Pfl/igers Arch (1993) 422: 364- 370

Joumal of Ph 010gy �9 Springer-Verlag 1993

Agonist-induced inhibition of inositol-trisphosphate-activated IK(Ca)

in NG108-15 neuroblastoma hybrid cells Jonathan Robbins

Department of Pharmacology, University College London, London WC1E 6BT, UK

Received August 18, 1992/Received after revision September 15, 1992/Accepted September 17, 1992

Abstract. IK(Ca) activated by intracellular ionophoresis of inositol trisphosphate (IP3) or pressure-applied acetyl- choline was inhibited by bradykinin and acetylcholine in NG108-15 cells transfected with ml receptors. The inhibition of the IP3-evoked current was complete at 10 gM acetylcholine. This inhibition was not seen if the current was evoked by intracellular ionophoresis of cal- cium ions. Only receptors the activate the phospho- inositide system in these cells produced this inhibition, i.e. transfected muscarinic ml and m3 and bradykinin receptors, but not muscarinic m2, m4 or adrenergic e2 receptors. This inhibition was not sensitive to pertussis toxin or staurosporine. The concentrations of acetyl- choline needed to inhibit the evoked current were identi- cal to those needed to raise intracellular calcium but tenfold less than those needed for the agonist to activate IK(Ca). In a normal calcium-containing superfusate, recov- ery from inhibition required around 8 rain (half-time 4 min) after removal of acetylcholine. When the exper- iment was performed in calcium-free medium no recovery was seen after 8 min washing in drug-free solution, but complete recovery was seen within 3 min (half-time 1.5 min) after adding calcium. Responses to repeated pressure applications of acetylcholine could be reversibly inhibited by acetylcholine and bradykinin. It seems, then, that there is no direct action of acetylcholine or bradykinin on the IK(Ca) channels themselves but that concentrations below those needed to activate IK(c.) can empty and inhibit the IP3-sensitive calcium store. This may provide a mechanism for heterologous desensitiza- tion for phospholipase-C-linked receptor-mediated re- sponses.

Key words: Acetylcholine - IK~Ca) -- IP3 -- Calcium stores - Muscarinic receptors - NG108-15 cells - Bradykinin - Desensitization

Introduction

In NG108-15 neuroblastoma x glioma hybrid cells, ago- nists that stimulate receptors coupled to phospholipase

C are capable of generating a Ca 2 +-activated K § current (/K(ca)). This response can be induced by bradykinin [3, 11, 23] and, in cells transformed to express ml or m3 muscarinic receptors, by acetylcholine [9, 16]. This effect probably results from the generation of inositol 1,4,5- trisphosphate (IP3) and the subsequent release of Ca / § from intracellular stores, and can be replicated by the intracellular ionophoretic injection of IP3 or Ca 2 § ions [4, 11,261.

In the present experiments I have studied the interac- tion between the external application of agonists for phospholipase-C-linked receptors and intracellular appli- cation of IP 3. I find that agonist concentrations sub- threshold to those that activate IK(C,) strongly inhibit the response to intracellular ionophoresis of IPa and present evidence suggests that this results from a depletion of the IPa-releasable Ca 2 § stores. This provides a mechanism for heterologous desensitization of IP3-mediated re- sponses mediated by phospholipase-C-linked receptors.

Materials and methods

The tissue culture, receptor transfection and electrophysiological recording methods have been described in detail elsewhere [25, 26]. Briefly, cells were superfused at 35 ~ C in a gassed (95% Oz/5% CO2) solution containing (mM) NaC1 120, KC1 3, HEPES 5, NaHCO3 22.6, glucose 11.1, MgC12 1.2, CaC12 2.5 and tetrodotoxin 0.5 gM, pH 7.36. For a calcium-free solution CaClz was omitted, MgC12 increased to 5 mM and EGTA (0.1 raM) added, pH 7.36. The whole- cell variant of the patch-clamp technique was used in discontinuous mode (Axoclamp 2, Axon Instruments, Calif., USA). Electrodes (series resistances 4 - 8 Mr2) were filled with a solution containing (mM) potassium acetate 90, KC120, HEPES 40, MgC12 0.15, EGTA 3 and CaCI2 1, ([Ca] = 40 nM), pH adjusted to 7.4 with KOH.

A second sharp microelectrode (25-40 MO, when filled with 1 M tripotassium citrate) was inserted into the cell and used to ionophorese 1,4,5-inositol trisphosphate (IP3) tetralithium salt (Calbiochem, Nottingham UK; 200 gM, --5 to -40 nA, 100- 500 ms) or CaC12 (BDH, Dorset, UK; 200 mM, + 10 to + 60 nA, 100-500ms). Cells were clamped at between -30mV and -40 mV and IK(c~) was activated by (a) intracellular ionophoresis of IPa, (b) intracellular ionophoresis of Ca 2 +, (c) pressure applications (Neurophore, Medical Systems Corp., New York, USA) of acetyl-

ACh

C IP 3

l

0.5hA

lnA

BK

Ca2+

10s

365

Fig. 1 A - D. Activation of IK(C,) by agonists and intra- cellular ionophoresis of inositol trisphosphate (IP3) and calcium ions in ml-transfected cells. Pressure ap- plications of (A) acetylcholine (ACh, 1 mM, 69 kPa, 300 ms) and (B) bradykinin (BK, I gM, 69 kPa, 300 ms). Intracellular ionophoresis of (C) IP3 (200 gM, - 2 0 hA, 300 ms) and (D) Ca/+ (200 mM, 20 nA, 300 ms). The cell in A was voltage-clamped at - 4 0 mV and in B - D at - 3 0 mV. Upper traces, recorded current; lower traces, pressure/ionophoretic current signal

(i) Control ACh (100/zM)

IP3 ~ ) i ~ . : f ~ ~ .

(ii) ACh (I00/zM)

Wash

__~L I1.0~,

10 s

Fig. 2A, B. Effect of ACh on IK(ca) evoked by [P3 and Ca 2 + intracellular ionophoresis in ml-transfected NG108-15 cells. A IK<c~) evoked by intracellular ionophoresis of IPa (200 ~tM, - 4 0 hA, 500 ms) at A. Bath appli- cation of ACh (100 gM) produces (after a de- lay) an outward current in its own right and inhibits the evoked IK(ca)- This effect is revers- ible on returning to a drng-free solution (Wash). B IK(ca) evoked by intracellular ionophoresis of calcium ions (200 mM Ca 2 +, + 20 hA, 300 ms) at A. Although ACh pro- duces a large outward current it has no effect on the amplitude of the evoked IK(ca). Both cells were voltage-clamped at - 30 mV

choline (ACh; 1 mM, 34-69 kPa, 100-300 ms) or bradykinin (1 gM, 34-69 kPa, 100-300 ms) and (d) bath application of ACh (Sigma, Dorset, UK; 0.1 -- 100 gM), or bradykinin (Sigma; 10 gM). Other compounds used were caffeine (Sigma; 10mM) and noradrenaline (Sigma; 10 gM). The ionophoretic and pressure ap- plication commands were given once per minute.

Intracellular calcium measurements were performed as detailed previously [27] using cells voltage-clamped at - 4 0 mV and filled by microetectrode dialysis of Indo-I tetrapotassium salt (0.1 mM; Calbiochem). Calcium concentrations were calculated from a dual- wavelength emission ratio on line, using PC-based software (CALGRAB-2, Dr. S.J. Marsh, Department of Pharmacology, University College London).

Results

Outward current could be induced by pressure appli- cat ion o f ACh and bradykinin , or by intracellular ionophoresis o f IP3 and Ca 2 + ions. Examples o f current responses to all o f these stimuli are shown in Fig. 1.

Initial experiments were under taken on m l - D N A - transfected cells, using a s tandard bath-appl ied concen- t rat ion o f 100 g M A C h to stimulate receptors maximal ly (see [25]). As reported previously [9, 16], this generates an ou tward current, due to the fo rmat ion o f IP3, release o f intracellular calcium and act ivat ion o f Ca 2 +-depen- dent K + channels. Figure 2 shows that, when ba th appli- cations o f A C h were interposed between repeated ionophore t ic injections o f IP3, the response to IP 3 was strongly depressed (Fig. 2A). In a sample o f ten cells, the mean depression was 99.7 _ 0.3%.

2+ Effect on Ca -activated IK(Ca)

This inhibit ion o f the IP3-act ivated IK(Ca ) might have re- sulted f rom an impaired response o f the IKtCa) channels to the release o f Ca 2 +. To test this, effects o f ACh on equivalent currents induced by intracellular injections o f C a : + ions were used. As illustrated in Fig. 2 B, there was

366

control ACh (100,uM)

( i i ) aCh (IO0/~M) BK (IO#M)

m 2 2 ~ . . . . k . . ~

Off) ACh (100#M)

m 3 ~ 4j ~ ~

1.0 nA

[0.5 aA

o.5 aA

wash

L_

Fig. 3 A - C . Effect of ACh on the IP3- evoked IK(ca) in cells transfected with differ- ent muscarinic receptors. A ml-transfected cell shows the typical response seen in Fig. 2; IP3 injections (200 gM, -30 hA, 100 ms) at � 9 B m2-transfected cell: ACh (100 gM) produces no outward current nor inhibition of the evoked IK(ca). Subsequent application ofbradykinin (10 gM), induces an outward current and inhibits the re- sponse to IPa; IP3 injections (200 gM, -20 nA, 300 ms) at � 9 C In an m3-trans- fected cell ACh activates I~:(ca) and inhibits the evoked current as with the ml-trans- fected cells; IP3 injections (200 gM, -20 hA, 100 ms) at �9 ; the agonist-evoked outward current is attenuated as the re- sponse reaches the maximum deflection of the pen recorder. All cells voltage-clamped at - 30 mV

no significant impairment of the effect of Ca 2 § ions. In five cells the mean depression produced by 100 gM ACh was - 1.4 _+ 3.3%. Likewise, bradykinin, which also in- hibited responses to IP3 (see below) failed to inhibit re- sponses to Ca 2+ ions (3.2 _+ 12.4%; n = 5). Hence the reduced response to IP3 was not due to a failure of re- leased Ca 2 § to activate IK(c,).

Receptor specificity

To test whether inhibition of the response to IP3 was specific to receptors linked to phospholipase C, the inter- action of ACh and IP3 was examined in subclones trans- fected with DNA for other muscarinic receptors (Fig. 3). In these cells, ml and m3 receptors couple to phospho- lipase C whereas m2 and m4 receptors do not (see [9]). Application of ACh to cells transfected with m3 receptors also induced an outward current and subsequently in- hibited the response to IP3, by 98.8 + 1.2% (Fig. 4). In contrast, no outward current, and no significant inhi- bition of the response to IP3, could be detected in cells transfected with DNA for m2 receptors (Figs. 3 B and 4). Since all the cells possess an endogenous m4 receptor, this latter observation implies that activation of m4 receptors also had no effect.

Bradykinin receptors are also coupled to phospho- lipase C in NG108-15 cells [11, 35]. Bath application of 10 gM bradykinin also induced an outward current and inhibited the response to IP3 (Figs. 3 B and 4). In contrast, noradrenaline (which activates e2 receptors in NG108-15 cells: [6]) had no effect (Fig. 4).

Thus, the inhibitory effect appears specific to those receptors that couple to phospholipase C. In these cells, activation of phospholipase C and consequent pro- duction of inositol phosphates appear to be mediated by a pertussis-toxin-insensitive G protein [9, 10, 18]. In

~C

L cl o a3

1.3 D_ 'S

110 ( 1 0 ) (5) (5) ( 8 ) ( 7 )

90

70

50

30

10

- - l O

ml(m4) m2(m4) m3(m4) BK NAd

Fig, 4. Inhibition of IPa-evoked outward current by stimulation of different receptors. Inhibition as a percentage (+ SEM) of predrug response amplitude. Note that all NG108-15 cells express endoge- nous m4 muscarinic receptors: ml (m4), ml-transfected muscarinic receptors; m2(m4), m2-transfected muscarinic receptors; m3(m4), m3-transfected muscarinic receptors. BK, action of bradykinin on endogenous bradykinin receptors. NAd, action of noradrenaline on e2-adrenergic receptors. The effects were measured in response to ACh (100 gM), bradykinin (10 gM) or noradrenaline (10 gM). Numbers in brackets indicate number of cells tested

agreement with this, inhibition of IP3-activated Ilqc,) by ACh was not prevented by pretreatment for 24 h with 500 ng/ml pertussis toxin.

Stimulation ofphospholipase C produces two putative second messengers - IP3 and diacylglycerol [1]. The latter activates protein kinase C (PKC; [17]). Activation of PKC has been implicated in the desensitization of bradykinin-induced IP3 production [8] and Ca 2 § release [14]. The role of PKC in the inhibitory effect of ACh was tested by the application of staurosporine, an inhibitor of PKC [29]. Staurosporine (2 ~M) did not affect the inhibitory action of 100 txM ACh (n = 4). Further, it has been previously shown that phorbol dibutyrate (a PKC activator) actually increased rather than reduced the cur-

i control A C h (10/zM)

: l !

wash

:i

!I : ' /

~ ~--_..__-

A C h (1/~M) B ii +

LI !1

'i

10s { 1.0 nA

Fig. 5 A, B. Concentration dependence of ACh on the inhibition of the IP3-evoked IK(ca) and activation of the outward current in ml- transfected cells. The effect of 10 ~tM ACh (A) and 1 IxM ACh (B) on IP3-evoked 1K(ca). Note the activation of outward current and complete inhibition of the evoked response at 10 ktM but the lack of an outward current with a large inhibition of the evoked current at 1 gM ACh. IP3 injections (200 IxM, -20 nA, 500 ms) at A, Holding potential = - 30 mV

rent [4] and I have confirmed a similar effect of phorbol dibutyrate (1 gM) under the present recording conditions (data not shown).

Quantitative relation between agonist-induced activation of I~(ca) and inhibition of lP3-activated I~(c~)

Figure 5 shows examples of the relation between the concentrations of ACh required to activate I~:(c,) and those that inhibited the subsequent response to ionopho- retically injected IP3. I t is clear that ACh inhibited the response to IP3 at substantially lower concentrations than those required to produce an outward current. The mean ICso against the response to IP3 was 0.8 gM (confidence limits 0 . 7 - 1 . 1 gM), whereas the EC5o for the current activation was 10.5 gM (confidence limits 9.8 - 11.2 gM), see below.

100 nM ACh

367

500-

400 -

300

200

i00 -

0

1 #M ACh

300 nM ACh

0/~M ACh _ _

Fig. 6. Intracellular calcium changes in response to bath applied ACh at the concentrations shown. The cell was voltage-clamped at -40 mV and the internal recording solution was as stated in Materials and methods but EGTA was replaced by 0.1 mM BAPTA plus 0.1 mM Indo-1 and CaC12 was omitted

180.00.60. ~ "

0 " ' " " �9 I I 0.01 0 .10 1.00 10.00 100.00 1000 .00 acet:ytcho[ine concentration (p,M)

Fig. 7. Concentration/response curves of the inhibition of the TP3- evoked response ( �9 activation of the outward current ( , ) and change in intracellular concentration (- �9 I~ - .). y-axis is percentage inhibition (+ SEM) for the IPa-evoked current, normalised percent- age (_+ SEM) amplitude for the agonist-activated current and nor- malised calcium rise (_+ SEM) to the response to 10 gM ACh. Four to eight cells for each point. All curves are least-squares fits to the data using the equation y = lOOx/(x + K), where y = percentage inhibition/amplitude, x = agonist concentration (IxM) and K is a constant. Mean values for K used in these curves were: ( � 9 0.8 rtM, (A) 10.5 gM and (~) 0,9 ~tM

Role of calcium ions

Calcium fluorescence measurements of the elevation of [ Ca2 +]i by ACh (Fig. 6) corresponded closely to the con- centration/response curve for the inhibitory effect of ACh on the IPa-induced IKtca) (Fig. 7). The ECso for the nor- malised calcium rise was identical to that of the inhibition of the IP3-induced Ilqc,) at 0.9 gM (confidence limits 0 . 7 -1 .1 gM).

This suggests that ACh might act by releasing C a 2 +

f rom IPa-sensitive C a 2 + stores and thereby depleting the stores. To test this, I a t tempted to deplete the Ca 2 + stores with caffeine: this inhibits IP3-induced Ca 2+ release in some cells [13, 20, 34] but not in others [5, 22, 28]. How- ever, exposure to 10 m M caffeine did not produce any outward current, nor did it produce any significant inhi-

bition ( - 1 5 . 5 _+ 15.4%; n = 4) of the amplitude of IK(c,) produced by IP3.

I f inhibition of the effect of IP3 resulted f rom depletion of calcium stores, then recovery f rom inhibition should be dependent on refilling the stores. In NG108-15 cells, refilling of Ca 2+ stores after release by IPa seems to require an influx of extracellular Ca 2 + [4, 24]. Hence, the effect of extracellular Ca 2 + on the time course of recovery was determined (Fig. 8). In normal external solution (containing 2.5 m M calcium) recovery f rom inhibition occurred with a half-time of about 4 min, and was com- plete within 8 min (Fig. 8 A). In contrast, in the absence of extracellular Ca 2 +, no recovery at all could be detected after 8 min: however, on restoring external Ca 2+, the response to IP3 recovered completely within 3 min, with a half-time of around 1.5 min (Fig. 8B).

3 6 8

A mo-

B 80- O-

E e 6 0 -

4 0 - t_

o

2 0 -

r j

EL 0 o

- 2 0

,_T l

T f 1

/ i .

i

[ [ I I I I I I I I

0 1 2 3 4 5 6 7 8 9 time (rains)

1 0

B

100

.~_ 8 0 .

E u 60-

~- 4 0 -

2 0 .

E o o

c -20

�9 IT.I r O . / ~ ,

I I I I I I I I I 0 1 2 ; 4 5 6 7 8 9 ll01t11121131411511617

time (rains)

Fig. 8 A, B. Time course of recovery from inhibition by ACh of the IP3-evoked Ire(ca) in ml-transfected cells. A Normalised current amplitude (+ SEM, n = 6 cells); application of ACh (100 gM) indi- cated by solid bar. The superfusate contained CaClz (2.5 raM) at all times. B as A but the initial part of the experiment was performed in a calcium-free solution (0 mM CaC12, 5 mM MgC12 and 100 gM EGTA). ACh (100 IxM) was applied for the period indicated by the solid bar. The cells were washed in a calcium-free solution, then returned to a calcium-containing medium for the period indicated by the open bar (n = 7 cells)

Effect o f acetylcholine and bradykinin on agonist-induced IK(c.)

Since the outward current induced by ACh or bradykinin can be ascribed to the generation of IP3 and the conse- quent release of Ca / § (see Introduction), subthreshold concentrations of acetylcholine might be expected to in- hibit the subsequent Ca2+-dependent K § current re- sponses to larger doses of ACh in the same manner that it inhibits responses to IP3 injections. By repetitive pressure applications of ACh I could demonstrate this phenom- enon. Figure 9A shows the total inhibition of the agonist(ACh)-induced response by a concentration of ACh that did not evoke an outward current in its own right (0.3 gM). This was demonstrated in a total of four cells, where subthreshold doses of ACh (0 .3-0 .6 gM) produced no outward current but inhibited the ACh- evoked IK{c,) by 88.0 + 9.0%. At a maximal concen- tration of ACh (100 gM, Fig. 9B) the mean inhibition was 100.0 + 0.0% in six cells and the rate of recovery from the inhibition was of the same order as that seen with the IP3-evoked current (6.0 + 0.4 min). Bradykinin (10 gM) also inhibited the response to pressure-applied

ACh, by 98.6 + 1.4% (n = 7); the inhibition recovered in 4.7 _+ 0.6 min (Fig. 9C).

Discussion

The principal observations in the present experiments are that (a) in NG108-15 cells transfected with ml mAChR DNA, ACh inhibits the activation of IK(c,) by IP3, (b) that this inhibitory effect is restricted to receptors that stimulate phospholipase C, and (c) that it occurs at con- centrations of agonist substantially below those that themselves activate Irqc,). Since there was no comparable inhibition of the response to intracellularly injected Ca 2 § ions, it is not due to an inhibition of the K + channels to the Ca z§ released by IP3, but must instead be due to inhibition of the ability of IP3 to release Ca 2 +

Since inhibition was produced by agonists that stimu- late phospholipase C, two mediators seem possible- diacylglycerol or IPa itself. Diacylglycerol and the conse- quent activation of PKC have been implicated in the desensitizing action of acetylcholine in lacrimal acinar cells [32]. However, this appears not to be responsible for the inhibition of the response to IP3, since inhibition was not prevented by the PKC inhibitor staurosporine and PKC activation by phorboldibutyrate actually increased the current [4]. Instead I suggest that the inhibition is due to the formation of IP3 and subsequent desensitization and/or depletion of IP3-sensitive Ca / + stores.

There are a number of possible mechanisms for this effect. (a) It is known that calcium can inhibit calcium release by IP3 [2, 7, 19, 21] but I do not think this effect is likely to explain all the data, as the time course of recovery of the measured intracellular calcium rise is in the order of seconds (Fig. 6) whereas the inhibitory effect takes minutes to recover (Fig. 8). (b) Another mechanism for inhibiting calcium release is phosphorylation of the IP3 receptor by kinases, particularly protein kinase A [31]. However the high concentrations of staurosporine that I have used would preclude this as both PKC and PKA activity would be inhibited [29]. (c) A further possi- ble mechanism recently described [15] (but see [30]) is that the IP3-sensitive calcium store reduces its sensitivity to IP3 as the store loses its calcium. I suggest that the IP3 injections only release a small part of the store. However the more prolonged application of agonist might release much more calcium, even at low concentrations, and therefore the store becomes less sensitive to IP3. Over time in a calcium-containing medium the store refills and its sensitivity to IP3 returns. In a calcium-free solution the store cannot refill, therefore its sensitivity to IP3 does not return. The principal evidence in favour of this is that (a) the concentrations of ACh that inhibited the response to IP3 are in accord with those that elevated intracellular [Ca2+], and (b) recovery from inhibition required the presence of extracellular Ca 2+, presumably in order to replete the Ca 2 + stores. Indeed there is now direct evi- dence, from mouse pancreatic acinar cells, that the Ca 2 + released by muscarinic stimulation is all extruded from the cytosol to the outside of the cell [33].

Control

A + ACh (0.3/~M)

B ACh (100/~M)

c BK (10/xM)

Wash

10 s 0.5 nA

j \

369

Fig. 9 A - C . Homologous and heterologous desensitization of agonist- evoked response in ml-transfected NG108-15 cells. A Pressure-applied ACh (1 mM, 69 kPa, 300 ms, one every 60 s) at �9 , with the addition of bath-applied 0.3 IxM ACh (subthreshold concentration for activation of the outward current, see Fig. 7). ACh responses (1 mM, 39 kPa, 300 ms, every 60 s) at �9 and the bath appli- cation of maximal concentrations of ACh (B) and bradykinin (C). Cell in A was volt- age-clamped at - 3 0 mV and - 4 0 mV in B and C

This would imply that, in such a system, a subthres- hold concentration of agonist for any phospholipase- C-linked receptor may effectively inactivate the effector response to a subsequent application of any agonist with an effect mediated through the formation of IP3 and release of Ca z +. This certainly appears to be the case in the NG108-15 cells, since subthreshold concentrations of ACh inhibit the outward current induced by ACh and both ACh and bradykinin can inhibit the response to ACh (Fig. 9). It is also worth noting that the inclusion of IP3 (100 gM) in the whole-cell recording electrode in- hibits both the C a 2 + rise and the outward current acti- vated by ACh in ml-transfected NG108-15 (J. Robbins, S. J. Marsh and D. A. Brown, unpublished observations). This then provides an alternative mechanism for hetero- logous desensitization to those previously described (e. g., [12]), which act at the level of the receptor-induced stimu- lation of phospholipase C.

Acknowledgements. Supported by the U K Medical Research Coun- cil. I thank Y. Vallis for expert tissue-culture assistance, Professors D. A. Brown and B. Alger and Dr. J. A. Sim for helpful discussions and Dr. S. J. Marsh for help with the fluorescence measurements.

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