on Aortic Contraction and Relaxation in Rats1

Transcription

1 yjfl.3555/5i)/21424)355$(fl./o T#{248}JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright 198 by The American 5ociety for Pharmacology and Experimental Therapeutics VoL 214. No. 2 Prinied in U.S.A. Effects of Different Stages of Aortic Coarctation Hypertension on Aortic Contraction and Relaxation in Rats1 FONG M. LAI, TARAK TANIKELLA, LUCIEN THIBAULT, PETER S. CHAN and PETER CERVONI American Cyanamid Company, Medical Research Division, Lederle Laboratories, Cardiovascular Biological Research Department, Pearl River, New York Accepted for publication May 8, 198 ABSTRACT Lal, Fong M., Tarak Tanikella, Lucien Thibault, Peter S. Chan and Peter Cervoni: Effects of different stages of aortic coarctation hypertension on aortic contraction and relaxation in rats. J. Pharmacol. Exp. Ther. 214: , 198. Contractile responses to norepinephrine, serotonin and potassium (K) and relaxant responses to isoproterenol and papaverine were studied in vitro with spirally cut thoracic aortic strips from aortic coarcted hypertensive rats (AHR) 2, 6, 1 4 and 28 days postoperatively and compared to time-matched, shamoperated normotensive controls. At every stage after coarctation, the rats developed hypertension with elevated plasma renin activity. In response to stimulation by norepinephrine and serotonin, aortic strips from 2 to 28 day AHR developed the same tension as controls, whereas aortas of 6 and 1 4 day AHR had reduced maximal responses. For K-stimulated aortic The vascular reactivity of the spirally cut rat aorta has been studied from animals with genetic hypertension, such as spontaneously hypertensive rats (SHR), and from experimentally induced hypertension, such as deoxycorticosterone acetate (DOCA)-salt hypertensive rats and renal hypertensive rats (RHR). Although many studies have been done with these animals, published data on the responsiveness of vascular tinsues to norepinephrmne (NE) or potassium chloride (K) are conflicting. The contractility of aortic strips from SHR has been found to decrease (Spector et al., 1969; Shibata et al., 1973) or not to change (Hallback et al., 1971) in response to stimulation with NE as compared to strips from normotensive controls. Aortic strips from DOCA-salt or renal hypertensive rats have been found to develop either the same or less tension (Redleaf and Tobian, 1958) or develop more tension (Gordon and Nogueira, 1962) in their responsiveness to NE. On the other hand, a reduced vasorelaxant ability to vasodilators was reported in SHR and RHR which suggested that vascular relaxant mechanisms may be impaired in both types of hypertension (Cohen and Berkowitz, 1976). Despite studies of the various Received for publication November 1, I Parts of these results have been presented at the 7th International Congress of Pharmacology, Paris, 1978 and The American Society for Pharmacology and Experimental Therapeutics Fall Meeting, Houston, Texas, August 13 to 17, strips, maximal contractile force was decreased at 6 day AHR only. Relaxation by isoproterenol and papaverine in serotonincontracted aortas was the same in AHR and normotensive controls 2 and 28 days postoperatively but was reduced at 6 and 1 4 days. The demonstrated changes of vascular contractility and relaxation in AHR is a hypertensive stage-dependent phenomenon. It is speculated that 6 and 1 4 days after coarctation the diminished relaxant ability of the aortas helps to maintain the elevated blood pressure and the diminished sensitivity to contractile stimulants is a protective mechanism in response to the elevated blood pressure. The return of normal contraction and relaxation to the agonists in the chronic stage of hypertension may possibly reflect an adaptive change to the prolonged stimulus of the elevated blood pressure that aortic tissue had undergone in order to maintain normal physiologic functions. models of hypertension, the change in vascular reactivity to vasoconstrictors or vasodilators at different stages in the development of hypertension in any model has received little attention. The present studies describe the aortic responsiveness to various vasoconstrictors and vasodilators during the development of hypertension after complete ligation of the abdominal aorta between the origin of two renal arteries. The reactivity of aortic strips from hypertensive rats was compared to their respective time-matched, sham-operated normotensive controls at various time periods after aortic coarctation. Methods Male Sprague-Dawley rats (27-3 g) purchased from Charles River Breeding, Inc., Wilmington, MA were used in the present study. Induction of hypertension. Hypertension was induced by complete ligation of the aorta between the two renal arteries according to the method of Rojo-Ortega and Genest (1968) with minor modification of the surgical procedure. In brief, the methohexital (Brevital sodium, Eli Lilly and Company, Indianapolis, IN)-anesthetized rats were placed on the right side of the body. A 2- to 3-cm incision was made in the lateral abdominal wall and a retractor was placed in the incision. The kidney was gently pushed aside to locate the renal artery and the aorta was ligated with no. silk suture. The abdominal muscle was closed with suture and the skin was closed with wound clips. Sham-operated rats went through the same surgical procedures except the aortas were not ligated with suture. After surgery, the rats were returned to cages

2 198 Hypertension and Vascular Reactivity 389 and maintained on Purina Laboratory Chow and tap water ad libitum until use. The experiments were carried out at 2, 6, 14 and 28 days after aorta coarctation and the degrees of hypertension for these periods were referred to as acute, early, middle and chronic stages of hypertension, respectively. Blood pressure measurement. Arterial blood pressure was measured directly through a PE-5 catheter which was implanted into the ascending aorta through the left carotid artery and exteriorized at the back of the neck. The surgical implantation of the catheter was performed under phenobarbital anesthesia 24 hr before blood pressure recording. Blood pressure was measured in conscious, unrestrained animals which were moving about freely in the cage. Plasma renin activity (PRA) determination. After the recording of blood pressure, 3 ml of blood was withdrawn from the arterial catheter into chilled tubes which contained disodium EDTA (3.4 mm). The blood sampling was usually performed between 8 and 1 A.M. The blood samples were centrifuged at 1, rpm for 15 mm at 4#{176}C. Aliquots of 1 nil of plasma were kept frozen until assay (samples were usually assayed within 2 weeks). The PRA was measured by the radioimmunoassay method ofhaber et al. (1969) by using GammaCoat- [ 25IJPlasma Renin Activity Kit (Clinical Assays, Cambridge, MA). Briefly, the angiotensin I generation medium consisted of 1 ml of thawed plasma, 1 tl of phenylmethylsuffonyl fluoride solution and 1 It1 of maleate buffer (.7 M; ph 6). The reaction mixture was then incubated for 3 mm at 37#{176}C and the enzymatic reaction was stopped by placing the tubes in ice-cold water. The generated angiotensin I was quantitated by radioimmunoassay in GammaCoat tubes. Duplicate determinations were performed for each incubation. The results were expressed as nanograms of angiotensin I generated per milliliter of plasma per hour of incubation. Aortic strip preparation. After blood sampling, rats were decapitated. The thoracic aorta was removed, placed in Krebs solution and cleared of blood, fat and connective tissues. The aorta was helically cut to equivalent lengths (3 cm) and widths (2.5-3 mm) by the method of Furchgott and Bhadrakom (1953). Strips were suspended in a 1-nil organ bath containing an oxygenated (95% 2-5% C2) Krebs solution of the following compositions (millimolar): NaC1, 12.6; KC1, 4.6; CaCl2. 2H2, 2.3; KH2PO4, 1.1; Mg54.7H2, 1.1; NaHCO3, 24.9; and dextrose, 1L5. An initial tension of 1 g was applied to each aortic strip. The strips were allowed to equilibrate for 2 hr before drugs were added to the bath. Changes in tension were recorded with a force-displacement transducer (Grass FT 3) coupled to a Grass polygraph. Concentrationresponse curves to NE, K and serotonin (5-HT) were conducted by cumulative additions of the compounds. At the end of the experiment, the tissues were carefully removed, blotted with ifiter paper and weighed. Relaxation experiments were carried out by the procedure of Cohen and Berkowitz (1974). An equivalent degree of tension was first produced with 5-HT in each tissue. When the contraction reached a steady state, the vasodilators were added cumulatively. Relaxation of the contracted tissue back to the base line represented 1% relaxation. The tissues which were exposed to both NE and K were not used again to test for 5-HT contraction or for relaxation. The relaxation results were generated from the tissues exposed only to 5-HT. Statistical analysis. Student s t test for unpaired dat.a was used to compare the data between hypertensive and normotensive animals. Statistical significance was taken when P was less than.5. Chemicals. The sources of the drugs used were as follows: 1-isoproterenol bitartrate, l-norepinephrine bitartrate, Sigma Chemical Company, St. Louis, MO; serotonin creatinine sulfate complex, Calbiochem, San Diego, CA; papaverine hydrochloride, Regis Chemical Company, Morton Grove, IL; and potassium chloride, Fisher Scientific Company, Fair Lawn, NJ. All doses are expressed as free base equivalent concentrations. Results Effects of am-tic coarctation on body weight, PRA and arterial blood pressure. Weight gain was much lower in the hypertensive rats than in the sham-operated controls; therefore, the body weights of the hypertensive rats were less than those of the control rats at any stage of hypertension (table 1). The complete constriction of the abdominal aorta caused a rapid increase in arterial blood pressure. Two days after the aortic constriction, the mean arterial blood pressure of the aorta coarcted animals had increased to 47% above the corresponding sham-operated controls. Rats made hypertensive by this procedure maintained this high pressure or reached even higher levels through the remainder of the 28-day experiment. PRA was also markedly elevated in the hypertensive animals. As summarized in table 1, the hypertensive rats had 6 to 14 times higher PRA than that of the sham-operated controls at different stages of hypertension. It should be noted that, although PRA was somewhat lower in samples obtained from rats after 28 days of hypertension compared to the earlier hynertensive stages, the hypertensive rats stifi had significantly greater PRA than that of time-matched, sham-operated controls. Contractile responses of aortic strips to NE. Cumulative concentration-response curves to NE were recorded in aol-tic strips from hypertensive rats and time-matched, shamoperated normotensive control rats at different stages of hypertension (fig. 1). At the acute state of hypertension, 2 days after coarctation, the aortic strips from hypertensive and normotensive rats developed the same degree of contractile force. However, 6 days after coarctation, the maximal force developed by the aortas of the hypertensive rats was reduced as compared with that of the control rats. Fourteen days after surgery, the reduced contractility to NE stifi existed in aortic strips from the hypertensive rats. At the chronic state of hypertension, 28 days after coarctation, the reduced maximal contraction to NE in the tissues from the hypertensive rats was no longer observed. Contractile response of aortic strips to K. The ability for developing maximal tension in response to K for the aortic strips from hypertensive and normotensive rats was measured in the same tissue used for NE contraction (fig. 2). Aortas of rats in the acute phase of hypertension and the control animals developed the same degree of tension. Six days after aortic coarctation, the maximal contractile force in response to K generated by the aortas from the hypertensive rats was depressed as compared to that of the controls. However, as hypertension progressed toward the middle (day 14) or the more chronic (day 28) stages of hypertension, the aortas of hypertensive rats regained their ability to develop the maximal tension to K as compared to their respective controls. Effects of the stage of hypertension on aortic tissue weight. Table 2 summarizes the data on tissue weights of aortas used in the NE and K contraction studies. Weights of the aortic strips of hypertensive and normotensive control rats increased with age (day 2 to 28). As early as 2 days after aortic coarctation, aortas from hypertensive rats weighed more than the controls and consistently weighed more than their respective controls in all stages of hypertension examined. Contractile responses of aortic strips to 5-HT. To determine if the stages-associated change in contractility to NE and K was specific only to the neurotransmitter or the depolarizing agent, the contraction produced by 5-HT was examined in the aortas from rats in acute, early, middle and chronic stages of hypertension. As shown in figure 3 during the acute stage of hypertension, aortas from normotensive and hypertensive rats developed the same degree of tension to 5-HT. As the hypertension proceeded to early and middle stages, the aortas from

3 39 Vol. 214 TABLE 1 Effects of aortic coarctation on body weight, PRA and development of arterial hypertension in rats Days after Surgery NR HR NR HR NR HR NR HR Body weight (g) ± ± ± 8.2 (16) ± ± ± ± ± 9.9 MABP (mm Hg) 19.4 ± ± 2.8** ± 4. (16) 182. ± ± ± ± ± 7.O PRA (ng Al/mi! 3.7 ± ± ± ± ± ± ± 1.1 hry (16) 31.2 ± 56 rm, sham-operated normotensive controls; HR, aorta-coarcted hypertensive rats. The number in parentheses is the total number of animals usedin both contraction and relaxation studies. The values are expressed as means ± S.E. b MABP, mean arterial blood pressure which was Calculated from the equation of diastolic pressure + one-third of pulse pressure. Al, angiotensin I.. Statistically different from the values of their respective time-matched, sham-operated controls (P <.5)..s Statistically different from the values of their respective time-matched, sham-operated controls (P <.1) #{149}-#{149}NORMOTENSIVE o-o HYPERTENSIVE DAY2 ief DAViS o : 1, DAYS DAY-2S 1#{149} ios. 1-S NOREPINEPHR$NE MOLAR) Fig. 1. Cumulative concentration-response curves for the contractile effect of NE in aortic strips from time-matched normotensive and hypertensive rats. Days after coarctation are indicated in the upper left-hand corner of curves. Each point represents the mean ± SE. of strips from 6 to 1 animals. Asterisks indicate those points significantly different at P <.5(), D <.1( #{149}) or P <.1 ( #{149} hypertensive rats developed significantly less tension than that of their corresponding controls. However, at the chronic state of hypertension, the aortas from hypertensive animals developed as much tension as that of the controls. Relaxant responses of aortic strips to isoproterenol and papaverine. 5-HT elicited a concentration-dependent contraction in aortic strips of control and hypertensive rats at different stages of hypertension as shown in figure 3. Contraction in response to 1 to 3 &M of 5-HT was therefore used to maintain an equivalent tension before the addition of the relaxant agonists. The addition of isoproterenol to the 5-HT-contracted aortas produced a concentration-dependent relaxation (fig. 4). Lsoproterenol-induced relaxation was equal in aortas taken from day 2 control and hypertensive rats except at the highest concentration of isoproterenol. At day 6, the aortas of the hypertensive rats showed a decreased ability to relax in response to isoproterenol. At 14 days, the aortas of the hypertensive rats were less responsive to isoproterenol than the sham-operated, time-matched control aortas despite a reduction in responsiveness of the latter to isoproterenol. At day 28, the aortas of the hypertensive and the time-matched control rats also showed a decreased responsiveness to isoproterenol. The variability in the responses of these preparations was considerably greater than in the preparations in the early phases of hypertension and thus there was no significant difference in the responsiveness of the aortas of the 28 day hypertensive rats and their time-matched, sham-operated controls. The same pattern also held true for papaverine-induced relaxation in the different stages of hypertension (fig. 5). Discussion The complete ligation of the abdominal aorta between the two renal arteries induces an acute, severe and sustained hypertension associated with an elevation of PRA in rats. The effect on blood pressure is in agreement with the results of Rojo-Ortega and Genest (1968). Fernandes et al. (1976) also showed that PRA reached a peak at 5 days after aortic coarctation but had returned to control level by 3 days despite an elevated blood pressure. In the present study, the elevation of PRA in hypertensive rats was observed 2 days after coarctation, reached a peak at day 6 and, although lower on day 28 than on

4 198 Hypertension and Vascular Reactivity I_ #{149}-#{149} NORMOTENSIVE o-o HYPERTENSIVE DAY2 DAV4 S I- S U. Oh TABLE 2 DAViS I I I Ii 1 io#{149} I I I I I I t I L DAY-fl -J 1#{149} 1#{149} io#{149} io#{149} KCI (MOLAR) Fig. 2. Cumulative concentration-response curves for the contractile effect of K in aortic strips from time-matched normotensive and hypertensive rats. The tissues in this study were the same as used in figure 1. Each point represents the mean ± S.E. of strips from 6 to 1 animals. Asterisks indicate those points significantly different at P < 5(), P <.1( ) or P <.1( #{149} Aortlc strip weight of rats at different stages of hypertension At the end of the experiment, In response to the stimulation by NE and K, aortic strips were carefully removed from the bath. blotted with filter paper and weighed. Aortic Strip Weighr Days after Surgery E9 PIg Sham-operated normotensive rats 13.5 ±.42 (6) 13.1 ±.46 (1) ±.89 (8) 18.6 ±.99 (8) Aorta-coarcted hypertensive rats ±.67 (6) 17.5 ±.5 (1)W ±.78 (8) ±.97 (8) a The values are expressed as mean ± S.E. for the number of tissues indicated in parentheses. S p <.5; P <.2; P <.1. day 6 and 14, did not return to control level by day 28 (table 1). The fact that elevated PRA was observed in every stage of coarctation hypertension suggests the involvement of the reninangiotensin system in the development and maintenance of this model of experimental hypertension. Recently, Garst et al. (1979) demonstrated that vascular renin activity was significantly increased, whereas PRA was reduced in the chronic twokidney Goldblatt hypertensive rat suggesting that arterial wall renin may play a role in the maintenance of this model of chronic renal hypertension. Whether vascular renin activity plays a role in the maintenance of blood pressure in the chronic stage of coarctation hypertension remains to be elucidated. In the present studies, the changes of both contractile and relaxant responses of rat aortas have been examined as a function of the stages of hypertension. The ability of the aortic strips to generate maximal tension in response to NE, 5-HT and K was found to vary with the stage of hypertension. For instance, in response to stimulation by NE and 5-HT, the maximal contractile force of aortic strips from hypertensive rats was found to be the same as controls on day 2 and 28 and less on day 6 and 14. For K-stimulated tissues, the maximal contractile force was decreased only at day 6 after aortic coarctation. Changes in electrolyte composition of the artery wall or morphological changes of vascular smooth muscle cells such as distortion and loss of filaments, accumulation of membranebound vacuoles have been proposed to account for the decrease in maximal tension development in vascular tissues from hypertensive rats (Hollander et al., 1968; Gardner and Matthews, 1969). The mechanism(s) for the inability to develop maximal tension in the hypertensive rat was not investigated in the present stiidy. The possibility that the regained ability for development of maximal contraction to agonist.s 28 days after aortic coarctation was due to the increased aortic weight was considered. Increased aortic tissue weight in hypertensive rats was observed as early as 2 days after coarctation and the weight continuously

5 392 Lai et al. Vol #{149} NORMOTENSIVE 1 OO- G-O HYPERTENSIVE DAY S- 5&- S a a 4 a V- 5O 2Sf- L.. L L DAY 14 DAY 21 - A I.._ 11 1,1O#{149} 1*1O 1*1O#{149} 11O#{149} 1,1O- I-.. o-o NMOTENSVE HYPRRTIN$IVE SEROTONIN L (MOLAR). i L. Fig. 3. Cumulative concentration-response curves for the contractile effect of 5-HT in aortic strips from time-matched normotensive and hypertensive rats. Days after coarctation are indicated in the upper lefthand corner of each set of curves. The tissues in this study were obtained from another group of rats and were used for the relaxation experiments. Each point represents the mean ± S.E. of four to six animals. - Asterisks indicate those points significantly different atp<.5. OAY#{149}14 DAY-SI.W.W ISOPROTERENOL Fig. 4. Cumulative concentration-response curves for the relaxant effect of isoproterenol in aortic strips from time-matched normotensive and hypertensive rats. Days after coarctation are indicated on top of each pair of curves. Each point represents the mean ± S.E. of four to six animals. Asterisks indicate those points significantly different at P <.5() or P <.1( ). IMOLANI increased through day 28 of the experiment (table 2). Aortic tissue with greater weight in 6 and 14 day hypertensive rats not only failed to develop more tension but, in fact, generated less tension than their respective controls with lesser weights. Thus, it is unlikely that the normal maximal contraction developed in day 28 hypertensive rats was due to increased aortic weight. The force developed by smooth muscle in response to agonist stimulation depends on its initial tension (or length) (Gordon

6 198 Hypertension and Vascular Reactivity 393 DAY-2 DAYS DAY-iS DAY-fl 2 So 5-#{149} NORMOTENSIVE -a HYPERTENSIVE 1 I J L I. J j 1,1#{149} 1*1O 1*1O. 1*1O O ,1 11O llsr PAPAVERINE Fig. 5. Cumulative concentration-response curves for the relaxant effect of papaverine in aortic strips from time-matched normotensive and hypertensive rats. The tissues used in figure 4 were used in this study. Days after coarctation are indicated on the top of each pair of curves. Each point represents the mean ± SE. of four to six animals. Asterisks indicate those points significantly different at P <.5(1 or P <.1( 1. and Nogueira, 1962). However, length-passive tension studies did not demonstrate a difference in passive stiffness between aortic strips obtained from normotensive and hypertensive rats (SHR, RHR and DOCA-salt hypertensive rats) (Halloway and Bohr, 1973). In addition, Shibata et al. (1973) reported that aortic strips from hypertensive and prehypertensive SHR showed the same extensibility to applied tension (1.5 g) as their normotensive controls. Although there was no determination of the optimal tension for each strip in this study, the applied tension of 1 g was nearly optimal for active tension development in rat aorta as documented in the literature. Therefore, the difference in the maximal tension developed by the tissues at day 6 and 14 of hypertension cannot be attributed to their initial tensions and lengths. The possibility exists that the change in the NE response in aortas from day 6 and day 14 hypertensive rats could be due to alterations in the amine uptake mechanism or in the sensitivity of beta adrenergic receptors resulting in a decreased vasoconstrictor response to NE. If either of these mechanisms were operative then the NE concentration-response curves should be done in the presence of an inhibitor of the amine uptake mechanism (e.g., cocaine) or a beta adrenergic receptor blocking agent (e.g., propranolol). However, it has been shown that rat thoracic aorta has a relatively poor adrenergic innervation compared to other mammalian species such as cat, guinea pig or rabbit (Maling et al., 1971; Kuchii et al., 1973). Neither cocaine (Maling et al., 1971; Kuchii et al., 1973) nor propranolol (Cohen and Wiley, 1977) affected the NE concentration-response curve in rat aortic strips. Thus, it is unlikely that the difference in the maximum response to NE at day 6 and 14 of hypertension is due to an alteration in the tissue disposition of NE. The results of the present study from the early and middle hypertensive stages in rats confirm the observations of Spector et al. (1969) and Shibata et al. (1973) that the contractile responses of aortic strips from hypertensive rats to NE were less than that of strips from controls. In addition, our data from the acute and chronic stages of hypertension are also in agreement with the observations ofhallback et al. (1971) and Redleaf and Tobian (1958) that aortic strips from hypertensive rats (MOLAR) develop as much tension as those of normotensive animals. Therefore, the conflicting findings by other investigators using different types of hypertensive rats (SHR, RHR or DOCA-salt hypertensive rats) may be due to the use of animals at different stages of hypertension and to a lack of comparison with the proper normotensive control rats. For example, Clineschmidt et al. (197) reported that aortic strips from matched Wistar normotensive and SHR from NIH developed identical tension in response to NE but that aortas of Wistar normotensive rats from another breeding facility developed about twice as much tension as the SHR strain. These data support the contention that proper controls are needed to make meaningful comparisons. The finding of a time element during the course of hypertension might explain, in part, the conflicting data in the literature regarding the contractility of vascular smooth muscle of hypertensive and normotensive rats. The stage-associated changes in contractile responses also holds true in the relaxation studies. These results suggest that vascular reactivity may vary during different stages of hypertension and a proper normotensive control must be used simultaneously in order to provide a meaningful comparison. The observed stage-associated changes in contraction and relaxation of aortic smooth muscle do not necessarily apply to other small arteries and arterioles. The isolated perfused mesenteric artery or the hindquarter blood vessels of SHR show a greater reactivity to NE and 5-HT (Haeusler and Finch, 1972; Lais and Brody, 1975). The isolated renal arteries of SHR also show increased sensitivity to NE (Folkow et a!., 197). On the other hand, there was no observable difference in the contractile response of isolated perfused renal arteries to 5-HT of SHR and controls (Haeusler and Finch, 1972). Thus, no definitive conclusion on the reactivity ofvascular smooth muscle in hypertension could be drawn from studies by using these small arteries. Whether the discrepancy is also due to the stage of hypertension remains to be studied. The reduced relaxant ability of the aortas at 6 and 14 days after coarctation may help maintain the elevated blood pressure and the diminished sensitivity to contractile stimulants is a protective mechanism in response to elevated blood pressure. The return of normal contractility to NE, 5-HT and K and

7 394 Laietal. Vol. 214 relaxation to isoproterenol and papaverine in the chronic stages of hypertension may possibly reflect that the aortic vascular tissue has undergone an adaptive change to the prolonged stimulus of the elevated blood pressure in order to maintain normal physiological functions. In summary, in this study it was demonstrated that 1) PRA was elevated in every stage of hypertension induced by aortic coarctation between the two renal arteries of the rat; 2) the contraction and relaxation of aortic vascular tissue was decreased during the early and middle stages of hypertension and the tissue then regained its ability to fully contract and relax in the chronic stage of hypertension; and 3) when vascular reactivity (or other parameters) is to be studied, it is necessary to examine tissues obtained from various stages of hypertension. References CLINESCHMIDT, B. V., GELLER, R. G., GOVSER, W. C. AND SJOF.RDSMA, A.: Reactivity to norepinephrine and nature of the alpha adrenergic receptor in vascular smooth muscle of a genetically hypertensive rat. Eur. J. Pharmacol. 1: 45-5, 197. COHEN, M. L. AND BERKowITz, B. A.: Age-related changes in vascular responsiveness to cyclic nucleotides and contractile agonists. J. Pharmacol. Exp. Ther. 191: , COHEN, M. L AND BsRKowrrz, B.: Decreased vascular relaxation in hypertension. J. PharmacoL Exp. Ther. 196: , COHEN, M. L. AND WILEY, K. S.: Specific enhancement of norepinephrine-induced contraction in rat veins after beta adrenergic antagonists. J. Pharmacol. Exp. Ther. 21: , FERNANDES, M., ONarri, G., WEDER, A., DYKYJ, R, Gouw, A. B., Kin, K. AND SWARTZ, C.: Experimental model of severe renal hypertension. J. Lab. Cliii. Med. 87: , Fouow, B., Hu.BAcK, M., LUNDGREN, Y. AND WEIss, L: Structurally based increase of flow resistance in spontaneously hypertensive rats. Acts PhysioL Scand. 79: , 197. FURCHGOI-r, R. F. AND BHADRAXOM, S.: Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs. J. PharrnacoL Exp. Ther. 18: , GARDNER, D. L AND MATFHEWS, M. A.: Ultrastructure of the walls of small arteries in early experimental rat hypertension. J. Pathol. BacterioL 97: 51-62, GARST, J. B., KOLETSKY, S., WISENBAUGH, P. E., HADADY, M. AND MAI-FHEWS, D.: Arterial wall renin and renal venous renin in the hypertensive rat. Clin. Sd. (Oil.) 58: 41-46, GORDON, D. B. AND NOGUEIRA, A.: Increased vascular reactivity in experimental hypertension. Circ. Res. 1: , HABER, E, KOERNER, T., PAGE, L B., KILMAN, B. AND PERNODE, A.: Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. Chin. EndocrinoL Metab. 29: , HAEUSLER, G. AND FINcH, L: Vascular reactivity to 5-hydroxytryptamine and hypertension in the rat. Naunyn-Schmiedeberg s Arch. Pharmacol. 272: , HALLBACK, M., LUNDGREN, Y. AND WEISS, L: Reactivity to noradrenalune of aortic stripe and portal veins from spontaneously hypertensive and normotensive rats. Acts PhysioL Scand. 81: , HALLOWAY, E. T. AND BOHR, D. F.: Reactivity of vascular smooth muscle in hypertensive rats. Circ. Res. 33: , HOLLANDER, W., KRAMSCH, D. M., FARNELANT, M. AND M*ixwir, I. M.: Arterial wall metabolism in experimental hypertension of coarctation of the aorta of short duration. J. Cliii. Invest. 47: , KucHIS, M., SHIBATA. S. AND Mmii, J.: Pharmacological nature of adrenergic innervation in rat aorta. Comp. Gen. Pharmacol. 4: , LAIS, L T. AND BRODY, M. J.: Mechanism ofvascular hyperresponsiveness in the spontaneou8ly hypertensive rat. Circ. Eas. 36/37: suppl. 1, , MALING, H. M., FLEISCH, J. H. AND SAUL, W. F.: Species differences in aortic responses to vasoactive amines: The effects of compounds 48/8, cocaine, reserpine and 6-hydroxydopamine. J. Pharmacol. Exp. Ther. 176: , REDLEAF, P. D. AND TOBIAN, L: The question of vascular hyperresponsiveness in hypertension. Circ. Res. 6: , ROJO-ORTEGA, J. M. AND Gxiarr, J.: A method for production of experimental hypertension in rats. Can. J. PhysioL PharmacoL 46: , SHIBATA, S., KURAHASHI, K. AND KUcHII, M.: A possible etiology of contractility impairment of vascular smooth muscle from spontaneously hypertensive rats. J. Pharmacol. Exp. Ther. 185: , SPECTOR, S., FLEISCH, J. H., MALING, H. M. AND BRODIE, B. B.: Vascular smooth muscle reactivity in normotensive and hypertensive rats. Science (Wash. DC) 188: , Send reprint requests to: Dr. Fong M. Lai, American Cyanamid Company, Medical Research Division, Lederle Laboratories, Cardiovascular Biological Research Department, Pearl River, NY 1965.