Regional
Hemodynamic
,Q1-Blocker,
and
Effects
of Betaxolol,
Atenolol in Conscious Hypertensive Rats
a New
Selective
Spontaneously
Tetsuo Shoji, Yasuharu Aki, Kunisuke Horiuchi, Toshiaki Tamaki, Hong He, Akira Nishiyama, Hideyasu Kiyomoto, Yoshihide Fujisawal, Hiroshi Iwao and Youichi Abe Department of Pharmacology and 'Research Equipment Center, Kagawa Medical School, Kagawa 761-07, Japan Received
May
11, 1992
Accepted
August
21, 1992
ABSTRACT Betaxolol is a new fl-blocker that has been reported to have 8,-selectivity, and it is de void of both membrane stabilizing action and intrinsic sympathomimetic action. The effects of betaxolol on systemic and regional hemodynamics were examined in conscious spontaneously hypertensive rats (SHR) by a microsphere method and compared with the effects of atenolol. A single oral administra tion of betaxolol at 1 and 10 mg/kg decreased the mean arterial pressure in a dose-dependent manner. At the same doses, atenolol also showed a similar but weak hypotensive effect. Both of these drugs at the high dose decreased cardiac output and heart rate and at the low dose, did not. Total peripheral resistance decreased by only betaxolol at the low dose. Betaxolol showed a tendency to normalize the hemodynamic abnormalities which were observed in the kidney, spleen and gastrointestinal tract of SHR, while atenolol did not. It should be noted that betaxolol increased the flow rate in the kidney, which may be explained by its direct vasodilatory action on renal blood vessels. In conclusion, betaxolol showed an antihypertensive action at the doses of 1 and 10 mg/kg, exhibited the characteristics of a ,81 blocker and produced preferable effects on regional hemodynamics in SHR. Keywords:
Regional
hemodynamics,
Microsphere,
Betaxolol, (±)-1-[4-[2-(cyclopropylmethoxy)ethyl] phenoxy]-3-(isopropylamino)-2-propanol hydrochloride, is a new highly selective 8-blocker that has been re ported to be devoid of a blocking, membrane stabiliz ing and intrinsic sympathomimetic actions (1-4). This drug possesses good plasma bioavailability after oral administration and has a long plasma half-life (5, 6). Although the pharmacodynamic and antihypertensive effects of betaxolol have already been reported (1, 4, 6), its effects on each organ vascular bed, along with hypotension, are unknown. We and others have re ported the differences in regional hemodynamics be tween spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). These studies show that all organs do not equally contribute to the elevation of to tal peripheral resistance (TPR) in SHR and that some of the organs, such as the kidney, gastrointestinal tract and spleen, contribute greatly to the increased TPR (7 10). It would be interesting to know whether betaxolol normalizes the hemodynamic abnormality observed in
Betaxolol,
Atenolol,
Spontaneously
hypertensive
rat
SHR. The present study was designed to clarify whether be taxolol can counteract the basic hemodynamic abnor mality of hypertensive rats in addition to providing a characteristic profile of the drug in relation to its anti hypertensive effect. The effects of betaxolol on systemic and regional hemodynamics were examined and com pared with those of atenolol, a selective 81-blocker (11 13), in conscious SHR by a radioactive microsphere method. MATERIALS AND METHODS Animals and experimental preparation Thirteen to 15-week-old male SHR (Charles River, Japan) were used in this investigation. They were placed on a normal diet until the time of this experi ment. Under a light sodium pentobarbital anesthesia, polyethylene catheters (PE-50) were placed in the fem oral artery and left ventricle via the right carotid artery.
These catheters were filled with heparinized saline (100 IU/ml) and exteriorized through a cutaneous tunnel at the back of the neck after confirming the tip locations by pressure tracings. The animals were allowed to re cover for 24 hr before initiating the experimental proce dures. The experiments were performed in a tempera ture controlled room at 23°C. The catheterized SHR were placed in a plastic cham ber. The femoral arterial catheter was connected to the pressure transducer, and systemic arterial pressure was continuously recorded on a multichannel polygraph (NEC-San-ei, Japan). All of the following experiments were performed under unanesthetized and unrestricted conditions.
dose injection of pentobarbital. The brain, lungs, heart, liver, spleen, kidneys, stomach, intestines (small and large intestine), skin and hindlimb skeletal muscle were removed and weighed. Due to the relatively low blood flow to the skin and skeletal muscle, at least 5 g of these tissues were removed. The skin and skeletal mus cle samples contained 300-400 microspheres, while all other tissues contained over 500 microspheres. The activ ities of samples were analyzed by a gamma scintillation counter. The cardiac output was calculated as follows:
Measurements of systemic and regional hemodynamics Pilot experiments were performed to determine the appropriate point in time for measuring the regional hemodynamic effects of betaxolol and atenolol. In 3 rats of each group, betaxolol and atenolol were orally administered at doses of 1 and 10 mg/kg. We observed the hypotensive effects continuously up to 8 hr after the administration of the drugs. The hypotensive effects appeared within 1 hr and peaked at 3 4 hr. Persistent hypotension was observed for more than 8 hr. No signi ficant change in either blood pressure or heart rate were observed in the control group which was adminis tered distilled water. Based on these results, the measurements of cardiac output and organ blood flow were carried out at 4 hr after the administration of each drug. Radioactive microspheres were utilized to measure the cardiac output and regional blood flow, according to the methods of Tsuchiya et al. (14) and Ishise et al. (15). Radionuclide-labeled microspheres (141Ce, New England Nuclear, U.S.A.), 15 ± 3 ,um in diameter, were used. Following a 60 min stabilization period for the rats to adapt to the chamber, either betaxolol or atenolol at doses of 1 or 10 mg/kg was orally administered (betax olol: n = 10 for 1 mg/kg and n = 9 for 10 mg/kg; ate nolol: n = 9 for 1 mg/kg and n = 10 for 10 mg/kg). Distilled water was administered in the control group (n = 19). The systemic blood pressure and heart rate were stabilized 4 hr after the above administration, and then 0.25 ml of saline containing 75,000 microspheres was in jected into the left ventricle. The injection procedures were carried out over a 15-sec period. Arterial blood samples for reference blood were obtained using a with drawal pump at a rate of 0.65 ml/min starting im mediately before the injection of the microspheres and ending 60 sec later. After termination of the micro sphere injection, the animal was sacrificed by an over
The total injected radioactivity was obtained by sub tracting the residual radioactivity from the radioactivity before injection. Absolute organ blood flow was calcu lated as follows:
Statistical analyses In the present paper, the values are expressed as the mean ± S.E.M. Organ blood flow is expressed as ml/g of organ/min, and organ vascular resistance is express ed as mmHg/ml/g of organ/min. The data were evalu ated by Student's t-test, where P values less than 0.05 were regarded as significant. Chemicals The betaxolol used in this study was gift from Mitsu bishi Chemical Industries, Ltd. (Japan). The atenolol was purchased from Sigma Chemical Co. (U.S.A.). For the oral administration to rats, betaxolol and atenolol were dissolved in distilled water. The drug solution or distilled water was orally administered at 5 ml/kg. RESULTS Effects of betaxolol on systemic and regional hemodynam ics in SHR The effects of betaxolol, 4 hr after administration, on mean arterial pressure (MAP), cardiac output, heart rate and TPR in SHR are tabulated in Table 1. Betax olol at either dose produced significant hypotension. While the low dose of betaxolol failed to reduce cardiac output and heart rate, the high dose reduced both param eters significantly. As a result, the calculated TPR was reduced by the low dose but was not changed by the high dose.
Table tensive
The
regional
hemodynamic
2.
Effects
of
betaxolol
on
organ
effects
induced
by betax
olol are summarized in Table 2, which shows the organ blood flow. The calculated organ vascular resistances are shown in Fig. 1. A low dose of betaxolol signifi cantly increased the flow rate in the kidney, but did not affect the flow rates in the other organs. The high dose of betaxolol decreased the flow rates in the brain, heart, skeletal muscle and skin, along with a reduction in cardiac output. The increased flow rate in the kidney was observed at the high dose, as well as at the low dose. Calculated cantly decreased
organ vascular resistances were signifi in the kidney and gastrointestinal
tract, along with hypotension by either dose. Vascular resistance in the kidney was decreased remarkably by both doses. The high dose increased vascular resistance in the skeletal skin.
muscle,
blood
flow
in conscious
spontaneously
hyper
rats
but not in the brain,
heart
and
Effects of atenolol on systemic and regional hemodynam ics in SHR The effects of atenolol, 4 hr after the administration, on systemic hemodynamics in SHR are shown in Table 3. Although atenolol administered orally at 1 mg/kg tended to reduce the blood pressure, it was not signifi cant. The atenolol at 10 mg/kg decreased significantly by about 20 mmHg. The low dose of atenolol reduced the heart rate, while the high dose reduced both heart rate and cardiac output. TPR was not changed by ate nolol. The effects of atenolol on organ blood flow are tabu lated in Table 4. The low dose of atenolol decreased the flow rates in the portal region and skin. The high dose of atenolol decreased the flow rates in the heart, gastrointestinal tract and skeletal muscle, but did not change it in the kidney. Organ vascular resistances in the liver, intestines and skin were increased by both doses (Fig. 2).
Fig. 1. Effects of betaxolol (1 and 10 mg/kg, p.o.) on organ vascular resistance in conscious spontaneously hypertensive rats. * indicates significant difference between the control and the betaxolol group (P < 0.05). 0 Control; Betaxolol, 1 mg/kg; Betaxolol, 10 mg/kg.
Table
3.
Effects
Table tensive
of atenolol
4.
Effects rats
of
on
systemic
atenolol
on
hemodynamics
organ
blood
in conscious
flow
in conscious
spontaneously
spontaneously
hypertensive
hyper
rats
Fig. 2. Effects of atenolol (1 and 10 mg/kg, p.o.) on organ vascular resistance in conscious spontaneously hypertensive rats. * indicates significant difference between the control and the atenolol group (P < 0.05). [1 Control; ® Atenolol, 1 mg/kg; El Atenolol, 10 mg/kg.
DISCUSSION The effects of the new selective /81-blocker betaxolol on systemic and regional hemodynamics were examined and compared with those of atenolol in conscious SHR. Betaxolol showed a dose-dependent hypotensive effect in SHR following an oral administration of 1 and 10 mg/kg. Atenolol also showed the same hypotensive effect, but it was weaker than that of the betaxolol. These findings are consistent with the previous report (4). It has been reported that other /3-blockers did not reduce or required relatively high doses to reduce MAP in a single administration in hypertensive rats (16-19). Thus it seems that betaxolol, among 8-blockers, has a unique pharmacological property concerning anti hypertensive action. The low dose of betaxolol signifi cantly reduced TPR, while the high dose and atenolol at either dose did not. The lack of change in TPR by both drugs at the high dose may be explained by the re duced cardiac output and heart rate due to the cardio inhibitory effects of the drug. Betaxolol, at the low dose, did not reduce heart rate. This may have been due to reflex tachycardia induced by potent hypotension, which masks its cardioinhibitory effect. At the same dose, atenolol did not reduce MAP but reduced heart rate. These results indicate that betaxolol may have other effects in addition to N1-adrenoceptor blocking action, such as the direct vasodilatory effect.
We and others have previously reported the differ ence between systemic and regional hemodynamics in SHR and WKY (7-10, 18, 19). These reports showed that some of the organs, such as the kidney, gastroin testinal tract and spleen, contributed greatly to the in creased TPR in SHR. The present report provides a re gional hemodynamic profile of betaxolol's effects on these abnormal hemodynamics observed in SHR. Both betaxolol and atenolol, at high doses, reduced the flow rate in many organs. It seems that there are not large differences between them. Hypotension or reduced car diac output may account partially for the results. At the low dose, atenolol tended to reduce the flow rate more than betaxolol in some organs. Since there are differ ences in their systemic effects, a comparison of organ vascular resistance between them is available. Betaxolol at the low dose showed reductions in organ vascular re sistances in the spleen, kidney and gastrointestinal tract. These organs are considered to be closely related to the high TPR in SHR. The flow rate increased, although the blood pressure was reduced, especially in the kidney. This indicates that betaxolol produced renal vasodilatation. On the other hand, atenolol tended to increase the vascular resistance in some organs. Bolt and Saxena (20) have reported that atenolol showed modest effects on organ flow rates in conscious renal hypertensive rabbits, and in the kidney, the flow rate or vascular conductance did not change. Considering these
differences, it is difficult to explain betaxolol's organ vascular resistance reduction effects simply by a 91 adrenoceptor blocking action. It has been reported that propranolol and others tend to decrease renal blood flow in clinical use and animal experiments (21, 22). Betaxolol, however, increased the flow rate in the kidney. There is a question as to how betaxolol causes this increase. It is known that the in hibition of a-adrenoceptors or the stimulation of ( adrenoceptors can increase renal blood flow. The in hibition of presynaptic ,8-adrenoceptor, causing an in hibition of the release of noradrenaline (23, 24), and of the renin-angiotensin system (25) may be also consi dered. However, betaxolol and atenolol have the same properties as a 81-blocking agent (11). Therefore, the difference between their renal actions can not be ex plained by the above factors. It can be considered that betaxolol may have other, yet unknown, vasodilating actions including a direct action. Both of the drugs, at the high dose, reduced the flow rate in the skeletal muscle. Betaxolol increased the vascular resistance in the skeletal muscle, while atenolol tended to increase it as well. Although betaxolol is a highly selective ,81-blocker (2), this may be explained by the fact that betaxolol, at the high dose, exerts a blockade of 82-adrenoceptors in the vascular bed of skeletal muscle (26). Since the flow rate in the skeletal muscle in SHR is higher than that in WKY (10), it might be considered that the flow rate was merely re versed to the WKY level by betaxolol. It is known that a /8-blocker does not exert its hy potensive effect powerfully in SHR by a single adminis tration when compared with other species. Warrington et al. (27) indicated that betaxolol had a more powerful antihypertensive effect than others, but Roudaut et al. (28) did not find any difference between betaxolol and atenolol. In the present results, there are interesting points which show that betaxolol decreased MAP by a single administration, at the relatively low dose, and in creased the flow rate in the kidney in contrast to other ,8-blockers. Thus, betaxolol can be considered to be a ,81-blocker with unknown vasodilating mechanisms.
stimulation of spinal cord, in spontaneously hypertensive rats. Br. J. Pharmacol. 78, Supp. 28P (1983) 3 Cavero, I. and Lefevre-Borg, F.: Antihypertensive activity of betaxolol in conscious spontaneously hypertensive rats. Br. J. Pharmacol. 78, Supp. 141P (1983) 4 Cavero, I., Lefevre-Borg, F., Manoury, P. and Roach, A.: In vitro and in vivo pharmacological evaluation of betaxolol, a new, potent, and selective 61-adrenoceptor antagonist. In Betaxolol and Other ,81-Adrenoceptor Antagonists, Edited by Morselli, P.L., Kilborn, J.R., Cavero, I., Harrison, D.C. and Langer, S.Z., p. 31-42, Raven Press, New York (1983) 5 Cadigan, P., London, D. and Petecost, B.: Cardiovascular effects of single oral doses of the new beta-adrenoceptor blocking agent betaxolol (SL 75212) in healthy volunteers. Br. J. Clin. Pharmacol. 9, 569-575 (1980) 6 Warrington, S., Turner, P., Kilborn, J., Bianchetti, G. and Morselli, P.: Blood concentrations and pharmacodynamic effects of betaxolol (SL 75212) a new beta-adrenoceptor an tagonist after oral and intravenous administration. Br. J. Clin. Pharmacol. 10, 449-452 (1980) 7 Tadepalli, A., Walsh, G. and Tobia, A.: Normal cardiac out
8
9
10
11
12 13
14
Acknowledgments We are grateful to Stacey Laumann for the proof reading of this manuscript and J. Fujita and A. Tanaka for their secretarial ser vices.
15
REFERENCES
16
1 Boudot, J., Cavero, I. and Fenard, S.: Preliminary studies on SL 75212, a new potent cardioselective 8-adrenoceptor an tagonist. Br. J. Pharmacol. 66, 445P (1979) 2 Cavero, I.: Betaxolol inhibits pressor responses to electrical
17
put in the conscious young spontaneously hypertensive rat: evidence for high oxygen utilization. Life Sci. 15, 1103 -1114 (1974) Ferrone, R., Walsh, G., Tsuchiya, M. and Frohlich, E.: Comparison of hemodynamics in conscious spontaneous and renal hypertensive rats. Am. J. Physiol. 236, H403-H408 (1979) Fukui, K., Tamaki, T. and Yamamoto, A.: Regional blood flow and cardiac responses to the a-adrenoceptor blocker terazosin in spontaneously hypertensive rats. Asian Pac. J. Pharmacol. 2, 7 12 (1987) Abe, Y., Sakamoto, W., Fukui, K., Iwao, H., Takada, T . and Yamamoto, K.: Effects of angiotensin converting enzyme inhibitor (SA-446) on regional hemodynamics in renal and spontaneously hypertensive rats. Arch. Int. Pharmacodyn. Ther. 292, 237-247 (1988) Gross, F.: Growing points of fl-blocking agents. In Betaxolol and Other 81-Adrenoceptor Antagonists, Edited by Morselli, P.L., Kilborn, J.R., Cavero, I., Harrison, D.C. and Langer, S.Z., p. 1 12, Raven Press, New York (1983) Barrett, A.: The pharmacology of atenolol. Postgrad. Med. J. 53, Supp. 3, 58-64 (1977) Johansson, B.: Effects of atenolol, metoprolol, and pamatolol on cardiac and vascular beta-adrenoceptors in the rat. J. Car diovasc. Pharmacol. 1, 287-298 (1979) Tsuchiya, M., Ferrone, R., Walsh, G. and Frohlich, E.: Re gional blood flow measured in conscious rats by combined Fick and microsphere methods. Am. J. Physiol. 235, H357 H360 (1978) Ishise, S., Pegram, B., Yamamoto, J., Kitamura, Y. and Frohlich, E.: Reference sample microsphere method: cardiac output and blood flows in conscious rat. Am. J. Physiol. 239, H443 H449 (1980) Regoli, D.: Pressor action of beta blocking agents in rats. Can. J. Physiol. Pharmacol. 48, 481-489 (1970) Roba, J., Lambelin, G. and De Schaepdryver, A.: Antihyper tensive activity of four fi blocking agents in spontaneously hypertensive rats. Arch. Int. Pharmacodyn. Ther. 200, 182 190(1972)
18
19
20
21 22
23
24
Clark, B.: Pharmacology of beta-adrenoceptor blocking agents, In Beta-Adrenoceptor Blocking Agents, Edited by Saxena, P.R. and Forsyth, R.P., p.45-76, North-Holland Publishing Co., Amsterdam (1976) Sugawara, K. and Ozaki, M.: Acute hypotensive effect in conscious rats and catecholamine releasing action from adre nal medulla by propranolol. In Proceedings of a Symposium on Antihypertensive Mechanisms of Propranolol, Edited by Sokabe, F., p. 40-49, Life Science Publishing Co., Ltd., Tokyo (1981) Bolt, G. and Saxena, P.: Interaction of atenolol with the sys temic and regional hemodynamic effects of hydralazine in conscious renal hypertensive rabbits. J. Pharmacol. Exp. Ther. 230, 205-213 (1984) Epstein, M. and Oster, J.: Beta-blocker and the kidney. Minerva Electr. Metab. 8, 237 (1982) Bernstein, K. and O'Connor, D.: Antiadrenergic anti hypertensive drugs: their effect on renal function. Annu. Rev. Pharmacol. 24, 105 120 (1984) Westfall, T., Peach, M. and Tittermary, V.: Enhancement of the electrically induced release of norepinephrine from the rat portal venin. Eur. J. Pharmacol. 58, 67-74 (1979) Langer, S. and Galzin, A.: ,8-Adrenoceptors and noradren ergic neurotransmission: effects of betaxolol on the stimu lation-evoked release of 3H-norepinephrine in isolated rat
atria and rabbit hypothalamic slices. In Betaxolol and Other ,81-Adrenoceptor Antagonists, Edited by Morselli, P.L., Kilborn, J.R., Cavero, I., Harrison, D.C. and Langer, S.Z., 25
26
27
28
p. 21-30, Raven Press, New York (1983) Milavec-Krizman, M., Evenou, J., Warner, H., Berthold, R. and Stoll, A.P.: Characterization of beta-adrenoceptor sub types in rat kidney with new highly selective ,81 blockers and their role in renin release. Biochem. Pharmacol. 34, 3951 3957 (1985) Gilman, A.G., Rall, T.W., Nies, A.S. and Taylor, P.: The Pharmacological Basis of Therapeutics, Pergamon Press, New York (1990) Warrington, S., Taylor, E. and Kilborn, J., Comparison of pharmacodynamic effects of betaxolol with atenolol, practo lol, and propranolol given intravenously. In Betaxolol and Other 81-Adrenoceptor Antagonists, Edited by Morselli, P.L., Kilborn, J.R., Cavero, I., Harrison, D.C. and Langer, S.Z., p. 109 122, Raven Press, New York (1983) Roudaut, R., Billes, M., Wicker, P., Clementy, J. and Dallocchio, M.: Comparative study of betaxolol and atenolol in hypertension. In Betaxolol and Other ,81-Adrenoceptor Antagonists, Edited by Morselli, P.L., Kilborn, J.R., Cavero, I., Harrison, D.C. and Langer, S.Z., p.325-332, Raven Press, New York (1983)
Hemodynamic
,Q1-Blocker,
and
Effects
of Betaxolol,
Atenolol in Conscious Hypertensive Rats
a New
Selective
Spontaneously
Tetsuo Shoji, Yasuharu Aki, Kunisuke Horiuchi, Toshiaki Tamaki, Hong He, Akira Nishiyama, Hideyasu Kiyomoto, Yoshihide Fujisawal, Hiroshi Iwao and Youichi Abe Department of Pharmacology and 'Research Equipment Center, Kagawa Medical School, Kagawa 761-07, Japan Received
May
11, 1992
Accepted
August
21, 1992
ABSTRACT Betaxolol is a new fl-blocker that has been reported to have 8,-selectivity, and it is de void of both membrane stabilizing action and intrinsic sympathomimetic action. The effects of betaxolol on systemic and regional hemodynamics were examined in conscious spontaneously hypertensive rats (SHR) by a microsphere method and compared with the effects of atenolol. A single oral administra tion of betaxolol at 1 and 10 mg/kg decreased the mean arterial pressure in a dose-dependent manner. At the same doses, atenolol also showed a similar but weak hypotensive effect. Both of these drugs at the high dose decreased cardiac output and heart rate and at the low dose, did not. Total peripheral resistance decreased by only betaxolol at the low dose. Betaxolol showed a tendency to normalize the hemodynamic abnormalities which were observed in the kidney, spleen and gastrointestinal tract of SHR, while atenolol did not. It should be noted that betaxolol increased the flow rate in the kidney, which may be explained by its direct vasodilatory action on renal blood vessels. In conclusion, betaxolol showed an antihypertensive action at the doses of 1 and 10 mg/kg, exhibited the characteristics of a ,81 blocker and produced preferable effects on regional hemodynamics in SHR. Keywords:
Regional
hemodynamics,
Microsphere,
Betaxolol, (±)-1-[4-[2-(cyclopropylmethoxy)ethyl] phenoxy]-3-(isopropylamino)-2-propanol hydrochloride, is a new highly selective 8-blocker that has been re ported to be devoid of a blocking, membrane stabiliz ing and intrinsic sympathomimetic actions (1-4). This drug possesses good plasma bioavailability after oral administration and has a long plasma half-life (5, 6). Although the pharmacodynamic and antihypertensive effects of betaxolol have already been reported (1, 4, 6), its effects on each organ vascular bed, along with hypotension, are unknown. We and others have re ported the differences in regional hemodynamics be tween spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). These studies show that all organs do not equally contribute to the elevation of to tal peripheral resistance (TPR) in SHR and that some of the organs, such as the kidney, gastrointestinal tract and spleen, contribute greatly to the increased TPR (7 10). It would be interesting to know whether betaxolol normalizes the hemodynamic abnormality observed in
Betaxolol,
Atenolol,
Spontaneously
hypertensive
rat
SHR. The present study was designed to clarify whether be taxolol can counteract the basic hemodynamic abnor mality of hypertensive rats in addition to providing a characteristic profile of the drug in relation to its anti hypertensive effect. The effects of betaxolol on systemic and regional hemodynamics were examined and com pared with those of atenolol, a selective 81-blocker (11 13), in conscious SHR by a radioactive microsphere method. MATERIALS AND METHODS Animals and experimental preparation Thirteen to 15-week-old male SHR (Charles River, Japan) were used in this investigation. They were placed on a normal diet until the time of this experi ment. Under a light sodium pentobarbital anesthesia, polyethylene catheters (PE-50) were placed in the fem oral artery and left ventricle via the right carotid artery.
These catheters were filled with heparinized saline (100 IU/ml) and exteriorized through a cutaneous tunnel at the back of the neck after confirming the tip locations by pressure tracings. The animals were allowed to re cover for 24 hr before initiating the experimental proce dures. The experiments were performed in a tempera ture controlled room at 23°C. The catheterized SHR were placed in a plastic cham ber. The femoral arterial catheter was connected to the pressure transducer, and systemic arterial pressure was continuously recorded on a multichannel polygraph (NEC-San-ei, Japan). All of the following experiments were performed under unanesthetized and unrestricted conditions.
dose injection of pentobarbital. The brain, lungs, heart, liver, spleen, kidneys, stomach, intestines (small and large intestine), skin and hindlimb skeletal muscle were removed and weighed. Due to the relatively low blood flow to the skin and skeletal muscle, at least 5 g of these tissues were removed. The skin and skeletal mus cle samples contained 300-400 microspheres, while all other tissues contained over 500 microspheres. The activ ities of samples were analyzed by a gamma scintillation counter. The cardiac output was calculated as follows:
Measurements of systemic and regional hemodynamics Pilot experiments were performed to determine the appropriate point in time for measuring the regional hemodynamic effects of betaxolol and atenolol. In 3 rats of each group, betaxolol and atenolol were orally administered at doses of 1 and 10 mg/kg. We observed the hypotensive effects continuously up to 8 hr after the administration of the drugs. The hypotensive effects appeared within 1 hr and peaked at 3 4 hr. Persistent hypotension was observed for more than 8 hr. No signi ficant change in either blood pressure or heart rate were observed in the control group which was adminis tered distilled water. Based on these results, the measurements of cardiac output and organ blood flow were carried out at 4 hr after the administration of each drug. Radioactive microspheres were utilized to measure the cardiac output and regional blood flow, according to the methods of Tsuchiya et al. (14) and Ishise et al. (15). Radionuclide-labeled microspheres (141Ce, New England Nuclear, U.S.A.), 15 ± 3 ,um in diameter, were used. Following a 60 min stabilization period for the rats to adapt to the chamber, either betaxolol or atenolol at doses of 1 or 10 mg/kg was orally administered (betax olol: n = 10 for 1 mg/kg and n = 9 for 10 mg/kg; ate nolol: n = 9 for 1 mg/kg and n = 10 for 10 mg/kg). Distilled water was administered in the control group (n = 19). The systemic blood pressure and heart rate were stabilized 4 hr after the above administration, and then 0.25 ml of saline containing 75,000 microspheres was in jected into the left ventricle. The injection procedures were carried out over a 15-sec period. Arterial blood samples for reference blood were obtained using a with drawal pump at a rate of 0.65 ml/min starting im mediately before the injection of the microspheres and ending 60 sec later. After termination of the micro sphere injection, the animal was sacrificed by an over
The total injected radioactivity was obtained by sub tracting the residual radioactivity from the radioactivity before injection. Absolute organ blood flow was calcu lated as follows:
Statistical analyses In the present paper, the values are expressed as the mean ± S.E.M. Organ blood flow is expressed as ml/g of organ/min, and organ vascular resistance is express ed as mmHg/ml/g of organ/min. The data were evalu ated by Student's t-test, where P values less than 0.05 were regarded as significant. Chemicals The betaxolol used in this study was gift from Mitsu bishi Chemical Industries, Ltd. (Japan). The atenolol was purchased from Sigma Chemical Co. (U.S.A.). For the oral administration to rats, betaxolol and atenolol were dissolved in distilled water. The drug solution or distilled water was orally administered at 5 ml/kg. RESULTS Effects of betaxolol on systemic and regional hemodynam ics in SHR The effects of betaxolol, 4 hr after administration, on mean arterial pressure (MAP), cardiac output, heart rate and TPR in SHR are tabulated in Table 1. Betax olol at either dose produced significant hypotension. While the low dose of betaxolol failed to reduce cardiac output and heart rate, the high dose reduced both param eters significantly. As a result, the calculated TPR was reduced by the low dose but was not changed by the high dose.
Table tensive
The
regional
hemodynamic
2.
Effects
of
betaxolol
on
organ
effects
induced
by betax
olol are summarized in Table 2, which shows the organ blood flow. The calculated organ vascular resistances are shown in Fig. 1. A low dose of betaxolol signifi cantly increased the flow rate in the kidney, but did not affect the flow rates in the other organs. The high dose of betaxolol decreased the flow rates in the brain, heart, skeletal muscle and skin, along with a reduction in cardiac output. The increased flow rate in the kidney was observed at the high dose, as well as at the low dose. Calculated cantly decreased
organ vascular resistances were signifi in the kidney and gastrointestinal
tract, along with hypotension by either dose. Vascular resistance in the kidney was decreased remarkably by both doses. The high dose increased vascular resistance in the skeletal skin.
muscle,
blood
flow
in conscious
spontaneously
hyper
rats
but not in the brain,
heart
and
Effects of atenolol on systemic and regional hemodynam ics in SHR The effects of atenolol, 4 hr after the administration, on systemic hemodynamics in SHR are shown in Table 3. Although atenolol administered orally at 1 mg/kg tended to reduce the blood pressure, it was not signifi cant. The atenolol at 10 mg/kg decreased significantly by about 20 mmHg. The low dose of atenolol reduced the heart rate, while the high dose reduced both heart rate and cardiac output. TPR was not changed by ate nolol. The effects of atenolol on organ blood flow are tabu lated in Table 4. The low dose of atenolol decreased the flow rates in the portal region and skin. The high dose of atenolol decreased the flow rates in the heart, gastrointestinal tract and skeletal muscle, but did not change it in the kidney. Organ vascular resistances in the liver, intestines and skin were increased by both doses (Fig. 2).
Fig. 1. Effects of betaxolol (1 and 10 mg/kg, p.o.) on organ vascular resistance in conscious spontaneously hypertensive rats. * indicates significant difference between the control and the betaxolol group (P < 0.05). 0 Control; Betaxolol, 1 mg/kg; Betaxolol, 10 mg/kg.
Table
3.
Effects
Table tensive
of atenolol
4.
Effects rats
of
on
systemic
atenolol
on
hemodynamics
organ
blood
in conscious
flow
in conscious
spontaneously
spontaneously
hypertensive
hyper
rats
Fig. 2. Effects of atenolol (1 and 10 mg/kg, p.o.) on organ vascular resistance in conscious spontaneously hypertensive rats. * indicates significant difference between the control and the atenolol group (P < 0.05). [1 Control; ® Atenolol, 1 mg/kg; El Atenolol, 10 mg/kg.
DISCUSSION The effects of the new selective /81-blocker betaxolol on systemic and regional hemodynamics were examined and compared with those of atenolol in conscious SHR. Betaxolol showed a dose-dependent hypotensive effect in SHR following an oral administration of 1 and 10 mg/kg. Atenolol also showed the same hypotensive effect, but it was weaker than that of the betaxolol. These findings are consistent with the previous report (4). It has been reported that other /3-blockers did not reduce or required relatively high doses to reduce MAP in a single administration in hypertensive rats (16-19). Thus it seems that betaxolol, among 8-blockers, has a unique pharmacological property concerning anti hypertensive action. The low dose of betaxolol signifi cantly reduced TPR, while the high dose and atenolol at either dose did not. The lack of change in TPR by both drugs at the high dose may be explained by the re duced cardiac output and heart rate due to the cardio inhibitory effects of the drug. Betaxolol, at the low dose, did not reduce heart rate. This may have been due to reflex tachycardia induced by potent hypotension, which masks its cardioinhibitory effect. At the same dose, atenolol did not reduce MAP but reduced heart rate. These results indicate that betaxolol may have other effects in addition to N1-adrenoceptor blocking action, such as the direct vasodilatory effect.
We and others have previously reported the differ ence between systemic and regional hemodynamics in SHR and WKY (7-10, 18, 19). These reports showed that some of the organs, such as the kidney, gastroin testinal tract and spleen, contributed greatly to the in creased TPR in SHR. The present report provides a re gional hemodynamic profile of betaxolol's effects on these abnormal hemodynamics observed in SHR. Both betaxolol and atenolol, at high doses, reduced the flow rate in many organs. It seems that there are not large differences between them. Hypotension or reduced car diac output may account partially for the results. At the low dose, atenolol tended to reduce the flow rate more than betaxolol in some organs. Since there are differ ences in their systemic effects, a comparison of organ vascular resistance between them is available. Betaxolol at the low dose showed reductions in organ vascular re sistances in the spleen, kidney and gastrointestinal tract. These organs are considered to be closely related to the high TPR in SHR. The flow rate increased, although the blood pressure was reduced, especially in the kidney. This indicates that betaxolol produced renal vasodilatation. On the other hand, atenolol tended to increase the vascular resistance in some organs. Bolt and Saxena (20) have reported that atenolol showed modest effects on organ flow rates in conscious renal hypertensive rabbits, and in the kidney, the flow rate or vascular conductance did not change. Considering these
differences, it is difficult to explain betaxolol's organ vascular resistance reduction effects simply by a 91 adrenoceptor blocking action. It has been reported that propranolol and others tend to decrease renal blood flow in clinical use and animal experiments (21, 22). Betaxolol, however, increased the flow rate in the kidney. There is a question as to how betaxolol causes this increase. It is known that the in hibition of a-adrenoceptors or the stimulation of ( adrenoceptors can increase renal blood flow. The in hibition of presynaptic ,8-adrenoceptor, causing an in hibition of the release of noradrenaline (23, 24), and of the renin-angiotensin system (25) may be also consi dered. However, betaxolol and atenolol have the same properties as a 81-blocking agent (11). Therefore, the difference between their renal actions can not be ex plained by the above factors. It can be considered that betaxolol may have other, yet unknown, vasodilating actions including a direct action. Both of the drugs, at the high dose, reduced the flow rate in the skeletal muscle. Betaxolol increased the vascular resistance in the skeletal muscle, while atenolol tended to increase it as well. Although betaxolol is a highly selective ,81-blocker (2), this may be explained by the fact that betaxolol, at the high dose, exerts a blockade of 82-adrenoceptors in the vascular bed of skeletal muscle (26). Since the flow rate in the skeletal muscle in SHR is higher than that in WKY (10), it might be considered that the flow rate was merely re versed to the WKY level by betaxolol. It is known that a /8-blocker does not exert its hy potensive effect powerfully in SHR by a single adminis tration when compared with other species. Warrington et al. (27) indicated that betaxolol had a more powerful antihypertensive effect than others, but Roudaut et al. (28) did not find any difference between betaxolol and atenolol. In the present results, there are interesting points which show that betaxolol decreased MAP by a single administration, at the relatively low dose, and in creased the flow rate in the kidney in contrast to other ,8-blockers. Thus, betaxolol can be considered to be a ,81-blocker with unknown vasodilating mechanisms.
stimulation of spinal cord, in spontaneously hypertensive rats. Br. J. Pharmacol. 78, Supp. 28P (1983) 3 Cavero, I. and Lefevre-Borg, F.: Antihypertensive activity of betaxolol in conscious spontaneously hypertensive rats. Br. J. Pharmacol. 78, Supp. 141P (1983) 4 Cavero, I., Lefevre-Borg, F., Manoury, P. and Roach, A.: In vitro and in vivo pharmacological evaluation of betaxolol, a new, potent, and selective 61-adrenoceptor antagonist. In Betaxolol and Other ,81-Adrenoceptor Antagonists, Edited by Morselli, P.L., Kilborn, J.R., Cavero, I., Harrison, D.C. and Langer, S.Z., p. 31-42, Raven Press, New York (1983) 5 Cadigan, P., London, D. and Petecost, B.: Cardiovascular effects of single oral doses of the new beta-adrenoceptor blocking agent betaxolol (SL 75212) in healthy volunteers. Br. J. Clin. Pharmacol. 9, 569-575 (1980) 6 Warrington, S., Turner, P., Kilborn, J., Bianchetti, G. and Morselli, P.: Blood concentrations and pharmacodynamic effects of betaxolol (SL 75212) a new beta-adrenoceptor an tagonist after oral and intravenous administration. Br. J. Clin. Pharmacol. 10, 449-452 (1980) 7 Tadepalli, A., Walsh, G. and Tobia, A.: Normal cardiac out
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Acknowledgments We are grateful to Stacey Laumann for the proof reading of this manuscript and J. Fujita and A. Tanaka for their secretarial ser vices.
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