Intracellular calcium changes in mice Leydig cells are dependent on calcium entry through T-type calcium channels

Abstract

Luteinizing hormone (LH) regulates testosterone synthesis in Leydig cells by inducing an intracellular increase in cAMP concentration. LH also increases the intracellular calcium concentration ([Ca2+]i), dependent on the presence of Ca2+ in the extracellular medium ([Ca2+]e) for its effect. Despite these evidences, the identity of a pathway for calcium entry has remained elusive and the relationship between cAMP and [Ca2+]i has been questioned. Here we show that mice Leydig cells do have an inward Ca2+ current carried by T-type Ca2+ channels. In 10 mm [Ca2+]e, the currents start to be activated at -60 mV, reaching maximal amplitude of 1.8 +/- 0.3 pA pF(-1) at -20 mV. Currents were not modified by Ba2+ or Sr2+, were suppressed in Ca2+-free external solution, and were blocked by 100 microm nickel or 100 microm cadmium. The Ki for Ni2+ is 2.6 microm and concentrations of Cd2+ smaller than 50 microm have a very small effect on the currents. The calcium currents displayed a window centred at -40 mV. The half-voltage (V0.5) of activation is -30.3 mV, whereas the half-voltage steady-state inactivation is -51.1 mV. The deactivation time constant (taudeactivation) is around 3 ms at -35 mV. Confocal microscopy experiments with Fluo-3 loaded cells reveal that both LH and dibutyryl-cAMP (db-cAMP) increase [Ca2+]i. The db-cAMP induced calcium increase was dependent on Ca2+ influx since it was abolished by removal of extracellular Ca2+ and by 400 microm Ni2+. [Ca2+]i increases in regions close to the plasma membrane and in the cell nucleus. Similar effects are seen when Leydig cells are depolarized by withdrawing K+ from the extracellular solution. Altogether, our studies show that Ca2+ influx through T-type Ca2+ channels in the plasma membrane of Leydig cells plays a crucial role in the intracellular calcium concentration changes that follow binding of LH to its receptor.

Figure 4. LH and cAMP increase intracellular calcium concentration

A, time course of the fluorescence changes induced by addition (arrow) of 1 microg/ml LH to the solution superfusing 4 cells (lines represent different cells). Total number of cells analysed was 67 in 5 different preparations. Fluorescence increases sharply after treatment with LH and lasts for several minutes. Fluorescence was baseline subtracted and normalized to the maximum fluorescence measured in each cell (F_norm). B, a series of surface plot pictures from a cell exposed to LH at the indicated times (number on top of each picture, in seconds). C, time course of fluorescence changes before and after application of 400 μm db-cAMP to 5 different cells, from a total of 20 analysed in at least 4 preparations.

Figure 6. Influx of calcium is needed for the effect of cAMP

A, blocking calcium channels with 400 μm Ni²⁺ impairs the effect of cAMP shown in Fig. 5. Ni²⁺ and cAMP were present in the superfusing solution from the beginning of the measurement up to 37 s. Fluorescence only increased after Ni²⁺ was washed out from the bathing solution (horizontal bars at the top). Different traces illustrate the behaviour of different cells (24 analysed cells). B, representative surface plots for a cell treated with Ni²⁺ and cAMP (upper row: 0, 17 and 35 s) and after removal of Ni²⁺ from the bathing solution (lower row: 60, 107 and 180 s).

Figure 1. Calcium currents in Leydig cells

A, representative current traces elicited by depolarizing potentials from a holding potential of −80 mV to +40 mV in 10 mV steps. Note the typical criss-crossing of the traces. The trace at −20 mV is indicated. B, average I–V relationship for the peak currents obtained at each potential, normalized by the capacitance of the cell, in standard external solution (○) and in the presence of 400 μm db-cAMP (•). The continuous line is the fit of a Boltzmann function to the experimental points, giving V_0.5 = −31.8 ± 1.4 mV, k = 5.5 ± 0.3 mV and G_max = 2.0 ± 0.2 nS in control condition and V_0.5 = −34.7 ± 1.8 mV, k = 4.1 ± 0.5 mV and G_max = 2.6 ± 0.2 nS in the presence of cAMP (means ± s.e.m., n = 43). C, holding the cell at a more depolarized potential completely inactivates the current. We chose to depolarize from −90 mV to −30 mV and then from −50 mV to +10 mV because at +10 mV potential L-type currents can be activated (see voltage protocol under the current traces). All traces were obtained with 10 mm Ca²⁺ as charge carrier. The capacitive transients were suppressed from the traces for presentation purposes only.

Figure 2. Characteristics of the inward currents

A, average I–V relationships for currents measured at the indicated calcium concentrations. Points are means ± s.e.m. (n = 7). The continuous line shows a fit of the Boltzmann function to the experimental points. Increasing the calcium concentration shifts V_0.5 from −38.5 ± 0.8 for 2 mm Ca²⁺ to −37.0 ± 0.2 for 10 mm Ca²⁺, to −29.6 ± 1.3 for 20 mm Ca²⁺ and to −23.5 ± 0.7 mV for 40 mm Ca²⁺, without major changes in the voltage dependency of the currents. B, an average I–V plot for currents measured in the presence of 10 mm Ca²⁺ (V_0.5 = −31.7 ± 1.6 mV, k = 3.7 ± 0.3 mV) or 10 mm Ba²⁺ (V_0.5 = −29.9 ± 0.9 mV; k = 3.8 ± 0.3 mV) or 10 mm Sr²⁺ (V_0.5 = −32.8 ± 1.1 mV; k = 3.4 ± 0.4 mV) (n = 6). C, the inactivation time constants, obtained by fitting a single exponential function to the decay phase of the currents obtained at different potentials, in the presence of 10 mm calcium or 10 mm Ba²⁺. Note that the constants are equal in both conditions, have the same voltage dependency and are independent of the presence of calcium. D, representative traces showing the absence of current when Ca²⁺ is withdrawn from the external solution, or is blocked by 100 μm Cd²⁺ and 100 μm Ni²⁺.

Figure 3. Electrophysiological properties of the T-type calcium currents in Leydig cells

A, activation of the channels (○) was analysed by applying voltage pulses from −80 mV to 0 mV in 10 mV steps. Resulting peak currents measured at each voltage were normalized with respect to the maximum observed value (I/I_max). Continuous line represents the fit of a Boltzmann function to the experimental points giving V_0.5 = −30.2 ± 0.5 mV and k = 4.9 ± 0.6 mV. Inactivation properties were analysed by applying prepulses from −100 mV to −10 mV in steps of 5 mV, for 2 s, and then to a test pulse of −20 mV. The normalized currents (I/I_max) are plotted against the pre-pulse voltage. A Boltzmann relation was fitted to these points (means ± s.e.m., n = 6) resulting in V_0.5 = −51.1 ± 0.7 mV and k = 6.2 ± 0.4 mV. The inset shows the calculated relative window current (I_win percentage) computed as the product of the activation and the inactivation fits. Calculations did not take into consideration leakage currents. B, deactivation properties of the calcium currents. Upper traces show the tail currents elicited by a short pre-pulse of 10 ms duration from −100 mV to −20 mV, and then observing their deactivation with pulses ranging from −100 mV to −10 mV in steps of 10 mV. Tail currents were fitted with a single exponential and the decay time constants were plotted against the pulse voltage (lower plot). Experimental points (means ± s.e.m.; n = 7) are well fitted by a single exponential showing a shallow voltage dependency as expected from T-type calcium channels. Calcium at 10 mm was used as charge carrier in all experiments.

Figure 5

External calcium is needed for the db-cAMP effect Fluorescence changes against time for several cells (different traces representative of 30 cells) before and after addition of 10 mm Ca²⁺ (arrow) to the solution superfusing the cells. db-cAMP at 400 μm was present in the superfusing solution all the time. Note that db-cAMP alone did not induce any rise in fluorescence. The fast increase occurs only after addition of calcium. F_norm has the same meaning as described in Fig. 4.

Figure 7

Depolarization of the cell membrane increases [Ca²⁺]_i A, removal of K⁺ from the extracellular [K⁺]_e solution (arrow) depolarizes the membrane potential in Leydig cells. The magnitude of the depolarization, estimated from the increase in fluorescence of diBAC₄(3) is around 20–30 mV. The slow time course reflects the gradual block of the electrogenic Na⁺/K⁺ pump. Fluorescence points were collected at every 1.4 s. B, depolarization of the cell membrane, by removal of extracellular K⁺ (arrow) induces increase in [Ca²⁺]_i. C, nickel blocks the effect of the depolarization on [Ca²⁺]_i. Nickel (200 μm) was added to the superfusing solutions at the beginning of the experiment. Arrow 1 indicates the time when [K⁺]_e was made equal to zero. Arrow 2 indicates the time when Ni²⁺ was washed out of the external solution maintaining [K⁺]_e = 0. Fluorescence measurements were done every 0.8 s. Each trace in the figures represents measurements made on different cells.