Selective Enhancement of L-Type Calcium Currents by Corticotropin in Acutely Isolated Rat Amygdala Neurons

Materials and Methods

Cell Preparation.

The techniques used to prepare brain slices and cell dissociation were similar to those described previously (Wang et al., 1996). In brief, 10- to 18-day-old, male Sprague-Dawley rats were decapitated and transverse slices (500 μm) were cut from tissue block of the brain using a Vibroslice (Campden Instruments, Silbey, UK). Lateral and basolateral amygdala regions were visualized under a stereomicroscope and dissected from the brain slices with a scalpel. The pieces of brain tissue containing the amygdala were transferred to 10 ml of PIPES saline solution in a spinner flask. The temperature was maintained at 32 ± 1°C and 2–3 mg of protease XIV (Sigma, St. Louis, MO) was added. The amygdalar minislices were stirred gently at a rate sufficient to prevent them from settling, and the solution was bubbled continuously with 95% O₂/5% CO₂. The PIPES saline solution contained 120 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 25 mM glucose, and 20 mM PIPES, pH 7.0.

One hour later, minislices were washed with 10 ml of PIPES saline solution (three times) and transferred for storage to a holding chamber filled with continuously bubbled PIPES saline solution at room temperature. When needed, a slice was transferred to 1 ml of external solution and the neurons were dissociated by trituration using a fire-polished Pasteur pipette with a ∼1-mm tip diameter. The suspension of dissociated amygdalar neurons then was transferred to a 0.5- to 1-ml recording chamber mounted on an Olympus IMT-2 inverted microscope (Olympus, Tokyo, Japan). Neurons were allowed to settle on poly-l-lysine-coated coverslips.

Electrophysiological Recordings.

Whole-cell Ca²⁺ currents were recorded using a patch-clamp amplifier (Axopatch 200A; Axon Instruments, Foster City, CA) and filtered at 10 kHz. Patch pipettes were pulled from borosilicate glass and fire polished with a resistance of 2 to 5 MΩ. The external solution contained 3 mM CaCl₂, 10 mM glucose, 120 mM tetraethylammonium hydrochloride (TEA), 5 mM 4-aminopyridine, and 10 mM PIPES; 2 μM tetrodotoxin was always added. Pipette solution contained 2 mM MgCl₂, 10 mM PIPES, 10 mM glucose, 20 mM TEA, 100 mM Cs methanesulfonate, 10 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP, 20 mM phosphocreatine, and 0.2 mM leupeptin. All the experiments were performed at room temperature (22 to 25°C). After establishing a gigaseal, the membrane underlying the pipette was ruptured by a gentle suction to obtain whole-cell recording. Experimental tests were initiated when the size of I_Ca became stable, which usually took ∼5 min. When studying the effect of corticotropin on the voltage-dependent, steady-state activation, we used the voltage ramps and Ba²⁺ as the charge carrier (3 mM CaCl₂ was replaced by 5 mM BaCl₂ in the external solution).

Online and offline data acquisition and analysis were accomplished using a DigiData 1200 interface (Axon Instruments) between a Axopatch 200B preamplifier and a PC 486 computer using pClamp 6.0 software program. Current traces (not including those of the current-voltage curves) shown in the figures were the averaged responses of two identical voltage steps elicited consecutively at 7-s intervals. Statistical significance was determined at the level ofp ≤ 0.05 using paired or unpaired Student'st tests. All data were expressed as mean ± S.E.M.

Drugs.

Corticotropin and its analogs were obtained from Sigma. Nimodipine and ω-CgTX were obtained from Research Biochemicals (Natick, MA). ω-AgTX and tetrodotoxin (TTX) were obtained from Calbiochem (La Jolla, CA). Corticotropin and neurotoxins were dissolved in water and were aliquoted and frozen. Each of the stocks was diluted to the appropriate concentrations in the external solution immediately before the experiment. Nimodipine was dissolved in dimethyl sulfoxide and protected from light. Final dilution of nimodipine resulted in dimethyl sulfoxide concentration of 0.1%, which, when delivered alone, had no effect on calcium currents.

Results

These data were obtained from ≥100 neurons acutely dissociated from rat lateral and basolateral areas. From various cell shapes obtained after trituration, we selected only medium- or small-size neurons with a pyramidal or stellate morphology. No obvious differences in properties of these two cell types were observed, and the results were pooled. Under our recording conditions, low voltage-activated Ca²⁺ current was rarely seen.

Effect of Corticotropin.

Whereas corticotropin_1–39 is the naturally occurring neuropeptide processed from POMC, corticotropin_1–24 has the full biological activity of the 39-amino acid molecule, possessing both corticotropic and neurotropic characteristics (Hol et al., 1995). Small corticotropin fragments, especially the heptapeptide sequence corticotropin_4–10 common to corticotropin and melanotropin, have potent neurotropic and behavioral effects and evidence has accumulated to show that they are also physiologically produced. In the present study, we mainly applied these two peptides (1–24 and 4–10) to investigate their actions on the VDCC. When elicited by 200-ms step depolarization from a holding potential of −70 mV, a whole-cell I_Ca was normally activated around −40 mV, peaked at −10 mV and reversed between +30 to +40 mV (Wang et al., 1996; Huang et al., 1998). Figure1A illustrates examples of the effects of corticotropin on I_Ca. Application of 1 μM corticotropin_4–10 or corticotropin_1–24 increased the amplitude of I_Ca. The ability of corticotropin analogs to potentiate I_Ca was plotted as a function of concentrations tested (Fig. 1B). Mean values were calculated from six applications at the concentrations tested. The mean experimental data was fitted with a sigmoid curve (r² = 0.964 for corticotropin_1–24 and 0.989 for corticotropin_4–10). The half-maximal effective concentrations for corticotropin_1–24 and corticotropin_4–10 were estimated to be 65 and 176 nM, respectively.

The increase in I_Ca occurred within 1 min of application and this effect was persistent after the washout of corticotropin within the recording period of experiment. To see whether the lack of reversibility is caused by the washout of some cytoplasmic constituent during whole-cell dialysis or a slow washout of corticotropin, sharp electrode recordings from basolateral amygdala neurons were made in brain slices treated with TTX (1 μM) and TEA (10 mM). Previous work has shown that somatic Ca²⁺-dependent action potential could be evoked under this condition (Wang et al. 1997). Figure 1C shows that, in artificial cerebrospinal fluid containing TTX and TEA, depolarizing current step command (300 ms, 0.8 nA) evoked a high threshold Ca²⁺-spike. Superfusion of corticotropin_1–24 (1 μM) to the slice prolonged the duration of Ca²⁺-dependent action in a reversible manner. The durations measured at half-peak amplitude were 35.0 ± 2.5 ms in control, 54.3 ± 4.2 ms in the presence of corticotropin (n = 6, p < 0.01 paired t test) and 39.5 ± 2.7 ms 40 min after the washout of drug.

The voltage-dependent effect of corticotropin_1–24 was analyzed in current-voltage (I-V) relationships in the presence and absence of 1 μM corticotropin_1–24. As shown in Fig.2A, corticotropin increased I_Ca to a greater extent at negative potentials (≤−10 mV) than at more depolarized membrane potentials (0− +40 mV) and the increase was not accompanied by a shift in the current-voltage relationship (peak I_Ca voltages were −9 ± 1 mV in control and −8 ± 1 mV in corticotropin,n = 6). The I-V relationship was also studied with voltage ramps using 5 mM Ba²⁺ as the charge carrier (Fig. 2B). Currents were evoked by voltage ramps from −70 to +50 mV (0.33 mV/ms); consistent with the above observation, corticotropin_1–24 did not shift the I-V relationship (n = 5).

The possibility that corticotropin_1–24 may affect the voltage dependence of steady-state inactivation was examined. In this series of experiments, I_Ca was elicited by step commands to 0 mV from holding potentials ranging from −80 mV to 0 mV. The inactivation curve was fitted with the following Boltzmann equation: I / I_max = 1/{1 + exp[(V − V_1/2 / k)]}, where V_1/2 is the potential at which I / I_max = 0.5, and k is the slope factor of the curve. As shown in Fig. 3, corticotropin_1–24 did not shift the voltage-dependent steady-state inactivation. The values for V_1/2 and k in control and corticotropin were −21.2 ± 1.9 mV and −19.2 ± 1.4 mV and −20.8 ± 2.0 mV and −16.6 ± 1.5 mV (n = 4), respectively. Taken together, these data suggest that corticotropin-induced enhancement of I_Ca is not caused by a shift in the voltage dependence of steady-state activation or inactivation.

We also investigated the effect of a C-terminal fragment of corticotropin, corticotropin_18–39 (CLIP), on the I_Ca. Corticotropin_18–39also enhanced I_Ca, with a less maximal effect of ∼30% (Fig. 1B). If corticotropin_18–39 and corticotropin_4–10 were exerting their effects via the same receptor, then saturating concentration of corticotropin_4–10 should occlude the modulation produced by corticotropin_18–39. Indeed, this is the case. As shown in Fig. 4A, application of corticotropin_18–39 (1 μM) increased the I_Ca by 33.1 ± 3.2% (n = 6), but subsequent addition of corticotropin_4–10 (1 μM) in the presence of corticotropin_18–39 only increased the I_Ca by 3.3 ± 3.5% (n = 6), which was significantly less than that produced by corticotropin_4–10 alone (51.3 ± 1.1%,n = 6, p < 0.01). We also performed the reverse experiments in which corticotropin_4–10 was applied before the addition of corticotropin_18–39. Shown in Fig. 4B is a time course of these experiments in which peak current evoked by a voltage step to −10 mV was plotted as a function of time. Representative current traces were shown in the figure inset. Initially, application of corticotropin_4–10 (1 μM) increased the I_Ca by 44.7 ± 2.4% (n = 6). Subsequently, corticotropin_18–39 (1 μM) had little or no effect (2.0 ± 1.8%, n = 6, p > 0.1).

Types of Ca²⁺ Channels Modulated.

Rat amygdala neurons express several pharmacologically distinct subtypes of high-threshold Ca²⁺ channels (Foehring and Scroggs, 1994; Yu and Shinnick-Gallagher, 1997). To determine which Ca²⁺ channel subtypes were modulated, corticotropin was applied after selective block of populations of Ca²⁺ channels. Figure5A shows that application of nimodipine (1 μM), a selective L-type Ca²⁺ channel blocker, reduced the whole-cell I_Ca by 29.3 ± 1.8% (n = 6). However, subsequent application of corticotropin_1–24 (1 μM) in the presence of nimodipine failed to cause an enhancement (1.8 ± 1.4%,n = 6). If corticotropin acts selectively on the L-type channels, then nimodipine should be able to abolish the component of corticotropin-mediated enhancement. Figure 5B shows that application of corticotropin_1–24 (1 μM) increased the I_Ca to 170.8 ± 3.6% (n = 6) of control; subsequent addition of nimodipine (1 μM) reduced the I_Ca to 69.4 ± 4.0% (n = 6) of the initial control, a value similar to that without corticotropin treatment (70.2 ± 2.6%, n = 6, p> 0.1). These results suggest that corticotropin selectively acts on the L-type Ca²⁺ channels.

To further exclude the involvement of N- and P/Q-type Ca²⁺ channels in the action of corticotropin, we applied submaximal concentrations of ω-CgTX and ω-AgTX simultaneously. Application of ω-CgTX (100 nM) plus ω-AgTX (200 nM) reduced the whole-cell I_Ca to 70.2 ± 2.6% (n = 6) of control. As shown in Fig.6, in the presence of ω-CgTX + ω-AgTX, corticotropin_4–10 still increased I_Ca to 114.3 ± 3.6% (n = 6) of the initial control, suggesting that the effect of corticotropin is not influenced by ω-CgTX + ω-AgTX. Taken together, these data suggest that the primary effect of corticotropin on the I_Ca is to enhance dihydropyridine-sensitive L-type currents.

Involvement of PKA Pathway.

Because corticotropin receptor is known to be coupled positively to adenylyl cyclase (Florijn et al., 1993), experiments were performed to determine whether the cAMP-dependent protein kinase (PKA) was involved in mediating the I_Ca potentiation. Forskolin (25 μM), an adenylyl cyclase activator, mimicked the effect of corticotropin, increasing the I_Ca by 38.4 ± 5.0% (n = 6). Similarly, application of membrane-permeant analog of cAMP, (S_p)-cAMPS (50 μM), caused a 62.6 ± 2.7% (n = 6) enhancement (Fig.7A). Consistent with these results, phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) also enhanced the effect of subsaturating concentration of corticotropin_1–24. As depicted in Fig. 7A, corticotropin_1–24 (10 nM) alone increased the I_Ca by 31.3 ± 3.8% (n = 6). However, in the presence of IBMX (50 μM), the same concentration of corticotropin_1–24 increased the I_Ca by 53.3 ± 6.4% (n = 8). The difference is statistically significant (p < 0.05).

(R_p)-cAMPS, a PKA regulatory site antagonist, was dissolved into the pipette solution (50 μM). Figure7B shows that inclusion of intracellular (R_p)-cAMPS markedly reduced I_Ca potentiation in response to corticotropin_4–10 (1 μM); corticotropin increased the I_Ca by only 13.3 ± 1.8% (n = 9, p < 0.01). In another three experiments, cells were preincubated with (R_p)-cAMPS (100 μM) for at least 2 h before whole-cell recordings were made with normal pipette solution. Under this condition, the effect of corticotropin was also markedly reduced, causing an increase of only 9.8 ± 2.1% (n = 3). Similar result was obtained when neurons were pretreated with KT 5720 (1 μM), a PKA catalytic site antagonist. Corticotropin_4–10 (1 μM) increased the I_Ca by only 11.4 ± 1.4% (n= 7) in KT 5720-treated neurons compared with 51.3 ± 1.1% (n = 6, p < 0.01) without KT 5720 treatment (Fig. 7B). Thus, the action of corticotropin is reduced by the treatments that inhibit PKA.

To investigate whether an ongoing phosphorylation-dephosphorylation process mediated by PKA and a serine-threonine phosphatase is involved in corticotropin action on the Ca²⁺ currents, we studied the role of phosphatase in the corticotropin enhancement of I_Ca. In these experiments, the membrane permeant inhibitor of protein phosphatase 1 (PP1) and PP2A, okadaic acid, was applied. As illustrated in Fig. 7A, in the presence of okadaic acid (1 μM), corticotropin_1–24 (10 nM) increased the I_Ca from 31.3 ± 3.8% to 60.9 ± 3.8% (n = 6, p < 0.05).

Discussion

The present results show for the first time that corticotropin enhances voltage-dependent Ca²⁺ currents in brain neurons and that this increase is mediated through dihydropyridine-sensitive L-type channels. The effect was concentration-dependent, with estimated EC₅₀values of 65 and 176 nM and a maximal increase of 50 to 75% for corticotropin_1–24 and corticotropin_4–10, respectively. The mimicry of corticotropin effect by active analogs suggests a receptor-mediated mechanism. The effect of corticotropin probably involves a cAMP- and protein kinase-dependent mechanism because the increase was mimicked by forskolin and (S_p)-cAMPS and was markedly reduced in neurons intracellularly dialyzed with (R_p)-cAMPS or extracellular perfusion of KT 5720. Moreover, inhibition of the cAMP-degradative enzyme phosphodiesterase potentiated the corticotropin-induced enhancement of I_Ca. The enhancement of corticotropin-mediated modulation by inhibition of PP1/PP2A with okadaic acid further supports the hypothesis that protein phosphorylation was involved.

Enhancement of Ca²⁺ Currents by Corticotropin in the Amygdala Neurons.

Corticotropin and corticotropin-like peptides originate from the precursor molecule POMC. POMC is processed by tissue-specific enzymes to yield small peptides, such as corticotropin, α-melanotropin, β-melanotropin, γ-melanotropin, and endorphins, collectively termed melanocortins (MC). Five MC receptors have been identified so far, of which MC3, MC4, and MC5 were located in the brain (Hol et al., 1995; van Rijzingen et al., 1996). Detailed neuroanatomical mapping by in situ hybridization demonstrated that MC3 and MC4 were expressed in the hypothalamus and limbic system including the amygdala. MC3 differs from MC4 in that it specifically recognizes peptides with the corticotropin_4–10 core sequence. All peptides with the corticotropin_4–10 core are approximately equipotent for MC3 receptor; on the other hand, corticotropin_4–10 is 100 times less potent than corticotropin_1–24 in activating the MC4 receptor (Gantz et al., 1993). Thus, although it is now impossible to distinguish between the MC receptor subtypes without developing specific antagonists, based on ED₅₀ values, our data are more likely to be consistent with mediation through an MC3 receptor subtype.

In brain tissues, the concentrations of corticotropin_1–24 needed to elicit a half-maximal response ranged from 10 to 100 nM (Hol et al., 1995), which is comparable with the results reported herein but is ∼1000-fold higher than those in endocrine cells (Enyeart et al., 1996). The difference is probably caused by the differential expression of subtype receptors in different tissues. In situ hybridization analysis revealed that adrenal cortex expressed MC2 receptors (Mountjoy et al., 1992).

The increase in I_Ca could be caused by a shift of the current-voltage relationship in the negative direction by corticotropin, which would cause more channels to open at negative potentials, thereby enhancing the I_Ca. Alternatively, corticotropin could increase I_Caby shifting voltage-dependent steady state inactivation to a more depolarized level. In the present study, neither the peak of the current-voltage relationship nor the voltage-dependent steady-state inactivation were affected by corticotropin. These results suggest that corticotropin-induced enhancement of I_Ca was not caused by a shift in the voltage dependence of steady-state activation or inactivation. Further study with single-channel recordings is needed to determine whether corticotropin-induced increase in I_Ca results from an opening of more calcium channels and/or an increase in single-channel conductance.

L-Type Ca²⁺ Channel Is the Target of Modulation.

At least four different high-threshold, voltage-dependent Ca²⁺ currents (L-, N-, P/Q-, and R types) have been identified in the amygdala neurons based on pharmacological criteria (Foehring and Scroggs, 1994; Yu and Shinnick-Gallagher, 1997;Huang et al., 1998). In the present study, sequential application of nimodipine and corticotropin revealed that the effect of corticotropin on I_Ca was completely blocked by nimodipine. Conversely, pretreatment of neurons with corticotropin did not affect the inhibitory effect of nimodipine; nimodipine not only completely abolished the component of corticotropin-mediated enhancement but also reduced the I_Ca to ∼69% of the initial control, a value not significantly different from that without corticotropin pretreatment. In contrast to the clear participation of L-type channels, blockade of N-type Ca²⁺ channels with ω-CgTX and P/Q-type channels with ω-AgTX did not reduce the corticotropin response. Thus, corticotropin acts selectively on the dihydropyridine-sensitive L-type Ca²⁺ channels. Possible modulation of Ca²⁺ channels by corticotropin has been reported in several systems. For instances, in a rat model of volume-controlled hemorrhagic shock, corticotropin produced a sustained restoration of arterial pressure, pulse pressure and respiratory function and these effects were inhibited by N- and L-type Ca²⁺ channel blockers (Guarini et al., 1993). Intracerebroventricular administration of corticotropin induced a peculiar symptomatology characterized by recurrent episodes of stretching, yawning, and penile erection that could be blocked by ω-CgTX, implying the involvement of N-type Ca²⁺channels (Argiolas et al., 1990).

PKA-Mediated Modulation of L-type Currents.

The MC receptors belong to the superfamily of G protein-coupled receptors and are positively coupled to adenylyl cyclase. Early studies in adrenal cortex showed that increasing intracellular cAMP mimicked the stimulation of corticosteroid production, indicating that there was a direct functional coupling between the corticotropin receptor, cAMP production, and corticosteroid synthesis (Halkerston, 1975). In brain tissues (striatal and septal slices), an increase in cAMP after corticotropin treatment has also been observed (Wiegant et al., 1979;Florijn et al., 1993). In the present study, we have demonstrated that forskolin, but not 1,9-dideoxyforskolin, mimics the effect of corticotropin. Similarly, activation of PKA by (S_p)-cAMPS results in a potentiation. The effect of subsaturating concentration of corticotropin_1–24 is significantly enhanced in the presence of phosphodiesterase inhibitor or protein phosphatase inhibitor. Furthermore, modulation of I_Ca by corticotropin was blocked by (R_p)-cAMPS and KT 5720, the respective PKA regulatory and catalytic site antagonists. These results suggest that PKA mediates the enhancement of I_Ca by corticotropin in the amygdala neurons.

It is well established that stimulation of adenylyl cyclase or PKA leads to phosphorylation of cardiac L-type Ca²⁺channels, resulting in an increase in contractility and heart rate. However, the selective potentiation of L-type Ca²⁺ currents in central neurons by neuromodulators is limited in the literature. In neostriatal neurons recorded in the presence of phosphatase inhibitors, dihydropyridine-sensitive L-type currents were enhanced by dopamine D₁ agonists or cAMP analogs (Surmeier et al., 1995). An enhancement of L-type currents by β-adrenergic agonists and cAMP analogs has been reported in hippocampal neurons (Gray and Johnston, 1987; Kavalali et al., 1997).

Functional Implications.

In central neurons, L-type Ca²⁺ channel plays a critical role in the synthesis of new proteins important for the expression of long-lasting potentiation (L-LTP) of synaptic transmission and long-term memory. The generation of L-LTP requires a sufficiently large influx of Ca²⁺ through voltage-dependent L-type Ca²⁺ channels and de novo RNA transcription and protein synthesis. L-LTP and L-LTP-associated increases in cAMP response element-mediated gene expression are attenuated by L-type Ca²⁺ channel blockers (Impey et al., 1996). Up-modulation of I_Ca reported here implicates that corticotropin may influence behaviors in mammals. Indeed, this hypothesis was supported by the behavioral studies showing that corticotropin enhanced the acquisition and retention of passive avoidance as well as delayed extinction of pole-jumping avoidance (Greven and de Wied, 1977; Fekete and de Wied, 1982). Furthermore, deficits in spatial orientation induced by an acute administration ofN-methyl-d-aspartate receptor antagonist could be counteracted by corticotropin analog (Spruijt et al., 1994).