Brain Research, 594 (1992) 197-204 © 1992 Elsevier Science Pablishers B.V. All rights reserved 0006-8993/92/$05.00

197

BRES 18188

cAMP-mediated expression of inwardly rectifying potassium channels in cultured mouse Schwann cells T e t s u r o Konishi Department of Neurology, Utano Nauonal Hospital, Nanaaki, Kyoto (Japan) (Accepted 26 May 1992)

Key words: Schwann cell; Potassium channel: Cyclic AMP; Cycloheximide; Patch-clamp

Voltage-gated ionic currents were recorded from freshly dissociated or cultured mouse Schwann cells obtained from neonatal sciatic nerves by the whole-cell variation of the patch-clamp technique. Schwann cells virtually lost inwardly rectifying potassmm (K,r) currents within 2 days after nerve transection or in culture conditions of neonatal sciatic nerves confirming the previous results that axonal signals were suggested to play an important role in the expression of functional K,r channels. To see the effects of adenosine 3',5'-monophosphate (cAMP) analogues or forskolin on the expression of Ktr channels in cultured Schwann cells, these agents were added to the culture medium 4 days after the start of the culture, when Ktr currents were almost eliminated from cultured Schwann cells. Cultured Schwann cells restored the expression of Kir currents by co-culture with agents which elevate intraceilular cAMP level. The dose-response of 8-(4-chlorophenylthio)(CPT) cAMP for the incidence of the expression of Kit currents showed a steep increase in the percentage of cells with Kir currents between 0.02 and 0.1 mM of external CPT cAMP and approximately two thirds of cells had K,r currents i, higher concentrations of more than 0.1 mM of CPT cAMP after 4 days of incubation. After removal of CPT cAMP from the culture media after 4 days of incubation, K,r currents disappeared from cells within 2 days. The simultaneous application of cycloheximide (1 ~g/ml), an inhibitor of protein synthesis, with CPT cAMP suppressed the expression of K,r currents for up to 6 days of incubation. After the cessation of cycloheximide treatment for 3 days, the expression of K ~r currents were restored with a time lag of at least 3 days from the application of a cAMP analogue. These findings suggest that cAMP is an intracellular second messenger of axon-glial interactions for the expression of functional Kit channels in Schwann cells.

INTRODUCTION In axon-glial interactions, axonal signals not only regulate the differentiation of myelin-forming Schwann cells to form myelin ~'2s but also regulate non-myelinating Schwann cells to express galactocerebroside (GC), which is a major component of the myelin sheath, since non-myelinating cells lose GC expression after a loss of axonal contact and restore it after nerve regeneration 8. The induction of GC in cultured cells by an increase of Schwann cell adenosine 3',5'-monophosphate (cAMP) 21 suggests that axon-glial interaction leads to an elevation of intracellular cAMP levels and thus to Schwann cell differentiation. Expression of a 170 kDa protein found in myelin 2°, of a myelin specific protein Po ~s and a myelin basic protein, and corresponding mRNAs ~3,

are induced by exposure to cAMP analogues or forskolin. The presence of inwardly rectifying potassium (Kir) currents, which are bariur,.sensitive, in both neonatal myelin.associated !1,26 and non-myelin-associated 12 Schwann ceils and the virtual absence of these K~r currents in cells within a few days after the start of culture I! indicate that axonal contact plays an important role in the expression of K ir currents in Schwann cells. These observations also suggest tllat cAMP might be an intracellular second messenger of axon-glial interactions for the expression of K~r currents similar to the expression of myelin specific proteins. In a test of this hypothesis, cAMP analogues or forskolin was added to the culture medium, and voltage-gated ionic currents were recorded from cultured Schwann cells.

Correspondence: T. Konishi, Department of Neurology, Utano National Hospital, 8-Ondoyama-cho, Narutaki, Kyoto 616, Japan.

198 multipolar fibroblasts, by a whole-cell variation of the patch electrode technique7 with the L/M-EPC-7 patch clamp system (Listelectronic, FRG) at room temperature (22-24°C). For the identification of cells to be recorded, the tips of the patch pipettes were filled with 1% (w/v) Lucifer yellow CH (Aldrich, USA) dissolved in the pipette solution m, which enabled selection of cells without dye-coupling. The patch pipettes had a reststance of 4-8 M.O and were filled wRh a solution contaming (in raM) KCi 121, CaC! 2 1, MgC! 2 2, HEPES I0, EGTA 11 and KOH 29 (pH, 7.4). The composition of the standard external solution (in raM) was NaCI 145, KCi 5, CaCI 2 2.5, MgC! 2 I, HEPES 10 and NaOH 4 (pH, 7.4). When K + concentrations in the standard solutmn were changed, KCi was substituted for equimolar amounts of NaCI. Schwann cel;s placed on a coverslip with a volume of less than 0.2 ml of a solution were superfused at a rate of 10 ml/min. The resting membrane potentials (Vm) of cells were measured after establishment of the whole-cell recording. The liquid junction potentials between the standard external and internal solutions, which were less than 3 mV tl, were not corrected in this study. Series resistance compensation was not used. After pipette capacitance was cancelled in the cell-attached mode, the size of cell capacitance was read directly from the C-SLOW knob of the List amplifier after cancellation of the whole-cell capacitative currents elicited by 10 mV of depolarizing voltage step from a holding potential (HP) of - 8 0 mV. The magnitude of K,r currents was obtained as barium-sensitive inward currents elicited by -145 mV of hyperpolarizing voltage steps (HP; - 8 0 mV) by subtraction of a recording in the standard solution containing 2 mM BaCI 2 front that in the standard solution t t.).,.. The minimum level of detection of Ktr currents was 20 pA in this study. Current density was calculated by dividing the magnitude of K,r currents by whole-cell capacitance. The numerical data represent the mean + S.D. Statistical evaluation was done by Student's t-test or X 2 test.

The results show that cAMP analogues or forskolin can restore the expression of K,~ currents in cult,.red Schwann cells. MATERIALS AND METHODS

Preparattot)s Freshly dissociated Schwann cells were obtained, as described previously tt, from neonatal sciatic nerves of l-3-day-old mice (C57BL6 or Balb/c). In some experiments sciatic nerves of neonatal mice at 1st day after birth were transected at the site of sciatic notch under ether anaesthesia. Segments of sciatic nerves at mid thigh portion, which was distal to the transected sites, were excised from decapitated mice. These nerve segments were treated with 0.3% coUagenase (w/v) (Type I, Sigma, USA) dissolved in minimum essential medium (MEM) Earle's salts (Gibco, USA) containing 10% (v/v) fetal calf serum (Gibco) for 15 rain in a CO., incubator at 37°C. The treated nerve segments, 3-4 mm in length, were teased apart on coverslips with needles so that single nerve fibers were discernible under Hoffman modulation optics (×400, Nikon, Japan). These preparatmns were served for recordings within 4-6 h after dissociation. The day when newborn mice were first found in the morning was designated as day I after birth. Cultured Schwann cells were obtained from enzymatically dissociated excised sciatic nerve,, of l-2-day-old mice (Balb/c) and were cultured on polyq-lysine (Sigma, USA) coated round coverslips as described prcvmusly " '). Each coverslip contained I x 104 cells. The cells were incubated in Dulbecco's Modified Eagle Medium (Gibco, USA) containing 10% (v/v) fetal calf serum (Gibco), 100 U / m l penicillin and 100 ,u.g/ml streptomycin in a humidified atmosphere of 5e;, CO.,-95e,~, air at 37°C. cAMP derivatives (8-(4-chlorophenylthio) adenosine Y,5'-monophosphate (CPT cAMP), N~,O"'-dibutyryl adenosine 3',5'-mono. phosphate (DB cAMP)), cAMP, N:,O:'-dihutyryl guam)sine 3',5'monophosphate (DB cGMP) and hutyl.'tte were purchased from Sigma and were added to the medium ahout 96 h after the start of culture. These drugs were dissolved in a volume of distilled water before addition which constituted 5C; of the w)lum= of the culture medium and wits passed through a 0.22 # m pore filter (MILLEX-GS, Millipore Corporation, USA), Forskolin (I rag) (Sigma) was dissolved in I ml of ethanol and diluted by the culture medium to a fin.d concentration, The contn)l culture medium was replaced by fresh medium at the same incubation period,

RESULTS

Elimination of K~r currents in Schwann cells after nerve transection or in culture conditions of" neonatal sciatic ner+,es Both freshly dissociated non-myelin-associated and myelin-associated Schwann calls from neonatal sciatic nerves at 1-3 days after birth showed Kit currents =2 (Fig. 1). The magnitude of K+r currents elicited by a hyperpolarizing voltage step of -145 mV (HP; - 8 0

Recording aml soh+tions Ionic currents were recorded from Schwann cells, which have a distinct bipolar morphology and were readily identified from any flat

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Fig. I. Recordings of inwardly rectifying potassium (K,r) currents elicited by - 145 mV of a hyperpolarizing voltage step with 1 s duration (holding putential (HP): - 80 mV) before (!) and after (2) external application of 2 mM BaCi a in freshly dissociated Schwann cells obtained from neonatal sciatic nerves, a: myelin-associated Schwann cells from 2-day-old newborn mouse, b: non-myelin-associated Schwann cells from I-day-old mouse, c: Schwann cells obtained ! day after nerve transection of neonatal mouse, d; Schwann cells obtained 2 days after nerve transection. K,r currents were obtained by subtracting tracing 2 from tracing I (lower tracings). K,r currents were absent in d.

199

mV) were 0.24 + 0.39 n A (curren: density, 17.8 +_ 23.8

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p A / p F ; n = 14) in non-myelin-associated cells and 1.48 + 0.63 n A (current density, 79.9 + 29.5 p A / p F ; n = 16) in myelin-associated cells with cell capacitance less than 30 pF. These results of magnitude and density of K i, currents of Schwann cells in neonatal nerves were obtained from cells previously described t2. Since myelin-associated Schwann cells were distinguished from non-myelin-associated cells by the presence of myelin u n d e r Hoffman modulation optics in this study, non-myelin-associated cells might include cells with very thin myelin, not visible u n d e r a microscope, or cells with promyelin fibers. The magnitude and density of K,r currents of neonatal myelin-associated Schwann cells was significantly higher than that of non-myelinassociated cells ( P < 0.001). The incidence of cells with K,r currents at the distal segments of sciatic nerves, which were transected at the 1st day after birth, decreased to 40% ( 6 / 1 5 ) at one day after transection and the magnitude of K,r currents of these 6 cells were 0.13 + 0.12 nA (current density, 15.6 + 14.6 p A / p F ; n = 6). The incidence of cells with Kir currents further decreased to 7% ( 1 / 1 5 ) at 2 days after nerve transection. in culture conditions, the incidence of cells with K,r currents decreased markedly and only 4 (13.3%) out of

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Fig. 2. Time course of disappearance of K,r currents from cultured

Schwann cells (o) and reappearance or suppress,on of these currents upon treatment of the cultures with 1 mM 8-(4-chlorophenyithto)adenosine 3',5'-mor.ophosphate (CPT cAMP) (e) or both 1 mM CPT cAMP and cycloheximide(1 /~g/ml)(ll~ at 96 h of culture (arrow). The removal of CPT cAMP from the medium after 96 h apphcatton caused rapid elimination of K~r currents from the cultured cells within 48 h (Q). The percentage of cells with K,r currents (ordinate) was obtained from total number of 16-20 cells, (30 cells at 24 h), on two or more occasions. Abscissa: days of culture from the start of plating. Significant difference of incidence compared to the control (2/110) obtained from cells at 5-10 days of incubation mdicated by asterisk (P < 0.01 by X2 test). 30 cells showed K ir currents at one day after the start of culture (Fig. 2). The magnitude of K,, currents of these 4 cells were 0.06 + 0.05 nA (4.0 :i: 4.0 p A / p F ) . Kit currents were almost eliminated in cells within 2 days after the start of culture but there remained a

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Voltage ( m V ) Fig. 3. Recordings of currents Ca, b) and their current-voltage plots (c) of peak currents obtained from a cultured Schwann cell after 5 days of incubation with 1 mM CPT cAMP before (1, o) and after (b, *) bath application of 2 mM BaCi2. Currents were elicited by 20 mV voltage steps ( H P ; - 80 mV); some voltage steps are indicated in the figure. Hyperpolarization-activatedinward currents, which are barium sensitive, showed a sag at - 160 mV of a hyperpolarizingvoltage step.

200 cAMP, K,r currents disappeared from the cultured cells within two days (© in Fig. 2), suggesting that continuously elevated intracellular cAMP is necessary to express K,r currents similar to the cAMP-dependent induction of galactocerebroside (GC) 22. The simultaneous application of cycloheximide (1 ~g/ml), an inhibitor of protein synthesis, with 1 mM CPT cAMP suppressed the expression of inward K + currents for up to 6 days of incubation ( • in Fig. 2). After 6 days of incubation with CPT cAMP and cycloheximide, the number of spindle-shaped cells slightly decreased to 85.7 + 8.0% (n = 3) of cells counted before addition of these agents in a given area of the coverslips by the method previously described 9. The current-voltage relationship of voltage-gated currents were investigated in a cell after 5 days of incubation with 1 mM CPT cAMP. Depolarizationactivated outward currents and hypcrpolarizationactivated inward currents were elicited by 20 mV voltage steps from a HP of - 8 0 mV (Fig. 3). Magnitudes of depolarization-activated outward currents elicited by depolarizing voltage steps of 20 and 40 mV from a HP of - 8 0 mV were smaller than those of hyperpolarization-aetivated inward currents by the same degree of hyperpolarizing voltage steps showing a presence of

small number of cells with K,, currents after more than two days of incubation (Fig. 2). In the control culture at 5-10 days of incubation, 1.8% (2/110) of cells showed K , currents, the magnitude of which were 50 and 80 pA (3.3 and 5.5 p A / p F ) and this incidence of cells with K,~ currents was used as a control for later evaluation. These results showed that K,r currents virtually disappeared from Schwann cells within a few days after nerve transection or in cultured conditions of neonatal sciatic nerves.

Restoration of K,r currents in cultured cells treated with agents which elet'ate intracelhdar cAMP level When l mM of CPT cAMP, which is a membrane permeating analogue of cAMP, was added to the medium 96 h from the start of the culture, K, r currents restored in cultured cells. The incidence of cells with K , currents gradually increased and peaked 4 days after the addition of I mM CPT cAMP (o in Fig. 2). A significant difference of the incidence of cells with K~r currents was noted more than 48 h after the treatment of CPT cAMP ( , in Fig. 2). When the medium was replaced with a fresh medium containing no cAMP analogues after 96 h of incubation with l mM CPT

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Voltage ( m V ) Fig. 5. Recordings of currents (a, b) and their current-voltage plots (c) of peak currents obtained from a cultured Schwann cell after 5 days of incubation with 1 mM cAMP before Ca, o) and after bath application of 2 mM BaCI 2 (*), or both BaCI 2 and 2 mM quinine hydrochloride (b, e). Currents were elicited by 20 mV voltage steps (HP; - 80 mV); some voltage steps are indicated in the figure. Only quinine-sensitive voltage-gated outward currents, which are barium- resistent, were elicited.

TABLE !

Restoration of K . currents in cultured Schwann cells by co.culture with agents which elevate intracellular cAMP level Kit currents were investigated from cells after 4 days of incubation with adenosine Y,5'-monophosphate (cAMP) analogues (8.(4.chlorophenylthio) (CPT) cAMP, N*,O2°-dibutyryl (DB) cAMP), forskolin, cAMP, N:,O2'-dibutyryl guanosine 3',5'-monophosphatc (DB cGMP), bulylate, distiih:d water (5% of volume) or standard medium, These agents were added at 96 h of culture. Percentage of ceils with KIt currents was obtained from cells on two or more occasions, Number of cells for current density is indicat~.'d in parentheses. Control result~ were obttdned from cells at 5-10 days of incubation in the standard medium.

Treatment CPT cAMP (2 mM) (1 mM) (0.5 mM) (0.1 mM) (0.05 mM) (0.02 mM) (0.01 mM) DB cAMP (1 mM) (0.1 mM) Forskolin (200/tM) (20 ~M) cAMP (1 raM) DB cGMP (1 mM) Butylate (1 mM) Distilled water (5%) Control

Magnitudes of inward K + currents (hA) (range)

Current density (pA / pF) Or)

* * * * *

0.175:0.10 (0.02-0.30) 0.22 5:0.35 (0.02-1.30) 0.195:0.30 (0.02-0.87) 0.14:i:0.10 (0.03-0.24) 0.105:0.10(0.02-Ir).25)

26.5 + 18.1 (10) 15.4 + 13.5 (10) 19.5 5:30.8 (7) 14.1 5:12.5 (7) 9.45:9.8 (4)

6/17 (35) * 0/15 (0)

0.155:0.11 (0.02-0.36)

26.85:24.7 (4)

6/18 (33) * 0/15 (0) 0/20 (0) 1/18 (6) 0/18 (0) 0/16 (0) 2/110 (1.8)

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Number of cells with inward K + currents (%) 11/17 12/18 7/15 8/15 4/20 0/15 0/15 0/18

(65) (67) (47) (53) (20) (0) (0) (0)

* Significant difference of incidence compared to the control (P < 0.01 by X2 test).

2~ inward rectification of inward currents elicited by hyperpolarizing voltage steps, in this cell hyperpolarization-activated inward currents exhibited inactivation following a peak at a hyperpolarizing voltage step of - 160 mV, as has been observed in freshly dissociated neonatal myelin-associated cells ~t. External application of 2 mM BaCI 2 eliminated the inward currents leaving leakage currents and reduced the magnitude of outward currents (Fig. 3b). The residual outward currents were voltage-gated outward K + currents, which were blocked by external application of 2 mM quinine hydrochloride, as observed in freshly dissociated neonatal myelin-associated cells *~ (not illustrated), in another cell cultured with 1 mM CPT cAMP for 6 days, inward rectification of inward K ÷ currents elicited by hyperpolarizing voltage steps was obvious in a high K ÷ (40 mM K ÷) solution (Fig. 4). Thus, CPT cAMP restored functional K,, channels, which are barium sensitive. However, the presence of inward rectification of inward K + currents observed in cultured cells treated with CPT cAMP was different from barium-sensitive K+r currents observed in freshly dissociated neonatal myelin-associated cells, which showed an almost linear current-volt. age relationship without obvious inward rectification even in a high K ÷ solution I~, in a cell cultured for 5 days with a medium containing 1 mM cAMP, only quinine-sensitive voltage-gated outward currents, which are barium resistant, were elicited (Fig. 5). These quinine-sensitive outward currents were the voltage-gated outward K + currents observed in cultured Schwann cells '~'~, it is concluded that CPT cAMP restored functional K~r channels in cultur~.d Schwann cells and continuous elevation of intraccllular cAMP was required for the expression of these K~r currents. Only substances which would be expected to increase intracellular cAMP levels restored the expression of K~, currents examined after 4 days of incubation with drugs which were added to the medium on day 4 of culture (Table l). in their potency of expression of K,, currents by cAMP analogues, CPT cAMP was more potent than dibutyryl (DB) cAMP similar to its potency on the activation of cAMP-dependent protein kinase in rat hepatocytes ~*. A high dose of DB cAMP or forskolin was required to restore the expression of K,, currents, which is a similar condition to the requirement of a rather high dose of these drugs for "~ the induction of GC expression in rat Schwann cells"~I 2-. The dose-response of CPT cAMP for the expre,;,+ionof K,, currents showed a steep increase in the incidence of positive cells with K,, currents between 0.02 and 0.1 mM of CPT cAMP and two thirds of cells became positive with concentrations of CPT cAMP more than 0.1 raM. There was no apparent relationship between

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Fig. 6. Time course of reappearance of K,, currents when CPT cAMP was added to cells preincubated with both cycloheximide (! ~.g/ml) and CPT cAMP (1 mM) for 72 h .~tarting on day 4 of culture. 1 mM CPT cAMP was added 0 (e). 2 (o), 24 ( 4 ) or 48 h (D) after washout (arrow). Ordinate: percentage of cells with K,, currents obtained from total number of 15-17 cells on two or more occasions. Abscissa: days of culture from the start of plating. Significant difference of incidence compared to the control (at 5-10 days of incubation) indicated by asterisk (P < 0.01 by X2 test).

the concentration of applied CPT cAMP and the magnitude of K~r currents or their current densities due to a large intrinsic variability especially in the latter rarameter. Resting membrane potentials (Vm) of cells with K~r currents for 4 days of incubation in solution containing CPT cAMP (0.05-2 raM) were -69.2 :t: 5.9 mV (n - 42) and were significantly more negative than V,~ of cells which lacked K~r currents (-53.6:1:10.8 mV, n - 4 3 ) (P