Prostaglandins and pacemaker activity in isolated guinea pig SA node

PROSTAGLANDINS

PROSTAGLANDINS AND PACEMAKER ACTIVITY IN ISOLATED GUINEA PIG SA NODE Kenneth William Richard

R. Courtney, T. Colwell, A. Jensen.

Ph.D. Ph.D. Ph.D.

Cardiovascular Pharmacology Laboratory Department of Toxicology SRI International Menlo Park, California 94025

ABSTRACT Prostaglandins PGE2, PGE1, PGF2,, and PGAl substantially increase automaticity in SA-nodal, right atrial preparations excised from guinea pigs. This natural pacemaker tissue is sensitive to nanomolar doses of PG with, for example, 10m8 M PGE,, increasing SA rate by about 20%. If these preparations are pretreated with 2 PM indomethacin, a blocker of endogenous prostaglandin synthesis, then spontaneous rate drops and subsequent rate increases due to PGE2 administration can be more easily demonstrated. Guinea pig pacemaker tissue differs from similar rabbit tissue not only in that it is directly responsive to in that PGE2 does not depress the absolute response PGE2, but also to transmural stimulation (adrenergically mediated rate increase). The positive chronotropic responses to PGE2 also occur when the guinea pig tissue is pretreated in 0.6 PM propranolol, which causes blockade of beta-adrenergic receptors. The pacemaker myocardium in the guinea pigs thus appears to be directly stimulated by exogenous PGE2 at very low doses. The observation that 2 PM indomethacin reduces SA-nodal rate suggests the presence of a very sensitive, functionally important, PGE-like system which modulates heart rate in this mammalian species. INTRODUCTION Prostaglandins (PGs) modulate the sympathetic effector system in some mammalian hearts. Recent reports have stressed the role of PGs as modulators of the sympathetic neurotransmission process in rabbit heart using both sympathetic nerve stimulation (I) and transmural stimulation schemes (2). Results of these studies have indicated that PGs act primarily to reduce transmitter release from nerve terminals, that is, that they exert a primarily presynaptic effect on Reported here are positive chronothe nerve-effector junction (3). tropic effects of PGE2 on guinea pig sinoatrial (SA) node preparations, The fact that effects that appear to be postsynaptically mediated. PGE2 is effective in nanomolar doses, a dose range many times smaller than those used in a previous report of such PGE2 actions on guinea

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PROSTAGLANDINS

pig atria (4), suggests the presence of a very sensitive PGE-like system in this mammalian species. Experiments a prostaglandin synthesis inhibitor, also support this

endogenous with indomethacin, hypothesis.

METHODS Two New Zealand rabbits (2 to 3 kg) and 12 guinea pigs (300 to 400 g) were sacrificed by a blow to the neck and bled, and their hearts were excised. A segment of tissue containing the SA node and attached right atria1 myocardium was dissected and mounted on a Grass strain gauge (Model FT03C) in a Plexiglas tissue bath, Contractile tensions developed by atria1 muscle attached to a lever arm of a Grass Model FT03C force transducer triggered a Grass Tachograph (Model 7P4), giving a continuous monitoring of SA rate. The tissue bath and the Tyrode’s solution were maintained at 34 f lo C and bubbled with 35% oxygen and 5% carbon dioxide. The composition of the Tyrode’s solution was as follows (in mM): 137 NaCl, 3 KCl, 1.8 CaCl2, 1.1 MgCl2, 12 NaHC03, and 5.5 glucose. Tyrode’s solutions flowed through the 5-ml tissue bath at rates of 2 to 5 ml/min. The autonomic blocking agents used were atropine sulfate (Baker Chemical Company) and propranolol-HCl (Inderal, Ayerst) at doses of 1 mg/liter (1.4 MM) and 0.15 mg/liter (0.6 PM), respectively. The norepinephrine (L-arterenol HCl) source was Sigma, PGE;! was obtained from Ono Pharmaceutical, and PGE1, PGF2, and PGAl from Upjohn. We used the transmural stimulation (TS) scheme of Amory and West (5), which permits assessment of the function of autonomic effector junctions for heart rate control without requiring the dissection of an SA preparation with an intact functional nerve (e.g., Ref. 6). A digital stimulator (F. Haer, Pulsar 6i) delivered 2-set-long trains 10 Hz unless otherwise indicated; of brief pulses (pulse frequency, pulse amplitude, 8 to 10 volts; pulse duration, 0.3 msec). These stimulating procedures activated the postganglionic autonomic nerve fibers without exciting the SA cells directly, thereby causing the release of both acetylcholine and norepinephrine, which modulate The muscarinic blocking agent atropine sulfate pacemaker activity. was present in all experiments at 1.4 PM, effectively eliminating cholinergic modulation of automaticity in these preparations. RESULTS We first repeated the essential features of the study by Park, and Vincenzi (2) on PGE2 effects on autonomic transmission. Dyer, We .found that the adrenergic system of SA rate control was depressed, using as the other investigators had rabbit preparations and the TS scheme (5) for assessing the efficacy of this adrenergic effector junction. Figure 1 shows that 3.5 PM PGE, depressed the rate response to ~-HZ transmural stimulation. The response to 50-Hz transmural stimulation was not depressed to the same extent, which agrees with observations in previous reports on the interaction of stimulation frequency and these PGE2 effects (2,7). Basal discharge rate was

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reduced slightly all in another

in this preparation

preparation exposed

to

(Figure I) and did 2 uM PGE2.

not

change

at

3.5 /JM PGE2

E

200

7 z

LT 160

a a ld ZJ

0 TIME

Figure

I

Rabbit sinoatrial rate is monitored using right atria1 tension recordings to trigger a ratemeter. PGE, (3.5 PM) does not affect rate (shaded region) but does suppress the response of this preparation to ~-HZ transmural stimulation; such stimulation increased rate by 25% before the prostaglandin was applied. lndomethacin (2 PM) also present.

Then we conducted similar experiments using the more stable guinea pig SA node preparation, which gave very different results (Figure 2, left) with the same drug and dose. Basal rate increased significantly (from 215 to 250 beats/min in this example) when the SA nodal tissue was treated with 2 MM PGE2, and the absolute rate response to TS was slightly potentiated. The absolute positive chronotropic response to TS did not substantially change in several other experiments with guinea pig preparations; Table 1, column (d) [c.f. column (b)], documents these results for six preparations. The impression that the adrenergic response was depressed, achieved by expressing the TS response as percentage change over prestimulus rate while in PGE;! [column (e)], was always accounted for by large increases in basal rate rather than by reductions in the absolute adrenergic response. These basal rate

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RATEMETER SCALE-bpm

r

A

;

300

B qm

‘00

E 250

-

+TS

0.0

$ 5

.O

SAR o-0 \ 0.0.

LT ;z’ E 1;; 200:k? 65

2 FM PGE2

2 uM PGE, 0

Figure

454

2

r / / /I

-TlME

Guinea pig sinoatrial rate is increased by 2 $4 PGE, perfusion. 2A - Ratemeter record before (left) and during (right) PGE2 exposure. Absolute rate response to transmural stimulation (arrow) is slightly increased during prostaglandin exposure. 28 - Basal rate and rate increase response to transmural stimulation is plotted against time, with PGE2 exposure trials indicated by cross-hatched areas both before (left) and during (right) treatment with 2 PM indomethacin, an endogenous prostaglandin synthesis inhibitor applied 75 minutes earlier.

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Table

1.

Effects

of

(4

PGE2 on Transmural

(cl

b)

basal rate 1.0”

peak to

response TS

w

Pre-PGE2 exposure rates normalized rates averaged 184 beats/min.

**

Mean f

for

six

Rate

Responses

(d)

2 PM PGE2 rate 1.24 f .06

*

S.E.M.

Stimulation

to

(4

peak response to TS in PG 1.34 f .06

allow

data

(d)/(c) 1.08 f

averaging,

.03

actual

preparations

increases occurred within a few minutes after perfusion lines were switched to allow PGE2 entry into the bath, and the rate increase persisted during up to 15-min exposures to PGE2. More than 60 min was required for wash-out of the PGE effects, making it difficult to do detailed dose/response studies on any given preparation. Endogenous PG synthesis and release may be contributing to basal rate in these excised guinea pig preparations. To test this hypothesis, we exposed guinea pig SA tissue to 2 PM indomethacin, a potent inhibitor of PGE synthesis (8). Figure 2B shows an example of such an experiment; basal rate dropped from 215 to 177 beats/min in this example. After indomethacin pretreatment, exogenous PGE2, applied at the same 2 FM concentration, still produced an increase in rate to about the same level as in the first trial (260 beats/min). The response to exogenous PGE2 was enhanced by pretreatment with indomethacin; this enhancement is accounted for by depressed spontaneous rate in the presence of indomethacin (see Table 2). Table 2 documents these chronotropic effects of PGE2. Doses of PGE2 in the uM range are supramaximal for this rate effect so trials at both 1 and 2 pM have been pooled for statistical purposes. If a first order binding scheme is fitted to the results of Table 2 (in indomethacin),the half-maximaldoseof PGE2 is found to be about 3 nM.

increase automaticity Prostaglandins PGE1, PGFp,, and PGAl also of sinoatrial tissues excised from guinea pigs, with lOme M concentrations increasing rate by about 30% (in indomethacin). We have already described very different results to exogenous PGE2 application for guinea pig SA preparations compared with rabbit preparations. Hedqvist (3) has suggested that PGEs act at a

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PROSTAGLANDINS

Table

Before

2.

Comparison of PGEz Effects on Guinea Pig Before and During lndomethacin Treatment

indomethacin

treatment: 1.01

Control l-2 During

2 $4

SA Rate

indomethacin

x lo+

M PGEz

1.19

f

.06

(4)

.93

t

.a3

(6)

f

.07 (2)

treatment:

Control 1 x lO-g

M PGEz

1.01

1 x lo-*

M PGE2

1.17 + .05 (2)

l-2 x lo+

M PGE2

* Rates normalized to allow data averaging, number of preparations in parentheses.

1.23 f .04 (6)

mean rate

190 beats/min,

presynaptic site to inhibit the release of sympathetic neurotransmitters, basing his concepts largely on experiments with rabbit heart. Since guinea pig preparations respond so differently to PGEz, we designed several experiments to localize the response site in the guinea pig system. Experiments involving applications of exogenous norepinephrine and propranolol (which blocks the beta-receptor for norepinephrine) were therefore used to help identify the locus of the PGE;, response. Figure 3 shows that propranolol, at a dose that effectively blocked norepinephrine receptors, did not block the rate-increasing response to PGE2, confirming similar observations by Bhagat et al. (4). Also, exposure of this preparation to a maximal dose of PGE2 (2 uM) did not render the tissue insensitive to further rate-increasing effects of exogenous norepinephrine. DISCUSSION Many recent publications and reviews dealing with the mechanisms whereby PGEs modulate autonomic neurotransmission in the heart have implicated a presynaptic rather than postsynaptic locus for PGE actions (1,2,3). For instance, PGEl and PGE2 depress the positive chronotropic response to sympathetic nerve stimulation in isolated rabbit heart (I). On the other hand, direct effects of PGEz on rabbit heart rate, in the absence of sympathetic nerve stimulation, have not been observed (1,3). We have verified the correctness of these reports using excised rabbit SA nodal preparations (Figure 1) and have repeated the studies on guinea pig preparations. The

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220

2 /JM PGE,

2 PM PGE2

+ 0.6 PM PROPRANOLOL

l

0

+o’

O-0

\

12 min 0 TIME

Figure

3

The beta-blocker propranoioi does not block the rate response to 2 pM PGE2. The rate response to application of 2 ug norepinephrine to the s-ml bath volume is indicated by the filled circles; this tissue can still respond to exogenous norepinephrine when a maximal dose Rate responses to exogenous norepiof PGE2 is present. nephrine nearly mimicked the rate increases due to lndomethacin transmurai stimulation in ail trials. pretreatment, 2 vt4.

results with guinea pig preparations are substantially different in that (I) prostagiandins clearly increase SA rate, and (2) PGE2 does not depress the absolute adrenergic response (Figure 2, Table 1). and Usardi (9) have also shown that chronotropic Berti, Lentati, responses to PGEl differ between rabbit and guinea pig preparations in the same way that we found responses to PGE2 to differ, and Bhagat et al. (4) have shown that uMoiar doses of PGE2 increase guinea pig heart rate. Likewise, Kaumann and Birnbaumer (ii) have shown that VMoiar doses of PGEl increase kitten sinoatriai rate. These results, and ours using much smaller concentrations of PG,

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should serve to caution us about accepting present presynaptic mode1 s for PGE action on autonomic effector junctions as complete or sufficiently general to allow predictions of PG effects in humans. The information on guinea pig preparations presented in Table 1 shows that PGE appears to reduce the adrenergic response to transHowever, basal rate [Table 1, column (c)] increases mural stimulation. in PGE2 account for the apparent TS response reduction indicated in column (e), not an absolute reduction in the adrenergic rate response The authors do not consider this to TS [compare column (d) to (b)]. a “response inhibition”, although other experimenters might argue this point based on a report of column (e) information only. The exquisite sensitivity of these guinea pig pacemaker preparations to prostaglandins has not been adequately spelled out in previous publications. Supramaximal doses of prostaglandin E2 will increase SA rate by some 20% before indomethacin treatment and by more than 30% after incubation in 2 uM indomethacin. We estimate a half-effective dose of PGE2 of only 3 nM, which approaches PGE levels Prostaglandins PGE1, PGExe, and PGA,, found in human plasma (10). when applied at the 10 nM dose level, also have strong positive If the hypothesis is advanced that indomethacin chronotropic effects. reduces SA rate because it eliminates endogenous prostaglandins, then the results of Table 2 show that about 1 nM PGE2 wi 11 restore SA rate to that observed before indomethacin treatment; that is, 1 nM PGE2 may be equivalent in chronotropic effect to prostaglandin presumably All of these observations suggest lost by indomethacin treatment. a sensitive and important postsynaptic role for endogenous PGE2 in some mammalian species. Because rabbit and guinea pig preparations differed markedly in to test their responses to exogenous PGEs, we though it important the hypothesis that the site of action of the PGEs might be the postOur ‘first determination was of synaptic beta-adrenergic receptor; Figure 3 showed that whether propranolol occludes the PGE2 response. propranolol does not block the heart rate increase due to PGE2 perfusion since the rate increased by 26% in the presence of propranolol compared with 22% without (indomethacin pretreatment). This result and those of others (4) would suggest that PGEz-induced release of norepinephrine (a presynaptic effect) may not be responsible for the However , propranolol usually observed PGE-induced rate increase. slows basal rate in guinea pig (but not rabbit) preparations, presumably because of elimination of background beta-receptor activation by endogenous catecholamines; this circumstance might make operation of any rate-increasing factor easier. Our second question was, does a maxima1 dose of PGE2 OCOUPY all the receptor sites involved in this response so that the tissue is not capable of responding to exogenously applied norepinephrlne? to norepinephrine was still Figure 3 showed that some rate response pogsible under these circumstances;

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PROSTAGLANDINS

Klein are

and

Levey

sensitive

when

to

activated,

both

in myocardium, would account It

is

also

show

an

suggested

to

regarding pig

and act

increasing results of

intriguing

guinea

have

receptors

thereby for the

differences PGE2;

(12)

norepinephrine

note

increased of

to

cyclic

cyclic

receptor

and

and

reports Levey

of

others

report

those

AMP after

from

PGEl

responses

certain

and

(4,ll).

species

AHP accumulation

not

types

that,

cyclic AMP accumulation Such a receptor system

rate. 3 and

Klein

but

separate

respectively,

increase

heart Figure

that

preparations

accumulation

that

PGEs,

other

PGE2

to species

treatments.

ACKNOWLEDGMENTS

The this

This research authors thank

was Rita

supported Malik for

by NIH Grants her substantial

HL

19066 and contributions

HL

19076. to

study. REFERENCES

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