Com/.~. Biochem. Physiol. Vol. 96C, No. 1, pp. 147-149, 1990 Printed in Great Britain

0306-4492/90 53.00 + 0.00 0 1990 Pergamon Press plc

CONTROL OF HEART RATE IN THE ADULT, AQUATIC NOTOPHTHALMUS

VIRIDESCENS VIRIDESCENS

RUTHANNE B. PITKIN* and KATHRYN MADIO BONNET?

Biology Department, Allegheny College, Meadville, PA 16335, U.S.A.

(Received 17 January 1990)

Abstract-I. We investigated the role of the autonomic nervous system in the control of the heart rate using an isolated heart preparation.

2. Addition of the parasympathetic blocker, atropine, to the organ bath resulted in an increase in heart rate as expected.

3. Addition of the sympathetic blocker, ergotamine, to the organ bath showed no change in the heart rate.

4. Addition of the sympathtic blocker, propranolol, to the organ bath resulted in the expected decrease in heart rate.

5. Both the sympathetic and parasympathetic nervous systems appear to play a role in the control of the heart rate.

INTRODUCTION

Although the role of the sympathetic and para- sympathetic branches of the autonomic nervous sys- tem have been investigated in anurans (Ask, 1983; Brady, 1964; Lillo, 1979; Stene-Larsen and Helle, 1978, 1979), the control systems of caudate hearts have not received much attention. The aim of this study was to try to define the role of the autonomic nervous system in the control of heart rate in the red-spotted newt using isolated hearts.

Specialized cardiac pacemaker cells in the sinus venosus of the amphibian heart, like the sinoatrial node of the mammalian heart, initiate the heart beat (Brady, 1964). Fibers of the autonomic nervous system are responsible for modulating the rate at which the heart beats. In frogs, sympathetic nerve impulses increase the heart rate while para- sympathetic impulses decrease the heart rate (Brady, 1964). The fibers of the sympathetic nervous system are adrenergic, i.e. they release catecholamines at junctions with effector cells, but parasympathetic fibers are cholinergic, releasing acetylcholine. Ac- cording to Nilsson (1983), the amphibian heart is innervated by parasympathetic vagal fibers and by fibers of the sympathetic chain ganglia. These two types of fibers join near the cranium to form the vagosympathetic trunks. The branches of these nerve trunks enter the heart at the sinus venosus (Nilsson, 1983). Sterne-Larson and Helle (1978) report that in Rana temporaria there is a dense network of sympathetic terminals on the sinus venosus and the ventricular side of the auriculoventricular groove, as well as throughout the atria.

Present addresses: *Department of Biology, Shippensburg University, Shippensburg, PA 17257, U.S.A. Telephone: (717) 532-1401, and tWright State University of Medi- cine, Dayton, OH 45435, U.S.A.

Lillo (1979) attempted to define the nature of the autonomic control of the heart rate of Rana cates- beiana during submergence and emergence using atropine sulfate, a vagal blocker; propranolol HCl, a beta adrenergic blocker; and phentolamine HCl, an alpha adrenergic blocker. Lillo (1979) found that atropine produced a long-lasting increase in heart rate; propranolol produced a prolonged heart rate decrease; and phentolamine resulted in a decrease in arterial pressure and initiated profound, irreversible, bradycardia. During submergence trials, the bullfrog showed a bradycardia that quickly returned to or was higher than the presubmergence level during emergence (Lillo, 1979). Adult red-spotted newts also show a bradycardia upon submergence in normoxic and hypoxic water, but the recovery was slower than for the frogs upon emergence (Pitkin, 1980).

The abolition of the submergence bradycardia with atropine led Lillo (1979) to conclude that brady- cardia in bullfrogs is dependent on parasympathetic cardiac inhibition. With the sympathetic blockers, propranolol and phentolamine, the heart rate de- creases in both the nonsubmergence and the sub- mergence trials. Using the nonsubmergence results, Lillo (1979) reports that presubmergence heart rate levels reflect a balance in the actions of both divisions of the autonomic nervous system.

Ferlan (1984) performed a study similar to that of Lillo (1979), using adult, aquatic red-spotted newts, Notophthalmus viridescens viridescens. Instead of monitoring heart rate and blood pressure via a cannula, Ferlan implanted two stainless steel wire electrodes in each subject to record heart rate data. Neural blocking tests were conducted using intra- peritoneal injections of the same drugs and using forced submergence and emergence techniques simi- lar to those used by Lillo (1979). Unlike Lillo (1979), Ferlan found significant changes in the nonsubmerg- ence drug tests only in the atropine-treated group.

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148 RUTHANNE B. PITKIN and KATHRYN MADIO BONNET

Propranolol and phentolamine had no significant effect on the heart rate in the newts in Ferlan’s study. Although problems in methodology prevented her from making conclusions about the role of the auto- nomic nervous system in heart rate control in newts during submergence and emergence, Ferlan did con- clude that atropine is an effective vagal blocker in newts.

This study used isolated heart preparation because they eliminate factors such as hormonal influences, pressure responses, and central nervous activity. To test for parasympathetic activity, the hearts were exposed to the competitive vagal antagonist, atro- pine. Blocking the vagal receptors is expected to lead to a heart rate increase. To test for sympathetic activity, the hearts were exposed to ergotamine, an alpha sympathetic receptor antagonist or propranolol, a beta adrenergic receptor antagonist. Blocking the sympathetic receptors is hypothesized to result in a decrease in heart rate.

MATERIALS AND METHODS

Thirty adult, aquatic red-spotted newts were acquired from Berkshire Biological, Florence, MA, in January 1987. All newts were kept in an environmentally controlled room at 15°C k 5°C with fluorescent lighting under a 168, 1ight:dark photoperiod. The newts were kept in an aerated aquarium containing 5 cm of dechlorinated tap water and a few rocks for climbing. The newts were fed red worms regularly.

Prior to surgery, each newt was anesthetized by sub- mersion in a 0.1% solution of MS222 (ethyl-m- amino-benzoate, Sigma E 1626) with pH maintainedVnear 7 with TRISMA 7.4 (Siama T-8508). When the newt was unresponsive, its heart was surgically removed. The bulbus arteriosus was cut and tied off with cotton thread. The heart was completely severed from the body by making cuts at the base of the vagus and the sinus venosus. After the heart was removed, a small wire hook, made from a size 0.15 insect pin, was passed through the tip of the ventricle.

A 20 ml organ bath (Harvard Bioscience) was used to test and maintain each isolated newt heart. The Frog Ringers I solution (Oakley and Schaffer, 1978) in the organ bath was maintained at 15°C with a Lauda/Brinkman circulating water bath. The Ringers was aerated throughout each drug trial. The heart was anchored in the organ bath using the thread from the bulbus arteriosus. To record the heart rate, a thread from the hook in the ventricle was attached to a Grass FT.03 force displacement transducer connected to a 7Pl22 DC amplifier within a Grass polygraph.

Only one drug was tested on each heart. Each drug trial consisted of three 15 min phases. During the first phase, the heart rate was monitored while the heart was in Frog Ringers. In the second phase, 5 ml of the Ringers solution was drained and 5 ml of a stock solution of one of the drugs was added. The concentrations of the drugs in the organ bath were 8 mg/l of atropine sulfate (Sigma A 0257) 8 mg/l of propranolol hydrochloride (Sigma P 0884). and 10 mg/l of ergotamine tartrate (Sigma E 6875). After monitoring the heart rate with the drug in the solution, the organ bath was drained and refilled with fresh amphibian Ringers solution. The heart rate in Ringers solution alone was then monitored in phase three of the drug trial.

Five random 60 set readings were taken for each phase of each trial and were averaged to give the number of heart beats per min. After collecting the heart rate data for all 10 specimens in each drug group, the heart rates for each drug were analyzed using a one-way analysis of variance with

repeated measures, followed by a Neuman-Keuls Multiple Comparisons Test, using the Psycho-Stats computer pro- gram (Anderson, 1986). A p value of less than 0.05 was considered significant.

RESULTS

The mean heart rate (*SD.) during atropine exposure (47.8 f 2.7 beats/min) was significantly higher than the mean heart rates either before (43.2 _+ 2.5 beats/min) or after (42.6 _+ 2.6 beats/min) atropine exposure (ANOVA, F2,,49 = 1571, p < 0.001). The Neuman-Keuls analysis also revealed a signifi- cant difference in the mean heart rates before and after atropine exposure.

The mean heart rate during propranolol exposure (37.7 * 10.1 beats/min) was significantly lower than the mean heart rates either before (42.5 f 8.5 beats/min) or after (41.8 f 8.6 beats/min) propranolol exposure (F2,,49 = 53.9; p c: 0.001) and Newman-Keuls.

There were no significant differences in the mean heart rates in the ergotamine trials (F2,,49 = 0.522; p > 0.05). The mean heart rates before, during, and after ergotamine exposure were 44.2 f 6.8, 45.0 f 2.3 and 44.6 &- 2.3 beats/min, respectively.

The mean heart rates in amphibian Ringers at 15°C before drug exposure in the atropine, propranolol, and ergotamine groups (43.2 + 2.5, 42.5 + 8.5 and 44.4 f 6.8 beats/min, respectively) are similar to mean heart rates of intact newts calculated to be 39.2 from the regression equation of Pitkin (1980).

DISCUSSION

Atropine in the heart bath led to the same tachy- cardia response in newt hearts as reported by Lillo’s (1979) study on intact bullfrogs and in Ferlan’s (1984) study on intact newts. These results indicate that atropine effectively blocks the vagal receptors of the parasympathetic nervous system of N. v. viridescens. The significant differences in mean heart rate before and after atropine exposure are probably due to the very small variability in the data which result from averaging the five samples of the records for each newt, and then averaging heart rates for the 10 different newts.

The effectiveness of sympathetic alpha and beta adrenoceptor blockers is influenced by many vari- ables such as temperature, metabolic state, season of the year, and species (Stene-Larsen and Helle, 1979). In this study, exposing the heart to the general beta blocker, propranolol, did result in the expected decrease in heart rate, but the alpha blocker, ergotamine, had no effect. The significant decrease with propranolol is similar to the results with bull- frogs (Lillo, 1979). Stene-Larsen and Helle (1978) also report that propranolol inhibited the chrono- tropic response to atropine in isolated atrial strips of hearts from R. esculenta, R. pipiens and R. tempo- raria. However, Ferlan (1984) did not observe a decrease in mean heart rate when she injected newts with propranolol. This difference between Ferlan’s fall-collected newts and the results of this study’s

Autonomic control of heart rate in newts 149

winter-collected newts may have been due to dosage differences or seasonal variations.

There can be many interpretations of the lack of effect of ergotamine. Gilman et al. (1980) state that ergot alkaloids not only act as alpha adrenergic blockers but can be partial agonists as well. Some other possible explanations are that the alpha sym- pathetic receptors in newts may not be affected by ergotamine, an improper dosage of ergotamine may have been used, or there may be a small or insignifi- cant population of alpha receptors in newt hearts. In his study on the nature of adrenergic receptors in lower vertebrates, Ask (1983) examined the effects of phenylephrine and adrenalin, two sympathetic agon- ists, on the heart rates of frog atria1 strips. Ask found that the effects of these agonists could not be blocked by the alpha blocking agent phentolamine. Ask con- cluded that the alpha receptors in the atria of the frog have a minor, if any, role in heart rate control in frogs. Ask’s findings in frogs agree with the results of this study on newts.

Although there are many uncertainties about exactly what a drug added to a bath does to the heart, the tachycardia response to the parasympathetic blocker, atropine, indicates that in N. v. viridescent the parasympathetic division of the autonomic nervous system does play a role in the control of the heart rate. The bradycardia in response to the addition of propranolol supports our hypothesis that there is some role for the sympathetic fibers as well.

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