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Differential sensitivity to cocaine in spontaneously hypertensive and wistar-Kyoto rats
Life Sciences, Vol. 45, pp. 223-232 Printed in the U.S.A.
Pergamon Press
DIFFERENTIAL SENSITIVITY TO COCAINE IN SPONTANEOUSLY HYPERTENSIVE AND WISTARKYOTO RATS Y. Ishizuka, R.W. Rockhold, K. Kirchner*, B. Hoskins and I.K. Ho Department of Pharmacology and Toxicology and * Department of Medicine University of Mississippi Medical Center 2500 N. State St. Jackson, MS 39216 (Received in final form May 12, 1989) Summary
Physiological, pharmacological and toxicological responses t o two regimens of cocaine administration were compared between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. An initial experiment examined renal excretory and hemodynamic function in response to an acute volume load in anesthetized SHR and WKY following subaeute cocaine treatment (20 mg/kg, s.c., twice a day for 9 days). Anticipated renal responses to volume loading were obtained but the responses of cocaine-treated SHR and WKY did not differ from vehicle-treated rats. A second group of experiments compared responses to continuous i.v. infusions of cocaine (1.25 mg/kg.min). In freely moving animals, no differences were noted between SHR and WKY in the increases in mean blood pressure (MBP) and heart rate 0 t R ) produced during cocaine infusion. The elapsed time-to-onset of convulsions (To) elicited by cocaine was similar in both strains. However, when rats were subjected to restraint during the infusion period, pressor and tachycardic responses were observed to be significantly less in WKY than in SHR or in freely moving rats of either strain. Restraint also differentially affected rectal temperature (RT) responses to cocaine. Hypothermie responses to cocaine were observed in all WKY. Both hypothermic and hyperthermic responses were observed in SHR. A significant correlation was demonstrated between the Tc and the maximal change in RT produced during cocaine infusion. Division of SHR into two arbitrary groups was made, based on the direction of cocaine-induced change in RT. A significant (p<0.01) shortening of the Tc was obvious in SHR (8 of 15) in whom cocaine produced a hyperthermia. These animals were designated SHR H. The mean value for Tc in those SHR which demonstrated a lowering in RT (SHRL; 7 o f 15) in response to cocaine was similar to that for WKY. Moreover, the SHR H evidenced significantly greater increases in HR, but not MBP, to cocaine infusion thandid SHR L. The results indicate that restraint stress causes expression of a significant heterogeneity in the RT response of SHR to cocaine. The magnitude and direction of the RT responses are negatively correlated with sensitivity to the convulsive effects of cocaine in SHR. Stress may modify toxic responses to cocaine by interactions with body temperature homeostasis.
Cocaine is a tropane alkaloid whose toxic effects have been noted following both medicinal and recreational use. Convulsions, hyperthermia and respiratory depression are of concern in acute overdosage. The circulatory toxicity of cocaine can be pronounced. Tachycardia, pressor responses, acceleration of coronary artery disease, dysrhythmias and myocardial infarction have been frequently reported (1-4), indicating that effects of cocaine on the cardiovascular system are of major concern. The etiology of cocaine-induced cardiovascular toxicity remains uncertain. Until recently, it was suspected that adverse circulatory and cardiac responses occurred secondary to cocaine-mediated stimulation of sympathetic nervous system neurotransmission (5, 6). Thus, cocaine has been shown to potentiate and prolong the cardiac responses to epinephrine infusion in dogs (7, 8). Intravenous administration of cocaine to human subjects has been 0024-3205/89 $3.00 + .00 Copyright (e) 1989 Pergamon Press plc
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repeatedly demonstrated to produce a dose-related increase in mean blood pressure and heart rate (9, 10). However, Pitts et al. (11) compared the cardiovascular effects of cocaine under different anesthetic conditions in the rat and reported that some effects of cocaine might be elicited by mechanisms other than potentiation of sympathetic nervous responses. Moreover, Trulson et al. (12) reported that cardiac function was severely impaired following chronic cocaine administration. The spontaneously hypertensive rat (SHR) is a model of genetic hypertension useful in determining genetic differences in susceptibility to the toxic actions of cocaine. For example, Watanabe et al. (13) demonstrated that SHR were less sensitive to the hepatotoxic actions of cocaine than were age-matched normotensive Wistar-Kyoto (WKY) rats, a difference that was suggested to be pharmacodispositional in origin. Additionally, we have reported that differences exist between SHR and WKY in terms of resistance to the convulsive and lethal effects of acute cocaine administration (14). The present study was initiated to further define the relative sensitivity of SHR and WKY to cocaine. Circulatory, renal, convulsive and body temperature responses to varying regimens of cocaine treatment were compared in the two strains. Methods General Procedures: Male SHR and WKY, 11-12 weeks of age, were purchased from Taconic Farms, Inc.. Rats were housed, prior to experimental use, in plastic cages (3-4/cage) under controlled conditions of temperature (22° C), humidity (50-55%) and lighting (12 hr light: 12 hr dark) and given rat chow (Purina Laboratory Chow) and tap water, ad libitum. Surgical procedures were performed under halothane anesthesia (2-4% in medical grade oxygen). Polyethylene catheters were inserted 4 cm into the abdominal aorta (PE-50) and vena cava (PE-10) by way of left femoral vessels. These catheters were filled with heparinized saline (10 U/ml), exteriorized at the nape of the neck and sealed until use. Animals were housed subsequently in individual hanging wire cages. Experimental protocols were initiated two to four days following catheter implantation. Renal Studies: Two groups (n=6) each of SHR and WKY were placed in individual wire metabolic cages and allowed free access to food and water during an acclimation period of 48 hr. Rats then received twice daily (08:30 and 16:30) s.c. injections of either cocaine hydrochloride (20 mg/kg as base) or 0.15 M NaC1 vehicle for a period of 9 days. Rats from cocaine and vehicle injected groups of each strain were pair-fed to prevent large differences in weight gain. Ten days following the initiation of the injection sequence, animals were anesthetized with Inactin (100 mg/kg body weight, i.p.; Promonto GMBH, Hamburg, F.R.G.) and placed on a heated animal table. Rectal temperatures, monitored by telethermometer (YSI Corp., Yellow Springs, OH), were maintained between 36.5 and 38° C. A tracheotomy was performed and polyethylene catheters (PE-50) introduced into the right jugular vein for polyfructosan (Inutest, Laevosan GaseUschaft, Linz, Austria) and paraaminohippuric acid (PAIl; Sigma Chemical Co., St. Louis, MO) infusions and into the left jugular vein for volume expansion. A PE-50 catheter was introduced into the right femoral artery for blood sampling and continuous blood pressure monitoring by means of a P23 DC transducer (Statham Laboratories, Hato Rey, PR) and a Grass Model 7D polygraph. A flanged PE-50 catheter was placed in the bladder through a midline suprapubic incision. From the start of surgery, 5% polyfructosan and 1% PAH in Ringer's solution (in mEq/l: Na 140, CI 115, HCO3 30 and K 5) was given by constant i.v. infusion at 3 ml/hr for 20 min and thereafter at 1.5 ml/hr. Following a 30 min post-surgical stabilization period, a 45 min urine collection period was initiated to determine baseline renal function and electrolyte excretion rates. Urine was collected under oil for determination of inulin, sodium, potassium, chloride and osmolar concentrations. Upon completion of this hydropenic period, animals underwent acute extracellular volume expansion by administration of Ringer's solution in a volume equivalent to 10% body weight given over 60 rain. The infusion rate was reduced to a rate equal to urine output at the end of the volume expansion period and this rate maintained for the duration of the experiment. A second 45 min experimental period was then initiated and the measurements and collections described for the hydropenic period were repeated. Rats were sacrificed by exsanguination at the end of the experiment and blood collected for determination of serum electrolytes. Kidneys were removed, blotted to remove excess blood and weighed. Sodium and potassium concentrations in serum and urine were measured with a Model 143 Flame Photometer (Instrumentation Laboratory, Lexington, MA). Chloride concentration in serum and urine was analyzed amperometrically (Buchler Instruments, Fort Lee, NJ). Inulin concentration in urine and plasma was determined by the diphenylamine method of Walser et al. (15). Serum and urine osmolalities were determined with a freezing point osmometer (Advanced Instruments, Needham Heights, MA). The PAH concentration in plasma and urine was determined by the method of Waugh and Beall (16). Determination of urinary flow rate and the concentration of inulin, sodium and chloride in the blood and urine
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Effects
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p e r m i t t e d calculation of w h o l e kidney g l o m e r u l a r filtration r a t e ( G F R ) a n d u r i n a r y excretion of s o d i u m and chloride a c c o r d i n g to s t a n d a r d expressions. Effective renal b l o o d flow ( E R B F ) was calculated as: E R B F = [ ( U p A H . V ) / P P A H ] / ( 1 - H c t ) w h e r e U p A H and P P A H a r e the concentrations of P A I l in urine and s e r u m , respectively. T h e u r i n e flow r a t e in # l / m i n is V a n d H c t is the h e m a t o c r i t d e t e r m i n e d at the e n d of the collection period. Inulin clearance, P A H and absolute u r i n a r y electrolyte excretion r a t e s w e r e all expressed p e r g r a m kidney weight. TABLE I Effect of Subacute Cocaine (20 mg/kg, b.i.d., s.c., for 9 days) or Vehicle (0.9% NaCI) on the Response of SHR and WKY to 10% Volume Expansion with Ringer's Bicarbonate Solution. MBP
Flow
C1N
ERBF
UNa.V
FeNa
UK.V
FeK
UcI.V
FeCI
Uosm
122 +2
5.1 +1.8
1028 +64
5.2 +0.3
146 +112
0.09 +0.02
953 +119
22.1 +2.6
128 +36
0.10 +0.03
1442 +131
Volume 120 Expansion +__2
28.2" +9.6
1031 +101
6.5* +0.6
4009* +1060
2.47" +0.58
1615" +274
40.6* +3.7
4430* +1002
3.77* +0.84
782* +172
124 +_.3
4.1 +0.9
959 +60
5.1 +0.3
114 +71
0.08 +0.04
730 +108
21.0 +112
167 +112
0.15 +0.09
1554 +123
Volume 118 Expansion +4
16.6" +4.4
986 +60
6.2' +0.2
2711" +1019
1.71" +0.57
1310' +193
33.0 +4.5
3327" +1293
2.87" +0.97
1~* +239
174
3.4
876
4.6
345
0.25
807
24.8
164
0.18
1434
+6
+0.3
+59
+0.3
+132
+0.08
+121
+3.1
+72
+0.09
+157
38.1" +13.6
1043 +83
6.3* +0.9
8376* +2421
5.59* +1.88
1739" _.+150
45.1" +6.7
8492* +2243
7.24* +2.40
814' +104
165 +7
4.4 +0.7
1079 +99
5.7 +0.6
423 +90
0.28 +0.06
1002 +94
25.9 +6.0
27.8 +74
0.23 +0.05
1506 +226
Volume 159 Expansion +4
34.7" +6.5
1268 +179
7.5" +0.5
7763* +1408
4.64* +1.30
1899" +__131
41.6" +5.6
8104" +1410
6.48" +1.87
749" +62
WKY SALINE (n=6) Control
COCAINE (n=6) Control
SHR
SALINE (n = 6) Control
Volume 165 Expansion +7 COCAINE (n =5) Control
Number of animals in each group is indicated in parentheses. Mean values + 1 S.E.M. are presented. Column title abbreviations and units are MBP = mean arterial blood pressure (ram Hg), Flow = urine flow (~l/min/g), CIN = urinary inulin clearance (~l/min/g), ERBF = effective renal blood flow (ml/min/g), UNa.V = urinary sodium excretion (mEq/min/g), FeNa = fractional sodium excretion (%), UK.V = urinary potassium excretion (mEq/min/g), FeK = fractional potassium excretion (%), U c r V = urinary chloride excretion (mEq/min/g), FeCI = fractional chloride excretion (%), Uosm = urinary osmolality (mOsm/kg H20 ). All values are expressed per gram of wet kidney weight at the time of sacrifice, unless indicated otherwise. Asterisks denote statistically significant (p<0.05) differences between control and volume expanded groups.
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Experiments in freely moving animals: Rats were housed in wooden recording chambers (30.5 x 29.5 x 44.5 cm) at least 30 min prior to drug administration. Blood pressure was monitored by means of a Cobe R transducer connected to a Grass model 7D polygraph. Mean arterial blood pressure (MBP) was obtained by electronic damping of the pulsatile blood pressure signal. Heart rate (HR) was determined electronically from the pulse interval and displayed on the polyg~.aph. Cocaine hydrochloride (1.25 mg/kg.min as base, Sigma Chemical Co., St. Louis, MO) was infused continuously i.v. using a Sage Model 353 pump. Continuous measurements of MBP and HR as well as the time from the start of infusion until the onset of convulsions (Tc) were recorded. Experiments in restrained animals: Animals were placed in plastic restraining cages and a lubricated temperature probe inserted 4 cm into the rectum and held in position by adhesive tape. Drug infusion was commenced following an acclimation period of at least 30 min. Rectal temperature (RT) was recorded every 5 min by a Model BAT-12 (Sensortek Inc.) electronic thermometer while MBP and HR were recorded continuously. Ambient temperatures during the infusion experiments were 22-24 ° C. Determinations of LDs0: The LD50 values for cocaine were determined in both freely moving and restrained SHR and WKY according to the Up and Down method of Dixon (17). Cocaine was administered by bolus i.v. injection through indwelling femoral catheters.
Statistical analysis: Analysis of variance for repeated measures (one- or two-way for appropriate groups) was performed on experimentally derived values. Neuman-Keuls or multiple "t"-tests were performed to differentiate group means if significant interactions were found by analysis of variance. Results Renal response to acute volume expansion: Resting values for MBP averaged 174 +_ 6 and 165 + 7 mm Hg in anesthetized saline-treated and cocaine-treated SHR, respectively and 122 + 2 and 124 + 3 mm Hg in salinetreated and cocaine-treated WKY, respectively. Acute volume expansion with Ringer's bicarbonate solution (10 % of body weight) did not alter MBP in any of the 4 groups. The anticipated effects of volume expansion were observed. Significant increases in urine flow, effective renal plasma flow and urinary excretion of sodium, potassium and chloride were noted (Table I). Urine osmolality decreased upon volume expansion. Urinary excretion of sodium, but not of potassium or chloride, was significantly greater in SHR when compared to WKY, regardless of the treatment regimen. Neither resting values nor the maximum changes evoked by volume expansion were altered by subacute cocaine treatment in either SHR or WKY (Table I). Cardiovascular responses to cocaine: Resting values of MBP averaged 162 + 4 mm Hg and 160 + 4 mm Hg in freely moving (n=12) and restrained (n=12) SHR, respectively and 113 + 4 and 116 + 3 mm Hg in freely moving (n=12) and restrained (n=10) WKY, respectively. Resting values for HR were significantly (p<0.01) higher in restrained SHR (413 + 7 beats/rain) than in freely moving SHR (366 + 8 beats/min) or WKY in either condition (352 + 7 and 375 + 8 beats/min; freely moving and restrained).
TABLE II Maximum Initial Changes in MBP and HR following Cocaine Infusion in SHR and WKY. Conditions
MBP (mm Hg)
HR (beats/min)
WKY
freely moving restrained
(12) (10)
33 + 3 23 +2**
22 + 3 13 + 4*
SHR
freely moving restrained
(12) (12)
37 + 3 33 + 2 a
27 + 2 24 + 1a
Asterisks denote significant differences (*: p<0.05, **: p<0.01) from freely moving WKY. a: p<0.05 compared to restrained WKY. Numbers in parentheses refer to the number on animals in each group. The i.v. infusion of cocaine produced initial increases in MBP and HR in all animals (Fig. 1). The maximum increases in MBP and HR produced by cocaine were significantly less in restrained WKY than in
Vol. 45, No. 3, 1989
Effects of Cocaine on SHR
227
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FIG. 1 Percent changes in mean blood pressure (a) and heart rate (b) from the start of cocaine infusion (1.25 mg/kg.min.) until the onset of convulsions in SHR and WKY. The number of animals included in each group was: freely moving SHR (O), 12; restrained SHR (A), 12; freely moving WKY (O), 12 and restrained WKY(&), 10. The abscissa represents the elapsed time from onset of infusion until onset of convulsions, divided into 10 equal periods. Asterisks signify differences (*:p<0.05, **:p<0.01) at the indicated time between restrained groups (see text). freely moving WKY. No differences were noted in the responses of SHR under the two conditions. However, the initial increases in MBP and HR of restrained SHR were significantly greater than those recorded in restrained WKY (Table II). Continuous cocaine infusion produced a return to baseline or a gradual decrease in HR following the initial tachycardic response while MBP showed a progressive elevation until the onset of convulsive activity. Again, the increases in MBP noted in restrained WKY were significantly less than those observed in other groups (Fig. 1). There was a greater fall in HR in WKY than in SHR. The maximum percentage change in HR (excluding the initial period of increase) was significantly greater in WKY than in SHR, respectively, under both freely moving (-0.12 + 0.03% vs -0.004 + 0.4%, p<0.05) and restrained (-0.14 + 0.05% vs -0.04 + 0.02%, p < 0.05) conditions.
LD50 of cocaine: Restraint significantly altered the LD50 for cocaine in WKY but not in SHR. The mean value-s (+ 1 S.E.M.: n=6 for all groups) for LD50 were 13.01 (+ 0.46) and 12.30 (+ 0.43) for freely moving WKY and SHR, respectively. Restraint increased the LD50 in WKY to 16.17 ( + 0.57, p<0.01) but did not significantly alter the value in SHR (10.62 + 0.37).
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TABLE III Effects of Cocaine Infusion on Rectal Temperature in Restrained Animals. Resting body temperature (°C)
Changes in body temperature (°C)
WKY
37.89 + 0.07 (11)
-1.10 + 0.10 (11)
SHR
39.13 + 0.10 (15)**
-0.94 + 0.18 (SHRL, 7) +0.48 _ 0.12 (SHRH, 8)
**: p<0.01 compared to corresponding group. Numbers in parentheses refer to the number of animals in each group. Rectal temperature was increased in 8 SHR(SHRH) and decreased in 7
SIaRCSHRt).
o
2.000 1.500 1.000
0.500 0.000
-0.500 o _1.000 r.~
r...l
o -1.500 ~ -2.000 u 0
5
10
15
20
9.5
30
35
40
45
50
TIME-T0-0NSET OF CONVULSIONS (rain.)
FIG. 2 Correlation between body temperature response and time-to-onset of convulsions following cocaine infusion (1.25 mg/kg.min) in restrained SHR. The regression coefficient was -0.805 (p < 0.001). Relationship between body temperature and responses to cocaine: Rectal temperature, when measured under restraint, was significantly higher in SHR than in WKY (Table III). Body temperature was not measured in freely moving animals. A decrease in RT was noted in all restrained WKY following cocaine infusion. In contrast, the maximal change in RT varied in both magnitude and direction in restrained SHR. When the maximal change in RT following cocaine infusion was plotted against the Tc, a significant, negative correlation became evident (Fig. 2; regression coefficent = -0.805; p<0.001). No significant correlation was evident in data from WKY. The data from SHR suggested the possibility of division of SHR into those animals in whom the maximal cocaine-induced change in RT was positive (i.e., an increase in body temperature; tentatively designated SHRH) and those in whom the maximal change was negative (a lowering of body temperature; SHRI. ). Using this division, the mean values for maximal RT changes in SHR H and SHR L are also given in Table III. The values for maximum change in RT ranged from +0.10 to + 1.10 in SHR H and -0.10 to -1.50 in SHR L. No differences were detected between SHR H and SHR L in terms of body weight (264.3 -+ 8 "6 vs 266 "3 + 7.6 g, respectively) or pre-infusion values for MBP (159 + 6 vs 161 + 4 mmHg, respectively), HR (419 + 11 vs. 403 + 7 beats/min, respectively) and RT (39.16 + 0.17 vs 39.09 + 0.09 °C, respectively). In addition,
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Effects of Cocaine on SHR
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TABLE IV Time-to-onset of Convulsions following Cocaine Infusion in SHR and WKY. Conditions freely moving
restrained
WKY
28.52 +_.0.19 (12)
31.69 + 0.20 (11)
SHR
28.89 ± 0.10 (12)
30.68 + 2.36 (SHRL, 7) 15.55 +_ 1.12 (SHRH, 8)**
**:p<0.01 compared to all other groups. animals in each group.
Numbers in parentheses refer to the number of
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FIG. 3 Percent changes in mean blood pressure (a) and heart rate (b) from the start of infusion (1.25 mg/kg.min.) until the onset of convulsions in restrained SHR. The number of animals included in each group was: body temperature increasing group ( O , SHRjrI), 7 and body temperature decreasing group ( 0 , SHRL), 5. The abscmsa represents the elapsed time from onset of infusion until onset of convulsions, divided into 10 equal periods. Asterisks signify differences (*:p<0.05, **:p<0.01) at the indicated times between groups. regression analysis was unable to show significant correlations between Tc and either initial RT, HR, MBP or body weight.
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Effects of Cocaine on SHR
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No differences were noted in MBP between SHR L and SHR H during cocaine infusion. However, HR was significantly elevated during cocaine infusion in SHI~H as compared to the response in SHR L (Fig. 3). Moreover, the Te was reduced by 50% in SHR H compared to that in SHR L (Table IV). The Te in SHR L was similar to that in freely moving SHR and WKY and in restrained WKY (Table IV).
Discussion Recent evidence suggests a genetic component for the differences in responses to cocaine between the closely related SHR and WKY (13, 14). The results of the present study more completely define differences between physiological, pharmacological and toxicological responses to cocaine in these two rat strains. It is clear that subacute treatment with cocaine alters neither resting arterial blood pressure, renal hemodynamics nor renal excretory function in either strain, at least under InactinR anesthesia. The results did confirm that saline-induced volume expansion stimulates enhanced sodium excretion in SHR (18). Dibona and Sawin (19) have shown that efferent renal sympathetic nerve stimulation lowers urine flow and excretion of sodium and chloride in the absence of changes in renal hemodynamics. Others have demonstrated an elevated basal renal sympathetic nerve traffic in SHR, which mediates the tendency for sodium retention noted in SHR (20) and may help explain the etiology of hypertension in these animals (20-23). Saline loading exerts greater inhibitory actions over efferent renal sympathetic nerve traffic in SHR, which has been postulated to explain the enhancement of sodium excretion under these circumstances (20). Others have noted an enhancement of sympathetically mediated cardiac (24) and vascular (25) responses in animals treated acutely with cocaine, but little data are available concerning either renal responses or responses following subacute cocaine treatment regimens. Thus, the effect of cocaine on the renal responses to acute volume expansion were of considerable interest. Responses to this physiological challenge were found not to differ between cocaine and vehicletreated SHR or WKY in the present study. These results indicate that subacute cocaine treatment exerts minimal effects on normal physiological function within the kidney. The effects of acute cocaine treatment on renal responses remain to be studied. Little difference was noted in the pharmacological responses of the cardiovascular system to acute cocaine infusion between freely moving SHR and WKY. Intravenous cocaine infusion increased HR and MBP similarly in both strains. The Tc following cocaine infusion also did not differ between SHR and WKY in the freely moving state. The cardiovascular effects of cocaine are widely believed to be the result of sympathomimetic actions (5, 6, 24, 25) and a number of authors have reported evidence which indicates that sympathetic nervous activity is enhanced in the SHR, at least in the developmental stage of hypertension (2629). Cocaine treatment, in the present study, did not produce significantly greater changes in MBP, HR or renal hemodynamics and excretory function in freely moving SHR as opposed to WKY. These data might indicate that there is little difference between the two strains in sympathetic cardiovascular or renal outflow, under our conditions, or that cocaine has little effect to augment whatever differences in endogenous sympathoadrenal outflow may exist between SHR and WKY. However, it is also possible that compensatory alterations in hemodynamic function (cardiac output, regional vascular resistance, changes in vasoactive hormones) acted to mask differential effects of cocaine on sympathetically-mediated responses. Using a paradigm of restraint stress, differential actions of cocaine do become obvious in the areas of cardiovascular function, susceptibility to convulsions and body temperature regulation. Minor differences in cardiovascular sensitivity to cocaine are detectable between SHR and WKY, with restraint appearing to reduce the pressor and tachycardic responses of WKY, rather than augment those of SHR. This is surprising, since SHR are generally more responsive to pressor stimuli, including opioid peptides (30-32), cholinergic agonists (33, 34), noradrenergic agonists (34) and stress (21, 22, 35), than WKY (30-32). However, SHR have been shown to exhibit impairments in baroreflex responsiveness (36-38). Cocaine has been suggested to lower heart rate through vagal activation by others (39). It is conceivable that cocaine-induced pressor responses trigger compensatory vagal activation which acts to reduce the maximal pressor effect and lower heart rate in WKY. Impaired baroreflex responsiveness in SHR would minimize these effects. Restraint was also found to significantly increase the LD50 for cocaine in WKY but not SHR. The mechanism by which restraint stress expresses these differences between SHR and WKY has not been explored directly. However, stress alters the activity of many central nervous pathways, including that of central opioid peptide systems (40-41). Kiritsy-Roy et al. (41) noted that intra-hypothalamic injections of an opiate agonist were able to blunt, through vagal activation, the tachycardia produced by restraint stress. Moreover, restraint has been shown to enhance morphine-induced bradycardia in the rat (42). We have recently demonstrated interactions between cocaine
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Effects of Cocaine on SHR
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and central opiate receptors in SHR and WKY (43). The possibility should be considered that stress-induced alterations in central neurotransmitter systems, perhaps including opioidergic pathways, can modify the cardiovascular and toxic actions of cocaine. It is also possible that restraint stress can reduce responsiveness through desensitization of peripheral catecholamine receptors. Yamaguchi e t a / . (44) demonstrated desensitization of vascular alpha and cardiac betal-adrenoceptors in normotensive rats following repeated immobilization stress or repeated isoproterenol injections. While we cannot exclude this as a factor, it must be pointed out that the stress levels are not comparable in the present study. Our animals were introduced into ventilated plexiglass tubes, in which rotational and forward and backward movement was possible at all times, and a period of no more than 30 min of restraint preceded initiation of cocaine infusion. Moreover, since both sympathetic (20-22) and hormonal (45) responses to stress are known to be exaggerated in SHR compared to those in WKY, it is difficult to explain a reduction in WKY responsiveness by this mechanism. Perhaps more interesting is the finding of a sub-group of SHR which responds to i.v. cocaine with an increase in body temperature. A hypothermic response to cocaine administration was observed in all WKY and approximately half of the SHR (SHRL) examined in the present study. The remaining SHR (SHRH) exhibited a mild hyperthermia which could be correlated with significant alterations in both cardiac and convulsive responses to cocaine. The available literature indicates that the effect of cocaine on body temperature in the rat is modified by as yet poorly defined factors. Acute administration of cocaine has been reported to increase (46, 47), decrease (48) or have no effect (49) on body temperature in this species. Wiechman and Spratto (46) demonstrated that the response of body temperature to cocaine was dependent on ambient temperature and could be modified by concomitant opiate receptor activation with morphine. Our data indicate that a heterogenons response of body temperature to cocaine is characteristic of the SHR genotype and can be demonstrated during restraint. Moreover, while under restraint, the SHR H demonstrate an increased tachycardia and a lower threshold for cocaine-induced convulsions when compared to either SHRI, or WKY. Tachycardia is a well-recognized sequela to hyperthermia and thus the elevation in heart rate noted in SHR H may result, in part, from the hyperthermia. In addition, Peterson and Hardinge (50) have reported a positive correlation between elevation in body temperature and sensitivity to the acute toxic effects of cocaine in mice. The response of body temperature to cocaine may be the critical variable determining the differential responses noted between SHR H and SHR L in the present study. Specific evidence linking cocaine-induced thermoregulatory andcirculatory aberrations with central opioid peptide systems must await further experimentation. The results from this investigation indicate that substantive differences exist between SHR and WKY in their responsiveness to acute cocaine administration. These differences are most evident under conditions of acute stress, a finding which suggests that stress is a factor which interacts with different genotypes to expose otherwise latent susceptibilities to cocaine-induced toxicity. Determination of the mechanisms which mediate the differences in susceptibility should provide data which will aid in the understanding of differences in human responses to cocaine. Acknowled2ement
The present study was supported by a grant from NIDA (1 RO1 DA04264). References 1.
2. 3. 4.
5. 6.
10.
L.L. CREGLER and H. MARK, Am. J. Cardiol. 57, 1185-1186 (1986). D.W. MATHIAS, Am. J. Med. 81, 675-677 (1986). R.J. WEISS, Am. Heart J. ~ 793 (1986). J.L. ROD and R.P. ZUCKER, Am. J. Cardioi. ~ , 161 (1987). C.C. CHIUEH and I.J. KOPIN, J. Pharmacol. Exp. Ther. 205, 148-154 (1978). J.M. RITCHIE and N.M. GREENE, in The Pharmacological Basis of Therapeutics. (Eds. A. G. Gilman, L. S. Goodman, T. W. Rail and F. Murad), p. 302-321, Macmillan press, New York (1985). Y. MASUDA, Y. MATSUDA and M.N. LEVY, J. Pharmacol. Exp. Ther. 215, 20-27 (1980). H. RUBEN and L.E. MORRIS, J. Pharmacol. Exp. Ther. ~ 55-64 (1952). M.W. FISCHMAN, C.R. SCHUSTER, L. RF.~NEKOV, J.F.E. SHICK, N.A. KRASNEGOR, W. FENNELL and D.X. FREEDMAN, Arch. Gen. Psych. ~ 983-989 (1976). M.W. FISCHMAN, C.R. SCHUSTER, J. JAVAID, Y. HATANO and J. DAVIS, J. Pharmacol. Exp. Ther. ~ 677-682 (1985).
232
11. 12. 13. 14. 15. 16, 17. 18. 19, 20. 21. 22. 23. 24. 25.
26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.
43. 44. 45. 46.
47. 48. 49. 50.
Effects
of Cocaine
on SHR
Vol.
45, No. 3, 1989
D.K. PITTS, C.E. UDOM and J. MARWAH, Life Sci. 40, 1099-1111 (1987). M.E. TRULSON, L.R. EPPS and J.C. JOE, Acta Anat. 12_..99,165-168 (1987). H.K. WATANABE, B. HOSKINS and I.K. HO, Alcoh. Drug Res. 7, 363-370 (1987). R.W. ROCKHOLD, B. HOSKINS and I.K. HO, Fed. Proc. 46, 402 (1987). M. WALSER, D.G. DAVIDSON and J. ORLOFF, Clin. Invest. 34, 1520-1523 (1955). W.H. WAUGH and P.T. BEALL, Kidney Int. 5, 429-436, (1974). WJ. DIXON, Am. Stat. Ass. J. 1965, 967-978 (1965). M.M. MULLINS and R.O. BANKS, Am. J. Physiol. 23_!, 1364-1370, (1976). G.F. DIBONA and L.L. SAWIN, Am. J. Physiol. 24__33,F576-F580 (1982). P. THOREN, Clin. Exp. Hypertension, A__9_9,259-279 (1987). J.P. KOEPKE and G.F. DIBONA, Hypertension, 7, 350-356 (1985a). J.P. KOEPKE and G.F. DIBONA, Hypertension, _7,356-363 (1985b). R. DIETZ, A. SCHOMIG, H. HAEBARA, J.F.E. MANN, W. RASCHER, J.B. LUTH, N, GRUNHERZ and F. GROSS, Circ. Res. 43, I98-I106 (1978). Y. MATSUDA, Y. MASUDA, B. BLATFBERG and M.N. LEVY, Eur. J. Pharmacol. 63, 25-33 (1980). M.A. BAYORH, Z. ZUKOWSKA-GROJEC and I..1. KOPIN, J. Clin. Pharmacol. 23, 24-31 (1983). W.V. JUDY, W.R. MURPHY, A.M. WATANABE, H.R. BESCH, JR., D.P. HENRY and P.L. YU, in Spontaneous Hypertension: Its Pathogenesis and Complications, (Ed. K. Okamoto) p. 167-180, Springer-Verlag, New York (1977). K. OKAMOTO, S. NOSAKA, Y. YAMORI and M. MATSUMOTO, Jap. Heart J. _8, 168-180 (1967). K. NAKAMURA and K. NAKAMURA, Naunyn-Schmeid. Arch. Pharmacol. 299, 143-148 (1977). T. KUBO, Can. J. Physiol. Pharmacol. 57, 59-64 (1979). R.W. ROCKHOLD, J.T. CROP'TON and L. SHARE, Clin. Sci. 59, 235s-2378 (1980). R.W. ROCKHOLD, J.T. CROFTON and L. SHARE, Hypertension, 3, 410-415 (1981). G. FEUERSTEIN, R.L. ZERBE and A.I. FADEN, Hypertension, 5, 663-671 (1983). T. KUBO and M. TATSUMI, Naunyn-Schmeid. Arch. Pharmacol. 306, 81-83 (1979). W.E. HOFFMAN, P.G. SCHMID and M.I. PHILLIPS, J. Pharmacol. Exp. Ther. 206, 644-651 (1978). J. YAMAMOTO, M. NAKAI and T. NATSUME, Hypertension, 9, 362-370 (1987). M.C. ANDERSEN, S. KURAOKA and A.M. BROWN, Circ. Res. 47, 821-828 (1980). W.R. SAUM, A.M. BROWN and F.H. TULEY, Circ. Res. 39, 497-505 (1976). J.H. COOTE and Y. SATO, Circ. Res. 40, 571-577 (1977). G.R. GAY, Ann. Emerg. Med. 11, 562-572 (1982). M. NORDIN, P. MORAT and M. ZAINORA, Clin. Exp. Pharmacol. Physiol. 14, 303-308 (1987). J.A. KIRITSY-ROY, N.M. APPEL, F.G. BOBBITT and G.R. VAN LOON, J. Pharmacol. Exp. Ther. 23__.99,814-822 (1986). K.S. SCHWARZ, J. PERIS and C.L. CUNNINGHAM, Alcohol Drug Res. 7, 327 (1987). Y. ISHIZUKA, R.W. ROCKHOLD, B. HOSKINS and I.K. HO, Life Sci. 43, 2275-2282 (1988). I. YAMAGUCHI, T. TORDA, F. HIRATA and I.J. KOPIN, Am. J. Physiol. 240, H691-H696 (1981). K.H. BERECEK, G. COSHATT, A.J. NARKATES, S. OPARIL, K.M. WILSON and J. ROBERTSON, Hypertension, 11, 1144-1147 (1988). B.E. WlECHMAN and G.R. SPRATTO, Pharmacol. 25, 308-319 (1982). E. COSTA, A. GROPPETrI and M.K. NAIMZADA, Br. J. Pharmacol. 44, 742-751 (1972). I. FAHIM, M. ISMAIL and O.H. OSMAN, W. Afr. J. Pharmacol. Drug Res. 2, 48-56 (1975). G.T. PRYOR, F.F. LARSEN and S. HUSAIN, Pharmacol. Biochem. Behav. 8, 295-318 (1978). D.I. PETERSON and M.G. HARDINGE, J. Pharm. Pharmacol. 19, 810-814 (1967).
Pergamon Press
DIFFERENTIAL SENSITIVITY TO COCAINE IN SPONTANEOUSLY HYPERTENSIVE AND WISTARKYOTO RATS Y. Ishizuka, R.W. Rockhold, K. Kirchner*, B. Hoskins and I.K. Ho Department of Pharmacology and Toxicology and * Department of Medicine University of Mississippi Medical Center 2500 N. State St. Jackson, MS 39216 (Received in final form May 12, 1989) Summary
Physiological, pharmacological and toxicological responses t o two regimens of cocaine administration were compared between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. An initial experiment examined renal excretory and hemodynamic function in response to an acute volume load in anesthetized SHR and WKY following subaeute cocaine treatment (20 mg/kg, s.c., twice a day for 9 days). Anticipated renal responses to volume loading were obtained but the responses of cocaine-treated SHR and WKY did not differ from vehicle-treated rats. A second group of experiments compared responses to continuous i.v. infusions of cocaine (1.25 mg/kg.min). In freely moving animals, no differences were noted between SHR and WKY in the increases in mean blood pressure (MBP) and heart rate 0 t R ) produced during cocaine infusion. The elapsed time-to-onset of convulsions (To) elicited by cocaine was similar in both strains. However, when rats were subjected to restraint during the infusion period, pressor and tachycardic responses were observed to be significantly less in WKY than in SHR or in freely moving rats of either strain. Restraint also differentially affected rectal temperature (RT) responses to cocaine. Hypothermie responses to cocaine were observed in all WKY. Both hypothermic and hyperthermic responses were observed in SHR. A significant correlation was demonstrated between the Tc and the maximal change in RT produced during cocaine infusion. Division of SHR into two arbitrary groups was made, based on the direction of cocaine-induced change in RT. A significant (p<0.01) shortening of the Tc was obvious in SHR (8 of 15) in whom cocaine produced a hyperthermia. These animals were designated SHR H. The mean value for Tc in those SHR which demonstrated a lowering in RT (SHRL; 7 o f 15) in response to cocaine was similar to that for WKY. Moreover, the SHR H evidenced significantly greater increases in HR, but not MBP, to cocaine infusion thandid SHR L. The results indicate that restraint stress causes expression of a significant heterogeneity in the RT response of SHR to cocaine. The magnitude and direction of the RT responses are negatively correlated with sensitivity to the convulsive effects of cocaine in SHR. Stress may modify toxic responses to cocaine by interactions with body temperature homeostasis.
Cocaine is a tropane alkaloid whose toxic effects have been noted following both medicinal and recreational use. Convulsions, hyperthermia and respiratory depression are of concern in acute overdosage. The circulatory toxicity of cocaine can be pronounced. Tachycardia, pressor responses, acceleration of coronary artery disease, dysrhythmias and myocardial infarction have been frequently reported (1-4), indicating that effects of cocaine on the cardiovascular system are of major concern. The etiology of cocaine-induced cardiovascular toxicity remains uncertain. Until recently, it was suspected that adverse circulatory and cardiac responses occurred secondary to cocaine-mediated stimulation of sympathetic nervous system neurotransmission (5, 6). Thus, cocaine has been shown to potentiate and prolong the cardiac responses to epinephrine infusion in dogs (7, 8). Intravenous administration of cocaine to human subjects has been 0024-3205/89 $3.00 + .00 Copyright (e) 1989 Pergamon Press plc
224
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repeatedly demonstrated to produce a dose-related increase in mean blood pressure and heart rate (9, 10). However, Pitts et al. (11) compared the cardiovascular effects of cocaine under different anesthetic conditions in the rat and reported that some effects of cocaine might be elicited by mechanisms other than potentiation of sympathetic nervous responses. Moreover, Trulson et al. (12) reported that cardiac function was severely impaired following chronic cocaine administration. The spontaneously hypertensive rat (SHR) is a model of genetic hypertension useful in determining genetic differences in susceptibility to the toxic actions of cocaine. For example, Watanabe et al. (13) demonstrated that SHR were less sensitive to the hepatotoxic actions of cocaine than were age-matched normotensive Wistar-Kyoto (WKY) rats, a difference that was suggested to be pharmacodispositional in origin. Additionally, we have reported that differences exist between SHR and WKY in terms of resistance to the convulsive and lethal effects of acute cocaine administration (14). The present study was initiated to further define the relative sensitivity of SHR and WKY to cocaine. Circulatory, renal, convulsive and body temperature responses to varying regimens of cocaine treatment were compared in the two strains. Methods General Procedures: Male SHR and WKY, 11-12 weeks of age, were purchased from Taconic Farms, Inc.. Rats were housed, prior to experimental use, in plastic cages (3-4/cage) under controlled conditions of temperature (22° C), humidity (50-55%) and lighting (12 hr light: 12 hr dark) and given rat chow (Purina Laboratory Chow) and tap water, ad libitum. Surgical procedures were performed under halothane anesthesia (2-4% in medical grade oxygen). Polyethylene catheters were inserted 4 cm into the abdominal aorta (PE-50) and vena cava (PE-10) by way of left femoral vessels. These catheters were filled with heparinized saline (10 U/ml), exteriorized at the nape of the neck and sealed until use. Animals were housed subsequently in individual hanging wire cages. Experimental protocols were initiated two to four days following catheter implantation. Renal Studies: Two groups (n=6) each of SHR and WKY were placed in individual wire metabolic cages and allowed free access to food and water during an acclimation period of 48 hr. Rats then received twice daily (08:30 and 16:30) s.c. injections of either cocaine hydrochloride (20 mg/kg as base) or 0.15 M NaC1 vehicle for a period of 9 days. Rats from cocaine and vehicle injected groups of each strain were pair-fed to prevent large differences in weight gain. Ten days following the initiation of the injection sequence, animals were anesthetized with Inactin (100 mg/kg body weight, i.p.; Promonto GMBH, Hamburg, F.R.G.) and placed on a heated animal table. Rectal temperatures, monitored by telethermometer (YSI Corp., Yellow Springs, OH), were maintained between 36.5 and 38° C. A tracheotomy was performed and polyethylene catheters (PE-50) introduced into the right jugular vein for polyfructosan (Inutest, Laevosan GaseUschaft, Linz, Austria) and paraaminohippuric acid (PAIl; Sigma Chemical Co., St. Louis, MO) infusions and into the left jugular vein for volume expansion. A PE-50 catheter was introduced into the right femoral artery for blood sampling and continuous blood pressure monitoring by means of a P23 DC transducer (Statham Laboratories, Hato Rey, PR) and a Grass Model 7D polygraph. A flanged PE-50 catheter was placed in the bladder through a midline suprapubic incision. From the start of surgery, 5% polyfructosan and 1% PAH in Ringer's solution (in mEq/l: Na 140, CI 115, HCO3 30 and K 5) was given by constant i.v. infusion at 3 ml/hr for 20 min and thereafter at 1.5 ml/hr. Following a 30 min post-surgical stabilization period, a 45 min urine collection period was initiated to determine baseline renal function and electrolyte excretion rates. Urine was collected under oil for determination of inulin, sodium, potassium, chloride and osmolar concentrations. Upon completion of this hydropenic period, animals underwent acute extracellular volume expansion by administration of Ringer's solution in a volume equivalent to 10% body weight given over 60 rain. The infusion rate was reduced to a rate equal to urine output at the end of the volume expansion period and this rate maintained for the duration of the experiment. A second 45 min experimental period was then initiated and the measurements and collections described for the hydropenic period were repeated. Rats were sacrificed by exsanguination at the end of the experiment and blood collected for determination of serum electrolytes. Kidneys were removed, blotted to remove excess blood and weighed. Sodium and potassium concentrations in serum and urine were measured with a Model 143 Flame Photometer (Instrumentation Laboratory, Lexington, MA). Chloride concentration in serum and urine was analyzed amperometrically (Buchler Instruments, Fort Lee, NJ). Inulin concentration in urine and plasma was determined by the diphenylamine method of Walser et al. (15). Serum and urine osmolalities were determined with a freezing point osmometer (Advanced Instruments, Needham Heights, MA). The PAH concentration in plasma and urine was determined by the method of Waugh and Beall (16). Determination of urinary flow rate and the concentration of inulin, sodium and chloride in the blood and urine
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Effects
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p e r m i t t e d calculation of w h o l e kidney g l o m e r u l a r filtration r a t e ( G F R ) a n d u r i n a r y excretion of s o d i u m and chloride a c c o r d i n g to s t a n d a r d expressions. Effective renal b l o o d flow ( E R B F ) was calculated as: E R B F = [ ( U p A H . V ) / P P A H ] / ( 1 - H c t ) w h e r e U p A H and P P A H a r e the concentrations of P A I l in urine and s e r u m , respectively. T h e u r i n e flow r a t e in # l / m i n is V a n d H c t is the h e m a t o c r i t d e t e r m i n e d at the e n d of the collection period. Inulin clearance, P A H and absolute u r i n a r y electrolyte excretion r a t e s w e r e all expressed p e r g r a m kidney weight. TABLE I Effect of Subacute Cocaine (20 mg/kg, b.i.d., s.c., for 9 days) or Vehicle (0.9% NaCI) on the Response of SHR and WKY to 10% Volume Expansion with Ringer's Bicarbonate Solution. MBP
Flow
C1N
ERBF
UNa.V
FeNa
UK.V
FeK
UcI.V
FeCI
Uosm
122 +2
5.1 +1.8
1028 +64
5.2 +0.3
146 +112
0.09 +0.02
953 +119
22.1 +2.6
128 +36
0.10 +0.03
1442 +131
Volume 120 Expansion +__2
28.2" +9.6
1031 +101
6.5* +0.6
4009* +1060
2.47" +0.58
1615" +274
40.6* +3.7
4430* +1002
3.77* +0.84
782* +172
124 +_.3
4.1 +0.9
959 +60
5.1 +0.3
114 +71
0.08 +0.04
730 +108
21.0 +112
167 +112
0.15 +0.09
1554 +123
Volume 118 Expansion +4
16.6" +4.4
986 +60
6.2' +0.2
2711" +1019
1.71" +0.57
1310' +193
33.0 +4.5
3327" +1293
2.87" +0.97
1~* +239
174
3.4
876
4.6
345
0.25
807
24.8
164
0.18
1434
+6
+0.3
+59
+0.3
+132
+0.08
+121
+3.1
+72
+0.09
+157
38.1" +13.6
1043 +83
6.3* +0.9
8376* +2421
5.59* +1.88
1739" _.+150
45.1" +6.7
8492* +2243
7.24* +2.40
814' +104
165 +7
4.4 +0.7
1079 +99
5.7 +0.6
423 +90
0.28 +0.06
1002 +94
25.9 +6.0
27.8 +74
0.23 +0.05
1506 +226
Volume 159 Expansion +4
34.7" +6.5
1268 +179
7.5" +0.5
7763* +1408
4.64* +1.30
1899" +__131
41.6" +5.6
8104" +1410
6.48" +1.87
749" +62
WKY SALINE (n=6) Control
COCAINE (n=6) Control
SHR
SALINE (n = 6) Control
Volume 165 Expansion +7 COCAINE (n =5) Control
Number of animals in each group is indicated in parentheses. Mean values + 1 S.E.M. are presented. Column title abbreviations and units are MBP = mean arterial blood pressure (ram Hg), Flow = urine flow (~l/min/g), CIN = urinary inulin clearance (~l/min/g), ERBF = effective renal blood flow (ml/min/g), UNa.V = urinary sodium excretion (mEq/min/g), FeNa = fractional sodium excretion (%), UK.V = urinary potassium excretion (mEq/min/g), FeK = fractional potassium excretion (%), U c r V = urinary chloride excretion (mEq/min/g), FeCI = fractional chloride excretion (%), Uosm = urinary osmolality (mOsm/kg H20 ). All values are expressed per gram of wet kidney weight at the time of sacrifice, unless indicated otherwise. Asterisks denote statistically significant (p<0.05) differences between control and volume expanded groups.
226
Effects
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Vol. 45, No.
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Experiments in freely moving animals: Rats were housed in wooden recording chambers (30.5 x 29.5 x 44.5 cm) at least 30 min prior to drug administration. Blood pressure was monitored by means of a Cobe R transducer connected to a Grass model 7D polygraph. Mean arterial blood pressure (MBP) was obtained by electronic damping of the pulsatile blood pressure signal. Heart rate (HR) was determined electronically from the pulse interval and displayed on the polyg~.aph. Cocaine hydrochloride (1.25 mg/kg.min as base, Sigma Chemical Co., St. Louis, MO) was infused continuously i.v. using a Sage Model 353 pump. Continuous measurements of MBP and HR as well as the time from the start of infusion until the onset of convulsions (Tc) were recorded. Experiments in restrained animals: Animals were placed in plastic restraining cages and a lubricated temperature probe inserted 4 cm into the rectum and held in position by adhesive tape. Drug infusion was commenced following an acclimation period of at least 30 min. Rectal temperature (RT) was recorded every 5 min by a Model BAT-12 (Sensortek Inc.) electronic thermometer while MBP and HR were recorded continuously. Ambient temperatures during the infusion experiments were 22-24 ° C. Determinations of LDs0: The LD50 values for cocaine were determined in both freely moving and restrained SHR and WKY according to the Up and Down method of Dixon (17). Cocaine was administered by bolus i.v. injection through indwelling femoral catheters.
Statistical analysis: Analysis of variance for repeated measures (one- or two-way for appropriate groups) was performed on experimentally derived values. Neuman-Keuls or multiple "t"-tests were performed to differentiate group means if significant interactions were found by analysis of variance. Results Renal response to acute volume expansion: Resting values for MBP averaged 174 +_ 6 and 165 + 7 mm Hg in anesthetized saline-treated and cocaine-treated SHR, respectively and 122 + 2 and 124 + 3 mm Hg in salinetreated and cocaine-treated WKY, respectively. Acute volume expansion with Ringer's bicarbonate solution (10 % of body weight) did not alter MBP in any of the 4 groups. The anticipated effects of volume expansion were observed. Significant increases in urine flow, effective renal plasma flow and urinary excretion of sodium, potassium and chloride were noted (Table I). Urine osmolality decreased upon volume expansion. Urinary excretion of sodium, but not of potassium or chloride, was significantly greater in SHR when compared to WKY, regardless of the treatment regimen. Neither resting values nor the maximum changes evoked by volume expansion were altered by subacute cocaine treatment in either SHR or WKY (Table I). Cardiovascular responses to cocaine: Resting values of MBP averaged 162 + 4 mm Hg and 160 + 4 mm Hg in freely moving (n=12) and restrained (n=12) SHR, respectively and 113 + 4 and 116 + 3 mm Hg in freely moving (n=12) and restrained (n=10) WKY, respectively. Resting values for HR were significantly (p<0.01) higher in restrained SHR (413 + 7 beats/rain) than in freely moving SHR (366 + 8 beats/min) or WKY in either condition (352 + 7 and 375 + 8 beats/min; freely moving and restrained).
TABLE II Maximum Initial Changes in MBP and HR following Cocaine Infusion in SHR and WKY. Conditions
MBP (mm Hg)
HR (beats/min)
WKY
freely moving restrained
(12) (10)
33 + 3 23 +2**
22 + 3 13 + 4*
SHR
freely moving restrained
(12) (12)
37 + 3 33 + 2 a
27 + 2 24 + 1a
Asterisks denote significant differences (*: p<0.05, **: p<0.01) from freely moving WKY. a: p<0.05 compared to restrained WKY. Numbers in parentheses refer to the number on animals in each group. The i.v. infusion of cocaine produced initial increases in MBP and HR in all animals (Fig. 1). The maximum increases in MBP and HR produced by cocaine were significantly less in restrained WKY than in
Vol. 45, No. 3, 1989
Effects of Cocaine on SHR
227
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FIG. 1 Percent changes in mean blood pressure (a) and heart rate (b) from the start of cocaine infusion (1.25 mg/kg.min.) until the onset of convulsions in SHR and WKY. The number of animals included in each group was: freely moving SHR (O), 12; restrained SHR (A), 12; freely moving WKY (O), 12 and restrained WKY(&), 10. The abscissa represents the elapsed time from onset of infusion until onset of convulsions, divided into 10 equal periods. Asterisks signify differences (*:p<0.05, **:p<0.01) at the indicated time between restrained groups (see text). freely moving WKY. No differences were noted in the responses of SHR under the two conditions. However, the initial increases in MBP and HR of restrained SHR were significantly greater than those recorded in restrained WKY (Table II). Continuous cocaine infusion produced a return to baseline or a gradual decrease in HR following the initial tachycardic response while MBP showed a progressive elevation until the onset of convulsive activity. Again, the increases in MBP noted in restrained WKY were significantly less than those observed in other groups (Fig. 1). There was a greater fall in HR in WKY than in SHR. The maximum percentage change in HR (excluding the initial period of increase) was significantly greater in WKY than in SHR, respectively, under both freely moving (-0.12 + 0.03% vs -0.004 + 0.4%, p<0.05) and restrained (-0.14 + 0.05% vs -0.04 + 0.02%, p < 0.05) conditions.
LD50 of cocaine: Restraint significantly altered the LD50 for cocaine in WKY but not in SHR. The mean value-s (+ 1 S.E.M.: n=6 for all groups) for LD50 were 13.01 (+ 0.46) and 12.30 (+ 0.43) for freely moving WKY and SHR, respectively. Restraint increased the LD50 in WKY to 16.17 ( + 0.57, p<0.01) but did not significantly alter the value in SHR (10.62 + 0.37).
228
Effects
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on SHR
Vol.
45, No.
3, 1989
TABLE III Effects of Cocaine Infusion on Rectal Temperature in Restrained Animals. Resting body temperature (°C)
Changes in body temperature (°C)
WKY
37.89 + 0.07 (11)
-1.10 + 0.10 (11)
SHR
39.13 + 0.10 (15)**
-0.94 + 0.18 (SHRL, 7) +0.48 _ 0.12 (SHRH, 8)
**: p<0.01 compared to corresponding group. Numbers in parentheses refer to the number of animals in each group. Rectal temperature was increased in 8 SHR(SHRH) and decreased in 7
SIaRCSHRt).
o
2.000 1.500 1.000
0.500 0.000
-0.500 o _1.000 r.~
r...l
o -1.500 ~ -2.000 u 0
5
10
15
20
9.5
30
35
40
45
50
TIME-T0-0NSET OF CONVULSIONS (rain.)
FIG. 2 Correlation between body temperature response and time-to-onset of convulsions following cocaine infusion (1.25 mg/kg.min) in restrained SHR. The regression coefficient was -0.805 (p < 0.001). Relationship between body temperature and responses to cocaine: Rectal temperature, when measured under restraint, was significantly higher in SHR than in WKY (Table III). Body temperature was not measured in freely moving animals. A decrease in RT was noted in all restrained WKY following cocaine infusion. In contrast, the maximal change in RT varied in both magnitude and direction in restrained SHR. When the maximal change in RT following cocaine infusion was plotted against the Tc, a significant, negative correlation became evident (Fig. 2; regression coefficent = -0.805; p<0.001). No significant correlation was evident in data from WKY. The data from SHR suggested the possibility of division of SHR into those animals in whom the maximal cocaine-induced change in RT was positive (i.e., an increase in body temperature; tentatively designated SHRH) and those in whom the maximal change was negative (a lowering of body temperature; SHRI. ). Using this division, the mean values for maximal RT changes in SHR H and SHR L are also given in Table III. The values for maximum change in RT ranged from +0.10 to + 1.10 in SHR H and -0.10 to -1.50 in SHR L. No differences were detected between SHR H and SHR L in terms of body weight (264.3 -+ 8 "6 vs 266 "3 + 7.6 g, respectively) or pre-infusion values for MBP (159 + 6 vs 161 + 4 mmHg, respectively), HR (419 + 11 vs. 403 + 7 beats/min, respectively) and RT (39.16 + 0.17 vs 39.09 + 0.09 °C, respectively). In addition,
Vol. 45, No. 3, 1989
Effects of Cocaine on SHR
229
TABLE IV Time-to-onset of Convulsions following Cocaine Infusion in SHR and WKY. Conditions freely moving
restrained
WKY
28.52 +_.0.19 (12)
31.69 + 0.20 (11)
SHR
28.89 ± 0.10 (12)
30.68 + 2.36 (SHRL, 7) 15.55 +_ 1.12 (SHRH, 8)**
**:p<0.01 compared to all other groups. animals in each group.
Numbers in parentheses refer to the number of
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tO
PERIOD
FIG. 3 Percent changes in mean blood pressure (a) and heart rate (b) from the start of infusion (1.25 mg/kg.min.) until the onset of convulsions in restrained SHR. The number of animals included in each group was: body temperature increasing group ( O , SHRjrI), 7 and body temperature decreasing group ( 0 , SHRL), 5. The abscmsa represents the elapsed time from onset of infusion until onset of convulsions, divided into 10 equal periods. Asterisks signify differences (*:p<0.05, **:p<0.01) at the indicated times between groups. regression analysis was unable to show significant correlations between Tc and either initial RT, HR, MBP or body weight.
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No differences were noted in MBP between SHR L and SHR H during cocaine infusion. However, HR was significantly elevated during cocaine infusion in SHI~H as compared to the response in SHR L (Fig. 3). Moreover, the Te was reduced by 50% in SHR H compared to that in SHR L (Table IV). The Te in SHR L was similar to that in freely moving SHR and WKY and in restrained WKY (Table IV).
Discussion Recent evidence suggests a genetic component for the differences in responses to cocaine between the closely related SHR and WKY (13, 14). The results of the present study more completely define differences between physiological, pharmacological and toxicological responses to cocaine in these two rat strains. It is clear that subacute treatment with cocaine alters neither resting arterial blood pressure, renal hemodynamics nor renal excretory function in either strain, at least under InactinR anesthesia. The results did confirm that saline-induced volume expansion stimulates enhanced sodium excretion in SHR (18). Dibona and Sawin (19) have shown that efferent renal sympathetic nerve stimulation lowers urine flow and excretion of sodium and chloride in the absence of changes in renal hemodynamics. Others have demonstrated an elevated basal renal sympathetic nerve traffic in SHR, which mediates the tendency for sodium retention noted in SHR (20) and may help explain the etiology of hypertension in these animals (20-23). Saline loading exerts greater inhibitory actions over efferent renal sympathetic nerve traffic in SHR, which has been postulated to explain the enhancement of sodium excretion under these circumstances (20). Others have noted an enhancement of sympathetically mediated cardiac (24) and vascular (25) responses in animals treated acutely with cocaine, but little data are available concerning either renal responses or responses following subacute cocaine treatment regimens. Thus, the effect of cocaine on the renal responses to acute volume expansion were of considerable interest. Responses to this physiological challenge were found not to differ between cocaine and vehicletreated SHR or WKY in the present study. These results indicate that subacute cocaine treatment exerts minimal effects on normal physiological function within the kidney. The effects of acute cocaine treatment on renal responses remain to be studied. Little difference was noted in the pharmacological responses of the cardiovascular system to acute cocaine infusion between freely moving SHR and WKY. Intravenous cocaine infusion increased HR and MBP similarly in both strains. The Tc following cocaine infusion also did not differ between SHR and WKY in the freely moving state. The cardiovascular effects of cocaine are widely believed to be the result of sympathomimetic actions (5, 6, 24, 25) and a number of authors have reported evidence which indicates that sympathetic nervous activity is enhanced in the SHR, at least in the developmental stage of hypertension (2629). Cocaine treatment, in the present study, did not produce significantly greater changes in MBP, HR or renal hemodynamics and excretory function in freely moving SHR as opposed to WKY. These data might indicate that there is little difference between the two strains in sympathetic cardiovascular or renal outflow, under our conditions, or that cocaine has little effect to augment whatever differences in endogenous sympathoadrenal outflow may exist between SHR and WKY. However, it is also possible that compensatory alterations in hemodynamic function (cardiac output, regional vascular resistance, changes in vasoactive hormones) acted to mask differential effects of cocaine on sympathetically-mediated responses. Using a paradigm of restraint stress, differential actions of cocaine do become obvious in the areas of cardiovascular function, susceptibility to convulsions and body temperature regulation. Minor differences in cardiovascular sensitivity to cocaine are detectable between SHR and WKY, with restraint appearing to reduce the pressor and tachycardic responses of WKY, rather than augment those of SHR. This is surprising, since SHR are generally more responsive to pressor stimuli, including opioid peptides (30-32), cholinergic agonists (33, 34), noradrenergic agonists (34) and stress (21, 22, 35), than WKY (30-32). However, SHR have been shown to exhibit impairments in baroreflex responsiveness (36-38). Cocaine has been suggested to lower heart rate through vagal activation by others (39). It is conceivable that cocaine-induced pressor responses trigger compensatory vagal activation which acts to reduce the maximal pressor effect and lower heart rate in WKY. Impaired baroreflex responsiveness in SHR would minimize these effects. Restraint was also found to significantly increase the LD50 for cocaine in WKY but not SHR. The mechanism by which restraint stress expresses these differences between SHR and WKY has not been explored directly. However, stress alters the activity of many central nervous pathways, including that of central opioid peptide systems (40-41). Kiritsy-Roy et al. (41) noted that intra-hypothalamic injections of an opiate agonist were able to blunt, through vagal activation, the tachycardia produced by restraint stress. Moreover, restraint has been shown to enhance morphine-induced bradycardia in the rat (42). We have recently demonstrated interactions between cocaine
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and central opiate receptors in SHR and WKY (43). The possibility should be considered that stress-induced alterations in central neurotransmitter systems, perhaps including opioidergic pathways, can modify the cardiovascular and toxic actions of cocaine. It is also possible that restraint stress can reduce responsiveness through desensitization of peripheral catecholamine receptors. Yamaguchi e t a / . (44) demonstrated desensitization of vascular alpha and cardiac betal-adrenoceptors in normotensive rats following repeated immobilization stress or repeated isoproterenol injections. While we cannot exclude this as a factor, it must be pointed out that the stress levels are not comparable in the present study. Our animals were introduced into ventilated plexiglass tubes, in which rotational and forward and backward movement was possible at all times, and a period of no more than 30 min of restraint preceded initiation of cocaine infusion. Moreover, since both sympathetic (20-22) and hormonal (45) responses to stress are known to be exaggerated in SHR compared to those in WKY, it is difficult to explain a reduction in WKY responsiveness by this mechanism. Perhaps more interesting is the finding of a sub-group of SHR which responds to i.v. cocaine with an increase in body temperature. A hypothermic response to cocaine administration was observed in all WKY and approximately half of the SHR (SHRL) examined in the present study. The remaining SHR (SHRH) exhibited a mild hyperthermia which could be correlated with significant alterations in both cardiac and convulsive responses to cocaine. The available literature indicates that the effect of cocaine on body temperature in the rat is modified by as yet poorly defined factors. Acute administration of cocaine has been reported to increase (46, 47), decrease (48) or have no effect (49) on body temperature in this species. Wiechman and Spratto (46) demonstrated that the response of body temperature to cocaine was dependent on ambient temperature and could be modified by concomitant opiate receptor activation with morphine. Our data indicate that a heterogenons response of body temperature to cocaine is characteristic of the SHR genotype and can be demonstrated during restraint. Moreover, while under restraint, the SHR H demonstrate an increased tachycardia and a lower threshold for cocaine-induced convulsions when compared to either SHRI, or WKY. Tachycardia is a well-recognized sequela to hyperthermia and thus the elevation in heart rate noted in SHR H may result, in part, from the hyperthermia. In addition, Peterson and Hardinge (50) have reported a positive correlation between elevation in body temperature and sensitivity to the acute toxic effects of cocaine in mice. The response of body temperature to cocaine may be the critical variable determining the differential responses noted between SHR H and SHR L in the present study. Specific evidence linking cocaine-induced thermoregulatory andcirculatory aberrations with central opioid peptide systems must await further experimentation. The results from this investigation indicate that substantive differences exist between SHR and WKY in their responsiveness to acute cocaine administration. These differences are most evident under conditions of acute stress, a finding which suggests that stress is a factor which interacts with different genotypes to expose otherwise latent susceptibilities to cocaine-induced toxicity. Determination of the mechanisms which mediate the differences in susceptibility should provide data which will aid in the understanding of differences in human responses to cocaine. Acknowled2ement
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