Mechanisms involved in the cardiovascular responses to opioid products of proenkephalin in the anaesthetised rat

Gen Pharmac Vol 23, No 2, pp 269-277, 1992 0306-3623/92 $5 00+000 Pnnted m Great Bntam All rights reserved Copyright © 1992 Pergamon Press plc

MECHANISMS INVOLVED IN THE CARDIOVASCULAR RESPONSES TO OPIOID PRODUCTS OF PROENKEPHALIN

IN THE ANAESTHETISED RAT

HELEN DOUGLAS and IAN KITCHEN* School of Biological Sciences, Receptors and Cellular Regulation Research Group, UmversRy of Surrey,

Gmldford, Surrey GU2 5XH, England [Tel (0483) 300800, Fax (0483) 576978]

(Recewed 9 July 1991)

Abstract--I Cardiovascular effects of opmid peptide products of procnkephahn, [Met] enkephahn (ME), [Leu] enkephahn (LE), [Met] enkephalyl Arg6-Phe 7 (MEAP) and [Met] enkephalyl Arg6-GlyLLeu s (MEAGL) have been studied m urethane-anaesthetlsed rats

2 ME, LE, MEAP and MEAGL produced vasodepresslon and bradycardm mediated by #-opioid receptors

3 Atypical responses to MEAP were observed m a quarter of the animals studied showmg tachyearcha and pressor effects This response was probably due to the release of the dlpepUde Arg-Phe which exerted its effects at sympathetic ganglia

4 Studies with the peptldase mhlbltors captopnl and bestatln showed a dlfferentml potentmUon of the cardiovascular effects of the proenkephahn products by mhlbmon of anglotensln converting enzyme and ammopepUdase

5. The effects of vagotomy, pRhmg and studies with atropine, and N-methyl levallorphan were used to demonstrate that, for all four proenkephahn peptldes, cardiovascular effects were medmted by peripheral oploid receptors and transmission to the CNS via vagal afferents

INTRODUCTION

One of the earliest reported observaUons on the biological acUwty of oplolds, besides analgesia, was the demonstration of potent cardloresplratory effects produced by morphine, In more recent years studies with morphine and several synthetic oploid have confirmed effects on both peripheral and central cardiovascular reacUwty, though wRh notable species differences (for review see Holaday, 1983). The discovery of three oploid pepUde precursors (proenkephahn, prodynorphm and prooplome- lanocortm) producing several biologically active oplold peptides (Rossler, 1982) has raised the posslbdRy that some of these peptides rmght be revolved m cardiovascular control. The dlstnbutmn of proenkephahn products m CNS areas involved in cardiovascular regulaUon (Khachatunan et aL, 1985) and peripherally m the heart (Lang et al., 1983) and the adrenal glands (Hook and Llston, 1987) make these peptldes transmitter candidates m the regulation of cardiovascular function

In the rat, there ~s evidence for two of the proenkephahn products, [Met] enkephahn (ME) and [Leu] enkephahn (LE) of modulatory effects on the heart (Eiden and Ruth, 1982) blood vessels (Ruth et al., 1984) and from whole ammal studies effects on the CNS (Wet et al , 1980) Little attention has been paid to the cardiovascular effects of the other major proenkephahn products [Met] enkephalyl Arg6-Phe 7 (MEAP) and [Met] enkephalyl ArgLGlyT-Leu 8 (MEAGL) and since the oplold receptor acUvatlon profile of the four proenkephahn products is disslm-

*To whom all correspondence should be addressed

llar (Paterson et al., 1983) we have camed out a comparaUve study of cardsovascular effects of all four peptides m the anaesthetlsed rat and elucidated the mechanisms underlying their m vivo cardiovascular effects m companson wRh op~oid receptor selective agomsts.

METHODS

Operatwe procedures and car&ovascular recording Adult male Wtstar albmo rats (UntversRy of Surrey strain

225-275 g) were anaestheUsed vnth 25% (w/v) urethane (1 6g/kg, Lp) and lard on their dorsal side on a heated ammai operating table Rectal temperature was maintained at 37 + I°C The trachea, left jugular veto and nght caroUd artery were cannulated after a 15 mm equthbratton penod, and experimental studies were begun after a further 30 rain period Drugs were administered m 0 I ml sahne (0 9%) by intravenous bolus into the jugular vem and washed through ~th a further 0 2 ml sahne The carotid artery was connected to a Statham P231D blood pressure transducer (primed with hepanmsed sahne, 100 umts/ml) and artenal pressure and integrated heart rate were recorded on a Grass Model 79D polygraph

Vagotomy and ptthmg In some expenments after responses to opiold agomsts

had been obtained, bilateral vagotomy was performed by locating and cutting both vagus nerves at a point close to the exposed caroUd artenes A 15 mm period follovang sectioning was allowed before repeating drug adrmms- tratlon

For the preparation of pithed ammals rats were anaesthetlsed vdth ether and the trachea cannulated Ptthmg was achieved by the msertton of a short steel tube (13 s w g, 4 ¢m long) through the orbR and via the foramen magnum to the level of the first couple of vertebrae Through thts trocar a

269

270 HELEN DOUGLAS and IAN KITCHEN

Table 1 Cardmvascular parameters m intact bfiatorally vagotomlsed and puhed rats

Blood pressure

Heart rate Systohc Dtastohc pO 2 pCO 2 HCOf Base excess Treatment (bpm) (mmHg) (mmHg) pH (mmHg) (mmHg) (retool/I) (retool/I)

Control 357+_9 7 2 + 2 56+_2 73+_003 7 0 + 4 6 42+-27 21+-07 - 5 5 + 0 7 Bilateral vagotomy 425+_18" 60+_2* 52+_2* 72+_003 75+_64 5 1 + 5 3 " 24+_08 - 5 3 + 0 8 Ptthmg 213+16" 63+_2* 53+_2* 73+_002 167+_23 5 1 + 3 7 " 20+_08 - 5 7 + _ 0 5

Values represent mean + SEM of at least 5 observaUons at 90 mm after each procedure Stausucal analysts performed by two way ANOVA followed by Duncans test on repeated measures made at 30 mm intervals, *P < 0 05

teflon tube (0 16 mm dla, 20cm long) with a fine steel tube (26 s w g, 24 cm long) mstde tt, was rapidly passed down the spinal cord Immediately, the animal was arttficlally venti- lated via a respiratory pump (connected to a pure 02 cyhnder) set to dehver 1 ml O2/100 g body weight at a rate of 50 strokes/rain Thirty mmutes were allowed before drug admlntstratlon

Blood gas analysts

Studtes of changes m blood gases, pH, derived bicarbonate concentration and base excess were made in anaesthetlsed rats, vagotomlsed and ptthed animals For measurements of these parameters, 400/al samples were taken from the carotid artery anaerobically into hepartnlsed plastac synnges vta a 3 way tap Samples were m~xed vigorously for 20 sec and analysed immedtately using a IL413 pH/blood gas analyser (Instrumentation Labora- tories, Warnngton) After blood gas analysis, 5/zl of re- sidual blood was taken Into a haematoertt m~crocaptllary tube, mtcrocentnfuged for 5 mm (Compur M 1100) to deter- mine haematocrtt Samples were taken every 30 mm over a 2 hr period

Drug and dosing protocols

[Met]-Enkephahn (ME), [Leu]-enkephahn (LE) [Met]- enkephalyl Arg6-Phe 7 (MEAP) [Met]-enkephalyl Arg6-Gly 7- Leu s (MEAGL) [D-AIa 2, Me-Phe 4, Gly-olS]-enkephahn (DAGO) and [D-Pen z, o-Pen S] enkephalm (DPDPE) as acetate salts were purchased from Cambndge Research Btochemtcals, and morphme sulphate from May and Baker, Dagenham Atropine sulphate, bestatm, captopnl, urethane, sodium mtroprusstde and acetylchohne chloride were purchased from Sigma, Dorset, noradrenahne bttar- trate from Sterling Research Laboratories, Gmldford, propranolol hydrochlonde from ICI, Macclesfield, and phentolamme mesylate from Ctba, Horsham The followmg drugs were gifts naloxone hydrochlortde (Dupont), N- methyl levallorphan methane sulphonate (Sanofi) and US0488H (Upjohn) Drugs were admmlstered on a 4 mm dose cycle unless their duration exceeded this time hmtt where subsequent doses were gwen 1 mm after return to predrug responses No more than two dose-response curves were performed m a single preparation and only one op~old agonlst was admlntstered to an lndtvldual antmal Studtes were carried out as pmred experiments and where antagontsts or pepttdase mhtbttors were employed these were admmtstered 15 mm before dosmg regtmes were repeated

RESULTS

Control studws

The heart rate, blood pressure, blood pH, pO2, pCO2 and bicarbonate concentration d~d not change slgmficantly over a 2 hr period in the absence of drug admimstraUon In bilaterally vagotonused ammals only heart rate was sigmficantly altered up to 60 mm after vagotomy, wRh pCO2 levels being elevated at 90mln and blood pressure slgmficantly lower In pithed ammals, heart rate and blood pressure was slgmficantly lower over a 90 nun penod and there were concomitant elevations m pCOz and pO2 (Table 1)

Cardiovascular responses to proenkephahn products

Representatwe traces of the cardmvascular responses to each proenkephahn product are shown m Fig 1 and their quantRatwe assessment m Figs 2 and 3 For all proenkephahn products lmmedmte vasodepressor and bradycardlc effects were observed after mtravenous admnustraUon (30-300/zg/kg), the changes m dmstohc pressure bemg more marked than the decrease m systohc The duration of the effects showed vanabfllty between each peptlde with responses to ME and M E A P being twice as long as the response to LE and M E A G L (Vasodepressor response (300 #g/kg), ME, 126 _ 11 sec, LE, 77 + 18 sec, MEAP, 114 + 15 sec, M E A G L , 68 _ 14 sec. Bradycardm (300#g/kg) ME, 1 5 8 _ 15see, LE, 58 + 13 sec; MEAP, 156 + 12 see, M E A G L , 65 _ 19 sec.) Equivalent cardiovascular effects were observed following jugular or femoral veto adnums- traUon of the peptldes and repeated dose response curves (up to 3 t~mes) showed no tachyphylax~s Cardiovascular responses to D A G O (3-30/zg/kg) and morphine (30-150/ag/kg) were quahtatwely slnu- lar to those observed with proenkephahn pepUdes but with a longer duration o f effect The J-selectwe agomst D P D P E (75-300/lg/kg) and the x-agonlst U50488H (75-300#g/kg) had no slgmficant effects on blood pressure or heart rate (data not shown)

Naloxone (1 mg/kg) totally abohshed the cardiovascular responses to all proenkephahn pepUdes and to morphme and D A G O and had no effects itself on blood pressure and heart rate up to 2 5 mg/kg

Cardiovascular measurements and stattstwal tests

Cardiovascular effects were quantified by comparison of systohc and diastolic pressure and of integrated heart rate at the time of maximum change with pre-dosmg values Mean artenal pressure was calculated as the dtastohc pressure + 1/3 pulse pressure Paired t-tests were used for companson of cardiovascular effects and analysis of ttme and treatment on blood gas parameters was analysed using ANOVA followed by Duncan's Multiple Range test

Atypical cardiovascular responses to MEAP

In about 25% of rats an atypical cardiovascular profile to M E A P was observed At doses above 30/~g/kg transient bradycardla and vasodepressor responses were followed by marked tachycardla and pressor effects (Figs 1 and 4) The decrease m blood pressure and heart rate was antagomsed by naloxone (1 mg/kg) whilst the tachycardla and pressor responses were unaffected (Fig 5). Phentolamme (300/ag/kg) and propranolol (300ttg/kg) abolished

bpm

mmHg

4OO

2OO

7o

Cardmvascular effects of proenkephahn peptades

[Met] enkephalin

t 4,

s 30 7S 150 300

271

bpm

mmHg

400

20O 100 [

70

[Leu] enkephahn

-I-- F - " -T" " - - ! • J . 4,

S 30 7S 150 300

bpm

mmHg

400

200 I

,oo [ 7O

[Met] enkephalyl - arg s - phe 7 (a) --~-, , • - ~ ~-__

s 30 75 15o 300

bpm

mmHg

°° E 200

100 E 70

r 1"

[Met] enkephalyl - arg e - phe 7 (b)

s 30 75 150 300

bpm

400 [Met] enkephalyl - arg s - gly 7 - Leu 8

100 mmHg [

70

s 30 75 150 300

Fig 1. Representatwe traces of cardiovascular responses to proenkephalin products Traces show the effects on heart rate (bpm) and blood pressure (mmHg) The control saline response is shown as s, and the numbers represent doses in #g/kg The effects of each proenkephahn product were stuched in separate

preparations, The two traces for MEAP represent (a) typical and Co) atypical responses

the presser response and tachycardm rcspectwdy. Intravenous administration of the dipeptide Arg-Phc (30-300 #g/kg) produced a dose related increase in blood pressure and m heart rate (Fig. 4), effects which were unaffected by naloxone (1 mg/kg) but abolished

by phentolamme (300 #g/kg) and propanolol (300/~g/kg) respectively Hexamethomum (12 rag/ks) also blocked the Arg-Phe response. HPLC analysis of samples of MEAP and Arg-Phe confirmed the purity of these peptides (data not shown).


5 p ME LE MEAP

' i "ii - g C

30 pg/kg

g [ ] 150 IJ.g/kg -20 i 75 pg/kg

B 3 0 0 p.glkg

MEAGL

iJ Fig 2 Effect of proenkephahn products on mean arterml pressure The effects of each proenkephahn product were studies in separate animals (n = 10 for each peptlde) Histograms represent mean change in mean arterial pressure (mmHg) + SEM Mean restmg arterial pressure for each pepttde (ME 74 + 2, LE 78 + 2, MEAP 80 _+ 2, MEAGL 73 + 2 mmHg) One way ANOVA of the effects of each proenkephahn

product, P < 0 01 for all peptldes

Effects of captoprd and bestatm

Captopnl at doses of 500/~g/kg and above produced a sustained decrease m mean arterial pressure wzth accompanying reflex tachycardta which s~gmficantly attenuated responses to several vasodepressor drugs A dose of 200/~g/kg captopd had no slgmficant effects on cardmvascular responses per se and did not attenuate responses to sodmm nitropruss~de This dose was chosen for studies with proenkephahn

peptldes. The duraUon of the cardiovascular responses to ME, MEAP, M E A G L but not LE were s~gmficantly potentiated m the presence of captoprd (Table 2) al though the magnitude of the response to all four peptzdes remained unaffected Captopnl (200 #g/kg) had no effect on morphine responses and slgmficantly attenuated vasopressor responses to anglotensm I (data not shown).

Bestatln (0 5-10 mg/kg) had no effect on resting heart rate or mean arterial pressure. Doses of

A

E Q .

¢1

¢1

C

¢1

p.

O

10

0

-10

-20

-30

-40

-50

-60

-70

ME LE MEAP MEAGL

[ ] Sahne

L~15o pg/kg

[ ] Q,ko

• 300 .g,kQ

Fig 3 Effect of proenkephahn products on heart rate The effects of each proenkephahn product were studied m separate animals (n = 10 for each peptlde) H~stograms represent mean change m heart rate (bpm) + SEM. Mean restmg heart rate for each peptide (ME 383 + 13, LE 358 + 8, MEAP 377 + 8, MEAGL 367 _+ 13 bpm) One way ANOVA of the effects of each proenkephahn product, P < 0 01 for

all peptldes

Cardtovascular effects of proenkephahn pept~des 273

(a) MEAP (b) Arg-Phe

40

2O

i: C

1 -

[ ] Iso • 75 itg/kg

• 300 Ixg/kg

Fig 4 Cardiovascular effects of (a) [Met] enkephalyl-Arg 6- Phe 7 and (b) Arg-Phe Upper histograms represent the mean change m mean arterial pressure + SEM and lower histograms the mean change in heart rate + SEM of 5 observations The effects of each peptlde were studted m separate ammals Mean resting heart rates and mean arterial press-

ures ranged from 340-374 bpm and 70-79 mmHg

0.5-5mg/kg had no significant effects on proenkephalin products At 10 mg/kg bestatm potentiated the magnitude of the vasodepressor response to ME and LE but was without effect on MEAP and MEAGL (Table 2). There were no effects on the bradycardia or on the duration of the responses

The combined effect of captopnl and bestatm was to potentiate both the duration and magnitude of cardiovascular responses to all proenkephahn products by a similar degree to that achieved following the administration of either peptidase inhibitor (Table 2)

had no slgmficant effects on the vasodepressor responses (Fig. 6)

In the pithed rat, proenkephahn products, DAGO and morphine had no effects on blood pressure or heart rate Cardiovascular reactwlty of pithed animals was confirmed by demonstrating responses to acetylchohne, noradrenahne and sodium nitroprus- side

N-Methyl levallorphan (NML) slgmfieantly attenuated responses to proenkephalin products at 2mg/kg and completely abohshed cardiovascular effects to these peptldes, DAGO and morphine at 5 mg/kg (Fig 7) The parent compound levallorphan at 1 mg/kg had slmdar effects

D I S C U S S I O N

In agreement with prewous studies for [Met] and [Leu] enkephahn (We] et al, 1980) and enkephalin

MEAP dose 30 75 150 300

2 5 - 2; E E 2 0 -

D 15 - - ¢J

- - 10- - L .

C

~ 0 - -

E c_

e ,

~ -10 -

3 0 -

~ 2 0 - - &

¢-

_e ~ - 1 0 -

e -

~ - 2 0 - - 0

- 3 0 - -

Effect of bilateral vagotomy, atropine, pithing or N- methyl levallorphan

Bdateral vagotomy totally abolished the cardiovascular effects of the proenkephahn products, DAGO and morphine. Intravenous administration of atropine (300/~g/kg), a dose that antagonised the cardiovascular response to acetylcholine (10/~g/kg), significantly increased heart rate. At- ropine antagonised the bradycardia produced by proenkephalin products, DAGO and morphine but

Fig 5 Effect of naloxone on atypical eardlovascular responses to [Met] enkephalyl Arg~-Phe 7. Upper histograms represent the mean change m mean arterial pressure + S E M and lower histograms the mean change m heart + SEM of 5 observations. The histograms (l l) and (B) represent response to MEAl) m the absence and presence of naloxone (1 mg/kg) respectwely. Numbers show doses of MEAP (~g/kg). Responses were obtmned 15rmn after administration of naloxone Mean resting heart rates and mean arterial pressures ranged from 339-367 bpm and

69--80 mmHg


¢.

8~

o~.~

g l e . ~

analogues (Willette et al , 1982, Martynova and Medvedev, 1986) m urethane-anaesethetised rats, naloxone reversible vasodepressor and bradycardic effects were observed for all four procnkephalin peptldes after intravenous administration. The duration of their effects were markedly &fferent, with ME and MEAP exhibmng more prolonged effects than LE or MEAGL The eqmvalent effects of morphine and DAGO and the lack of effect of <5 and r selective drugs, DPDPE and US0488H, points to a #-receptor mediation of this cardmvascular effect despite classlficatton of LE and ME as 6-prefemng hgands (Paterson et al , 1983).

In about a quarter of preparatmns atypical responses to MEAP were observed These effects were not ewdent for the other peptldes m studies over a 2 yr period and m more than 600 preparatmns. The atypical tachycardm and pressor effects of MEAP are

A

-r E 5 E

ME LE MEAP MEAGL DAGO Mo~)hine -o1 ]u [ -s

~ -10 C I g •

E -15 "T C " ' . ~

~ -20 C

e -

o

2O

Q. o

" -20 I::

" - 4 0 c

t ~

, ,C

o --8o

ME LE M E A P MEAGL DAGO Moq~hlne

Fig 6 Effects of atropme on cardmvascular responses to proenkephahn products, DAGO and morplune Upper tns- tograms represent the mean change m mean arterial pressure + SEM and lower Mstograms the mean change m heart rate + SEM of 5 observations m separate animals for each agomst The lustograms (L';'J) and (D) represent responses before and 15 mm after atropine admmlstraUon (300 #g/kg) respectively, each animal serving as its own control Full dose ranges were studied, although for brewty only the highest doses are shown (ME, LE, MEAP, MEAGL-- 300 #g/kg, DACK)---30/zg/kg, morphine---75/~g/kg) Mean resting heart rates and mean artenal pressures ranged from

337-380 bpm and 72-80 mmHg

Cardiovascular effects of proenkephahn pept~des 275

E S E

" 0

r -

~ -10

e -

~ -20

ME LE MEAP MEAGL DAO0 Mo~hine

ME LE MEAP MEAGL DAGOMo~hme

0 A

E O . t , i

m -20

¢0

" -40 F. 0

-60 ¢-

-80

Fig 7 Effect of N-methyl levallorphan or cardiovascular responses to proenkephahn, DAGO and morphine Upper histograms represent the mean change m mean arterial pressure + SEM and lower h~stograms the mean change m heart rate + SEM of 4 observations m separate ammals for each agomst The histograms (~) and (I-7) represent responses before and 15mm after N-methy levallorphan adnumstratlon (5 mg/kg) respectively, each ammal serving as Rs own control Full close ranges were studied but for brevRy only the lughest doses are shown (ME, LE, MEAP, MEAGL, 300 #g/kg, DAGO--30/z g/kg, morphine 75pg/kg) Mean resting heart rates and mean arterial

pressure ranged from 365-400 bpm and 76-81 mmHg

not oploid receptor medmted but revolve adreno- ceptors as evidenced by the studies with naloxone, phentolamme and propranolol. These non-oploid receptor medmted effects were mimicked by the dlpeptlde Arg-Phe and raise the possibility of rapid enzymaUc cleavage of the heptapeptlde m vwo HPLC analysis of MEAP and Arg-Phe confirmed that e x vtvo degradation had not occurred. Tins suggests that differential degrading capainhtles may exist witinn ammals and the observation that tins atypzcal response predonunantly occurred in litter mates suggests that genetically controlled expression of a particular cleaving enzyme may be responsible. Atypical cardiovascular responses to MEAP have also been reported m the dog (Sanders and Giles, 1985) and cleavage of Arg-Phe from MEAP has been shown in rat brain synaptosomes (Marks et a l , 1982).

It ~s hkely that the transient modest carchovascular effects of proenkephahn products are due to rap~d degradation followmg intravenous admm]strataon. Tins is supported by potentiating effects of the an- glotensin converting enzyme ininintor captopd, and Rs' more marked effect on responses of the hepta- and octapepude agrees with antinoc]ceptlve studies (Chou et ai., 1984) Others have suggested captopnl potenti- ates vasodepressor responses wa other mechanisms as stable enkephahn analogues have been shown to have enhanced actwRy in the presence of captopril (Geh et al., 1986) Our lack of effect upon morpinne or DAGO confhcts with tins proposal. It is worthy of note that atypical responses to MEAP were not observed in the peptidase minintor studies with captopnl and the known protectzon of the Arg-Phe cleavage by tins minintor (Yang et al., 1982) might suggest that enhanced expression of anglotensm converting enzyme ~s responsible for the atypical responses to MEAP

Potentmtzon of ME and LE responses by the ammopept]dase mhlintor confirm the importance of tins degradauon to enhance m wvo effects (Chaillet et a l , 1983) The lack of effect of bestatm and MEAP and MEAGL shows the d~fferent cleavage suscept~- bilmes of the extended enkephahn sequences, and conflicts with reports of potentmtmn of MEAP and MEAGL reduced antmociceptlon by bestatin (Melistrom et al., 1987) Lack of synergistic effects of captopnl and bestatm is supported by studies of captopnl and bestatm on MEAP antmoc~ception (Chou et a l , 1984)

D~fferent effects of vagotomy on responses to opmlds m anaesthetlsed rats have been reported m the hterature For example, cardiovascular effects of fentanyl are abohshed and ethylketocylazocine is unaffected (Gautret and Schmltt, 1985) whilst etorphine produces a pressor rather than a depressor effect after vagotomy (Roquebert and Delgoulet, 1988). Whilst tins latter response has been reported for an enkephahn analogue (Randlch and Callahan, 1986) most studies with pepUdes show vagotomy abolishes the cardiovascular effects (Wdlette and Sapru, 1982; Wei et a l , 1980). The results of our study confirm that responses to all four proenkephahn products are dependent on an intact vagus clearly pointing to vagal afferents and/or efferents being revolved in medmt~on of the cardiovascular responses. The an- tagomsUc effects of atropine upon bradycardia shows that direct effects of proenkephalin products on the heart do not contribute to ~ts cardmvascular acUons confirming our prewous work with Isolated atrial preparations (Douglas and Kitchen, 1988).

The use of the quaternary antagomst N-methyl levallorphan at a dose specific for blocking opioid receptors in the penphery (Douglas and IOtchen, 1990; Bianchetti et a l , 1985) suggests that the cardmvascular responses to all proenkephahn products are medmted by peripheral opiold receptors. The lack of effect of all the peptides in pRhed animals accords with work on other opio]ds (Eimerl and Feuerstein, 1986) and suggests that peripheral actions at opioid receptors are indirectly mediated vaa the CNS.

In conclusion, card]odepressant effects of ME, LE, MEAP and MEAGL show quahtatwe and quan- titative s~milant~es However, these responses are


differentially influenced by pepUdase mhlbmon and atypical responses to MEAP have been observed probably due to cleavage of the dipeptlde Arg-Phe All four proenkephahn products exert cardiovascular effects wa peplpheral #-opiold receptor rotes but their end blologmal response is dependent on the CNS funcUomng.

SUMMARY

The cardmvascular effects of the four major opioid peptlde of proenkephahn (ME, LE, MEAP and MEAGL) were studied m urethane-anaestheUsed rats Blood pressure and integrated heart rate were measured using a pressure transducer connected to a carotid artery cannula In addition arterial blood gases were momtored m samples taken at repeated intervals after pharmacological mampulatmns

Intravenous administration of all proenkephahn products produced a dose-related decrease in mean arterial pressure and heart rate of a similar magm- tude. The response was abolished by naloxone demonstrating that it was opioid receptor mediated and was qualitatively mmlar to that produced by the g-oplold receptor agomst DAGO The involvement of g-receptors in the response was further supported by the lack of effect of the 6-receptor selective agonist DPDPE and the x-receptor selectwe agomst U50488H

An "atypical response" to MEAP was exhibited m 25% of preparations studied This consisted of a transmnt mmal bradycardla and decrease a mean arterial pressure followed by a marked tachycardm and mcrease in mean arterial pressure The tachycardm and pressor response was mimicked by Arg-Phe and antagomsed by propranolol and phentolamme respectively but unaffected by naloxone Further, both components of the response were abohshed by hexamethonmm which suggests that the effect is due to Arg-Phe stimulation of sympathetic gangha The "atypical response" was seen in litter mates pointing to a genetically controlled differential capability to enzymatically degrade the heptapepude and generate the pharmacologically active dipeptide.

The cardiovascular responses to proenkephahn products were potentiated following pretreatment with the anglotensln converting enzyme inhibitor, captopnl or the anunopeptldase inhibitor, bestatm The degree of potentiation was dlslmilar for the proenkephalin products, with ME and LE potency most enhanced by bestatm and the duration of the response to MEAP and MEAGL most enhanced by captopril.

Pharmacologacal manipulations such as the use of atropine, and the quaternary opiold antagonist N- methyl levallorphan and surgical procedures includ- ing bilateral vagotomy and pithing were employed to examine the mechanisms revolved in the cardiovascular responses to each proenkephahn product. It was demonstrated that the response to all proenkephalln peptides was vagally dependent, mediated by peripheral opioid receptors but dependent on functioning of the CNS. The cardiovascular response to proenkephalin products arises from peripheral stimulation of opioid receptors and probable transmission via vagal afferents to the brain, resulting in the

production of centrally mediated vasodepressmn and atropme-sensmve bradycardm

Acknowledgements--H D is supported by an SERC stu- dentshtp We are grateful to Dupont, Sanofi and Upjohn for the gifts of drugs

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Mechanisms involved in the cardiovascular responses to opioid products of proenkephalin in the anaesthetised rat