Regulatory mechanisms involved in the activation of bradykinin-induced membrane currents in PC12 cells

ELSEVIER

Neuroscience Letters 195 (1995) 37-40

NEUHOSCIEHC[ tEllEgS

Regulatory mechanisms involved in the activation of bradykinin-induced membrane currents in PC 12 cells Alexandre Bouron, Harald Reuter* Department of Pharmacology, University of Bern, Friedbi&lstrasse49, CH-3010Bern, Switzerland

Received 18 May 1995; revised version received 26 June 1995; accepted 26 June 1995

Abstract

Whole-cell patch-clamp measurements were made in nerve-growth-factor (NGF)-treated PC12 cells. External application of bradykinin (BK) activated an outward and an inward current which could be separated by using KC1- or CsCl-containing pipette solutions. The slowly activating inward current could be induced by BK independently of the filling of intracellular Ca 2÷ stores. By using GDP-/3-S in the pipette medium, we showed that BK-induced outward and inward currents were differentially regulated through Gprotein-sensitive and -insensitive mechanisms, respectively. While the outward current was inhibited by GDP-fl-S, the inward current was not affected. Our results show that occupancy of BK receptors activates different signaling pathways for the induction of outward and inward currents. Keywords: PC12; Bradykinin; G-Proteins; Membrane currents

The nonapeptide bradykinin (BK) exerts a wide range of cellular actions, such as increasing vascular permeability, promoting vasodilatation or inducing leukocyte attraction. In addition, BK also stimulates neuronal cells, notably sensory neurons, involved in pain reactions [ 1,4]. The rat pheochromocytoma cell line PC12 expresses BK receptors [8]. The binding of BK to its receptors activates, among several signaling pathways, phospholipase C and, thereby, the hydrolysis of phosphatidylinositol bisphosphate resulting in the production of inositol-l,4,5trisphosphate (IP3) and the release of Ca 2+ from intracellular stores [5,18,19]. In addition, the peptide affects membrane excitability by producing a biphasic change in membrane currents. It activates an hyperpolarizing Ca 2+dependent K ÷ current and a voltage-independent depolarizing inward cationic current that is, at least partially, carried by Ca 2+ [11,13]. The outward current is triggered by Ca 2+ released from intracellular stores [2], while little is known about the regulation of the BK-stimulated inward current. We have investigated regulatory mechanisms of BK-activated currents in nerve growth factor (NGF)-differentiated PC12 cells. We could show that BK * Corresponding author, Tel.: +41 31 6323281; Fax: +41 31 3027230.

activates outward and inward currents via G-proteinsensitive and -insensitive mechanisms, respectively. Properties of the rat pheochromocytoma cell line PC 12 have been described elsewhere [11,12,16]. Cells were plated on poly-L-lysine coated cover slips at a density of 4 x 104 cells/ml in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum (HS) (Boehringer, Rotkreuz, Switzerland), 100 units/ml penicillin and 100/tg/ml streptomycin (Gibco, Basel, Switzerland). Morphological differentiation was induced by adding 100 ng/ml NGF (7S fraction, Sigma, St Louis, USA) to a culture medium containing 2.5% HS. Membrane currents were recorded in the whole-cell configuration of the patch-clamp technique [6] with a List EPC-7 patch-clamp amplifier (List Electronics, Germany). Currents were low-pass filtered at 50 Hz and digitized at 0.5 kHz. Cell membrane capacitance was measured in order to express the current density in pA/pF [ 16]. The current amplitude was calculated as the difference between the peak current and baseline current at the holding potential. Experimental conditions and procedures have been described in a previous study [11]. Briefly, BK-activated inward currents were studied with a pipette solution containing (in mM): CsCI 115, Na2BAPTA 10, MgCi2 1, CaCI 2 6, Na2ATP 1, HEPES 10

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A. Bouron, H. Reuter / Neurosctence Letters 195 (1995) 37~t0

(pH 7.2 with CsOH). The concentration of free intracellular calcium, [Ca2÷]i, was calculated according to Zhou and Neher [20], with a dissociation constant of B A P T A of 225 nM. The concentrations of B A P T A and Ca 2+ adjusted [Ca2+] i to 380 nM. The external solution contained (in mM): NaC1 150, KC1 5, CaCI2 10, MgC12 1, HEPES 10 (pH 7.4 with NaOH). BK-activated outward currents were recorded with a KCI- instead of a CsCl-containing pipette solution. Several batches o f PC12 cells were used throughout this study and we noticed that there was some variability in current densities between different cell cultures. Thus, the control values were obtained separately for each batch o f cells and for each experimental protocol. Unless stated otherwise, outward and inward currents were stimulated by the external application of 500 nM B K and the responses are referred as 'control responses'. Since B K could not be washed out, cells could be exposed to the peptide only once. Bradykinin was purchased from Boehringer M a n n h e i m A G (Rotkreuz, Switzerland), the bradykinin antagonists, (D-Phe7)-bradykinin and (DArg,Hyp2.3,D-Phe7)-bradykinin, from Bachem Feinchemikalien A G (Bubendorf, Switzerland), the PKC inhibitor P K C ( 1 9 - 3 1 ) from Peninsula Labs (Basel, Switzerland). All other chemicals and reagents were purchased from S i g m a Chemie (Buchs, Switzerland). Statistical data were given as mean _+ SEM with the number of cells tested expressed by n. Differences among groups were estimated by using a Student's t-test for unpaired groups. Differences were considered significant at P < 0.05. W h e n PC12 cells were perfused with a CsC1containing pipette solution, in order to block outward K ÷ currents, B K activated an inward current, partially carried by Ca 2÷ ions [11,13]. Activation of this current was associated with an increase in membrane conductance. This is shown in Fig. 1A where the cell was voltage-clamped at - 5 0 m V and the current was activated by addition of 500 nM BK, and hyperpolarizing clamp steps o f 20 mV were applied for 200 ms. The increase in size of the corresponding currents indicates the conductance change. The inward current is activated by B K in a concentration dependent manner. Cells were stimulated with 100, 500 or 1000 nM BK. The mean current-densities were 0.6 _+ 0 . 2 p A / p F ( n = 6 ) , 1 . 1 _ 0 . 1 p A / p F ( n = 8 ) , and 2.1_+ 0.5 p A / p F (n = 7), respectively (Fig. 1B). BK produces its actions by activating, at least, two types o f B K receptors (B1 and B2), although the B 1-type seems to be poorly expressed under physiological conditions [4]. In order to find out which type of receptor is involved in activation of the inward current, we have used the specific B2-antagonist (D-Phe7)-BK. When applied externally for 1-4 min prior to BK addition, 4 / z M (DPheT)-BK inhibited the BK-induced inward current by about 50% (Fig. 1C). This concentration of the B2antagonist corresponds to about three times its K a value [15,19]. The non-specific B K antagonist (D-Arg, Hyp 2,3,

D-Phe7)-BK (1/zM), however, completely blocked the BK-induced inward current. This indicates that in our P C I 2 clone the current is not activated by B2-receptors only. Since BK activates phospholipase C and, hence, PKC stimulation, we have also studied the effect of the PKC inhibitor peptide PKC(19-31). When added into the pipette solution at a concentration of 2/~M, the peptide had no significant effect on BK-induced inward current, indicating that this signaling pathway is not involved in activation of this current (Fig. 1D). Thapsigargin, which depletes intracellular Ca 2+ stores by inhibiting Ca2+-ATPases in some excitable and nonexcitable cells, activates a voltage-independent Ca 2+ inward current (Ic~Ac) that resembles in several respects the BK-activated inward current described here [7,9,21]. Thapsigargin also empties intracellular Ca 2+ stores in our PC12 cell clone, as indicated by fura-2 measurements (B.F.X. Reber, pers. commun.). However, in the presence of a CsCl-containing pipette solution, we failed to detect any thapsigargin-activated inward currents. A subsequent addition o f BK stimulated the usual inward current (Fig.

A

B

100

500

I000

Bradykinin (nM)

D

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i 0

BK

(D-PheT)-BK (D-Arg,Hyp,D*PheI-BK

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oi

Control

PK C( 19-31 )

Fig. 1. Concentration-response relationship of BK on the BK-activated inward current. (A) BK-induced inward current superimposed by 20 mV hyperpolarizing steps (200 ms, every 17 s; holding potential: 50 mV, 500 nM BK). The dotted horizontal line represents the holding current before the addition of BK (arrow head). (B) Columns show the effects of different BK concentrations on BK-induced inward currents. The experiments were performed on PC12 cells belonging to the same cell culture. (C) Inhibition of the inward current by the B2 type antagonist (D-PheT)-BK(4/~M) and by the non-specific BK antagonist (DArg,Hyp2"3,D-Phe7)-bradykinin(1/~M). Drugs were externally applied (2-4 min) before BK. The current density values were obtained by dividing, for each cell, the peak current amplitude (pA) by the cell membrane capacitance (pF). The number of cells tested is indicated for each group. (D) Effect of the PKC inhibitor PKC(19-31) on the BKinduced inward current. PKC(19-31) (2 laM) was added into the pipette solution. PC12 cells dialysed for 2-4min before BK application. *P < 0.05, **P < 0.01. The horizontal arrow indicates the zero current level.

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A. Bouron, H. Reuter / Neuroscience Letters 195 (1995) 37-40

2A). Since the pipette m e d i u m contained 380 nM free Ca 2+, the channels may not have sensed depleted Ca 2÷ stores [9]. Therefore, we also perfused PC12 cells for 2 4 min with a Ca2+-free pipette m e d i u m containing 5 10 m M B A P T A and then added thapsigargin at concentrations o f 50, 200, or 1000 nM for 2-11 min. W e failed to detect any thapsigargin-induced inward current under these conditions. Our results are different from those in other PC12 clones, where thapsigargin seemed to elicit a Ca 2÷ influx in fura-2 loaded cells [3]. In our clone, however, B K activated the current independently of the filling of the stores. This is in contrast to the BK-activated inward current described in bovine aortic endothelial cells [17]. As previously shown, the inward current became very small or disappeared in Ca2+-free solutions [11,13] indicating that this ion is a major charge carrier. The external application of 100/zM Zn 2+ (or Cd 2÷, not shown) completely blocked the BK-activated inward current within a few seconds (Fig. 2B). This effect is more potent than that on voltage-activated Ca 2+ channels, since 100/~M Zn 2+ inhibited whole-cell Ba 2+ currents through voltageactivated Ca 2÷ channels by only 23 _+ 1% (n = 5). W h e n PC12 cells were dialysed with a KCI- instead of a CsCl-containing pipette solution, the external application o f 500 nM B K (Fig. 3A, arrow head) activated a large transient outward current followed by the small inward current described above. W e added GDP-/3-S into the pipette solution, in order to uncouple B K receptors from the biochemical cascade that is responsible for IP 3induced release of Ca 2÷ and leads to the activation of the outward current [13]. W h i l e 1 m M GDP-fl-S inhibited the outward current by - 6 0 % , 4 m M GDP-/5-S blocked - 9 5 % of it (Fig. 3B). However, these experiments do not exclude a direct effect o f GDP-fl-S on K + channels. Thus, in another series of experiments, K ÷ channels were activated by a caffeine-induced release of Ca 2÷ [11,13]. Caffeine releases Ca 2÷ from IP3-insensitive stores that are blocked by ryanodine [14]. Ca 2+ release from these stores does not involve G-proteins. PC12 cells were first depolarized by KC1 (80 mM) in order to fill caffeine-sensitive Ca 2+ stores [12]. Under these conditions, caffeine (30 mM) evoked a transient outward current (2.1 _+ 0.3 pA/pF, n = 6) that A

B

Zn 2 +

Tit v

Fig. 2. The BK-induced inward current is not activated by depleted intracellular Ca2+ stores. (A) Thapsigargin (Th, 200 nM) was applied for 45 s prior to BK addition (500 nM, arrow head). (B) 500 nM BK (arrow head) induced the development of an inward current which was completely inhibited by 100/zM Zn2+. The horizontal arrows indicate the zero current level.

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B 120

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.

.

.

.

.

.

-~

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Fig, 3. Effects of GDP-fl-Son bradykinin-activated outward and inward currents. NGF-treated PCI2 cells were dialysed with a KCI- (A,B) or CsCl-containing pipette solutions (C,D). Applications of 500 nM bradykinin (BK) are indicated by arrow heads (A,C). The dotted horizontal lines represent the holding current before the addition of BK. The columns illustrate the effects of 1 and 4 mM GDP-/3-S added into the pipette solution on BK-activated outward (B) and inward current densities (D), after normalisation. *P < 0.05, **P < 0.01. The harrow horizontal indicates the zero current level. resembled the BK-induced current [13]. With 4 m M GDP-fl-S in the pipette medium, caffeine gave rise to an outward current of comparable amplitude as in the controis (2.7 _+0.9 pA/pF, n = 5, P > 0.05). W e conclude that GDP-fl-S inhibits Ca2+-dependent K ÷ currents by uncoupling BK receptors from the IP3-induced release of Ca 2+ from intracellular stores, rather than by direct inhibition of Ca2+-activated K ÷ channels. By contrast, in the presence of CsC1 in the pipette solution, the inward current remained unaffected by the intracellular application of 1 or 4 m M GDP-fl-S (Fig. 3C,D). In agreement with this, GTP, GTP-y-S and PTX were ineffective in modulating the BK-activated inward current [11]. In conclusion, the data we have obtained show that B K activates outward and inward currents through G-protein sensitive and insensitive regulatory mechanisms, respectively. The release of Ca e+ from IP3-sensitive Ca 2÷ stores controls the development of the BK-activated outward current. Depletion of the stores, however, has no effect on activation of the inward current by BK. In a previous study [11], it has been shown that the BK-induced inward current became larger with increasing steady-state [Ca2+]i. It could also be shown that CGS 9343B, a non-specific Ca2+/calmodulin dependent protein kinase (Ca2+/CaM PK) inhibitor, reduced the amplitude of this inward current in a concentration-dependent manner. In addition, we have used KN-62, a Ca2÷/CaM-PK II inhibitor, which produced the same effect (not shown). These results suggest that the BK-induced inward cationic current is under the control of a Ca2+/CaM-PK activity.

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A. Bouron, H. Reuter / Neuroscience Letters 195 (1995) 37~I0

W e a r e p a r t i c u l a r l y g r a t e f u l to M s C B e c k e r a n d M r s P e c c i o l i f o r e x p e r t t e c h n i c a l a s s i s t a n c e w i t h t h e cell cultures. T h i s w o r k w a s s u p p o r t e d b y t h e S w i s s N a t i o n a l Science Foundation (grant 31-29862.90).

[12]

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