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Presynaptic modulation of the release of noradrenaline from electrically stimulated bicuspid valve leaflet of the rabbit heart
Journal of the Autonomic Nervous System, 35 (1991) 99-106
99
,'2 1991 Elsevier Science Publishers B.V. 0165-1838/91/$03.50
JANS 01184
Presynaptic modulation of the release of noradrenaline from electrically stimulated bicuspid valve leaflet of the rabbit heart G.T. Somogyi
1, J.R.
K e a s t ~ a n d E.S. Vizi 2
I Department of Pharmacology', University of Pittsburgh, Pittsburgh, Pennsyh'ania, U.S.A., and -"Institute of Experimental Medicbw, Hungarian Academy of Sciences, Budapest, Hungary (Received 17 January 1991) (Revision received and accepted 3 April 1991)
Key words: Bicuspid valve; Noradrenaline release; a2-Adrenoceptor; Muscarinic receptor; Presynaptic modulation Abstract The bicuspid (mitral) valves were obtained from male albino New Zealand rabbits. The noradrenaline (NA) content (12.93 + 1.14 n m o l / g ) of the valve tissue was determined by high pressure liquid chromatography (HPLC) combined with electrochemical detection. After incubation with tritiated N A for 45 rain, the tissues were m o u n t e d in perfusion baths and superfused with Krebs solution at a constant perfusion rate. After a 90 rain washing period, the tissues were stimulated three times (SI; $2; $3) at a frequency of 1 or 10 Hz, and the release of NA was expressed as the stimulus-induced overflow of radioactivity. Using a constant n u m b e r of impulses, the release of N A was significantly higher when the frequency applied was 10 Hz than in the case of 1 Hz. The release of N A was inhibited by stimulating the presynaptic a2-adrenoceptors with xylazine or by stimulating the presynaptic muscarinic receptors with oxotremorine. Yohimbine (1 /*M) not only overcame the effect of the a2-adrenoceptor stimulation caused by xylazine, but increased it over the control level, whereas atropine blocked the inhibitory effect of oxotremorine. It is concluded that the adrenergic nerves in the valve tissue release NA in a frequency-dependent fashion, and the release of N A can be modulated through presynaptic a 2- and muscarinic receptors. This is the first case that neurochemical evidence was obtained showing that N A is released from the mitral valve and is subject to presynaptic modulation.
Introduction
The heart has a dense noradrenergic innervation, and release of NA from sympatfietic axon terminals can be detected in the isolated perfused heart [7,15] and in isolated atrial tissue [6,12,14,18,19,24].
Correspondence: E.S. Vizi, D e p a r t m e n t of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, H-1450 Budapest, Hungary.
The modulation of NA release from the noradrenergic nerve terminals has been extensively studied [22,23]. It has been shown that the release of NA from the sympathetic axon terminals in the right atrium can be regulated through presynaptic muscarinic [3,6,8,12,15,18,19,27], and c%-receptors [23] located in the membrane of the presynaptic nerve endings. Although the characteristics of automaticity, cellular electrical properties, and autonomic innervation [28] of atrioventricular valves have been extensively studied [2-5,13,17,28,30], no information is available on
100
the release of NA and its prejunctional modulation. Thus, the present investigation was carried out to study physiological and pharmacological characteristics of the release of NA from the bicuspid (mitral) valves of the rabbit heart and its modulation through different prejunctional receptors. A preliminary report of this study was presented at the 10th Annual Meeting of the European Neuroscience Association in Marseille in 1986 [20].
Materials and Methods
Preparation and incubation Male New Zealand rabbits of 2-3 kg were sacrificed by injecting air into the marginal ear vein. The heart was removed from the chest and the bicuspid (mitral) valves were prepared. The valves were suspended in an organ bath of 1 ml in Krebs solution (mmol/l: NaC1 113, KCI 4.7, CaC12 2.5, MgSO4 1.2, N a H C O 3 25, K H 2 P O 4 1.2, glucose 11.5) containing 300 /xM ascorbic acid, 30 # M N a 2 E D T A and 10 /xCi/ml 7,8-[~H] noradrenaline (S.A.: 40 C i / m m o l , Amersham, U.K.). The tissues were incubated for 45 min at 37°C and bubbled with a mixture of 95% 0 2 and 5% CO 2. Perfusion After the incubation period, the valves were suspended in a perfusion bath and superfused at rate of 1.0 m l / m i n with Krebs solution containing ascorbic acid and N a z E D T A . After a 90 rain washing period, 3 min effluents were collected for 87 rain. The agonists and antagonists to be tested were kept in the perfusion solution for 15 rain before the third stimulation period. Stimulation technique Field stimuli were delivered by a two-channel electrical stimulator (Eltron, Hungary) through platinum electrodes inserted from the top and the bottom of the perfusion bath. The stimuli were of supramaximal voltage and of 1 ms duration. The tissues were stimulated 3 times (S 1, S 2, S 3) delivering in each case 180 shocks with a frequency of 1 Hz. In a few experiments 10
Hz and 18(t shocks were applied, and in thesw cases the tissue was stimulated for 1,'; ~.
Calculation of the evoked release of radioactitqty An aliquot of 1 ml of the effluent was dissolved in 5 ml of mixture of toluene (2l)00 ml) and Triton X-100 containing 2 g of PPO and 6 g of POPOP. The radioactivity in the effluents was measured by using liquid scintillation counting with an LKB spectrometer. The measured counts were corrected to absolute activity by using an external standard technique. The data in these experiments were expressed as the absolute amount of radioactivity in B q / g (disintegrations per s per g tissue), or as fractional release values. The fractional release is the percent ratio of the released amount of radioactivity and the stored amount of radioactivity in the tissue. Determination of noradrenaline content The NA content was determined by high pressure liquid chromatography (HPLC) combined with electrochemical detector. The HPLC system consisted of a high performance pressure pump (Labor MIM) with a pulse dampener, a sample injector, and a Bondapak C18 HPLC column (10 /zm). For electrochemical detection an amperometric detector (Eltron, Hungary) was applied with a glassy carbon electrode set at +0.72 V against a Ag/AgCI reference electrode. The output from the electrochemical detector was recorded on a Servogor (Goerz, Austria) compensographic recorder. The mobile phase consisted of 0.05 m o l / l sodium acetate/citric acid buffer adjusted to pH 4.0, containing 4.5 /xM heptanc sulfonic acid. The mobile phase was filtered through a 0.45 ~xm Millipore filter and degassed under vacuum. The flow rate was maintained at 1.0 ml/min. NA standards were diluted freshly in 0.2 m o l / l perchloric acid-0.11 retool/1 ascorbic acid from the stock solution stored frozen in 1 m o l / l HC1. Visualization of catecholamines using histofluorescence methods Cardiac tissues were obtained from three rabbits sacrificed as described above. The entire bicuspid valve was removed along with small re-
lO1 gions of the a d j a c e n t tissues, including c h o r d a e t e n d i n e a e and p a p i l l a r y muscles. T h e site of att a c h m e n t of the valve to the a t r i a l wall c o n s i s t e d of a mixture of d e n s e c o n n e c t i v e tissue a n d cardiac muscle. T h e d i s s e c t e d valves w e r e w a s h e d briefly in o x y g e n a t e d K r e b s solution, p i n n e d out fiat in a Petri dish lined with Sylgard a n d were fixed for 2 - 4 h at 4°C in a m i x t u r e of 4 % p a r a f o r m a l d e h y d e s o l u t i o n in 0.1 M p h o s p h a t e buffer, p H 7.2 a n d 0.5% g l u t a r a l d e h y d e [9]. T h e s p e c i m e n was t h e n i m m e r s e d in the s a m e solution c o n t a i n i n g 30% sucrose ( w / v ) for a f u r t h e r hour. T h e valve was d i s s e c t e d free from p a p i l l a r y muscle a n d fibrous tissue a n d cryostat sections (28 /xm). F r o m two a n i m a l s only half of the valve was s e c t i o n e d a n d the r e m a i n i n g s e g m e n t viewed as a w h o l e m o u n t . Sections w e r e m o u n t e d on glass slides c o a t e d with p o l y - L - o r n i t h i n e a n d h e a t e d for 10 min at 45°C. Valve s e g m e n t s to be viewed u n s e c t i o n e d were also m o u n t e d flat on slides and t r e a t e d similarly. Slides w e r e t h e n d i p p e d briefly in xylene a n d c o v e r s l i p p e d , using E n t e l l a n ( M e r c k ) as t h e m o u n t a n t . C a t e c h o l a m i n e s w e r e i d e n t i f i e d in sections using a Leitz m i c r o s c o p e e q u i p p e d with a P l o e m p a k a n d a D filter cube.
Statistical analysis" T h e statistical significance of the results was d e t e r m i n e d by p a i r e d t-test, S t u d e n t ' s t-test a n d o n e - w a y analysis of v a r i a n c e ( A N O V A ) followed by D u n n e t t ' s test. In all cases P < 0.05 was cons i d e r e d significant.
Results
Morphological evidence for the presence of noradrenergic nert,e fibers in the bicuspid value B l u e - g r e e n f l u o r e s c e n c e , r e p r e s e n t i n g catec h o l a m i n e - c o n t a i n i n g nerve fibers, was f o u n d in all tissues e x a m i n e d . T h e r e w e r e d i f f e r e n c e s in the d e n s i t y a n d d i s t r i b u t i o n of t h e s e fibers between each of the areas. E x a m p l e s t a k e n from each r e g i o n are shown in Fig. 1 a n d the results arc s u m m a r i z e d in Fig. 2. T h e g r e a t e s t d e n s i t y of this i n n e r v a t i o n was found in the t r a n s i t i o n a l zone of the valve n e a r its
Fig. 1. Diagram showing relative density of catecholaminecontaining nerve fibers in various cardiac tissues, the papillary muscle, chordae tendineae, bicuspid valve and transitional zone (i.e. the site of attachment of the valve to the atrial wall). The valve tissue is shaded. The greatest density of innervation amongst these tissues is of the transitional zone, with intermediate innervation at the adjacent edge of the valve. Fluorescent nerve fibers are more sparse in the papillary' muscle, and rare or absent in most of the chordae tendineae and remainder of the valve.
site o f a t t a c h m e n t to the atrial wall. M a n y varicose f l u o r e s c e n t nerve fibers w e r e f o u n d t h r o u g h o u t this tissue, as fine single units a n d in small b u n d l e s (Fig. 2a). T h e g r e a t e s t d e n s i t y of nerve fibers was f o u n d at the o u t e r m a r g i n of the valve, in the t r a n s i t i o n a l zone (Fig, 2 a - c ) , but fluorescent fibers b e c a m e s p a r s e r until b e i n g a b s e n t at d i s t a n c e s g r e a t e r than 1 m m from this edge. T h e i n n e r v a t i o n of the p a p i l l a r y muscle was of interm e d i a t e density b e t w e e n these two valve regions. F l u o r e s c e n t varicose nerve fibers in this region
102
were associated with blood vessels a n d also with cardiac muscle. A few single nerve fibers were f o u n d associated with the c h o r d a c t e n d i n e a e , some of which travelled on the surface of the t e n d i n e a e for a short distance.
The endogenous noradrenaline attd tritium content of the bicuspid valve T h e e n d o g e n o u s N A c o n t e n t in the bicuspid valve m e a s u r e d by H P L C c o m b i n e d with electrochemical detection was 12.93 _+ 1.14 n m o l / g wet tissue (n = 15). T h e average N A c o n t e n t of the atrial tissue 11.49_+ 1.9 n m o l / g tissue (n = 8), was not significantly different from that of the bicuspid valve ( S t u d e n t ' s t-test; P > 0.1) (Table
1). T h e tritium c o n t e n t of the valve tissues incubated with [3H]-NA (13.3 # C i / m l ) at the end of the 90 min washing period was 1.27 -+: {1.09 M B q / g (SEM, n = 18) (Table I). The uptake of NA in the valve was in the same o r d e r of m a g n i t u d e as in the g u i n e a pig atrium [24].
The evoked release of tritium by electrical stimulation of I and 10 Hz D u r i n g the collection period three consecutive s t i m u l a t i o n s of 180 impulses were given either at I or 10 Hz, starting after the 8th, 36th. a n d 66th rain of the collection (S~, S 2, S 3). T h e effiux of radioactivity increased due to s t i m u l a t i o n and it r e t u r n e d to the level of the s p o n t a n e o u s effiux
Fig. 2. Examples of fluorescent catecholamine-containing nerve fibers: (a). Bicuspid valve, whole mount preparation. Nelwork ot single varicose nerve fibers, a region of valve and close to the .junction with the cardiac muscle; (b). Bicuspid valve, section. Micrograph shows (on the left) a region close 1o the transitional zone with many nerve fibers and a decrease in innelx'ation density with increasing distance (towards the right); (c). Transitional zone. Dense array of brightly-stained fibers amongst the muscle cells; and (d). Chordae tendineae, whole mount. Brighl, varicose nerve fibers are seen on and just under the surface. Calibration: B a r = 50 ,am.
103 TABLE
7000
1
('omparison ~,fthe ?v~4 content and uptake Of the guinea pig and rabbit atrium atut rabbit bicuslfid valve. Tissue
NA content nmol/g
NA uptake " MBq/g
4500
Guinea pig atrium 42.90+ 1.7 ;' 1.7 +_1/.2 ;' Rabbit atriuin 11.49 +_ 1.9 (n 8) Rabbit bicuspid valve 12.93+_ 1.14 (n 15) 1.27+_0.09 (n = 25)
o~l
66 0
T T
200C
The data were determined by HPLC combined with electrochemical detection. The NA content between the rabbit atrium and bicuspid wdve was not statistically significant ( P > 0.2, Student's t-lest). " NA uptake was measured as the tritium content of the tissue at the beginning of the collection period. l, Data taken from Sugimori et al [24].
within 12-15 rain (Fig. 3). The calculated areas below the increased effiux curve due to 1 Hz stimulation during the S~, S > and S 3 were 3802 +_ 562, 3681.+560, and 3619_+ 220 B q / g , respectively (mean -+ SEM, n = 4). The values are not significantly different from each other ( A N O V A , P < 0.1). The $ 2 / S 1 and S ~ / S 2 ratios were 0.98 -+ 0,01 and 1.04 +_ 0.16, respectively, not significantly different from 1.0 ( P = 0.1; paired t-test). When the release of N A was evoked by 10 Hz stimulation, the evoked releases were 6641 _+ 777, 6365 -+ 488, and 6914 +_ 927 B q / g , respectively (mean _+ SEM, n = 4), not significantly different from each other ( A N O V A , P = 0.11. The S x / S 1 and $ 3 / S 2 ratios were 0.99 -+ 0.1 and 1.05 -+ 0.10,
TABLE
0
30
60
90
rain
Fig. 3. The effect of 1 and 10 Hz stimulation on the NA release from the bicuspid wdve. The released amount of tritium expressed in B q / g was ploned against the lime. The tissue was stimulated three times (SI; $2: S~) from the hth, 36th and 6bth min of the collection period for 3 rain. • • 1 Hz, 1(1 ttz stimulation.
not significantly different from 1.0 (paired t-test P > 0.1). The newly taken-up N A can be released from the bicuspid valve in a frequency dependent fashion. The volley output was 63.36 Bq/g/volley at 1 Hz stimulation and 11.06 B q / g / v o l l e y at 10 Hz.
Effect of stimulation and / or inhibition qf prejunctional a-receptors Stimulation of the presynaptic c~2-receptors by 1 /,tM xylazine resulted in a significant decrease of the released amount of NA (Table II). Yohimbine (1 ~ M ) given before the S 3, in addition to xylazine, significantly increased the release of N A above the control value, producing an $ 3 / S 2 value
I1
EJfect q( o~, and muscarine receptor agonists at d atttagonists' on the release of [¢H]-noradrenaline fi'om isolated rabbit mitral t'alces.
1. Control 2. + Xylazine, 1 /xM 3. + Yohimbine, 1 ~ M 4. + Yohimbine, 1 IzM, + Xylazine, 1 /,tM 5. Control 6. + Oxotremorine, 0.1 ,v.M 7. +Atropine, 1 /,tM 8. +Atropine, 1 >M, +Oxotremorine, 0.1 p,M
n
[ 3tt]-noradrenaline release, $ 3 / S 2
5 5 4 5 5 4 5 4
0.99 4 0.39 + 1.6(/+ 1.71 + /).98 + 1t.55 + 1.14 + 1.14 +
0.(12 (1.1)6 0.11 tl.26 0.02 0.1/9 (L15 0.14
P
2:1 < 0.(Jl 3 : 1 < 11.01 4:2 < 0.111 6:5 < (1.111 7:5 > 0.05 8:5 < 0.111
The drugs were added into the perfusion fluid 15 min before S 3. n: number of experiments: P: significance calculated by A N O V A followed by Dunnett's test.
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significantly higher than the control. Yohimbine (1 >M), applied alone in the perfusion solution, increased the NA outflow and the measured $3/S 2 value was 1.60 _+ 0.11 (n = 4) significantly higher than the control.
Effect of stimulation and~or inhibition of presynaptic muscarinic receptors Stimulation of the prejunctional muscarinic receptors with 0.1 /~M oxotremorine resulted in a significant decrease of the released amount of NA (Table II). Atropine (1 p,M) given before the S 3, in addition to oxotremorine, increased the release of NA above the control value producing an $ 3 / S 2 value significantly higher than the control. Atropine (1 /.tM) applied alone in the perfusion solution increased the NA outflow and the measured $ 3 / S 2 value was 1.14 _+ 0.15 (n = 5) not significantly higher than the control ( P > 0.05) (Table II).
Discussion
The atrioventricular valves in the hearts of mammalian species contain sparsely arranged cardiac fibers in direct continuity with the working atrial myocardium [2,3,5,30]. Studies performed in mitral valves have demonstrated the possibility that the valves may represent a site of ectopic impulse formation in the intact heart [17]. Therefore, the neurovegetative innervation of this region might play an important role in affecting automaticity. In the present paper evidence was obtained that the bicuspid (mitrai) valves of the rabbit heart contain a significant amount of endogenous NA, and in addition take up [3H]-NA from the physiological media. What is even more important, NA can be released from the sympathetic nerve terminals of valve tissue and the release is subject to presynaptic modulation. The release of NA has been measured both from the whole heart preparation and from the isolated atrium of different species (rat heart: [8], guinea pig atrium: [12], rabbit atrium: [16,18,19,22]). This is the first case that the release of NA is measured from the valves. Similarly with the whole heart or atrium
preparation, the release evoked by electrical stim.ulation was inhibited by xylazine, a potent , . agonist, in the valve tissue as well. The c~e antagonist, yohimbine, antagonized the inhibitory effect of xylazine. However, the resulting release after the ~2 antagonist was higher than the control release. When yohimbine was applied alone in the perfusion solution, the release of NA was also increased to the same extent (Fig. 3). From these results it is concluded that the release of NA from the bicuspid valve is under a significant tonic control of the endogenous NA, and the inhibition caused by the release of endogenous NA is switched off by yohimbine, i.e. there is a negative feedback modulation. The release of NA can also be diminished by stimulating the presynaptic muscarinic receptors of the noradrenergic nerve endings of the heart [7,12,16,18,19]. In the present experiments, oxotremorine also resulted in a significant inhibition of the release of NA. Atropine overcame this inhibitory effect. However, atropine applied alone did not significantly increase the release of NA. This finding shows that the release of NA under these present circumstances is not under any cholinergic control, although the axon terminals are equipped with muscarinic receptors. However, our data do not preclude the possibility of presynaptic interaction between vagal nerve and sympathetic fibers. In fact it would be expected that under appropriate conditions (i.e. in response to enhanced vagal tone) the release of NA is tonically inhibited by ACh released from adjacent vagal nerve endings [25,26]. In this case vagal stimulation reduces automacy of the valve cells, not only through a direct action counteracting the effect of NA on the cells, but in an indirect way, inhibiting the release of NA from the axon terminals present in the valve. There is another possibility of how NA released from the valves behaves. It might have an effect on the membrane of the platelets. Activation of the c~2-adrenoceptors in human platelets is the factor which may help to initiate platelet aggregation [1]; this effect can be inhibited by selective c~2 antagonist [10]. The physiological and pathophysiological significance of these findings is not yet clear. It may be possible that,
105
under pathological conditions, N A released from the valve tissue may be involved in the aggregation of platelets in the close vicinity of the valve, promoting formation of thrombus inside the heart, in the valve area.
13 14
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106 modulation of transmitter release, Neuroscience 31 (1989) 259-267. 28 Williams, T.H., Mitral and tricuspid valve innervation. Brit. Heart J., 26 (1964) 105-115. 29 Wit, L.A., Fenolgio, J.J.Jr., Wagner, M.B. and Bassett, L.A., Electrophysiological properties of cardiac muscle in
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,'2 1991 Elsevier Science Publishers B.V. 0165-1838/91/$03.50
JANS 01184
Presynaptic modulation of the release of noradrenaline from electrically stimulated bicuspid valve leaflet of the rabbit heart G.T. Somogyi
1, J.R.
K e a s t ~ a n d E.S. Vizi 2
I Department of Pharmacology', University of Pittsburgh, Pittsburgh, Pennsyh'ania, U.S.A., and -"Institute of Experimental Medicbw, Hungarian Academy of Sciences, Budapest, Hungary (Received 17 January 1991) (Revision received and accepted 3 April 1991)
Key words: Bicuspid valve; Noradrenaline release; a2-Adrenoceptor; Muscarinic receptor; Presynaptic modulation Abstract The bicuspid (mitral) valves were obtained from male albino New Zealand rabbits. The noradrenaline (NA) content (12.93 + 1.14 n m o l / g ) of the valve tissue was determined by high pressure liquid chromatography (HPLC) combined with electrochemical detection. After incubation with tritiated N A for 45 rain, the tissues were m o u n t e d in perfusion baths and superfused with Krebs solution at a constant perfusion rate. After a 90 rain washing period, the tissues were stimulated three times (SI; $2; $3) at a frequency of 1 or 10 Hz, and the release of NA was expressed as the stimulus-induced overflow of radioactivity. Using a constant n u m b e r of impulses, the release of N A was significantly higher when the frequency applied was 10 Hz than in the case of 1 Hz. The release of N A was inhibited by stimulating the presynaptic a2-adrenoceptors with xylazine or by stimulating the presynaptic muscarinic receptors with oxotremorine. Yohimbine (1 /*M) not only overcame the effect of the a2-adrenoceptor stimulation caused by xylazine, but increased it over the control level, whereas atropine blocked the inhibitory effect of oxotremorine. It is concluded that the adrenergic nerves in the valve tissue release NA in a frequency-dependent fashion, and the release of N A can be modulated through presynaptic a 2- and muscarinic receptors. This is the first case that neurochemical evidence was obtained showing that N A is released from the mitral valve and is subject to presynaptic modulation.
Introduction
The heart has a dense noradrenergic innervation, and release of NA from sympatfietic axon terminals can be detected in the isolated perfused heart [7,15] and in isolated atrial tissue [6,12,14,18,19,24].
Correspondence: E.S. Vizi, D e p a r t m e n t of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, PO Box 67, H-1450 Budapest, Hungary.
The modulation of NA release from the noradrenergic nerve terminals has been extensively studied [22,23]. It has been shown that the release of NA from the sympathetic axon terminals in the right atrium can be regulated through presynaptic muscarinic [3,6,8,12,15,18,19,27], and c%-receptors [23] located in the membrane of the presynaptic nerve endings. Although the characteristics of automaticity, cellular electrical properties, and autonomic innervation [28] of atrioventricular valves have been extensively studied [2-5,13,17,28,30], no information is available on
100
the release of NA and its prejunctional modulation. Thus, the present investigation was carried out to study physiological and pharmacological characteristics of the release of NA from the bicuspid (mitral) valves of the rabbit heart and its modulation through different prejunctional receptors. A preliminary report of this study was presented at the 10th Annual Meeting of the European Neuroscience Association in Marseille in 1986 [20].
Materials and Methods
Preparation and incubation Male New Zealand rabbits of 2-3 kg were sacrificed by injecting air into the marginal ear vein. The heart was removed from the chest and the bicuspid (mitral) valves were prepared. The valves were suspended in an organ bath of 1 ml in Krebs solution (mmol/l: NaC1 113, KCI 4.7, CaC12 2.5, MgSO4 1.2, N a H C O 3 25, K H 2 P O 4 1.2, glucose 11.5) containing 300 /xM ascorbic acid, 30 # M N a 2 E D T A and 10 /xCi/ml 7,8-[~H] noradrenaline (S.A.: 40 C i / m m o l , Amersham, U.K.). The tissues were incubated for 45 min at 37°C and bubbled with a mixture of 95% 0 2 and 5% CO 2. Perfusion After the incubation period, the valves were suspended in a perfusion bath and superfused at rate of 1.0 m l / m i n with Krebs solution containing ascorbic acid and N a z E D T A . After a 90 rain washing period, 3 min effluents were collected for 87 rain. The agonists and antagonists to be tested were kept in the perfusion solution for 15 rain before the third stimulation period. Stimulation technique Field stimuli were delivered by a two-channel electrical stimulator (Eltron, Hungary) through platinum electrodes inserted from the top and the bottom of the perfusion bath. The stimuli were of supramaximal voltage and of 1 ms duration. The tissues were stimulated 3 times (S 1, S 2, S 3) delivering in each case 180 shocks with a frequency of 1 Hz. In a few experiments 10
Hz and 18(t shocks were applied, and in thesw cases the tissue was stimulated for 1,'; ~.
Calculation of the evoked release of radioactitqty An aliquot of 1 ml of the effluent was dissolved in 5 ml of mixture of toluene (2l)00 ml) and Triton X-100 containing 2 g of PPO and 6 g of POPOP. The radioactivity in the effluents was measured by using liquid scintillation counting with an LKB spectrometer. The measured counts were corrected to absolute activity by using an external standard technique. The data in these experiments were expressed as the absolute amount of radioactivity in B q / g (disintegrations per s per g tissue), or as fractional release values. The fractional release is the percent ratio of the released amount of radioactivity and the stored amount of radioactivity in the tissue. Determination of noradrenaline content The NA content was determined by high pressure liquid chromatography (HPLC) combined with electrochemical detector. The HPLC system consisted of a high performance pressure pump (Labor MIM) with a pulse dampener, a sample injector, and a Bondapak C18 HPLC column (10 /zm). For electrochemical detection an amperometric detector (Eltron, Hungary) was applied with a glassy carbon electrode set at +0.72 V against a Ag/AgCI reference electrode. The output from the electrochemical detector was recorded on a Servogor (Goerz, Austria) compensographic recorder. The mobile phase consisted of 0.05 m o l / l sodium acetate/citric acid buffer adjusted to pH 4.0, containing 4.5 /xM heptanc sulfonic acid. The mobile phase was filtered through a 0.45 ~xm Millipore filter and degassed under vacuum. The flow rate was maintained at 1.0 ml/min. NA standards were diluted freshly in 0.2 m o l / l perchloric acid-0.11 retool/1 ascorbic acid from the stock solution stored frozen in 1 m o l / l HC1. Visualization of catecholamines using histofluorescence methods Cardiac tissues were obtained from three rabbits sacrificed as described above. The entire bicuspid valve was removed along with small re-
lO1 gions of the a d j a c e n t tissues, including c h o r d a e t e n d i n e a e and p a p i l l a r y muscles. T h e site of att a c h m e n t of the valve to the a t r i a l wall c o n s i s t e d of a mixture of d e n s e c o n n e c t i v e tissue a n d cardiac muscle. T h e d i s s e c t e d valves w e r e w a s h e d briefly in o x y g e n a t e d K r e b s solution, p i n n e d out fiat in a Petri dish lined with Sylgard a n d were fixed for 2 - 4 h at 4°C in a m i x t u r e of 4 % p a r a f o r m a l d e h y d e s o l u t i o n in 0.1 M p h o s p h a t e buffer, p H 7.2 a n d 0.5% g l u t a r a l d e h y d e [9]. T h e s p e c i m e n was t h e n i m m e r s e d in the s a m e solution c o n t a i n i n g 30% sucrose ( w / v ) for a f u r t h e r hour. T h e valve was d i s s e c t e d free from p a p i l l a r y muscle a n d fibrous tissue a n d cryostat sections (28 /xm). F r o m two a n i m a l s only half of the valve was s e c t i o n e d a n d the r e m a i n i n g s e g m e n t viewed as a w h o l e m o u n t . Sections w e r e m o u n t e d on glass slides c o a t e d with p o l y - L - o r n i t h i n e a n d h e a t e d for 10 min at 45°C. Valve s e g m e n t s to be viewed u n s e c t i o n e d were also m o u n t e d flat on slides and t r e a t e d similarly. Slides w e r e t h e n d i p p e d briefly in xylene a n d c o v e r s l i p p e d , using E n t e l l a n ( M e r c k ) as t h e m o u n t a n t . C a t e c h o l a m i n e s w e r e i d e n t i f i e d in sections using a Leitz m i c r o s c o p e e q u i p p e d with a P l o e m p a k a n d a D filter cube.
Statistical analysis" T h e statistical significance of the results was d e t e r m i n e d by p a i r e d t-test, S t u d e n t ' s t-test a n d o n e - w a y analysis of v a r i a n c e ( A N O V A ) followed by D u n n e t t ' s test. In all cases P < 0.05 was cons i d e r e d significant.
Results
Morphological evidence for the presence of noradrenergic nert,e fibers in the bicuspid value B l u e - g r e e n f l u o r e s c e n c e , r e p r e s e n t i n g catec h o l a m i n e - c o n t a i n i n g nerve fibers, was f o u n d in all tissues e x a m i n e d . T h e r e w e r e d i f f e r e n c e s in the d e n s i t y a n d d i s t r i b u t i o n of t h e s e fibers between each of the areas. E x a m p l e s t a k e n from each r e g i o n are shown in Fig. 1 a n d the results arc s u m m a r i z e d in Fig. 2. T h e g r e a t e s t d e n s i t y of this i n n e r v a t i o n was found in the t r a n s i t i o n a l zone of the valve n e a r its
Fig. 1. Diagram showing relative density of catecholaminecontaining nerve fibers in various cardiac tissues, the papillary muscle, chordae tendineae, bicuspid valve and transitional zone (i.e. the site of attachment of the valve to the atrial wall). The valve tissue is shaded. The greatest density of innervation amongst these tissues is of the transitional zone, with intermediate innervation at the adjacent edge of the valve. Fluorescent nerve fibers are more sparse in the papillary' muscle, and rare or absent in most of the chordae tendineae and remainder of the valve.
site o f a t t a c h m e n t to the atrial wall. M a n y varicose f l u o r e s c e n t nerve fibers w e r e f o u n d t h r o u g h o u t this tissue, as fine single units a n d in small b u n d l e s (Fig. 2a). T h e g r e a t e s t d e n s i t y of nerve fibers was f o u n d at the o u t e r m a r g i n of the valve, in the t r a n s i t i o n a l zone (Fig, 2 a - c ) , but fluorescent fibers b e c a m e s p a r s e r until b e i n g a b s e n t at d i s t a n c e s g r e a t e r than 1 m m from this edge. T h e i n n e r v a t i o n of the p a p i l l a r y muscle was of interm e d i a t e density b e t w e e n these two valve regions. F l u o r e s c e n t varicose nerve fibers in this region
102
were associated with blood vessels a n d also with cardiac muscle. A few single nerve fibers were f o u n d associated with the c h o r d a c t e n d i n e a e , some of which travelled on the surface of the t e n d i n e a e for a short distance.
The endogenous noradrenaline attd tritium content of the bicuspid valve T h e e n d o g e n o u s N A c o n t e n t in the bicuspid valve m e a s u r e d by H P L C c o m b i n e d with electrochemical detection was 12.93 _+ 1.14 n m o l / g wet tissue (n = 15). T h e average N A c o n t e n t of the atrial tissue 11.49_+ 1.9 n m o l / g tissue (n = 8), was not significantly different from that of the bicuspid valve ( S t u d e n t ' s t-test; P > 0.1) (Table
1). T h e tritium c o n t e n t of the valve tissues incubated with [3H]-NA (13.3 # C i / m l ) at the end of the 90 min washing period was 1.27 -+: {1.09 M B q / g (SEM, n = 18) (Table I). The uptake of NA in the valve was in the same o r d e r of m a g n i t u d e as in the g u i n e a pig atrium [24].
The evoked release of tritium by electrical stimulation of I and 10 Hz D u r i n g the collection period three consecutive s t i m u l a t i o n s of 180 impulses were given either at I or 10 Hz, starting after the 8th, 36th. a n d 66th rain of the collection (S~, S 2, S 3). T h e effiux of radioactivity increased due to s t i m u l a t i o n and it r e t u r n e d to the level of the s p o n t a n e o u s effiux
Fig. 2. Examples of fluorescent catecholamine-containing nerve fibers: (a). Bicuspid valve, whole mount preparation. Nelwork ot single varicose nerve fibers, a region of valve and close to the .junction with the cardiac muscle; (b). Bicuspid valve, section. Micrograph shows (on the left) a region close 1o the transitional zone with many nerve fibers and a decrease in innelx'ation density with increasing distance (towards the right); (c). Transitional zone. Dense array of brightly-stained fibers amongst the muscle cells; and (d). Chordae tendineae, whole mount. Brighl, varicose nerve fibers are seen on and just under the surface. Calibration: B a r = 50 ,am.
103 TABLE
7000
1
('omparison ~,fthe ?v~4 content and uptake Of the guinea pig and rabbit atrium atut rabbit bicuslfid valve. Tissue
NA content nmol/g
NA uptake " MBq/g
4500
Guinea pig atrium 42.90+ 1.7 ;' 1.7 +_1/.2 ;' Rabbit atriuin 11.49 +_ 1.9 (n 8) Rabbit bicuspid valve 12.93+_ 1.14 (n 15) 1.27+_0.09 (n = 25)
o~l
66 0
T T
200C
The data were determined by HPLC combined with electrochemical detection. The NA content between the rabbit atrium and bicuspid wdve was not statistically significant ( P > 0.2, Student's t-lest). " NA uptake was measured as the tritium content of the tissue at the beginning of the collection period. l, Data taken from Sugimori et al [24].
within 12-15 rain (Fig. 3). The calculated areas below the increased effiux curve due to 1 Hz stimulation during the S~, S > and S 3 were 3802 +_ 562, 3681.+560, and 3619_+ 220 B q / g , respectively (mean -+ SEM, n = 4). The values are not significantly different from each other ( A N O V A , P < 0.1). The $ 2 / S 1 and S ~ / S 2 ratios were 0.98 -+ 0,01 and 1.04 +_ 0.16, respectively, not significantly different from 1.0 ( P = 0.1; paired t-test). When the release of N A was evoked by 10 Hz stimulation, the evoked releases were 6641 _+ 777, 6365 -+ 488, and 6914 +_ 927 B q / g , respectively (mean _+ SEM, n = 4), not significantly different from each other ( A N O V A , P = 0.11. The S x / S 1 and $ 3 / S 2 ratios were 0.99 -+ 0.1 and 1.05 -+ 0.10,
TABLE
0
30
60
90
rain
Fig. 3. The effect of 1 and 10 Hz stimulation on the NA release from the bicuspid wdve. The released amount of tritium expressed in B q / g was ploned against the lime. The tissue was stimulated three times (SI; $2: S~) from the hth, 36th and 6bth min of the collection period for 3 rain. • • 1 Hz, 1(1 ttz stimulation.
not significantly different from 1.0 (paired t-test P > 0.1). The newly taken-up N A can be released from the bicuspid valve in a frequency dependent fashion. The volley output was 63.36 Bq/g/volley at 1 Hz stimulation and 11.06 B q / g / v o l l e y at 10 Hz.
Effect of stimulation and / or inhibition qf prejunctional a-receptors Stimulation of the presynaptic c~2-receptors by 1 /,tM xylazine resulted in a significant decrease of the released amount of NA (Table II). Yohimbine (1 ~ M ) given before the S 3, in addition to xylazine, significantly increased the release of N A above the control value, producing an $ 3 / S 2 value
I1
EJfect q( o~, and muscarine receptor agonists at d atttagonists' on the release of [¢H]-noradrenaline fi'om isolated rabbit mitral t'alces.
1. Control 2. + Xylazine, 1 /xM 3. + Yohimbine, 1 ~ M 4. + Yohimbine, 1 IzM, + Xylazine, 1 /,tM 5. Control 6. + Oxotremorine, 0.1 ,v.M 7. +Atropine, 1 /,tM 8. +Atropine, 1 >M, +Oxotremorine, 0.1 p,M
n
[ 3tt]-noradrenaline release, $ 3 / S 2
5 5 4 5 5 4 5 4
0.99 4 0.39 + 1.6(/+ 1.71 + /).98 + 1t.55 + 1.14 + 1.14 +
0.(12 (1.1)6 0.11 tl.26 0.02 0.1/9 (L15 0.14
P
2:1 < 0.(Jl 3 : 1 < 11.01 4:2 < 0.111 6:5 < (1.111 7:5 > 0.05 8:5 < 0.111
The drugs were added into the perfusion fluid 15 min before S 3. n: number of experiments: P: significance calculated by A N O V A followed by Dunnett's test.
104
significantly higher than the control. Yohimbine (1 >M), applied alone in the perfusion solution, increased the NA outflow and the measured $3/S 2 value was 1.60 _+ 0.11 (n = 4) significantly higher than the control.
Effect of stimulation and~or inhibition of presynaptic muscarinic receptors Stimulation of the prejunctional muscarinic receptors with 0.1 /~M oxotremorine resulted in a significant decrease of the released amount of NA (Table II). Atropine (1 p,M) given before the S 3, in addition to oxotremorine, increased the release of NA above the control value producing an $ 3 / S 2 value significantly higher than the control. Atropine (1 /.tM) applied alone in the perfusion solution increased the NA outflow and the measured $ 3 / S 2 value was 1.14 _+ 0.15 (n = 5) not significantly higher than the control ( P > 0.05) (Table II).
Discussion
The atrioventricular valves in the hearts of mammalian species contain sparsely arranged cardiac fibers in direct continuity with the working atrial myocardium [2,3,5,30]. Studies performed in mitral valves have demonstrated the possibility that the valves may represent a site of ectopic impulse formation in the intact heart [17]. Therefore, the neurovegetative innervation of this region might play an important role in affecting automaticity. In the present paper evidence was obtained that the bicuspid (mitrai) valves of the rabbit heart contain a significant amount of endogenous NA, and in addition take up [3H]-NA from the physiological media. What is even more important, NA can be released from the sympathetic nerve terminals of valve tissue and the release is subject to presynaptic modulation. The release of NA has been measured both from the whole heart preparation and from the isolated atrium of different species (rat heart: [8], guinea pig atrium: [12], rabbit atrium: [16,18,19,22]). This is the first case that the release of NA is measured from the valves. Similarly with the whole heart or atrium
preparation, the release evoked by electrical stim.ulation was inhibited by xylazine, a potent , . agonist, in the valve tissue as well. The c~e antagonist, yohimbine, antagonized the inhibitory effect of xylazine. However, the resulting release after the ~2 antagonist was higher than the control release. When yohimbine was applied alone in the perfusion solution, the release of NA was also increased to the same extent (Fig. 3). From these results it is concluded that the release of NA from the bicuspid valve is under a significant tonic control of the endogenous NA, and the inhibition caused by the release of endogenous NA is switched off by yohimbine, i.e. there is a negative feedback modulation. The release of NA can also be diminished by stimulating the presynaptic muscarinic receptors of the noradrenergic nerve endings of the heart [7,12,16,18,19]. In the present experiments, oxotremorine also resulted in a significant inhibition of the release of NA. Atropine overcame this inhibitory effect. However, atropine applied alone did not significantly increase the release of NA. This finding shows that the release of NA under these present circumstances is not under any cholinergic control, although the axon terminals are equipped with muscarinic receptors. However, our data do not preclude the possibility of presynaptic interaction between vagal nerve and sympathetic fibers. In fact it would be expected that under appropriate conditions (i.e. in response to enhanced vagal tone) the release of NA is tonically inhibited by ACh released from adjacent vagal nerve endings [25,26]. In this case vagal stimulation reduces automacy of the valve cells, not only through a direct action counteracting the effect of NA on the cells, but in an indirect way, inhibiting the release of NA from the axon terminals present in the valve. There is another possibility of how NA released from the valves behaves. It might have an effect on the membrane of the platelets. Activation of the c~2-adrenoceptors in human platelets is the factor which may help to initiate platelet aggregation [1]; this effect can be inhibited by selective c~2 antagonist [10]. The physiological and pathophysiological significance of these findings is not yet clear. It may be possible that,
105
under pathological conditions, N A released from the valve tissue may be involved in the aggregation of platelets in the close vicinity of the valve, promoting formation of thrombus inside the heart, in the valve area.
13 14
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