Cycle length dependence of the chronotropic effects of adrenaline, acetylcholine, Ca2+ and Mg2+ in the Guinea-pig sinoatrial node

Journal of the Autonomic NeroousSystem, 11 (1984)349-366 Elsevier

349

JAN 00394

Cycle length dependence of the chronotropic effects of adrenaline, acetylcholine, C a 2+ and M g 2+ in the Guinea-pig sinoatrial node * Tobias Opthof, Berend de Jonge, Branca Schade, Habo J. Jongsma and Lennart N. Bouman Department of Physiology, Academic Medical (?enter, Meibergdreef 15, 1105 A Z Amsterdam (The Netherlands) (Received March 12th, 1984) (Revised version received July 9th, 1984) (Accepted July 24th, 1984)

Key words: sinoatrial node - pacemaker shifts - chronotropic responses - cycle length dependence

Abstract

Ca (1.1-5.5 mM) has a positive chronotropic action on isolated right atria of the guinea-pig. The magnitude of the response depends on the cycle length. Magnitude and cycle length dependence of the Ca response are independent of beta-blockade by propranolol. Mg (0.6-6.0 mM) has a negative chronotropic action. At 6.0 mM it interferes with responses to adrenaline and acetylcholine by preventing pacemaker shifts. Adrenaline has a positive chronotropic action in a cycle length dependent manner. A shift of pacemaker dominance under the influence of adrenaline to an identical site in all preparations (as in the rabbit) was not observed. However, pacemaker shifts in the presence of adrenaline do occur and they are always directed towards the inferior part of the node. Acetylcholine has a negative chronotropic action, independent of cycle length. Acetylcholine also induces pacemaker shifts. Contrary to the pacemaker shifts caused by adrenaline, the new, acetylcholine-induced pacemaker center, has an identical site in all preparations. This was previously observed in the rabbit too. The acetylcholine-induced center is located about 1 mm * This investigation is part of the research program of the Netherlands Organization for the Advancement of Pure Research (ZWO). Correspondence: Tobias Opthof, Department of Physiology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. 0165-1838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

350

inferior from the primary center. During exposure to acetytcholine different action potentials may be recorded at the epi- and endocardial side of the preparation, but only close to the Ach-induced center. The acetylcholine-induced center is located at the epicardial side. The response to acetylcholine predominates over the response to adrenaline. All results are discussed in comparison with our previous findings in the rabbit.

Introduction

The magnitude of chronotropic responses of the sinoatrial node is subject to variability of both biological and experimental origin. It is well-known that considerable differences may exist in the effect of the same concentration of substances in different species. It is clear, on the other hand, that also differences in experimental procedures, such as the composition of (su)perfusion fluids in experiments with isolated hearts or right atrium preparations may introduce variability in results. This last variability is thus not of biological origin. In previous papers we have described the chronotropic responses to an increase of the Ca concentration [16,21,23], an increase of the Mg concentration [21-23] and to the addition of adrenaline (Adr) [17,18,24] and acetylcholine (Ach) [17,18,24] in the rabbit sinoatrial node. In the present study we want to compare these results with data obtained in the guinea-pig sinoatrial node. Equal results may point to general characteristics of the rodent or mammalian sinoatrial node, whereas different results may point to species peculiarities. The chronotropic response to an increase of the Ca concentration has been reported to be positive in the guinea-pig sinoatrial node [27-29,32]. The mode of action of this Ca effect is not very clear. Schaer [28] showed that the beta-blocker pronethalol did not abolish the chronotropic response to Ca, but pronethalol itself had a negative chronotropic effect. The chronotropic response to an increased Ca concentration depends on the spontaneous cycle length (CL) in the rabbit [21]. This CL-dependence means that the decrease in CL, due to the increase of the Ca concentration is only small when the spontaneous CL is short, but that this decrease in CL is larger, when the spontaneous CL is longer. If the same is true in the guinea-pig, then it is not clear whether the results of Schaer [28] must be regarded as an inhibition of the chronotropic response to Ca or not. In the present study we focused on CL-dependence of the chronotropic action of Ca in the guinea-pig and on the influence of beta-blockade by propranolol. Mg has been shown to have a relatively simple action in the rabbit sinoatrial node as regards its chronotropic response. Its effect is linear with the concentration and shows no CL-dependence [21]. Its influence on the chronotropic responses to Adr and Ach seems more intricate. It was discussed previously that this interference with the chronotropic responses to Adr and Ach is due to the fact that an increase in the Mg concentration retards latent pacemakers more than the primary pacemaker (i.e. the leading pacemaker under standard conditions) [22,23].

351

Further, the action of Adr and Ach on guinea-pig sinoatrial function has been investigated. In the rabbit it is known that pacemaker dominance shifts during sympathetic [35,39] as well as during parasympathetic stimulation [3,36,39]. Also the addition of Adr and Ach causes shifts of pacemaker dominance in the rabbit [17,18,24]. Both during simultaneous sympathetic and parasympathetic stimulation and during the simultaneous addition of Adr and Ach the deceleration due to the cholinergic effects predominates over the acceleration due to the adrenergic effects [8,10,17,18,24]. The chronotropic response to Adr turned out to be dependent on the spontaneous CL in the rabbit sinoatrial node [24]. It was the aim of this study to investigate the response of the guinea-pig sinoatrial node to an increase in the concentration of divalent cations (Mg and Ca) and to the addition of Adr and Ach both with respect to the chronotropic effects and to the site of pacemaking. The results are compared with data from the rabbit.

Methods

Preparation Guinea-pigs were decapitated and the hearts were excised. The preparation [1] was superfused with a balanced salt solution of the following millimolar composition: NaCI 130.6; KC1 5.6; N a H C O 3 24.2; CaC12 2.2 (except in the Ca experiments, it then was 1.1 mM); MgC12 0.6; glucose 11.1; sucrose 13.2. The solution was equilibrated with 95% O 2 and 5% CO 2, p H was 7.4. The preparation was superfused in a tissue bath by 1200 m l / h and the experimental temperature was kept constant at 38°C within 0.1°C. The Ca concentration could be raised by mixing small volumes (3.6 or 12 m l / h ) of 440 mM CaC12 with the fluid perfusing the tissue bath. The Mg concentration was raised by mixing 12 m l / h of 540 mM MgCI 2 with the fluid perfusing the tissue bath. Beta-blockade was established by adding 1.7 x 10 -7 M propranolol. We checked the efficacy of this blockade in each experiment by measuring the response to 6 x 10 7 M Adr prior and after the addition of propranolol. The blockade of the response to Adr was always over 95%. i-Adrenaline bitartrate (Adr) and acetylcholine bromide (Ach) were administered by mixing small amounts (3,6,12 or 30 m l / h ) of concentrated solutions with the fluid perfusing the tissue bath. The final concentrations were 1.5-] 5 × 10 7 M Adr and 1 5 0 x 1 0 -6 M A c h .

Recording of electrical activity A bipolar surface electrogram was derived from the crista terminalis and the tachogram was recorded continuously. Chronotropic effects are expressed as shortening or prolongation of CL in milliseconds + 1 S.E. and are analyzed by standard statistics (Student's t-test and covariance analysis). Transmembrane potentials were recorded using the conventional glass microelectrode technique. Activation maps were made as described previously [1]. Activation times (SACT) of the cells were measured from Vhfamp (i.e. the potential halfway the maximum diastolic potential

352

(MDP) and the top of the action potential) during the upstroke of the action potential till the moment of the steepest deflection of the atrial electrogram. A negative SACT means that the cell activates prior to the right atrium. A positive SACT means that the cell is activated after the discharge of the right atrium. In this study, the pacemaker celt group dominating under standard conditions will be referred to as the primary pacem.aker. The action potential duration (APD) was measured from the moment of Vhfamp during the upstroke till the moment of Vhramp during the repolarization of the action potential (APD50). The action potential duration was further measured (APD100) from the moment of Vhfamp during the upstroke till the moment of the m a x i m u m diastolic potential (MDP). The rate of diastolic depolarization was measured during the first 100 ms after the moment of the MDP. All signals were stored on magnetic tape (AMPEX FR 1300, frequency response 0-5000 Hz) for off-line computer analysis.

Results Calcium

The positive chronotropic effect of Ca has a maximum between 5 and 10 mM [28,29,32]. In the experiments the Ca concentration was raised from 1.1 to 2.2. from 1.1 to 3.3 and from 1.1 to 5.5 raM. The sequence of these changes was varied. In the same experiments propranolol was given to obtain beta-blockade. This blockade was preceded by measuring the response to 6 × 10 -7 M Adr. It turned out that 1.7 x 10 -7 M propranolol inhibited the response to 6 x 10 -7 M Adr by more than 95%. This blockade was effective after 30 min. Propranolol itself had no chronotropic effect. Next. during exposure to propranolol, we measured once again the responses to the changes in the Ca concentration. It turned out that the chronotropic effect was CL-dependent at all tested Ca concentrations (Fig. 1). The response to 2.2 m M was significantly smaller than the responses to 3.3 and 5.5 mM. The difference between the responses to 3.3 and 5.5 m M was not significant (covariance analysis). In the presence of propranolol the effects were identical, i.e. covariance analysis 20 0 ._/2,00

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Fig. 1. The c h r o n o t r o p i c response to Ca. The C L at 1.1 m M C a was t a k e n as reference (0 ms). The C a c o n c e n t r a t i o n was raised from 1.1 to 2.2 (e), from 1.1 to 3.3 (~) or from 1.1 to 5.5 m M (U). T h ¢ C L at 1.1 m M was 255 + 30.3 (S.D,) ms (n = 48). 2.2 m M Ca: Y = - 0.30 X + 65.5, n = 16, r = - 0.783, P < 0.0005; 3.3 m M Ca: Y = - 0 . 4 0 X+79.7, n=18, r=-0.719, P < 0 . 0 0 0 5 ; 5.5 m M Ca: Y = - 0 . 7 1 X+151.2, n = 1 4 , r = - 0 . 7 2 7 , P < 0.005.

353

showed no differences between the responses with and without propranolol. Neither the magnitude of the chronotropic action of Ca, nor its CL-dependence is influenced by a concentration of propranolol effective in blocking the chronotropic effect of 6 x 10 -v M Adr. The action of Ca is, therefore, not mediated by adrenergic mechanisms.

Magnesium Mg prolongs the intrinsic CL of latent pacemakers more than the intrinsic CL of the primary pacemaker in the rabbit [22,23]. Therefore, Mg does not induce pacemaker shifts in this species [22,23]. Mg has a prominent negative chronotropic effect, which is linear with the concentration in the rabbit [21]. In the present experiments, 6.0 mM Mg increased the CL by 120 _+ 8.3 ms (n = 4), which is more than in the rabbit [23]. We observed in 4 experiments that the activation pattern under standard conditions changed after the addition of 6 × 10 7 M Adr. The leading pacemaker site moved from the primary pacemaker to an Adr-induced pacemaker. The increase of the Mg concentration from 0.6 to 6.0 mM, in the presence of 6 x 10 -7 M Adr, re-established the original activation pattern that was observed prior to the addition of Adr. The responses to 1 and 2 x 10 - 6 M Ach were potentiated during exposure to 6.0 mM Mg, while the response to 6 x 10 -7 M Adr was diminished. The explanation for this phenomenon is that 6.0 mM Mg prolongs the intrinsic CL of the latent pacemakers more than the intrinsic CL of the primary pacemaker. This prevents the Ach-induced center to take over dominance during exposure to Ach. This uncovers the larger response of the primary pacemaker to Ach compared with the response of the Ach-induced center. In the same way Adr-induced centers are prevented from taking over dominance during exposure to Adr. This uncovers the smaller response of the primary pacemaker to Adr compared with the response of Adr-induced centers. This mechanism was previously hypothetized with respect to data in the rabbit taken from the literature [22]. Fig. 2 demonstrates the effect of Mg in a continuous impalement of one cell. In panel A, the cell is leading under standard conditions, in panel B it has lost dominance by the action of 6 x 10 -v M Adr. In panel C, dominance is regained by the beginning action of Mg. It is obvious that this cell has regained dominance by the action of Mg during exposure to Adr long before this Mg concentration (6.0 mM) has exerted its complete chronotropic effect. The photograph in panel C has been taken just after the site of pacemaker dominance has shifted back. The very long time needed for Mg to exert its full effect (30 min) is an argument for an intracellular mode of action.

Adrenaline Fig. 3 shows the positive chronotropic effect of Adr. The effect depends strongly on the basic CL (lowest significance level 0.0005). The effect differs also for the 4 tested Adr concentrations (co-variance analysis). Under 15 x 10 -v M Adr the CL is 182 _+ 3.5 (S.D.) ms (n = 13), i.e. the variability in CL between different preparations has almost disappeared. This variability is approximately 13% under standard conditions. We never observed a guinea-pig sinoatrial preparation beating with shorter CL than 175 ms.

354

With the same techniques it appeared far more difficult to make stable impalem e n t s in the presence of A d r in the guinea-pig than in the rabbit. Although pacemaker shifts i n d u c e d by A d r do occur, we have not established a typical A d r - c e n t e r like that which has been reported in the r a b b i t [17,18,24]. Sites leading in the presence of A d r were all well outside the area of central nodal cells, which we checked by light microscopy [1]. In general, the site of p a c e m a k i n g does not shift until 1.5 × 10 -7 M Adr. At higher c o n c e n t r a t i o n s shifts occur and when they occur, they are always directed towards the inferior part of the preparation. However, the exact location of the leading center in the presence of A d r with respect to the distance to the crista terminalis was very variable. We also observed preparations in which the p r i m a r y pacemaker m a i n t a i n e d d o m i n a n c e even at 15 × t 0 -7 M Adr. In the rabbit the site of p a c e m a k i n g definitely shifts to the tail of the node d u r i n g

i

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mV 200 ms Fig. 2. Continuous impalement of a primary pacemaker cell. The activation pattern of this preparation was mapped. Upper traces: atrial electrograms. Lower traces: action potentials. A: the cell leads by 10 ms under standard conditions. CL 224 ms, amplitude 69 mV, SACT - 10 ms, Vmax 5.6 V/S, MDP - 60 mV, APD50 71 ms, APD100 124 ms, rate of diastolic depolarization 141 mV/s. B: the same, continuously impaled cell lost dominance by the action of 6 × 10-7 M Adr. CL 196 ms, amplitude 74 mV, SACT + 5 ms, ~/rnax16.7 V/S, MDP -63 mV, APD50 67 ms, APD100 111 ms, rate of diastolic depolarization 163 mV/s. C: the cell has regained dominance during 6 X 10 - 7 M Adr and the beginning action of 6.0 mM Mg. CL 233 ms, amplitude 7t mV, latency - 1 0 ms, ~rm8x 7.8 V/s, MDP -61 mV, APD50 69 ms, APD100 119 ms, rate of diastolic d c p o l ~ t i o n 140 mV/s. The photograph was taken long before the steady-state in the presence of 6.0 mM Mg is established, just after the moment of the pac4maker shift, CL has only prolonged by 9 ms; in steady-state it was prolonged by 114 ms.

355 exposure to 6 X 10-7 M A d r [17,18,24]. Since we did not observe a typical A d r - c e n t e r we will focus on the responses of the p r i m a r y pacemaker to Adr. A variance test on 500 consecutive CLs showed that the b e a t - t o - b e a t r h y t h m is stable, although less stable than u n d e r s t a n d a r d c o n d i t i o n s when we measured a coefficient of variation (100 x S . D . / m e a n CL) of 0.1%. In the presence of 6 x 10 7 M A d r we measured once 0.2% a n d once 2.3%. The b i n w i d t h in this test was 4 ms. Regardless whether a pacemaker shift occurred or not, both Vmax a n d the rate of diastolic depolarization increase in the p r i m a r y pacemaker cells d u r i n g exposure to 6 x 10 7 M Adr. F u r t h e r m o r e A P D 5 0 a n d A P D 1 0 0 both shorten. The influence on the action potential a m p l i t u d e is not constant. It increases in the range 0 - 6 mV. In the rabbit it decreases [18]. Latent pacemaker cells show both increase and decrease in action potential a m p l i t u d e after the a d d i t i o n of Adr, varying from site to site a n d from p r e p a r a t i o n to preparation. In the p r i m a r y pacemaker, the M D P hyperpolarizes between 0 a n d 4 mV. Vmax increases more when a pacemaker shift occurs. The influence of the discharge of the primary pacemaker by a latent pacemaker that has become d o m i n a n t d u r i n g exposure to Adr, then becomes clear (Fig. 4A). Fig. 4 shows examples of a p r i m a r y pacemaker losing (panel A) a n d m a i n t a i n i n g (panel B) d o m i n a n c e during exposure to Adr.

Acetylchofine Fig. 5 shows the negative c h r o n o t r o p i c response to Ach. The response does not d e p e n d on the basic CL, which was also observed in the rabbit [24]. The response has the same m a g n i t u d e as the response in the rabbit in the c o n c e n t r a t i o n range till 5 x 10 6 M Ach. Fig. 6 shows a selection of data from Fig. 5. We selected 8 p r e p a r a t i o n s in which we had measured the response to 3 or more Ach c o n c e n t r a tions. It can be seen that a Ach c o n c e n t r a t i o n between 5 a n d 50 x 10 -6 M causes a

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Fig. 3. The chronotropic response to Adr. The concentrations are 1.5 (e), 3 (A), 6 (X) and 15 X l0 7 M (m). The CL under standard conditions before each addition of Adr was taken as reference. The mean CL under standard conditions was 244+ 31.1 (S.D.) ms (n = 60). 1.5 x 10 - 7 M Adr: Y = -0.90 X + 199.7, n = 9 , r = -0.916, P < 0.0005; 3x10 -7 M Adr: Y= -0.86 X+175.1, n=13, r =-0.916, P < 0.0005; 6x10 -7 M Adr: Y = - 0 . 7 6 X+137.8, n=25, r=-0.899, P<0.0005; 15x10 7 M Adr: Y = - I . 0 1 X + 184.8, n = 13, r = -0.986, P < 0.0005. The 4 regression lines differ significantly (covariance analysis, lowest level of significance0.025).

356

marked increase of CL. One preparation shows this phenomenon already between 1 and 2 x 10 -6 M and another between 2 and 5 x 10 -6 M. At 50 x 10 -6 M Ach, CL was approximately doubled in all preparations. The process of diastolic depolarization is then retarded in such a way that a blockade in the impulse generation occurs. The large value of SE at the concentrations 10, 20 and 27.5 x 10-6 M Ach (Fig. 5) is caused by the fact that this blockade in impulse generation occurs in the concentration range 5-50 x 10-6 M Ach varying from preparation to preparation. The doubling of CL was caused by blockade in impulse generation and not by poor impulse conduction from the sinoatrial node towards the atrium. However, at concentrations lower than 5 x 10 -6 M, parts of the sinoatriai node may show 2:1 intranodal blocking phenomena. This is observed after the shift of pacemaker dominance from the primary pacemaker to the new site (Ach-induced center). Under such circumstances the conduction from this Ach-induced center towards the atrium is not disturbed, but the part of the node that comprises the primary pacemaker may show 2 : 1 blocking phenomena. We never observed such phenomena in the rabbit. The beat-to-beat rhythm under Ach was checked by a variance test on 500

I

200 m s

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Fig. 4. Primary pacemaker centers with and without Adr. Upper traces: atrial electrograms superimposed on the moment of the steepest deflection of the left electrograms. The longer CLs belong to the standard conditions, the shorter CLs were recorded in the presence of Adr. Lower traces: action potentia!s recorded under standard conditions and in the presence of Adr. The action potentials were superimposed on the moment of the steepest deflection of the left atrial electrograms (upper traces). A: primary center losing dominance during exposure to 6 x 1 0 - 7 M Adr. CL 224 (standard) and 195 (AdO ms, amplitude 65 and 71 mV, SACT - 1 0 and + 5 ms, Vmax 5.3 and 1%3 V / s , M D P - 5 8 and - 6 2 mV, APDS0 72 and 65 ms, APD100 124 and 111 ms, rate of diastolic depolarization 139 and 165 m V / s . B: primary center maintaining dominance in the presence of 6 x 1 0 - 7 M Adr. CL 270 (standard) and 210 (Adr) ms, amplitude 68 and 72 mV, SACT - 8 and - 9 ms, ~m~ 5.9 and 7.5 V / s , M D P - 6 2 and - 6 6 mV; APD5G 79 and 70 ms, APD100 130 and 114 ms, rate of diastollic depolarization 126 and 181 m V / s .

357

consecutive CLs. Its coefficient of variation during exposure to 50 × 10 6 M Ach was 0.6%. So the CL was still stable, although less than under standard conditions (oA%). Completely different from the response to Adr, the response to Ach is accompanied by a shift of pacemaker dominance to a site of constant location in all preparations. This shift is in the same direction as in the rabbit, i.e. inferiorly, somewhat towards the crista terminalis. The major difference with the rabbit is that the Ach-induced pacemaker center is not located within the light microscopically defined peripheral nodal cells, but that it, like the primary pacemaker, is located within the light microscopically defined central nodal cells [1]. The Ach-induced center has, during exposure to Ach, much lower Wmaxvalues than in the rabbit. For the primary pacemakers under standard conditions the inverse is true. because the V,,,~, of the primary pacemaker is about 2 V / s in the rabbit (n = 25) and about 6 V / s in the guinea-pig (n = 40) (unpublished results). Very often the Ach-induced center is localized in the area where the peripheral nodal cells [1] are overlaying the central nodal cells. This small area is located at the lateral (crista terminalis) side of the node. In those cases different action potentials at the endo- and epicardial side of the preparation are recorded. During exposure to Ach, the leading, slowly rising action potentials are always at the epicardial side of the preparation. Fig. 7 shows A CYCLE

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Fig. 5. The chronotropic response to Ach. The CL under standard conditions was taken as reference. The number of observations is between parentheses. All values differ significantly from zero (lowest level of significance 0.005). Fig. 6. Selection of data from Fig. 5. Eight preparations were selected in which the responses to 3 or more different Ach concentrations were measured. The figure shows that Ach can increase the CL up to 70-80 ms. A further increase of the Ach concentration will result in about a doubling of the CL under standard conditions. This figure shows why the SE at 2 and 5 x 10 -6 M Ach are larger than at 1 x 10 -6 M A c h in Fig. 5, although the number of observations at 5 x 10 -6 M is nearly twice the number of observations at 1 × 10 -6 M in Fig. 5. It also shows why especially the values of SE are large at 10, 20 and 27.5 × 10 -6 M.

358

the response of primary pacemakers cells from 4 preparations to the addition of 2, 4 and 5 x 10 -6 M Ach. There is a very large decrease of DDR, but also the APD50 and action potential amplitude decrease (Fig. 7). Most of these effects were already significant at 2 × 10 -6 M Ach. From the fact that the SACT turns from negative to positive values, it can be understood that a concentration of less than 2 × 10 - 6 M Ach is enough to bring about a shift of the site of pacemaker dominance. Concomitant with this shift we observed an increase of ~'max of the primary pacemaker, although this rise was not significant, because one of the 4 primary pacemaker cells showed an enormous increase of Vmax, which turned the results statistically insignificant. The impalement was maintained throughout the experiment, i.e. after the measurement of the response to any Ach concentration, the standard conditions were restored and the response to another concentration was tested. Table I gives the action potential parameters of the Ach-induced center and the primary center both in the presence of 5 × 10 - 6 M Ach (the data of the primary pacemaker parameters are the same as the data of Fig. 7 at 5 × 10 - 6 M Ach). In these 4 experiments (Table I), the shift occurred in the inferior-lateral direction. We localized the Ach-induced pacemaker electrophysiologically at the epicardial side of the preparation. In two experiments we recognized the Ach-induced pacemaker by light microscopy (1) in the central nodal zone with endocardially overlaying peripheral nodal cells (as stated above). The shifts occurred over 1.3 + 0.08 (S.E.) mm (n = 4). Fig. 8 shows that two cells within the same sinoatrial node may respond very different to the same intervention. These differences do not only concern the magnitude of the responses, but also the sign of the responses. Fig. 8A shows the action potentials of the primary pacemaker and Fig. 8B shows the action potentials of the Ach-induced center. The Ach-induced center was located 1.2 mm inferiorly • DDR (mVsl~ ~, A P D qms) • Amplitude ~mV) =l Vmax I V.s"1J • SACT (msJ

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Fi 8. ?.. The continuous impalement of 4 primary counters both under standard conditions and in the presence of 5 x 10 -6 M A c h . Two of these 4 cells were also measure,d at 4 x 10 -6 M A c h and 3 of these 4 were also measured at 2 x 10,6 MAch. ~', significantly different from standard conditions (lowest level of significance 0.05). A negative value for the SACT m ~ n s that the impaled cell discharges ahead of the atrium, a positive value means that t h e cell discharges after the atrium. The impalements were also continuous between the measurement of the different Ach concentrations.

359 TABLE I THE PRIMARY PACEMAKER A N D THE Ach-INDUCED PACEMAKER U N D E R THE INF L U E N C E OF Ach The primary and Ach-induced pacemakers were impaled in the same preparations at 5 x 10- 6 M Ach. All parameters differed significantly (lowest level of significance 0.05) except V,,,~.

Amplitude (mV) Diastolic depolarization rate ( m V / s ) .... ( V / s ) APD50 (ms)

Primary pacemaker (n = 4)

Ach-induced pacemaker (n = 4)

54_+ 3.2

70_+ 6.8

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Fig. 8. Continuous impalement of the primary center (A) and the Ach-induced center (B) under standard conditions and in the presence of 8 x 10 -6 M Ach (superimposed at the moment of the first (left) atrial electrogram). The Ach-induced center was located 1.2 mm inferiorly from the primary center. Upper traces: atrial electrograms superimposed on the moment of the steepest deflection of the left electrograms. The short CL belongs to the standard conditions. The longer CL (more than twice the CL under standard conditions) was recorded in the presence of 8X 10 -6 M Ach. Lower traces: action potentials recorded under standard conditions and in the presence of 8 x 10 -6 M Ach. The action potentials were superimposed on the moment of the steepest deflection of the left atrial electrograms (upper traces) A: primary center. CL 211 (standard) and 453 (Ach) ms, amplitude 79 and 59 mV, SACT - 8 and + 1 ms, V.... 6 and 9 V / s , APD50 79 and 51 ms, APD100 122 and 103 ms, D D R 208 and 31 m V / s , MDP - 6 4 and - 6 5 mV. B: Ach-induced center. CL 223 (standard) and 478 (Ach) ms, amplitude 66 and 69 mV, SACT + 1 and - 1 3 ms, ~',.0~,, 8 a n d 4 V / s , APD50 73 and 71 ms, APD100 133 and 120 ms, D D R 132 and 59 m V / s , MDP - 56 and - 5 9 mV.

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from the primary center. The cells were continuously impaled under standard conditions an d during the impalement Ach was added (8 x 10 - 6 U ) . The shorter CI_ (see the atrial electrogram, upper trace) was observed during standard conditions and the longer CL (more than twice the CL under standard conditions) was observed in the presence of Ach. Apart from the very large increase of CL caused by Ach (cf. Fig. 6) it is clear that the action potential amplitude decreases substantially in the primary pacemaker (from 79 to 59 mV, Fig. 8A), whereas it increases in the Ach-induced center (from 66 to 69 mV, Fig. 8B). It can be seen that the primary pacemaker loses dominance, due to Ach (Fig. 8A), whereas the Ach-induced center turns dominant, due to Ach (Fig. 8B). The D D R decreases substantially in both cells, but it decreases from 208 to 31 m V / s in the primary pacemaker, whereas it decreases only from 132 to 59 m V / s in the Ach-induced center. Both the APD50 and the APD100 were little affected in the Ach-induced center, whereas the decrease in APD50 was substantial in the primary pacemaker. The MDP hyperpolarizes by 3 mV in the Ach-induced center. It is unaffected in the primary center. From Fig. 8A it can be seen that the take-off potential is much more hyperpolarized in the primary center in the presence of Ach than it was under standard conditions. This is accompanied by an increase of Vm~x from 6 to 9 V/s. The opposite happens in the Ach-induced center; Vm,x decreases from 8 to 4 V / s by the action of Ach. The differences between the action potential parameters in Fig. 8 (8 x 10- ~ M Ach) are in good agreement with the data of Table 1 (5 x 10 - 6 M Ach). A cetylcholine and adrenaline In the guinea-pig the response to Ach predominates over the response to Adr. In the presence of Ach, the response to 3, 6 and 15 x 10 - 7 M Adr was only - 5 _+ 3.2 (n = 5), - 1 5 + 3.4 (n = 10) and - 2 2 + 6.1 (n = 5) ms, respectively, which is significantly smaller (Student's t-test for paired observations; lowest"significance level 0,025) than when Adr alone is present (Fig. 3), We never observed that Adr was able to overcome a Ach-induced shift, nor was it ever able to shift the site of dominant pacemaking to another (third) location. The same was previously reported in the rabbit [18,24].

Discussion Calcium The response of the guinea-pig sinoatrial node to Ca is positive chronotroplc, as previously reported [27-29,32]. The CL-dependence of the effect (Fig. 1) explains in our opinion why larger responses are reported in some papers [27,29] compared with other papers [28,32], because the authors used preparations with different spontaneous CLs. There is controversy in the literature with respect to the mode of action of the chronotropic Ca response. It appears that an increase in the Ca concentration does potentiate the effects of vagal [37] and sympathetic nerve stimulation [34] in the rabbit. Such experiments are lacking in other species, presumably because they are very difficult to perform. The controversy concerns both the results of reserpine

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treatment and the influence of beta-blockers like pronethalol and propranolol. First, reserpine treatment does not affect the response to Ca in the dog (whole heart preparation, refs. 12, 30, 31) and it gives conflicting results in the rabbit (atrium preparation, refs. 19 and 38 vs 14 and 15). Second, the results of beta-blockade by pronethalol or propranolol are also not very consistent. The problem here is that these substances themselves may introduce chronotropic responses. Pronethalol increases the CL in the guinea-pig (atrium preparation, ref. 28). Propranolol increases the CL in the rabbit according to Jurevics and Carrier [14], but it does not according to Freeman and Turner [9] (atrium preparations). However, these studies were performed at different temperatures. Propranolol has also no significant effect in the dog (ref. 2, in vivo study). Since the chronotropic response to Ca is Ck-dependent in rabbit [21] and guinea-pig (Fig. 1, this study) and since this response is also dependent on the temperature (ref. 21, rabbit; unpublished results, guinea-pig), all these literature data become very confusing. A decrease of the chronotropic response to Ca, due to propranolol, is reported in the dog (whole heart preparation, ref. 12), but not in the rabbit (atrium preparation, ref. 14). Pronethalol did not change the chronotropic response to Ca in the guinea-pig (atrium preparation, ref. 28), but in this last study pronethalol had increased the CL substantially, prior to the increase of the Ca concentration. Since we showed (Fig. 1) that longer CLs are related with larger chronotropic responses to Ca, it is not clear whether or not the conclusion of Schaer [28] - - the chronotropic response to Ca is independent of beta-blockade - is correct. The literature upon this topic is obscured by the use of different species, different types of preparations, different experimental temperatures, different composition of (su)perfusion fluids and different methods in obtaining beta-blockade, i.e. different substances and, particularly, different concentrations. A comparison of our beta-blockade study with propranolol in the guinea-pig right atrium preparation with the previous study of Schaer (ref. 28, same species, same preparation, same temperature, propranolol instead of pronethalol, in very different concentrations) leads to the conclusion that we did not observe a chronotropic effect of propranolol itself (1.7 × 10 -7 M =0.05 mg/1), whereas Schaer [28] did observe a negative chronotropic effect of pronethalol (1.0 and 5.0 mg/l). We presume that this difference is caused by the different concentrations. Hachisu and Koeda [11] have shown, in isolated rabbit atria, that atrial rate was decreased by the application of beta-blockers at a 100-1000 times higher dose of beta-blocking activity. Covariance analysis showed no differences between Ca responses with and without propranolol. We conclude that the chronotropic action of Ca is a direct one without interference of adrenergic mechanisms in the guinea-pig. We did the same observations in the rabbit. If the results are compared with the Ca responses in the rabbit (ref. 21, Fig. 1C) it becomes clear that the same responses in the guinea-pig occur at shorter CLs. Variability exists concerning the site of the primary pacemaker in different preparations [4,24], Variability exists also in the relative contribution of the 3 time-dependent pacemaker currents (if, isi and i~) to the process of diastolic depolarization [7]. We consider the CL-dependence of the Ca response as the reflection of these variabilities, i.e. faster preparations are less responsive to Ca because currents other than i si a r e the most relevant to their pacemaking process,

362

while slower preparations show the opposite. In these terms it may be stressed that there seems no disagreement between Brown et al. [7] and Irisawa and Noma [13] concerning the fact that isi becomes more important for the process of diastolic depolarization when the diastole and thus the CL becomes longer.

Magnesium The response to Mg is somewhat larger but in the same range as in the rabbit. Since we showed previously that this response may be considered as independent of the response to Ca [21] we focused now on the previously predicted interference with autonomic responses [22]. Fig. 2 shows the action of Mg in cancelling a pacemaker shift to a latent pacemaker. The main effect of Mg, i.e. decrease of the rate of diastolic depolarization, is obvious in all parts of the sinoatrial node. However, these effects are smallest in the primary center [22,23]. The difference in intrinsic theoretical CL (i.e. the CL of the different pacemakers if they were allowed to beat at their own CL without being discharged by the primary pacemaker) between primary and latent pacemakers is thus enlarged. By this mechanism the chances for pacemaker shifts to occur, as a result of other interventions such as the addition of Adr or Ach, diminish. Mg thus not only interferes with the occurrence of pacemaker shifts, but also with the magnitude of the chronotropic responses to other interventions [21-23]. In the case of Fig. 2, little Mg was already effective in cancelling a pacemaker shift due to the action of Adr (CL Fig. 2C - CL Fig. 2A = 9 ms; the full response in steady-state amounted to 114 ms).

Adrenaline The response to Adr is smaller in the guinea-pig sinoatrial node than in the rabbit. The response to 6 × 10 -7 M is, averaged, - 4 8 + 5 . 6 (S.E.) ms in the guinea-pig (taken from data of Fig. 3) and - 104 + 7.0 (S.E.) ms in the rabbit [24]. However, the averaged spontaneous CL, prior to the addition of Adr was 244 ms in the guinea-pig sinoatrial node and 398 in the rabbit [24]. Bgth in the rabbit [24] and in the guinea-pig (Fig. 3) the response to Adr is CL-dependent. Adr increases isi, i K and if, which was measured in voltage clamp experiments with small preparations from the rabbit sinoatrial node [6,7]. If it is assumed (1) that the relative contribution of isi, i K and if to the process of diastolic depolarization is different in preparations with shorter and longer spontaneous CL and if it is further assumed (2) that Adr does not increase those 3 pacemaker currents equally, then one can understand that the chronotropic response to Adr is different in preparations with a shorter and longer spontaneous CL. Adr increases Vmax, hyperpolarizes MDP and increases the action potential amplitude of the guinea-pig primary pacemaker (Fig. 4). On the contrary, Adr decreases the action potential amplitude of the rabbit primary pacemaker [18]. We consider such a difference between species as an argument for a different impact of one or more membrane currents for the generation of action potentials in these two species. Another difference between rabbit and guinea-pig is the absence of an Adr-induced center with a constant intranodal location in the latter. Adr (6 × 10 -7 M) definitely induces a pacemaker shift towards the tail of the node in the rabbit

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[17,18,24], where the cells have larger action potential amplitudes and higher Vm~x values [18]. In the guinea-pig we observed variable results. If shifts occurred they were always in the inferior direction, but the cells leading during exposure to Adr were not constant with respect to location from preparation to preparation. We verified by light microscopy [1] that the Adr-induced shifts took place to atrial cells. So we consider the function of Adr in the guinea-pig sinoatrial node to accelerate the primary center. Unlike the rabbit, a further acceleration by Adr cannot be achieved under sinoatrial node control. Above a critical Adr concentration, the pacemaker will shift from the primary center to a location outside the sinoatrial node. This presumably makes the guinea-pig sinoatrial node, compared with the rabbit, less susceptible for in vivo frequency regulation via the sympathetic part of the autonomic nervous system. The background may be a less specialized innervation, or a less specialized distribution of beta-receptors within the guinea-pig sinoatrial node compared with the rabbit.

Acetylcholine The response of the guinea-pig sinoatrial node to Ach concentrations lower than 5 × 10 -6 M (Fig. 5) is equal to the response in the rabbit [18]. However, there are also marked differences: (1) intranodal blocking phenomena occur. This is not observed in the rabbit. The Ach-induced center is located within the light microscopic central nodal cells [1]. In the rabbit it is located within the peripheral nodal cells [18,24]. (2) Ach reduces several electrophysiological parameters in the primary center of the guinea-pig substantially (Figs. 7, 8A). These changes are absent in the rabbit or they are only minor effects [18]. This last difference may point to a relatively specialized vagal innervation of the guinea-pig primary pacemaker compared with the rabbit. The response of the guinea-pig sinoatrial node to concentrations higher than 5 x 10 - 6 M Ach is different from the rabbit sinoatrial node. In the guinea-pig there seems, contrary to the rabbiL to be some kind of a critical Ach concentration. When this critical concentration is exceeded, it seems that the diastolic depolarization is not steep enough to reach the threshold-level and it seems like one out of each two cycles is dropped (Figs. 6, 8A and B). When this happens, the onset of the diastolic depolarization is steeper than the last part (see the action potentials in the presence of Ach in Fig. 8A and especially Fig. 8B). Ach is believed to bring about its negative chronotropic effect mainly by interaction with an Ach-induced K-specific current [25,26], not to be confused with the time-dependent pacemaker current i K. The Ach effect o n isi is still a matter of controversy. In the voltage clamp experiments of Noma and Trautwein [20] i~, is not affected. Intranodal differences in responsiveness to both Ach addition [18,24] and vagal stimulation [33] exist (see for review Brown [5]). From our experiments (Figs. 7 and 8) it appears that Ach is able to bring about different effects not only in magnitude, but also in sign within one and the same sinoatrial node (Fig. 8A and B). In the primary center, slow response action potentials disappear and are changed into short action potentials without plateau phase and with an increased upstroke velocity. Amplitude, APD50 and D D R are decreased substantially (Fig. 8A). In the

364 A c h - i n d u c e d center (Fig. 8B), however, the only big change is the decrease of D D R . W e assume that in the discussion a b o u t the interference of Ach with i ~, the choice of the preparation, i.e. which part of the n o d e is selected for a small p r e p a r a t i o n for voltage clamp experiments, is of vital importance. O u r experiments permit no conclusions to be d r a w n as to whether the Ach effect in Fig. 8A is caused by an increase i n K ÷ - c o n d u c t a n c e a n d / o r a decrease in i~i. The p r e d o m i n a n c e of the action of Ach over the action of A d r is the same as in the rabbit [17,18,24].

Acknowledgements The advice a n d help of Elly Besselsen, W i m Bleeker, A n t o n v a n G e n t , H a r m Louwes, A l b e r t Mackaay, A r n o l d Meijer, Willem Schreurs a n d Ben Treytel are gratefully acknowledged. A n t o n i van G i n n e k e n is t h a n k e d for his critical reading of the manuscript.

Abbreviations Ach, acetylcholine; Adr, adrenaline; APD50, action potential duration from 50% depolarization till 50% repolarization (ms); APD100, action potential duration from 50% depolarization till 100% repolarization (ms); CL, cycle length (ms); DDR, diastolic depolarization rate (mV/s); if, inward membrane current activating on hyperpolarization [5]; i K, time-dependent outward membrane current [5]; isi , slow inward membrane current [5]; MDP, maximum diastolic potential (mV); SACT, sinoatrial conduction time (time between the moment of 50% depolarization of a cell, impaled by a microelectrode, till the moment of the steepest deflection of the (extracellular) atrial electrogram (ms); S.D., standard deviation; S.E., standard error of the mean; Vhfamp,.membranepotential halfway the maximum diastolic potential and the top of the action potential (mV)i Vmax~maximum dV/dt of the upstroke of the action potential (V/s).

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