Effects of nerve stimulation on the uptake of norepinephrine by the perfused spleen of the cat

EUROPEAN JOURNAL OF PHARMACOLOGY 17 (1972) 25-33. NORTH-HOLLAND PUBLISHING COMPANY

EFFECTS

OF NERVE

OF NOREPINEPHRINE

STIMULATION

BY THE PERFUSED

ON THE UPTAKE SPLEEN OF THE CAT

H. YAMAMOTO * and S.M. K I R P E K A R Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, U.S.A.

Received 15 March 1971

Accepted 10 September 1971

H. YAMAMOTO and S.M. KIRPEKAR, Effects of nerve stimulation on the uptake of norepinephrine by the perfused spleen of the cat, European J. Pharmacol. 17 (1972) 25-33. 3H-norepinephrine (3H-NE) was infused intra-arterially at a constant rate of 1, 5 or 50 ng/min into the cat spleen, perfused with Krebs-bicarbonate solution. In the course of 3H-NE infusion, splenic nerves were stimulated at frequencies of 5 and 30 sec -1 for 1 min and total norepinephrine (NE), as well as 3H-NE of the perfusate collected during stimulation, was measured; nerves were also stimulated after infusion of aH-NE. During infusion at different rates, 55% of the infused radioactivity was recovered in the venous perfusate. Of the radioactivity so recovered, about 70% was due to NE. Perfusing the spleen at varying flow rates (2-13 ml/min) did not appreciably affect recoveries. During stimulation at a frequency of 30 sec -1, there was a net increase in aH-NE output over the background level. In spleens perfused with either low calcium or high magnesium Krebs solution, the increase in 3H-NE outflow in response to nerve stimulation during 3H-NE infusion was considerably reduced, but the specific activity of the released amine was not appreciably altered. Specific activities of NE released by nerve stimulation during infusion of 3H-NE were comparable or only slightly higher than those obtained after stopping the infusion of the amine. Retention of 3H-NE during its infusion was also not significantly different in stimulated and nonstimulated portions of the same spleen. It is concluded that increase in 3H-NE outflow during stimulation is probably due to enhanced release and that nerve stimulation does not appreciably affect the uptake of infused 3H-NE. Spleen Autonomic nervous system

Norepinephrine uptake

1. INTRODUCTION A number o f investigators have studied the influence of enhanced nervous activity on the uptake o f infused NE. Gillis and co-workers, in a series o f papers (Gillis, 1963; Gillis et al., 1966; Gillis and Schneider, 1967), have shown that increased sympathetic activity in the cardio-accelerator fibers o f the cat is accompanied by a greater retention o f infused radioactive NE. Similarly, Chang and Chiueh (1969) and Palaic and Panisset (1968) have reported in* Postdoctoral Fellow on U.S. Public Health Service grant, No. HE-05237. Present address: Department of Pharmacology, Wakayama Medical College, Wakayama, Japan.

Nerve stimulation Release

creased uptake o f radioactive NE in the rat submaxillary gland and guinea pig vas deferens while the sympathetic nerves to these organs were stimulated at high rates. However, Blakeley and Brown (1964), who infused relatively high concentrations o f NE into the blood-perfused cat spleen, concluded that impulses in the splenic nerves actually impaired the uptake o f the infused amine. More recently, Haggendal and Malmfors (1969) and Palaic and Panisset (1969) reported that the NE uptake was depressed in salivary glands and guinea pig vas deferens. The present investigation was undertaken to determine whether enhanced sympathetic activity in the spleen influences the removal o f inflused NE. In order to determine whether nerve stimulation is indeed ac-

26

H. Yamamoto, S.M.Kirpekar, Uptake of norep&ephrine

companied by a change in uptake of infused NE, an ideal arrangement would be to suppress the release of NE without affecting the conduction of impulses. This can be easily achieved by perfusing the spleen either with reduced calcium or increased magnesium solutions, since in these solutions the NE output is substantially depressed without interfering with conduction (Hukovic and Muscholl, 1962; Burn and Gibbons, 1964; Boultin, 1967; Kirpekar and Misu, 1967). We have studied the removal of infused radioactive NE by the spleen, perfused with normal Krebs solu~' tion as well as with solutions with reduced calcium or increased magnesium. Our results indicate that stimulation of the sympathetic nerves during infusion does not cause any significant change in the removal of infused NE by the spleen. A preliminary report of this work has been published (Yamamoto and Kirpekar, 1968).

2. MATERIALS AND METHODS The technique of perfusing the spleen of the cat has been previously described (Kirpekar and Wakade, 1968). Briefly, cats were anesthetized with ether followed by chloralose. The spleen was isolated and perfused in situ with Krebs-bicarbonate solution a t 35°C after isolating its arterial and venous supply. Both adrenals were removed. The spleen was perfused by means of a pump at a constant volume output of about 7 ml/min unless otherwise indicated. Perfusion pressure was recorded as a measure of peripheral resistance. 3H-DL-NE (specific activity 1.9 Ci/mM) was infused into the splenic artery at a constant rate of 1, 5 or 50 ng/min, in a volume of 0.05 ml, by placing a cannula in the hepatic artery so that the tip of the cannula was at the junction of the hepatic artery with the main coeliac artery. In most experiments, 3H-NE was infused at a concentration of 5 ng/min. In the course of infusion, the splenic nerves were stimulated at frequencies of 5 or 30 sec -1 for 1 min with supramaximal voltage. Stimulations were applied at 10 min intervals. Venous samples were collected for 1 rain during each stimulation period and for 1 min midway between stimulation periods. These samples were analyzed for both total NE and radioactive NE. In some experiments, the spleen was perfused with Krebs solution throughout the entire infusion period of 3H-NE;

the total infusion period was about ~ hr. In other experiments Krebs solution was replaced by either low calcium or high magnesium solution midway through the period of infusion of 3H-NE. Low calcium solution contained only 0.2 mM Ca 2÷, as compared with 2.5 mM Ca 2+ in the normal Krebs solution, while high magnesium solution contained 12 or 18 mM Mg 2÷, as against 1.2 mM Mg 2÷ in the normal Krebs solution. After termination of the 3H-NE infusion, the spleen was further perfused for 30 min, and the splenic nerves were stimulated as in the previous perfusion periods. In order to determine the effect of nerve stilnulation on uptake, one portion of the spleen was acutely denervated by cutting the sympathetic nerves running along with one of the principal branches of the splenic artery which bifurcates at the hilum. Sympathetic nerves running with the other branch were left untouched. It was thus possible to stimulate the sympathetic nerves only to half the portion of the spleen without stimulating the other half, which served as a control. 3H-NE was infused at a constant rate of 20ng/min for 20min. During the last 10min of 3H-NE infusion, splenic nerves were continuously stimulated at a frequency of 30 sec-1. At the end of the 3H-NE infusion and nerve stimulation, the spleen was perfused with Krebs solution for 15 rain. Large portions of the spleen supplied by the respective branches of the splenic artery were immediately removed to determine their NE and 3H-NE contents. Similar experiments were also done using low calcium Krebs solution.

2.1. Assay of catecholamines Total radioactivity in the venous effluent was measured by adding 0.5 or 1 ml directly to 10 ml of scintillation fluid. A Packard Tricarb Scintillation counter was used with an automatic external reference standard for applying correction for quenching. NE was estimated by fluorometric assay procedure (Anton and Sayre, 1962). Net increase in total radioactivity or 3H-NE output after nerve stimulation was determined by subtracting the background activity of the same volume of solution in the absence of nerve stimulation from the total output of radioactivity or a H-NE during nerve stimulation. In this paper specific activity of stimulation samples is defined as follows: Net increase in 3H-NE × 100 Specific activity = Total NE released

H. Yamamoto, S.M.Kirpekar, Uptake of norepinephrine

27

3. RESULTS

-13 -12

o

3.1. Venous recovery o f intra-arterially-infused 3H-NE from the perfused spleen 3H.NE was infused intra-arterially at three different rates, 1, 5 and 50 ng/min. Fig. 1 shows the average recoveries for a period of about 90 min. The recovery of total radioactivity in the venous outflow remained fairly constant from the 10th min onward, and was about 55% of the infused radioactivity at each of the three infusion rates. In order to study the effect of flow, the recovery of infused NE was studied at varying flow rates (fig. 2). 3H-NE was continuously infused at a rate of 5 ng/min for 80 min, and the perfusion rate of Krebs solution was varied from about 2 to 13 ml/min in the same experiment. The recovery of total radioactivity in the venous outflow at each of these perfusion rates was compared to that obtained during a standard perfusion rate of 7 ml/min. Fig. 2 shows that the recovery of radioactivity at different perfusion rates was fairly constant and was about 55% of the infused amount of 3H-NE. Generally, with low flow rates the concentration of radioactivity in the venous effluent

~)

2ot/ ~.~

o

-o- 1

,

O_

o~

o

o,

o

-I|

o

:

•

-i.

•

-I0

•

ID

>~

i

s

_zE

o

¢

>-~

o

7 •

•

8*

4

<

~=

z~ w

3

>1

o

:

2

o

| i

o i

v

i

i

i I

= I

0

I0 min. N'~NE

INFUSION

TIME (rain.)

Fig. 2. Relationship between perfusion rate and the recovery of infused aH-NE. 3H-NE was infused at a constant rate of 5 ng/min for 80 min. From about the 10th min onward, the recovery of radioactivity remained constant. Data are obtained from 4 experiments. The spleen was initially perfused with the standard rate of 7 ml/min and the perfusion rate was varied from 2 ml to 13 ml/min in the same experiment. Righthand ordinate shows the venous outflow per min (open circle), and the lefthand ordinate shows the recovery of total radioactivity in the venous outflow in 1 min (filled circle). With low perfusion rates, there is an increase in NE concentration in the venous samples, so that the total 3H-NE recovered per min remains fairly constant. proportionately increased, and with high flow rates it was reduced, so that the recoveries per min at different flow rates remained constant.

o o

,50 n 9 per rain.

io

30

50

ro

r-g

Io

~'

H 3-

o~ m i~, ~

~'

30

50

*

3.2. The effect of nerve stimulation on the recovery of 3H-NE

ro

J'

N E I N F U S I O N RATE 5 n g per mln.

I i-

,o

2o

,'o

H a-

NE I N F U S I O N T I M E ( r a i n . )

,o

,o

6o

,o

6o

,o

Fig. 1. Recovery of ~H-NE in the splenic venous effluent at various times during arterial infusion of 3H-NE in normal cats. Three infusion rates were used. In each graph the average recovery of radioactivity per rain is plotted against the infusion time. The data for an infusion rate of 5 ng/min are obtained from 4-11 experiments and those for 50 ng/min are obtained from 1-4 experiments.

The relationship between the recovery of radioactivity appearing in the venous outflow, the change in perfusion pressure, and the mean venous flow rate during nerve stimulation is shown in fig. 3. During each stimulation period, the venous radioactivity increased over the basal rate of recovery of infused 3H-NE; this was accompanied by a rise in perfusion pressure and an increase in venous outflow. Venous outflows in response to nerve stimulation which were usually maximum at the beginning of the experiment, tapered off with subsequent stimulation periods. Even though stimulation frequencies of 5 and 30 sec-1 were used, we mostly relied on results obtained at the higher frequency, since only at this frequency was it possible to determine fluorometrically the NE content of the venous sample with cer-

H. Yamamoto, S.M.Kirpekar, Uptake of norepinephrine

28

8"

-18

-17 -16

7"

-15 ~¢ -14

o -.I

6-

-12

-II o~ mE

-,o 5-

-9 -8

0

Z == tlJ

n n n n

> Z

O. c_ OE ~ ~

-7

4

n

~ _J I..0 F-

0

I"1

n

6

-6 -s

2_ ,oo ~ # _ I-

[ I'o

2 '0

3'0

: go

H3-NE

5 'o

6 0'

INF'USION

f , l : / 7 0'

8 0'

TIME

(rain.)

9 0'

I 0' 0

' I10

'-40~ ' 120

Fig. 3. A typical experiment showing the effect of nerve stimulation on the appearance of radioactivity in the venous sample during a continuous infusion of 3H-NE. Open circles represent total radioactivity in the venous samples, and inverted signs (n) represent venous outflows per min. Abscissa shows the aH-NE infusion time. Bottom record shows perfusion pressure responses due to nerve stimulation. In this experiment aH-NE was infused at 5 ng/min for 120 min. Nerves were stimulated alternately at stimulation frequencies of 5 and 30 sec - ' after 30 min. For other details refer to the text. tainty and thus calculate the specific activity of the released transmitter• Net increases in 3H-NE and NE values obtained at 30 sec- I in this experiment were used to calculate the specific activities of the released transmitter (fig. 4). It can be seen that as the infusion of 3H-NE was continued, the specific activity of the released transmitter gradually increased during the subsequent stimulation periods, as shown by the rising slope of the line. 3.3. The effect o f nerve stimulation on the recovery o f 3H-NE during perfusion with modified Krebs solution Experiments with high magnesium or low calcium Krebs solution were undertaken with a view that in

this medium, nerve impulses could still reach the nerve endings but would not be able to release the neurotransmitter (Katz and Miledi, 1965). If the increase in radioactivity obtained after nerve stimulation in normal spleen is due to release of stored 3H-NE, then this increase should be absent or considerably reduced in spleens perfused with modified Krebs solutions, without affecting the specific activity of the released transmitter. It was first established that perfusion of the spleen with high magnesium (12 or 18 mM) or low calcium (0.2 mM) Krebs solution did not influence the recovery of 3H-NE; it was about 55% of the infused amount of 3H-NE (9 experiments). The results obtained in three experiments during perfusion of the

H. Yamamoto, S.M.Kirpekar, Uptake of norepinephrine I"i H~-NE NET INCREASE ( ng/min.] I:1:1NE (100 n g / m i n . ) @ SPECIFIC ACTIVITY

J

J 45

I I 60 75 95 105 H~NE I N F U S I O N TIME [rain.)

29

with low calcium Krebs solution, which was preceded and followed by perfusion with normal Krebs solution. In each experiment, the point representing the specific activities o f the first and the last stimulation samples obtained during perfusion o f normal Krebs solution were joined by a straight line. In all five experiments, the points obtained during perfusion with low calcium solution fell on the lines or above them. The lines represent the rate o f change o f specific activities o f the released NE in normal Krebs solution without the intervening perfusion with low calcium Krebs. Comparable results were obtained with high magnesium Krebs solution (fig. 5c). During perfusion with either low calcium or high magnesium solution the release o f NE was markedly depressed.

I15

Fig. 4. Data are obtained from the experiment of fig. 3 at the stimulation frequency of 30 sec- ~ only. Net increases in NE and 3H-NE contents of venous samples after nerve stimulation are shown by hatched and open columns, and the specific activity (net increase in 3H-NE/total NE X 100) of each sample at each stimulation period by an open circle. A straight line through these open circles represents the rate of rise of specific activity of released NE during a continuous infusion of 3H-NE.

spleen with normal Krebs solution are shown in fig. 5a. Specific activities o f released NE increased linearly with time, as represented b y the straight lines, and the slopes o f these lines varied from experiment to experiment. In fig. 5b are shown the specific activities of the stimulation samples taken during perfusion

3.4. Time-course of net aH-NE increase during nerve stimulation The net increase in 3H-NE during nerve stimulation could conceivably be due to saturation o f the reuptake mechanism b y endogenous NE released b y nerve stimulation. To study this possibility, the experiment shown i n table 1 was carried out. aH-NE was infused into the spleen over a period o f 2.5 hr. Nerves were stimulated for 60 sec at a frequency o f 30 sec- 1 during the infusion, and then again after the end o f the infusion. Venous samples were collected in two portions o f 30 sec each, and the net increases in 3 H-NE and the total NE content o f the samples were determined. It is obvious that the rate o f release o f endogenous NE was considerably lower during the second 30 sec collection period as compared to the

Table 1 Time-course of net increase in 3H-NE outflow during nerve stimulation and its correlation to endogenous NE release* (with or without 3H-NE infusion). a H-NE infusion (5 ng/min) During infusion 30 min after infusion

Stimulation time

Net 3H-NE increase (ng)

Total NE (ng)

Ratio percentage (3H-NE/total NE)

30 see 30 see

13.90 4.31

873.12 253.50

1.59 (F)** 1.70 (S)

1.07

30 see 30 see

4.79 2.52

421.56 184.61

1.14 (F) 1.36 (S)

1.19

* Nerves were stimulated at 30 sec-~ for 1 min, and samples were collected in two 30 sec portions. ** F: first; S: second.

S/F**

H. Yamamoto, S.M.Kirpekar, Uptake of norepinephrine

30

Normal

High Mg "i"+

Low Ca + +

®

®

®

6-

5O&

4-

o

i "S I

I

I

I

I

I

3o o •

•

2-

I-

|

20 40 60 80 100120140

1

I

I

I

I

I

I

20 40 60 80 I00 120 140

I

I

l

U

I

I

I

20 40 60 80 moo 12014C

H~NE INFUSION TIME (rain.) Fig. 5. Rate o f change of specific activities in normal, low calcium and high magnesium Krebs solution is plotted against 3H-NE infusion time. In any particular group, different symbols are used for different experiments. Filled symbols in the same experiment show the specific activities in low calcium and high magnesium solutions. All values are obtained at a stimulation frequency o f 30 sec - 1

first collection period; this trend was also observed in the net increase of aH-NE during the two collection periods. The ratios of net aH-NE increase to total NE were not significantly different during the two collection periods, in spite of the much greater release during the first 30 sec collection period. If the stimulation was repeated some 30min after the end of 3 H-NE infusion, there was a greater increase in both a H-NE and total amine released during the first than the second 30 sec collection period. However, the specific activities of the released transmitter in the two collection periods were comparable, and less than those obtained during infusion of aH-NE. Similar analyses were made in five experiments, and the average S/F ratios (specific activity during the second 30 sec collection period/specific activity during the. first 30 sec collection period) during infusion of aH-NE was 1.22 + 0.11 and 0.91 + 0.09 without infusion.

3.5. Comparison of the specific activities o f the released N E by nerve stimulation, with or without infusion of 3H-NE A very simple assessment of the effect of nerve stimulation on uptake of 3H-NE is to determine the specific activities of NE released by nerve stimulation during and after an infusion of aH-NE. If the uptake of infused NE is influenced by nerve stimulation, then this should be reflected in the specific activities of the samples obtained during aH-NE infusion. In experiments shown in table 2, the two stimulation samples were separated by about 30 min, and 3H-NE infusion was stopped soon after obtaining the first stimulation sample. Specific activities of NE in stimulated samples taken during infusion of 3H-NE were only slightly higher than those obtained without a H-NE infusion, but the values were not significantly different.

H. Yamamoto, S.M.Kirpekar, Uptake of norepinephrine Table 2 Comparison of specific activities (net a H-NE increase/total NE × 100) of NE released during nerve stimulation* (with or without 3H-NE infusion). During infusion (D)

After infusion (A)

0.98 3.10 0.86 3.61 1.25 1.42 1.62 2.70 Average

0.98 2.29 0.49 3.54 0.85 1.10 1.20 3.18

A/D 1.00 0.74 0.57 0.98 0.68 0.77 0.74 1.18 0.83±0.07

* Nerves were stimulated at 30 sec-~ for 1 rain and SH-NE was infused at 5 ng/min.

~Low

Co÷ + K r e b s ~

I,I

w D.

o

3.6. Effect o f nerve stimulation on the uptake o f

3H-NE Fig. 6 shows the effect o f rest and nerve stimulation on the uptake o f infused aH-NE. Stimulation o f the nerves at this frequency reduced uptake in a normally innervated spleen compared to the rested portion o f the same spleen. However, these differences were not significant because o f the considerable variation in different experiments. In innervated portions o f the spleen, nerve stimulation produced contraction, whereas the acutely denervated portion was relaxed. Since contraction might prevent aH-NE from reaching the sympathetic nerves in this portion o f the spleen it was decided to use low Ca 2÷ solution (0.1 mM), which would block release but not affect uptake of 3H-NE and conduction along the nerves. Stimulating the nerves in low Ca 2. Krebs solution did not appreciably affect retention o f 3H-NE as compared to the rested portion of the spleen. On the basis o f these experiments, it is concluded that the retention o f infused NE b y the innervated portion o f the spleen was either reduced or not affected b y nerve stimulation.

(8)

(A) z bJ J O. (n 2"

u

31

n

30IS

REST

3o/s

REST

Fig. 6. Effect of nerve stimulation on the retention of infused NE. One portion of the spleen was acutely denervated (rest), while the nerves to the other portion were stimulated. ~H-NE (20 ng/min) was infused for 20 rain, and during the latter half of the 3H-NE infusion period, nerves were stimulated at 30 see-~ for 10 rain. At the end of infusion and nerve stimulation, the spleen was perfused with normal Krebs solution for 15 min, and large portions of the stimulated and rested spleen were removed fox NE and 3H-NE determinations. Experiments in fig. 6B were done in low calcium Krebs solution (0.1 raM). Infusion of 3H-NE and stimulation of the nerves were carried out in low calcium Krebs solution.

4. DISCUSSION In the present experiments, aH-NE was infused at a steady rate into the perfused spleen and the effects o f nerve stimulation on its removal were studied. Even though the rates o f infusion o f aH-NE into the spleen were much lower than those previously used (Gillespie and Kirpekar, 1966), the percentage recovery o f total radioactivity, as well as aH-NE, was very similar. It would therefore appear that the NE removal mechanism in spleen can only remove a constant fraction o f the infused NE over a very wide range o f concentrations. Stimulation of the splenic nerves at various frequencies during the course o f 3H-NE infusion usually resulted in the appearance o f large increases in radioactivity in the venous perfusate over the background recovery, this increase being maximal at 30 sec-1. A number o f observations made in this paper showed that the increase was primarily due to release o f a H-NE which was taken up b y the adrenergic nerves during the course o f aH-NE infusion, and partly due to prevention o f uptake o f infused NE. During perfu-

32

H. Yamamoto, S.M.Kirpekar, Uptake of norepinephrine

sion with low calcium or high magnesium solutions, the release o f the endogenous transmitter was markedly depressed with a simultaneous decrease in the release of aH-NE. In many experiments, specific activities o f NE released in modified Krebs solution were above the expected control values (fig. 5). In calculating the specific activities o f stimulation samples in modified Krebs solution, the fraction of infused 3H-NE normally recovered in the venous effluent under resting conditions was subtracted from the stimulation sample. If one assumes that sympathetic activity enhances uptake, then the calculated specific activities o f the stimulation samples during perfusion with modified Krebs solution should have been very low or even negative since the release had been largely blocked. However, in no instance were the specific activities of the released NE lower than the expected control values. Secondly, if uptake had been increased due to enhanced nerve activity, the calculated specific activity of the released NE during the course o f 3 H-NE infusion ought to have been lower and not higher than those o f the succeeding samples obtained without the infusion of 3H-NE. In fact, these values were slightly higher (table 2). It is therefore concluded that in the spleen, sympathetic activity does not enhance uptake of infused NE. The results o f experiments in which retention of 3H-NE was measured in the stimulated and unstimulated portions o f the same spleen also showed that enhanced nervous activity does not influence the uptake o f infused NE. Several factors may account f o r the increase in a H-NE content of the venous perfusate induced b y nerve stimulation during NE infusion. These are: release o f 3H-NE, prevention of uptake of infused NE, and saturation o f the uptake process b y the increased NE concentration in the vicinity o f the nerve endings. Results shown in table 1 do not support the idea that saturation o f the uptake process b y released NE can account for the increase. That the presence o f extra aH-NE was only partly due to prevention of uptake is indicated b y the fact that the specific activities of the released transmitter at the terminal period o f infusion were only slightly greater than those o f the post-infusion period. Secondly, the results obtained from experiments in modified Krebs solution and those presented in table 2 also suggest that uptake of infused NE was only slightly affected during enhanced nervous activity. However, Blakeley

and Brown (1964), Haggendal and Malmfors (1969) and Palaic and Panisset (1969) have shown impairment of uptake process during nerve stimulation. Even though the results in this paper suggest that a large part o f the excess NE is mainly due to release, we cannot completely rule out the possibility that a small portion is due to prevention o f uptake.

ACKNOWLEDGMENTS The authors are grateful to Professor Robert F. Furchgott for his keen interest in this work. This work was supported by a U.S. Public Health Service grant, No. HE-05237.

REFERENCES Anton, A.H. and D.F. Sayre, 1962, A study of the factors affecting the aluminum oxide-trihydroxyindoleprocedure for the analysis of catecholamines, J. Pharmacol. Exptl. Therap. 138,360-375. Blakeley, A.G.H. and G.L. Brown, 1964, The effect of nerve stimulation on the uptake of infused noradrenaline by the perfused spleen, J. Physiol., London 172, 19P-21P. Boullin, D.J., 1967, The action of extracellular cations on the release of the sympathetic transmitter from peripheral nerves, J. Physiol., London 189, 85-99. Burn, J.H. and W.R. Gibbons, 1964, The part played by calcium in determining the response to stimulation of sympathetic postganglionic nerves, Brit. J. Pharmacol. 22, 540-548. Chang, C.C. and C.C. Chiueh, 1969, Modification of noradrenaline incorporation by nerve activities in the rat submaxillary gland, J. Physiol., London 203,145-157. Gillespie, J.S. and S.M. Kirpekar, 1966, The uptake and release of radioactive noradrenaline by the splenic nerves of cats, J. Physiol., London 187, 51-68. Gillis, C.N., 1963, Increased retention of exogenous norepinephrine by cat atria after electrical stimulation of the cardioaccelerator nerves, Biochem. Pharmacol. 12, 593-595. Gillis, C.N. and F.H. Schneider, 1967, Frequency dependent potentiation by various drugs at the chronotropic response of isolated cat atria to sympathetic nerve stimulation, Brit. J. Pharmacol. 30, 541-553. Gillis, C.N., F.H. Schneider, L.S. Van Orden and N.J. Giarman, 1966, Biochemical and microfluorometric studies of norepinephrine redistribution accompanying sympathetic nerve stimulation, J. Pharmacol. Exptl. Therap. 151, 46-54. Haggendal, J. and T. Malmfors, 1969, The effect of nerve stimulation on the uptake of noradrenaline into the

H. Yamamoto, S.M.Kirpekar, Uptake o f norepinephrine adrenergic nerve terminals, Acta Physiol. Scand. 75, 28-32. Hukovic, S. and E. Muscholl, 1962, Die NoradrenalineAbgabe aus den isoUerten Kaninchen herzen bei sympathischer Nervenreizung und ihre pharmakologische Beeinflussung, Arch. Exptl. Pathol. Pharmakol. 224, 8 1 - 9 6 . Katz, B. and R. Miledi, 1965, The effect of calcium on acetylcholine release from motor nerve terminals, Proc. Roy. Soc. B. 1 6 1 , 4 9 6 - 5 0 3 . Kirpekar, S.M. and Y. Misu, 1967, Release of noradrenaline by splenic nerve stimulation and its dependence on calcium, J. Physiol., London 188, 2 1 9 - 2 3 4 .

33

Kirpekar, S.M. and A.R. Wakade, 1968, Factors influencing noradrenaline uptake by the perfused spleen of the cat, J. Physiol., London 1 9 4 , 6 0 9 - 6 2 6 . Palaic, D. and J.C. Panisset, 1968, Effect of angiotensin and nerve stimulation on the uptake of 3H-norepinephrine in guinea-pig vas deferens, Fed. Proc. 27 (2), 712. Palaic, D. and J.C. Panisset, 1969, Inhibition of the noradrenaline uptake in guinea-pig vas deferens by continous nerve stimulation. J. Pharm. Pharmacol. 2 1 , 3 2 8 - 3 2 9 . Yamamoto, H. and S.M. Kirpekar, 1968, Effects of nerve stimulation on the primary uptake of norepinephrine (NE) by the perfused spleen, Pharmacologist 10, 195.