Intrathecal St-587: Effects on Nociceptive Reflexes ...

0022.3565/92/2632-0428$03.OO/O THE JOURNAL 0? PHARMACOLOGY Copyright C 1992 by The American

EXPERIMENTAL

AND

Society

Vol. 283, No.2 Printed in U.S.A.

THERAPEUTICS

for Pharmacology

and Experimental

Therapeutics

Intrathecal St-587: Effects on Nociceptive Pressure in the Rat1’2 W. LOOMIS

CHRISTOPHER School

of Pharmacy,

Accepted

Memorial

KANTHA

and

UnWersIty

Reflexes

and Blood

D. ARUNACHALAM

of Newfoundland,

St. John’s,

Newfoundland,

Canada

A1B 3V6

June 5, 1992

for puation

The purpose of this study

was to determine the effect of i.t. St injection) on tail-flIck (iT) latency and paw pressure (PP) withdrawal threshold In conscious rats, and the effect of i.t. (mldthoracic injection) and i.v. St 587 on blood pressure in urethane-anesthetized rats. lkilike i.t. methoxamine (alpha agonist), which produced antinociception, l.t. St 587 (0.5-30 tg) decreased TF latency and PP threstdd below base line. Hyperalgesia was also produced by i.t. Wy 271 27 (alpharselective antagonist). At l.t. doses of St 587 > 3 g, there was an apparent but incomplete return of TF latency and PP threshold toward base line. Pretrestment with I.t. prazosin (2.5 g) enhanced the hyperalgesic effect of 30 gig, but not 1 9 of St 587. Intrathecal St 587 and Wy 27127 each antagonized the antinociceptive effect of i.t. guanfacine (alpha2 agonist) in the TF and PP tests. Intravenous St 587 produced a dose-dependent pressor effect 587

(lumbar

The processing and transmission of nociceptive information in the spinal cord is subject to modulation by many different neuronal systems. The spinal cord is also an important site for integration of sympathetic nerve activity and cardiovascular regulation. Among the vast array of neuronal systems which contribute to the spinal modulation of sensory and cardiovascular activity (e.g., monoamines, acetyicholine, amino acids and various peptides), bulbospinal serotonergic and NA neurons are the most extensively characterized (see reviews by MCCall, 1988; Proudfit, 1988). Developments in our understanding of the pharmacology of NA modulation have been facilitated by agents which selectively affect NA neurotransmission, including agonists and antagonists for each subtype of the aipha and beta adrenoceptor. Among the a4ha1-selective agonists, phenylephrine and methoxamine have been most frequently used. More recently, St 587 has been reported to be a highly Received for publication 1 This work was supported 2 Portions of these data Federation the 34th

of Biological meeting ofthe

Ontario,

Canada

(1991).

September

10,

by the Medical

were

Societies Canadian

presented

in Halifax, Federation

1991. Research

at the

Nova

Council

33rd meeting Scotia,

ofBiological

of Canada. of the Canadian

Canada

Societies

and at Kingston,

(1990), in

by pretreatment wfth i.v. prazonin (0.14 in the EDSO), but weakly antagonized by 1 .5-fold increase in the EDro). ST587 also produced a dose-dependent pressor response after i.t. injection which was antagonized by i.t. prazosin (10 pig) or i.v. hexamethonlum (1 0 mg/kg). The resufts Indicate that: St 587 is a highly selective agorst for alpha adrenoceptors with no agonist activity at perIpheral or spinal aipha2 adrenoceptors; i.t. St 587 Is an effective antagonist of spinal alpha2 adrenoceptors; that was antagonized mg/kg; 52-fold increase Wy 27127 (0.5 mg/kg;

the ratio of alpha, agonist/alpha2 antagonist

activity

increases

with the I.t. dose; and St 587 has a spinal locus of action after i.t. injection in the rat. The blockade of eipha2 adrenoceptors, in addition to agonist activity at aipha1 adrenoceptors, must be considered in the interpretation of experimental results with i.t. St 587.

selective Kobinger

a4pha

adrenoceptor agonist (De Jonge et at., 1981; Pichier, 1982). Radioligand binding studies indicate that St 587 has a very high affinity for sites labeled with agomsts or antagonists with known selectivity for the a4phai adrenoceptor (De Jonge et aL, 1983; Timmermans et aL, 1984; Megens et aL, 1986). Consistent with these binding data are and

pharmacological studies which have examined the cardiovascular, behavioral and motor effects of ST-857 after systemic administration to rats, cats and rabbits (Timmermans et aL, 1984; Hayes et aL, 1986; Menon et at., 1986; Stumpfand Pichler, 1988). The collective results of these in vivo experiments in& cate that St 587 is a selective agonist at atpha adrenoceptors in both the periphery and the central nervous system. St 587 is also unique among the alpha1-selective agonists in being very lipid soluble (De Jonge et aL, 1981). On the basis of these characteristics, systemically administered St 587 has been used to investigate the functional role of central aphai adrenoceptors in laboratory animals. The potency and selectivity of St 587 as an aipha agonist suggest that it could be useful in pharmacological studies of spinal NA function after i.t. administration. Indeed, St 587 was

AVP, arginine vasopressin; bpm, beats per minute; Cl, confidence interval; DM80, dimethyl sulfoxide; DRC, dose-response curve; IML, intermedlolateral cell column; MAP, mean arterial pressure; NA, noradrenergic; PP. paw pressure; SPN, sympathetic preganglionic neuron; TF, tall-flick; St 587, 22loro-5-thfluoromethyl-phenyl-iminomidazoline; BP, blood pressure; HR, heart rate. ABBREVIATIONS:

428

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ABSTRACT

1992

Intrathscal

Methods Animals and surgery. All procedures in this study were approved by the Animal Care Committee of Memorial University of Newfoundland in accordance with the Guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (350-450 g at the time of experimentation) were obtained from Charles River Inc. (St. Constant, Canada). Rats were housed in group cages for at least 72 h before surgery; room temperature was maintained at 25#{176}C with a 12-h light/ dark cycle (lights on at 7:00 A.M.). Purina rodent laboratory chow and tap water were provided ad libitum. Intrathecal catheters were constructed from stretched PE-lO tubing and implanted under halothane anesthesia (Loomis et aL, 1987). The i.t. catheter was inserted through a slit in the cisternal membrane to extend to either the rostral end of the lumbar enlargement or the midthoracic level of the spinal cord. The exposed tip of the catheter was externalized on the top of the head and sealed with a stainless steel plug. Animals with lumbar i.t. catheters were housed individually, allowed to recover for a minimum of4 days and subsequently used in behavioral experiments. Animals with thoracic i.t. catheters were used immediately after surgery in the cardiovascular experiments. The location of the spinal tip of the catheter was visually determined in animals randomly selected at the end of the experiment. Behavioral experiments. After surgery and during recovery, rats with i.t. catheters were observed for evidence of impaired gait and hindlimb weakness. Only those animals exhibiting normal motor function and having normal base-line responses in the TF (D’Amour and Smith, 1941) and PP withdrawal (Randall and Selitto, 1957) tests were

In the TF test, a cutoff of 10 sec was used to avoid tissue damage, and the intensity of the lamp was adjusted to yield appropriate baseline latencies (see below). In the PP test, mechanical pressure was applied to the dorsal surface of the hind paw until a withdrawal response or withdrawal attempt was observed (cut-off of 750 g UgoBasile apparatus). Rats were acclimatized to the IT and PP apparatus before the experiment and each rat was used in a maximum of two experiments (minimum of 96 h between experiments). The mean of three consecutive TF and PP determinations was used at each time point. Base-line responses were determined on the day before the experiment and immediately before i.t. injection, and behavioral testing was carried out between 9:00 A.M. and 1:30 P.M. Cardiovascular experiments. Under urethane anesthesia (1.2 g/ kg, i.p.), cannulas were surgically placed in the carotid artery and the external jugular vein for the recording of BP and i.v. drug injection, respectively. Body temperature was maintained at 37#{176}C throughout the experiment using a thermostatically controlled heating blanket (Harvani Instruments, Montreal, Canada). BP and HR were continuously monitored using a Statham pressure transducer coupled to a Gould 8000S Recorder which was equipped with an ECG/Biotach amplifier. Drugs were injected only after BP and HR had stabilized (usually 30 mm after surgery). For i.v. injection, drugs were administered in a used.

maximum

volume

of 0.20

ml/kg

and

flushed

with

saline.

For

i.t.

injection, drugs were administered in a volume of 5.0 Ml followed by a saline flush. The i.t. solutions were gradually infused over 30 sec using a hand-held 10-jzl syringe to minimize pressure changes in the subarachnoid space. To determine the selectivity of St 587 as an agonist at apha1 and a4,ha2 adrenoceptors, animals were pretreated with the selective antagonists, prazosin (ajplw1) and Wy 27127 (alpha,). Wy 27127 is a disulphonamido-benzoquinolizine compound which is equipotent with idazoxan in blocking aphaz adrenoceptors in isOlated smooth muscle preparations (Bill et at., 1986). However, its aipha:aipha1 selectivity is considerably greater than idazoxan. The i.v. dose of Wy 27127 (0.5 mgI kg) used in the present investigation was estimated from doses of idazoxan in conscious and anesthetized rats (Harland and Brown, 1988). Drugs. The following drugs were generously provided by their manufacturers: St 587 nitrate (Boehringer-Ingelheim, Canada Ltd., Burlington, Ont., Canada); Wyeth 27127 (Wyeth Ltd., Philadelphia, PA); guanfacine HC1 (Sandoz Canada Inc., Dorval, P.Q., Canada); prazosin HC1 (Pfizer Canada Inc., Kirkland, P.Q., Canada); and methoxamine HC1 (Burroughs Welcome Ltd., Kirkland, P.Q., Canada) AVP and urethane were purchased from Sigma Chemical Co. (St. Louis, MO). All drugs were dissolved in sterile saline except prazosin, which was dissolved in DMSO. Drug doses are expressed as the salt. Data analysis. All data are expressed as the mean ± S.E.M. The results of the BP experiments are presented as the maximum change in MAP [(systolic pressure + diastolic pressure)/2J as compared to base line, which was determined immediately before each drug injection. Behavioral data are expressed as percent of base-line response, absolute TF latency in seconds, or percent inhibition (P1), which was calculated using the formula: P1 = 100 X (1 - agonist effect in presence of antagonist/agonist effect in vehicle control). ED values and 95% confidence intervals for the dose-response curves were calculated using the method of Tallarida and Murray (1986). Statistical analysis among multiple groups was determined using one-way analysis of variance followed by Newman-Keuls test for a significant F ratio. Student’s t test was used for comparison between two groups.

Results of i.v. St 587 on MAP in anesthetized rats. To the selectivity of St 587 as an agonist at peripheral a4,hai adrenoceptors, we determined the effect of St 587 on systolic and diastolic BP in urethane-anesthetized rats. Baseline MAP and HR, determined before i.v. drug injection for all rats, were 81 ± 1.9 mm Hg and 335 ± 9.8 bpm, respectively. All Effect

confirm


recently used to characterize the alpha adrenoceptors modulating BP in the spinal cord of anesthetized rats (Kubo et aL, 1987). However, the utility ofSt 587 as a pharmacological probe of alpha activity in the spinal cord may be complicated by a report that St 587 also blocks peripheral and central alpha adrenoceptors (Pichier and Kobinger, 1985a). In the spinal cord, posthynaptic a4ha adrenoceptors mediate the inhibitory effects ofbulbospinal NA neurons on nociceptive sensory transmission and sympathetic preganglionic discharge (Fleetwood-Walker et aL, 1985; Guyenet and Cabot, 1981). To the extent that these NA fibers are tonically active, the blockade of spinal apha adrenoceptors by i.t. St 587 could yield biological responses qualitatively similar to those evoked by St 587-induced activation of ajpha adrenoceptors. Thus, results with it St 587 could be subject to misinterpretation because of these concurrent pharmacological effects. Moreover, if the activity ratio ofSt 587 (e.g., alpha agonism: alpha antagonism) were to vary with the dose, the problem of data interpretation would be compounded. Such a difference in activity has been suggested with i.v. St 587 (Pichier and Kobinger, 1985a). It is also unknown if St 587 remains localized in the subarachnoid space after it injection. Given its high lipid solubility, i.t. St 587 may undergo vascular redistribution to peripheral and supraspinal sites of action. This is an important consideration when drugs are used experimentally for the purpose of localized spinal effects. To our knowledge, there have been no systematic studies of St 587 after administration into the spinal subarachnoid space of conscious or anesthetized animals. Indeed, there are few reports at all of its use in the spinal corcL The purpose of the present study was to investigate the effects of i.t. St 587 on behavioral responses to thermal and mechanical nociceptive stimuli in conscious rats, and on BP in anesthetized rats. The pressor effect of i.v. St 587 was also determined to verify the selectivity of St 587 as a alpha adrenoceptor agonist.

429

St 587 In the Rat

430 doses

Loomis

and Arunachalam

of i.v. St 587 produced

diastolic

BP,

which

was

Vol. 263

an immediate accompanied

rise in systolic by

reflex

and

bradycardia.

MEAN ARTEMAL 100

ii 1

ise

10

Dose

i

$1457

1. Log DRC illustrating the change in MAP induced by St 587 (SI) in urethane-anesthetized, normotensive rats. Doses of St 587 were injected iv. after saline (no antagonist), or 1 5 mm after pretreatment with the alpha2 antagonist Wyeth 27127 (WV; 500 ,g/kg, i.v.), the alpha1 antagonist prazosin (PZ; 140 ,zg/kg, i.v.) or the combination of prazosin and Wyeth (PZ; 140 ag/kg + WY; 500 ag/kg, i.v.). Data are presented as the mean ± S.E.M. of 6 to 26 rats. Fig.

TABLE

10

15

0 10

ED,,

95% Cl pg/kg

St 587 Wy + St 587 Pz + St 587 Pz+Wy+St587

/2

Ii

St 587. Treatment

15.0 22.9 777

13.5-1 6.6 19.2-27.3 553-1090

985

800-1212

URE

a. 20

1

ED values and 95% CI for i.v. St-587 alone and after alpha adrenoceptor blockade on the presser response in urethaneanesthetized rats Rats were pretreated with the alpha adrenoceptor antagonist by iv. InjeCtiOn mm before

MEAN ARTERL 40

20

N*hsc

80

i#{243}o

2

81457

a) in MAP induced by it. St 587 (SI) alone (#{149}), or I 5 mm after pretreatment with it. prazosmn (10 pg; A) or i.t. DMSO (I) in urethane-anesthetized, normotensive rats. Data are presented as the mean ± S.D. of 5 to I I rats. Intrathecal St 587 (1 pg) had no significant effect on MAP. Fig. 2. Change


The i.v. injection of saline had no effect on cardiovascular activity. As shown in figure 1, the i.v. injection of St 587 to naive and antagonist-pretreated rats produced a dose-dependent increase in MAP. Pretreatment with the selective apha antagonist, Wy 27127 (0.5 mg/kg i.v.), shifted the St 587 DRC to the right with a coresponding 1.5-fold increase in the EDo (table 1). In contrast, pretreatment with the aipha adrenoceptor antagonist prazosin (0.14 mg/kg, i.v.) yielded a 52-fold increase in the ED of St 587 (table 1). The marked, parallel shift to the right of the St 587 DRC by prazosin (fig. 1) was not significantly enhanced by the addition of Wy 27127 (0.5 mg/kg, i.v.) (fig. 1). The ED of St 587 after pretreatment with Wy 27127 + prazosin was not significantly different from prazosin alone, as indicated by the overlapping CI in table 1. With doses of St 587 > 6 tgg, a secondary increase in BP and HR was observed beginning about 1 mm after i.v. injection (data not shown). The time course of this effect was similar to the time course of cardiovascular changes induced by i.t. St 587. The delayed increase in BP and HR produced by i.v. St 587 was probably a central effect (spinal and/or supraspinal) after its distribution from the systemic circulation into the central nervous system. Cardiovascular effects of i.t. St 587. To investigate the spinal action of St 587 on BP and HR, the drug was injected into the midthoracic subarachnoid space of urethane-anesthetized rats. Base-line MAP and HR, determined before i.t. drug injection for all rats, were 83 ± 1.9 mm Hg and 339 ± 7.6 bpm,

respectively. There was a slow and sustained rise in BP and HR beginning 1 to 2 mm after i.t. injection of St 587, as compared to the stable BP and HR recordings after i.t. saline. Occasionally, a transient hypotensive effect ( 3 gig, there was an apparent but incomplete return of TF latency and PP threshold toward base line, suggesting both a reversal of hyperalgesia and a difference between low and high doses of i.t. St 587. It should be noted that hyperalgesia was also observed after the i.t. injection of the selective alpha antagonist, Wy 27127 (figs. 4 and 5). In contrast, i.t. methoxamine (10 tg) significantly increased TF latency (fig. 4) with no evidence of cutaneous hyperreflexia or spontaneous tail movements, motor effects that are observed with higher doses of i.t. methoxamine. These data indicate that unlike methoxamine, the predominant effect of i.t. St 587 in behavioral tests of nociception is hyperalgesia due to blockade of spinal apha adrenoceptors.

Effect and

of prazosin pretreatment on i.t. St-587 in the PP tests. To determine if the difference between low I.

TAIL FLICK 150

*

s.

7,

#5S

lii

S

I

z

1#{149}

LI. 81457

1

duced to adjust the mean baseline response to 4.9 ± 0.3 sec in these experiments. Data are ex-

#{149}1

!;100

pressed as the mean ± S.E.M. of 6 to 1 1 animals; ‘indtes a significant difference from both DM80 and prazosin alone at P < .001 . The hyperalgesic effect of 1 ,g of St 587 alone is significantly greater than that of 30 g alone. Insert: DRC illustrating the hyperalgesic effect of it. St 587 in the IF test; mean base-line response = 2.63 ± 0.19 sec (mean ± S.E.M.; n = 54). ‘mdi-

* * *

-

MXDMSO

PZ

a

Fig. 4. The effect of it. methoxamine (Mx; 10 ,g), DMSO (vehicle), prazosin(PZ; 2.5 ,g), Wyeth 27127 (Wy; 1 g) or St-587 (ST; 1 .0 or 30 ,g) alone, and prazosin followed by St-587 (ST + PZ) on IF latency. The maximum change from base line (expressed as percent of control) was determined from time course experiments. Lamp intensity of the TF apparatus was re-

WY

T

STPZ+ST1 Ijig

I ST

PZ+5T1 30 jig

cates

a

significant

from base line

at

difference

P