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Anesthesia-induced alteration of small vessel responses to norepinephrine
European Journal of Pharmacology, 44 (1977) 0 Elsevier/North-Holland Biomedical Press
331
331-337
ANESTHESIA-INDUCED ALTERATION OF SMALL VESSEL RESPONSES TO NOREPINEPHRINE FREDERICK
N. MILLER and DAVID L. WIEGMAN
Microcirculatory Systems Research Group, Dalton Research Center, and Departments of Pharmacology Physiology, University of Missouri School of Medicine, Columbia, Missouri 65201, U.S.A.
and
Received 2 December 1976, revised MS received 27 April 1977, accepted 2 May 1977 F.N. MILLER and D.L. WIEGMAN, Anesthesia-induced alteration of small vessel responses to norepinephrine, EUropean J. Pharmacol. 44 (1977) 331-337. Closed-circuit television microscopy was used to quantitate the in vivo response of small arteries (‘100 pm) and small veins (2150pm) to topically applied norepinephrine in the rat cremaster muscle. Rats were anesthetized with pentobarbital(50 mg/kg), or urethane (1200 mg/kg) or a combination of urethane (800 mg/kg) and chloralose (60 mg/kg). Complete concentration-response curves were obtained for an artery and vein pair in each rat and pD2 values (-log EDso) were used to evaluate the vascular sensitivity to norepinephrine. Both the artery and the vein in urethane-anesthetized animals had decreased sensitivity to norepinephrine in comparison to the vessels of animals anesthetized with pentobarbital or urethane-chloralose. Pretreatment with cocaine (10-s M) significantly increased the sensitivity of both the artery and vein in pentobarbital-anesthetized animals but did not affect the vessels in urethane--chloralose-anesthetized animals. These results are consistent with two opposing effects of the urethane--chloralose combination. The first is an increased sensitivity to norepinephrine via blockade of neuronal uptake and the second is a decreased sensitivity of norepinephrine via a vascular inhibitory effect of urethane. Microcirculation
Norepinephrine
Anesthesia
Adrenergic receptor
1. Introduction The use of television microscopy to quantify in vivo effects of catecholamines on the microvasculature has led to the concept of differential effects of drugs at various microcirculatory levels. Altura (1971) found in the mesentery of pentobarbital-anesthetized rats that norepinephrine produced greater maximal constriction of small arteries than small veins and that the small arteries were more sensitive to the constrictor effects of norepinephrine than the small veins. In contrast, we (Miller and Harris, 1975) determined in unanesthetized rats, that norepinephrine produced the same maximal constriction in small arteries and veins in the S.C. wing tissue. We also found that the small artery was less sensitive than the small vein apparently because neuronal uptake of norepinephrine was greater in the small artery. Previously, we have shown in the bat wing preparation that small arteries and veins dilate during pento-
barbital anesthesia (Harris et al., 1971). Furthermore, although these small vessels constrict significantly in response to bilateral carotid artery occlusion during urethane or cu-chloralose anesthesia, they do not respond during pentobarbital anesthesia (Harris et al., 1972). Collectively these data suggest the hypothesis that pentobarbital anesthesia may alter the microvascular effects of norepinephrine and/ or norepinephrine disposition mechanisms and thus contribute to the observed differences in drug effects at different microcirculatory levels. The present study examines this hypothesis by quantitating the effects of norepinephrine on small blood vessels in the cremaster muscle of rats which were anesthetized with different agents. 2. Materials and methods Individual in vivo concentration response curves were obtained in 5 groups of Sprague-
332
Dawley rats (90-171 g) to quantitate the constrictor effects of norepinephrine hydrochloride on the diameters of a small artery (-1001.tm) and vein (=150 pm) in the cremaster muscle. One group of animals (X + S.E.M.) (138 + 21 g) was anesthetized with sodium pentobarbital, 50 mg/kg (PB). A second group (158 + 4 g) was anesthetized with a combination of urethane 800 mg/kg and a-chloralose 60 mg/kg, (U-Cl). The third group of animals (136 + 10 g) was anesthetized with urethane, 1200 mg/kg (U). The fourth (163 + 22 g) and fifth (124 + 9 g) groups were anesthetized with pentobarbital (50 mg/kg) and urethane--chloralose (800 mg/kg and 60 mg/kg) respectively and in addition the cremaster muscle of these animals was treated with cocaine hydrochloride (10e5 M) before the effects of norepinephrine were observed (PB-cot and U-Cl-cot). Anesthetics were administered i.p. and supplements were given as needed to maintain approximately a surgical level, plane 1, of anesthesia. Except for anesthesia, the animal preparation was the same for all groups. The scrotal sac was opened and the cremaster muscle of the right testicle was carefully dissected free of surrounding tissues. Major nerves and blood vessels supplying the cremaster were left intact. The muscle was slit on a median line and spread with sutures over a cover glass in the bottom of a custom built Plexiglas bath (7 cm diameter, 60 ml capacity). The bath was filled with a modified Krebs solution containing (mM) : NaCl 113, dextrose 11.6, KC1 4.7, MgS04 - 7Hz0 1.2, KH2P04 1.2, CaClz 2H20 2.6 and NaHCO, 25. Bath temperature was controlled at 31°C by means of an immersed insulated heating coil and the pH of the bath was maintained at 7.2 by bubbling with carbon dioxide and nitrogen. The rat was positioned on a stage of a compound trinocular microscope so that light could be transmitted through the cremaster muscle. A small artery and vein pair in the cremaster were observed using a 12X water immersion lens. The images of these vessels
F.N. MILLER,
D.L. WIEGMAN
were projected through a closed circuit television system at about 1500 times magnification. The artery and vein diameters were measured on a calibrated monitor screen at 30-set intervals with a mm ruler. The left femoral artery was cannulated for measurement of mean arterial blood pressure via a Statham P23De pressure transducer and a Brush polygraph. Three S.C. needle electrodes provided an electrocardiogram recording for calculations of heart rate. Blood pressure and heart rate were measured every minute. A small heating pad with a rheostat control was placed under the rat to maintain rectal temperature at 36°C. Back temperature was kept below 41°C to insure that overheating did not result in a false sign for additional anesthetic. Rectal, back and bath temperatures were recorded at 5-min intervals with thermister probes and a Bailey Bat-4 instrument. Prior to each experiment, stock serial dilutions of norepinephrine were prepared in 0.1% ascorbic acid. After a 5-min period of control data, a quantity of norepinephrine stock solution was added to the Krebs solution which bathed the exteriorized rat cremaster muscle to give the desired molar concentration of norepinephrine in the bath. The effect of each concentration of norepine: phrine was observed for 10 min to insure attainment of a maximal response. Each norepinephrine response period was followed by a 20-min recovery period (no data collection) during which the bath solution was changed five times with fresh Krebs solution. 5-7 concentrations of norepinephrine were administered in each experiment to obtain a complete concentration-response curve for each vessel. In the PB-cot and U-Cl-cot groups, the effect of blockade of neuronal uptake by cocaine (10m5 M) on the response to nore-. pinephrine was studied. After the initial 5-min control period, cocaine was added to the bath. for 15 min before the application of the first concentration of norepinephrine. Thereafter, a 10m5 M cocaine-Krebs solution was used for the bathing solution.
SMALL
VESSEL
RESPONSES
TO NOREPINEPHRINE
Data for vessel diameters was smoothed with a center weighted three-point digital filter to minimize random errors which might occur while marking and measuring diameters on the television monitor (Harris et al., 1970). Smoothed data was normalized by expressing each data point as a percent of the average value during the control period (equal to 100%) which immediately preceded the application of each concentration of drug. The molar concentration of norepinephrine which produced 50% of the maximal percent constriction was graphically determined from the individual concentration response curves for each animal. These values were converted to pD2 (-log EDso) values (Miller et al., 1948) for a measure of the sensitivity of the vessels to norepinephrine. Analysis of variance was used to determine if differences were present among the group means. Duncan’s new multiple range test with correction for unequal sample size (Kramer, 1956) was used to determine which group means were significantly different. A paired t-test was used to compare the maximal effects and pD* values of the arteries to those of the veins in each group. A significance level of p < 0.05 was used for all tests. Each animal was accepted for this study on the basis of predetermined criteria: mean blood pressure greater than 80 mm Hg, heart rate greater than 240 bpm, rectal temperature between 35 and 37”C, and a cremaster preparation with good arterial, venule and capillary blood flow. In addition, variation in the average control values (vessel diameters, blood pressure, and heart rate) from dose to dose could not exceed 20%. : 3. Results Norepinephrine produced concentration dependent constrictions of the small vein and artery. Pig. 1 shows the results of one experiment for the effects of topically applied norepinephrine on small artery and vein diameters and on mean arterial blood pressure and heart
333
, rate. Increasing concentrations of norepinephrine produced progressively greater small vessel constriction until maximal constriction was reached at a concentration of 30 X lo-’ M. During the recovery period the vessels returned to control diameters. Topical application of norepinephrine had no significant effect on heart rate or blood pressure in any of the 5 groups of animals. A comparison of control data for the three anesthetic groups without cocaine (table 1, first 3 rows) demonstrates that there were no significant differences @ > 0.05) in mean arterial blood pressure, heart rate or control artery and vein diameters between PB and U-Cl animals. Urethane-anesthetized animals (U) had a lower mean arterial blood pressure than animals anesthetized with either U-Cl or PB. In addition, the U group had significantly larger arteries than U-Cl animals and a tendency toward larger veins and arteries than either U-Cl or PB animals. Calculation of correlation coefficients indicated that there were no significant correlations between vessel diameter and pD* value for either the small vein or the small artery in any of the groups. Topical application of cocaine to the cremaster muscle (table 1, bottom 2 groups) did not affect the mean arterial blood pressure, heart rate or control diameters of the arteries or veins in pentobarbital (PB vs PBcoc) or urethane--chloralose (U-Cl vs U-Cl-cot) animals. In both PB-coc and U-Cl-cot, the addition of cosine had no significant effect on vessel control diameters. There were no statistically significant differences @ > 0.05) in the maximal constriction produced by norepinephrine in the arteries (165% constriction) or in the veins (-40% constriction) among the 3 groups of animals without cocaine (table 2, first 3 rows). However, within each group, maximal constrictions in the arteries were significantly greater (p < 0.05) than those in the veins (65%, 62% and 66% versus 41%, 47% and 38% respectively). The sensitivity of both the arteries and veins to norepinephrine were similar in PB
F.N. MILLER, D.L. WIEGMAN
334
NOREPINEPHRINE
(M x IO-’ 1
1.0
30.0
100.0
t
t
t
‘\ 80 t
;_
L t
Lf
60
t
ARTERY L
I_J
40 t 120
x
100 450 410 370 r I
t
0
100
50
TIME
OF
150
EXPERIMENT
200
(minutes)
Fig. 1. Mean arterial blood pressure, heart rate and small artery and vein diameters are given for one experiment in which the rat was anesthetized with pentobarbital. For each concentration of norepinephrine indicated at the top of the graph there was a 5-min control period, addition of norepinephrine to the bath (indicated by arrows) to give the desired molar concentration, and a lo-min response period. During the 20-min recovery periods, no data were collected while vessels returned to near control diameters.
TABLE 1 Data II + S.E.M. for the control values in each of the 5 experimental groups. Animals were anesthetized with pentobarbital (PB), urethane--chloralose (U-Cl) or urethane (U). In 2 additional groups cocaine (cot) was added to the cremaster bath (PB-cot and U-Cl-cot). Group
n
Mean arterial blood pressure (mm Hg)
Heart rate (bpm)
Control artery diameter (pm)
Control vein diameter (rm)
PB U-Cl U PB-cot u-Cl-cot
7 10 8 7 10
104* 97 + 85 * 105 * 98 +
429 402 400 434 375
102 97 114 114 97
156 152 164 155 168
3 2 2 3 4
* * + * ?
12 23 26 13 25
+ * + + +
5 4 5 5 6
f 11 f 6 f 8 * 8 f 8
Statistical analyses (p < 0.05). (1) Comparisons among the 3 anesthetic groups without cocaine (PB, U-Cl, U): mean arterial pressure, U < PB, U-Cl; artery diameter, U-Cl < U. (2) Comparisons between anesthetic groups without and with cocaine (PB, PB-cot; and U-Cl, U-Cl-cot): no significant differences.
335
SMALL VESSEL RESPONSES TO NOREPINEPHRINE TABLE 2
Data represent ?I r S.E.M. for the effects of norepinephrine on small arteries and veins in the rat cremaster. Animals were anesthetized with pentobarbital (PB), urethane-hloralose (U-Cl) or urethane (U). In 2 additional groups cocaine (cot) was added to the cremaster bath (PB-cot and U-Cl-cot). Group
PB U-Cl U PB-cot u-Cl-cot
n
7 10 8 7 10
Vein
Artery Maximal percent constriction
PD,
65 * 62+ 66* 63+ 66 +
6.4 6.7 6.1 7.5 6.9
2 2 2 2 3
* 2 + * +
0.16 0.17 0.10 0.20 0.14
Statistical analyses (p < 0.05). (1) Comparisons among anesthetic vein pD2, U < U-Cl. (2) Comparisons between anesthetic groups U-Cl, U-Cl-cot): artery pD2, PB < PB-cot; vein pD2, PB < PB-cot. arteries (A) and veins (V): maximal percent constriction, V < A for
and U-Cl (no statistical differences in pD2 values). However, the U group had lower pD2 values (p < 0.05) in both the small artery and vein than the U-Cl group (the statistical comparison of artery and vein pD2 values between U and PB gave 0.05 < p < 0.10). The addition of cocaine did not alter the sensitivity or maximal percent constriction of the vessels in urethane-hloralose-anesthetized animals (U-Cl vs U-Cl-cot). In contrast, in pentobarbital-anesthetized animals (PB vs PB-cot), the addition of cocaine resulted in a significant increase in sensitivity to norepinephrine in both the small artery and the small vein; however, there was no effect of cocaine on maximal percent constriction.
4. Discussion This study quantitates in vivo changes in small vessel diameter as a response to graded concentrations of topically applied norepinephrine in the cremaster muscle of rats anesthetized with pentobarbital, urethane-chloralose or urethane. Since this method of application did not produce any response in heart rate or blood pressure there was neither drug induced pressure changes nor reflex sympa-
Maximal percent constriction
pD2
41 * 47 f 382 37 k 38+
7.0 7.1 6.5 7.6 7.1
4 4 2 4 2
+ f. * r *
0.16 0.06 0.07 0.17 0.14
groups (PB, U-Cl, U): artery pD2, U < U-Cl; without and with cocaine (PB, PB-COC; and (3) Comparisons within each group between all groups; pD2, A < V for PB, U-Cl and U.
thetic nerve activity to influence the small vessel diameter during norepinephrine application. In each of the 5 groups of animals (PB, PBcot, U-Cl, U-Clcoc, and U) maximal arterial constriction (165% was significantly greater than maximal venous constriction (540%). These data are consistent with those of Altura (1971) in the mesentery of the anesthetized rat but contrast with our data (Miller and Harris, 1975) in the S.C. wing tissue of the unanesthetized bat where we found no significant difference in maximal constriction of small arteries (57 f 6%) and veins (67 f 3%). Our vascular data (pD2 values, table 2) also indicate that anesthetics may differentially influence the sensitivity of the microvasculature to norepinephrine without affecting the maximal contractile response. The order of sensitivity to topically applied norepinephrine for both the small artery and vein was U-Cl 2 PB > U. Since there were no differences in the maximal percent constriction for either vessel among our three anesthetic groups, decreased sensitivity to norepinephrine in the artery and vein of the urethane group cannot be attributed to ‘depression’ of vascular smooth muscle. Neuronal uptake of norepinephrine should
F.N.
336
decrease the effective concentration of any given dose of norepinephrine at the receptor site of an innervated vessel and therefore shift the concentration-response curve to the right and give a smaller pD2 value. In PB, U-Cl, and U animals without cocaine, the small artery was significantly less sensitive (paired comparisons) than the small vein to norepinephrine (table 2). However, in the presence of cocaine which blocks neuronal uptake and should therefore increase the pD2 values of innervated vessels, there was no difference in artery and vein sensitivity to norepinephrine in PB and U-Cl animals. Thus, with PB anesthesia neuronal uptake (PB vs PB-coc) appears to have a greater effect on the pD2 of the small artery (6.4 f 0.16 vs 7.6 + 0.17) than on the pD2 of the small vein (7.0 f 0.16 vs 7.6 + 0.17) although the sensitivity of both vessels is increased. In the U-Cl group, cocaine produces only a slight (not significant) increase in vascular sensitivity of the artery (6.7 ? 0.17 vs 6.9 f 0.14) and no change in that of the vein (7.1 + 0.06 vs 7.1 + 0.14). The data in both pentobarbitaland urethane-chloralose-anesthetized animals supports previous data in the bat wing (Miller and Harris, 1975) that neuronal uptake has a greater influence on vascular sensitivity of the artery than of the vein of a small vessel pair. The minimal effect of cocaine in the urethane-chloralose-anesthetized rats suggests that functionally, there was substantial blockade of neuronal uptake already present with this combination of anesthetics. If blockade of neuronal uptake by urethane-chloralose was the only important influence of this anesthetic combination on the small vessel response to norepinephrine, the pD2 values for U-Cl-cot and PB-cot should be the same. However, in our data the pD2 values for PBcot are significantly greater than those for U-Clcoc. These data, then, indicate that there is an additional mechanism which acts to inhibit small vessel responses to norepinephrine during anesthesia with this combination of urethane and chloralose. Our data from the urethane group support
MILLER.
D.L. WIEGMAN
the hypothesis that urethane anesthesia has an inhibitory effect on vascular responses. Both mean arterial blood pressure and small vessel sensitivity were lower in urethane-anesthetized animals than those in urethane--chloralose-anesthetized animals. A mechanism for this inhibition may involve high levels of renin (Pettinger et al., 1975) or altered calcium metabolism (Peng et al., 1972) - factors associated with urethane anesthesia. These results are consistent with other data which suggest that urethane (Brezenoff, 1973) inhibits and chloralose (Monroe, 1963) augments cardiovascular responses. Thus, during urethanechloralose anesthesia there appear to be at least two opposing mechanisms which affect the vascular responses to norepinephrine. One of these appears to be an ‘inhibitory effect’ which is produced by urethane. The second appears to represent an ‘excitatory effect’ on small vessel sensitivity through an action of chloralose and/or the chloralose-urethane combination to functionally block neuronal uptake of norepinephrine.
Acknowledgements Dr. Wiegman is a recipient of a National Research Service Award, HL 05184 from the Public Health Service. This research was supported in part by Public Health Service Grants HL 12614 and 13207. The authors wish to express their thanks to David Andersen, John Krstansky, Wayne Lamm, Walter Parrish and Randall Sheller for their fine technical assistance.
References Altura, B.M., 1971, Chemical and humoral regulation of blood flow through the precapillary sphincter, Microvasc. Res. 3, 361. Brezenoff, H.E., 1973, Cardiovascular responses to noradrenaline in the rat before and after administration of various anesthetics, Brit. J. Pharmacol. 49, 565. Harris, P.D., E.K. Greenwald and P.A. Nicoll, 1970, Neural mechanisms in small vessel response to hemorrhage in the unanesthetized bat, Amer. J. Physiol. 218, 560. Harris, P.D., L.F. Hodoval and D.E. Longnecker,
TO NOREPINEPHRINE
337
1971, Quantitative analysis of microvascular diameters during pentobarbital and thiopental anesthesia in the bat, Anesthesiology 35, 337. Harris, P.D., L.F. Hodoval and D.E. Longnecker, 1972, Microvasculature responses to carotid occlusion after pentobarbital, urethane, and a-chloralose , Microvasc. Res. 4, 325 (abstr.). Kramer, C.Y., 1956, Extension of multiple range tests to group means with unequal numbers of replications, Biometrics 12, 307. Miller, F.N. and P.D. Harris, 1975, Sensitivity of subcutaneous small arteries and veins to norepinephrine, and epinephrine and isoproterenol in the unanesthetized bat, Microvasc. Res. 10, 340.
Miller, L.C., T.J. Becker and M.L. Tainter, 1948, The quantitative evaluation of spasmolytic drugs in vitro, J. Pharmacol. 92, 260. Monroe, R.R., G.V. Balis and E. Ebersberger, 1963, The hypnotic effects of alpha and beta chloralose in rats, Curr. Therap. Res. 5, 141. Peng, T.-C., C.W. Cooper and P.L. Munson, 1972, The hypocalaemic effect of urethane in rats, J. Pharmacol. Exptl. Therap. 182, 522. Pettinger, W.A., K. Tanaka, K. Keeton, W.B. Campbell and S.N. Brooks, 1975, Renin release, an artifact of anesthesia and its implications in rats, Proc. Sot. Exptl. Biol. Med. 148, 625.
SMALL VESSEL RESPONSES
331
331-337
ANESTHESIA-INDUCED ALTERATION OF SMALL VESSEL RESPONSES TO NOREPINEPHRINE FREDERICK
N. MILLER and DAVID L. WIEGMAN
Microcirculatory Systems Research Group, Dalton Research Center, and Departments of Pharmacology Physiology, University of Missouri School of Medicine, Columbia, Missouri 65201, U.S.A.
and
Received 2 December 1976, revised MS received 27 April 1977, accepted 2 May 1977 F.N. MILLER and D.L. WIEGMAN, Anesthesia-induced alteration of small vessel responses to norepinephrine, EUropean J. Pharmacol. 44 (1977) 331-337. Closed-circuit television microscopy was used to quantitate the in vivo response of small arteries (‘100 pm) and small veins (2150pm) to topically applied norepinephrine in the rat cremaster muscle. Rats were anesthetized with pentobarbital(50 mg/kg), or urethane (1200 mg/kg) or a combination of urethane (800 mg/kg) and chloralose (60 mg/kg). Complete concentration-response curves were obtained for an artery and vein pair in each rat and pD2 values (-log EDso) were used to evaluate the vascular sensitivity to norepinephrine. Both the artery and the vein in urethane-anesthetized animals had decreased sensitivity to norepinephrine in comparison to the vessels of animals anesthetized with pentobarbital or urethane-chloralose. Pretreatment with cocaine (10-s M) significantly increased the sensitivity of both the artery and vein in pentobarbital-anesthetized animals but did not affect the vessels in urethane--chloralose-anesthetized animals. These results are consistent with two opposing effects of the urethane--chloralose combination. The first is an increased sensitivity to norepinephrine via blockade of neuronal uptake and the second is a decreased sensitivity of norepinephrine via a vascular inhibitory effect of urethane. Microcirculation
Norepinephrine
Anesthesia
Adrenergic receptor
1. Introduction The use of television microscopy to quantify in vivo effects of catecholamines on the microvasculature has led to the concept of differential effects of drugs at various microcirculatory levels. Altura (1971) found in the mesentery of pentobarbital-anesthetized rats that norepinephrine produced greater maximal constriction of small arteries than small veins and that the small arteries were more sensitive to the constrictor effects of norepinephrine than the small veins. In contrast, we (Miller and Harris, 1975) determined in unanesthetized rats, that norepinephrine produced the same maximal constriction in small arteries and veins in the S.C. wing tissue. We also found that the small artery was less sensitive than the small vein apparently because neuronal uptake of norepinephrine was greater in the small artery. Previously, we have shown in the bat wing preparation that small arteries and veins dilate during pento-
barbital anesthesia (Harris et al., 1971). Furthermore, although these small vessels constrict significantly in response to bilateral carotid artery occlusion during urethane or cu-chloralose anesthesia, they do not respond during pentobarbital anesthesia (Harris et al., 1972). Collectively these data suggest the hypothesis that pentobarbital anesthesia may alter the microvascular effects of norepinephrine and/ or norepinephrine disposition mechanisms and thus contribute to the observed differences in drug effects at different microcirculatory levels. The present study examines this hypothesis by quantitating the effects of norepinephrine on small blood vessels in the cremaster muscle of rats which were anesthetized with different agents. 2. Materials and methods Individual in vivo concentration response curves were obtained in 5 groups of Sprague-
332
Dawley rats (90-171 g) to quantitate the constrictor effects of norepinephrine hydrochloride on the diameters of a small artery (-1001.tm) and vein (=150 pm) in the cremaster muscle. One group of animals (X + S.E.M.) (138 + 21 g) was anesthetized with sodium pentobarbital, 50 mg/kg (PB). A second group (158 + 4 g) was anesthetized with a combination of urethane 800 mg/kg and a-chloralose 60 mg/kg, (U-Cl). The third group of animals (136 + 10 g) was anesthetized with urethane, 1200 mg/kg (U). The fourth (163 + 22 g) and fifth (124 + 9 g) groups were anesthetized with pentobarbital (50 mg/kg) and urethane--chloralose (800 mg/kg and 60 mg/kg) respectively and in addition the cremaster muscle of these animals was treated with cocaine hydrochloride (10e5 M) before the effects of norepinephrine were observed (PB-cot and U-Cl-cot). Anesthetics were administered i.p. and supplements were given as needed to maintain approximately a surgical level, plane 1, of anesthesia. Except for anesthesia, the animal preparation was the same for all groups. The scrotal sac was opened and the cremaster muscle of the right testicle was carefully dissected free of surrounding tissues. Major nerves and blood vessels supplying the cremaster were left intact. The muscle was slit on a median line and spread with sutures over a cover glass in the bottom of a custom built Plexiglas bath (7 cm diameter, 60 ml capacity). The bath was filled with a modified Krebs solution containing (mM) : NaCl 113, dextrose 11.6, KC1 4.7, MgS04 - 7Hz0 1.2, KH2P04 1.2, CaClz 2H20 2.6 and NaHCO, 25. Bath temperature was controlled at 31°C by means of an immersed insulated heating coil and the pH of the bath was maintained at 7.2 by bubbling with carbon dioxide and nitrogen. The rat was positioned on a stage of a compound trinocular microscope so that light could be transmitted through the cremaster muscle. A small artery and vein pair in the cremaster were observed using a 12X water immersion lens. The images of these vessels
F.N. MILLER,
D.L. WIEGMAN
were projected through a closed circuit television system at about 1500 times magnification. The artery and vein diameters were measured on a calibrated monitor screen at 30-set intervals with a mm ruler. The left femoral artery was cannulated for measurement of mean arterial blood pressure via a Statham P23De pressure transducer and a Brush polygraph. Three S.C. needle electrodes provided an electrocardiogram recording for calculations of heart rate. Blood pressure and heart rate were measured every minute. A small heating pad with a rheostat control was placed under the rat to maintain rectal temperature at 36°C. Back temperature was kept below 41°C to insure that overheating did not result in a false sign for additional anesthetic. Rectal, back and bath temperatures were recorded at 5-min intervals with thermister probes and a Bailey Bat-4 instrument. Prior to each experiment, stock serial dilutions of norepinephrine were prepared in 0.1% ascorbic acid. After a 5-min period of control data, a quantity of norepinephrine stock solution was added to the Krebs solution which bathed the exteriorized rat cremaster muscle to give the desired molar concentration of norepinephrine in the bath. The effect of each concentration of norepine: phrine was observed for 10 min to insure attainment of a maximal response. Each norepinephrine response period was followed by a 20-min recovery period (no data collection) during which the bath solution was changed five times with fresh Krebs solution. 5-7 concentrations of norepinephrine were administered in each experiment to obtain a complete concentration-response curve for each vessel. In the PB-cot and U-Cl-cot groups, the effect of blockade of neuronal uptake by cocaine (10m5 M) on the response to nore-. pinephrine was studied. After the initial 5-min control period, cocaine was added to the bath. for 15 min before the application of the first concentration of norepinephrine. Thereafter, a 10m5 M cocaine-Krebs solution was used for the bathing solution.
SMALL
VESSEL
RESPONSES
TO NOREPINEPHRINE
Data for vessel diameters was smoothed with a center weighted three-point digital filter to minimize random errors which might occur while marking and measuring diameters on the television monitor (Harris et al., 1970). Smoothed data was normalized by expressing each data point as a percent of the average value during the control period (equal to 100%) which immediately preceded the application of each concentration of drug. The molar concentration of norepinephrine which produced 50% of the maximal percent constriction was graphically determined from the individual concentration response curves for each animal. These values were converted to pD2 (-log EDso) values (Miller et al., 1948) for a measure of the sensitivity of the vessels to norepinephrine. Analysis of variance was used to determine if differences were present among the group means. Duncan’s new multiple range test with correction for unequal sample size (Kramer, 1956) was used to determine which group means were significantly different. A paired t-test was used to compare the maximal effects and pD* values of the arteries to those of the veins in each group. A significance level of p < 0.05 was used for all tests. Each animal was accepted for this study on the basis of predetermined criteria: mean blood pressure greater than 80 mm Hg, heart rate greater than 240 bpm, rectal temperature between 35 and 37”C, and a cremaster preparation with good arterial, venule and capillary blood flow. In addition, variation in the average control values (vessel diameters, blood pressure, and heart rate) from dose to dose could not exceed 20%. : 3. Results Norepinephrine produced concentration dependent constrictions of the small vein and artery. Pig. 1 shows the results of one experiment for the effects of topically applied norepinephrine on small artery and vein diameters and on mean arterial blood pressure and heart
333
, rate. Increasing concentrations of norepinephrine produced progressively greater small vessel constriction until maximal constriction was reached at a concentration of 30 X lo-’ M. During the recovery period the vessels returned to control diameters. Topical application of norepinephrine had no significant effect on heart rate or blood pressure in any of the 5 groups of animals. A comparison of control data for the three anesthetic groups without cocaine (table 1, first 3 rows) demonstrates that there were no significant differences @ > 0.05) in mean arterial blood pressure, heart rate or control artery and vein diameters between PB and U-Cl animals. Urethane-anesthetized animals (U) had a lower mean arterial blood pressure than animals anesthetized with either U-Cl or PB. In addition, the U group had significantly larger arteries than U-Cl animals and a tendency toward larger veins and arteries than either U-Cl or PB animals. Calculation of correlation coefficients indicated that there were no significant correlations between vessel diameter and pD* value for either the small vein or the small artery in any of the groups. Topical application of cocaine to the cremaster muscle (table 1, bottom 2 groups) did not affect the mean arterial blood pressure, heart rate or control diameters of the arteries or veins in pentobarbital (PB vs PBcoc) or urethane--chloralose (U-Cl vs U-Cl-cot) animals. In both PB-coc and U-Cl-cot, the addition of cosine had no significant effect on vessel control diameters. There were no statistically significant differences @ > 0.05) in the maximal constriction produced by norepinephrine in the arteries (165% constriction) or in the veins (-40% constriction) among the 3 groups of animals without cocaine (table 2, first 3 rows). However, within each group, maximal constrictions in the arteries were significantly greater (p < 0.05) than those in the veins (65%, 62% and 66% versus 41%, 47% and 38% respectively). The sensitivity of both the arteries and veins to norepinephrine were similar in PB
F.N. MILLER, D.L. WIEGMAN
334
NOREPINEPHRINE
(M x IO-’ 1
1.0
30.0
100.0
t
t
t
‘\ 80 t
;_
L t
Lf
60
t
ARTERY L
I_J
40 t 120
x
100 450 410 370 r I
t
0
100
50
TIME
OF
150
EXPERIMENT
200
(minutes)
Fig. 1. Mean arterial blood pressure, heart rate and small artery and vein diameters are given for one experiment in which the rat was anesthetized with pentobarbital. For each concentration of norepinephrine indicated at the top of the graph there was a 5-min control period, addition of norepinephrine to the bath (indicated by arrows) to give the desired molar concentration, and a lo-min response period. During the 20-min recovery periods, no data were collected while vessels returned to near control diameters.
TABLE 1 Data II + S.E.M. for the control values in each of the 5 experimental groups. Animals were anesthetized with pentobarbital (PB), urethane--chloralose (U-Cl) or urethane (U). In 2 additional groups cocaine (cot) was added to the cremaster bath (PB-cot and U-Cl-cot). Group
n
Mean arterial blood pressure (mm Hg)
Heart rate (bpm)
Control artery diameter (pm)
Control vein diameter (rm)
PB U-Cl U PB-cot u-Cl-cot
7 10 8 7 10
104* 97 + 85 * 105 * 98 +
429 402 400 434 375
102 97 114 114 97
156 152 164 155 168
3 2 2 3 4
* * + * ?
12 23 26 13 25
+ * + + +
5 4 5 5 6
f 11 f 6 f 8 * 8 f 8
Statistical analyses (p < 0.05). (1) Comparisons among the 3 anesthetic groups without cocaine (PB, U-Cl, U): mean arterial pressure, U < PB, U-Cl; artery diameter, U-Cl < U. (2) Comparisons between anesthetic groups without and with cocaine (PB, PB-cot; and U-Cl, U-Cl-cot): no significant differences.
335
SMALL VESSEL RESPONSES TO NOREPINEPHRINE TABLE 2
Data represent ?I r S.E.M. for the effects of norepinephrine on small arteries and veins in the rat cremaster. Animals were anesthetized with pentobarbital (PB), urethane-hloralose (U-Cl) or urethane (U). In 2 additional groups cocaine (cot) was added to the cremaster bath (PB-cot and U-Cl-cot). Group
PB U-Cl U PB-cot u-Cl-cot
n
7 10 8 7 10
Vein
Artery Maximal percent constriction
PD,
65 * 62+ 66* 63+ 66 +
6.4 6.7 6.1 7.5 6.9
2 2 2 2 3
* 2 + * +
0.16 0.17 0.10 0.20 0.14
Statistical analyses (p < 0.05). (1) Comparisons among anesthetic vein pD2, U < U-Cl. (2) Comparisons between anesthetic groups U-Cl, U-Cl-cot): artery pD2, PB < PB-cot; vein pD2, PB < PB-cot. arteries (A) and veins (V): maximal percent constriction, V < A for
and U-Cl (no statistical differences in pD2 values). However, the U group had lower pD2 values (p < 0.05) in both the small artery and vein than the U-Cl group (the statistical comparison of artery and vein pD2 values between U and PB gave 0.05 < p < 0.10). The addition of cocaine did not alter the sensitivity or maximal percent constriction of the vessels in urethane-hloralose-anesthetized animals (U-Cl vs U-Cl-cot). In contrast, in pentobarbital-anesthetized animals (PB vs PB-cot), the addition of cocaine resulted in a significant increase in sensitivity to norepinephrine in both the small artery and the small vein; however, there was no effect of cocaine on maximal percent constriction.
4. Discussion This study quantitates in vivo changes in small vessel diameter as a response to graded concentrations of topically applied norepinephrine in the cremaster muscle of rats anesthetized with pentobarbital, urethane-chloralose or urethane. Since this method of application did not produce any response in heart rate or blood pressure there was neither drug induced pressure changes nor reflex sympa-
Maximal percent constriction
pD2
41 * 47 f 382 37 k 38+
7.0 7.1 6.5 7.6 7.1
4 4 2 4 2
+ f. * r *
0.16 0.06 0.07 0.17 0.14
groups (PB, U-Cl, U): artery pD2, U < U-Cl; without and with cocaine (PB, PB-COC; and (3) Comparisons within each group between all groups; pD2, A < V for PB, U-Cl and U.
thetic nerve activity to influence the small vessel diameter during norepinephrine application. In each of the 5 groups of animals (PB, PBcot, U-Cl, U-Clcoc, and U) maximal arterial constriction (165% was significantly greater than maximal venous constriction (540%). These data are consistent with those of Altura (1971) in the mesentery of the anesthetized rat but contrast with our data (Miller and Harris, 1975) in the S.C. wing tissue of the unanesthetized bat where we found no significant difference in maximal constriction of small arteries (57 f 6%) and veins (67 f 3%). Our vascular data (pD2 values, table 2) also indicate that anesthetics may differentially influence the sensitivity of the microvasculature to norepinephrine without affecting the maximal contractile response. The order of sensitivity to topically applied norepinephrine for both the small artery and vein was U-Cl 2 PB > U. Since there were no differences in the maximal percent constriction for either vessel among our three anesthetic groups, decreased sensitivity to norepinephrine in the artery and vein of the urethane group cannot be attributed to ‘depression’ of vascular smooth muscle. Neuronal uptake of norepinephrine should
F.N.
336
decrease the effective concentration of any given dose of norepinephrine at the receptor site of an innervated vessel and therefore shift the concentration-response curve to the right and give a smaller pD2 value. In PB, U-Cl, and U animals without cocaine, the small artery was significantly less sensitive (paired comparisons) than the small vein to norepinephrine (table 2). However, in the presence of cocaine which blocks neuronal uptake and should therefore increase the pD2 values of innervated vessels, there was no difference in artery and vein sensitivity to norepinephrine in PB and U-Cl animals. Thus, with PB anesthesia neuronal uptake (PB vs PB-coc) appears to have a greater effect on the pD2 of the small artery (6.4 f 0.16 vs 7.6 + 0.17) than on the pD2 of the small vein (7.0 f 0.16 vs 7.6 + 0.17) although the sensitivity of both vessels is increased. In the U-Cl group, cocaine produces only a slight (not significant) increase in vascular sensitivity of the artery (6.7 ? 0.17 vs 6.9 f 0.14) and no change in that of the vein (7.1 + 0.06 vs 7.1 + 0.14). The data in both pentobarbitaland urethane-chloralose-anesthetized animals supports previous data in the bat wing (Miller and Harris, 1975) that neuronal uptake has a greater influence on vascular sensitivity of the artery than of the vein of a small vessel pair. The minimal effect of cocaine in the urethane-chloralose-anesthetized rats suggests that functionally, there was substantial blockade of neuronal uptake already present with this combination of anesthetics. If blockade of neuronal uptake by urethane-chloralose was the only important influence of this anesthetic combination on the small vessel response to norepinephrine, the pD2 values for U-Cl-cot and PB-cot should be the same. However, in our data the pD2 values for PBcot are significantly greater than those for U-Clcoc. These data, then, indicate that there is an additional mechanism which acts to inhibit small vessel responses to norepinephrine during anesthesia with this combination of urethane and chloralose. Our data from the urethane group support
MILLER.
D.L. WIEGMAN
the hypothesis that urethane anesthesia has an inhibitory effect on vascular responses. Both mean arterial blood pressure and small vessel sensitivity were lower in urethane-anesthetized animals than those in urethane--chloralose-anesthetized animals. A mechanism for this inhibition may involve high levels of renin (Pettinger et al., 1975) or altered calcium metabolism (Peng et al., 1972) - factors associated with urethane anesthesia. These results are consistent with other data which suggest that urethane (Brezenoff, 1973) inhibits and chloralose (Monroe, 1963) augments cardiovascular responses. Thus, during urethanechloralose anesthesia there appear to be at least two opposing mechanisms which affect the vascular responses to norepinephrine. One of these appears to be an ‘inhibitory effect’ which is produced by urethane. The second appears to represent an ‘excitatory effect’ on small vessel sensitivity through an action of chloralose and/or the chloralose-urethane combination to functionally block neuronal uptake of norepinephrine.
Acknowledgements Dr. Wiegman is a recipient of a National Research Service Award, HL 05184 from the Public Health Service. This research was supported in part by Public Health Service Grants HL 12614 and 13207. The authors wish to express their thanks to David Andersen, John Krstansky, Wayne Lamm, Walter Parrish and Randall Sheller for their fine technical assistance.
References Altura, B.M., 1971, Chemical and humoral regulation of blood flow through the precapillary sphincter, Microvasc. Res. 3, 361. Brezenoff, H.E., 1973, Cardiovascular responses to noradrenaline in the rat before and after administration of various anesthetics, Brit. J. Pharmacol. 49, 565. Harris, P.D., E.K. Greenwald and P.A. Nicoll, 1970, Neural mechanisms in small vessel response to hemorrhage in the unanesthetized bat, Amer. J. Physiol. 218, 560. Harris, P.D., L.F. Hodoval and D.E. Longnecker,
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1971, Quantitative analysis of microvascular diameters during pentobarbital and thiopental anesthesia in the bat, Anesthesiology 35, 337. Harris, P.D., L.F. Hodoval and D.E. Longnecker, 1972, Microvasculature responses to carotid occlusion after pentobarbital, urethane, and a-chloralose , Microvasc. Res. 4, 325 (abstr.). Kramer, C.Y., 1956, Extension of multiple range tests to group means with unequal numbers of replications, Biometrics 12, 307. Miller, F.N. and P.D. Harris, 1975, Sensitivity of subcutaneous small arteries and veins to norepinephrine, and epinephrine and isoproterenol in the unanesthetized bat, Microvasc. Res. 10, 340.
Miller, L.C., T.J. Becker and M.L. Tainter, 1948, The quantitative evaluation of spasmolytic drugs in vitro, J. Pharmacol. 92, 260. Monroe, R.R., G.V. Balis and E. Ebersberger, 1963, The hypnotic effects of alpha and beta chloralose in rats, Curr. Therap. Res. 5, 141. Peng, T.-C., C.W. Cooper and P.L. Munson, 1972, The hypocalaemic effect of urethane in rats, J. Pharmacol. Exptl. Therap. 182, 522. Pettinger, W.A., K. Tanaka, K. Keeton, W.B. Campbell and S.N. Brooks, 1975, Renin release, an artifact of anesthesia and its implications in rats, Proc. Sot. Exptl. Biol. Med. 148, 625.
SMALL VESSEL RESPONSES