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Effect of α1-adrenoceptor blockade on the development of hypertension in the spontaneously hypertensive rat
European Journal of Pharmacology, 211 (1992) 263-268
263
© 1992 Elsevier Science Publishers B V. All rights reserved 0014-2999/92/$05.00
EJP 52246
Effect of a l-adrenoceptor blockade on the development of hypertension in the spontaneously hypertensive rat Julie R. J o n s s o n a, R i c h a r d J. H e a d b a n d D e r e k B. F r e w i n
a
a Department of Chnwal and Experimental Pharmacology, Unwersttyof Adelaide, Adelaide, Austraha and b Hypertenston Research Umt, Dwtston of Human Nutntton, Commonwealth Sctenttfic and Industrial Research Orgamsatton (CSIRO), Adelatde, Austraha
Received 11 April 1991, revised MS recewed 24 September 1991, accepted 29 October 1991
There is a large body of evidence to suggest that the sympathetic nervous system plays a critical role in the development of hypertension and vascular medial hypertrophy in the spontaneously hypertensive rat (SHR). The synthesis of a water soluble, specific al-adrenoceptor antagonist (terazosin) has permitted an examination of the influence of al-adrenoceptors on those two phenomena. Thus, in the present study, terazosin (43 mg/kg per day) was administered to SHR and Wistar-Kyoto (WKY) rats from 4.5 to 12 weeks of age, and a number of assessments made in vitro and in vivo. In the SHR, the development of hypertension was not prevented by terazosin. The drug did not influence blood pressure in the WKY. This was despite the fact that animals which had been chronically treated with terazosin displayed marked a-adrenoceptor blockade in vivo. The response of systolic blood pressure to tyramine and noradrenaline was significantly reduced in animals which had been chronically treated with terazosin. In both the SHR and WKY, chronic administration of terazosin did not influence vascular concentrations of 3-methylhistidine, a biochemical marker for contractile proteins and vascular medial hypertrophy. The results therefore argue against a role of t~l-adrenoceptors in the development of hypertension and vascular medial hypertrophy in the SHR. Hypertension; 3-Methylhlstidine; Terazosin; a-Adrenoceptor blockade; Spontaneously hypertensive rat (SHR)
I. Introduction
The pathogenesis of hypertension in the spontaneously hypertensive rat (SHR) is unknown. However, there are findings which implicate the sympathetic nervous system. These include evidence of increased sympathetic nerve activity in the S H R (Iruchijima, 1973; Judy et al., 1976; Judy and Farrell, 1979) and increased innervation of the vasculature in the S H R When compared with the normotensive W i s t a r - K y o t o rat (WKY) (Head et al., 1985; Lee et al., 1983; Scott and Pang, 1983; Head, 1989). The peripheral sympathetic innervation of the rat occurs after birth (Slotkin et al., 1988), and in the SHR, just prior to the development of hypertension. In the S H R there are concurrent changes in the cellular morphology of the vasculature which lead to the development of vascular medial hypertrophy (Lee and Smeda, 1985). It could be logically argued that the phase of developing hypertension in this species of rat is mediated via the sympathetic nervous system as has been suggested by Lee et al. (1987). This
influence would extend to vascular smooth muscle and include changes in the nature and content of contractile proteins. The key adrenoceptor subtype involved in responses of the vasculature to sympathetic nerve stimulation is the at-subclass (Van Zwieten, 1988). Thus, it could be proposed that in the S H R the exaggerated sympathetic neural influence is mediated through al-adrenoce ptors. In the current study we have explored this hypothesis further by examining the effect of a~-adrenoceptor blockade, with the selective at-antagonist terazosin, on the development of hypertension and upon cardiovascular responses in the SHR. The concentration of 3-methylhistidine, a biochemical marker for contractile proteins and vascular medial hypertrophy (Jonsson et al., in press), was also determined in blood vessels from animals subjected to a l - a d r e n o c e p t o r blockade during the developing phase of hypertension in the SHR.
2. Materials and methods 2.1. A n i m a l s and treatments
Correspondence to: D.B. Frewm, Department of Chnlcal and Experimental Pharmacology, University of Adelaide, G.P.O. Box 498, Adelaide 5001, Austraha. Tel. 61.8.228 5193, fax 61.8.223 2076.
Male S H R and W K Y which were obtained from the Glenthorne Division of the Commonwealth Scientific
264 and Industrial Research Organisation (CSIRO), were from 4 to 5 weeks of age at the commencement of the experiments. The animals were housed in wire-bottomed cages (4-8 rats per cage) and fed standard rat chow for the duration of the study. Terazosin-treated animals received this agent in their drinking water (43 mg/kg per day) from 4.5 to 12 weeks of age. The drug solution was freshly prepared in animal house water every 2-4 days and the concentration of terazosin adjusted, depending on the volume per body weight consumed by rats in each cage. Control rats also received a fresh supply of animal house water every 2-4 days. From 5-12 weeks of age, blood pressure was monitored using a photoelectric tail cuff pulse detector (IITC, Life Sciences, CA).
2.2. 3-Methylhistidine The 3-methylhistidine content of the mesenteric artery and its branches, aorta and caudal artery from a subgroup of 12 week old control and treated animals was determined by HPLC (Wassner et al., 1980) as previously described (Jonsson et al., in press). The rats were sacrificed by decapitation. The tissues were removed, frozen in liquid nitrogen and then stored at -20°C until homogenization in 0.05 M Na2HPO 4, containing 2 M NaC1 and 2 mM EDTA at pH 7.4. The protein content of tissue homogenates was measured by the method of Lowry et al. (1951). The protein in each sample was hydrolysed in 6 M HC1 at ll0°C for 24 h. The hydrolysate was dried under vacuum, reconstituted with 5% perchloric acid, filtered and then derivatized with fluorescamine as described by Wassner et al. (1986). The heat and acid stable derivative was injected directly into the HPLC apparatus. This consisted of a Waters chromatography pump, a Waters WISP 710B automatic injector, a ~Bondapack C/s column, a Perkin-Elmer LS-5 Luminescence Spectrometer and a Houston Instruments Omniscribe recorder. The excitation and emission wavelengths were 365 and 460 nm respectively. Elution was performed using 30% Acetonitrile in 10 mM NaPO 4, pH 7.4.
2.3. In utuo experiments The blood pressure responses to tyramine, noradrenaline, and terazosin were monitored intra-arterially in a sub-group of 12 week old control and terazosin-treated rats. The rats were anaesthetised with Brietal/Nembutal (2: 1, 0.3-0.5 ml per 100 g body weight). The carotid artery was cannulated (polyethylene tubing, internal diameter 0.5 ram, outside diameter 1 mm) for recording of arterial pressure, while the jugular vein was cannulated (polyethylene tubing, internal diameter 0.4 mm, outside diameter 0.8 mm) for
administration of drugs. Arterial pressure was monitored using a Statham P23 pressure transducer and a Grass Model 7B polygraph recorder. In each experiment, the schedule for drug administration was as follows: tyramine (0.25 mg/kg), noradrenaline (0.0125, 0.025, 0.125, 0.25, 1.25, 2.5 /zg/kg), terazosin (0.25 mg/kg), followed by a repeat of the same tyramine and noradrenaline doses. The injection volume was always 0.1 ml, and the blood pressure was always allowed to return to baseline between each injection.
2. 4. Drugs and chemicals Except where indicated below, all chemicals used in the present study were of standard laboratory grade. Freshly filtered Millipore water was used in the preparation of all solutions. Drug solutions for the in vivo experiments were prepared in saline (0.9% NaCI). HiPerSolv methanol was purchased from BDH, Poole, U.K., for HPLC. Fluorescamine, 3-methylhistidine, noradrenaline and tyramine were purchased from Sigma Chemical Company, St. Louis, MO, U.S.A. Nembutal (pentobarbitone sodium, 60 mg/ml) was obtained from Abbott Laboratories Pty. Ltd., Sydney, Australia. Brietal (Methylhexital sodium, 10 mg/ml) was purchased from Eli Lilly (Australia) Co., West Ryde, Australia. Supplies of terazosin pure substance were provided by Abbott Laboratories Pty. Ltd., Chicago, IL, U.S.A.
2.5. Statistical analyses Data are presented as means + S.E.M. Statistical analyses were performed using the Student's t-test for paired data, the one-way analysis of variance or the two-way analysis of variance, (as appropriate). A P value of < 0.05 was considered to be significant.
3. Results
3.1. Blood pressure and physical characteristics The systolic blood pressures of 12 week old SHR and WKY measured by tail cuff and the intra-arterial route are presented in table 1. Although blood pressures under anaesthesia were lower than those in conscious animals for all treatment groups, there was a significant correlation between the two recording methods (r = 0.92, P < 0.0001). Chronic administration of terazosin from 4.5 to 12 weeks of age failed to prevent the development of hypertension in the SHR and did not influence blood pressure in the WKY. Terazosin did not significantly alter body weight or the heart to body weight ratio in either rat strain.
265 TABLE 1 Data from the 12-week old S H R and WKY. Treated animals received terazosm (43 m g / k g per day) m their drmking water, while control ammals did not. The data are represented as m e a n s + S E.M T h e n u m b e r s of animals in each group are shown in parentheses. Control
Treated
P
Tall cuff BP ( m m Hg)
SHR WKY P
181 _+ 3(12) 125 _+ 3 (12) < 0 01
172 +_3(11) 127 _+3 (12) < 0.01
ns ns
Intra-artenal BP(mmHg)
SHR WKY P
172 _+ 5 (5) 97 _+10(4) < 0.001
157 + 6 (5) 98 _+3(5) < 0.01
ns ns
Body weight (g)
SHR WKY P
285 + 5 (12) 333 + 4 (12) < 0.001
292 _+4 (11) 330 _+6 (12) < 0.001
ns ns
Heart/body weight ratio (mg/kg)
SHR WKY P
3.73+ 0.11 (9) 3.26 + 0.07 (9) < 0.01
3.54+-0.05 (5) 3.32 + 0.06 (6)
20
0.5
00
SHR
ns ns
WKY
Fig. 1 The 3-methylhlstldme concentration m the m e s e n t e n c artery and Its branches from control [] and terazosm-treated [] 12-week old animals. The data represent the means + S E.M. The n u m b e r of animals in each experimental group was; control S H R 12, terazosintreated S H R 9, control W K Y 8; terazosin-treated W K Y 6. * P < 0 01
ns
3.2. 3-Methylhistidine The 3-methylhistidine concentration in the mesenteric artery and its branches was significantly higher in control (untreated) SHR when compared with that of control (untreated) WKY (fig. 1). In both the SHR and WKY, chronic treatment with terazosin did not influence 3-methylyhistidine concentration in this blood vessel. (A similar profile was seen in the aorta and the caudal artery - data not shown).
3.3. In vivo experiments The intra-arterially recorded systolic blood pressure responses to tyramine are shown in fig. 2. In both the SHR and WKY, the response to tyramine was significantly reduced in animals which had been chronically treated with terazosin, when compared with control animals. Acute i.v. administration of terazosin to control animals significantly reduced the response to tyramine to the level seen in chronically treated animals. However, in animals which had previously been treated with terazosin from 4.5 to 12 weeks of age, acute administration of terazosin did not influence the response to tyramine. A similar profile was seen in the response to noradrenaline in both the SHR (fig. 3) and WKY (WKY data not shown). There was a significantly greater response to noradrenaline in control (untreated) animals when compared with chronically treated animals. Acute administration of terazosin reduced the response to noradrenaline in control animals but did not influence the response to this agent in chronically treated animals. In both the SHR and WKY, acute i.v. administration of terazosin significantly reduced systolic blood
pressure in control animals which had not previously been exposed to this agent (table 2). This reduction in blood pressure was simultaneous with administration of the agent. However, in rats which had previously been chronically treated with terazosin for 7.5 weeks, acute administration of the drug did not influence systolic blood pressure (table 2).
4. Discussion
Despite the proposal that the sympathetic nervous system makes a substantial contribution to the genesis of hypertension, in the present study, oh-adrenoceptor blockade with the selective al-antagonist terazosin failed to prevent the development of hypertension in 50 , ----~
40
T "T- .
2(
T
control
treated
control
treated
SHR Fig. 2. Intra-artenally monitored (I.a.) systohc blood pressure response to i v admimstrat~on of tyramine (0.25 m g / k g ) in 12-week old ammals before and after the acute admlmstratlon of terazosin (0 25 m g / k g ) . [] control before i.v. terazosm, ! control after LV terazosin; [] terazosin-treated before LV. terazosin, [] terazosln-treated after i v terazosm The data represent the m e a n s + S . E . M . The n u m b e r of animals in each experimental group was; control S H R 5; terazosm-treated S H R 5; control W K Y 4; terazosin-treated W K Y 5 * P<001
266 8O
6C
~
40
~,
30 20 1[ I
!
i
0.0125 0.025
i
0.125 0.25 NA (pg/kg)
i
1.25
I
2.5
Fig. 3. I.a. momtored systol,c blood pressure response to i.v. administration of noradrenahne in 12-week old SHR before and after the acute administration of terazosin (0 25 mg/kg), o Control before ,.v. terazosin, • control after LV. terazosin; zx terazosin-treated before LV.terazosm; • terazosm-treated after ,v. terazosm. The data represent the means+S.E.M The number of an,mals ,n each experimental group was 5 * P < 0.05, control before Lv. terazosm &fferent from all other groups.
the SHR. This seems all the more remarkable in view of the fact that animals which had received terazosin over the 7.5-week period displayed marked a-adrenoceptor blockade. The findings in the present study are in contrast with the observations that chronic a tadrenoceptor antagonism effectively lowers blood pressure in adult S H R with established hypertension (Kyncl, 1986; Sanchez et al., 1989a) and in man (Dauer, 1986). As indicated in the Introduction, there is strong evidence to support an enhanced innervation and increased sympathetic nerve activity in the S H R which are both thought to play a role in either the initiation or maintenance of hypertension in this model. Further support for this proposal arises from the observations that either chemical or immuno sympathectomy prevents the development of hypertension in the SHR (Cutilletta et al., 1977; Finch et al., 1973; Lee et al., 1987). From a functional standpoint, sympathectomy TABLE 2 I.a. momtored systolic blood pressure (mm Hg) before (Pre) and after (Post) the Lv. admm,stration of terazosin (0.25 mg/kg) m 12 week old control and terazosm-treated animals. The data are represented as means+S.E.M. The number of ammals in each group is shown in parentheses. Pre
Post
P
SHR
Control Treated P
167+5(5) 144+9(5) ns
114+ 11 (5) 144+ 8(5) ns
< 0.01 ns
WKY
Control Treated P
92 + 3 (4) 90 + 6 (5) ns
78 + 3 (4) 89 + 6 (5) ns
< 0.01 ns
removes the endogenous vasoconstrictor noradrenaline from its vascular effector cells. Consequently it would be anticipated that prolonged treatment with an aadrenoceptor antagonist would achieve a similar effect and thus retard (or prevent) the development of hypertension as seen with sympathectomy. The results of the present study have not supported this view and suggest that al-adrenoceptor blockade does not prevent the development of hypertension in the SHR. A similar conclusion was reached by Sanchez et al. (1989b) following observations made after the administration of the selective oq-adrenoceptor antagonist prazosin to young SHR. However, the conclusions made by those authors could be questioned on the basis of certain shortcomings relating to the pharmacokinetic characteristics of prazosin. For example, the relatively short half-life of prazosin, together with once daily administration of the drug, may not have permitted a continuous and prolonged antagonism of ~l-adrenoceptors. Thus it could be argued that the blood pressure of SHR treated with this agent was still influenced by increased sympathetic nerve activity. The choice of terazosin as the selective al-adrenoceptor antagonist overcomes some of these difficulties. Firstly, the greater water solubility of terazosin (relative to prazosin) permitted the administration of the drug in 'the drinking water. Thus terazosin was available to the rats during the entire duration of the experiment and was not restricted to a single daily i.p. dose as was the case with prazosin in the study by Sanchez et al. (1989b). Secondly, the half-life of terazosin is approximately 12 h, which is 3 - 4 times longer
267 than that of prazosin (Sonders, 1986). Finally, and most importantly, we examined several pharmacological parameters to demonstrate that the animals which had received terazosin did in fact display significant aadrenoceptor blockade. For example, the responses to tyramine and noradrenaline were significantly reduced in animals which had been subjected to chronic administration of terazosin. It could be argued that the a~-adrenergic responses were not impaired throughout the developmental phase of hypertension in the SHR and that the decreases in adrenergic responses which were seen at the end of the treatment period was due to the progressive accumulation of terazosin. However, this seems unlikely in view of the fact that short-term treatment of SHR with terazosin results in an immediate drop in blood pressure following administration of the agent (Kyncl, 1986). Collectively, the results of the present study and those of the study by Sanchez et al. (1989b), argue against a role for al-adrenoceptors in the development of hypertension in the SHR. A puzzling feature of the present study was the failure of the acute administration of terazosin to decrease blood pressure in animals which had previously been chronically treated with the drug. This finding was in contrast with the significant blood pressurelowering effect of terazosin in animals which had not had prior exposure to the drug. It has been proposed (Warnock and Docherty, 1986) that under conditions where al-adrenoceptor-mediated vascular tone is impaired (e.g. sympathectomy or al-adrenoceptor blockade) there is an increase in a2-adrenoceptor-mediated vascular tone. Thus it might be argued that the failure of terazosin to reduce blood pressure in animals which had previously been chronically treated with the drug can best be explained by a shift in reliance from a~- to a2-adrenoceptor-mediated control of blood pressure. However this explanation is not supported by the results of the present study, or the findings of Sanchez et al., (1989b). The responses to noradrenaline (which can occupy both o~1- and a2-adrenoceptors) were shown in the present study to be markedly reduced in animals which had been chronically treated with terazosin. An increase in a2-adrenoceptor activity would be associated with an restoration of responses to noradrenaline. Secondly, in the study of Sanchez et al. (1989b), concomitant administration of the a2-adrenoceptor antagonist yohimbine, with the al-adrenoceptor antagonist prazosin, failed to prevent or retard the development of hypertension in the SHR. Thus the development of hypertension in SHR which have been subjected to chronic a-adrenoceptor blockade involves the intervention of other mechanisms, as yet unknown, which play a key role in the initiation of hypertension. The amino acid 3-methylhistidine is found uniquely in actin and myosin and thus is a marker for contractile proteins. We have previously shown that the concen-
trations of 3-methylhistidine are selectively elevated in the blood vessels of the SHR, when compared with vessels from WKY (Jonsson et al., in press). 3-Methylhistidine concentrations in non-vascular tissue (e.g. skeletal muscle or vas deferens) are similar in the two rat strains. Thus, 3-methylhistidine can be regarded as a biochemical marker for the vascular medial hypertrophy which has previously been demonstrated using morphological and morphometric techniques (Lee and Smeda, 1985). Chronic administration of the angiotensin converting enzyme inhibitor captopril to young SHR prevented the development of hypertension, and significantly, restored the 3-methylhistidine levels in the blood vessels to levels that were comparable to those seen in the WKY (Jonsson et al., in press). In the present study, 3-methylhistidine levels in blood vessels from the SHR were not influenced by chronic treatment with terazosin, which suggests that the enhanced 3-methylhistidine content in the vessels from SHR is not, in a simple fashion, related to the activity and function of a~-adrenoceptors. Lee et al. (1987) have proposed that sympathetic nerves exert a t r o p h i c influence on vascular smooth muscle in the SHR based on observations that neonatal sympathectomy prevented the development of vascular medial hypertrophy in the mesenteric artery. Thadani and Schanberg (1979) found that the ot1adrenoceptor agonist phenylephrine, but not the /3adrenoceptor agonist isoprenaline, increase~ aortic ornithine decarboxylase activity. (Ornithine decarboxylase is the rate limiting enzyme for polyamine synthesis and thus is regarded as a marker for cellular hypertrophy and hyperplasia.) On the basis of in vitro studies which examined the growth characteristics of cardiac myocytes, it has been proposed that noradrenaline acting through al-adrenoceptors , can induce hypertrophy of these ceils (Simpson, 1983). However, the results of the present study suggest that the trophic influence of sympathetic nerves upon vascular smooth muscle in the developing phase of hypertension in the SHR is not an al-adrenoceptor mediated phenomenon. Thus, the question arises as to the precise nature of the trophic influence of sympathetic nerves. Is it noradrenaline acting through a mechanism distinct from the currently accepted a- and /3-adrenoceptors, or some other factor, yet to be identified, which is released from the nerves? In conclusion, although an important role is well established for the sympathetic nervous system in the development of hypertension in the SHR, the results presented herein argue against the involvement of a~adrenoceptors in the phenomenon. Further studies are needed to better define the relationship between sympathetic nerves and certain other mediators with respect to the changes which occur in the vasculature of the SHR.
268
Acknowledgements Terazosm pure substance was kmdly supphed by Dr. R. Klabunde and Dr. J. Kyncl, from Abbott Laboratories Pty. Ltd, Chicago, IL, USA
References Cutilletta, A F , L. Ermoff, A Heller, J Low and S. Oparil, 1977, Development of left ventncular hypertrophy in young spontaneously hypertenswe rats after peripheral sympathectomy, Clrc Res 40, 428 Dauer, A.D., 1986, Terazosln: An effectwe once-daffy monotherapy for the treatment of hypertension, Am. J. Med 80 (Suppl 5B), 29 Finch, L, M Cohen and W D Horst, 1973, Effects of 6-hydroxydopamlne at birth on the development of hypertensmn m the rat, Life SCL 13, 1403 Head, R.J., 1989, Hypernoradrenerglc lnnervatmn' its relationship to functional and hyperplastic changes in the vasculature of the spontaneously hypertenswe rat, Blood Vessels 26, 1. Head, R.J., L.A. Cassls, R L. Robinson, D P Westfall and R.E. Stltzel, 1985, Altered catecholamme contents m vascular and nonvascular tissues in genetically hypertensive rats, Blood Vessels 22, 196. Iruchijima, J., 1973, Sympathetic &scharge rate in spontaneously hypertensive rats, Jap Heart J. 14, 350. Jonsson, J.R., D.B. Frewm and R J Head, Chronic captoprd reverses the enhanced vascular concentrations of 3-methylhlstldme in the spontaneously hypertensive rat, Blood Vessels (m press) Judy, W.V. and S K. Farrell, 1979, Arterial baroreceptor reflex control of sympathetic nerve actwlty in the spontaneously hypertensive rat, Hypertensmn 1,605 Judy, W.V., A.M. Watanabe, D.P Henry, H.R. Besch, W.R. Murphy and G M Hockel, 1976, Sympathetic nerve actwlty. Role m regulatmn of blood pressure m the spontaneously hypertensive rat, Circ. Res. 38 (Suppl 3), 21. Kyncl, J J., 1986, Pharmacology of terazosm, Am J Med. 80 (Suppl. 5B), 12. Lee, R.M K.W and J.S. Smeda, 1985, Primary versus secondary structural changes of the blood vessels in hypertension, Can. J. Physml. Pharmacol. 63, 392 Lee, R.M.K.W, R.E Garfield, J.B. Forrest and E.E Daniel, 1983,
Morphometnc study of structural changes in the mesenterlc blood vessels of spontaneously hypertenswe rats, Blood Vessels 20, 57 Lee, R.M.K.W., C.R. Tnggle, D W T. Cheung and M.D. Coughhn, 1987, Structural and functional consequence of neonatal sympathectomy on the blood vessels of spontaneously hypertenswe rats, Hypertension 10, 328 Lowry, O.H., N.J. Rosebrough, A.L. Farr and R J Randell, 1951, Protein measurement with the Fohn reagent, J Biol Chem 193, 265 Sanchez, A , A. Torres and J Salz, 1989a, Renal a-adrenoceptor density and blood pressure m SHR rat chronically exposed to prazosm a n d / o r yohlmbme, Chn. Exp Hyper. Theory Pract A 11 (1), 119. Sanchez, A , A. Torres and J Saiz, 1989b, a-Blockade on blood pressure and on cardmc noradrenaline content in SHR and DOCA-salt rats, J. Pharm. Pharmacol. 41,545. Scott, T.M. and S.C. Pang, 1983, The correlation between the development of sympathetic innervatton and the development of medial hypertrophy m jejunal arteries m normotensive and spontaneously hypertenswe rats, J. Auton. Nerv. Syst. 8, 25. Simpson, P, 1983, Noreplnephrme-stlmulated hypertrophy of cultured rat myocar&al cells is an alpha 1 adrenergic response, J. Chn Invest 72, 732 Slotkln, T A., C Lau, R.J. Kavlock, J A. Gray, L. Orband-Mdler, K L Queen, F.E Baker, A M Cameron, L Antohck, K. Halm, M Bartolome and J Bartolome, 1988, Role of sympathetic neurons m biochemical and functional development of the kidney: neonatal sympathectomy with 6-hydroxydopamlne, J Pharmacol Exp. Ther 246, 427. Sonders, R.C., 1986, Pharmacoklnetlcs of terazosm, Am J. Med 80 (Suppl 5B), 20 Thandani, P.V and S.M. Schanberg, 1979, Effect of stress and sympathetic activity on rat car&ac and aortic ornlthlne decarboxylase actwlty, Life Sci. 25, 1009. Van Zwieten, P.A., 1988, Antlhypertenswe drugs interacting with aand fl-adrenoceptors, Drugs 35 (Suppl. 6), 6 Warnock, P and J.R Docherty, 1986, Further investigation of the sites of vascular alpha 1 and alpha 2 adrenoceptors in the anaesthehsed spontaneously hypertenswe rat, J Car&ovasc Pharmacol 8, 67 Wassner, S.J., J.L. Schhtzer and J B Ll, 1980, A rapid, sensitive method for the determination of 3-methylhlstldme levels in urine and plasma using high pressure liquid chromatography, Anal Blochem 104, 284.
263
© 1992 Elsevier Science Publishers B V. All rights reserved 0014-2999/92/$05.00
EJP 52246
Effect of a l-adrenoceptor blockade on the development of hypertension in the spontaneously hypertensive rat Julie R. J o n s s o n a, R i c h a r d J. H e a d b a n d D e r e k B. F r e w i n
a
a Department of Chnwal and Experimental Pharmacology, Unwersttyof Adelaide, Adelaide, Austraha and b Hypertenston Research Umt, Dwtston of Human Nutntton, Commonwealth Sctenttfic and Industrial Research Orgamsatton (CSIRO), Adelatde, Austraha
Received 11 April 1991, revised MS recewed 24 September 1991, accepted 29 October 1991
There is a large body of evidence to suggest that the sympathetic nervous system plays a critical role in the development of hypertension and vascular medial hypertrophy in the spontaneously hypertensive rat (SHR). The synthesis of a water soluble, specific al-adrenoceptor antagonist (terazosin) has permitted an examination of the influence of al-adrenoceptors on those two phenomena. Thus, in the present study, terazosin (43 mg/kg per day) was administered to SHR and Wistar-Kyoto (WKY) rats from 4.5 to 12 weeks of age, and a number of assessments made in vitro and in vivo. In the SHR, the development of hypertension was not prevented by terazosin. The drug did not influence blood pressure in the WKY. This was despite the fact that animals which had been chronically treated with terazosin displayed marked a-adrenoceptor blockade in vivo. The response of systolic blood pressure to tyramine and noradrenaline was significantly reduced in animals which had been chronically treated with terazosin. In both the SHR and WKY, chronic administration of terazosin did not influence vascular concentrations of 3-methylhistidine, a biochemical marker for contractile proteins and vascular medial hypertrophy. The results therefore argue against a role of t~l-adrenoceptors in the development of hypertension and vascular medial hypertrophy in the SHR. Hypertension; 3-Methylhlstidine; Terazosin; a-Adrenoceptor blockade; Spontaneously hypertensive rat (SHR)
I. Introduction
The pathogenesis of hypertension in the spontaneously hypertensive rat (SHR) is unknown. However, there are findings which implicate the sympathetic nervous system. These include evidence of increased sympathetic nerve activity in the S H R (Iruchijima, 1973; Judy et al., 1976; Judy and Farrell, 1979) and increased innervation of the vasculature in the S H R When compared with the normotensive W i s t a r - K y o t o rat (WKY) (Head et al., 1985; Lee et al., 1983; Scott and Pang, 1983; Head, 1989). The peripheral sympathetic innervation of the rat occurs after birth (Slotkin et al., 1988), and in the SHR, just prior to the development of hypertension. In the S H R there are concurrent changes in the cellular morphology of the vasculature which lead to the development of vascular medial hypertrophy (Lee and Smeda, 1985). It could be logically argued that the phase of developing hypertension in this species of rat is mediated via the sympathetic nervous system as has been suggested by Lee et al. (1987). This
influence would extend to vascular smooth muscle and include changes in the nature and content of contractile proteins. The key adrenoceptor subtype involved in responses of the vasculature to sympathetic nerve stimulation is the at-subclass (Van Zwieten, 1988). Thus, it could be proposed that in the S H R the exaggerated sympathetic neural influence is mediated through al-adrenoce ptors. In the current study we have explored this hypothesis further by examining the effect of a~-adrenoceptor blockade, with the selective at-antagonist terazosin, on the development of hypertension and upon cardiovascular responses in the SHR. The concentration of 3-methylhistidine, a biochemical marker for contractile proteins and vascular medial hypertrophy (Jonsson et al., in press), was also determined in blood vessels from animals subjected to a l - a d r e n o c e p t o r blockade during the developing phase of hypertension in the SHR.
2. Materials and methods 2.1. A n i m a l s and treatments
Correspondence to: D.B. Frewm, Department of Chnlcal and Experimental Pharmacology, University of Adelaide, G.P.O. Box 498, Adelaide 5001, Austraha. Tel. 61.8.228 5193, fax 61.8.223 2076.
Male S H R and W K Y which were obtained from the Glenthorne Division of the Commonwealth Scientific
264 and Industrial Research Organisation (CSIRO), were from 4 to 5 weeks of age at the commencement of the experiments. The animals were housed in wire-bottomed cages (4-8 rats per cage) and fed standard rat chow for the duration of the study. Terazosin-treated animals received this agent in their drinking water (43 mg/kg per day) from 4.5 to 12 weeks of age. The drug solution was freshly prepared in animal house water every 2-4 days and the concentration of terazosin adjusted, depending on the volume per body weight consumed by rats in each cage. Control rats also received a fresh supply of animal house water every 2-4 days. From 5-12 weeks of age, blood pressure was monitored using a photoelectric tail cuff pulse detector (IITC, Life Sciences, CA).
2.2. 3-Methylhistidine The 3-methylhistidine content of the mesenteric artery and its branches, aorta and caudal artery from a subgroup of 12 week old control and treated animals was determined by HPLC (Wassner et al., 1980) as previously described (Jonsson et al., in press). The rats were sacrificed by decapitation. The tissues were removed, frozen in liquid nitrogen and then stored at -20°C until homogenization in 0.05 M Na2HPO 4, containing 2 M NaC1 and 2 mM EDTA at pH 7.4. The protein content of tissue homogenates was measured by the method of Lowry et al. (1951). The protein in each sample was hydrolysed in 6 M HC1 at ll0°C for 24 h. The hydrolysate was dried under vacuum, reconstituted with 5% perchloric acid, filtered and then derivatized with fluorescamine as described by Wassner et al. (1986). The heat and acid stable derivative was injected directly into the HPLC apparatus. This consisted of a Waters chromatography pump, a Waters WISP 710B automatic injector, a ~Bondapack C/s column, a Perkin-Elmer LS-5 Luminescence Spectrometer and a Houston Instruments Omniscribe recorder. The excitation and emission wavelengths were 365 and 460 nm respectively. Elution was performed using 30% Acetonitrile in 10 mM NaPO 4, pH 7.4.
2.3. In utuo experiments The blood pressure responses to tyramine, noradrenaline, and terazosin were monitored intra-arterially in a sub-group of 12 week old control and terazosin-treated rats. The rats were anaesthetised with Brietal/Nembutal (2: 1, 0.3-0.5 ml per 100 g body weight). The carotid artery was cannulated (polyethylene tubing, internal diameter 0.5 ram, outside diameter 1 mm) for recording of arterial pressure, while the jugular vein was cannulated (polyethylene tubing, internal diameter 0.4 mm, outside diameter 0.8 mm) for
administration of drugs. Arterial pressure was monitored using a Statham P23 pressure transducer and a Grass Model 7B polygraph recorder. In each experiment, the schedule for drug administration was as follows: tyramine (0.25 mg/kg), noradrenaline (0.0125, 0.025, 0.125, 0.25, 1.25, 2.5 /zg/kg), terazosin (0.25 mg/kg), followed by a repeat of the same tyramine and noradrenaline doses. The injection volume was always 0.1 ml, and the blood pressure was always allowed to return to baseline between each injection.
2. 4. Drugs and chemicals Except where indicated below, all chemicals used in the present study were of standard laboratory grade. Freshly filtered Millipore water was used in the preparation of all solutions. Drug solutions for the in vivo experiments were prepared in saline (0.9% NaCI). HiPerSolv methanol was purchased from BDH, Poole, U.K., for HPLC. Fluorescamine, 3-methylhistidine, noradrenaline and tyramine were purchased from Sigma Chemical Company, St. Louis, MO, U.S.A. Nembutal (pentobarbitone sodium, 60 mg/ml) was obtained from Abbott Laboratories Pty. Ltd., Sydney, Australia. Brietal (Methylhexital sodium, 10 mg/ml) was purchased from Eli Lilly (Australia) Co., West Ryde, Australia. Supplies of terazosin pure substance were provided by Abbott Laboratories Pty. Ltd., Chicago, IL, U.S.A.
2.5. Statistical analyses Data are presented as means + S.E.M. Statistical analyses were performed using the Student's t-test for paired data, the one-way analysis of variance or the two-way analysis of variance, (as appropriate). A P value of < 0.05 was considered to be significant.
3. Results
3.1. Blood pressure and physical characteristics The systolic blood pressures of 12 week old SHR and WKY measured by tail cuff and the intra-arterial route are presented in table 1. Although blood pressures under anaesthesia were lower than those in conscious animals for all treatment groups, there was a significant correlation between the two recording methods (r = 0.92, P < 0.0001). Chronic administration of terazosin from 4.5 to 12 weeks of age failed to prevent the development of hypertension in the SHR and did not influence blood pressure in the WKY. Terazosin did not significantly alter body weight or the heart to body weight ratio in either rat strain.
265 TABLE 1 Data from the 12-week old S H R and WKY. Treated animals received terazosm (43 m g / k g per day) m their drmking water, while control ammals did not. The data are represented as m e a n s + S E.M T h e n u m b e r s of animals in each group are shown in parentheses. Control
Treated
P
Tall cuff BP ( m m Hg)
SHR WKY P
181 _+ 3(12) 125 _+ 3 (12) < 0 01
172 +_3(11) 127 _+3 (12) < 0.01
ns ns
Intra-artenal BP(mmHg)
SHR WKY P
172 _+ 5 (5) 97 _+10(4) < 0.001
157 + 6 (5) 98 _+3(5) < 0.01
ns ns
Body weight (g)
SHR WKY P
285 + 5 (12) 333 + 4 (12) < 0.001
292 _+4 (11) 330 _+6 (12) < 0.001
ns ns
Heart/body weight ratio (mg/kg)
SHR WKY P
3.73+ 0.11 (9) 3.26 + 0.07 (9) < 0.01
3.54+-0.05 (5) 3.32 + 0.06 (6)
20
0.5
00
SHR
ns ns
WKY
Fig. 1 The 3-methylhlstldme concentration m the m e s e n t e n c artery and Its branches from control [] and terazosm-treated [] 12-week old animals. The data represent the means + S E.M. The n u m b e r of animals in each experimental group was; control S H R 12, terazosintreated S H R 9, control W K Y 8; terazosin-treated W K Y 6. * P < 0 01
ns
3.2. 3-Methylhistidine The 3-methylhistidine concentration in the mesenteric artery and its branches was significantly higher in control (untreated) SHR when compared with that of control (untreated) WKY (fig. 1). In both the SHR and WKY, chronic treatment with terazosin did not influence 3-methylyhistidine concentration in this blood vessel. (A similar profile was seen in the aorta and the caudal artery - data not shown).
3.3. In vivo experiments The intra-arterially recorded systolic blood pressure responses to tyramine are shown in fig. 2. In both the SHR and WKY, the response to tyramine was significantly reduced in animals which had been chronically treated with terazosin, when compared with control animals. Acute i.v. administration of terazosin to control animals significantly reduced the response to tyramine to the level seen in chronically treated animals. However, in animals which had previously been treated with terazosin from 4.5 to 12 weeks of age, acute administration of terazosin did not influence the response to tyramine. A similar profile was seen in the response to noradrenaline in both the SHR (fig. 3) and WKY (WKY data not shown). There was a significantly greater response to noradrenaline in control (untreated) animals when compared with chronically treated animals. Acute administration of terazosin reduced the response to noradrenaline in control animals but did not influence the response to this agent in chronically treated animals. In both the SHR and WKY, acute i.v. administration of terazosin significantly reduced systolic blood
pressure in control animals which had not previously been exposed to this agent (table 2). This reduction in blood pressure was simultaneous with administration of the agent. However, in rats which had previously been chronically treated with terazosin for 7.5 weeks, acute administration of the drug did not influence systolic blood pressure (table 2).
4. Discussion
Despite the proposal that the sympathetic nervous system makes a substantial contribution to the genesis of hypertension, in the present study, oh-adrenoceptor blockade with the selective al-antagonist terazosin failed to prevent the development of hypertension in 50 , ----~
40
T "T- .
2(
T
control
treated
control
treated
SHR Fig. 2. Intra-artenally monitored (I.a.) systohc blood pressure response to i v admimstrat~on of tyramine (0.25 m g / k g ) in 12-week old ammals before and after the acute admlmstratlon of terazosin (0 25 m g / k g ) . [] control before i.v. terazosm, ! control after LV terazosin; [] terazosin-treated before LV. terazosin, [] terazosln-treated after i v terazosm The data represent the m e a n s + S . E . M . The n u m b e r of animals in each experimental group was; control S H R 5; terazosm-treated S H R 5; control W K Y 4; terazosin-treated W K Y 5 * P<001
266 8O
6C
~
40
~,
30 20 1[ I
!
i
0.0125 0.025
i
0.125 0.25 NA (pg/kg)
i
1.25
I
2.5
Fig. 3. I.a. momtored systol,c blood pressure response to i.v. administration of noradrenahne in 12-week old SHR before and after the acute administration of terazosin (0 25 mg/kg), o Control before ,.v. terazosin, • control after LV. terazosin; zx terazosin-treated before LV.terazosm; • terazosm-treated after ,v. terazosm. The data represent the means+S.E.M The number of an,mals ,n each experimental group was 5 * P < 0.05, control before Lv. terazosm &fferent from all other groups.
the SHR. This seems all the more remarkable in view of the fact that animals which had received terazosin over the 7.5-week period displayed marked a-adrenoceptor blockade. The findings in the present study are in contrast with the observations that chronic a tadrenoceptor antagonism effectively lowers blood pressure in adult S H R with established hypertension (Kyncl, 1986; Sanchez et al., 1989a) and in man (Dauer, 1986). As indicated in the Introduction, there is strong evidence to support an enhanced innervation and increased sympathetic nerve activity in the S H R which are both thought to play a role in either the initiation or maintenance of hypertension in this model. Further support for this proposal arises from the observations that either chemical or immuno sympathectomy prevents the development of hypertension in the SHR (Cutilletta et al., 1977; Finch et al., 1973; Lee et al., 1987). From a functional standpoint, sympathectomy TABLE 2 I.a. momtored systolic blood pressure (mm Hg) before (Pre) and after (Post) the Lv. admm,stration of terazosin (0.25 mg/kg) m 12 week old control and terazosm-treated animals. The data are represented as means+S.E.M. The number of ammals in each group is shown in parentheses. Pre
Post
P
SHR
Control Treated P
167+5(5) 144+9(5) ns
114+ 11 (5) 144+ 8(5) ns
< 0.01 ns
WKY
Control Treated P
92 + 3 (4) 90 + 6 (5) ns
78 + 3 (4) 89 + 6 (5) ns
< 0.01 ns
removes the endogenous vasoconstrictor noradrenaline from its vascular effector cells. Consequently it would be anticipated that prolonged treatment with an aadrenoceptor antagonist would achieve a similar effect and thus retard (or prevent) the development of hypertension as seen with sympathectomy. The results of the present study have not supported this view and suggest that al-adrenoceptor blockade does not prevent the development of hypertension in the SHR. A similar conclusion was reached by Sanchez et al. (1989b) following observations made after the administration of the selective oq-adrenoceptor antagonist prazosin to young SHR. However, the conclusions made by those authors could be questioned on the basis of certain shortcomings relating to the pharmacokinetic characteristics of prazosin. For example, the relatively short half-life of prazosin, together with once daily administration of the drug, may not have permitted a continuous and prolonged antagonism of ~l-adrenoceptors. Thus it could be argued that the blood pressure of SHR treated with this agent was still influenced by increased sympathetic nerve activity. The choice of terazosin as the selective al-adrenoceptor antagonist overcomes some of these difficulties. Firstly, the greater water solubility of terazosin (relative to prazosin) permitted the administration of the drug in 'the drinking water. Thus terazosin was available to the rats during the entire duration of the experiment and was not restricted to a single daily i.p. dose as was the case with prazosin in the study by Sanchez et al. (1989b). Secondly, the half-life of terazosin is approximately 12 h, which is 3 - 4 times longer
267 than that of prazosin (Sonders, 1986). Finally, and most importantly, we examined several pharmacological parameters to demonstrate that the animals which had received terazosin did in fact display significant aadrenoceptor blockade. For example, the responses to tyramine and noradrenaline were significantly reduced in animals which had been subjected to chronic administration of terazosin. It could be argued that the a~-adrenergic responses were not impaired throughout the developmental phase of hypertension in the SHR and that the decreases in adrenergic responses which were seen at the end of the treatment period was due to the progressive accumulation of terazosin. However, this seems unlikely in view of the fact that short-term treatment of SHR with terazosin results in an immediate drop in blood pressure following administration of the agent (Kyncl, 1986). Collectively, the results of the present study and those of the study by Sanchez et al. (1989b), argue against a role for al-adrenoceptors in the development of hypertension in the SHR. A puzzling feature of the present study was the failure of the acute administration of terazosin to decrease blood pressure in animals which had previously been chronically treated with the drug. This finding was in contrast with the significant blood pressurelowering effect of terazosin in animals which had not had prior exposure to the drug. It has been proposed (Warnock and Docherty, 1986) that under conditions where al-adrenoceptor-mediated vascular tone is impaired (e.g. sympathectomy or al-adrenoceptor blockade) there is an increase in a2-adrenoceptor-mediated vascular tone. Thus it might be argued that the failure of terazosin to reduce blood pressure in animals which had previously been chronically treated with the drug can best be explained by a shift in reliance from a~- to a2-adrenoceptor-mediated control of blood pressure. However this explanation is not supported by the results of the present study, or the findings of Sanchez et al., (1989b). The responses to noradrenaline (which can occupy both o~1- and a2-adrenoceptors) were shown in the present study to be markedly reduced in animals which had been chronically treated with terazosin. An increase in a2-adrenoceptor activity would be associated with an restoration of responses to noradrenaline. Secondly, in the study of Sanchez et al. (1989b), concomitant administration of the a2-adrenoceptor antagonist yohimbine, with the al-adrenoceptor antagonist prazosin, failed to prevent or retard the development of hypertension in the SHR. Thus the development of hypertension in SHR which have been subjected to chronic a-adrenoceptor blockade involves the intervention of other mechanisms, as yet unknown, which play a key role in the initiation of hypertension. The amino acid 3-methylhistidine is found uniquely in actin and myosin and thus is a marker for contractile proteins. We have previously shown that the concen-
trations of 3-methylhistidine are selectively elevated in the blood vessels of the SHR, when compared with vessels from WKY (Jonsson et al., in press). 3-Methylhistidine concentrations in non-vascular tissue (e.g. skeletal muscle or vas deferens) are similar in the two rat strains. Thus, 3-methylhistidine can be regarded as a biochemical marker for the vascular medial hypertrophy which has previously been demonstrated using morphological and morphometric techniques (Lee and Smeda, 1985). Chronic administration of the angiotensin converting enzyme inhibitor captopril to young SHR prevented the development of hypertension, and significantly, restored the 3-methylhistidine levels in the blood vessels to levels that were comparable to those seen in the WKY (Jonsson et al., in press). In the present study, 3-methylhistidine levels in blood vessels from the SHR were not influenced by chronic treatment with terazosin, which suggests that the enhanced 3-methylhistidine content in the vessels from SHR is not, in a simple fashion, related to the activity and function of a~-adrenoceptors. Lee et al. (1987) have proposed that sympathetic nerves exert a t r o p h i c influence on vascular smooth muscle in the SHR based on observations that neonatal sympathectomy prevented the development of vascular medial hypertrophy in the mesenteric artery. Thadani and Schanberg (1979) found that the ot1adrenoceptor agonist phenylephrine, but not the /3adrenoceptor agonist isoprenaline, increase~ aortic ornithine decarboxylase activity. (Ornithine decarboxylase is the rate limiting enzyme for polyamine synthesis and thus is regarded as a marker for cellular hypertrophy and hyperplasia.) On the basis of in vitro studies which examined the growth characteristics of cardiac myocytes, it has been proposed that noradrenaline acting through al-adrenoceptors , can induce hypertrophy of these ceils (Simpson, 1983). However, the results of the present study suggest that the trophic influence of sympathetic nerves upon vascular smooth muscle in the developing phase of hypertension in the SHR is not an al-adrenoceptor mediated phenomenon. Thus, the question arises as to the precise nature of the trophic influence of sympathetic nerves. Is it noradrenaline acting through a mechanism distinct from the currently accepted a- and /3-adrenoceptors, or some other factor, yet to be identified, which is released from the nerves? In conclusion, although an important role is well established for the sympathetic nervous system in the development of hypertension in the SHR, the results presented herein argue against the involvement of a~adrenoceptors in the phenomenon. Further studies are needed to better define the relationship between sympathetic nerves and certain other mediators with respect to the changes which occur in the vasculature of the SHR.
268
Acknowledgements Terazosm pure substance was kmdly supphed by Dr. R. Klabunde and Dr. J. Kyncl, from Abbott Laboratories Pty. Ltd, Chicago, IL, USA
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