knockout mice and endothelin A receptor null - Clinical Science

4 downloads 0 Views 99KB Size Report
Blood pressure of endothelin-3 null (k/k) knockout ... ETA receptor (k/k) mice were measured using the servo null pressure ..... an endothelin receptor antagonist.
48S

Clinical Science (2002) 103 (Suppl. 48), 48S–52S (Printed in Great Britain)

Blood pressure of endothelin-3 null (k/k) knockout mice and endothelin A receptor null (k/k) knockout mice under anaesthesia Tomoyuki KUWAKI*†, Toru ISHII†, Kihwan JU‡, Masashi YANAGISAWA§ and Yasuichiro FUKUDA† *Department of Molecular and Integrative Physiology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan, †Department of Autonomic Physiology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan, ‡Department of Biomedical Engineering, School of Engineering, The City University of New York, NY 10031, U.S.A., and §Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75235, U.S.A.

A

B

S

T

R

A

C

T

Blood pressure (BP) and heart rate (HR) in endothelin-3 (ET-3) null (k/k) knockout mice and ETA receptor (k/k) mice were measured using the servo null pressure measuring technique under halothane anaesthesia. In infant ET-3 (k/k) mice (2–3 weeks old), mean BP and HR were 55p2 mmHg and 436p30 beats/min respectively. These values were not different from those in age-matched wild-type mice (53p3 mmHg and 430p18 beats/min respectively). Baroreflex sensitivity, which was calculated as the slope of the relationship between systolic BP and RR interval on an ECG, was also similar in ET-3 (k/k) mice (0.84p0.20 ms/mmHg) and wild-type mice (1.07p0.38 ms/mmHg). ETA receptor (k/k) mice were obtained by caesarean section on the expected day of delivery and tracheotomized, so that they would live for more than 24 h. Mean BP and HR in ETA receptor (k/k) mice were 15p1 mmHg and 333p6 beats/min respectively. These values were not different from those in age-matched, similarly treated wildtype mice (16p3 mmHg and 308p10 beats/min respectively). Baroreflex sensitivity in the newborn ETA receptor (k/k) mice (0.45p0.15 ms/mmHg) and wild-type mice (0.31p0.06 ms/mmHg) were very low compared with the values in infant wild-type mice, but not different between the mutant mice and their littermates. Moreover, HR in awake ETA receptor (k/k) mice (396p13 beats/min) was not different from that in wild-type mice (409p13 beats/min). These results show that the ETA receptor and ET-3 are not involved in cardiovascular regulation, at least during the very early life of the mice. A possible involvement of the ETA receptor in BP regulation, if any, seems to occur at later times and/or in some pathological settings.

INTRODUCTION A deficiency of endothelin-1 (ET-1) [1] or of ETB receptors [2,3] has been shown to induce hypertension in several animal models of genetically engineered rodents.

However, possible roles for ET-3 and ETA receptors in blood pressure (BP) regulation are still unclear, due to a lack of the appropriate animal models and measurement techniques. Both ET-3 [4] and ETA receptor [5] null knockout mice die early in life, and until now there had

Key words : baroreflex, blood pressure, endothelin, halothane anaesthesia, mice. Abbreviations : BP, blood pressure ; ET, endothelin ; HR, heart rate ; SBP, systolic blood pressure. Correspondence : Professor Tomoyuki Kuwaki, at Department of Molecular and Integrative Physiology (e-mail kuwaki!med.m.chiba-u.ac.jp).

# 2002 The Biochemical Society and the Medical Research Society

Blood pressure of endothelin null mice

been no adequate method for measuring BP in very small animals, such as newborn mice. To overcome these problems, we have recently established a method to measure BP in developing mice using a servo null technique [6]. The objective of the present study was to examine possible roles of ET-3 and ETA receptors in BP regulation by measuring BP, heart rate (HR) and autonomic baroreflex control.

METHODS ET-3 null (k/k) knockout mice and ETA receptor null (k/k) knockout mice ET-3 null (k\k) mutated knockout mice with the genetic background 129\Sv were generated as reported previously, and genotypes were confirmed by PCR on DNA extracted from a tail biopsy [4]. Mice used in this study were 2–3 weeks old ET-3 (k\k) mice (n l 6) and wild-type [ET-3 (j\j)] mice of the 129\Sv strain (n l 9). We selected this age because baroreflex control in mice matures between postnatal days 7 and 14 [6]. The disease-free period for ET-3 (k\k) mice before they developed megacolon varied widely, between 1 and 12 weeks of age [4]. However, there were no apparent symptoms of Hirchsprung’s disease in the mice that we used in the present study. Body weight [9.8p1.0 g in ET-3 (k\k) and 10.5p0.9 g in ET-3 (j\j) mice] was not significantly different between the two groups. ETA receptor null (k\k) knockout mice were generated by a similar gene targeting technique [5]. Since these mice would be expected to die soon after birth due to mechanical obstruction of the upper airway, they were delivered by caesarian delivery on the expected day of delivery and tracheotomized to secure the airway [7]. ETA receptor (k\k) mice treated in this way remained alive for at least 24 h [5]. Wild-type [ETA receptor (j\j)] littermates were treated in the same manner. The body weight of ETA receptor (k\k) mice (1.23p0.05 g ; n l 9) was not different from that of the ETA receptor (j\j) mice (1.32p0.08 g ; n l 12). All experimental procedures were in accordance with the ‘ Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences ’, as recommended by the Physiological Society of Japan.

Measurement of BP and HR In accordance with our recently established method [6], the animals were anaesthetized with halothane and their BP was measured by the servo null technique (model 900A ; WPI, Sarasota, FL, U.S.A.) from the femoral artery through a glass pipette filled with 1 M NaCl and 50 units\ml heparin. The halothane concentration (1.5–2.0 % during surgery and " 1.0 % during BP

measurement) was decided on the basis of results from our previous experiments [6]. Pin electrodes for the measurement of ECG were also attached to the root of the legs. HR was calculated using a tachometer triggered by either BP or an ECG signal. Rectal temperature was maintained between 34 mC and 36 mC in newborn mice [ETA receptor (k\k) and ETA receptor (j\j)] and between 36 mC and 38 mC in juvenile mice [ET-3 (k\k) and ET-3 (j\j)]. In each animal, BP and ECG were measured for more than 20 min, at least 30 min after the completion of surgery. In a different set of newborn animals, an ECG (but not BP) was measured in the awake condition from clip electrodes attached to the legs under local anaesthetic (lidocaine jelly). The animal was kept in a warm (32–35 mC) chamber, and the ECG was measured for 20 min after a 30 min stabilization period.

Data processing and evaluation of baroreflex sensitivity BP and ECG signals were fed into an analogue-to-digital converter (Mac Lab\16s ; AD Instruments, Castle Hill, NSW, Australia) at a sampling frequency of 200 Hz for BP and 4000 Hz for ECG, and then to a Macintosh computer. Systolic BP (SBP), mean BP, diastolic BP, pulse pressure and RR interval for each heartbeat were obtained using CHART data-acquisition\analysis software (AD Instruments). The baroreflex was analysed by the naturally occurring sequence method, as described in our previous report [6]. In brief, recordings were scanned by a computer program written in our laboratory to identify spontaneous sequences of three or more consecutive beats in which SBP progressively increased or decreased by more than 1 mmHg\beat. Of these SBP sequences, those associated with baroreflex-driven lengthening or shortening of the RR interval ( 0.5 ms) were selected and defined as baroreflex sequences. A linear regression analysis between SBP and RR interval was applied to each baroreflex sequence, and the slope of the regression line was calculated. An averaged value of the slopes in a mouse was taken as the sensitivity (gain) of baroreflex in the animal. We also calculated the baroreflex effectiveness index, defined as the ratio between the number of baroreflex sequences and the total number of SBP ramps, regardless of the possible occurrence of a concomitant reflex change in RR interval [8].

Statistical analysis Data are expressed as meanspS.E.M. Tukey–Kramer’s multiple-comparison method was used to detect possible differences between genotypes and between ages. A probability level of P 0.05 was considered significant. # 2002 The Biochemical Society and the Medical Research Society

49S

50S

T. Kuwaki and others

Figure 1 Typical tracings of BP measured by the servo null method and simultaneous ECG from electrodes attached to the legs of mice under halothane anaesthesia

Upper panels : sample recordings from a newborn (postnatal day 0 ; P0) ETA receptor null (AR−/−) mouse and a control wild-type mouse (AR+/+). Lower panels : recordings from a juvenile (postnatal day 14 ; P14) ET-3 null (ET3−/−) mouse and a wild-type littermate (ET3+/+).

RESULTS AND DISCUSSION BP and HR in ET-3 (k/k) mice and ETA receptor (k/k) mice To examine the possible roles of ET-3 and ETA receptors in cardiovascular regulation, we measured BP and HR in ET-3 (k\k) mice and ETA receptor (k\k) mice. The servo null pressure system supplied stable, good-quality recordings of BP and ECG in newborn and juvenile mice (Figure 1). From recordings of BP and ECG of " 20 min for each animal, SBP, mean BP, diastolic BP and HR were calculated ; values are summarized for 5–9 animals in each group in Figure 2. Although age-dependent differences in BP and HR in the wild-type mice were apparent, as was the case in our previous study [6], there were no differences in either parameter between ETA receptor (k\k) and ETA receptor (j\j) mice, or between ET-3 (k\k) and ET-3 (j\j) mice. In the newborn mice, tracheotomy seemed not affect BP or HR, since there was no difference between tracheotomized wildtype mice in the present study and intact wild-type mice in our previous study with regard to either mean BP (16p3 and 19p2 mmHg respectively) or HR (308p10 and 330p10 beats\min respectively). To examine possible effects of halothane anaesthesia on cardiovascular parameters, HR was also measured in unanaesthetized ETA receptor (k\k) and (j\j) mice. Again, there was no difference between ETA null (396p13 beats\min ; n l 4) and wild-type (409p13 beats\min ; n l 7) newborn mice, while both # 2002 The Biochemical Society and the Medical Research Society

Figure 2 Comparison of BP and HR between ETA receptor null (AR−/−) mice and wild-type littermates (AR+/+), and between ET-3 null (ET3−/−) mice and wild-type controls (ET3+/+)

ETA receptor null mice (n l 5) and their wild-type controls (n l 5) were obtained by caesarian delivery on the expected day of birth and tracheotomized to secure the airway. ET-3 null mice (n l 6) and their wild-type controls (n l 9) were assessed at 2–3 weeks old. Data are meanspS.E.M. Note that there was no significant difference between mutants and their controls in SBP, mean BP, diastolic BP or HR. On the other hand, BP and HR increased with age in the wild-type mice.

Blood pressure of endothelin null mice

Figure 3

Arterial baroreceptor reflex in ETA receptor null (AR−/−) mice and ET-3 null (ET3−/−) mice

Left panel : number of SBP ramps in which SBP increased or decreased progressively for three or more consecutive beats. Middle panel : baroreflex effectiveness index, defined as the ratio between the number of SBP ramps associated with reflex changes in the RR interval (baroreflex sequences) and the total number of SBP ramps, regardless of the possible occurrence of concomitant reflex change. Right panel : baroreflex gain, calculated as the slope of the regression line between SBP and RR interval during the baroreflex sequences. Data are meanspS.E.M. There were no genotype-dependent differences in the number of ramp sequences, baroreflex effectiveness index or baroreflex gain. An age-dependent difference in baroreflex gain was observed. values were significantly higher than those measured under anaesthesia [in different sets of mice : 333p6 beats\min in ETA null mice (n l 5) and 308p 10 beats\min in wild-type mice (n l 5)]. Thus the effect of anaesthesia on cardiovascular parameters seems to be independent of ETA receptor genotype.

Baroreflex control of HR in ET-3 (k/k) mice and ETA receptor (k/k) mice Since ET and ET receptors are expressed in a part of the central and peripheral nervous system that subserves the arterial baroreceptor reflex [9], we next analysed baroreflex function in ETA receptor (k\k) and ET3 (k\k) mice. Although the mice were anaesthetized, there were considerable spontaneous fluctuations in SBP, and pressure ramps appeared 50–150 times per 1000 heart beats (Figure 3). Of these SBP ramps, 7–10 % were associated with reflex prolongation or shortening of the RR interval, and hence were classified as a baroreflex sequence. Baroreflex gain in the tracheotomized newborn wildtype mice (0.31p0.06 ms\mmHg) was smaller than that in juvenile wild-type mice (1.07p0.38 ms\mmHg), but not different from that in intact newborn wild-type mice (0.37p0.09 ms\mmHg). These results in wild-type mice were coincided well with our previous observations [6]. In comparisons between ETA receptor (k\k) and ETA receptor (j\j) mice, and between ET-3 (k\k) and ET-3 (j\j) mice, there were no genotypedependent differences in the number of ramp sequences, the baroreflex effectiveness index or baroreflex gain

(Figure 3). The normal baroreceptor HR reflex in ETA receptor (k\k) mice shows a striking contrast with the attenuated chemoreceptor\ventilatory reflex in this mutant [5]. Meanwhile, both the baroreflex (the present study) and the chemoreflex [10] were normal in ET-3 (k\k) mice, indicating the importance of the ET-1 (but not ET-3)\ETA receptor signalling system in respiratory regulation. These results support our hypothesis that the long-term abnormality in respiratory regulation in ET-1deficient mice may secondarily affect their BP [11]. It may be useful to point out that BP in adult ETA receptor ‘ half-deficient ’ (j\k) mice was not different from that in wild-type controls [11]. A role for the ETA receptor in the regulation of BP, if any, may well achieved by a small population of the receptors, and this may explain why BP in normal animals tolerated ETA receptor blocking agents in some reports [12]. This point should be studied further using conditional-knockout animal models.

Conclusion We conclude that the ETA receptor and ET-3 are not involved in cardiovascular regulation, at least during the very early life of mice. A BP regulatory function of the ETA receptor, as suggested by many pharmacological experiments using adult animals [13], may occur later in life and\or become apparent only in some pathological settings.

ACKNOWLEDGMENTS Part of this work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, # 2002 The Biochemical Society and the Medical Research Society

51S

52S

T. Kuwaki and others

Science, Culture and Sports, Japan, and grants from the Shimadzu Science Foundation and the Japan Foundation of Cardiovascular Research.

REFERENCES 1

2

3

4

5

Kurihara, Y., Kurihara, H., Suzuki, H. et al. (1994) Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature (London) 368, 703–710 Ohuchi, T., Kuwaki, T., Ling, G. Y. et al. (1999) Elevation of blood pressure by genetic and pharmacological disruption of endothelin-B receptor in mice. Am. J. Physiol. 276, R1071–R1077 Gariepy, C. E., Ohuchi, T., Williams, S. C., Richardson, J. A. and Yanagisawa, M. (2000) Salt-sensitive hypertension in endothelin-B receptor-deficient rats. J. Clin. Invest. 105, 925–933 Baynash, A. G., Hosoda, K., Giaid, A. et al. (1994) Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79, 1277–1285 Clouthier, D. E., Hosoda, K., Richardson, J. A. et al. (1998) Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development 125, 813–824

# 2002 The Biochemical Society and the Medical Research Society

6

Ishii, T., Kuwaki, T., Masuda, Y. and Fukuda, Y. (2001) Postnatal development of blood pressure and baroreflex in mice. Auton. Neurosci. Basic Clin. 94, 34–41 7 Kuwaki, T., Cao, W. H., Kurihara, Y. et al. (1996) Impaired ventilatory responses to hypoxia and hypercapnia in mutant mice deficient in endothelin-1. Am. J. Physiol. 270, R1279–R1286 8 Di Rienzo, M., Parati, G., Castiglioni, P., Tordi, R., Mancia, G. and Pedotti, A. (2001) Baroreflex effectiveness index : an additional measure of baroreflex control of heart rate in daily life. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R744–R751 9 Kuwaki, T., Kurihara, H., Cao, W. H., Kurihara, Y., Yazaki, Y. and Kumada, M. (1997) Physiological role of brain endothelin in the central autonomic control : from neuron to knockout mouse. Prog. Neurobiol. 51, 545–579 10 Nakamura, A., Kuwaki, T., Kuriyama, T., Yanagisawa, M. and Fukuda, Y. (2000) Normal ventilation and ventilatory responses to chemical stimuli in juvenile mutant mice deficient in endothelin-3. Respir. Physiol. 124, 1–9 11 Kuwaki, T., Ling, G. Y., Onodera, M. et al. (1999) Endothelin in the central control of cardiovascular and respiratory functions. Clin. Exp. Pharmacol. Physiol. 26, 989–994 12 Li, J. S. and Schiffrin, E. L. (1995) Effect of chronic treatment of adult spontaneously hypertensive rats with an endothelin receptor antagonist. Hypertension 25, 495–500 13 Schiffrin, E. L. (1999) Role of endothelin-1 in hypertension. Hypertension 34, 876–881