Evidence against cyclic adenosine 3′,5′-monophosphate (AMP) mediation of noradrenaline depression of cerebral cortical neurones

Brain Research, 60 (1973) 411--421

411

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

EVIDENCE AGAINST CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE (AMP) MEDIATION OF NORADRENALINE DEPRESSION OF CEREBRAL CORTICAL NEURONES

N. LAKE, L. M. J O R D A N AND J. W. PHILLIS*

Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg (Canada) (Accepted March 12th, 1973)

SUMMARY

Microiontophoretic studies were carried out to investigate the interaction between noradrenaline (NA) and adenyl cyclase in the cerebral cortex of cats, rats and guinea pigs. The predominant effect of NA was one of depression, and there were no differences among the species as to the numbers of NA-sensitive cells or the depressant potency of NA. This strong depressant action of NA was not mimicked by cyclic AMP or dibutyryl cyclic AMP tested on the same neurones. Prostaglandins of the E series effected antagonism of NA depression in a minority of neurones. Phosphodiesterase-inhibiting drugs, such as aminophylline and papaverine, enhanced the depression produced by NA, but were observed to have depressant actions of their own even on neurones not affected by NA. Furthermore, prostaglandins were observed to be more effective antagonists of aminophylline, rather than NA depressions. These findings suggest that the depressions produced by aminophylline and NA may be unrelated, that enhancement of NA depression by aminophylline may be merely summation of their individual inhibitory effects, and that aminophylline and prostaglandins interact owing to pharmacological actions other than those involving adenyl cyclase. Our evidence suggests that the depressant action of NA on cerebral cortical neurones is not mediated by cyclic AMP.

INTRODUCTION

Evidence for a neurotransmitter role for noradrenaline (NA) in the cerebral cortex is provided by histofluorescence studies1,16,is which reveal the presence of a large number of fine NA-containing fibres and terminal varicosities in all cortical layers. Cerebral cortical neurones are depressed by microiontophoretic applications of NA 15,26,29 when care is taken to eliminate the possibility of excitation caused by * Present address: Department of Physiology, College of Medicine, Saskatoon, Canada.

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ejection of hydrogen ions from acidic solutions of NA15,25. Rat cerebellar Purkinje cells are also depressed by microiontophoretic applications of NA 21-23, and Bloom and associates have provided evidence that this inhibitory transmitter-like action of NA is mediated by cyclic AMP 2x-23, in analogy with the action of catecholamines in the periphery 6,19. Impetus to carry out a species comparison of the actions of NA arose from the work of Forn and Krishna 14. They investigated the ability of exogenous NA to stimulate the formation of cyclic AMP in cerebral cortical slices from various species. Of particular interest to us are the data for the cat, the rat and the guinea pig. NA is most effective in the rat. The difference between control and NA-stimulated formation of cyclic AMP is highly significant for the cat and rat, but the difference is not statistically significant for the guinea pig. Similar findings have been reported by other laboratories~7, 34,3s. Whenever increases in cyclic AMP produced by NA in guinea pig cortex were observed 2s,36, they were of a much smaller magnitude (60-70 ~ of control) than the increases produced in rat cerebral cortex (400~ of control). From the Forn and Krishna study one might expect to see differences between rats, cats and guinea pigs as to the actions of NA. If one assumes the hypothesis that the effects of NA are mediated by cyclic AMP, then one might expect to observe more NA-sensitive cells, or a higher potency of NA in the cerebral cortex of rats as compared to cats or guinea pigs. The objectives of these investigations were to examine the effects of NA on cerebral cortical neurones of cats, rats and guinea pigs, and to determine if any of these actions may be mediated by cyclic AMP. For these studies we have repeated many of the experimental paradigms of Bloom and associates m,23,a9. Preliminary reports of some of our findings have appeared elsewhere 26,3°. METHODS

The animals were anaesthetized with methoxyflurane and nitrous oxide. Each animal was tracheotomized and breathed spontaneously. The rectal temperature was automatically maintained between 37 and 38 °C by means of a servo system. The effects of drugs, applied microiontophoretically, were assessed from extracellular records of neuronal firing of single cerebral cortical neurones in the somatosensory area, transcribed on a penrecorder. The techniques of current balancing at the electrode tip 35 and use of control sodium or chloride currents were utilized to distinguish genuine effects from current artefacts. The centre barrel of 7-barreUed micropipettes (overall tip diameters of 3-8 #m) was filled with 2 M NaCI for recording, while the outer barrels were filled by centrifugation immediately prior to use with the following substances: sodium L-glutamate (Baker) 0.2 M, pH 7.0; L-noradrenaline bitartrate (Sigma) 0.2 M, pH 5.0; aminophylline (Sigma) 0.2 M; papaverine hydrochloride (Marion) 0.1 M; cyclic adenosine 3',5'-phosphate (Calbiochem) 0.5 M, pH 6.0; N 6, O2"-dibutyryl cyclic adenosine 3',5'-phosphate (Calbiochem) 0.2 M, pH 6.5; prostaglandin E1 (Upjohn) 0.014 M, pH 7.8; prostaglandin E2 (Upjohn) 0.018 M, pH 6.5; sodium salt of prostaglandin E1 (Upjohn) 0.1 or 0.5 M, pH 7.0; sodium

NA DEPRESSIONAND CYCLIC AMP

413

RAT NA

CC 20

10

I

GUINEA

CAT

Nt::

1 ~in

I

PIG

NA

NA

CC

>5.\'~

.......

Fig. 1. The effects of iontophoretic applications of NA on the firing rates of cerebral cortical neurones of rats, cats and guinea pigs. Ratemeter records of neuronal firing, with the number of action potentials/see on the ordinate. Bars above records indicate the periods of application of 20 nA NA or 20 nA control anodal current (CC).

chloride, 2 M. To avoid the complications of the usual alcohol solvent prostaglandins were 'solubilized' in aqueous solution using ultrasound 21. These solutions were active when assayed on the rat fundus strip 1°,42. In addition, further studies which gave similar results were carried out using the water soluble sodium salt of prostaglandin E1 which allowed the use of a higher drug concentration a. Three rats were treated with 200 #g of 6-hydroxydopamine injected interstitially into the frontal cortex through a small hole in the skull. They were subsequently used in experiments 3-5 days later, and cortical tissue was removed at the end of the experiments and examined for NAcontaining fibres using the fluorescence histochemical technique 13.

TABLE I ACTIONS OF NORADRENALINE

ON CEREBRAL CORTICAL NEURONES

Species

Total cells

Excitation

Depression

No effect

Cat Rat Guinea pig

76 101 130

0 0 0

59 (78 %) 68 (67%) 98 (75 %)

17 (22 %) 33 (33 %) 32 (25 %)

414

N. LAKEet al.

RESULTS

Typical responses of cerebral cortical neurones in the 3 species to iontophoretic applications of NA are shown in Fig. 1. A brief (30 sec) application of 20 nA of NA caused a reduction in the firing rate which recovered fairly rapidly following the cessation of the drug current. This was a genuine depression caused by NA as control anodal currents of equal magnitude (CC) failed to influence the firing rate. The responses to NA were very similar for these species and there were no significant differences among the species as to the threshold iontophoretic current of NA required to produce depression ( c a t , 23 ± 2 nA; rat, 30 ± 4 nA; guinea pig, 26 ± 4 nA). A summary of the effects of NA on glutamate-driven or spontaneously firing neurones is given in Table I. The predominant action of NA was one of depression (70-80 % of neurones) in all species. There were no excitations observed at this pH, and the remaining ceils were either unresponsive to NA or showed only effects which could be mimicked by anodal current. Chi-square tests of contingency tables of the distributions of the effects revealed that there were no significant differences among the 3 species as to the effects of NA (P > 0.05). As suggested from studies of the rat cerebellar Purkinje cell by Bloom and associatesZ2,z3, 3a, experiments were carried out using the methylxanthine, aminophylline, one action of which is to inhibit phosphodiesterase 7, the catabolic enzyme for cyclic AMP. If the action of NA were mediated by cyclic AMP, then inhibition of phosphodiesterase should potentiate the effects of NA. The technique of intravenous theophylline administration 3a was considered to be unsuitable as theophylline given by this route caused a marked increase in blood pressure which was maintained for over an hour, thus complicating the interpretation of changes in neuronal sensitivities to drugs. For many cells, following the prior iontophoretic application for 1-3 min of a small dose of aminophylline (2-15 nA) the depression caused by a threshold dose of NA was increased. The results with aminophylline are summarized in Table II. Chisquare tests of contingency tables of the distribution of the effects revealed no significant differences among the species at the 0.01 level (P > 0.01); however, at the 0.05 level there were significantly fewer enhancements (P < 0.05) seen in guinea pigs than in the other species. A small number of trials with papaverine, a structurally unrelated drug which also inhibits phosphodiesterase, revealed no statistically significant differences between guinea pigs and the other species (P > 0.05) as to the numbers os neurones (67 %) showing enhancement of NA depression by papaverine. This data if TABLE II ENHANCEMENT OF NORADRENALINE DEPRESSION BY AMINOPHYLLINE

Species

Total cells

Enhancement

No effect

Cat Rat Guinea pig

14 19 29

11 (79%) 13 (68%) 11 (38 %)

3 (21%) 6 (32%) 18 (62 ~)

415

N A DEPRESSION AND CYCLIC A M P TABLE III ANTAGONISM OF NORADRENALINE DEPRESSION BY PROSTAGLANDIN

Species

Total cells

Antagonism

No effect

Cat Rat Guinea pig

12 15 22

5 (42%) 3 (20%) 4 (18%)

7 (58%) 12 (80%) 18 (82%)

consistent with cyclic A M P m e d i a t i o n o f the effects o f N A , b u t a m i n o p h y l l i n e was o b s e r v e d to have a very p o w e r f u l d e p r e s s a n t a c t i o n o f its o w n on all neurones, even on those which were unaffected by N A . P a p a v e r i n e also h a d a d e p r e s s a n t a c t i o n o f its own, b u t it was n o t as p o t e n t as t h a t o f a m i n o p h y l l i n e . The d e p r e s s a n t a c t i o n o f a m i n o p h y l l i n e was also o b s e r v e d on cortical neurones o f rats p r e t r e a t e d with 6h y d r o x y d o p a m i n e to d e s t r o y the N A - c o n t a i n i n g fibres 41. E x a m i n a t i o n o f cerebral cortical tissue using the fluorescent histochemical technique ta revealed the presence o f N A - c o n t a i n i n g fibres in c o n t r o l rats a n d absence o f these fibres in rats t r e a t e d with 6-hydroxydopamine. I n o t h e r p r e p a r a t i o n s , including the r a t cerebeUar cortex ~t, p r o s t a g l a n d i n s o f the

A

NA

P__G cC...

1 I

B

Min

I

NA

CC

AMINOPHYLLINE

NA

~

"" .......

'lllllllllllllll[lllJ,•

.X&\\~

pc,,#_ .c..c... p,,~G

Fig. 2. Interactions between prostaglandins, noradrenaline and aminophylline. Ratemeter records from a guinea pig cerebral cortical neurone (A and B, same neurone). Ordinate shows number of action potentials/sec. Periods of drug application are indicated by bars above the record. A: NA 20 nA with concurrent application of PG (prostaglandin El) 80 nA and CC (control cathodal current) 80 nA. B: NA 20 nA; CC (control anodal current) 20 nA; aminophylline 15 nA, with concurrent applications of PG 80 nA, CC (control cathodal current) 80 nA, and PG 80 nA; NA 20 nA.

416

N. LAKEet al.

E series have been found to antagonize the effects of NA. It has been suggested that prostaglandins may prevent hormonal activation of adenyl cyclase, thereby preventing hormone-induced increases in intracellular concentration of cyclic AMP2, z4. In view of these findings, Hoffer et al. 21 consider the antagonism by prostaglandins E1 and E2 of NA depression of Purkinje cell firing to be evidence for the mediation by cyclic AMP of the NA depression. Table III summarizes our findings with prostaglandins E1 and E2 and the sodium salt of El. The prostaglandins were most effective in the cat, where they antagonized NA depressions in 42 9/0 of the neurones; however, they were less effective in the rat and in the guinea pig. For the majority of neurones in the 3 species even high doses of the prostaglandins (up to 100 nA) were unable to antagonize threshold depressions caused by low doses of NA. Fig. 2 illustrates an interaction between aminophylline and prostaglandin (cf. refs. 39 and 40) which is typical of those investigated. The ratemeter records in A and B are from the same cerebral cortical neurone of a guinea pig. In A is shown an apparent antagonism of NA depression by prostaglandin, which is not genuine, however, as it is mimicked by an equal magnitude of cathodal current. In B is repeated the depression by NA which is not mimicked by a control anodal current. A low dose of aminophylline (15 nA) produces a marked depression of firing typical of the depressions seen also in cats and rats. A dose of prostaglandin equal to that given in A produces a genuine antagonism of the aminophylline depression (not mimicked by control current). The firing rate recovers rapidly after the cessation of the aminophylline current, and NA still has its former depressant action. This type of interaction was observed in 80 ~ of 30 neurones examined in the 3 species (Table IV). Prostaglandins E1 or E2 were significantly more effective in antagonizing depressions caused by aminophylline than depressions caused by NA for the cat (P < 0.01), the rat (P < 0.005) and the guinea pig (P < 0.05). One type of evidence which is taken as a positive indication of the mediation of the action of a substance by cyclic AMP is the mimicking of the effect of the substance by applications of exogenous cyclic AMP or its more lipid soluble and less rapidly metabolized dibutyryl derivative 33. The technique of automatic balancing of current at the tip of the micropipettes a5 was employed whenever tests with cyclic AMP or dibutyryl cyclic AMP were performed. Cyclic AMP (20-200 nA) was tested on 24 neurones in the rat cerebral cortex. A weak excitant effect was observed on some of these neurones and none were obviously depressed, even after prior application of the phosphodiesterase inhibitor 7, aminophylline. Applications of NA depressed 21 of TABLE IV ANTAGONISM OF AMINOPHYLLINE DEPRESSION BY PROSTAGLANDIN

Species

Total cells

Antagonism

No effect

Cat Rat Guinea pig

7 7 16

7 (100~) 6 (86~) 8 (50~)

0 1 (14~) 8 (50~)

417

NA DEPRESSION AND CYCLIC AMP TABLE V EFFECTS OF DIBUTYRYLCYCLIC A M P ON CEREBRALCORTICAL NEURONES

Species

Total cells

Excitation

Depression

No effect

Cat Rat Guinea pig

43 18 23

4 (9~) 1 (6~) 3 (13 %)

7 (16~) 2 (lifo) 1 (4~o)

32 ( 7 5 ~ ) 15 (83~o) 19 (83 ~ )

these 24 neurones. The remainder of the studies were carried out with dibutyryl cyclic AMP, and a summary of our findings is shown in Table V. All of the neurones tested with dibutyryl cyclic AMP were depressed by NA with threshold requirements of 20-30 nA of drug current. Though all these neurones were depressed by NA, depression by dibutyryl cyclic AMP was observed in only a small minority of cells even with doses of up to 200 nA, whether or not the current balancing technique was used. A small number of neurones were excited, but the majority was unaffected by dibutyryl cyclic AMP or showed only current effects. There was, therefore, little evidence to suggest a mimicking of the depressant action of NA by cyclic AMP or its dibutyryl derivative. DISCUSSION

The evidence presented in Table I demonstrates that the predominant action of NA on cerebral cortical cells of cats, rats and guinea pigs is one of depression of neuronal firing. The generality of this depressant action may have clinical implications, as recent investigations have demonstrated the presence of many NA-containing fibres and varicosities in the human cerebral cortexat. There were no statistically significant differences among the species as to the numbers of NA-sensitive neurones or the depressant potency of NA (as estimated from the threshold iontophoretic currents of NA which caused depression). This is somewhat surprising in view of the Forn and Krishna studyt4 which showed that NA was a very effective stimulant of cyclic AMP formation in cerebral cortical slices of the rat, less effective in the cat, and without effect in the guinea pig. This discrepancy is the first indication that the depressant action of NA may not be mediated by cyclic AMP. The comparison of' the effects of NA on neurones in cats, rats and guinea pigs is complicated by the possibility that not all NA depressions imply the corresponding existence of functional noradrenergic synapses and by the lack of certainty that the formation of cyclic AMP in vitro is an accurate index of the ability of synaptic transmitter to enhance cyclic AMP formation in vivo. Indeed, Schmidt et a l Y were unable to detect activation of adenyl cyclase by NA in vivo. Furthermore, the fact that increases in cyclic AMP produced by NA may occur mainly in glia8,t7 is another indication that activation of adenyl cyclase by NA in brain slices does not necessarily imply a role for cyclic AMP in synaptic transmission; thus iontophoretic studies of neuronal sensitivities give information which perhaps may be more specific.

418

N. LAKEet al.

The most direct tests of the hypothesis that cyclic AMP mediates the depressant actions of NA on cerebral cortical cells are provided in the experiments employing iontophoretic applications of cyclic AMP and dibutyryl cyclic AMP (Table V). It was found that even high doses of cyclic AMP or dibutyryl cyclic AMP failed to mimic the strong depressant action of NA on the same cells. The small number of depressions observed were much weaker than those produced by NA. The excitation of neurones by cyclic AMP may be accounted for by its calcium chelating action al, which can cause excitation of neurones lz. The majority of neurones, although depressed by NA, were not depressed by cyclic AMP or dibutyryl cyclic AMP, a finding which does not support the hypothesis that cyclic AMP mediates the actions of NA. Studies with the phosphodiesterase-inhibiting drugs, aminophylline and papaverine, showed that iontophoretic applications of these drugs tended to enhance the depressant action of NA (Table II). This finding is consistent with the mediation by cyclic AMP of NA depression; however, it is somewhat misleading in view of the powerful depressant action of aminophylline39 and the weaker depressant effect of papaverine. Strong depression by aminophylline was observed even in neurones which were not affected by NA. This suggests that the depressions produced by NA and aminophyUine may be unrelated, and that the enhancement of NA depression by aminophylline may be merely a summation of their individual inhibitory actions. The observation that aminophylline caused depression of neurones in the cortices of rats pretreated with 6-hydroxydopamine indicates that aminophylline probably does not act by releasing NA from nerve terminals3, 43. Experiments employing prostaglandins revealed only a weak antagonism of NA depression by these agents (27 ~o of all neurones, Table III). Since prostaglandins are thought to block the activation of adenyl cyclase by catecholaminesZ, z4, these findings do not support the hypothesis that cyclic AMP mediates the depressant action of NA. It was found that prostaglandins were good antagonists (80 ~ of all neurones, Table IV) of aminophylline depression. If the assumption is made that the depressions produced by aminophylline are due to its phosphodiesterase-inhibiting capability7, then a tonic background release of some substance which activates adenyl cyclase and whose action on this enzyme can be antagonized by prostaglandins must also be assumed. The fact that this activator of adenyl cyclase is not likely to be NA is clear from the finding that prostaglandins antagonized a significantly greater proportion of depressions produced by aminophylline than by NA, and from the fact that aminophylline depressed neurones regardless of whether or not they were influenced by NA. The failure of dibutyryl cyclic AMP 2~ and cyclic AMP to depress neurones which were strongly depressed by aminophylline, even after prior application of the phosphodiesterase inhibitor, makes it unlikely that cyclic AMP is involved in the depressant action of aminophylline. It is possible that aminophylline and the prostaglandins interact through effects on calcium rather than adenyl cyclase. For example, there is evidence that both these agents influence calcium movements2,5,10, and that Ca z+ antagonizes the actions of prostaglandins20. (The finding that prostaglandins affect the formation of cyclic AMP by cortical tissue slices in the same direction as phosphodiesterase inhibitors4 lends no

NA DEPRESSIONAND CYCLICAMP

419

support to the suggestion that their opposite effects on NA actions are mediated through cyclic AMP.) Furthermore, we have recently demonstrated3~ that known Ca 2+ antagonists reverse the depressions induced by NA; hence the most parsimonious interpretation is that the interactions observed between prostaglandins, aminophylline and NA involve calcium. In conclusion, our evidence suggests that, in contrast to the situation in the cerebellar cortex 22,23, the powerful depressant action of NA on cerebral cortical neurones of cats, rats and guinea pigs is not mediated by cyclic AMP. In addition, our observations of the effects and interactions of aminophylline and prostaglandins E1 and E2 suggest that the findings of studies on the central nervous system using these drugs should be interpreted with caution. ACKNOWLEDGEMENTS

This work was supported by the Medical Research Council of Canada. N.L. is a postdoctoral fellow of the Council. Dr. J. Pike of the Upjohn Company kindly supplied the samples of prostaglandins used in these studies. NOTE ADDED IN PROOF

In studies of cat brain stem neurones Anderson et al. (Brain Research, 49 (1973) 471-475) observed that cyclic AMP depressed many cells also depressed by NA; however, they state that data from studies with methylxanthines and prostaglandins offered 'little additional support for the hypothesis' that cyclic AMP mediated the depressant actions of NA. REFERENCES 1 AND~N, N.-E., FUXE, K., AND UNGERSTEDT,U., Monoamine pathways to the cerebellum and cerebral cortex, Experientia (Basel), 23 (1967) 838-839. 2 BERGSTROM,S., CARLSON,L. A., AND WEEKS,J. R., The prostaglandins: A family of biologically active lipids, Pharmaeol. Rev., 20 (1968) 1-48. 3 BERKOW1TZ,B. A., TARVER,J. H., AND SPECTOR,S., Norepinephrine release by theophylline and caffeine, Fed. Proc., 28 (1969) 415. 4 BERTI,F., TRABOCCm,M., BERNAREGGI,V., ANDFUMAGALLI,R., The effects of prostaglandins on cyclic-AMP formation in cerebral cortex of different mammalian species, Pharmacol. Res. Commun., 4 (1972) 253-259. 5 BIANCHI,C. P., Cell Calcium, Appleton-Century-Crofts, New York, 1968. 6 BUTCHER, R. W., HO, R. J., MENG, H. C., AND SUTHERLAND,E. W., Adenosine 3',5'-monophosphate in biological materials. II. The measurement of adenosine 3',5'-monophosphate in tissues and the role of cyclic nucleotide in the lipolytic response of fat to epinephrine, J. biol. Chem., 240 (1965) 4515-4523. 7 BUTCHER,R. W., AND SUTHERLAND,E. W., Adenosine 3',5'-phosphate in biological materials. I. Purification and properties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine Y,5'-phosphate in human urine, J. biol. Chem., 237 (1962) 1244-1250. 8 CLARK,R. B., ANDPERKINS,J. P., Regulation of adenosine 3',5'-cyclic monophosphate concentration in cultured human astrocytoma cells by catecholamines and histamine, Proc. nat. Acad. ScL (Wash.), 68 (1971) 2757-2760.

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35 SALMOIRAGHI,G. C., AND WEIGHT, F. F., Micromethods in neuropharmacology: an approach to the study of anesthetics, Anesthesiology, 28 (1967) 54-64. 36 SATTIN, A., AND RALL, T. W., The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',5'-phosphate content of guinea pig cerebral cortex slices, Molec. Pharmacol., 6 (1970) 13-23 37 SCHMIDT,M. J., HOPKINS,J. T., SCHMIDT,D. E., AND ROBISON,G. A., Cyclic AMP in brain areas: effects of amphetamine and norepinephrine assessed through the use of microwave radiation as a means of tissue fixation, Brain Research, 42 (1972) 465-477. 38 SHIMIZU,H., CREVELING,C. R., ANDDALY,J. W., The effects of histamine and other compounds on the formation of adenosine 3',5'-monophosphate in slices from cerebral cortex, J. Neurochem., 17 (1970) 4-41-~4~4. 39 SIGGINS,G. R., HOFFER,B. J., ANDBLOOM,F. E., Studies on norepinephrine-containing afferents in Purkinje cells of rat cerebellum. III. Evidence for mediation of norepinephrine effects by cyclic 3',5'-adenosine monophosphate, Brain Research, 25 (1971) 535-553. 40 SIGGINS,G. R., HOFFER, B., AND BLOOM, F. E., Prostaglandin-norepinephrin¢ interactions in brain: microelectrophoretic and histochemical correlates, Ann. N. Y. Acad. Sci., 80 (1971) 302-323. 41 UNGERSTEDT,U., 6-Hydroxydopamine induced degeneration of central monoamine neurons, Europ. J. PharmacoL, 5 (1968) 107-110. 42 VANE, J. R., A sensitive method for the assay of 5-hydroxytryptaminc, Brit. J. Pharmacol., 12 (1957) 344-349. 43 WESTFALL,D. P., AND FLEMING,W. W., Sensitivity changes in the dog heart to norepinephrine, calcium and aminophylline resulting from pretreatment with reserpine, or. Pharmacol. exp. Ther., 159 (1968) 98-106.