Effect of Atropine on Denervated Rabbit Ear Blood Vessels

http://www.paper.edu.cn

*Shu-Qin Liu, *Wei-Jin Zang, *Zeng-Li Li, *Xiao-Jiang Yu, and †Bao-Ping Li

similar to those of atropine and scopolamine. Henbane drugs Abstract: Surgical denervation of rabbit ear blood vessel beds was might exert multiple advantages in shock patients, but the va- combined with the isolated perfused rabbit ear technique to investi- sodilator effect is undoubtedly the main mechanism. Although gate the mechanism of atropine’s vasodilator action. Intramuscular many studies have been done throughout the world, the mecha- injection of atropine 0.2 mg/kg dilated the denervated blood vessels in the rabbit ear like innervated ones in vivo. Atropine at the maximal nisms of henbane drugs’ vasodilator action remain unclear. concentration (C )of3×10−6 to3×10−4 M did not increase ef- Atropine usually activates the central nervous system at max 8,9 fluent flow of the isolated perfused denervated rabbit ear under con- high doses. Scopolamine, in general, inhibits the central ner- −6 10–13 stant perfusion pressure, but chlorpromazine at a Cmax of 10 M and vous system at any dose. Anisodine slightly inhibits the acetylcholine (ACh) at 2.5 × 10−7 M significantly increased it and central nervous system compared with scopolamine.12,13 An- noradrenaline (NA) at 10−7 M significantly decreased it. Atropine at isodamine is so difficult to pass through the blood–brain bar- −7 −6 Cmax of3×10 M did not affect, but at3×10 M it abolished the rier that it might neither activate nor inhibit the central nervous −7 increase of the effluent flow induced by ACh 2.5 × 10 M. Atropine system.4,14 Therefore, the central nervous system is not the key at3×10−7 M did not affect it, but at 10−6,3×10−6, and 10−5 it signi- −7 pathway through which henbane drugs implement their vaso- ficantly alleviated the decrease of effluent flow induced by NA 10 dilator action. M. Because the increase of effluent flow of rabbit ear under constant Because atropine inhibits the activity of sweat glands perfusion pressure reflects vasodilation of the ear to some extent, the and usually increases body temperature, especially that of in- study suggests that atropine has no direct vasodilator action; its vaso- 15 dilator action is not attributed to blockade of M-cholinoreceptors lo- fants and small children, most investigators surmise that va- ␣ sodilation might be a compensatory reaction permitting the ra- cated on the vascular wall; however, the 1-adrenoceptor might be a 16,17 target site mediating atropine’s vasodilator action in vivo. diation of heat. However, animals that do not sweat, such as dogs, do not exhibit fever, but rather dilation of blood ves- Key Words: atropine, denervation, rabbit ear, vasodilation sels, after administration of atropine.16 Therefore, some inves- (J Cardiovasc Pharmacol™ 2004;43: 99–105) tigators also supposed that atropine has direct vasodilator action,16 but there is no experimental evidence. Vasodilation in response to atropine may be attributed to blockade of M- t has long been known that high doses of atropine can dilate cholinoceptors located on the vascular wall. Iblood vessels. In the 1960s, Chinese doctors repeated admin- Atropine reduces the vasocontraction induced by istration of high doses of atropine to salvage patients with bac- adrenaline (AD) or electrical stimulation of sympathetic nerve teremic shock, and mortalities were reduced as a result.1–3 in isolated perfused rabbit ear18 and increases the median le- Nowadays, atropine, scopolamine, and some other henbane thal dose (LD50) of AD for rabbits. Therefore, Luduena and drugs such as anisodamine and anisodine are important drugs Branin stated that atropine also blocked ␣-adrenoceptors in in the prevention of death in patients with bacteremic shock vivo.19 Therefore, we also investigated the question of whether and hemorrhagic shock.4–7 Anisodamine and anisodine are an- atropine-induced vasodilation in vivo be attributed to blockade ticholinergic (M-cholinoceptor antagonists) alkaloids origi- of ␣-adrenoceptors. We investigated the effects of atropine on nally extracted from Scopolia tangutica (a variety of datura) blood vessel in the rabbit ear after sympathetic and sensory and currently produced in China. Their chemical structures are denervation.

Received February 4, 2003; accepted July 21, 2003. MATERIALS AND METHODS *From the Department of Pharmacology, School of Medicine; and †Experi- mental Center of Chemistry, School of Science, Xi’an Jiaotong University, Appliances Xi’an, China. We used a digital drop recorder (Model YSD-4; Second Supported by the National Natural Science Foundation of China (Nos. Radio Factory, Bengbu Medical College, China) and an elec- 39970273; 30270554) and the State Education Commission of China (Nos. 20010698034, 01161). trical stimulator (Model sen-7203; Nihon Kohden, Japan). The Reprints: Prof. Wei-Jin Zang (e-mail: zwj@mail xjtu.edu.cn). gravity-feed apparatus used was designed and made by our Copyright © 2003 by Lippincott Williams & Wilkins laboratory to meet the need for keeping constant perfusion

J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004 99 中国科技论文在线 http://www.paper.edu.cn

Liu et al J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004

pressure in the experiment such that the change of perfusion moved into the chlorpromazine group, adjusting each group to liquid height is no more than 1 mm H2O when 300 mL liquid eight rabbits. leaves the 3000-mL bottle. In Vivo Study Drugs and Reagents The similar positions of dorsal auricular arterial trunks in Atropine sulfate, acetylcholine chloride, chlorproma- each rabbit’s left and right ears were marked on the surface of zine hydrochloride, noradrenaline bitartrate, prazosin hydro- skin after the hair was shaved around them, and the diameters chloride, and sodium pentobarbital were purchased from of the dorsal auricular arterial trunks were measured with a pair Sigma Chemical. Other drugs and reagents are products of of compasses and the ruler in a dissecting microscope. Every pharmaceutical companies and chemical companies in China. rabbit was fixed in a rabbit box for approximately 40 minutes All reagents were of analytic grade or the highest purity avail- (the room temperature was approximately 25°C). Atropine able. (0.2 mg/kg), chlorpromazine (2.0 mg/kg), or saline solution (0.2 mL/kg) was administered intramuscularly to the rabbits in Animals their respective groups. Atropine was dissolved in saline solu- New Zealand white rabbits were supplied by the Medical tion (1%) and chlorpromazine was dissolved in 0.1 mM HCl Experimental Animal Center at Xi’an Jiaotong University. and diluted with saline solution to 1%. Therefore, the injection Thirty rabbits weighing 2.0–2.3 kg (15 male) were used in the volume of each drug was equal (0.2 mL/kg). All drugs were experiment. Among these animals, 10 (five male and five fe- incubated to 37°C before injection. The changes of blood ves- male) that were sensitive to atropine (intramuscular injection sels in rabbit ears were observed, and the diameters of the dor- of atropine sulfate caused blood vessels in ears to dilate) were sal auricular arterial trunks were measured. selected as the atropine group. Others were divided into two The change in diameter of dorsal auricular arterial trunks weight- and sex-matched groups, one to receive chlorproma- was analyzed with the paired t test, respectively. The differ- zine and one to receive normal saline solution. ence of diameter change between left and right ears was analyzed with the group t test. Surgical Denervation Surgery was performed on all 30 rabbits in according to In Vitro Study the report of Morris and Bevan20 with slight revision. Every Model Confirmation rabbit was anesthetized by intraperitoneal injection of sodium This part of the experiment was performed with six pentobarbital (35 mg/kg). Hair was shaved bilaterally over model made animals (three male and three female) 1 day after identical areas before surgery. A medial incision was made in the in vivo study. The rabbits were anesthetized with sodium the neck and the right superior cervical ganglion and right cer- pentobarbital and fixed in rabbit boxes individually. After vical sympathetic preganglionic trunk were removed. The shaving, a 1-cm incision at the base of the medial border of the greater auricular nerve was exposed by a 1.5-cm incision on ear was made. Dorsal auricular artery and dorsal auricular the right side of the neck, and 1 cm was excised just proximal nerve were dissected from vein and other surrounding tissues, to the origin of the anterior and posterior branches. The inci- respectively. The artery was ligated at the base of the ear for sions were closed with four to six sutures. After a 1-cm inci- cannulation. The nerve also was ligated. A small incision was sion at the base of the medial border of right ear was made, the made on the dorsal auricular artery near the ligature with an dorsal auricular nerve, derived from the dorsal root of the sec- ophthalmologic scissors. An arterial cannula of 34°C Ringer ond cervical nerve, the auricular branch of the vagus, and the Locke solution containing heparin (10−4 U/L) was intubated facial nerve were exposed and excised. The anterior auricular into the artery. After the arterial cannula with the artery was artery was not touched. The incision was closed with four su- ligated, the ear was cut off rapidly and the base of the ear was tures. Sham operations were performed on the left side of the ligated with a cotton rope to prevent bleeding. After washing neck and the left ear, with resection of the auricular branches of the blood in the ear with Ringer Locke solution with use of a the vagus nerve and facial nerve. syringe, the isolated rabbit ear was perfused continuously, aer-

Sulfamylon powder was scattered on the surface of inci- ating with 95% O2 and 5% CO2 Ringer Locke solution (34°C; sions and animals were administered penicillin and streptomy- pH 7.3–7.4) through the arterial cannula under constant perfu- cin intramuscularly twice daily for 3 days postoperatively. sion pressure (60 cm Ringer Locke solution). Ringer Locke Animals were left to regain consciousness in a warm environ- solution was composed of (in mM): NaCl, 154.0; KCl, 5.5; ment and raised for 14 days. During this time, two rabbits in the CaCl2, 1.8; NaHCO3, 6.0; and glucose, 5.5. The effluent, com- atropine group, three in the chlorpromazine group, and one in posed of outflow from the dorsal auricular vein and the leak of the saline solution group died of infection or other undeter- interstitial fluid, was recorded with the digital drop recorder. mined reasons. Therefore, one rabbit from the saline group was The perfusion speed was controlled such that the effluent rate

J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004 Effect of Atropine on Denervated Blood Vessels in Rabbit Ear

was 19–21 drops (approximately 1.5 mL) per minute. After 15 3 × 10−7 or 3 × 10−6; ACh, 2.5 × 10−7; atropine, 3 × 10−7,10−6, minutes at equilibrium, electrical stimulation was applied to 3 × 10−6 or 10−5; and NA, 10−7. The effluent was recorded the dorsal auricular nerve through a pair of platinum electrodes immediately after addition of each pair of drugs. The maxi- (distance between the two poles was 2 mm). For stimulation, mum or minimum effluent was recorded as the representative square wave pulses of 2 milliseconds width were applied at a effect of the drugs. The change in effluent flow was deter- rate of 25 Hz for periods of 10 seconds. The stimulant voltage mined with the paired t test. The differences of effluent was 2–10 V according to different preparation. The effluent of changes between concomitant addition of atropine and ACh or the ear was recorded. Prazosin was then added into the drug atropine and NA and ACh or NA alone were analyzed with the kettle (10 mL) to make the maximum concentration (Cmax)in group t test. the perfusion liquid 10−5 M. The change in effluent flow of rabbit ears was analyzed RESULTS with the paired t test. The difference of effluent change between left and right ears was analyzed with the group t test. Response of Rabbit Ear Blood Vessels on Atropine In Vivo Effect of Atropine Alone on Effluent Flow of the Isolated One minute after intramuscular injection of atropine, the Denervated Rabbit Ear blood vessels in all eight rabbit ears dilated quickly, lasting The other eight rabbit right ears (denervated) were in- 10–25 minutes. There was no difference in change of dorsal cised and perfused as described. After perfusion with a steady auricular arterial trunk diameter between left and right ears (P speed (19–21 drops per minute) for 15 minutes, the same vol- > 0.05). In addition, all rabbits appeared excited and two ran ume of saline solution, atropine, chlorpromazine, acetylcho- out of their rabbit box. In chlorpromazine group, the blood ves- line (Ach), or noradrenaline (NA) were added into the drug sels obviously dilated 5 minutes after administration of the kettle individually, respectively. Different concentrations for drug; this lasted for 30–40 minutes. However, there was no difference of diameter change between left dorsal auricular ar- different drugs were prepared so that the Cmax of each drug in the perfusion liquid was (in M): chlorpromazine 10−6; atro- teries and right ones (P > 0.05; Table 1). In addition, all the pine, 3 × 10−6,3× 10−5, and 3 × 10−4; ACh, 2.5 × 10−7; and NA, animals appeared tranquilized. 10−7. As soon as addition of each drug (or each concentration, eg, atropine), the effluent rate was recorded until it rehabili- Changes in Effluent Flow of Isolated Perfused tated. The maximal or the minimal effluent flow was recorded Rabbit Ear After Electrical Stimulation of as the effect of the drug (concentration). After 5 minutes at Dorsal Auricular Nerve equilibrium, the next drug (or concentration) could be added. As shown in Table 2, the effluent of isolated perfused The change in effluent flow of rabbit ear was determined with left rabbit ears significantly decreased after electrical stimula- the paired t test. tion of the dorsal auricular nerve (P < 0.001). In the presence of a high concentration of prazosin (10−5 M) in the perfusion Effects of atropine on Acetylcholine-induced Vasodilation fluid, there was no significant change in rate of effluent flow and Noradrenaline-induced Vasocontraction in Isolated after electrical stimulation (P > 0.05). In the absence of prazo- Denervated Rabbit Ear sin, electrical stimulation of the cut end of the dorsal auricular Seven right rabbit ears were isolated and perfused as de- nerve of right ears (denervated) had no significant vasocon- scribed. After 15 minutes at equilibrium, atropine and ACh or strictor effect (P > 0.05); addition of prazosin did not signifi- atropine and NA were added into the drug kettle, respectively. cantly alter baseline flow in the denervated right ears (P >

The Cmax of drugs in the perfusion liquid were (in M): atropine, 0.05).

TABLE 1. Effect of Atropine on Diameter of Dorsal Auricular Arterial Trunk in Rabbit Ear In Vivo (mm)

Left Ears (Innervated) Right Ears (Denervated) Group Dose n Before After Difference Before After Difference Saline solution 0.2 mg/kg 8 0.9 ± 0.1 0.9 ± 0.1 0 ± 0.2 0.9 ± 0.1 1.0 ± 0.1 0 ± 0.1 Atropine 0.2 mg/kg 8 0.9 ± 0.1 1.1 ± 0.1 0.3 ± 0.1* 0.9 ± 0.1 1.1 ± 0.1 0.2 ± 0.1* Chlorpromazine 2.0 mg/kg 8 0.9 ± 0.1 1.1 ± 0.1 0.4 ± 0.2* 0.9 ± 0.1 1.3 ± 0.1 0.4 ± 0.1*

*P < 0.001. Data are expressed as means ± SD.

Liu et al J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004

TABLE 2. Changes in Effluent Flow of Isolated Perfused Rabbit Ear After Electrical Stimulation of Dorsal Auricular Nerve Remain (Drops per Minute)

Left Ears (Innervated) Right Ears (Denervated) Prazosin (1×10−5 M)n Before After Difference Before After Difference

Without 6 19.9 ± 1.2 10.9 ± 2.9 −9.0 ± 2.5* 20.2 ± 0.8 19.0 ± 1.3 −1.5 ± 0.8 With 6 19.8 ± 0.8 19.2 ± 1.0 −0.3 ± 1.2 20.0 ± 0.9 19.2 ± 1.0 −0.8 ± 0.8

*P < 0.001. Data are expressed as means ± SD.

Effluent Flow in Isolated Perfused Rabbit Ear The higher concentration of atropine (10−6 to 10−5 M) signifi- After Addition of Atropine Alone cantly decreased the reduction of effluent flow induced by As shown in Table 3, chlorpromazine (10−6 M) signifi- 10−7 M NA, compared with that seen with addition of NA cantly increased the flow of effluent of the isolated perfused alone (P < 0.05, P < 0.01). denervated rabbit ear (P < 0.001). ACh (2.5 × 10−7 M) also increased effluent flow (P < 0.001). NA (10−7 M) decreased DISCUSSION effluent flow as well. However, atropine by itself (3 × 10−6 M, Selection of the rabbit ear to explore the vasodilator ef- 3 × 10−4 M) did not significantly alter effluent flow of the iso- fect and involved mechanisms of atropine-induced vasodila- lated perfused denervated rabbit ear under constant perfusion tion is based on the following reasons: (1) vasculature in the pressure (P > 0.05). ear forms a prominent part of facial cutaneous vasculature, which means atropine-dilated facial blood vessels are obvious; Effect of Atropine on Change in Effluent Flow (2) the rabbit ear is large and thin, which makes changes, es- Induced by Acetylcholine or Noradrenaline in pecially in the blood vessel beds before and after stimulation, Isolated Perfused Denervated Rabbit Ear easy to observe; (3) and the most prominent advantage of the As shown in Table 4, atropine (3 × 10−7 M) did not affect model is the ability to observe direct vasodilator actions of the dilation of vasculature induced by ACh 2.5 × 10−7 M (P < drugs. 0.001). However, a higher concentration of atropine, 3 × 10−6 Bussel, in 1940, demonstrated that atropine could block M, did significantly inhibit vasodilation induced by ACh; the the effect of adrenaline to reduce the effluent flow of isolated flow of effluent was significantly reduced compared with that rabbit ear, and Luduena, in 1966, found that atropine could −7 seen with addition of ACh alone (P < 0.05). Atropine 3 × 10 increase the LD50 of adrenaline for rabbits, similar to phentol- M did not affect the vasoconstriction induced by NA 10−7 M amine, and therefore was the first to suggest that atropine compared with that seen with addition of NA alone (P > 0.05). might have an effect of blockade of ␣-adrenoceptors.19 How-

TABLE 3. Change in Effluent Flow of Isolated Perfused Denervated Rabbit Ear After Addition of Atropine (Drops per Minute)

Effluent of Denervated Ears

Drug Cmax (M)n Before After Difference Saline solution — 8 20.1 ± 0.8 19.8 ± 1.0 −0.4 ± 0.7 Chlorpromazine 1 × 10−6 8 20.0 ± 0.8 26.5 ± 1.5 6.5 ± 2.1* 3 × 10−6 8 19.3 ± 1.2 19.3 ± 1.0 0.3 ± 1.5 Atropine 3 × 10−5 8 19.3 ± 0.7 19.6 ± 1.3 0.4 ± 1.1 3 × 10−4 8 19.5 ± 0.9 19.6 ± 0.9 0.1 ± 1.1 Acetylcholine 2.5 × 10−7 8 19.4 ± 0.7 23.5 ± 1.6 4.1 ± 1.9* Noradrenaline 1 × 10−7 8 19.8 ± 0.8 12.2 ± 2.0 −7.7 ± 2.4*

*P < 0.001. Data are expressed as means ± SD.

J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004 Effect of Atropine on Denervated Blood Vessels in Rabbit Ear

TABLE 4. Effects of Atropine on Changes in Effluent Flow of Isolated Perfused Denervated Rabbit Ear Induced by Acetylcholine or Noradrenaline (Drops per Minute) Effluent of Denervated Ears

Drug Cmax (M)n Before After Difference Acetylcholine 2.5 × 10−7 8 19.4 ± 0.7 23.5 ± 1.6 4.1 ± 1.9* Atropine 3 × 10−7 7 20.4 ± 0.9 23.9 ± 2.4 3.5 ± 2.4† Acetylcholine 2.5 × 10−7 Atropine 3 × 10−6 7 20.0 ± 0.8 20.4 ± 1.1 0.4 ± 1.1‡ Acetylcholine 2.5 × 10−7 Noradrenaline 1 × 10−7 8 19.9 ± 1.2 10.9 ± 2.9 9.0 ± 2.5* Atropine 3 × 10−7 7 20.8 ± 0.8 11.9 ± 1.6 8.1 ± 2.0* Noradrenaline 1 × 10−7 Atropine 1 × 10−6 7 19.3 ± 0.9 12.6 ± 1.2 6.8 ± 1.5*‡ Noradrenaline 1 × 10−7 Atropine 3 × 10−6 7 19.3 ± 1.0 14.5 ± 2.7 4.8 ± 2.3*§ Noradrenaline 1 × 10−7 Atropine 1 × 10−5 7 19.6 ± 0.9 14.9 ± 1.7 4.6 ± 1.4*§ Noradrenaline 1 × 10−7

*P < 0.001. †P < 0.01. ‡P < 0.05. §P < 0.01. Data are expressed as means ± SD.

ever, the hypothesis has not been thoroughly verified, particu- endogenous ACh in the blood vessels of the ear can be ques- larly in investigations in denervated blood vessels. tioned. Histologic study21,22 and histochemical study20 had It has long been recognized that vasodilator nerves also demonstrated that there was NA in all blood vessels in the rab- supply the rabbit ear. Feldberg25 and Grant et al26 demon- bit ear. NA is undoubtedly the main vasocontractile substance strated that antidromic stimulation of sensory nerves arising released from noradrenergic terminals in rabbit ear blood ves- from the second and third cervical dorsal root, the trigeminal sels after stimulation. Purinergic transmitter23 and neuropep- nerve, and the vagus nerve could dilate the vasculature in the tide Y24 might be the cotransmitters in terminals of nor- ear. The transmitter(s) responsible for this antidromic vasodi- adrenergic axons. It has been suggested that sympathetic cho- lation has not been determined. Substance P20,31 and calcitonin linergic nerves mediate vasodilation of the small vessels in the gene–related peptide31 have been localized in sensory nerves rabbit ear,25,26 and it has been claimed that there is cholinergic in blood vessels of the rabbit ear. innervation in rabbit ear blood vessels after demonstration of The present study aimed to resolve whether atropine can acetylcholinesterase-positive perivascular nerve.22,27 In addi- directly dilate blood vessels. In addition, whether atropine- tion, there might be N1- and M-cholinoreceptors distributed on induced vasodilation can be attributed to blockade of M- the membrane of noradrenergic axon terminal (presynaptic re- cholinoreceptor or ␣-adrenoceptor located on the vascular wall ceptors), and the membrane also exhibited a weak but definite was also investigated. Therefore, the surgical denervation of acetylcholinesterase activity.27 The existence of M-choli- the rabbit ear model and isolated perfused rabbit ear technique noreceptor in endothelial cells and smooth muscle cells of were combined and applied in this study. Because the sensory blood vessels is well-known. The affinity of atropine to the nerve plays a certain role in regulation of the rabbit ear, in the endothelial cell M-cholinoreceptor is approximately 0.1% of present experiment, the auricular branch of the facial nerve that to the smooth muscle M-cholinoreceptor.28 However, it is was also resected. Complete postparasympathetic denervation not known what subtypes (M1,M2,M3,M4,orM5) are promi- (if it is true in rabbit ear) is difficult, and therefore resection of nent in endothelium and in smooth muscle. Because the histo- the auricular branch of the vagus is all we could attempt. chemical reaction of identifying acetylcholinesterase is not a It has been demonstrated with histochemical techniques specific marker for cholinergic nerves,29,30 the existence of that sympathetic and substance P inmmunoreactive sensory

Liu et al J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004

denervation of vasculature in the rabbit ear could be realized 2 gland, so atropine produced its vasodilator action on denervat- 20 ␣ weeks after denervative surgery. This study confirmed indi- ed rabbit ears in vivo. Because 1-adrenoceptor is a type of rectly the success of surgical sympathetic denervation by func- membrane protein that receives and handles messages from tional experiment: electrical stimulation of the remains of the endogenous neural transmitters such as NA, hormones such as dorsal auricular nerve did not decrease the effluent flow of the AD, or extraneous drugs such as phenylephrine, it is premature ␣ isolated perfused denervated rabbit ears. to claim atropine to be a competitor of 1-adrenoceptors lo- Because there is atropinesterase in some rabbits’ blood cated on the vascular wall with endogenous catecholamines plasma or liver,32 which can hydrolyze atropine and thereby such as NA and AD. The ventricular myocardial tissue, spleen make the animals insensitive to atropine, the rabbits who re- strips, and seminal vesicle of the rat will be investigated in ceived atropine were selected for in vivo study. functional experiments, and the radio-ligand binding method The increase in effluent flow of the isolated perfused will be used, in future studies. rabbit ear under constant perfusion pressure reflects the dilation of the vasculature in the ear to some extent. The effluent ACKNOWLEDGMENTS flow was controlled to 19–21 drops (approximately 1.5 mL) The authors thank Associate Professor You-Li Yu, Fac- per minute before addition of drug(s) to insure the increased ulty of Physics, School of Science, for helpful design of the potential of the vasodilator action of drug(s), especially direct gravity-feed apparatus; postgraduate Liang Cheng, Depart- vasodilator action. ment of Pharmacology, School of Medicine, for participation −6 −5 −4 Atropine at the Cmax of 3 × 10 ,3× 10 ,or3× 10 M of surgical operation; and Prof. Bing-Xiang Yuan, Department did not increase the effluent flow of isolated perfused dener- of Pharmacology, School of Medicine, for valuable revision of vated rabbit ear. This implies that atropine has no direct vaso- the article. dilator effect. This experiment also provides evidence of chlorpromazine’s direct vasodilator effect. REFERENCES It has been known that extraneous ACh alleviates con- tractile substance–induced contraction of blood vessel ring via 1. Qian C, Xu XL, Cai TZ. Therapeutic action of a comprehensive cure in which atropine plays the major role on toxic bacillary dysentery. Zhong- activation of M-cholinoreceptors located on endothelial cell, hua Yixue Zazhi. 1961;47:163–165. [article in Chinese] causing the synthesis and release of endothelium-dependent 2. Qian C, Cai TZ, Xu XL. The mechanisms and therapeutic effects of atro- relaxing factors such as nitric oxide.33,34 The action can be pine on toxic bacillary dysentery. Zhonghua Erke Zazhi. 1964;13: 363–366. [article in Chinese] eliminated by atropine. 3. Zhu SH, Yan QY, Gao YS. The knowledge and treatment about fulminate ACh can also cause contraction of endothelium- epidemic meningococcal meningitis. Zhonghua Neike Zazhi. 1966;14: removed blood vessel ring via activation of M-cholino- 19–21. [article in Chinese] 4. Anonymous. Pharmacologic effects of anisodamine. From the Chinese receptors located on vascular smooth muscle cells. In the Academy of Medical Sciences and Beijing Friendship Hospital. Chin Med present study, ACh 2.5×10−7 M increased effluent flow in the J. 1975;1:133–138. ear. This means the effect of ACh to activate endothelial M- 5. Anonymous. Changes in nail-fold microcirculation and amines in dis- eases manifesting acute microcirculatory disturbances. From the Chinese cholinoreceptor is stronger than its effect on vascular smooth Academy of Medical and Beijing Friendship Hospital. Chin Med J. 1975; muscle. Higher concentration of atropine blocks M- 1:216–224. cholinoreceptor in endothelium, which would alleviate the va- 6. Lin XR. Antishock drugs: vasodilating drugs. In: Song WX, Li Y, Sun YA, et al., eds. Practical Internal Medical Pharmacotherapeutics. Bei- sodilator effect of ACh. Because there is no direct evidence of jing: People’s Medical Publishing House, 2000:255–259. [text in Chi- endogenous ACh existing in rabbit ear blood vessels, the nese] physiologic value of M-cholinoreceptor in the vascular wall, 7. Li MT. Drugs acting on efferent nervous system: cholinoceptor blocking drugs. In: Chen RZ, Huang ZJ, eds. Therapeutic pharmacology: Beijing: especially in cutaneous blood vessel walls, is another interest- People’s Medical Publishing House, 2002;61–64. [text in Chinese] ing issue to investigate. This study suggests that the vasodila- 8. Felderberg W, Sherwood SL. Injection of drugs into the lateral ventricle tor effect of atropine is not related to blockade of M- of the cat. J Physiol. 1954;123:148–167. 9. Haley TJ, McComic WG. Pharmacological effects produced by intrace- cholinoreceptors in the vascular wall. rebral injection of drugs in the conscious mouse. Br J Pharmacol. 1957; Electrical stimulation of the dorsal auricular nerve in- 12:12–16. creased effluent flow of the isolated perfused left rabbit ear, an 10. Ostfeld AM, Argue A. Central nervous system effect of hyoscine in man. J Pharmacol Exp Ther. 1962;137:133–139. effect blocked by prazosin. This means that endogenous NA 11. Yue TL, Wang GF, Song ZY. The physiological dispositions of 3H- release is the key procedure while sympathetic nerves are ex- scopolamine and 3H-anisodamine. Acta Pharmaceutica. 1979;14: cited. As for the denervated ear, extraneous NA also induced 208–216. [article in Chinese] 12. Peng JZ, Jin LR, Chen XY, et al. Central effects of anisodamine, atropine, contraction of the blood vessel beds. The ability of atropine to anisodine and scopolamine after intraventricular injection. Acta Pharmar ␣ limit the vasocontractile effect of NA implies that 1- Sinica. 1983;4:81–87. [article in Chinese] adrenoceptor might be a target site that mediates high-dose 13. Anonymous. Pharmacologic effects of anisodine compared with scopolamine. From the Chinese Academy of Medical Science. Honghua Yixue atropine’s vasodilator action. The blood plasma contains AD Zazhi. 1975;11:795–798. and other catecholamines from the medulla of the adrenal 14. Anonymous. Adsorption, distribution and excretion of anisodamine.

J Cardiovasc Pharmacolீ • Volume 43, Number 1, January 2004 Effect of Atropine on Denervated Blood Vessels in Rabbit Ear

From the Chinese Academy of Medical Science and Beijing Friendship nergic as well as adrenergic responses of the rabbit ear artery. Eur J Phar- Hospital. Chin Med J 1973;1:274–278. [English abstract]. macol. 1990;176:117–125. 15. Unna KR, Glaser K, Lipton E, et al. Dosage of drugs in infants and chil- 25. Feldberg W. The peripheral innervation of the vessel of the external earof dren. I. Atropine. Pediatrics. 1950;6:197–207. the rabbit. J Physiol. 1926;61:518–529. 16. Weiner N. Atropine, scopolamine, and related antimuscarinic drugs: at- 26. Grant RT, Bland EF, Camp PD. Observations on the vessel and nerves of ropine, scopolamine, and related belladonna alkaloids. In: Gilman AG, the rabbit’s ear with special reference to the reaction to cold. Heart. 1932; Goodman LS, Gilman A, eds. Goodman and Gilman’s the Pharmacologi- 16:69–101. cal Basis of Therapeutics, 6th edition. New York: Macmillan, 1980: 27. Steinsland OS, Furchgott RF. Vasoconstriction of the isolated rabbit ear 121–132. artery caused by nicotine agonists acting on adrenergic neurons. J Phar- 17. Brown JH, Taylor P. Muscarinic agonists and antagonists. In: Hardman J, macol Exp Ther. 1975;193:128–137. Limbird LE, Gilman AG, eds. Goodman and Gilman’s the Pharmacologi- 28. Manjeet S, Sim MK. Atropine-and scopolanmine-resistant subtypes of cal Basis of Therapeutics, 10th edition. New York: McGraw-Hill, 2001: muscarinic receptors in the rabbit aorta. Eur J Pharmacol. 1989;174: 162–171. 99–165. 18. Bussel LJ. The relation of atropine to adrenaline and the sympathetic sys- 29. Barajas L, Wang P. Demonstration of acetylcholinesterase in the adrener- tem. J Pharmal. 1940;69:128–139. gic nerves of the renal glomerular arterioles. J Ultrastruct Res. 1975;53: 19. Luduena FP, Branin MJ. Adrenolytic activity of atropine, (t) – hyoscya- 244–253. mine, atroscine, homatropine, and related compounds. J Pharm Sci 1966; 55:280–284. 30. Papka RE, Furness JB, Della NG, et al. Depletion by capsaicin of sub- 20. Morris JL, Bevan RD. Proliferation of arteriovenous anastomoses in the stance P-immunoreactivity and acetylcholinesterase activity from nerve developing rabbit ear is enhanced after denervation. Am J Anat. 1986;176: fibers in the guinea pig heart. Neurosci Lett. 1981;27:47–54. 497–509. 31. Maynard KI, Saville VL, Burnstock G, et al. Sensory-motor neuromodu- 21. Waterson JG, Smale DE. Location of noradrenergic structures in the cen- lation of sympathetic vasoconstriction in the rabbit central ear artery. Eur tral artery of the rabbit ear. Aust J Exp Biol Med Sci. 1976;45:301–308. J Pharmacol. 1990;187:171–182. 22. Lijima T, Tagawa T. Adrenergic and cholinergic innervation of the arte- 32. Glick D, Glaubach S. The occurrence and distribution of atropinesterase riovenous anastomoses in the rabbit’s ear. Anat Rec. 1976;186:373–380. and the specificity of atropinesterase. J Gen Physiol. 1941;25:197–205. 23. Saville VL, Burnstock G. Use of reserpine and 6- Hydroxydopamine sup- 33. Drexler H. Factors involved in the maintenance of endothelial function. ports evidence for purinergic cotransmission in the rabbit ear artery. Eur J Am J Cardiol. 1998;82:35–45. Pharmacol. 1988;155:271–277. 34. Rubanyi GM. The role of endothelium in cardiovascular homeostasis and 24. Saville VL, Maynard KI, Burnstock G. Neuropeptide Y potentiates puri- disease. J Cardiovasc Pharmacol. 1993;22:S1–S14.