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Sensitivity of the Large Blood Vessels of the Rabbit to i-Epinephrine and i-Norepinephrine By J O H N A. BEVAN, M.B., B.S. With the technical assistance of J. V. Osher, Pli.B., B.A. T Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 jugular vein; the first 2 cm. of the common iliac and femoral arteries, the lower lobar pulmonary artery, and the anterior and posterior mesenterie arteries; the caudal half of the inferior vena cava; the abdominal aorta distal to the origin of the renal arteries; and the whole of the celiac artery and the adjacent part of its gastrohepatic branch. The width of the strips prepared from the largest arteries and from the veins was approximately 0.5 em.; from the medium-sized vessels, such ns the renal artery, 0.3 to 0.4 cm.; and from the smaller vessels, such as the saphena artery, 0.25 to 0.15 em. With available apparatus, it was possible to examine four preparations at a time. The thoracic aorta, which was used throughout as a standard, and three other vessels were removed from each animal and mounted in four separate baths. They were attached by ligatures to isotonic levers that wrote simultaneously on the same kymograph. These levers were counterbalanced to exert tensions between 0.5 to 3.0 Gin. on the preparations. A pilot experiment was conducted with a preparation from each anatomically distinct vessel to determine the load for optimum contraction. All preparations were mounted in bicarbonate buffered Krebs saline solution containing glucose 200 Mg. per cent w/v and disodium versenate 0.2 Mg. per cent w/v, gassed with 5 per cent carbon dioxide in oxygen and maintained at 39 C. A helical strip cut from a thin-walled vessel, such as a vein or small artery, presents certain practical problems when contractions are recorded by means of a lever writing on a smoked drum. The mass of muscle contained in a thin strip is small and the lowest tension acceptable with this method' of recording (0.5 Gm.) is probably greater than the optimum. As a result, the preparation appears greatly stretched and does not exhibit an acceptable magnitude of contraction. Such a strip tends to curl along its longitudinal axis. A simple muscle holder was designed to double the mass of tissue that might be contained within the tissue bath and to reduee this tendency to curl. The muscle strip was looped over a bar and each end attached 0.5 cm. apart to the holder. The attachment was by pressure along the entire width of the strip. This bar, a short piece of polyethylene tubing, was attached by silk thread H E LARGE arteries and veins do more than conduct blood to and from the heart. They act as central, elastic, volume reservoirs. Their walls contain vascular smooth muscle under nervous 1 and humoral control 2 and also many of the receptors that maintain homeostasis. The tone of this smooth muscle varies in response to pharmacological and humoral stimuli and, consequently, changes the activity of the associated volume or stretch receptors. 3 ' * This paper presents a comparison of some of the pharmacological parameters of the response to Z-epinephrine and Z-norepinephrine shown by the smooth muscle of the large arteries and veins in the rabbit. The results of this comparison suggest that there is a central group of vessels, both arteries and veins, that contain smooth muscle in which these parameters are identical. This smooth muscle is more sensitive to Z-norepinephrine than to Z-epinephrine. The smooth muscle of other arteries and veins is less sensitive to both these amines, but is more sensitive to /-epinephrine than to Z-norepinephrinp. Methods The details of the experimental method, unless otherwise stated, were similar to those previously described by Bevan.5 A total of 30 rabbits of both sexes, weighing 2.0 to 2.5 Kg., were used. Helically cut vessel strips were prepared from 17 of the largest- and medium-sized blood vessels of the rabbit. The following parts of the vascular tree were utilized: the whole of the braehial, facial, main pulmonary, renal, and saphena arteries and portal vein; the middle third of the common carotid artery, the thoracic aorta, and the external From the Department of Pharmacology, University of California at Los Angeles Medical Center, Los Angeles, California. Supported by Grant H-4301 from the U. S. Public Health Service. Receiver! for publication January 23, 1961. 700 Circulation Research, Volume IX, May 1961 SENSITIVITY OF LARGE BLOOD VESSELS Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 to the recording lever. The opposing surfaces of the vessel were sufficiently separated to allow adequate circulation of the bath solution. Preliminary experiments using standard strips from the thoracic aorta, showed that this method of mounting the preparation did not significantly alter the characteristics of the response to Z-epinephrine. Tissues were allowed to equilibrate in the Krebs saline solution two hours before use. Contractions were then elicited simultaneously from all four preparations every hour. When contractions were completed, the tissues were washed and then rested without tension until 20 minutes before the next contractions were elicited. Contractions to Z-epinephrine and Z-norepinephrine were measured alternately. A minimum of four groups of measurements (two of Z-epinephrine and 2 of Z-norepinephrine) were made with each preparation. A total of at least six responses to each drug was obtained for each anatomically distinct vessel. It has been shown that the maximum height of contraction to Z-epinephrine may be calculated from the responses to three successive submaxinial doses of the drug.5 This calculation depends on the previously shown fact" that experimental data satisfactorily represented by the probit transformation, which is true of the aorta strip response to Z-epinephrine, may be equally well fitted to the logistical distribution. From this relationship, it may be shown that if D is the drug concentration producing a response, E, and A and B are constants, then if 1/E is plotted against D~B, a. straight line is obtained with slope l/E max e A and intercept 1/Emax. The value for B is determined empirically by trial and error to give the best straight-line fit. The relationship between E and D is solved graphically to give E max . B is proportional to the slope of the dose-response curve. The sensitivity or median effective dose is obtained by extrapolation from a plot of logarithm dose and probit response. With the aorta strip, it has been shown that the maximum response to Z-epinephrine calculated in this manner is the same as the maximum response obtained experimentally using a large dose, 1 mg. or more of the drug. In these experiments, it was also confirmed that this relationship held with Z-norepinephrine and with helical preparations from other vessels. The bitartrate salts of the levo-isomers of epinephrine and norepinephrine were used throughout these experiments. An inference of significance was made when P < 0.05. Results The characteristics of response of 17 anatomically distinct vessels to Z-epinephrine and /-norepinephrine were determined and compared with those obtained from the thoracic Circulation Research, Volume IX. May 1961 701 aorta of the same animal at the same time. Comparisons were made of the median effective dose, the slope of the logarithm doseprobit response curve, and the ratio of the maximum heights of contraction to the two amines. By this method, some of the causes of variation, namely, that between individual animals and that due to the length of survival of the muscle strip, were reduced to a minimum. On the basis of these measurements, the vessels were divided into two groups: (a) those in which these pharmacological characteristics were identical within 95 per cent confidence limits7 with those of the thoracic aorta, and (b) a group in which they differed. The results of these measurements are illustrated graphically in figure 1. Vessels Containing Smooth Muscle with Pharmacological Characteristics Identical with Those of the Thoracic Aorta In these experiments, the mean median effective dose of Z-epinephrine for the thoracic aorta obtained three hours after the sacrifice of the animal was 0.0330 /ig./ml. and that for Z-norepinephrine was 0.0241 ^.g./ml. When the median effective doses for Z-norepinephrine and Z-epinephrine were compared in pairs from the same animal, there was a significant difference between them." The slopes of the logarithm dose-probit response curves and the maximum height obtained with the two sympathomimetic amines were identical. Within 95 per cent confidence limits, these parameters when measured in the following vessels were the same as in the thoracic aorta: abdominal aorta; the femoral, main pulmonary, and lobar pulmonary arteries; some of the preparations of the renal arteries; the external jugular vein; and the inferior vena cava. The smooth muscle of the common carotid and saphena arteries was identical in all respects except that the median effective dose of Z-norepinephrine was two to three times that for the thoracic aorta. The median effective dose for the smooth muscle of the external jugular vein was identical with that of the aorta, except that the slope of the logarithm doseprobit response curves for the response of the vein to both amines was more steep. 702 BEVAN FEMORAL MAIN li AORTA ABDOMINAL ARTERY PULMONARY INFERIOR VENA EXTERNAL RENAL ARTERY JUGULAR VEIN ARTERY ARTERY SAPHENA ARTERY PORTAL . EPINEPHRINE -I ILIAC 1 1 1 BRACHIAL 1 ARTERY ARTERY . NOREPINEPMRINE 1 VEIN COMMON i i i i i 1i li il !l i I t t i 1 ARTERY Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 POSTERIOR ANTERIOR CELIAC ARTERY CAVA CAROTID FACIAL 1 1 1 1 1 1 li AORTA PULMONARY LOBAR SLOPE SENSITIVITY VESSEL THORACIC MESENTERIC MESENTERIC ARTERY - 1 i l'i i i i ARTERY i i 1 ARTERY 1 C M.E.D. VESSEL 2 3 S i 10 20 M.E.D. THORACIC AORTA M.E.D.THORACIC AORTA i -050 -025 0 S L O P E v e s s E L - SLOPE THOR.CC AORTA M.E.D.THORACIC AORTA Figure 1 Summary of the sensitivity (median effective dose [M. E. D.]J and the slope of the doseresponse curve of the contractile response to \-norepinephrine and \-epinephrine in strips prepared from various vessels of the rabbit. The thoracic aorta was used as a standard. Vessels Containing Smooth Muscle with Pharmacological Characteristics Differing from Those of the Thoracic Aorta This group of vessels tested were less sensitive to Z-epinephrine and Z-norepinephrine than the thoracic aorta, and, in contrast to the previous group, were more sensitive to Z-epinephrine than Z-norepinephrine. This change was greatest in the ventral branches of the abdominal aorta, the anterior and posterior mesenteric, and the celiac arteries (fig. 1). Some strips of the renal arteries had properties similar to the thoracic aorta, others different. These latter preparations showed a greater sensitivity to Z-norepinephrine than /-epinephrine. The slope of the logarithm doseprobit response curve for both amines was significantly reduced in preparations from the anterior and posterior mesenteric, celiac, facial, and brachial arteries. This change of slope was most marked with the celiae artery. In figure 2, the logarithm dose-probit response curves are shown for the action of Z-epinephrine and Z-norepinephrine on the thoracic aorta and celiac artery of the same animal. It shows that the smooth muscle of the thoracic aorta is more sensitive to Znorepinephrine than Z-epinephrine, and that the slopes of the dose-response curves are parallel. The smooth muscle of the celiac artery is less sensitive to both amines, particularly Z-norepinephrine, and the slopes of the dose-response curves are more steep. Discussion This study suggests that there is a group of the larger arteries and veins that contain smooth muscle with the same parameters of response to Z-epinephrine and Z-norepinephrine : namely, the thoracic aorta; the abdominal aorta; the common carotid, femoral, main pulmonary, lobar pulmonary, and saphena arteries; some preparations of the renal arteries; the external jugular vein; and the inferior vena cava. The other vessels tested, the common iliac, brachial, facial, anterior and posterior mesenteric and celiac arteries, and the portal vein, are less sensitive and have Circulation Research, Volume IX. May 1961 703 SENSITIVITY OF LARGE BLOOD VESSELS Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 a steeper dose-response curve. "Whereas the vessels of the first group are more sensitive to Z-norepinephrine, those of the latter group are more sensitive to Z-epinephrine. The median effective dose is used as a measurement of sensitivity. Consequently, the sensitivity of vascular smooth muscle determined under the conditions of these experiments is independent of the magnitude of contraction. Sensitivity is also independent of tension.3 These differences in sensitivity may not be correlated with vessel diameter, for the femoral artery, although more sensitive than the posterior mesenteric, is of smaller diameter. Neither may they be correlated with vessel-wall thickness, for the wall of the anterior mesenteric artery is thicker and the sensitivitylower than the inferior vena cava, which contains smooth muscle with pharmacological properties identical to that of the aorta. No profitable conclusions may be drawn from a comparison of the absolute response of the smooth muscle from the different arterial strips. The maximum height of response, but not the sensitivity, is closely related to the tension applied,3 but with these strips the actual tension applied to the smooth muscle is not known. The maximum response is dependent not only on the dimensions of the tissue strips and the proportion of muscle within their walls, but also on the orientation of the fibers and the amount, type, and local characteristics of the connective-tissue elements. The embryonic cells that develop into the smooth muscle of the tunica media of the arterial and venous systems originate from local mesenchyme.s The older concept9—that most blood vessels develop as buds from the aorta-has now been proved wrong. It is of interest that the arteries and veins that develop from the paired dorsal aortae, the pharyngeal branchial arches, and the cardinal veins, all closely associated in development, tend to have the same sensitivity in the adult. The vessels that contain smooth muscle derived from mesoderm situated ventrally in the embryo, the anterior and posterior mesenterie and celiae arteries and the portal vein, have a Circulation Research, Volume IX. May 10G1 THORACIC AORTA & I EPINEPHRINE O 1NOREPINEPHRINE CEUAC ARTERY A • /EPINEPHRINE l.NOREPINEPHRINE MICROGRAMS OF AMINE ADDED TO 45 ml. BATH Figure 2 Relationship between contractile response and dose of 1-norepinephrine and \-epinephrine in strips prepared from the thoracic aorta and celiae artery. lower sensitivity than the preceding group. The muscle of the umbilical artery is also derived from ventral mesoderm, and in the adult some of this muscle is incorporated in the common iliac artery.10 This vessel has a lower sensitivity than the vessels proximal and distal to it, the abdominal aorta and the femoral artery. Changes in the mechanical properties of a vessel wall modify the activity of associated baro- or stetch receptors.11 All true pressoreceptors12'13 are found in the walls of the group of vessels that from this study appear to be most sensitive to the physiological amines. Consequent^, pressoreceptor activity will be affected by a wider range of amine levels than if they were situated in peripheral and less sensitive vessels. Summary Some of the pharmacological properties of the smooth muscle of 17 anatomically different vessels of the adult rabbit have been compared using helical strips under in vitro conditions. Using the median effective dose and the slope of the logarithm dose-probit response curve for Z-epinephrine and Z-norepinephrine as criteria, these vessels may be divided into two groups. The vascular muscle of the thoracic and abdominal aorta, the femoral, main pulmonary and lobar pulmonary arteries, some BBVAN 704 Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 preparations of the renal arteries and the inferior vena cava exhibit, within 95 per cent confidence limits, identical characteristics. They are more sensitive to Z-norepinephrine than £-epinephrine, and the slopes of the doseresponse curves to these amines are the same. The muscle of the common carotid and saphena arteries and the external jugular vein is very similar to this group, but not identical. The vascular muscle of the common iliac, brachial, facial, anterior and posterior mesenterie, and celiac arteries, and the portal vein is less sensitive, and the slope of the doseresponse curve more steep to these amines than the previous group. They are more sensitive to Z-epinephrine than Z-norepinephrine and the slopes of the dose-response curves for these amines are not the same. References 1. WECKMJN, N.: Local constriction and spasm of large arteries elicited by hypotlialamic stimulation. Experientia 16: 34, 1960. 2. WIGGERS, C. J., AND WECRIA, E.: Active changes in size and distensibility of the aorta during acute hypertension. Am. J. Physiol. 124: 603, 1938. 3. ALEXANDER, B. S.: Influence of constrictor drugs on the distensibility of the splanchnic venous system analyzed on the basis of an aortic model. Circulation Research 2: 140, 1954. 4. HEYMANS, C, AND VAN DEN HEUVEL-HEYMANS, G.: New aspects of blood pressure regulation. Circulation 4: 581, 1951. 5. BEVAN, J. A.: Use of the rabbit aorta strip in the analysis of the mode of action of Z-epinephrine on vascular smooth muscle. .T. Pharmacol. 129: 417, 1960. 6. FINNEY, D. J.: Statistical Method in Biological Assay. New York, Hafner Publishing Co., 1952. 7. DIXON, W. J., AND MASSEY, F. J.: Introduction to Statisticiil Analysis, ed. 2. New York, McGraw-Hill Book Co., Inc., 1957. 8. LE GROS CLARKE, W. E.: Tissues of the body, ed. 4. Oxford, Clarendon Press, 1958. 9. BREJIER, J. L.: Development of the aorta and aortic arches in rabbits. Am. J. Anat. 13: 11], 1912. 10. BAXTER, J. S.: Frazer 's Manual of Embroyology. London, Bailliere, Tindall and Cox, 1953. 11. PETERSON, L. H.: Regulation of blood vessels. Circulation 21: 749, .1960. 12. AVIADO, D. M., JR., AND SCHMIDT, C. F . : Reflexes from stretch receptors in blood vessels, heart and lungs. Physiol. Rev. 35: 247, 1955. 13. Boss, J., AND GREEN, J. H.: Histology of the common carotid baroceptor areas of the cat. Circulation Research 4: 12, 3956. Circulation Research, Volume IX, May 1901 Sensitivity of the Large Blood Vessels of the Rabbit to l-Epinephrine and l-Norepinephrine JOHN A. BEVAN Downloaded from http://circres.ahajournals.org/ by guest on April 29, 2017 Circ Res. 1961;9:700-704 doi: 10.1161/01.RES.9.3.700 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1961 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. 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