current was induced by adrenaline, qualitatively the same finding ...

Journal of Physiology (1992), 457, pp. 211-228 211With 10 figures

Printed in Great Britain

MODULATION OF /i-ADRENERGIC RESPONSES OF CHLORIDE ANDCALCIUM CURRENTS BY EXTERNAL CATIONS IN GUINEA-PIG

VENTRICULAR CELLS

BY FAIZ M. TAREEN*, ATSUYA YOSHIDA AND KYOICHI ONOtFrom the Department of Physiology, Faculty of Medicine, Kyushu University,

Higashi-Ku, 812 Fukuoka, Japan

(Received 24 October 1991)

SUMMARY

1. The catecholamine-induced Cl- current and the Ca2+ current were recorded inthe single ventricular cells of guinea-pig hearts, using the whole-cell patch clamptechnique combined with internal perfusion. Dependence of the fl-adrenergicresponses on external monovalent cations was investigated. The Cl- current wasrecognized by measuring the reversal potential of the agonist-induced current.

2. The amplitude of the Cl- current, activated by 1 /IM adrenaline or 0 01-0{1 fMisoprenaline, was decreased when the external Na' concentration ([Na+]O) wasreduced by replacement with Tris+. The conductance of the catecholamine-inducedCl- current was proportional to the logarithm ofthe [Na+]o over a range of 15-140 mM.When the conductance was plotted against the concentration of Tris+, a dose-dependent inhibition of the Cl- response by Tris+ was suggested with a half-maximum concentration of 95 mM.

3. The inhibitory effect of the Na+ substitute TEA' on the Cl- current was notaffected by either increasing the buffer for the internal Ca21 (10 mm BAPTA) or forthe pH (50 mm HEPES).

4. In the relationship between agonist concentration and the Cl- conductance, thehalf-maximum concentration (Ki) of isoprenaline was 0-013 JtM in the control Na+solution, and was shifted to 0 07, 0-08, 0.1 and 0 3 ,UM in the Li+, Cs+, TEA+ and Tris+external solutions, respectively. The maximum slope conductance was notsignificantly affected, except for a slight depression on the Tris+ solution. When thecurrent was induced by adrenaline, qualitatively the same finding was obtained; K,was 0-15 and 3-2 /tM in the Na+ and Tris+ solutions, respectively.

5. As a substitute for the external Na+, sucrose seemed to be inert. The activationof the inward Cl- current was conserved in the 300 mm sucrose solution ([Cl-]0 =8 mM) with a Kay value of 0-015 gM isoprenaline.

6. The Cl- current, when activated by either an external application of forskolin(0-2-10 pM) or an internal perfusion of cyclic AMP (100-500 /M), was not affected by

* Present address: Department of Anaesthesiology and Intensive Care Medicine, Faculty ofMedicine, Kyushu University, Fukuoka, Japan.

t To whom reprint requests should be addressed.MS 9838

F. M. TAREEN, A. YOSHIDA AND K. ONO

replacing external Na+ with other cations. Activation of the Cl- current by 0-2-5 /LMhistamine was also insensitive to a substitution of Na'. These findings indicate thatthe inhibition by the Na' substitute is at a point before the activation of GTP-binding protein.

7. The effects of Na' substitution were not affected by varying the Na'concentration (0-115 mM) in the internal solution, excluding an involvement of achange in the [Na+]i.

8. The enhancement of the Ca2+ current by isoprenaline was also modified by Na+substitutes. The use of Na+ substitutes shifted the dose-response relation forisoprenaline to higher doses.

9. These findings strongly suggest that monovalent cations interact with ,-adrenergic receptors on the external surface of the cell membrane to inhibit the /5-adrenergic response. This interaction is characterized by a rightward shift of thedose-response relations for ,-agonists. The Cl- channel itself is not dependent on theexternal cations.

INTRODUCTION

It is now well established that stimulation of the fl-adrenergic receptor activatesa time-independent Cl- current in cardiac ventricular cells. The response is mediatedby a cyclic AMP-dependent cascade, which leads from the agonist-receptor bindingto the final phosphorylation of the channel or related structure (Bahinski, Nairn,Greengard & Gadsby, 1989; Harvey & Hume, 1989; Harvey, Clark & Hume, 1990;Matsuoka, Ehara & Noma, 1990; Tareen, Ono, Noma & Ehara, 1991). This currentwas originally discovered by Egan, Noble, Noble, Powell, Twist & Yamaoka (1988),and these same authors then demonstrated that the response to catecholamines wasinhibited by removing Na+ from the bathing solution. Similar findings were alsoreported in subsequent studies (see also Harvey & Hume, 1990b). Furthermore,Matsuoka, Noma & Powell (1989) showed that the stimulation of the Ca2+ current byadrenaline was also partially inhibited by replacing Na+ with Li+. These findingssuggest that monovalent cations, in general, may modulate the /5-adrenergicresponse.

Recently a study on the cation dependence of the /3-adrenergic response of ionicchannels was published by Harvey, Jurevicius & Hume (1991). We also in-dependently conducted a systematic study, and found that the cation dependence ischaracterized by a rightward shift of the dose-response relations for ,8-agonists witha sequence of efficacy, Tris+ > TEA+ > Li', Cs' > Na+. The Cl- current, whenactivated by forskolin, cyclic AMP and histamine, is not affected by the substitutionof Na+. This finding is different from the observations reported by Harvey et al.(1991). We thus conclude that the Na+ substitutes inhibit the /-adrenergicstimulation by interacting with the ,-adrenergic receptor, most probably from theexternal side. The fact that the activation of the inward Cl- current is conserved inNa+-free sucrose solution indicates that the depletion of Na+ is not the primarycause, but that the Na+ substitutes, except for sucrose, interfere with the response.

212

CATION-DEPENDENT ACTIVATION OF A Cl- CURRENT

METHODS

Single cell preparationSingle ventricular cells were obtained from guinea-pig hearts using the enzymatic dissociation

technique as previously described (Powell, Terrar & Twist, 1980; Isenberg & Kldckner, 1982).Briefly, guinea-pigs (300-400 g) were anaesthetized with sodium pentobarbitone (50-70 mg/kg).The chest was opened and the aorta cannulated in situ to start the perfusion with normal Tyrodesolution. The heart was excised and mounted on a Langendorff-type perfusion apparatus. Theheart was then perfused with a Ca2+-free Tyrode solution for 10 min and finally with the samesolution containing 0 040% collagenase (Yakult, Tokyo, Japan or Sigma, type I) for 10-30 min.After the enzyme treatment, cells were dissociated in a high-K+, low-Cl- solution and stored in aculture medium (minimum essential Eagle's medium, Flow laboratories, Irvine, Scotland) at 30 0Cfor later use. All experiments were carried out at 35+ 0 5 'C.

SolutionsThe control Tyrode solution contained (mM): NaCl, 140; KCl, 54; CaCl2, 1-8; MgCl2, 05;

NaH2PO4, 033; glucose, 5 5; and the pH was adjusted to 7 4 with 5 0 mm HEPES-NaOH. Isolationof the Cl- current was facilitated by suppressing the membrane K+ conductance, Ca2+ current andthe Na+-K+ pump current. Also, inhibition of the Na+-Ca2+ exchange was crucial to avoid Ca 2

influx through the exchanger when the cell was superfused with a Na+-free external solution.Therefore, the standard external solution used to record the Cl- current was a Ca2+-free, K+-freesolution, which contained 140 mM NaCl, 2 mm MgCl2, 2 mm BaCl2, 1 /M nicardipine and 20 /LMouabain. The pH was adjusted to 7-4 with CsOH. The composition of the external solution forisolating Ca2` current was (mM): NaCl, 140; MgCl2, 0 5; CaCl2, 1-8; CsCl, 5-4; adjusted to pH 7-4with CsOH.

External solutions containing various monovalent cations were made by replacing NaCl withequimolar concentrations of tris-(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), tetra-ethylammonium chloride (TEA-Cl), LiCl, or CsCl. To examine the effects of removing cations,300 mm sucrose was substituted for NaCl in several experiments (Figs 6 and 7). In this particularexperiment, [Cl-]. was kept constant (8 mM) by using a solution containing 140 mm sodiumglutamate as a control. The pH of all external solutions was adjusted to 7-4 with 5 mm HEPES-CsOH.The composition of the standard internal solution for recording the Cl- current was (mM):

CsOH, 100; CsCl, 15; aspartic acid, 90 MgCl2. 5; TEA-Cl, 20; Tris-ATP, 5; and ethyleneglycol-bis-(/3-aminoethyl ether)-NNV',N'-tetraacetic acid (EGTA), 5; or 1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), 10. To ensure stabilization of [Ca2+]., BAPTA solution wasused in all experiments recording the Ca2+ current. The experimental results on the Cl- currentobtained by using BAPTA were not different from those using EGTA. The pH was adjusted to 7-4with 5 mM-HEPES-CsOH. When an involvement of internal Na+ was examined, Cs+ was partiallyor totally replaced with equimolar amounts of Na+ ([Na+]i = 15 or 115 mm, Fig. 9). The 50 mMHEPES internal solution contained (mm): CsOH, 55; CsCl, 15; aspartic acid, 45; MgCl2, 5; TEA-Cl, 20; Tris-ATP, 5; BAPTA, 10; and the pH was adjusted to 7-4 with CsOH.The liquid junction potential between the control Tyrode solution and each of other external

solutions was measured, assuming no junction potentials at the tip of the 3 mm KCl electrode. Thepotential of the control Tyrode solution was about -3 mV in junction to 140 mm Tris-HCl or TEA-Cl, and 0 mV in Na+, Li+ or Cs+ solution. I-V curves shown in the present study were not correctedfor the junction potential.

DrugsIsoprenaline, histamine and cyclic AMP were obtained from Sigma Chemical Co. (St Louis,

USA), adrenaline from Diaiichi Pharmaceutical Co. (Tokyo, Japan) and forskolin from CalbiochemCorporation (La Jolla, CA, USA).

Voltage clamp and recording techniqueA drop of cell suspension was added to the Tyrode solution in the recording chamber placed on

an inverted microscope. After the cells had settled on the glass bottom, the chamber wascontinuously perfused with the Tyrode solution. The dimension of the chamber was about 3 mm

213


in width and 20 mm in length. The depth of the saline was less than 2 mm and the rate of flow was3-4 ml/min. With this chamber shape, turbulence of the perfusate was avoided and the exchangewas usually completed within 10 s after an initial sign- of effect of the new solution.

Single ventricular cells were voltage clamped using the whole-cell configuration of the patchclamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The tip diameter of the glasssuction pipette was about 3 ,um and resistance was between 1 and 3 MCI when filled with the Tyrodesolution. To avoid a liquid junction potential between the pipette solution and the control Tyrodesolution, and also to facilitate formation of a gigaohm seal, the pipette was first filled with Ca2+-containing Tyrode solution. After establishing a gigaohm seal, the pipette solution was replacedwith internal solution by using an intrapipette perfusion device (Soejima & Noma, 1984). A briefstrong negative pressure was applied to the pipette interior to rupture the patch membrane. Afterequilibration of intracellular medium with pipette solution, the external solution was changed toan appropriate test solution. The reference electrode was usually Tyrode-agar with an integralAg-AgCl wire. In the experiments in which the external Cl- concentration was lowered (Figs 6 and7), a leaky 3 M KCl electrode was used instead of the Tyrode-agar to minimize changes in the liquidjunction potential at the tip of the reference electrode (Matsuoka et al. 1990).For recording the time-independent catecholamine-induced Cl- current, a ramp pulse (dV/dt

= + 0X46 V/s) was employed with a holding potential of -40 mV. The I-V relations were measuredduring the hyperpolarizing portion of the ramp pulse. The Ca2+ current was elicited by applyingsquare pulses of 300 ms duration to 0 mV after a short conditioning depolarization to -40 mVfrom a holding potential of -80 mV. The Na+ current was inactivated during 40 ms of the prepulseto -40 mV.

Current and voltage signals were stored on video tape through a PCM converter (SONY, PCM-501ES, Tokyo, Japan, modified for DC signal) for later computer analysis (NEC, PC98, XA, Tokyo,Japan). Na+ current spikes during the depolarizing portion of the ramp pulses were removed bycutting the high frequency signals at 100 Hz (48 dB/octave, NF, FV-625A, Tokyo, Japan) from allchart recordings shown here, with the exception of those in Fig. 9. Numerical data are given asmeans + S.D.

RESULTS

Inhibition of the catecholamine-induced Cl- current by reducing [Na+]oThe amplitude of the catecholamine-induced Cl- current was a continuous

function of the external Na+ concentration ([Na+]O) or of the concentration of thesubstitute. This is demonstrated in the experiment shown in Fig. 1. The holdingpotential was -40 mV and ramp pulses ranging from + 60 to - 130 mV were appliedevery 6 s. The application of 1 #UM adrenaline induced an inward shift of the holdingcurrent accompanied by a marked increase in the membrane conductance (Fig. IA).After the response reached a steady level, the [Na+]o was decreased from 140 to10 mM by replacement with Tris+, resulting in a depression of the adrenalineresponse. Subsequent rise in the [Na+]. to 50 and then to 140 mm increased themagnitude of the response up to the initial control level.The I-V relations in Fig. IB were determined by calculating an average of five

consecutive ramp pulses under each condition as indicated by corresponding symbolsin A. The adrenaline-induced Cl- currents were dissected by subtracting the controlcurrent (0) from those in the presence of adrenaline (Fig. 1 C). It is evident thatreducing the [Na+]o decreased the Cl- conductance without changing the reversalpotential of about -25 mV. Essentially the same findings were obtained when TEA'was used for substituting Na+ (n = 10).The depression of the response is not secondary to a possible change in intracellular

concentration of Ca2+ ([Ca2+]i) or H+. This is because we omitted Ca2+ from theexternal solution, and the use of 10 mm BAPTA instead of 5 mm EGTA in theinternal solution did not modulate the effect of the Na+ substitution on the Cl-

214


current (n = 17). The intracellular pH was buffered by the use of 50 mm HEPESwithout modulating the response; the Cl- conductance activated by 50 nMisoprenaline was decreased by 73-5 + 15-5 % when external-Na+ was replaced withTEA+ (n = 7), which is comparable to 78-8 + 16 % in the control run (5 mm HEPES)obtained in the same group of cells.

AAdrenaline (1 aM)

Tris+ (mM) 0 130 90 0Na+ (mM) 140, 10, 50 140

]0'5 nA

30 s0 A

BB ~~~~~~~~C

A-05>* -05n~~~~~~~ASO

-100 ;-100 -

l 50mV =| 50mV

Fig. 1. Effects of varying [Na+]o on adrenaline-induced Cl- current. A, current trace onthe chart recorder. The time of superfusing 1 /LM adrenaline, and various concentrations(mm) of external Na+ and Tris+ are indicated above the record. Frictional movements ofthe pen caused the jagged trace of the holding current in this chart recording. The currentdeflection due to the Na+ current was removed by cutting the high frequency signals at100 Hz. B, the I-V curves obtained from the negative limb of the ramp pulse wereaveraged for five consecutive measurements, indicated by the corresponding symbols inthe chart recording; 0, control; *, response to adrenaline in the presence of 140 mm Na+(0 mm Tris+); *, in 10 mM Na+ (130 mM Tris+); and A, in 50 mm Na+ (90 mm Tris+). C,the I-V curves of the adrenaline-induced current obtained by subtracting the average ofthe control I-V curve from the I-V curves obtained in the presence of 1 /M adrenaline.

Slope conductance was measured by calculating the regression line near thereversal potential of the Cl- current and conductances were normalized with respectto the response at 140 mm [Na+]O. The data were plotted against either the [Na+]o(Fig. 2A) or the concentration of substitute, Tris+ (Fig. 2B). The former plot mayrepresent the dose dependence of facilitation of the fl-response by Na+, and the latterthe inhibitory action of the substitute, which was considered to be the actualmechanism (see below). The least-squares fit of the Hill equation (continuous curvein Fig. 2B) gave a half-maximum concentration (Ki) of 95 mm and a Hill coefficientof 2-3.

It should be noted that the decrease of catecholamine response in Tris+ or TEA+solutions is not through binding of these chemicals to the muscarinic receptor sinceessentially the same results were obtained even after blocking the muscarinicreceptor by adding 10 #m atropine in the Tris+ or TEA+ solutions (not shown).

8 PHY 457

215

216 F. M. TAREEN, A. YOSHIDA AND K. ONO

A B100 - 100

80 N 80OR ~ ~ ~ ~ V A

° 60-A o 60C C

Q 40 40C. ~~~~~~~~C

o 20 A 0 20 AU U

0 1111i I1111 0 11 I 1111 I II

10 30 100 300 10 30 100 300Na' (mM) Tris' (mM)

Fig. 2. A, relationship between [Na+]o and the conductance of the adrenaline-induced Cl-current. The concentration of adrenaline was 1 /LM. Slope conductance around thereversal potential was obtained by the least-squares fit, and was normalized to thepercentage of the response in the external solution containing 140 mm NaCl. Differentsymbols indicate different cells. B, relationship between [Tris+]o and the conductance ofthe adrenaline-induced Cl- current. Data in A are replotted against [Tris+]0. Thecontinuous curve was drawn by the least-squares fit of the Hill equation. Conductance =1-1/(1 + (K,/[Tris+])n) with a half-maximum concentration (Ki) of 95 mm and a Hillcoefficient (n) of 2-3.

A __Tris-H CII NaCI ITris-HCI INaCIIsoprenaline (AmM) 01 10

*. ..

Inm 10lullllllfl ]_.nAA

1 min

B-1 nA

A:=~~~~~-

Fig. 3. Activation of the Cl- current by a high concentration of isoprenaline in Tris-HCIexternal solution. A, current trace on the chart recorder. The time superfusion of 041 and10 AM isoprenaline in either 140 mm Tris-HCl or NaCl external solution is indicated abovethe record. B, the I-V curves of the isoprenaline-induced current obtained by subtractingthe average of the four control currents (@, in Tris-HCl; and 0, in NaCl) from theaverages of the currents in the presence of isoprenaline (A, 0 1 AM in NaCl solution; A,0-1IM in Tris-HCl solution, and *, 10 /uM' in Tris-HCl solution), indicated by thecorresponding symbols in the chart recording.

Competition between the fl-stimulation and the Na+ substitutionAlthough the amplitude of the Cl- current induced by moderate concentrations of

isoprenaline was greatly decreased by replacing external Na' with Tris+, increasingthe concentration of isoprenaline restored the response. Figure 3A shows a


A

140 mM NaCI

B

140 mm Tris-HCI

-100

-05 0

nA10

; 0.1~v 0-2-~~~~~~~ -AP ,Qs nc %.F

I 50 mV

D 0-5 nA03

140 mM LiCI 0 3001

i-100 -0

.

1020-1

50 mV

C

140 mM TEA-Cl

-100

E

140 mM CsCI

-100

50 mV

Fig. 4. The I-V relations of the isoprenaline-induced current in solution containingchloride salts of various monovalent cations: 140 mm NaCl (A), Tris-HCl (B), TEA-Cl (C),LiCI (D) and CsCl (E). The drug was applied in a cumulative manner after the steadyresponse to the previous concentration was obtained. I-V relations were obtained bysubtracting the average of the five control I-V curves from the averages of I-V curves

obtained in the presence of isoprenaline. The concentrations of isoprenaline are indicatedto the right of each trace in /LM. A, B, C, D and E were obtained from different cells. I-Vrelation obtained in the presence of 1 /,M isoprenaline in the 140 mm NaCl solution isdenoted by *.

8-2

' 100 1

0-1

0-03

50 mV

1 nA 030*30.1

003:.- 00

50 mV

II -4_--

217

0


representative experiment. The replacement of external Na+ with Tris+ greatlyreduced the magnitude of the response to 0-1 /tM isoprenaline (A). Subsequentapplication of 10 ,UM isoprenaline induced a sizeable increase of the membraneconductance in the continuous presence of Tris'. A slight increase of the membraneconductance was observed on switching back to the Na+-containing externalsolution, indicating that the response to 10 #UM isoprenaline was still depressed in theTris+ solution.The difference I-V relations in Fig. 3B were obtained by subtracting the

background current in comparable ionic conditions. The configuration of the I-Vcurve remained quite similar during the interventions described above. A slightdifference in the reversal potential, -22 mV with Tris+ and -25 mV with Na+, couldbe explained by the differences of the liquid junction potential of these solutions atthe tip of the reference electrode. These findings confirm that the changes in themembrane conductance are all attributable to the catecholamine-induced Cl-current.

In order to characterize the competitive interaction between the Na+ substitutionand the fl-stimulation, the dose-response relationship for isoprenaline was de-termined in different cation solutions, e.g. Na+, Tris+, TEA+, Cs+ and Li+. Theexperimental time was shortened by applying the agonist in a cumulative manner,so that the spontaneous decay of the response was minimized. Figure 4 showsrepresentative I-V curves of the Cl- current. In all solutions, increasing theconcentration of isoprenaline increased the Cl- conductance. However, theconcentration range of isoprenaline required to obtain the response was obviouslyhigher in Tris+ and TEA+ solutions than in Na+, Li+ and Cs+ solutions. The reversalpotential was - 24-8 + 4*3 mV (N = 4) in Na+ solution, - 24-9 + 2-8 mV (N = 4) in Cs+solution and - 23-8 + 3-6 mV (N = 4) in Li+ solution, showing no significantdifference. The reversal potentials obtained in Tris+ and TEA+ solutions were slightlypositive (-22-9±2-8 mV, N= 6 in Tris+ and -22-4+336 mV, N= 5 in TEA+),probably due to the difference of the liquid junction potentials.

Slope conductance was measured around the reversal potential, and wasnormalized with respect to the response to 1 ,UM isoprenaline recorded in the same cellin the Na+ external solution. The results of several experiments are summarized inFig. 5. For each kind of cation five or six cells were tested. In the Na+ externalsolution (@), the relationship between the concentration of isoprenaline and theconductance of the Cl- current was fitted to the saturation kinetics, with a Hillcoefficient of about 2 and a half-maximum concentration (Ki) of 0-013 ,UM. Whenexternal Na+ was replaced with other monovalent cations, the dose-response curvewas shifted to higher doses of isoprenaline; Kl values were 0 07, 0-08, 041 and 0 3 /Min solutions containing 140 mm Li+ (A), Cs+ (O), TEA+ (E) and Tris+ (0),respectively. The value of the Hill coefficient and the maximum response were notmarkedly affected by monovalent cations, except in the Tris+ solution where themaximum response was also suppressed by about 25%.

Qualitatively the same finding was obtained with another, but less effectivefl-adrenergic agonist, adrenaline. In the Na+-containing external solution, the Cl-conductance increased over the concentration range 0 03-1 ItM with a Ki of 0 15 IM.Replacing Na+ with Tris+ increased the value of K1 to 3-2 #M (not illustrated). The

218

CAATION-DEPENDENT ACTIVATION OF A Cl- CURRENT

100T M

C~~~~~~N~

n ,,,,/vz >~~~TA- Tris-HCO0

0*001 0-01 0-1 1 10Isoprenaline (#sm)

Fig. 5. Dose-response relations between the C1- conductance and isoprenaline con-centration. The slope conductance around the reversal potential was measured by theleast-squares fit, and was normalized to a percentage of the response of 1 /LM isoprenalinein 140 mm NaCl solution. For each monovalent cation, five or six cells were tested.Different symbols indicate the mean values of Cl- conductance with different cations; *,140 mm NaCl, A, LiCl; O, CsCl; O, TEA-Cl; and 0, Tris HCl. Vertical bars indicate S.D.The smooth curves were drawn by the least-squares fit of the Hill equation. Conductance= 1/(1 + (K1/[drug])n), where n is the Hill coefficient and Ki the half-maximumconcentration. With NaCl, Kl = 0-013, n = 1 7. With LiCl, Ke = 00X7, n = 1 6. With CsCl,K1 = 0 08, n = 1-4. With TEA-Cl, K1 = 0 1, n = 1 5. With Tris-HCl, K1 = 0 3, n=19.

ANa-Glu Sucrose Na-Glu

Isoprenaline (sum). 0.001 0.01 0-1 1

]_ 0 - 5 ~~~~~~~~nA* v 30s

B C

-100 -100 50 mV*~~ ~ ~ ~~~-

U-0V +-1 nA -1 nA

Fig. 6. Activation of the C1- current in the cation-deficient sucrose-rich external solution.A, current trace on the chart recorder. The time superfusion of several concentrations ofisoprenaline, and either sodium glutamate (Na-Glu) or sucrose-rich (sucrose) externalsolution are indicated above the record. B, the averaged I-V curves obtained at the timesindicated by the corresponding symbols in the chart recording; 0O control in Na+-richsolution; *, control in sucrose-rich solution; A, *, and *, response to 0-001, 001 and0 1 M isoprenaline in sucrose-rich solution, respectively; V, response to 1 AM isoprenalinein Na+-rich solution. C, the I-V curves of the isoprenaline-induced current obtained bysubtracting the average of the control I-V curve from the I-V curves obtained in thepresence of 0 001, 0 01, 041 and 1 4tM isoprenaline.

219


Hill coefficient was not markedly affected. This result is in good agreement withthose obtained with isoprenaline.

Activation of the Cl- current in cation-deficient solutionThe parallel shift of the dose-response relation (Fig. 5) can be explained either by

assuming an inhibition of the response in a sequence of potency, Tris+ > TEA+ >

V

100 -

l 80

60 -

60Q 40

0 20

0-001 0-01 0-1 1Isoprenaline (4aM)

Fig. 7. Dose-response relations between the Cl- conductance and isoprenaline in thecation-deficient solution. The slope conductance around 20 mV was measured by theleast-squares fit, and was normalized to the response to 1 /lM isoprenaline. Differentsymbols indicate different cells. The continuous curve is the least-squares fit of the Hillequation. The details are the same as in Fig. 5; n = 1-4, K1 = 0-015 ftM.

Li+, Cs+ > Na+, or by a facilitation in a reverse sequence. In the latter case offacilitation, removal of cations will suppress the response of the Cl- current. In theformer case of inhibition, either no change or a leftward shift is expected in thedose-response curve. In the experiment shown in Fig. 6, NaCl in the externalsolution was replaced with 300 mm sucrose. To record a background current at agiven Cl- concentration (8 mM), the cell was superfused with 140 mm sodiumglutamate solution before and after the sucrose solution.On switching the bath solution from sodium glutamate (0) to sucrose solution

(M), the holding current was shifted outward accompanied with a decrease in themembrane conductance, most probably due to disappearance of a leak inward Na'current. Following the above phenomenon, a cumulative application of isoprenalineinduced a dose-dependent increase in the membrane conductance. The I-V relationsunder each condition were superimposed in Fig. 6B, and the isoprenaline-inducedcurrent in Fig. 6 C. As expected for the Cl- current, the difference current was mostlyinward over the potentials examined.The dose-response relation was constructed by measuring the slope conductance at

around +20 mV and the values were normalized with respect to the conductanceinduced by 1 ,uM isoprenaline (Fig. 7). The Hill equation gave a K, of 0-015 /Mwith aHill coefficient of 1-4. This value of K1 is comparable to 0-013 #tM obtained in thecontrol Na+ solution (see Fig. 5). Thus, we conclude that the substitutes for Na+ havean antagonistic action on the fl-adrenergic stimulation of the Cl- current.To examine whether Na+ itself has a cation-dependent inhibitory action on the

220

CATION-DEPENDENT ACTIVATION OF A C1- CURRENT

/3-adrenergic stimulation, we examined the effects of increasing the [Na']. from 140to 300 mm in the constant presence of 0 1/M isoprenaline. The amplitude of the Cl-current was consistently decreased to 40-70% control by this intervention (N = 3;not illustrated). If the twofold increase in osmolarity did not affect the currentresponse under the intracellular dialysis, the finding may suggest that Na' has aweak inhibitory action.

Effects of monovalent cations on various levels of cyclic AMP-dependent cascadereactions

In order to specify the site of action of monovalent cations, the Cl- current wasactivated by bypassing the f-receptor. In the experiment shown in Fig. 8A a, the Cl-current was induced by direct activation with the external application of forskolin.The subsequent replacement of Na' with TEA' slightly reduced the inwarddeflection of the current during the ramp pulses in a reversible manner. Forqualitative analysis, the Cl- current was dissected by subtracting the backgroundcurrents in the comparable ionic conditions from the records under the influence offorskolin (not illustrated). It was suggested that a slight decrease of the inwardcurrent in TEA+ solution was due to a change in the background Na+ conductance(see also Matsuoka et al. 1990) and that the Cl- current induced by 1 ftM forskolinwas not affected by the Na+ substitution. More systematic examination was thenconducted by measuring the dose-response relations for forskolin in different cationsolutions. The ordinate in the dose-response relation in Fig. 8B shows the slopeconductance of the Cl- current, which was normalized with respect to the responseto 10uM forskolin recorded in the same cell in the Na+ solution. Although theresponse to forskolin varied from cell to cell, the rightward shift of the dose-responsecurve was not observed when Na+ was substituted with Tris+ (0) or Li+ (A\). Thisis in striking contrast to the response to fl-agonists (Fig. 5) and suggests a site ofaction prior to the adenylate cyclase.The above view was supported by recording the cyclic AMP-induced Cl- current.

In the experiment in Fig. 8A b the internal perfusion of 500 JM cyclic AMP activatedthe Cl- current in TEA+ solution. Replacing TEA+with Na+ caused a slight increaseof membrane conductance which is attributable to the background Na+ conductanceas indicated in the initial part of the chart recording. The two Cl- currents obtainedby subtracting the background currents in comparable ionic conditions weresuperposable (not illustrated), suggesting that the Cl- current remained intact.The measurement of the dose-response relation (Fig. 5) indicated the necessity to

examine the response at moderate concentrations of agonists. Usually concentrationsof cyclic AMP higher than 100 /LM were required in the pipette solution, probably dueto the relatively high activities of phosphodiesterase. We therefore carefullyexamined the response in individual cells and used low concentrations of cyclic AMPto obtain submaximal responses of the Cl- current. It was a consistent finding thatthe Cl- current induced by 100-500 /tM cyclic AMP in Na+ solution was not decreasedby replacing Na+ with TEA+ (N = 3), Tris+ (N = 4), Li+ (N = 3) or Cs+ (N = 2).

221


TEA-Cu I NaCI ITEA-CL NaulForskolin (1 #M)

]-0,5 nA1 min

b

nAI-0.5

TEA-Cl I NaCI I TEA-ClHistamine (1 FM)

I NaCI

0

-0-5

B

0)

co4-0

0C-)mo

nA

05 1Forskolin (/EM)

Fig. 8. A, effects of monovalent cation on the C1- current induced by 1 #UM forskolin (a),internal dialysis of 500 /M cyclic AMP (b) and 1 /M histamine (c). Current traces on thechart recorder are shown. Application of the drugs and exchange of NaCl and TEA-Cl are

indicated above the record. B, dose-response of the Cl- current for forskolin. The slopeconductance was normalized to the response to 10 EM forskolin recorded in the same cell.Different symbols indicate different cations and the data obtained from the same cell are

connected (0, Na'; 0, Tris+; A, Li+).

222

A a

c

-- . - I .. - I -- . - I .. 0%1

- I . . - . - -


Effects of Na+ substitution on histamine-induced Cl- currentAlthough the histamine effect is mediated specifically via H2 receptors in cardiac

muscle, the H2 receptor shares a common cyclic AMP-dependent pathway with the/-adrenergic receptor, including the GTP-binding protein (Hescheler, Tang, Jastorff

TEA-TEA-CIINaCI Cl NaCI

Isoprenaline (50 nM)

05n

0 1 min

-100 -50 j* -

I,,^,7.,.,,, 100 mV

. L 0-5 nA

Fig. 9. Effects of intracellular Na+ on response to Na+ substitution. The recording pipettecontained 115 mm Na+ throughout the experiment. During the continuous presence ofisoprenaline (50 nM), the external NaCl was replaced with TEA-Cl (see inset). Thedifference I-V relations at the times indicated by symbols in the inset are shown in thegraph.

& Trautwein, 1987; Harvey & Hume, 1990 a). Thus, histamine provides a crucial toolfor examining whether the modulatory effect of monovalent cations is specific for thef-adrenergic receptor. We examined the Cl- current induced by various con-centrations of histamine (0l2-5 #M) and observed no obvious depression of theresponse on replacement of Na+ (N = 4). The Cl- current shown in Fig. 8A c wasactivated submaximally by 1 gM histamine. It is evident that the Cl- current is notaffected by replacing Na+ with TEA+.Based on the above findings, we conclude that monovalent cations affect ,-

stimulation prior to the GTP-binding protein, most probably on the level of the /3-adrenergic receptor.

Extracellular localization of the effect of Na+ substitutionAlthough the pipette solution contained no Na+, the presence of the background

Na+ conductance (Figs 3, 6 and 8A) might have caused an accumulation ofintracellular Na+. In the Na+-free solution the [Na+]i might secondarily decrease andthereby the f-response might have been decreased (Harvey & Hume, 1990b; Harveyet al. 1991). To minimize the effect of Na+ depletion, 115 mm Na+ was added in thepipette solution in the experiment shown in Fig. 9. Under this condition the outwardcurrent, which is most probably due to activation of the Na-activated K+ channels

223

F. M. TAREEN, A. YOISHIDA AND K. ONO

(Kameyama, Kakei, Sato, Shibasaki, Matsuda & Irisawa, 1984), markedly increasedimmediately after breaking the patch membrane in the normal Tyrode solution. Thisoutward current gradually decreased as intracellular K+ was replaced with Na', andcompletely disappeared when the intracellular medium was equilibrated with theNa'-containing pipette solution (not shown). After observing a steady backgroundconductance, the control background currents were recorded for external TEA' (K>)and Na' (0) solutions as shown in Fig. 9. During the continuous stimulation of theCl- current by 50 nM isoprenaline, external Na' was replaced with TEA+. Acomparison of the two I-V curves of the Cl- currents clearly indicated that thedepression of the isoprenaline response was well conserved. In an average of sixexperiments, the Cl- conductance was decreased by 74-7 + 10-9% by replacing Na+with TEA+, which is comparable to the decrease of 71-1 + 12-3% obtained with 0 mMNa+ pipette solution (N = 12), or to 669 + 14-4% with 15 mm Na+ pipette solution(N= 14).These findings make an involvement of the intracellular Na+ unlikely in the cation

dependence of the fl-stimulation observed in the present study. We conclude that thedepression of the ,8-stimulation is primarily due to an interaction of Na+ substituteswith the Preceptor from the external side of the cell membrane.

Cation-dependent enhancement of the Ca2+ current by isoprenalineThe /3-adrenergic enhancement of the Ca2+ current should share a common cascade

of reactions with the activation of the Cl- current (Kameyama, Hofmann &Trautwein, 1985). If so, the external monovalent cations will have the sameantagonistic action on the /3-adrenergic stimulation of the Ca21 current. To test thishypothesis, we recorded the Ca2± current in various cation solutions using the pulseprotocol shown in Fig 10A. The Na+ current was inactivated during the conditioningpulse to -40 mV, and the following depolarization to 0 mV induced the L-type Ca21current. K+ conductances were blocked with intracellular and extracellular Cs+.

Figure IOA shows representative records of the Ca2+ current. In the control Na+solution, the application of 0 03 JaM isoprenaline increased the amplitude of the Ca2+current by more than twofold (Fig. IOA a). The increments of the Ca2+ current weresignificantly smaller in Cs+ (b), Li+ (c), and Tris+ (d) solution. When the amplitude ofthe Ca2+ current was measured as a difference between the peak value and the currentjust before the end of the test pulse, it was increased in response to 30 nM isoprenalineby a factor of 2-15+0-54 in the Na' solution (N= 10), 100+011 in Li+ (N= 6),1 19+040 in Cs+ (N= 4) and 0-98+008 in Tris+ solution (N= 5).The depression of the Ca2+ channel response was consistently reversed by

increasing the agonist concentration. The dose-response relations were successfullyobtained in five experiments in different cation solution (Fig. lOB). The increase ofthe amplitude was measured at the test potential of 0 mV and normalized referringto the maximum response in each solution. It is evident that, as in the case of Cl-current, the substitution of external Na+ (0) with Cs+ (7) or Li+ (0) shifted thedose-response relation toward the higher doses. Although the measurement of thecomplete dose-response relation was difficult in TEA+ or Tris+ solution, thethreshold concentration for activation was higher than 0 05 ,/M (N = 10, not shown).

224

CATION-DEPENDENT ACTIVATION OF A C1 CURRENT

0A -40 B

Vm80~~~

d~ ~ C

C0CoL

..~~~~~~~~~~~~~~

50 ms

Fig. 10. A, effects of monovalent cations on the enhancement of the Ca2+ current inducedby isoprenaline. Myocytes were voltage clamped at the holding potential of-80 mV. Toinactivate Na+ current, conditioning pulses to -40 mV with a duration of40 ms precededtest pulses to 0 mV. Original current records in solution containing various monovalentcations; 140 mm Na+ (a), Cs' (b), Li' (c), and Tris+ (d). Superimposed currents are in theabsence (0) and presence of 30nm isoprenaline (M); a, b, c and d are obtained fromdifferent cells. B. dose-response relationships between isoprenaline concentration and theincrease in the Ca2+ current. The amplitude of the Ca2+ current was defined as thedifference between the peak value and the current just before the end of the test pulse.The increases in amplitude were normalized referring to the maximum response in eachsolution and were plotted against the isoprenaline concentration. Different symbolsindicate the different cations; 140 mm Na+ (-), Cs' (A) and Li' (0). The continuouscurve is a least-squares fit ofequation y = 1/(1 + (Ki/[drug]nf) to data obtained for 140 mmNaCl, with Kay = 0-012 Am, n = 1-3.

d

DISCUSSION

The amplitude of the Ca- current induced by Anagonists was decreased bysubstituting external Na+ with alkaline metal ions or organic cations in a dose-dependent manner. The Cl- current, when activated by forskolin, cyclic AMP or bystimulating the HM2receptor, was not affected by replacing external Na+ with othercations. If theHN2 receptor shares a common GTP-binding protein, it is concludedthat Na+ substitutes interact with the Preceptor to inhibit the n-adrenergicstimulation. Furthermore, adding Na+ in the pipette solution failed to diminish theinhibitory effect of the Na+ substitution. We therefore conclude that monovalentnations interact with the-p-adrenergic receptor on the external surface of the cellmembrane. This interaction is characterized by a rightward shift ofthe dose-responserelations for fthagonists. Essentially the same mechanism is suggested in theenhancement of the Ca2+ current by the u-adrenergic stimulation (Fig. 10).The insensitivity of the Cw- current to Na+ substitution when activated by cyclic

AMP is in agreement with the fdistngs of Matsuoka et al. (1990). However, some ofthe important experimental findings in the present study are different from those of

225


Harvey & Hume (1990b) and Harvey et al. (1991), and, therefore, our conclusion isdifferent from the other group. They showed that the responses of Cl- current, whenactivated by forskolin, 8-bromo cyclic AMP, catalytic subunit of protein kinase A,or 3-isobutyl- 1-methylxanthine, were all depressed by the Na+ substitution, and thatthe depression was weaker if the pipette solution contained 10 mm Na+. At presentwe have no definite explanation for the difference of our experimental results fromtheirs. One might argue that our cell isolation procedure involves the use of a high-K+, low-Cl- solution and culture media, which contain high concentrations of aminoacids. The pipette solutions contained CsOH and aspartic acid (see Methods), whileHarvey et al. (1990, 1991) used mainly CsCl. It is reported that amino acids canimprove the cell ability to regulate the [Na+]i (Chapman & Rodrigo, 1990). Thus, inour preparation Na+ might have been accumulated locally beneath the cellmembrane, which might explain the differences of the experimental findings betweenthe two groups. However, it should be emphasized that the depression of theresponse to fl-agonists was a consistent finding in the present study, and that it is auniversal phenomenon irrespective of the different experimental conditions describedabove (Egan et al. 1988; Bahinski et al. 1989; Harvey et al. 1990; Matsuoka et al.1990; Harvey et al. 1991). The above results may, therefore, indicate the presence ofa common mechanism, rather than the intracellular Na+.Although in the studies of Harvey et al. (1990, 1991) the time course of the effects

of the Na+ substitution was not thoroughly described, removing external Na+appeared to depress the response to isoprenaline with a relatively slower time coursecompared to the quick response to isoprenaline or acetylcholine applied to the samecell (Fig. 6 of Harvey et al. 1990). In the present study, we attempted to exchangesolutions within a short time span in order to confine our observation to the primaryeffects of the Na+ substitution. Complete exchange of the solution was confirmedduring the experiment by monitoring the original current on the oscilloscope. Thecomplete disappearance and reappearance of the Na+ current during the depolarizinglimb of the ramp pulses clearly reflected the changes in the [Na+]O. Furthermore, therecovery of the response was checked by bracketing the challenge of the Na+depletion with the control Na+ perfusion, so that the interference from run-down ordesensitization was avoided.The activation of the inward Cl- current was conserved when external Na+ was

replaced with sucrose. This excluded the possibility that the presence of external Na+is essential for the activity of the Cl- channel and/or for the activation of the C1-current by the fl-adrenergic stimulation. The Ki of 0-015 fM isoprenaline in thesucrose solution was comparable to 0-013 /M in the control Na+ solution. Under theassumption that sucrose is inert for fl-receptor, we therefore conclude that other Na+substitutes inhibit the fl-stimulation of the current. Na+ itselfmay have an inhibitoryaction, weaker than Li+ or Cs+, since increasing the [Na+]o up to 300 mm slightlydepressed the isoprenaline-induced Cl- current.The parallel shift of the dose-response relation suggests a competitive antagonism

between agonists and blocking molecules on the receptor in a simple system. Theagonists of the fl-adrenergic receptor have a protonated amine and the agonist-binding domain of the fl-adrenergic receptor protein has a negatively chargedcarboxyl group (Strader, Sigal, Candelore, Rands, Hill & Dixon, 1988; Strader,

226


Candelore, Hill, Dixon & Sigal, 1989). Thus, the external monovalent cations maycompete with /-adrenergic agonists for their binding sites, and/or bind to allostericsites to reduce the /3-adrenergic response. The actions of organic cations strongerthan those of alkaline metal ions might be consistent with this view. When thenegative charges of the binding site are neutralized by cations, the apparent affinityof fi-adrenergic agonists for the binding site might thus be reduced. Although thedose-response relations were applied to the Hill equation, the Hill coefficient doesnot necessarily implicate the stoichiometry of the agonist-receptor interaction.Further studies with the help of biochemical methods should be performed toelucidate the action of external cations on the agonist-receptor binding.

It was suggested that an intracellular Li+ concentration of only 0-6 mm inhibitsadenylate cyclase and/or GTP binding protein (Avissar, Schreiber, Danon &Belmaker, 1988). However, this mechanism seems unlikely to be involved in thepresent study, since adding 15 mm Li' in the pipette solution failed to detect anydepression of the fl-adrenergic response of the Cl- current (F. M. Tareen, A. Yoshida& K. Ono, unpublished observation). Rather, the inhibitory action on the ,3-adrenergic response seems to be common among alkaline metal and the alkalineearth metal cations, since K+, Rb+, and various divalent cations were also shown todecrease the activation of the Cl- current in the previous study (Matsuoka et al.1990).

We wish to thank Professor T. Ehara for his support in the initial series of experiments, ProfessorW. Trautwein and Professor A. Noma for their stimulating discussions and helpful advice, and DrB. T. Quinn for reading the manuscript. Thanks are also due to Professor J. Yoshitake, from theDepartment of Anaesthesiology, for providing one of us (F. M. Tareen) with the opportunity toconduct these studies. The secretarial service of Miss M. Fukushima is also greatly acknowledged.This work was supported by research grants from the Ministry of Education, Science and Cultureof Japan.

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