1 and 9 ± 1 mV for HVA channels, and −33 ± 1 and −34 ± 2 mV for T-type channels in control and NGF-treated cells, respectively. The values for k were 12.8 ± 0.9 and 13.1 ± 0.7 mV, and 8.3 ± 1.2 and 7.6 ± 0.7 mV, respectively.

Figure 4. NGF targets L-type Ca²⁺ channels

A, effects of including 1 μm nimodipine (NIM) in the bathing solution on Ca²⁺ currents recorded from control cells (left traces) and cells that were treated with NGF for 60 min before being examined in the continued presence of the growth factor (right traces). The pulse protocol is shown in the left panel. Scale bars apply to all traces, including those in C. B, average Ca²⁺ current values obtained from the indicated number of control and NGF-treated cells before and after application of nimodipine (NIM). ^*Significant difference from control values determined in the absence of the Ca²⁺ channel blocker. C, currents blocked by nimodipine in the examples shown in A. D, average amplitude of the L-type Ca²⁺ current in control and NGF-treated cells. These data were derived from the results in B. In each cell group, the mean value of current observed in the presence of nimodipine was subtracted from the current traces recorded in the absence of the Ca²⁺ channel blocker to yield the L-type current amplitude.

Figure 5. The effect of NGF on HVA Ca²⁺ current is rapidly reversible

A, relative Ca²⁺ current values in control cells and two cell groups that were exposed to NGF for 60 min at 37°C before the recordings. Test depolarizations of 10 ms to +20 mV were used. A treated cell group was voltage clamped in the continued presence of the growth factor (1 h NGF), whereas the other one was examined within 10–30 min after NGF removal (Wash). Each current measurement was divided by C_m and then converted to a percentage of the corresponding mean value of current density in control cells. This was done to facilitate comparison of the present data with those shown in Fig. 6, which were obtained from a different batch of cells. ^*Significant difference from controls. B, examples of Ca²⁺ currents recorded from one cell of each group.

Figure 6. Chronic NGF treatment stimulates the activity of both HVA and T-type Ca²⁺ channels

A, Ca²⁺ current density values obtained in response to 10-ms depolarizations to +20 mV in control cells and two cell groups treated with NGF for 72 h in culture. Recordings from treated cells were made in the presence (3 d NGF) or absence (Wash) of NGF in the bathing solution. Data are presented as in Fig. 5. B, representative Ca²⁺ current recordings from control and NGF-treated cells.

Figure 7. NGF increases the T-type Ca²⁺ current without affecting its voltage dependence

A, families of Ca²⁺ currents recorded from control and NGF-treated cells using the pulse protocol shown in the left panel. In this experiment, treated cells were exposed to NGF for 4 days and then examined in the absence of the growth factor (Wash after 4 d NGF). B, voltage dependence of the average Ca²⁺ current measured during 10-ms step depolarizations in control and NGF-treated cells (n = 7 for both). C, voltage dependence of tail current amplitude for HVA (fast tail) and T-type (slow tail) Ca²⁺ channels. Same cells and symbols as in B. Continuous lines correspond to Boltzmann fits to data points. The I_max values are given in the text. The values for V_1/2 were 10 ± 3 and 12 ± 1 mV for HVA channels, and −30 ± 2 and −32 ± 1 mV for T-type channels in control and NGF-treated cells, respectively. The values for k were 13.5 ± 0.7 and 12.9 ± 0.4 mV, and 7.7 ± 0.6 and 6.9 ± 0.5 mV, respectively.

Figure 8. Time course and block by actinomycin D of the NGF-induced increase in T-type current

A, typical T-type Ca²⁺ currents evoked by 200-ms test pulses to −30 mV in a control cell and a cell that was treated with NGF for 4 days. NGF was not included in the bathing solution. B, T-type current density as a function of the duration of NGF treatment. Data were derived from three different batches of cells, which are indicated by distinct symbols. The number of cells investigated is given next to each data point. As in panels C and D, the peak amplitude of the T-type current at −30 mV was divided by C_m and then normalized to the corresponding control value. C, slow decline in T-type current density after NGF withdrawal. The first point in this graph corresponds to the last point in B. The two additional data points were also obtained from the batch of cells that was exposed to NGF for 5 days. These cells were allowed to recover at 37°C for the indicated times before the recording session. D, T-type current density after 24-h exposures to NGF, 1 μm actinomycin D (Act D) or NGF in combination with actinomycin D (NGF + Act D). Nine or 10 cells were examined per group.

Figure 9. Comparison of isolated T-type Ca²⁺ currents in control and NGF-treated cells

A, representative traces showing increased T-type currents after a 4-day treatment with NGF compared to controls. Currents were evoked by 100-ms pulses to the indicated voltages. HVA Ca²⁺ channels were blocked by adding 1 μm nimodipine and 30 μm Cd²⁺ to the external recording solution. B, average peak amplitude of the T-type current measured in control and NGF-treated cells (n = 7 in both cases) as a function of V_m. C and D, voltage dependence of the conductance and inactivation time constant of T-type channels in same cells as in B. Ca²⁺ conductance was calculated by dividing peak current by V_m−V_rev, with V_rev (the reversal potential) equal to +50 mV. Continuous lines in C are Boltzmann functions with V_1/2 and k values as indicated; values for the maximal conductance are given in the text. E, inactivation curves for T-type channels in control and NGF-treated cells (n = 4 for both). The normalized peak amplitude of the T-type current at −20 mV is plotted against V_m during 500-ms prepulses. V_1/2 and k values derived from Boltzmann fits (continuous lines) were −45 ± 2 and 7.6 ± 0.3 mV, respectively, in control cells, and −46 ± 2 and 7.5 ± 0.6 mV, respectively, in NGF-treated cells.

Figure 10. The effects of NGF on Ca²⁺ currents are prevented by MC192, but not by tyrosine kinase inhibitors

A, summary of Ca²⁺ current measurements from control cells and seven groups of treated cells using 10-ms test pulses to +20 mV. Before the recordings, some cells were treated with 50 ng ml⁻¹ MC192, 50 nm K252a or 3 μm lavendustin A (LA) for 90–120 min at 37°C, or with 50 ng ml⁻¹ NGF for 60 min. Other cell groups were first incubated for 60 min with MC192, K252a or lavendustin A, and then exposed to NGF for a second 60-min period in the continued presence of those compounds. The external recording solution was supplemented accordingly. The number of cells examined is given above left-hand bars. B, examples of Ca²⁺ current recorded from the indicated cell groups. Same experiment as in A. C, peak T-type Ca²⁺ current density measured from the number of cells given above each bar using 200-ms test pulses to −30 mV. Cells were treated with the indicated compounds for 48 h, then examined in the presence of external recording solution without additions. D, examples of current traces obtained in experiment shown in C.