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Direct recording of total catecholamine secretion from the adrenal gland in response to splanchnic nerve stimulation in rats
Journal of Neuroscience Methods, 24 (1988) 39-43 Elsevier
39
NSM 00803
Direct recording of total catecholamine secretion from the adrenal gland in response to splanchnic nerve stimulation in rats Konosuke Kumakura *, Akio Sato and Harue Suzuki Department of Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan) (Received 16 March 1987) (Revised 3 November 1987) (Accepted 9 November 1987)
Key words: C a t e c h o l a m i n e ; Secretion; A d r e n a l gland; S p l a n c h n i c N e r v e ; S t i m u l a t i o n ; I n situ; Perfusion; Rat A new method for the continuous recording of catecholamine (CA) secretion from the adrenal gland in rats in response to splanchnic nerve stimulation was developed. The method consists of in situ perfusion of the adrenal gland and direct continuous recording of released total CA by an electrochemical detector (ECD). Splanchnic nerve stimulation evoked current responses in a stimulus frequency dependent manner. The amounts of total CA released was linearly related to peak amplitude and to the total integrated output current for various current responses, indicating that the ECD current response in this system represents a highly reliable parameter of the secretory process in the adrenal gland. This system has the advantage of directly and continuously monitoring total CA secretion from the adrenal gland in situ.
Introduction T h e m a j o r focus of recent i n v e s t i g a t i o n in this l a b o r a t o r y has b e e n o n the s t u d y of the secretion of a d r e n a l i n e a n d n o r a d r e n a l i n e f r o m the a d r e n a l g l a n d in r e s p o n s e to s o m a t i c a n d visceral afferent stimuli. I n these studies small v o l u m e s of b l o o d were w i t h d r a w n r e p e a t e d l y at r e g u l a r intervals ( a b o u t 2 0 - 3 0 times) f r o m the a d r e n a l vein of the s a m e anesthetized a n i m a l ( A r a k i et al., 1984; I t o et al., 1984). This m e t h o d e n a b l e d us to s t u d y the time course of the a d r e n a l m e d u l l a r y s e c r e t o r y r e s p o n s e to v a r i o u s stimuli in the s a m e a n i m a l w i t h o u t significant influences of h y p o v o l e m i a o n
* Visiting Professor in Project Department in this institute. Present address: Life Science Institute, Sophia University, 7-1 Kioicho, Chiyodaku, Tokyo 102, Japan. Correspondence: A. Sato, Department of Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan.
c a t e c h o l a m i n e ( C A ) secretion. These e x p e r i m e n t s were p o s s i b l e b e c a u s e o n l y small b l o o d samples, o f a b o u t 50 #1 each, were r e q u i r e d for each C A assay, d u e to the h i g h C A c o n c e n t r a t i o n in the a d r e n a l v e n o u s b l o o d . H o w e v e r , it has never b e e n p o s s i b l e to c o n t i n u o u s l y m e a s u r e a d r e n a l i n e a n d n o r a d r e n a l i n e secretion f r o m the a d r e n a l g l a n d in an in vivo e x p e r i m e n t d u e to the technical limitations to C A m e a s u r e m e n t s f r o m b l o o d s a m p l e s using c o n v e n t i o n a l m e t h o d s . Recently, G r e e n a n d P e r l m a n (1981) a n d K u m a k u r a et al. (1986) d e v e l o p e d a new m e t h o d to c o n t i n u o u s l y m e a s u r e t o t a l C A secretion f r o m c u l t u r e d a d r e n a l c h r o m a f f i n cells using a perfusion s y s t e m c o u p l e d to a n e l e c t r o c h e m i c a l d e t e c t o r ( E C D ) . I n their e x p e r i m e n t a l p r e p a r a t i o n s isolated a d r e n a l c h r o m a f f i n cells were t o t a l l y d i s c o n n e c t e d f r o m their a d r e n a l s y m p a t h e t i c nerves. It w o u l d b e a d v a n t a g e o u s if an a d r e n a l m e d u l l a r y p r e p a r a t i o n w i t h i n t a c t nerve connections c o u l d b e u s e d in their e x p e r i m e n t a l system.
0165-0270/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
40
Thus, the present experiment was performed in an attempt to apply the method of continuous recording of total CA to the in vivo preparations developed in this laboratory, in which there is an intact adrenal sympathetic nerve connection with the adrenal chromaffin cells of the anesthetized animal (Araki et al., 1984; Ito et al., 1984). For this purpose it was necessary to avoid disturbances from the blood for the rather selective detection of CA by ECD, therefore we perfused the adrenal gland in situ with a physiological solution through the adrenal artery.
of anesthetics (1/10 of initial doses) were administered i.v. to maintain anesthesia at a relatively constant level as judged by the recorded circulatory variables. Blood pressure was monitored continuously from the common carotid artery and maintained above 90 mm Hg (systolic) by infusion of 4% FicoU 70 (Pharmacia Fine Chemicals AB) in saline solution. The left adrenal gland was approached through an abdominal midline section. One catheter (catheter A in Fig. 1A; o.d. 1.35 mm, i.d. 0.8 ram) was inserted into the descending aorta through a small hole (a in Fig. 1A) and the tip of the catheter was kept near the branching point of the adrenal artery from the aorta. Both the proximal and distal portions of the aorta (b and c in Fig. 1A) were tied and occluded. All the remaining aorta branches not supplying the adrenal glands were occluded. Another catheter (catheter B in Fig. 1A; o.d. 0.61 mm, i.d. 0.28 ram) was inserted into the left adrenal vein according to the methods described by Araki et al. (1984). The Krebs-bicarbonate solution described by Wakade (1981) was employed for the perfusion. The composition of the solution was (mM); NaCl 118.4, KC1 4.7, CaC12 2.5, MgCI 2 1.18, NaHCO 3 25, KHEPO4 1.2, glucose 11.7. The solution was
Methods
In situ per/usion of the adrenal gland The present experiments were performed on urethane-chloralose (500 mg/kg and 50 mg/kg, i.p., respectively) anesthetized male Wistar rats. Respiration was maintained through a tracheal cannula connected to a respirator after immobilization with gallamine triethiodide (10 mg/kg, i.v.) which is known not to affect the catecholamine secretion from the adrenal medulla (Sumikawa et al., 1979). During the experiment, additional doses
adrenal ~ ~//TJ artery r ~ abdominalC~ / A V////A
etim. SPLN
B
S:alT::id pump
.0,=
,
heating I~
h ,_b._:,!=_.,V
r~
-, , , 1 1 " £ kidney
perfusate, I / ~ ~ Icatheter B to ECD
/
95% 02 5% CO2
ECD
electrode
drop I-- -I counter I
I
I
~lr outlet
Fig. 1. A: procedure for in situ adrenal perfusion and splanchnic nerve (SPLN) stimulation, as viewed from a ventral surgical approach. B: schematic diagram of the experimental pcrfusion system. ECD; electrochemical detector. Details of the procedure are described in the text.
41 bubbled with 95% 02 and 5% CO2 and adjusted to pH 7.4-7.5 and was kept at 37.0-38.0°C. The animal's body temperature monitored from the pharynx was also maintained between 37.0 and 38.0°C through use of an ir lamp and a DC current heating pad. The solution was delivered through the adrenal artery to the adrenal gland using a peristaltic pump system which had a twoway solenoid valve to generate pulsatile pressures (Fig. 1B) fluctuating between a minimum pressure of 70-110 mm Hg and a maximum pressure of 90-140 mm Hg (the pulse pressure was 20-30 mm Hg) with a frequency of 2 Hz as monitored by a pressure transducer. Flow rate for the perfusion was monitored by a drop counter and set at 130-200 #l/rain, which was nearly equivalent to the blood flow rate of the adrenal gland estimated from the adrenal venous plasma flow rate in the rat (50-240/~l/kg/min) reported by Araki et al. (1984). The splanchnic nerve ipsilateral to the perfused adrenal gland was severed centrally at the diaphragm level, and electrical stimulations were delivered to the distal end of the severed splanchnic nerve (Fig. 1A) which predominantly controls CA secretion from the adrenal gland (Araki et al., 1984).
Electrochemical monitoring of released catecholamine The perfusate emanating from the adrenal vein was directly introduced into the ECD for the detection of oxidizable substances, such as adrenaline and noradrenaline, released from the adrenal gland (Fig. 1B). The electrochemical detection of the released CA was performed using an ECD (Bioanalytical Systems, LC-4B amperometric detector) equipped with an electrochemical flow cell (TL-5A with a Teflon gasket of 50/~m thickness, Bioanalytical Systems). The electrode potential of the ECD was set at + 0.45 V vs the Ag/AgC1 reference electrode, and the reason for the selection of this electrode potential has been described previously (Kumakura et al., 1986). Moreover, neural peptides known to be co-released with CA from the adrenal gland cannot be detected electrochemically at this electrode potential (Bennett et al., 1981). The oxidation current signal was con-
tinuously recorded on a strip chart recorder. In some selected cases, adrenaline and noradrenaline concentration in the outlet perfusate from the ECD were measured using high performance liquid chromatography (HPLC) and ECD through a routine CA assay method (Ito et al., 1984). Results and Discussion
Electric current signals from the ECD in the perfusing system showed basal levels which were quite stable for long periods of up to 1-2 h after the splanchnic nerve was severed, as demonstrated between stimuli in Fig. 2A. These basal values ranged between 11 and 18 nA in the present preparations. The current signals increased immediately after electrical stimulation of the splanchnic nerve, as shown in Fig. 2A which demonstrate typical examples of frequency dependent responses with the stimulation for 20 s at 10 V (0.5 ms duration) at various frequencies. These increased current responses declined gradually to the resting level after the cessation of stimulation. When the nerve stimulation was prolonged for 3 min, the current responses usually reached their maximums around 20-30 s after their onsets. Then the response declined slightly and stabilized at levels which were below the maximal but above the basal levels during the continuation of the stimulation (Fig. 2B). The flow rate of the outlet perfusate was not changed during the stimulation (10 V, 10 Hz) for 20 s to 1 min, while it decreased by about 20% at the end of the stimulation for 3 min. Therefore, the stimulation for 20 s was employed for the following analysis. In Fig. 2C, relationships between peak amplitudes of the ECD currents and various stimulus frequencies of the splanchnic nerve, used for Fig. 2A, were plotted. The amount of adrenaline and noradrenaline secreted from the adrenal gland for 1 min after the onset of the corresponding responses were measured by H P L C / E C D (Fig. 2C). Algebraic summations of the amounts of adrenaline and noradrenaline measured were plotted as total CA (Fig. 2C). Both the magnitude of the current response and the amount of the released CA were increased by splanchnic nerve stimulation in a stimulus-frequency dependent manner
42
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I'°°"
B
A i/ or.a
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40 t,0 20
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-
2 3 5
,
,
,
,
10 20 30 50 Hz
',,
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!
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0
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20
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80 nA
Fig. 2. A, B: examples of ECD-monitored current responses (current-time curve) with splanchnic nerve stimulation for 20 s (A) or 3 min (B) at 10 V (0.5 ms duration) at various frequencies. The stimulation period is indicated by horizontal bars after compensation of time lag of the dead space (about 70/xl) between the adrenal gland and the ECD electrode in the perfusion system. C: the peak amplitude of ECD current (dosed circles, left ordinate) and the amount of adrenaline (closed triangles), noradrenaline (open triangles) and the total eatecho!amines (open circles) measured for 1 min (right ordinate) in each response in A are plotted as a function of stimulus frequency (abscissa). b, basal secretion without nerve stimulation. The peak amplitude is expressed as the amplitude (hA) above background current, which was obtained purely from the perfusing buffer. D: relationship between different parameters in the secretory response of the adrenal gland to splanchmc nerve stimulation. DI: amount of released total catecliolamine (ordinate) vs integrated output current ("area", abscissa). D2: peak amplitude (ordinate) vs integrated output current ("area", abscissa). D3: amount of released total catecholamine (ordinate) vs peak amplitude (abscissa). The values were from the particular experiment used for Fig. 2A and represent the amount of CA released in a 1 min collection period. The values expressed by o, (D and • indicate the responses to the 1st, 2rid and 3rd series of stimulations, respectively. A, C and D were taken from the same rat; B was taken from a different rat.
over the range of I to 20 Hz and plateaued around 20 Hz. The relationship between the CA secretion and stimulus-frequency is quite comparable to that in the conscious calf (Edwards et al., 1980) and the anesthetized rat (Togashi, 1983). From these data, a close relationship between the two parameters of output current and total CA release in response to splanchnic nerve stimulation was suggested. Therefore, we analyzed the relationship between output current and CA response to nerve stimulus by plotting these data in Fig. 2D. As we routinely collected outlet perfusate samples from the ECD electrode for 1 min to measure total CA secretion in each response, the total charge in each response for 1 min (Q = f I d t ) was estimated by graphically integrating the current-time curve as shown schematically by the
shaded area for the response to nerve stimulation at 50 Hz in Fig. 2A, and labelled as "area" on the abscissae of Fig. 2D. As shown in Fig. 2D, there was a linear relationship between the area and total released CA (Fig. 2D1), the area and the magnitude of peak amplitudes (Fig. 2192) as well as the peak amplitude and the released total CA (Fig. 2D3). These linear relationships were not appreciably affected by the repetition of a series of stimulations, as evidenced by our observation that the data from 3 repeated series of stimulations fitted a linear r6gression curve with only a small variation (Fig. 2D). Such a linear relationship between output ECD current and amount of CA in response to splanchnic nerve stimulation was confirmed in 6 other experiments (results not shown).
43
A
lower chest pinching.lmin
I -1
B
I 2
0
I 3
I 4 min
p h e n y l e p h r i n e 6)Jg,i v
I -1
I 0
I 1
1 2
[ 3
I 4
I 5
I .... 6 min
Fig. 3. Two examples of ECD-monitored current responses in two rats with their splanchnic nerves intact. A: effect on ECD-monitored current of lower chest skin pinching (2 cm2, 3 kg for 1 min). B: effect on ECD current of blood pressure increase (50 mm Hg) by i.v. injection of phenylephrine (6 #g).
From these results it is concluded that CA secretion from the adrenal gland in response to splanchnic nerve stimulation in this in situ preparation was reliably reflected by either the peak amplitude or by the integrated output current. Finaly, we examined the possibility of this system to continuously record adrenal CA secretion in rats with intact splanchnic nerves. When the splanchnic nerves were kept intact, the basal level of the electric current signal from the ECD was higher (32 to 64 nA) than that obtained after severing of the splanchnic nerve. Fig. 3A shows the increase in ECD current evoked by lower chest skin pinching (2 cm 2, 3 kg for 1 min), which has been shown to produce a reflex increase in CA secretion in the anesthetized rat (Araki et al., 1984). On the other hand, a blood pressure increase (40-80 mm Hg) through an intravenous injection of phenylephrine (4-10 ttg) produced a decrease in the ECD current (Fig. 3B). This evidence is in good agreement with the observations by Ito et al. (1984) that CA secetion from the adrenal gland, when tested intermittently, was reduced reflexly by similar procedure in the anesthetized rats. These effects were obtained when
the rat's systolic blood pressure was above 90 mm Hg, and until about 90 rain after starting the adrenal perfusion. In both cases the flow rate of the outlet perfusate was not changed during the stimulation. Therefore, the present method not only has the advantages of direct and continuous monitoring of CA secretion from the adrenal gland following splanchnic nerve stimulation, but could also be useful in intact preparations for the monitoting of the CA secretory responses to various physiological stimulations as well as pharmacological manipulations. Acknowledgements
The authors appreciate Dr. Y. Takahashi's contribution and Miss H. Hotta's technical help in this project. The authors are thankful for Mr. K. Yamamoto (BAS Co. Ltd. Tokyo) for providing the ECD system for measuring catecholamines. References Araki, T., Ito, K., Kurosawa, M. and Sato, A. (1984) Responses of adrenal sympathetic nerve activity and catecholamine secretion to cutaneous stimulation in anesthetized rats, Neuroscience, 12: 289-299. Bennett, G.W., Brazell, M.P. and Marsden, C.A. (1981) Electrochemistry of neuropeptides: a possible method for assay and in vivo detection, Life Sci., 29: 1001-1007. Edwards, A.V., Furness, P.N. and Helle, K.B. (1980) Adrenal medullary responses to stimulation of the splanchnic nerve in the conscious calf, J. Physiol. (Lond.), 308: 15-27. Green, D.J. and Perlman, R.L. (1981) On-line measurement of catecholamine secretion, Anal. Biochem., 110: 270-276. Ito, K., Sato, A., Shimamura, K. and Swenson, R.S. (1984) Reflex changes in sympatho-adrenal medullary functions in response to baroreceptor stimulation in anesthetized rats. J. Auton. Nerv. Syst., 10: 295-303. Kumakura, K., Ohara, M. and Sato, G.P. (1986) Real-time monitoring of the secretory function of cultured adrenal chromaffin ceils, J. Neurochem., 46: 1851-1858. Sumikawa, K., Kashimoto, T., Izumi, F., Yoshikawa, K. and Amakata, Y. (1979) Effects of muscle relaxants on catecholamine release from adrenal medulla, Res. Commun. Chem. Pathol. Pharmacol., 25: 205-214. Togashi, H. (1983) Central and peripheral effects of clonidine on the adrenal medullary function in spontaneously hypertensive rats, J. Pharmacol. Exp. Ther., 225: 191-197. Wakade, A.R. (1981) Studies on secretion of catecholamines evoked by acetylcholine or transmural stimulation of the rat adrenal gland, J. Physiol. (Lond.), 313: 463-480.
39
NSM 00803
Direct recording of total catecholamine secretion from the adrenal gland in response to splanchnic nerve stimulation in rats Konosuke Kumakura *, Akio Sato and Harue Suzuki Department of Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo (Japan) (Received 16 March 1987) (Revised 3 November 1987) (Accepted 9 November 1987)
Key words: C a t e c h o l a m i n e ; Secretion; A d r e n a l gland; S p l a n c h n i c N e r v e ; S t i m u l a t i o n ; I n situ; Perfusion; Rat A new method for the continuous recording of catecholamine (CA) secretion from the adrenal gland in rats in response to splanchnic nerve stimulation was developed. The method consists of in situ perfusion of the adrenal gland and direct continuous recording of released total CA by an electrochemical detector (ECD). Splanchnic nerve stimulation evoked current responses in a stimulus frequency dependent manner. The amounts of total CA released was linearly related to peak amplitude and to the total integrated output current for various current responses, indicating that the ECD current response in this system represents a highly reliable parameter of the secretory process in the adrenal gland. This system has the advantage of directly and continuously monitoring total CA secretion from the adrenal gland in situ.
Introduction T h e m a j o r focus of recent i n v e s t i g a t i o n in this l a b o r a t o r y has b e e n o n the s t u d y of the secretion of a d r e n a l i n e a n d n o r a d r e n a l i n e f r o m the a d r e n a l g l a n d in r e s p o n s e to s o m a t i c a n d visceral afferent stimuli. I n these studies small v o l u m e s of b l o o d were w i t h d r a w n r e p e a t e d l y at r e g u l a r intervals ( a b o u t 2 0 - 3 0 times) f r o m the a d r e n a l vein of the s a m e anesthetized a n i m a l ( A r a k i et al., 1984; I t o et al., 1984). This m e t h o d e n a b l e d us to s t u d y the time course of the a d r e n a l m e d u l l a r y s e c r e t o r y r e s p o n s e to v a r i o u s stimuli in the s a m e a n i m a l w i t h o u t significant influences of h y p o v o l e m i a o n
* Visiting Professor in Project Department in this institute. Present address: Life Science Institute, Sophia University, 7-1 Kioicho, Chiyodaku, Tokyo 102, Japan. Correspondence: A. Sato, Department of Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan.
c a t e c h o l a m i n e ( C A ) secretion. These e x p e r i m e n t s were p o s s i b l e b e c a u s e o n l y small b l o o d samples, o f a b o u t 50 #1 each, were r e q u i r e d for each C A assay, d u e to the h i g h C A c o n c e n t r a t i o n in the a d r e n a l v e n o u s b l o o d . H o w e v e r , it has never b e e n p o s s i b l e to c o n t i n u o u s l y m e a s u r e a d r e n a l i n e a n d n o r a d r e n a l i n e secretion f r o m the a d r e n a l g l a n d in an in vivo e x p e r i m e n t d u e to the technical limitations to C A m e a s u r e m e n t s f r o m b l o o d s a m p l e s using c o n v e n t i o n a l m e t h o d s . Recently, G r e e n a n d P e r l m a n (1981) a n d K u m a k u r a et al. (1986) d e v e l o p e d a new m e t h o d to c o n t i n u o u s l y m e a s u r e t o t a l C A secretion f r o m c u l t u r e d a d r e n a l c h r o m a f f i n cells using a perfusion s y s t e m c o u p l e d to a n e l e c t r o c h e m i c a l d e t e c t o r ( E C D ) . I n their e x p e r i m e n t a l p r e p a r a t i o n s isolated a d r e n a l c h r o m a f f i n cells were t o t a l l y d i s c o n n e c t e d f r o m their a d r e n a l s y m p a t h e t i c nerves. It w o u l d b e a d v a n t a g e o u s if an a d r e n a l m e d u l l a r y p r e p a r a t i o n w i t h i n t a c t nerve connections c o u l d b e u s e d in their e x p e r i m e n t a l system.
0165-0270/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
40
Thus, the present experiment was performed in an attempt to apply the method of continuous recording of total CA to the in vivo preparations developed in this laboratory, in which there is an intact adrenal sympathetic nerve connection with the adrenal chromaffin cells of the anesthetized animal (Araki et al., 1984; Ito et al., 1984). For this purpose it was necessary to avoid disturbances from the blood for the rather selective detection of CA by ECD, therefore we perfused the adrenal gland in situ with a physiological solution through the adrenal artery.
of anesthetics (1/10 of initial doses) were administered i.v. to maintain anesthesia at a relatively constant level as judged by the recorded circulatory variables. Blood pressure was monitored continuously from the common carotid artery and maintained above 90 mm Hg (systolic) by infusion of 4% FicoU 70 (Pharmacia Fine Chemicals AB) in saline solution. The left adrenal gland was approached through an abdominal midline section. One catheter (catheter A in Fig. 1A; o.d. 1.35 mm, i.d. 0.8 ram) was inserted into the descending aorta through a small hole (a in Fig. 1A) and the tip of the catheter was kept near the branching point of the adrenal artery from the aorta. Both the proximal and distal portions of the aorta (b and c in Fig. 1A) were tied and occluded. All the remaining aorta branches not supplying the adrenal glands were occluded. Another catheter (catheter B in Fig. 1A; o.d. 0.61 mm, i.d. 0.28 ram) was inserted into the left adrenal vein according to the methods described by Araki et al. (1984). The Krebs-bicarbonate solution described by Wakade (1981) was employed for the perfusion. The composition of the solution was (mM); NaCl 118.4, KC1 4.7, CaC12 2.5, MgCI 2 1.18, NaHCO 3 25, KHEPO4 1.2, glucose 11.7. The solution was
Methods
In situ per/usion of the adrenal gland The present experiments were performed on urethane-chloralose (500 mg/kg and 50 mg/kg, i.p., respectively) anesthetized male Wistar rats. Respiration was maintained through a tracheal cannula connected to a respirator after immobilization with gallamine triethiodide (10 mg/kg, i.v.) which is known not to affect the catecholamine secretion from the adrenal medulla (Sumikawa et al., 1979). During the experiment, additional doses
adrenal ~ ~//TJ artery r ~ abdominalC~ / A V////A
etim. SPLN
B
S:alT::id pump
.0,=
,
heating I~
h ,_b._:,!=_.,V
r~
-, , , 1 1 " £ kidney
perfusate, I / ~ ~ Icatheter B to ECD
/
95% 02 5% CO2
ECD
electrode
drop I-- -I counter I
I
I
~lr outlet
Fig. 1. A: procedure for in situ adrenal perfusion and splanchnic nerve (SPLN) stimulation, as viewed from a ventral surgical approach. B: schematic diagram of the experimental pcrfusion system. ECD; electrochemical detector. Details of the procedure are described in the text.
41 bubbled with 95% 02 and 5% CO2 and adjusted to pH 7.4-7.5 and was kept at 37.0-38.0°C. The animal's body temperature monitored from the pharynx was also maintained between 37.0 and 38.0°C through use of an ir lamp and a DC current heating pad. The solution was delivered through the adrenal artery to the adrenal gland using a peristaltic pump system which had a twoway solenoid valve to generate pulsatile pressures (Fig. 1B) fluctuating between a minimum pressure of 70-110 mm Hg and a maximum pressure of 90-140 mm Hg (the pulse pressure was 20-30 mm Hg) with a frequency of 2 Hz as monitored by a pressure transducer. Flow rate for the perfusion was monitored by a drop counter and set at 130-200 #l/rain, which was nearly equivalent to the blood flow rate of the adrenal gland estimated from the adrenal venous plasma flow rate in the rat (50-240/~l/kg/min) reported by Araki et al. (1984). The splanchnic nerve ipsilateral to the perfused adrenal gland was severed centrally at the diaphragm level, and electrical stimulations were delivered to the distal end of the severed splanchnic nerve (Fig. 1A) which predominantly controls CA secretion from the adrenal gland (Araki et al., 1984).
Electrochemical monitoring of released catecholamine The perfusate emanating from the adrenal vein was directly introduced into the ECD for the detection of oxidizable substances, such as adrenaline and noradrenaline, released from the adrenal gland (Fig. 1B). The electrochemical detection of the released CA was performed using an ECD (Bioanalytical Systems, LC-4B amperometric detector) equipped with an electrochemical flow cell (TL-5A with a Teflon gasket of 50/~m thickness, Bioanalytical Systems). The electrode potential of the ECD was set at + 0.45 V vs the Ag/AgC1 reference electrode, and the reason for the selection of this electrode potential has been described previously (Kumakura et al., 1986). Moreover, neural peptides known to be co-released with CA from the adrenal gland cannot be detected electrochemically at this electrode potential (Bennett et al., 1981). The oxidation current signal was con-
tinuously recorded on a strip chart recorder. In some selected cases, adrenaline and noradrenaline concentration in the outlet perfusate from the ECD were measured using high performance liquid chromatography (HPLC) and ECD through a routine CA assay method (Ito et al., 1984). Results and Discussion
Electric current signals from the ECD in the perfusing system showed basal levels which were quite stable for long periods of up to 1-2 h after the splanchnic nerve was severed, as demonstrated between stimuli in Fig. 2A. These basal values ranged between 11 and 18 nA in the present preparations. The current signals increased immediately after electrical stimulation of the splanchnic nerve, as shown in Fig. 2A which demonstrate typical examples of frequency dependent responses with the stimulation for 20 s at 10 V (0.5 ms duration) at various frequencies. These increased current responses declined gradually to the resting level after the cessation of stimulation. When the nerve stimulation was prolonged for 3 min, the current responses usually reached their maximums around 20-30 s after their onsets. Then the response declined slightly and stabilized at levels which were below the maximal but above the basal levels during the continuation of the stimulation (Fig. 2B). The flow rate of the outlet perfusate was not changed during the stimulation (10 V, 10 Hz) for 20 s to 1 min, while it decreased by about 20% at the end of the stimulation for 3 min. Therefore, the stimulation for 20 s was employed for the following analysis. In Fig. 2C, relationships between peak amplitudes of the ECD currents and various stimulus frequencies of the splanchnic nerve, used for Fig. 2A, were plotted. The amount of adrenaline and noradrenaline secreted from the adrenal gland for 1 min after the onset of the corresponding responses were measured by H P L C / E C D (Fig. 2C). Algebraic summations of the amounts of adrenaline and noradrenaline measured were plotted as total CA (Fig. 2C). Both the magnitude of the current response and the amount of the released CA were increased by splanchnic nerve stimulation in a stimulus-frequency dependent manner
42
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Fig. 2. A, B: examples of ECD-monitored current responses (current-time curve) with splanchnic nerve stimulation for 20 s (A) or 3 min (B) at 10 V (0.5 ms duration) at various frequencies. The stimulation period is indicated by horizontal bars after compensation of time lag of the dead space (about 70/xl) between the adrenal gland and the ECD electrode in the perfusion system. C: the peak amplitude of ECD current (dosed circles, left ordinate) and the amount of adrenaline (closed triangles), noradrenaline (open triangles) and the total eatecho!amines (open circles) measured for 1 min (right ordinate) in each response in A are plotted as a function of stimulus frequency (abscissa). b, basal secretion without nerve stimulation. The peak amplitude is expressed as the amplitude (hA) above background current, which was obtained purely from the perfusing buffer. D: relationship between different parameters in the secretory response of the adrenal gland to splanchmc nerve stimulation. DI: amount of released total catecliolamine (ordinate) vs integrated output current ("area", abscissa). D2: peak amplitude (ordinate) vs integrated output current ("area", abscissa). D3: amount of released total catecholamine (ordinate) vs peak amplitude (abscissa). The values were from the particular experiment used for Fig. 2A and represent the amount of CA released in a 1 min collection period. The values expressed by o, (D and • indicate the responses to the 1st, 2rid and 3rd series of stimulations, respectively. A, C and D were taken from the same rat; B was taken from a different rat.
over the range of I to 20 Hz and plateaued around 20 Hz. The relationship between the CA secretion and stimulus-frequency is quite comparable to that in the conscious calf (Edwards et al., 1980) and the anesthetized rat (Togashi, 1983). From these data, a close relationship between the two parameters of output current and total CA release in response to splanchnic nerve stimulation was suggested. Therefore, we analyzed the relationship between output current and CA response to nerve stimulus by plotting these data in Fig. 2D. As we routinely collected outlet perfusate samples from the ECD electrode for 1 min to measure total CA secretion in each response, the total charge in each response for 1 min (Q = f I d t ) was estimated by graphically integrating the current-time curve as shown schematically by the
shaded area for the response to nerve stimulation at 50 Hz in Fig. 2A, and labelled as "area" on the abscissae of Fig. 2D. As shown in Fig. 2D, there was a linear relationship between the area and total released CA (Fig. 2D1), the area and the magnitude of peak amplitudes (Fig. 2192) as well as the peak amplitude and the released total CA (Fig. 2D3). These linear relationships were not appreciably affected by the repetition of a series of stimulations, as evidenced by our observation that the data from 3 repeated series of stimulations fitted a linear r6gression curve with only a small variation (Fig. 2D). Such a linear relationship between output ECD current and amount of CA in response to splanchnic nerve stimulation was confirmed in 6 other experiments (results not shown).
43
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Fig. 3. Two examples of ECD-monitored current responses in two rats with their splanchnic nerves intact. A: effect on ECD-monitored current of lower chest skin pinching (2 cm2, 3 kg for 1 min). B: effect on ECD current of blood pressure increase (50 mm Hg) by i.v. injection of phenylephrine (6 #g).
From these results it is concluded that CA secretion from the adrenal gland in response to splanchnic nerve stimulation in this in situ preparation was reliably reflected by either the peak amplitude or by the integrated output current. Finaly, we examined the possibility of this system to continuously record adrenal CA secretion in rats with intact splanchnic nerves. When the splanchnic nerves were kept intact, the basal level of the electric current signal from the ECD was higher (32 to 64 nA) than that obtained after severing of the splanchnic nerve. Fig. 3A shows the increase in ECD current evoked by lower chest skin pinching (2 cm 2, 3 kg for 1 min), which has been shown to produce a reflex increase in CA secretion in the anesthetized rat (Araki et al., 1984). On the other hand, a blood pressure increase (40-80 mm Hg) through an intravenous injection of phenylephrine (4-10 ttg) produced a decrease in the ECD current (Fig. 3B). This evidence is in good agreement with the observations by Ito et al. (1984) that CA secetion from the adrenal gland, when tested intermittently, was reduced reflexly by similar procedure in the anesthetized rats. These effects were obtained when
the rat's systolic blood pressure was above 90 mm Hg, and until about 90 rain after starting the adrenal perfusion. In both cases the flow rate of the outlet perfusate was not changed during the stimulation. Therefore, the present method not only has the advantages of direct and continuous monitoring of CA secretion from the adrenal gland following splanchnic nerve stimulation, but could also be useful in intact preparations for the monitoting of the CA secretory responses to various physiological stimulations as well as pharmacological manipulations. Acknowledgements
The authors appreciate Dr. Y. Takahashi's contribution and Miss H. Hotta's technical help in this project. The authors are thankful for Mr. K. Yamamoto (BAS Co. Ltd. Tokyo) for providing the ECD system for measuring catecholamines. References Araki, T., Ito, K., Kurosawa, M. and Sato, A. (1984) Responses of adrenal sympathetic nerve activity and catecholamine secretion to cutaneous stimulation in anesthetized rats, Neuroscience, 12: 289-299. Bennett, G.W., Brazell, M.P. and Marsden, C.A. (1981) Electrochemistry of neuropeptides: a possible method for assay and in vivo detection, Life Sci., 29: 1001-1007. Edwards, A.V., Furness, P.N. and Helle, K.B. (1980) Adrenal medullary responses to stimulation of the splanchnic nerve in the conscious calf, J. Physiol. (Lond.), 308: 15-27. Green, D.J. and Perlman, R.L. (1981) On-line measurement of catecholamine secretion, Anal. Biochem., 110: 270-276. Ito, K., Sato, A., Shimamura, K. and Swenson, R.S. (1984) Reflex changes in sympatho-adrenal medullary functions in response to baroreceptor stimulation in anesthetized rats. J. Auton. Nerv. Syst., 10: 295-303. Kumakura, K., Ohara, M. and Sato, G.P. (1986) Real-time monitoring of the secretory function of cultured adrenal chromaffin ceils, J. Neurochem., 46: 1851-1858. Sumikawa, K., Kashimoto, T., Izumi, F., Yoshikawa, K. and Amakata, Y. (1979) Effects of muscle relaxants on catecholamine release from adrenal medulla, Res. Commun. Chem. Pathol. Pharmacol., 25: 205-214. Togashi, H. (1983) Central and peripheral effects of clonidine on the adrenal medullary function in spontaneously hypertensive rats, J. Pharmacol. Exp. Ther., 225: 191-197. Wakade, A.R. (1981) Studies on secretion of catecholamines evoked by acetylcholine or transmural stimulation of the rat adrenal gland, J. Physiol. (Lond.), 313: 463-480.