Regulatory Peptides, 31 (1990) 1-10

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Elsevier REGPEP 00960

Pressor responses in rats following intravenous dynorphin A(1_13 ) administration are blocked by AVP-V1 receptor antagonism Jim Thomhill 1, Linda Gregor ~ and Quentin Pittman 2 IDepartment of Physiology, University of Saskatchewan, Saskatoon and 2Neuroscience Research Group, University of Calgary, Calgary (Canada)

(Received 3 April 1990; revisedversion received 8 June 1990; accepted 11 June 1990) K e y words: Pressor response; Dynorphin A; Receptor antogonism; Arginine

vasopressin; Peptide, opioid; (Rat)

Summary Hemodynamic (blood pressure and heart rate) experiments were conducted in conscious and/or anesthetized male Sprague-Dawley (S.D.), heterozygous and homozygous Brattleboro rats given intravenous (iv) dynorphin A(~_13), arginine vasopressin (AVP), norepinephrine HC1, (NE) or sterile saline before and 10 rain after an iv bolus injection of a specific receptor antagonist. These receptor blockers (kappa receptor antagonist Mr2266, alpha adrenoceptor antagonist phentolamine HC1 or the AVP-V l receptor antagonist d(CH2)sTyr-(Me)AVP were given in equimolar concentrations (15 nmol/kg iv). In all conscious S.D. groups, iv injection of AVP (60 pmol/kg), NE (12.5 nmol/kg) and dynorphin AO_13) (60nmol/kg) evoked significant increases in mean arterial pressure (MAP) associated with concomitant bradycardia. The hemodynamic responses to 'both' AVP and dynorphin A(~_13) were blocked if given subsequent to AVP-V1 administration but not following phentolamine or Mr2266 pretreatment, The pressor and bradycardic responses of conscious heterozygous and homozygous Brattleboro rats after iv AVP or dynorphin again were only blocked by the AVP-V~ receptor antagonist. Anesthetized heterozygous and homozygous Brattleboro rats again showed pressor responses following iv AVP, NE or dynorphin Ao_13) but with slight or no associated bradycardia. The rise in blood pressure with AVP 'and' dynorphin Ao_~3) in these groups also was only blocked by the d(CH2)sTyr(Me)AVP antagonist. The results indicate that the pressor responses of rats given intravenous dynorphin

Correspondence: J. Thornhill, Department of Physiology, University of Saskatchewan, Saskatoon,

Saskatchewan S7N 0W0, Canada. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

A(1_13) involve the interaction of AVP-V 1 receptors and suggest a functional interaction of these two neuropeptides in the modulation of vascular tone.

Introduction

Intravenous administration of AVP to conscious or anesthetized heterozygous or homozygous diabetes insipidus rats evokes classical dose-related increases in blood pressure and reflex bradycardia due to vasoconstriction of vascular smooth muscle [1,2]. Binding studies have also indicated the presence of kappa opioid receptors on vascular smooth muscle [3,4] and dynorphin A(I_I3 ) has been shown to increase aortic tension in vitro in isolated rat descending thoracic aortae preparations [5]. Hemodynamic studies with dynorphin A have shown that intravenous dynorphin A(1_13) administration to anesthetized, artificially-ventilated rats caused hypotension and bradycardia, actions proposed to be caused by the activation of both central and cardiac kappa opioid receptors [6]. In conscious rats, we have found [7] using a wide range of doses (6.0 to 600 nmol/kg) that iv dynorphin A(1_13) caused a transient, dose-related increase in MAP and reflex bradycardia, responses partially or totally blocked by opiate (naloxone) or alpha receptor (prazosin or yohimbine) antagonists when given in #mol/kg doses. That study suggested that iv dynorphin A0_13) evoked a pressor response in rats due to an alpha receptor-mediated vasoconstrictor action. Recently it has been reported [8] that fetal lambs given iv dynorphin A(l_13) or AVP displayed pressor and bradycardic responses. Both effects by either peptide were blocked by the AVP-V~ receptor antagonist, d(CH2)sTyr-(Me)AVP inferring a functional interaction between dynorphin A and AVP-V 1 receptors within vascular smooth muscle. We wish to test that premise further in the rat animal model, where we already know (a) there exists a functional interaction of these two peptides to modulate renal responses [9] and (b) the hemodynamic responses in the rat to parenterally administered dynorphin A(l_13 ) and AVP are qualitatively similar to that seen in the fetal lamb preparation. Thus, the purpose of the present study is to use conscious and/or anesthetized volume control rats (Sprague-Dawley) or volume depleted animals (heterozygous and homozygous Brattleboro rats) to compare the hemodynamic responses following intravenous administration of AVP and dynorphin A(~_~3)of these groups and the possible receptor mechanisms which mediate the resultant blood pressure and heart rate responses.

Materials and Methods

General Male Sprague Dawley rats (approximately 300 g weight) and heterozygous and homozygous diabetic insipidus (DI) rats of the Brattleboro strain were obtained from Charles Rivers Laboratories (Montreal, Canada) and housed in a controlled environ-

mental room that was maintained at 21 °C, relative humidity of 30?o and had a 12 h on/off lighting schedule. These animals had free access to Purina Rat Chow and tap water. Both conscious and anesthetized animals were used for these hemodynamic studies. The catheters of the homozygous Brattleboro rats proved to be extremely difficult to keep patent over an extended period of time (days). In addition, these animals always had unstable control blood pressures before injections began, due to their high activity level. Thus, after obtaining data from only 2 of 12 conscious Brattleboro rats tested, we elected to use anesthetized homozygous and heterozygous Brattleboro rats for the remaining experiments (Figs. 3 and 4). For those hemodynamics studies done in conscious animals at least 7 days were allowed between surgery and the beginning of hemodynamic testing.

Surgicalprocedures Rats were anesthetized with pentobarbitol sodium (50 mg/kg ip) and femoral venous and arterial catheters (into the abdominal aorta) were inserted using PE 20 and 50 tubing, respectively. In conscious studies, catheters were externalised through the back

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Fig. 1. Mean arterial pressure (MAP) and mean heart rate (MHR) responses are shown over 5 min post iv administration for a group (n = 4) of 'conscious' male Sprague Dawley rats given sterile saline (0.40 ml volume, 0 ) , norepinephrine HCI (NE, 12.5 nmol/kg, A), arginine vasopressin (AVP, 60 pmol/kg, O ) or dynorphin Au_13) (60 nmol/kg, l l ) before (A,B) or 10 min after (C,D) intravenous administration of 15 nmol/kg of the specific AVP-V I receptor antagonist d(CH2)sTyr(Me)AVP. Symbols (*,t,#) denote significant changes (P < 0.05) in MAP or MHR following injection of NE, AVP or Dyn, respectively compared to preinjection control values.

of the neck and connected to stainless steel tubing, which was affixed to a polyester meshed headplate. These catherization procedures for hemodynamic testing of conscious rats have been used successfully and described in greater detail elsewhere [ 10]. Catheters were flushed dally with heparinized saline (100 IU/ml, Organon) to maintain patency.

Hemodynamic testing Hemodynamic (blood pressure and heart rate) responses were tested in conscious freely moving or anesthetized rats before and for 5 min after random intravenous bolus administration (total injected volume of 400 #1) of sterile saline, norepinephrine HC1 (NE), arginine vasopressin (AVP) or dynorphin m(]_]3 ) (Dyn). Each intravenous administration of sterile saline, peptide or amine was injected slowly over a period of 20 s and at least 30 min was allowed between successive intravenous drug injections when control pre-injection blood pressure and heart rate responses were again attained. Blood pressure was monitored using a Statham P23 ID pressure transducer in conjunction with a Grass 7B Polygraph. Blood pressure was calibrated using a Tycos manometer. A

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Fig. 2. Mean arterial pressure (MAP) and mean heart rate (MHR) responses are shown over 5 minutes post iv administration for a group (n = 4) of'conscious mate heterozygous Brattleboro rats' given sterile saline (0.40 ml volume, Q), norepinephrine HCI (NE, 12.5 nmol/kg, &), arginine vasopressin (AVP, 60 pmol/kg, 0 ) or dynorphin A(]_13) (60 nmol/kg, l ) before (A,B) or 10 min after (C,D) intravenous administration of 15 nmol/kg of the specific AVP-V] receptor antagonist d(CH2)sTyr(Me)AVP. Symbols (*,t, # ) denote significant changes (P < 0.05) in MAP or MHR following NE, AVP or Dyn, respectively, compared to preinjection control values.

Experimental protocol In the first set of experiments (Figs. 1 and 2) conscious Sprague Dawley or heterozygous or homozygous Brattleboro rats were given random intravenous injections of Dyn, AVP, NE (Sigma) or sterile saline (Abbott Laboratories) before and 10 min after an iv bolus injection of a specific receptor antagonist. Peptides or amine were dissolved in sterile saline. Blood pressure and heart rate responses following Dyn, NE, AVP or sterile saline were compared before and after one of the following specific receptor antagonists were given (15 nmol/kg): AVP-V t receptor antagonist, d(CHz)sTyr(Me)AVP; =] and ~2 adrenoceptor antagonist, phentolamine HC1 or the kappa opioid receptor antagonist, Mr2266. The dose of the AVP-V 1 antagonist chosen has been previously shown [ 11,12] to block the hemodynamic effects of AVP; the doses of the other antagonists were equimolar to the AVP antagonist. In a second set of experiments (Figs. 3 and 4) hemodynamic studies were monitored in heterozygous and homozygous Brattleboro rats anesthetized with sodium pentobarbitol (50 mg/kg ip). Again dynorphin A(]_13), NE, AVP or sterile saline, in the same doses as mentioned above, were given intravenously to these animals before and after an intravenous bolus injection of 15 nmol/kg of the specific AVP-V~ antagonist, d(CHz)sTyr(Me)AVP.

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Pressor responses in rats following intravenous dynorphin A(1-13) administration are blocked by AVP-V1 receptor antagonism.

Hemodynamic (blood pressure and heart rate) experiments were conducted in conscious and/or anesthetized male Sprague-Dawley (S.D.), heterozygous and h...
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