Effects of Higenamine on the Contractility of Aorta and Taenia Coli in Guinea-Pigs'

The Seoul Journal of Medicine Vol. 32, No. 4: 223-238, December 1991

Effects of Higenamine on the Contractility of Aorta and Taenia Coli in Guinea-pigs'

Sang Bok Suk, Kwang Wook KO, Sang Jin Lee,* Sang-lk Hwang* and Ki-Whan Kim*

Department of l'ediatrics and Department of Physiology, * Seoul National University College of Medicine, Seoul 110-460, Korea

=Abstract=Higenamine isolated from Aconiti tuber is known to have positive inotropic action through the adrenergic P-receptors. However, the effects of higenamine on the vascular smooth muscle and taenia coli have not yet been elucidated. The actions of higenamine were investigated on the activated aortic strips of guinea-pigs and rabbits by norepinephrine, high K-Tyrode's solution and electrical field stimulation, and also on the spontaneous contractions of guinea-pig taenia coli. Electrical activities and mechanical contractions were simultaneously recorded using the conventional suction-electrode method and single sucrose gap technique. All experiments were performed in tris-buffered Tyrode's solution which was aerated with 100% 02 and kept at 35t. Higenamine suppressed dose-dependently the norepinephrine-induced contraction. Propranolol potently antagonized the inhibitory effects of higenamine on rabbit aorta, while less potently on guinea-pig aorta. Higenamine seemed to suppress profoundly the component of intracellular Ca2+ mobilization in the contraction curve of norepinephrine in guinea-pig aorta. Higenamine did not seem to suppress the component of Ca2+ influx through the potential-sensitive Ca2+ channel in K-contracture of guinea-pig aorta. Higenamine suppressed both passive resting tension and active tension (phasic contraction) driven by electrical field stimulation (A.C. 60 Hz, 5-7 V/cm, duration 10 sec, interval 3 min) in guinea-pig aorta. Propranolol also rapidly suppressed both passive resting and active tensions. Higenamine dose-dependently suppressed the frequency and amplitude of spontaneous contraction, resulting in complete abolishment of it above lop6M concentration in guinea-pig taenia coli. Propranolol almost completely antagonized its effect. Higenamine reduced dose-dependently both burst frequency and spike frequency, and depolarized membrane potential in guinea-pig taenia coli. Propranolol almost completely blocked its effect. In conclusion the inhibitory action of higenamine to the norepinephrine-induced contraction might be produced by the depression of intracellular Ca2+ mobilization and Ca2+-influxthrough P-adrenoceptors in guinea-pig aorta. The Ca2+-influxthrough the potential-sensitive Ca2+ channel might not be suppressed by higenamine in guinea-pig aorta. The inhibitory effects of higenamine on the spontaneous contractions of guinea- pig taenia coli result from the decrease of burst frequency and spike frequency through the P-adrenoceptors.

Key Words: Higenamine, Aorta, Taenia coli, Norepinephrine-induced contraction, Electrical field stimulation, K-contracture, Spontaneous contraction

' This study was supported in part by a clinical research grant from Seoul National University Hospital (1 989). in order to understand the higenamine-induced re- INTRODUCTION sponses to the cardiovascular system. This study was undertaken to clarify the effects Higenamine is a substance which was isolated of higenamine on the Ca2+ transport through the from Aconiti tuber and named by Kosuge et a/. smooth muscle membrane using aorta and taenia in 1978. It is now known as Cl6HI7No3 HCI with coli. a structure of dl-1-(4-hydroxybenzy1)-6,7-dihydroxy- 1,2,3,4-tetra-hydroisoquinoline hydrochloride. MATERIALS AND METHODS The excitatory mechanism of higenamine was reported to be the inhibition of Na-K pump like Albino guinea-pigs of either sex, weighing 300 digitalis (Shin et a/. 1976). The inotropic action was g, were stunned and bled. Firstly, the taenia coli additively determined by the extracellular calcium were excised from the large intestine and then the concentration (Hong et a/. 1980).The positive ino- thoracic aorta from the thoracic cavity. The excised tropic action of higenamine on the rabbit left atrium tissues were managed to be used as experimental was reported to be due to the potentiation of cal- preparation in phosphate-buffered Tyrode's solu- cium influx through the cell membrane (Chang et tion at room temperature. The experiments were al. 1981). In the electrophysiological study of rabbit done in tris-buffered Tyrode's solution at 353. papillary muscle it was reported that higenamine The ionic compositions of phosphate-buffered increased the slow inward calcium currents (Kwon and tris-buffered Tyrode's solutions were as follows et a/. 1981). The action of higenamine studied on respectively (mM): phosphate-buffered Tyrode's, calcium transport and ATPase activity in the sarco- NaCl 147, KC1 4, CaCI2 2H20 2, MgCI2 = 6H20 1. plasmic reticulum (SR) was explained as the inhibi- 05, NaH2P04 2H20 0.42, Na2HP04- 12H20 1.81, tion of Mg-dependent Ca-ATPase that is essential glucose 5.5; tris-buffered Tyrode's, NaCl 147, KC1 for calcium transport into the SR as well as the 4, CaCI2 2H20 2, MgCI2 6H20 1.05, tris HCI 5, potentiation of calcium release from the SR, resul- glucose 5.5. The solutions were aerated with 100°/o ting in the increase of the intracellular calcium con- O2 and pH of the solution was maintained at 7.35. centration (Kim et a/. 1982). 1) The strips of thoracic aorta In the study of the effect of higenamine on the Pure thoracic aorta was isolated from peripheral adrenaline receptors of the cardiovascular system connective tissue in the preparation chamber and in rabbits, the inotropic action of higenamine was was made into a long strip by cutting helically (heli- explained as the stimulation of P-adrenoceptors cal strip). The strip, 10- 12 mm long and 1 - 2 mm (Park et a/. 1984; Kim et al. 1986). Higenamine in- wide was recovered at room temperature for 1 creased the heart rate by increasing sodium cur- hour. The mechanical contractions were recorded rent in the SA node of rabbits (Cho et al. 1986). in a vertical chamber (capacity, 100 ml) by Grass Higenamine was also reported to act on the cal- FT-03 force transducer connected to a Device cium current rather than the potassium current physiograph. (Rhee et a/. 1987). In in-vivo study of rabbits (Kim Before the main experiment the optimal length et a/. 1986) higenamine decreased blood pressure of the strip was determined from the length-tension and total peripheral resistance, and increased car- curve of the contractions evoked by electrical field diac output and heart rate. These effects were all stimulation (A.C. 60 Hz, 5-7 V/cm, duration 10 sec, blocked by pretreatment with propranolol. Atenolol interval 3 min). The basal tension was about 500 also lowered cardiac output and heart rate, and mg. High K-Tyrode's solution used in K-contracture hexamethonium lowered the heart rate and increa- experiment was made by replacing sodium ion with sed blood pressure. Most investigations concer- potassium ion isotonically. ning higenamine were focused on its effects on 2) The taenia coli the heart. However, the effects of higenamine on The taenia coli (longitudinal muscle) was isolated smooth muscle contractility must be elucidated, from the remaining circular muscle completely in the preparation chamber and was trimmed to the the electrical field stimulation because taenia coli experimental strip which was 10 mm long, 2 mm has spontaneous contractions. wide and 5 mg in wet weight. In the recording of electrical activities two me- The mechanical contractions were recorded by thods were used; extracellular (Fig. 1) and intracel- the same method as the aortic strip except lular recording using the single sucrose gap technique (Fig. 2). The extracellular recording was done in the horizontal chamber (capacity, 3 ml), where the strip was pinned out at one end for the recor- Recorder ding of action potentials and was connected to the force transducer via a thread ring at the other Suction electrode end for the simultaneous recording of contraction. Force The suction electrode was used for the extracellu- transducer - - - Smooth muscle lar recording of action potentials, attached to the Fig. 1. A schematic representation of the isometric serosal side of the strip. The perfusion rate of tris- contraction and electrical activity recording buffered Tyrode's solution was 6 mllmin and the system. The isometric contractions were re- optimal length for the strip was determined by the corded through a tension transducer from the length-tension diagram. The experimental chamber smooth muscle preparation. The electrical ac- for the single sucrose-gap method consisted of tivities were measured extracellularly by use three compartments: the normal Tyrode's compart- of suction electrode. ment where the isotonic contractions were recorded by force transducer, the sucrose compartment of 1 mm in width and the isotonic KC1 compartment where the electrical activity was recorded through AgIAgCI electrode.

Drugs used were as follows: atropine sulfate (Sigma) norepinephrine (Arterenol, Hochst) phentolamine (Regitine, CIBA) dl-propranolol (Sigma) tetrodotoxin (TTX, Sankyo)

Fig. 2. Diagram of the single sucrose-gap apparatus. In this experiment no statistics were used be- The muscle chamber consisted of three com- cause the result analysis was a qualitative compa- partments; NT for perfusion of normal Tyrode's rison between data groups, and the figures cited or test solution, sucrose compartment for per- in this article were representative ones selected fusion of isoosmotic sucrose solution, and KC1 after confirmation of the szme results. compartment for perfusion of isotonic KC1 Ty- rode's solution. A muscle strip is mounted as RESULTS shown, one end (KC1 compartment) fixed and the other (NT compartment) connected to a 1. The effect of higenamine on the contractility force transducer. The width of sucrose-gap of the aortic smooth muscle is 1 mm. Electrical activities are recorded Having no spontaneous contractions the strip through the two AgIAgCI recording electrodes of aorta was induced to contraction by norepine- connected to a preamplifier, CRO and recor- phrine, high K-Tyrode's solution or electrical field der. Mechanical contractions are measured stimulation. through the force transducer (FT) connected 1) The effect of higenamine on the norepine- to a recorder. phrine-induced contraction 5 min

t t 4 -30 5x10.'

Norepinephrine ( M )

Toe(o.'M HigenaminePropranolol 10'=M Phentolamine 5x10-~M Fig. 3. Dose-dependent contractile responses to noreprnephrlne (A) and the relaxing effects of higenamine on norepinephrine-induced contraction in guinea-pig aorta (B). (A) Norepinephrine was administered cumulatively to bathing Tyrode's solution. Sustained contraction was developed initially at the concentration of lo-' M and maximum tension was induced at M. (B) Hlgenamine suppressed the norepinephrine-induced contraction in a dose-dependent manner and propranolol blocked the relaxing effects of higenamine. -10 min -10 min

Norep~nephrine Norepinephrine (5xlO-'M) Higenamine W/NT (5~10~'~) Propranolol ( M 1 - MI Higenamine M) Fig. 4. Effect of pretreated propranolol on the relaxatior, response of higenamine to norepinephrine-induced contraction in guinea-pig aorta. The sustained contraction induced by norepinephrine (5X lop7M) was relaxed by 56% after higenamine M) was added to the bathing Tyrode's solution (left), but in the same propranolol-pretreated preparation higenamine relaxed the contraction by 41% (right).

When norepinephrine was administered cumula- and propranolol blocked the relaxing effect of tively to the bathing Tyrode's solution sustained higenamine (Fig. 38). contraction developed initially at the concentration The sustained contraction induced by norepine- of 10-7 M with maximum tension at 10-5 M. The phrine (5 X 1OP7 M) was relaxed by 56% after higen- value of ED50 was 2 X 10-6 M (Fig. 3A). Higenamine amine M) was added to the bathing Tyrode's suppressed the contraction induced by norepine- solution (Fig. 4, left). But in the same preparation phrine (5 X 10-7 M) in a dose-dependent manner pretreated with propranolol (10-5 M) higenamine 10 min --a cartrol (no propmmlol) e-. with pmpr-~ (10~~1

. .. Norepinephrine Norepinqhrim (1Q' M) (lQ*M) -10 min

+ EGTA 0.1 mM 500 mg

10 min

0L 4 1 I w/NT-1 W/NT lo-' t lo-' t to-' Norepinephrine Nompimphrine sxlO-' 5xUr' (lo-* M (10-*M) Concontration of Hipnomino (M) Fig. 6. Suppressicn of norepinephrine-induced con- Fig. 5. Dose-dependency of the relaxant effects of tractions of guinea-pig aortic strip by higen- higenamine on norepinephrine-induced con- amine in a Ca2+-freeTyrode's solution. Repea- traction, and the blocking effects of pretreated ted exposures of aortic strip to norepinephrine propranolol on the relaxing response of higen- developed contractions with same amplitude; amine. No tachyphylaxis was observable on the response of norepinephrine (A). In the presence relaxed norepinephrine-induced contraction by 41 of higenamine within the media, the compo- O/O (Fig. 4, right). nent of contraction curve produced by norepi- The dose-dependency of the relaxing effect of nephrine-induced intracellular Ca2+ mobiliza- higenamine on norepinephrine-induced contraction tion (C) was much suppressed, compared with was compared to that in cases pretreated with that in the absence of higenamine (B). propranolol (Fig. 5). Higenamine administered in the concentrations of M, 5X lop6M and lop5M 6B, C). Therefore, it was suggested that the relax- produced blocking effects of 35%, 60% and 65% ing effect of higenamine through P-receptors was respectively, while in the cases pretreated with pro- not only related to the inhibition of Ca influx through pranolol those effects were 10°/o, 30% and 35% receptor-operated Ca channel, but also related to respectively. the intracellular calcium mobilization. 2) The effect of higenamine on the intracellular cal- 3) Comparison between the effects of higenamine cium mobilization on guinea-pig aorta and those on rabbit Repeated exposures of the aortic strip to norepi- aorta nephrine did not diminish the amplitude of contrac- The response of the aortic smooth muscle to tions (no tachyphylaxis phenomenon) (Fig. 6A). norepinephrine was compared between guinea- Norepinephrine induced more small contraction in pigs and rabbits (Fig. 7). The value of EDso was the Ca-free Tyrode's solution containing higen- 2 X 10-6 M in the guinea-pig aorta, whereas it was amine than in pure Ca-free Tyrode's solution (Fig. lo-' M in the rabbit aorta, showing different sen- (%) H guinea-pig aorta pletely blocked the effect of higenamine on K-contracture (Fig. 10B). Therefore, it was suggested that the relaxing effect of higenamine was developed not through the potential-sensitive Ca-channel, but through the receptor-operated Ca-channel. 5) The effect of higenamine on the contraction induced by electrical field stimulation The strip was stimulated regularly with A.C. 60 Hz, 7 Vlcm for 10 sec every 2.5- 3 min. Both passive resting and active tensions were rapidly decreased by the addition of higenamine M and M) to bathing Tyrode's solution, and gradually recovered after washout with normal Tyrode's solution (Fig. 11A). But the phasic contractions supp- lo-' 10-~ ressed by higenamine were not restored to normal Concentration of norepinephrine (M ) level by the addition of propranolol to bathing Ty- Fig. 7. Dose-dependency of norepinephrine-induced rode's solution (Fig. 1 1B). contractions in guinea-pig aorta and rabbit The phasic contractions disappeared rapidly af- aorta. Developed tension was expressed as ter the administration of 3 blockers (TTX 3X 10-7 M, a percentage of maximum response induced phentolamine M, and atropine 10-6 M), and at the concentration of 2 X 10-5 M norepine- reappeared gradually after washout with normal phrine in guinea-pig aorta. Each point shows Tyrode's solution (Fig. 12A). The phasic contraction the meankS.E.M. for pooled data from 9 pre- driven by electrical field stimulation was suppres- parations. EDso was at a concentration of 2X sed by higenamine and completely disappeared 10-6 M norepinephrine in guinea-pig aorta, after the administration of phentolamine (Fig. 128). whereas that of rabbit aorta was at a concent- The phasic contractions evoked by electrical ration of 10-7 M norepinephrine. field stimulation were thought to be due to the release of neurotransmitters, especially norepinephrine from the tissue nerve endings. But the absence sitivity between animal species. Sustained contrac- of the effect of propranolol and the decrease of tions developed dose-dependently as the concen- passive resting tension could not be explained. tration of norepinephrine increased. These contractions were suppressed more severely in rabbits 2. The effect of higenamine on the spontaneous than in guinea-pigs and propranolol blocked com- contractions of the taenia coli pletely the relaxing effect of higenamine (Fig. 8). 1) The inhibitory effect of higenamine on the spon- The effects of pretreated and post-treated propra- taneous contractions nolol were shown in Fig. 9. Higenamine M) Higenamine suppressed the amplitude of spon- relaxed the contraction induced by norepinephrine taneous contractions and this effect was reversed M) by 78%. Complete recovery was observ- by washout with normal Tyrode's solution (Fig. 13). able after the administration of propranolol M). Higenamine exerted an inhibitory action on the am- Pretreated propranolol M) blocked comple- plitude and frequency of spontaneous contraction tely the relaxing effect of higenamine (lop6M). from the concentration of 1OP8 M dose-dependen- 4) The effect of higenamine on K-contracture tly and completely abolished it at the concentration Higenamine suppressed the K-contracture pro- of M (Fig. 14). The depressed contractility induced by 25 mM K-Tyrode's solution dose-depen- duced by higenamine (lo-' M) was antagonized dently (Fig. 10A) and pretreatment with propranolol by propranolol (10-6 M). However, phentolamine (5 X 10-6 M) and phentolamine (5 X 10-6 M) com- had no effect at all (Fig. 15). Rabbit aorta

0 0

10-~ 10- Norepinephrine (M )

5 min n

Norepinephrine (lo-' M )

Higenamine ( M ) Propranolol ( M Fhentolamine ( M) Fig. 8. Dose-dependent contractile responses to norepinephrine (A) and the relaxing effects of higenamine on norepinephrine-induced contraction in rabbit aorta (B). (A) The sustained contractions were potentiated dose-dependently in parallel with the increase in norepinephrine concentration administered cumulatively to bathing Tyrode's solution. ED50 was observed at the concentration of lo-' M and maximum tension developed at M norepinephrine. (B) Higenamine suppressed the norepinephrine-induced contraction in a dose-dependent manner, and post-treated propranolol blocked completely the relaxing effects of higenamine.

Rabbit aorta

5 min

f Norepinephrine ( M ) t Higenamine ( M ) lo-' ' 10-6 M f Propranolol ( M t Phentolamine ( M

5 min

-. -

Norepinephrine (lo-' M ) Propranolol M ) Higenamine M) Phentolamine M ) Fig. 9. Effects of post-treated (A) and pretreated (B) propranolol on the relaxation response of higenamine to norepinephrine-induced contraction in rabbit aorta. Higenamine M) relaxed the contraction induced by norepinephrine (lo-' M) by 78%, and complete recovery was observable after the administration of propranolol M) additionally (A). Pretreated propranolol blocked completely the relaxation response of higenamine to norepinephrine-induced contraction (B). 10 min n

10 min

- 7- 10 min

t W/NT K-25 t Phentolamine ( 5 ~10'~M ) I Pro~ranolol ( 5 ~10-~M 1

Concentration of external potassium (mM) Fig. 10. Effect of higenamine on K-contracture developed at 25 mM K-Tyrode's solution in guinea-pig aorta. Higenamine suppressed the K-contracture dose-dependently (A), whereas pretreated propranolol blocked completely the higenamine effect on K-contracture (B). Dose-dependency of K-contracture in guinea-pig aorta (C). 10 min

t 4 4 * W/NT Higenamine 10-6 1 lo-=M t Propranolol (lo-=M) -5 min

t 4 H igenamine (lo-' M ) Propranolol ( M )

Fig. 11 Effect of higenamine on the phasic contractions induced by electrical field stimulation in guinea-pig aortic strip. The strip was stimulated regularly with A.C., 60 Hz, 7 Vlcm for 10 sec every 2.5 minutes. Both passive resting tension and active tension were rapidly decreased by the addition of higenamine to bathing Tyrode's solution, and gradually recovered after washout with normal Tyrode's solution (A). The phasic contractions suppressed by higenamine were not restored to normal level by the addition of propranolol to bathing Tyrode's solution (A, B).

10 min

* t f t f W/NT 3 Blockers -10 min

, . f Higenamine W/NT Phentolamine (5~10-~M) (,O-eM)

Fig. 12. Characteristics of the phasic contractions induced by electrical field stimulation in guinea-pig aorta. Phasic contractions were induced regularly with A.C., 60 Hz, 7 Vlcm for 10 sec every 3 minutes. The phasic contractions disappeared rapidly after the administration of 3 blockers (TTX 3X lo-' M, phentolamine lop6M, and atropine lop6M), and reappeared gradually after washout with normal Ty- rode's solution (A). The phasic contractions driven by electrical field stimulation, which were suppressed by higenamine, disappeared rapidly after the administration of phentolamine (B). 5 min 10 min - n

t Higenamine t Washout with normal Tyrode ( W/NT M Fig. 13. Effects of higenamine on the spontaneous Higenamine contractions of guinea-pig taenia coli. Hi- 10 min . L 1 n genamine suppressed the amplitude of spontaneous contractions, and this action of higenamine was reversible; The higenamine-induced depression of contractility disappeared after washout with normal 0 O 'oh-, I Tyrode's solution. 10-'~

Higenamine Fig. 14. Dose-dependency of higenamine effects on the spontaneous contractions of guinea-pig taenia coli. Higenamine suppressed the frequency and the amplitude of spontaneous contractions dose-dependently, and blocked completely at the concentration of lop6 M.

10 min

Higenamine M Propranolol Washout with normal Tyrode (W/NT ) 10 min n

- t Higenamine Phentolamine ( 2 x M) (IO-~M) -Propranolol ( 2~10-~M ) Fig. 15. Effects of propranolol on the inhibitory action of higenamine to spontaneity in guinea-pig taenia coli. The depressed contractility induced by higenamine (lo-' M) was antagonized by the addition of propranolol M) to the bathing Tyrode's solution (A). However, phentolamine had no effects on the higenamine-induced depressed contractility of the taenia coli (B). I ]I~V (A) -- Control

1 min

-1 -1 1 mv (6) -- -. Higenamine

-1 min Fig. 16. Effects of higenamine on spontaneous electrical activity and contraction at the concentration of lo-' M in guinea-pig taenia coli. The parameters of electrical activity during the control period with respect to the interval between bursts of spike discharge and the spikes in a burst were compared with those obta~ned15 minutes after higenamine administration. The burst interval increased from control 98.4 seconds to 112.8 seconds. The number of spikes in a burst (spike frequency), however, was reduced from control 21 spikes to 11 spikes. The frequency and the amplitude of spontaneous contractions decreased as the burst interval and spike frequency were altered.

I I

]2mv Control v

-1 min

10 min after Higenamine adm. M

I 1 I 15 min after Higenamine adm.

Fig. 17. Effects of higenamine on spontaneous electrical activity and contraction at the concentration of lop6M in guinea-pig taenia coli. The burst interval was increased from control 85.2 seconds to 182.4 and 189.6 seconds, respectively, 10 and 15 minutes after the administration of higenamine M). The spike frequency was decreased from control 20 spikes to 18 and 14 spikes respectively. The frequency and the amplitude of spontaneous contractions decreased as the electrical parameters were changed. C],' I , I' m"

(A) - Control

1 min

Higenamine (2~10-'~)

Higenamine ( 2~lO-'M ) [Ll and propranolol M)

Fig. 18. Effects of propranolol on the higenamine-induced suppression of spontaneity in guinea-pig taenia coli. The spike frequency in a burst was decreased from the control 13 spikes to 8 spikes after higenamine (2X lo-' M) administration, and the depressed activity was recovered to 11 spikes by the addition of propranolol M). However, the slightly increased burst interval was nearly the same. The depressed amplitude of spontaneous contraction was increased after the addition of propranolol to higenamine-containing bathing Tyrode's solution.

4 H igenamine M )

4 Higenamine M) Fig. 19. Effects of higenamine on spontaneous electrical activity and contraction in guinea-pig taenia coli. Spontaneous electrical activity and contraction were simultaneously measured by use of the single sucrose-gap technique. Intentional overstretch induced continuous Ca2+-spikedischarge (A). Higen- amine M) blocked completely the continuous spike discharge (A), and regular spontaneous activities were also completely blocked by the addition of higenamine and membrane potential was gradually depolarized. 2) The effect of higenamine on the spontaneous suggesting that a part of the cardiotonic action electrical activity and contraction of higenamine was potentiation of calcium influx Two parameters of the electrical activity were through the cell membrane (Chang et a/. 1981). measured during the control period and 15 min Higenamine increased slow inward current, just after the administration of higenamine (Fig. 16). The like catecholamine, in the papillary muscle of rab- burst interval increased from control 98.4 sec to bits (Kwon et a/. 1981) and in the heart of pigs 112.8 sec and the number of spikes in a burst (Wang et a/. 1982). In the sarcoplasmic reticulum (spike frequency) was reduced from control 21 higenamine suppressed the reuptake of calcium spikes to 11 spikes. The frequency and the ampli- by inhibiting the activity of Ca-ATPase necessary tude of spontaneous contractions were decreased for Ca2+ transport and potentiated its release (Kim as the burst interval and the spike frequency of et a/. 1982). Higenamine was supposed to act on the spontaneous electrical activity decreased res- the adrenoceptors, considering the fact that the pectively. In the same way the burst interval was value of pA2 of higenamine to propranolol was si- increased from control 85.2 sec to 182.4 sec and milar to that of epinephrine to propranolol as being 189.6 sec, 10 and 15 min after the administration 8.58 and 7.50 respectively in the left atrium of rab- of higenamine (10-6 M) respectively, and the spike bits (Park et a/. 1984). frequency was decreased from control 20 spikes In in-vivo experiments with rabbits higenamine to 18 and 14 spikes respectively (Fig. 17). The ef- decreased blood pressure and total peripheral re- fect of higenamine was partially antagonized by sistance, and increased cardiac output and heart propranolol (Fig. 18). The spike frequency which rate. These effects were all blocked by propranolol was decreased from control 13 spikes to 8 spikes (Kim et al. 1986). From the above results higen- by higenamine was recovered to 11 spikes by the amine was supposed to act on P-adrenoceptors. addition of propranolol M). The depressed The contraction of the smooth muscle is known amplitude of spontaneous contraction was increa- to be generated in three mechanisms; excitation- sed in parallel with the increased spike frequency. contraction coupling (electrical activation), depola- However, the burst interval which was slightly inc- rization-induced contraction and pharmaco-me- reased was nearly the same as before. chanical coupling (non-electrical activation) by con- 3) The effect of higenamine on the membrane po- tracting agents such as norepinephrine, angioten- tential sin II, histamine and vasopressin and so on (Haeu- The spontaneous electrical activity was recor- sler 1972; Mekata and Nin 1972; Bohr 1973; Flec- ded simultaneously with the contraction using the kenstein 1977; Kim et a/. 1978; Bolton 1979). single sucrose-gap technique (Fig. 19). Intentional In this experiment, the contractions of the aortic overstretch of the strip induced continuous spike smooth muscle were generated by norepinephrine, discharges which were completely blocked by the depolarization driven by high K-Tyrode's solution addition of higenamine (lop6M) with development and electrical field stimulation. of the depolarization of the membrane potential. Norepinephrine was reported to generate con- In normal regular spontaneous activities higen- tractions without membrane depolarization in the amine ceased contractions and action potentials pulmonary artery (Su et a/. 1964) and in the carotid with gradual membrane depolarization. artery and the aorta of rabbits (Mekata et a/. 1972; Mekata 1974). In accordance with the above resu- DISCUSSION lts norepinephrine generated contraction without depolarization below the concentrations of lo-' M After the discovery of higenamine as a cardiac in the pulmonary artery of rabbits. However, it ge- stimulant, lots of investigations have been made nerated large contraction with depolarization and of this agent. Under treatment with lanthanum and decrease of membrane resistance above the con- verapamil in rabbit atrium higenamine recovered centrations of 2 X lo-' M. depressed cardiac contractility dose-dependently, Norepinephrine was supposed to generate contractions by calcium influx into the cell at low duce spike discharges resulting in large contrac- concentration and by calcium release from in- tion. The spike potentials of taenia coli have been tracellular calcium stores as well as calcium influx proved experimentally to be Ca spikes generated from outside the cell at high concentration (Cas- by Ca-entry from outside the cell (Brading et a/. teels et a/. 1977). It was also reported that norepi- 1969). nephrine induced contraction by releasing calcium The frequency of spontaneous contractions was from the cell membrane or intracellular calcium sto- supposed most likely to be determined by periodic res in the beginning of contraction and required fluctuation of electrogenic Na pump (Connor et a/. calcium supply through calcium influx for the main- 1974). The amplitude was related to the extent of tenance of it. Norepinephrine (10-' M- 1 M) Na efflux determined by ATP supply and to the produced steady tonic contractions (Fig. 3 and Fig. activity of electrogenic Na pump related to Ca 4) and it M) generated small contraction even (Prosser 1978). in Ca-free Tyrode's solution, supposedly through In this experiment, higenamine made a dose-de- calcium release from intracellular stores (Fig. 6). pendent decrease of frequency and amplitude of Higenamine was proved to act on two components the spontaneous contraction of taenia coli, which of the contraction, that is, the one maintained by could be interpreted to be due to the decrease Ca influx and the one induced initially by intracellular of the burst frequency and the spike frequency, Ca release. respectively. And it depolarized membrane poten- The relation between higenamine and the potential (Fig. 19). All these effects to the spontaneous tial-sensitive Ca channel was investigated (Fig. 10). contraction were blocked mostly by treatment with The contraction induced by 25 mM K-Tyrode's so- propranolol. From the above results higenamine lution was relaxed dose-dependently by higen- was supposed to have an effect on the slow wave amine. But the pretreatment with phentolamine and and the spike potential through P-adrenoceptors. propranolol completely blocked this effect. There- However, the phenomenon of membrane depolari- fore, it was supposed that higenamine did not affect zation could not be explained well by this experi- directly membrane potential depolarized by K-Ty- ment alone and required more investigat~on. rode's solution, but affected a process of releasing neurotransmitters from nerve endings embedded ACKNOWLEDGEMENT in tissue. Higenamine was, thus, estimated not to act on the potential-sensitive Ca channel. We would like to acknowledge the kind support Electrical field stimulation was supposed to in- of Professor CW Park for providing higenamine and duce the release of neurotransmitters from nerve advising in the process of the experiment and in endings because the phasic contraction induced the analysis of the results. by it was abolished by phentolamine (Fig. 12). Hi- genamine decreased both passive resting and ac- REFERENCES tive tensions and this effect was not recovered by propranolol. Bohr DF. Vascular smooth muscle updated. Circ. Res. There are slow rhythmic fluctuations of mem- 1973, 32: 665-672 brane potential called slow wave at intervals of Bolton TB. Mechanisms of action of transmitters and 1-3 min in the taenia coli of guinea-pigs. At the other substances on smooth muscle. Physiol. Rev. top of the fluctuation spike potentials are generated 1979, 59: 606-718 at the frequency of 1 spikelsec. The more the strip Brading A, Bulbring E, Tomita T. 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Acta Pharmacologica study on the adrenergic effect of higenamine in rabbit Sinica. 1982, 3: 1 12-1 17 cardiovascular system. Kor. Circ. J. 1986, 16: 1-18 3qqqp.j qqq] +)% yx)q]Aj Q+j?++% g2712 q4z3gAj 343 92 %A;) 7l-59 higenamineq 33 9 7j,%l;T- ggzq qg 4449 gqs 3 93)3~), 71 q 1$?q %-qq G!%%Z01 -$d-q01 5011 a 3 higenamineq q%zqq -1 713% H]iZ 948)%q. 2-20 q%q %gqTT~ norepinephrine, K-Tyrode's 9 ?!%x)qOS %g"]% -O q, $%% Sl-2 g%k%q 3%a9(7)~]$G)q +%*$% %A1 q] 71qS)71 4 aE4 9 $q+, 'H"olq g0-2 fi3sx