Tomoyuki Kuwaki

Elevation of blood pressure by genetic and pharmacological disruption of the ETB receptor in mice

Exogenously administered endothelin (ET) elicits both pressor and depressor responses through the ETA and/or the ETB receptor on vascular smooth muscle cells and ETB on endothelial cells. To test whether ETB has pressor or depressor effects under basal physiological conditions, we determined arterial blood pressure (BP) in ETB-deficient mice obtained by crossing inbred mice heterozygous for targeted disruption of the ETB gene with mice homozygous for the piebald ( s) mutation of the ETB gene ( ET Bs/s ). F1 ET B-/s and ET B+/s progeny share an identical genetic background but have ETB levels that are ∼[Formula: see text]and [Formula: see text], respectively, of wild-type mice ( ET B+/+ ). BP in ET B-/s mice was significantly higher, by ∼20 mmHg, than that in ET B+/s or ET B+/+ mice. Immunoreactive ET-1 concentration in plasma as well as respiratory parameters was not different between ET B-/s and ET B+/s mice. A selective ETB antagonist, BQ-788, increased BP in ET B+/s and ET B+/+ but not in ET B-/s mice. Pretreatment with indomethacin, but not with N G-monomethyl-l-arginine, can attenuate the observed pressor response to BQ-788. The selective ETA antagonist BQ-123 did not ameliorate the increased BP in ET B-/s mice. Moreover, BP in mice heterozygous for targeted disruption of the ETA gene was not different from that in wild-type controls. These results suggest that endogenous ET elicits a depressor effect through ETB under basal conditions, in part through tonic production of prostaglandins, and not through secondary mechanisms involving respiratory control or clearance of circulating ET.Exogenously administered endothelin (ET) elicits both pressor and depressor responses through the ETA and/or the ETB receptor on vascular smooth muscle cells and ETB on endothelial cells. To test whether ETB has pressor or depressor effects under basal physiological conditions, we determined arterial blood pressure (BP) in ETB-deficient mice obtained by crossing inbred mice heterozygous for targeted disruption of the ETB gene with mice homozygous for the piebald (s) mutation of the ETB gene (ETBs/s). F1 ETB-/s and ETB+/s progeny share an identical genetic background but have ETB levels that are approximately (1)/(8) and (5)/(8), respectively, of wild-type mice (ETB+/+). BP in ETB-/s mice was significantly higher, by approximately 20 mmHg, than that in ETB+/s or ETB+/+ mice. Immunoreactive ET-1 concentration in plasma as well as respiratory parameters was not different between ETB-/s and ETB+/s mice. A selective ETB antagonist, BQ-788, increased BP in ETB+/s and ETB+/+ but not in ETB-/s mice. Pretreatment with indomethacin, but not with NG-monomethyl-L-arginine, can attenuate the observed pressor response to BQ-788. The selective ETA antagonist BQ-123 did not ameliorate the increased BP in ETB-/s mice. Moreover, BP in mice heterozygous for targeted disruption of the ETA gene was not different from that in wild-type controls. These results suggest that endogenous ET elicits a depressor effect through ETB under basal conditions, in part through tonic production of prostaglandins, and not through secondary mechanisms involving respiratory control or clearance of circulating ET.

Impaired development of the thyroid and thymus in endothelin-1 knockout mice.

Summary: We have previously demonstrated that endothelin-1 (ET-1) is essential to the development of neural crest-derived craniofacial and cardiovascular structures. In this study we evaluated the intrauterine growth and development of the thyroid and thymus glands in Ednl-I- homozygous mice. Ednl-I- homozygous mice were smaller than their heterozygous or wild-type littermates (about 90% of normal body weight). The thyroid and thymus of Ednl −1 - homozygous mice were smaller than those of normal mice and were not fused in the midline, resulting in two separate lobules. The thymus of Ednl −1 - homozygous mice is not descended into the normal position. These results suggest that ET-1 is important for normal development of the glands in the neck region, as well as for systemic growth. The combination of abnormalities in Ednl-I- homozygous mice suggests that these mice might serve as an animal model for the human diseases DiGeorge syndrome and velocardiofacial syndrome.

Renal sympathetic nerve activity in mice: comparison between mice and rats and between normal and endothelin-1 deficient mice

Recently generated knockout mice with disrupted genes encoding endothelin (ET)-1 showed an elevation of arterial blood pressure (AP) and supplied an evidence for intrinsic ET-1 as one of the physiological regulators of systemic AP. Little is yet known, however, why deficiency of ET-1, which was originally found as a potent vasoconstrictor, led to higher AP in these mice. To address this apparent paradox, we first developed a method to measure renal sympathetic nerve activity (RSNA) in mice using rats as reference and successively compared it between normal and ET-1 deficient mice. RSNA was successfully recorded in urethane-anesthetized and artificially ventilated mice by a slight modification of the method used for rats. At basal condition, mean AP (MAP) and RSNA in ET-1 deficient mice (105+/-2 mmHg and 9.71+/-1.49 muVs, n=20) were significantly higher than those in wild-type mice (96+/-2 mmHg and 5. 07+/-0.70 muVs, n=25). Basal heart rate (HR) and baroreflex-control of HR was not significantly different between the two. On the other hand, resting RSNA, RSNA range, and maximum RSNA were significantly greater in ET-1 deficient mice, and thus MAP-RSNA relationship was upwards reset. Hypoxia-induced increase in RSNA was not different between ET-1 deficient (73.4+/-9.4%) and wild-type mice (91.2+/-12.0%), while hypercapnia-induced one was significantly attenuated in ET-1 deficient mice (18.8+/-3.6 vs. 39.1+/-5.2% at 10% CO2). These results indicate that endogenous ET-1 participates in the central chemoreception of CO2 and reflex control of the RSNA. Baroreceptor resetting and normally preserved hypoxia-induced chemoreflex may explain a part of the elevation of AP in ET-1 deficient mice.

Differential Central Modulation of the Baroreflex by Salt Loading in Normotensive and Spontaneously Hypertensive Rats

In salt-sensitive hypertensive animal models and human subjects compared with their salt-resistant counterparts, sympathetic activity is abnormally enhanced during a high salt diet. We examined whether salt loading differentially modulates the arterial baroreceptor reflex (ABR), a major control mechanism of arterial pressure and sympathetic vasomotor activity, in young normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Six-week-old WKY and SHR were fed a normal (0.66%) or high (8.00%) salt diet for 4 weeks. After the diet regimen, baseline levels of mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and the overall and central properties of the ABR were compared among the four groups of rats under halothane anesthesia. In WKY, a high salt diet did not affect baseline arterial pressure and RSNA but potentiated the ABR, as evidenced by an increase in the maximal slope of MAP-RSNA and MAP-heart rate relationships. In SHR, by contrast, salt loading accelerated hypertension and sympathetic overactivity and impaired the ABR. Salt-induced modulation of the ABR was associated with that of the central property, since reflex inhibition of RSNA by stimulation of the aortic depressor nerve was augmented in WKY and attenuated in SHR. These results suggest that differential modulation of the central mechanism subserving the baroreflex control of sympathetic activity at least partly accounts for the difference in salt sensitivity between WKY and SHR.

Effect of endothelin on vasomotor and respiratory neurons in the rostral ventrolateral medulla in rats

Abstract1. We have previously shown that intracisternal administration of endothelin-1 (ET-1) elicited cardiorespiratory responses acting on the ventral surface of the medulla oblongata (VSM) subjacent to the rostral ventrolateral medulla (RVLM). In this study, we examined whether vasomotor and respiratory neurons in RVLM participate in above-mentioned responses and whether those neurons respond to direct iontophoretic application of ET-1 and/or an ET-A receptor antagonist, FR139317.2. Unit activity of vasomotor, respiratory, or nociceptive neurons in RVLM was recorded together with arterial blood pressure (AP) and heart rate (HR) in urethane-anesthetized Sprague-Dawley rats.3. Intracisternal administration or topical application of ET-1 (0.1–1 pmol) to VSM caused excitation of the majority of vasomotor neurons (15/18) and respiratory neurons (10/11) but not in nociceptive neurons (0/7). Changes in neuronal activity were in similar time course with corresponding changes in AP and HR. Iontophoretic application of ET-1 to the vicinity of recording neuron caused excitation in 19 of 21 vasomotor neurons without affecting AP nor HR. Remaining two neurons were insensitive to ET-1. FR139317 did not affect basal activity of the vasomotor neurons but inhibited ET-1-evoked excitation. Twenty-four of 40 respiratory neurons were excited and 13 were inhibited by iontophoretic application of ET-1. Five of ET-1-excited respiratory neurons were inhibited by FR139317 alone while six of ET-1-inhibited neurons were not affected by FR139317 alone. In both cases, FR139317 inhibited the effect of simultaneously applied ET-1. Iontophoretic application of ET-1 excited only one out of 10 nociceptive neurons so far tested.4. These results support the view that intracisternally administered ET-1 alters activity of vasomotor and respiratory neurons in the RVLM, at least in part by acting directly on neurons themselves and hence causes systemic cardiorespiratory changes. Majority of vasomotor and respiratory neurons should express ET-A receptors and some respiratory neurons are under tonic excitatory control by ET-1.

Endothelin-1 modulates cardiorespiratory control by the central nervous system

In urethane-anesthetized, vagotomized and immobilized rats under artificial ventilation, an intracisternal injection of 0.1 pmol of endothelin-1 resulted in immediate increases, lasting for 3-15 min, in arterial pressure, heart rate and renal sympathetic nerve activity. Phrenic nerve activity and the rate of its burst activity (burst rate) also increased initially but subsequently decreased for 5-20 min. At doses of 1 or 10 pmol, the initial increases (phase I) were followed by a period of decreases in all variables, that lasted for 20-80 min, below the pre-injection level (phase II). Phrenic nerve activity often disappeared completely. All the variables usually returned to, or often exceeded, pre-injection levels (phase III). However, arterial pressure sometimes remained below control for at least 2 h. Topical application of endothelin-1 to the ventral surface of the medulla produced the same pattern of changes as with intracisternal injection. This particular response pattern was not generated by local administration to any other brain sites examined. In conclusion, intracisternally administered endothelin-1 modulates cardiorespiratory control by the central nervous system. The effect on the central respiratory control was especially powerful. The ventral surface of the medulla appears to play a crucial role in this modulation.

Endothelin-sensitive areas in the ventral surface of the rat medulla

In urethane-anesthetized rats, subregions of the ventral surface of the medulla (VSM) in which endothelin (ET) caused cardiorespiratory effects were mapped by topically applying 1 pmol of ET-1. Two distinct subregions, termed the rostral and caudal ET-sensitive areas, were identified. The rostral area was also sensitive to L-glutamate and glycine. It extended between the caudal end of the trapezoid body and the rootlet of the XIIth nerve partly overlying the pyramidal tract. In this position ET-1 caused the type I response consisting of an initial increase (excitatory component) in arterial pressure (AP), renal sympathetic nerve activity (RSNA), heart rate (HR), phrenic nerve activity (PNA) and the number of bursts of PNA (burst rate) followed by a sustained decrease (inhibitory component) in them. The caudal ET-sensitive area was located near the rootlet of the XIIth nerve. In this position ET-1 caused the type II response consisting of a decrease in PNA and an increase in burst rate. Part of this area responded to nicotine but not to glutamate or glycine. ET-3 (10 pmol) applied to the two ET-sensitive areas produced responses similar to those elicited by ET-1. The dose-response relationship was investigated by delivering ETs to the rostral area. The excitatory component of most of the variables was elicited at a dose of 1 fmol of ET-1 or 1 pmol of ET-3, whereas the inhibitory component was produced at 10 fmol of ET-1 or 10 pmol of ET-3. These results suggest that subregions of the rats VSM may participate in the central cardiorespiratory control by ET.

High calcium diet prevents baroreflex impairment in salt-loaded spontaneously hypertensive rats.

To investigate the role of the sympathetic control mechanism in the antihypertensive effect of dietary calcium supplementation, we examined whether a high calcium diet affected mean arterial pressure, renal sympathetic nerve activity, heart rate, and overall and central properties of the arterial baroreceptor reflex in salt-loaded young spontaneously hypertensive rats (SHR). Six-week-old SHR were fed either a normal (0.66%) or high (8.00%) salt diet with either a normal (1.17%) or high (4.07%) calcium content for 4 weeks. The arterial baroreceptor reflex was elicited with rats under halothane anesthesia by altering mean arterial pressure with nitroprusside or phenylephrine. The overall property of the arterial baroreceptor reflex was assessed by the median mean arterial pressure (MAP50) and maximal gain (Gmax) of the relation between mean arterial pressure and renal sympathetic nerve activity and between mean arterial pressure and heart rate. The central property of the arterial baroreceptor reflex was assessed by reflex inhibition of renal sympathetic nerve activity and heart rate elicited by electrical stimulation of the aortic depressor nerve. Compared with the control group fed a normal salt/normal calcium diet, the high salt/normal calcium group had significantly higher mean arterial pressure and renal sympathetic nerve activity but not heart rate. Moreover, the arterial baroreceptor reflex was impaired in the latter group, as evidenced by an increase in MAP50 and decrease in Gmax of the two relations and an attenuation of reflex inhibition of renal sympathetic nerve activity by aortic depressor nerve stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Power spectral analysis of arterial blood pressure in mice: comparison between normal and endothelin-1 gene knocked out mice

In mice heterozygous for endothelin-1 gene mutation and wild-type littermates power spectrum of systolic arterial pressure (SAP) was determined in a conscious stare by forward-backward autoregressive model with maximum entropy method. It was composed of low (LF) and high frequency components (HF). LF reflected sympathetic vasoconstrictor activity, whereas HF was inversely related to respiratory movement. In ET-1 heterozygous mice LF and HF were both significantly elevated than in wild-type mice. The authors conclude that power spectral analysis is a useful mathematical tool to study the autonomic cardiovascular control and respiration in mice and that sympathetic vasoconstrictor activity was increased in ET-1 heterozygous mice. The latter finding may account for observed elevation of arterial pressure (AP) in these mutant mice.

Development of an analysis system for 24-hour blood pressure and heart rate variability in the rat

Abstract We have developed a system to evaluate 24‐h changes of heart rate (HR) and blood pressure variability (BPV) in free moving rat. As a sensor to monitor electrocardiogram, we used the telemetry system. Analog data were sent to an analog to digital converter directly. The data were digitized at a sampling frequency of 1250 Hz and digitally stored on a computer. RR‐intervals (difference of successive RR in electrocardiogram) were calculated using custom designed programs. Circadian change of the blood pressure and heart rate variability were calculated. We suggest that the system is a powerful tool in the evaluation of circadian changes of hemodynamics and autonomic nervous function in free moving rats.