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Brain Research Bulletin, Vol. 35, No. I, pp. 15-22, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

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Autonomic and Sensory Cardiovascular Activities of Nonivamide: Intrathecal Administration

of Clonidine

YI-CHING LO,* JWU-LA1 YEH,* JIUNN-REN WU,t JWU-MAW YANG,* SHEUE-JIUN CHEN* AND ING-JUN CHEN*’

Departments of *Pharmacology and fpediatrics, Graduate Institute of Medicine, Kaohsiung Medical College, Kaohsiung, Taiwan 80708, ROC

$Chia-Nan Junior College of Pharmacy, Tainan, Taiwan, ROC

Received 19 March 1993; Accepted 18 March 1994

LO, Y.-C., J.-L. YEH, J.-R. WU, J.-M. YANG, S.-J. CHEN AND I.-J. CHEN. Autonomic and sensory cardiovascular activities ofnonivamide: Intruthecal administration of clonidine. BRAIN RES BULL 35(I) 15-22, 1994.-The effects of nonivamide on the cardiovascular system were examined and compared with the effects of substance P (SP) in rats. Intravenous (IV) injection (10 @kg) of nonivamide produced triphasic pressure responses (A; depressor, B; pressor, and C; depressor) and biphasic bra- dycardia responses (f; fast bradycardia and s; slow bradycardia). IA injection (10 &kg) into the epigastric artery caused hypo- tension and mild tachycardia. The effects of atropine, vagotomy, SP antagonist, propranolal, and clonidine on these responses were examined and mechanisms responsible for the nonivamide-induced responses are postulated as follows. A and f are due to vagal reflex resulting from the excitation of afferent sensory neurons in the heart and are parasympathetic efferent effects from the nucleus solitarius. B is involved in sympathetic activation, partly caused by the release of SP in the spinal cord. C is due to the vasodilatory effect of SP released from perivascular stores. s was diminished by vagotomy and is due to the bradycardiac effect of acetylcholine, released by SP, from cardiac stores. The activation of the autonomic system is inhibited by clonidine and involved in the wide spectrum of nonivamide-induced cardiovascular effects.

Nonivamide Capsaicin Clonidine Intrathecal Cardiovascular Autonomic Sensory

NONIVAMIDE (NVA, nonanoyl vanillylamide), a synthetic an- alog of capsaicin (CAP, 8-methyl-iV-vanillyl-nonenamide), was investigated to observe its influence on the autonomic and sen- sory nervous systems during cardiovascular responses. CAP, a natural product of red peppers, is known to activate sensory C- fibres and can produce a wide spectrum of pharmacological ef- fects (1,17,22), including inducing pain and causing the release of peptides such as substance P (SP) and the calcitonin gene- related peptide (CGRP) from primary sensory neurons (12,16,18,25). Intravenous administration of SP causes a de- crease in the blood pressure, which is dependent on endothelium and EDRP (6). Conversely, SP neurons descending from the ven- tral medulla to the intermediolateral (IML) cell column, which is the site of origin of sympathetic preganglionic neurons, mediate an excitatory sympathetic influence and may contribute to the increase in blood pressure and heart rate (27). This increased blood pressure is associated with the increased release of SP into the perfused spinal cord by excitatory amino acids (28).

changes caused by CAP were different between the above au- thors. Particularly, the experimental model used by Donnerer and Lembeck (11) to describe the inhibitory effect of clonidine, a central cardiovascular suppressive agent, on the CAP-induced depressor reflex but not its pressor response, remains controver- sial. NVA has been used in our laboratory to synthesize a series of NVA analogs for our study in the molecular modification of CAP (3 1). According to the above reports, to date, the autonomic cardiovascular effects of NVA and its released mediator SP have not been adequately described. The aims of this study are to determine the autonomic and sensory cardiovascular effects of NVA and compare the influence of different inhibitors on those effects. The findings will be compared to the effects of SP, and linked to two series of mechanisms. The results obtained from the NVA studies will be used for molecular modeling of non- pungent analogs of NVA and CAP (3 1,34).

METHOD

The IV NVA-induced biphasic bradycardia in the present in- Measurement of Blood Pressure and Heart Rate

vestigation was not described by Donnerer and Lembeck (9) or Chahl(4) for CAP. The classifications of triphasic blood pressure

Male Wistar rats weighing 250-350 g (provided by the Ex- perimental Animal Center, Cheng-Kung National University

’ To whom requests for reprints should be addressed.

15

16 LO ET AL.

30

20

IO

0

-10

-20

-30

-40

-50

-60

-70

-00

-40 -

-80 -

-120 t

-160 -

-200 -

-240 I

-280 -

-320 L

gIPl:Vogotomy+NVA (i.v.) El:Clonidine (i.t.)+NVA (i.v.)

FIG. 1~ Effects of intravenous injections of 10 &kg nonivam~de on blood pressure and heart rate in control and in rats after various treatments. Vertical lines represent SE (n = 6). Effects A, B, and C indicate the phases of blood pressure changes; effects f and s indicate fast and slow phases of bradycardiac effect, respectively (see text). ~Signi~cant~y different from controls, p < 0.05, **p < 0.01 (ANWA followed by Student’s t-test).

Medical College, Tainan, Taiwan) were anesthetized with so- dium ~ntob~ital (Nembutal, SO mg/kg IP). Following tracheal cannuiation, systemic blood pressure and heart rate were taken and recorded from a femoral artery with a transducer (Gould, Model P50, USA) connected with a pressure processor amplifier (Gould, Model 13-4615-52, USA) and recorder (Gould, series 8O#S, USA). Body temperature was maint~ned at a constant 37°C. NVA and CAP injections were prepared by diluting the stock solution (10% Tween SO, 10% ethanol, and 80% saline, v/ v) with normal saline. The final ethanol concentrations injected were below 0.1% (v/v). SP and SP antagonist were dissolved in normal saline for injection. Femoral veins were cannulated for IV injections and retrograde cannulation was performed on a su- perficial epigastric artery to facilitate IA NVA and IA SP injec- tions.

Pretreatments

NVA or SP was administered to rats 5 min after administra- tion of propranolol(0.3 mg/kg, IV> or SP antagonist (200 &kg, IV), and 15 min after pre~ea~ent with atropine (1 .O mgkg, IP). Bilateral cervical vagotomies were performed 1 h prior to the

experiments. Ail the presentments were given to separate groups of rats.

Intrathecal Perfusion of SP Antagonist and infusion of Clonidine

Clonidine (15 j&2.5 fig) and then saline (15 ~1) were infused through a po~ye~yIene catheter (8 cm long, PE 10) into the spinal cord subarachnoid space of rats using an electric gear-driven sy- ringe pump. The catheter was inserted through a cut in the cis- terna magna to the rostrai edge of the lumbar enlargement in the spinal level L5&6. SP an~gonist (1.0 nrno~~~ was perfused intrathecally for 5 min by a similar technique described in our previous report (32). NVA and SP were administered 7 min after the delivery of the SP antagonist (IO).

~rnrnu~oa~~~y of Intrathecaliy Released SP

Nonivamide was dissolved, diluted with artificial cerebrospi- nal fluid (CSF) as a 200 pM solution, and perfused intratbecally into anaestbesized rats by a technique described by Yaksh and Rody (30) and by Jham~das et al. (19) at the rate of ~~-~~~n for 6 x 20 min through the infiow ~~yethylene catheter (PI?- lo)

CARDIOVASCULAR EFFECTS OF NONIVAMIDE 17

f min E F

HR 100

C ---:::cy-:::c 500

btm/nin 3oo 300

FIG. 2. Effects of IA and IV injections of nonivamide (NVA) on blood pressure in control and in rats after various scents. (A) Control IV 50 &kg; (B) IV 10 @kg; (C) [D-Pro’, D-T~P~~]-SP (1 .O nmol, IT) pretreatment; (D) clonidine (2.5 pg, IT); (E), control IA 10 &kg; (F) IA 50 &kg; (G) [D-Pro’, D-TI$‘]-SP (I .O nmol, IT) pretreat- ment; (H) ctonidine (2.5 pg, IT). Effects A, B, and C indicate the phases of blood pressure changes; effects f and s indicate fast and slow phases of bradycardiac effect, respectively (see text).

of a two-channel peristaltic pump (Atta, SJ-1211, Japan). Those precannulated rats were mounted in a stereotaxic frame and the inflow catheter was then inserted about 8.0 cm through a cut in the cistema magna into the spinal subarachnoid space, with the tip of the catheter in the caudal lumbar region. An outflow cath- eter (PE-50) was placed below the CSF surface in the open cis- tema. Animals respired spontaneously. Control rats were per- fused in~athec~ly with artificial CSF at the same rate for 6 X 20 min before the injection of noniva~de. The success of intra- tbecal perfusion was confirmed by checking the the lumbar re- gion of the spinal cord for the stain, resulting from perfusing trypan blue solution (1%) after each experiment. The outflow of perfused CSF solution was collected in polypropylene tubes (20 ml) containing 0.2 ml acetic acid, then refrigerated promptly, and dried under vacuum. Obtained dried CSF was used to measure the amount of SP by radioimmunoassay (Peninsula Laboratories Inc, USA).

Substances

Drugs used were: capsaicin (Sigma), atropine {Sigma), pro- pranolol (Sigma), isoproterenol (Sigma), clonidine (Sigma), sub- stance P (Sigma), [D-Pro’, D-Trp7*9]SP (Sigma). All reagents were prepared immediately prior to use.

Statistical Evaluation of Data

All values in the text and figures are expressed as means ‘+ SE. Statistical differences were evaluated by Student’s t-test in unpaired samples and by paired t-test in paired samples. When- ever a control group was compared with more than one treated group, the one-way analysis of variance (ANOVA) was used. Probability values (P) smaller than 0.05 were considered to be significant. C~culation for the analysis was done with the aid of software run on an IBM PC-AT computer (29).

RESULTS

Blood Pressure and Heart Rate Changes Ajier Intravenous Injection

Acute intravenous administration of NVA (10 @kg) into Wistar rats induced a biphasic bradycardiac response: a fast (f) and a slow (s) phase bradycardia. In accordance with heart rate changes, a triphasic blood pressure response was observed: an initial transient fall (effect A), followed by an inte~~iate rise (effect B) and a subsequent delayed fall (effect C). The dose- response relationship of NVA (O.l- 100 pg/kg, IV) has been de- scribed in our previous report (33).

The intravenous injection of a lower dose of SP (2.0 peg) into rats showed both a depressor effect and tachycardia (changed from 372.2 it 13.6 to 401.5 + 12.0 beats/min,p < 0.05, n = 8), but a higher dose of SP (20 ,ug/kg) showed the depressor and bradycardia effect (Fig. 3).

Intravenous Injection and Different Pretreatments

As shown in Figs. 1 and 2, after separate pretreatment with IP atropine ( 1 .O mglkg), vagotomy, and intrathecal perfusion of SP antagonist-[D-Pro’, D-Try’*9l SP (1.0 nmo~ml), effect A of IV NVA (10.0 &kg) was ~ffe~ntly inhibited. Effects B and C were inhibited by IT SP antagonist (1.0 nmollml) and IT cloni- dine (2.5 yg/kg).

Effect f was inhibited by IP atropine (1.0 mg/kg) and vagotomy. Effect s was inhibited by IT SP antagonist (1.0 nmol/ml) and was manifested as mild tachycardia by vagot- omy.

IV SP antagonist (200 pg/kg) could not inhibit or even potentiate effects A, f, and s, even though it retained the hy- potensive and bradycardia effects of the partial agonist activ- ity of SP in the peripheral, not central, cardiovascular system (Fig. 6).

Pretreatment with vagotomy, lP atropine (1.0 mg/kg), and lT clonidine (2.5 pg) si~ific~dy inhibited the IV SP (2.0 &kg)-

18 LO ET AL.

SP(i.V.1 A S c

BP zoo I 200 1 ,200

mm w 100 Cl 100 C~ ,.,clsII

UR 500

C

500 500

bts/rin 3oo e 300 300 C-

SP (lJI.1 D E F

BP 200 PO0 200

y W 100 280- 100

nn 800

C

800

C

200

bta/rin 2oo -e

200 300 I?---

a m&a

FIG. 3. Effects of IA and IV injections of substance P (SP) on blood pressure in control and in rats after various treatments. (A) Control IV 2.0 &kg; (B) IV 20 ,z.#kg; (C) clonidine (2.5 pg. IT): (D) control IA 2.0 &kg; (Et [O- Pro’, R-Trp’~gl-SP (1 .O nmol, IT) pretreatment; (F) clonidine (2.5 pg, IT).

induced by~te~sive response, and propranolol (0.3 mglkg, IV) significandy inhibited the IV SP-induced hypotensive and tachy- cardia effects (Figs. 3 and 4).

I~tr~~~er~ai Injection and DiSferent Pretre~tment.~

A retrograde epigastric intraarterial injection of NVA ( 10 ,ug/ kg)-induced monophasic hypotension (changed from 138.3 c 3.6 to 110.2 f 2.4 mmIig, p < 0.05, n = 6) and a mild tachycardia response. This IA NVA-induced depressor effect was signifi- cantly reversed following intrathecal perfusion of a SP antagonist [D-Pro’, D-Trp7”] SP (I .O nmoYm1). After the treatment with IT clonidine (2.5 pg), the hy~tensive and tacbycardia effects in- duced by IA NVA were fnrtber reversed and inbib~ted (Figs. 2 and 5).

Intraarterial injection‘of SP (2.0 pg/Kg) induced tachycardia and biphasic (depressor and then delayed pressor) blood pressure responses. These effects were signi~c~dy inhibits by pretreat- ment with propranolol(0.3 mg/kg, IV) or clonidine (2.5 ~8, IT) (Figs. 3 and 5).

Immunoreactivity of Intrathecally Released SF

The stimulator effects of NVA on the release of W-like im- munoreactivity from the superfused spinal cord of anesthetized rats is shown in Fig. 7. In comparison with the control group, NVA (200 $vQ perfused for 20 min through the impl~ted PE- 10 catheter, released 202 i- 7.8 pg of SP-like i~uno~activity from the perfused outflow solution.

DISCUSSION

Previous studies reported that an in~avenous injection of CAP into rats induced bradycardia, aponea, and triphasic blood pres- sure changes: an initial short fall (effect A), an intermediate slight rise (effect B), and a subsequent delayed fall (effect C) in blood pressure (5,9,30,33). These cardiovascular reflexes have been traced to the excitation of a distinct ~p~ation of vagal afferent C fibers with chemosensitive endings in tbe lung. The existence

of cardiac chemosensitive receptors subserved by unmyelinated afferent fibers in the vagal and spinal sympathetic nerves has been demonstrated in dogs and cats. CAP has been reported to excite some chemosensitive vagal afferent endings in the heart of dogs; however, its acute action also involves the activation of sympathetic cardiac afferent neurons (26). On the other hand, CAP induces complex effects on the cardiovascular system, the nature of which varies with the dose and speed of administration. Effect B and the slow IV infusion of the CAP-induced pressor response were suggested to be mediated predo~n~dy by an adrenergic mechanism (4). Furthermore, the intratbecal adrnin- istration of a SP antagonist leads to autonomic, sensory, and mo- tor dys~n~tion (7). The IA CAP-induced depressor reflex is par- tially blocked by clonidine and is involved in alpha 2 adrenoceptor activity (1 i ). This depressor reflex response is sim- itar to that activated by SP (6), but contrasts with the pressor and tachycardia responses to SP (27). Our previous investigation sug- gested that adenosine receptors are involved in the CAP-evoked reflex fall in rat blood pressure (31).

Evidence for both autonomic and sensory components in the action of NVA was obtained in the present study. There- fore, it is proposed that biphasic bradycardia and triphasic blood pressure changes induced by the acute IV NVA are caused initially by the release of SP from afferent sensory neurons in the heart and nucleus tractus solitarii (NTS) in the CNS (34), resulting in the fast bradycardia and effect A, i.e., classic vagus reflex. The secondary release possibly occurs because SP in the spinal cord then activates the sympathetic pathway and released SP in the heart stimulates presynaptic parasympathetic neurons to reiease acetylcholine, as well as resulting in slow bradycardia (Fig. 2).

P~asympa~etic transmission by SP was reported previously by Chahl (3). Paka et al. showed that CAP depleted SP-immu- noreactivity and acetylcholinesterase activity from nerve fibers in the guinea pig heart (23). Dalsgaard et al. (1986) also found the distribution of SP i~unoreactive nerves in the guinea pig heart and demons~ted that systemic CAP treatment could cause a total loss of SP immunoreactive nerves (8). On the other hand,

CARDIOVASCULAR EFFEKTS OF NONIVA~I~E 19

10

I -10

I d -30

9 if I” -50

ii -70

60

*zm

d f! 40

p

= 6 20

g 10

0

0:sP (i&r*) ID Atmpine + SF (ix) ~:Vagotomy + SP (hf.) ~:Pmpmnoloi + SP (i.v.)

FIG. 4. Effects of in~avenous injections of 2.0 FgIkg substance P (SP) on btood pressure and heart rate in control and in rats after various treatments. Vertical lines represent SE (n = 6). *Significantly different from controls, p < 0.05, **p < 0.01 (ANOVA followed by Student’s t-test).

excitato~ amino acid injected into the ven~o~ateral medulla of rats resulting in an increase in blood pressure is associated with the increased release of SP into the spinal cord. The SP receptor in the IML cell column is dependent on the integrity of sympa- thetic ~stg~glionic neurons (27,28). CAP was reported to ex- cite some chemosensitive vagal afferent endings in the heart of dogs, but its acute action also involves sympathetic cardiac af- ferent neurons (26). In the isolated heart, both CAP and CGRP increase the contractile rate and force of the guinea pig atrium, but SP ( 10e6 M) does not influence it (14,21). The role of CGRP as a cardiac neurotransmitter in the action of CAP is nonadre- nergic and noncholinergic (21). Peptidenergic contributions to CAP-sensitive bradycardia in rats, such as positive chronotropic and inotropic effects of CGRP or vasomotor activity of SP, are controversial.

As shown in Fig. 1, IV NVA-induced biphasic bradycardia was inhibited by pretreatment with vagotomy and cholinocep- tor antagonist, as shown in our previous report (34). Under

this treatment, slow bradyc~dia was changed to mild tachy- cardia in vagotomized rats. NVA-induced triphasic blood pressure and biphasic bradycardia were particularly inhibited by IT elonidine. IV NVA-induced bradycardia and hypoten- sion may be attributed to the cardiovascul~ effects of SP re- leased in the heart and NTS (33), masking the tachycardia or sympathetic responses that result from the SP released in the spinal cord (27). That IV and IT SP antagonist potentiate the IV NVA-induced bradycardia may be due to a partial agonist activity of SP on the parasympathetic efferent pathway. How- ever, effect s was inhibited by IT SP antagonist. It was found that the pressor response produced by CAP is the adrenergic mechanism. Nevertheless, it is lacking a complete block by an alpha adrenergic blocker, which may be reiated to the release of neuro~ptide Y (15). Furthermore, CAP evokes a clear-cut sympathetic activation because the release of norepinephrine and neuropeptide Y, but not endothelin, can be clearly de- tected (24).

20 LO ET AL.

NVA

+

SP

-20 L Cl :NVA (Lo:) !EJ:[D-PrdE,O-Tr$qSP (i.t.)+NVA (ia.) m :Clonidine (i.t.)+NVA (La. WE :SP (i.0.) m:[D-Pr B m :Propronolol+SP (i.o.)

,D-TrpJ’qSP (i.t.)+SP (i.a.) D Clonidine (i.t.)+SP (Lo.)

FIG. 5. Effects of intraarterial injections of 10 &kg nonivamide (NVA) and 2.0 &kg of substance P (SP) on blood pressure and heart rate in control and in rats after various treatments. Vertical lines represent SE (n = 6). *Significantly different from controls, p < 0.05, **p < 0.01 (ANOVA followed by Student’s r-test).

Microinjection of NVA into the NTS induced both hypoten- sion and bradycardia in our previous reports (33), as did the in- jection of SP described by Lukovic et al. (20). Both are thought to contribute to the fast p~~ympa~etic efferent effect. A high dose of SP (20 &kg, IV) may partially penetrate into the central nervous system and stimulate the NTS, like the central effect of NVA or CAP, resulting in obvious bradycardia which is inhibited by atropine (2). In the present study, both vagotomy and atropine also significantly inhibited IV SP (2.0 &kg)-induced hypoten- sion, but not tachycardia (Fig. 4).

An epigastric retrograde intraarterial injection of NVA (10 ,ug/kg)-induced monophasic hypotension, as described by Donnerer and Lembeck (9). However, larger doses of IA NVA also showed biphasic bradycardia and triphasic blood pressure (Fig. 2). Varied responses between IV and IA NVA may be attributed to the following action sites: chemosensitive affer- ent endings in the heart, NTS, IML, and blood vessels. Redis-

tribution of NVA from the heart to the above sites after epi- gastric retrograde intraarterial injection of NVA may induce different cardiovascular responses.

SP-induced relaxation of the porcine coronary artery has been suggested to be involved in the release of EDRF from endothelial cells (6). The SF receptor in the intermediolateral cell column is dependent on the integrity of sympathetic postganglionic neu- rons. In the present study, IA SP (2.0 pg/kg> induced mild tachy- cardia and delayed pressor response, which were, however, sig- nificantly inhibited by propranolol and clonidine, conlirming the above results. Moreover, the fact that the resulting tachycardia was also prolonged to a sustained phase by the IA SP antagonist may be due to their partial additive agonist effect on the cardio- vascular system.

IA NVA-induced depressor response, inhibited by IT cloni- dine, indicates that a central alpha 2 adrenergic receptor is in- volved, as described for CAP (11). Effect B, or the pressor re-

CARDIOVASCULAR EFFECTS OF NONIVAMIDE 21

SP l lt8gonbt NVAfl @t&S)

1 mln i

FIG. 6. Typical records of NVA (IO &kg, IV~i~uc~ ~diov~~ul~ changes in rats pretreated with SP an~gonist (XXI &kg, IV). Left part: control; right part: pretreatment with SP antagonist. MBP: mean blood pressure; HR: heart rate.

sponse of IV NVA, which was described for CAP as being due to an adrenergic mechanism (4), was inhibited by IT clonidine in the present study. These facts indicate that sympathetic acti- vation may be caused by released SP in the central nervous sys-

i.IIl : Controt (A) m : NVA (B)

20 40 60 80 loo 120

Time after perfusion (min)

FlG. 7. The stimuiato~ effect of in~~~~ perfnsion of NVA (200 FM) on the release of SP-like immunoreactivity from the superfnsed spinal cord of anesthetized rats. Vertical lines represent SE (n = 5) *Signifi- cantly different from control, p < 0.05 (paired t-test).

tern, including in the spinal cord; this effect is also obviously increased after vagotomy (Fig. 1). Effect C of IV NVA may be due to the vasodilatory effect of SP released from perivascular stores and effect s may be due to the bradycardia effect of ace- tylcholine, released by SP, from cardiac stores (8,23). Those ef- fects are partly similar to effects resulting from exogenous SF. The fact that effect B of IV NVA was inhibited partly by IT SP antagonist and significantly by IV SP antagonist indicates that SP plays au important role in IV NVA-induced sympathetic car- diovascular responses. The IA SP-induced tachycardia and de- Iayed pressor response inhibited by IT clonidine is due to the decreased discharges in sympathetic ~stg~glionic fibers of car- diac nerves and blood vessels. This tachycardia is also inhibited by IV propranolol on the beta adrenoceptors of cardia muscle. However, it is also suggested that high vagus activity induced by IT clonidine may lead to ~minished IV NVA- and IV SP-in- duced depressor effects.

Finally, that intrathecal perfusion of NVA stimulates the re- lease of SP-like immunoreactivity in the outflow indicates the cardiovascular effects of NVA were partly mediated by the re- lease of SP in the spinal cord.

Effects of atropine, vagotomy, SP antagonist, and clonidine on NVA-induced responses were examined. Evidence in the present study indicates that adrenergic, cholinergic, and pep- tidenergic primary sensory mech~~sms are responsible for these responses. Clonidine, administrated in~athecally, is a powerful inhibitor in NVA-evoked cardiovascular reflexes.

We wish to thank Ms. Shu-Hui Lin for her excellent technical assis- tance. This work was supported by research grants from the National Science Council, Taiwan, ROC (NSC 80-0412-B037-79).

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