Vol. 87, Issue 4, 1266-1271, October 1999
INVITED REVIEW
Targeted deletion of the neutral endopeptidase gene alters
ventilatory responses to acute hypoxia in mice
H.
Grasemann1,
B.
Lu2,
A.
Jiao1,
J.
Boudreau1,
N. P.
Gerard2, and
G. T.
De
Sanctis1
1 Combined Program in Pulmonary
and Critical Care Medicine, Brigham and Women's Hospital and Harvard
Medical School, and 2 Ina Sue
Perlmutter Laboratory, Children's Hospital, Department of Medicine,
Beth Israel Deaconess Medical Center, and Harvard Medical School,
Boston, Massachusetts 02115
 |
ABSTRACT |
Neutral endopeptidase (NEP) is
one of the major endopeptidases responsible for the inactivation of
substance P in the carotid body, a neurotransmitter shown to be
important in the transduction of hypoxic stimuli.
Ventilatory responses to acute hypoxia were measured by indirect
plethysmography in unanesthetized, unrestrained wild-type mice and in
mice in which the NEP gene was deleted (NEP -/-). Ventilation was
measured while the animals breathed room air: 12%
O2 in
N2 and 8%
O2 in
N2. Deletion of the NEP gene
caused marked alterations in both the magnitude and composition of the hypoxic ventilatory response to both 8%
O2 in
N2 and 12%
O2 in N2, compared with the wild-type
mice (C57BL/6J) on the same genetic background as the NEP -/- mice.
Treatment of C57BL/6J mice with thiorphan, a NEP inhibitor, resulted in
a greater ventilatory response to 8%
O2 because of a significantly
greater shortening of expiratory time. The results of these studies
demonstrate that NEP plays an important role in modifying the
expression of the ventilatory response to acute hypoxia.
substance P; unanesthetized mice; control of breathing
| |
INTRODUCTION |
RESPIRATION IS ASSOCIATED with changes in both
PO2 and
PCO2 and pH in arterial blood. Two
chemoreceptors are responsible for sensing the blood-gas levels: one is
centrally located in the ventral medulla, and the other is peripherally located in the carotid and aortic bodies. The peripheral arterial chemoreceptors are responsible for the immediate increase in
ventilation produced by hypoxia in which the carotid bodies play a
pivotal role in responding to rapid changes in arterial
PO2. The increase in ventilatory
frequency with hypoxia is largely due to increases in afferent
discharge arising from the carotid body, as the decrease in both
inspiratory (TI) and
expiratory time (TE) with
hypoxia is significantly reduced after carotid body denervation in the
dog (15). The arterial chemoreceptors in the cat, rabbit, and other
animals contain several neuroactive substances, including biogenic
amines, enkephalins, and substance P (SP) (13, 27). Presently, we are
not aware of any studies that have examined the carotid body content of
biogenic amines and neuropeptides in the mouse.
SP, a tachykinin, has evoked considerable attention with regard to its
role in chemoreception (2, 4-7, 21, 22, 24). SP-like
immunoreactivity has been demonstrated in the carotid body (13, 22),
and intra-arterial administration of SP causes a dose-dependent
increase in carotid sinus nerve discharge in cats (18). Additionally,
administration of SP antagonists markedly attenuates the hypoxia- but
not CO2-induced excitation of the carotid body (23). Furthermore, administration of SP into the ventrolateral medulla oblongata has an excitatory effect on tidal volume (VT) and minute
ventilation (
E) (3).
Whereas numerous studies have investigated the role of neuropeptides in
chemoreception, the metabolic fate of these neuropeptides has only
recently been investigated in the carotid body. An extensive investigation of the biochemical, immunologic, and hydrolytic functions
of peptidases has recently been published by Kumar (11). In this study,
it was demonstrated that neutral endopeptidase (NEP) is the major
peptidase in the chemoreceptor tissue of the carotid body and is
believed to represent the major endopeptidase responsible for the
degradation of SP (11). The importance of NEP in setting the
sensitivity of the carotid body was demonstrated in experiments in
which close carotid body administration of the NEP inhibitor
phosphoramidon was found to significantly potentiate the carotid body
response to hypoxia (12). As these observations suggest that NEP may
play an important role in setting the level of sensitivity of the
carotid body to hypoxia, we investigated acute hypoxic ventilatory
responses in mice with a targeted deletion of the NEP gene (16). We
hypothesized that the targeted deletion of the NEP gene would result in
a diminished degradation of SP in the peripheral and/or central
chemoreceptors and an augmented ventilatory response to acute hypoxia.
Furthermore, we have also evaluated the effects of thiorphan, a NEP
inhibitor, on in vivo ventilatory responses to acute hypoxia.
| |
METHODS |
Animals.
Male C57BL/6J wild-type (Wt) mice, obtained from Jackson Laboratories,
that were 8-12 wk of age were compared with sex- and age-matched
NEP gene knockout mice (NEP -/-) (16) bred onto the same genetic
background (C57BL/6J).All NEP -/- mice were bred and raised in a
barrier facility. Sentinel animals housed in the same facility were
periodically screened for infections by routine serology. A separate
cohort of sex- and age-matched C57BL/6J mice was used in a second
series of experiments investigating the effects of thiorphan, a NEP
inhibitor, on acute hypoxic ventilatory responses.
Measurement of ventilatory parameters.
Ventilatory parameters were measured by barometric whole body
plethysmography (Buxco, Troy, NY). Pressure changes between the main
chamber containing the mouse and a reference chamber were detected by a
differential pressure transducer. The box pressure signal is caused by
the changes in lung volume and the resultant pressure associated with
the breathing cycle. A detailed description of the plethysmographic
system has recently been published by Hamelmann et al. (9). The signals
were recorded and analyzed by a dedicated computer from which the
following parameters were calculated: breathing frequency (f),
TI,
TE, and
VT. The system was calibrated
with a rapid injection of 200 µl of air into the main chamber, as
previously described (9). All calibrations were performed before the
initiation of the experiments.
Protocol for hypoxic exposures.
Mice were placed in the plethysmograph containing room air and allowed
to explore the chamber. After 5 consecutive days of conditioning, the
mice appeared calm and were entered into the experimental protocols.
Before exposure of hypoxic gas mixtures, ventilation was measured under
normoxic conditions for 5 min. After this, a mixture containing either
12% O2 in
N2 or 8%
O2 in
N2 was flushed through the animal
chamber. After 3 min of hypoxic challenge, ventilatory parameters were
recorded for the next 3 min at which point the recordings were stopped.
All ventilatory data sampled during hypoxia represent an average taken
during the 3-min recording period. The mice were monitored throughout the entire procedure for signs of stress, and none was observed.
In a second set of experiments, Wt (C57BL/6J) mice were placed in the
plethysmograph and monitored until they appeared calm. After baseline
recordings of ventilation were taken, control mice were injected with
0.1 ml ip of vehicle (PBS, pH = 7.4). After a 20-min period of time, a
mixture containing 8% O2 in
N2 was flushed through the animal
chamber, and hypoxic ventilatory responses were recorded as described
earlier. Thereafter, all mice were allowed to rest under normoxic
conditions for ~20-25 min before injection with the NEP
inhibitor. After the recovery period, the same cohort of mice was
subsequently injected with 100 mg/kg ip of
DL-thiorphan
(DL-3-mercapto-2-benzylpropanoyl-glycine;
Sigma Chemical) dissolved in a 0.1-ml volume of PBS. Pilot experiments revealed that this high dose of
DL-thiorphan was well tolerated: all mice appeared normal after the administration of the NEP inhibitor. After a 20-min period of time, a mixture containing 8%
O2 in
N2 was flushed through the animal
chamber, and hypoxic ventilatory responses were recorded.
Statistical analysis.
All results are presented as means ± SE. Statistical analysis was
performed with the JMP statistical package (SAS, Cary, NC). Intergroup
comparisons were assessed by a Wilcoxon/Kruskal-Wallis test for
nonparametric data and a Tukey Kramer honestly significant test for
parametric data. To assess treatment-induced effects on hypoxic
ventilation, comparisons of all ventilatory parameters with and without
pretreatment with the NEP inhibitor were made with paired
t-tests. Differences were judged
significant when P < 0.05.
| |
RESULTS |
Respiratory response to hypoxia comparing Wt and NEP -/- mice.
Mean ± SE values of f, VT,
and other respiratory parameters are listed in Table
1. A comparison of baseline values for all ventilatory parameters taken before the 12% hypoxic challenge revealed
a significantly smaller TE
(P = 0.011) and total cycle time
(TT)
(P = 0.041) in the NEP -/- group
compared with the Wt group. There were no other significant differences
in the baseline 1 parameters. A
comparison of ventilatory parameters measured during the subsequent
exposure to 12% hypoxia revealed significant differences between the
NEP -/- and Wt groups for VT,
TE, and E.
VT
(P = 0.041) and
E (P = 0.02) were significantly greater in the NEP -/- group compared with the
Wt mice, and TE
(P = 0.046) was significantly smaller
in the NEP -/- group.
A comparison of all ventilatory parameters between Wt and NEP -/-
groups breathing room air before 8% hypoxia revealed no statistically
significant differences, with one exception.
E was significantly higher in the NEP -/-
group during the second baseline measurements (normoxia) compared with
the Wt group (68 ± 10.1 vs. 42 ± 3.2 ml/min;
P = 0.02). Exposure to 8%
O2 resulted in a significant
increase in VT in both the Wt
and NEP -/- mice (P = 0.001 and 0.004, respectively). Additionally, exposure to 8%
O2 in
N2 resulted in a significant
increase in f in the NEP -/- mice (P = 0.01) but not in the Wt group. An intergroup comparison of f at 8%
O2 in
N2 revealed significantly greater
f (P < 0.0001) in the NEP -/- mice;
this was due to a shortening of both
TI
(P = 0.04) and
TE
(P = 0.002). An analysis of
E in the NEP -/- and Wt mice with 8%
O2 in
N2 revealed a significantly
greater response in the NEP -/- group
(P = 0.015). There were no significant differences in VT between the
NEP -/- and Wt mice at 8% O2 in N2. Interestingly, whereas there
were significant hypoxia-induced decreases in both
TI
(P = 0.04) and
TE
(P = 0.002) in the NEP -/- group, the
Wt mice did not increase their frequency response to 8%
O2 in
N2. Specifically, exposure to 8%
O2 in
N2 failed to decrease either
TI or
TE significantly in the Wt
group. The changes in TI,
TE, and
VT are graphically depicted in
the average spirogram shown in Fig. 1.
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Fig. 1.
Average spirogram (means ± SE) for untreated wild-type (Wt) mice
(n = 7;
A) and neutral endopeptidase
knockout (NEP -/-) mice (n = 7; B) during normoxia (21%
O2) and hypoxia (8%
O2).
VT, tidal volume;
TI, inspiratory time;
TE, expiratory time.
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|
Respiratory response to hypoxia after treatment with thiorphan.
Mean ± SE values of respiratory parameters for this set of
experiments are listed in Table 2. Exposure
to 8% O2 in
N2 significantly increased both
VT
(P = 0.0007) and
E (P = 0.002) in the vehicle-treated group; however, there was no
hypoxia-induced increase in f in this group. Comparison of all
ventilatory parameters between vehicle and thiorphan (100 mg/kg
ip)-treated animals at 8% O2
revealed significant thiorphan-induced changes in the ventilatory
responses to this acute hypoxic challenge, similar in pattern to those
observed for the comparison of the Wt and NEP -/- mice. Specifically, f (P = 0.0038) and
E (P = 0.04) were significantly increased; TE
(P = 0.0298) and
TT
(P = 0.0029) were significantly
shortened by the thiorphan treatment in the C57BL/6J mice. There were
no significant differences in TI
between the vehicle-treated mice with 8%
O2 in
N2 and after thiorphan treatment
under similar conditions of hypoxia. The average spirogram for
TI,
TE, and
VT under normoxic conditions and
at 8% O2 in
N2 hypoxia after treatment with
vehicle or 100 mg/kg thiorphan is shown in Fig.
2.
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Table 2.
Respiratory parameters measured in untreated (21% O2),
vehicle-treated (8% O2), and thiorphan-treated (100 mg/kg) wild-type mice during 8% O2 hypoxia
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Fig. 2.
Average spirogram (means ± SE) for untreated Wt mice during
normoxia (21% O2) and hypoxia
(8% O2) 20 min after
pretreatment with vehicle or 100 mg/kg ip thiorphan
(n = 7).
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| |
DISCUSSION |
The present study confirms our hypothesis that NEP is an important
regulator of the ventilatory response of the peripheral and/or central
chemoreceptors to acute hypoxia. These studies are the first to
demonstrate that the targeted deletion of the NEP gene by homologous
recombination alters both the magnitude and temporal components of the
ventilatory response to acute hypoxia. Ventilatory responses in the NEP
-/- mice were compared with Wt mice bred on the same genetic background
(C57BL/6J). The significantly greater ventilatory response to hypoxia
(8 and 12% O2 in
N2) in the NEP -/- mice vis a
vis the Wt mice resulted from differences in the temporal components of
the ventilatory response to hypoxia as well as differences in
VT. There were no significant
differences in ventilatory parameters between Wt and NEP -/- groups
breathing room air before the 8% hypoxic challenge
(baseline 2), except that
E was significantly higher in the NEP -/-
group vis a vis the Wt group. A comparison of the first baseline values
taken before the 12% hypoxic challenge (baseline
1) revealed a significantly smaller
TE
(P = 0.011) and
TT
(P = 0.041) in the NEP -/- group compared with the Wt group. There were no other significant differences in the baseline 1 parameters. Thus it
appears that targeted deletion of the NEP gene can significantly alter
baseline (normoxic) ventilatory patterns in the mouse. Whereas both NEP
-/- and Wt mice increased their
VT in response to severe
hypoxia, there was only a hypoxia-induced increase in f in the NEP -/-
mice. The significant increase in VT with no significant change in
frequency in the C57BL/6J Wt mice with hypoxia is dissimilar to the
acute ventilatory response to hypoxia reported by Tankersley et al.
(25) in the same genetic background. An analysis of the temporal
components of the ventilatory response in the C57BL/6J mice revealed
significant hypoxia-induced shortening of
TE but not
TI, a significant increase in f,
and a significant increase in VT
(25). It should be noted that a direct comparison of the hypoxic
protocols cannot be made because the level of hypoxic stimulation
differed between these two studies (10 vs. 8%
O2), as well as the fact that
3% CO2 was included in the gas
mixture. This inclusion of 3% CO2
in the hypoxic gas mixture in the Tankersley et al. (25) study may have
contributed to the increase in f noted in their study.
In a second series of experiments, administration of the NEP inhibitor
thiorphan was used to investigate its effect on ventilatory responses
to acute hypoxia in C57BL/6J mice. In these experiments, exposure to
8% O2 in
N2 significantly increased both
VT and
E in the vehicle-treated group with no
significant effect on f. When the same cohort of mice was administered
the NEP inhibitor, f and E were
significantly increased. The increase in f was largely due to a
thiorphan-induced shortening of
TE and, hence, TT. Interestingly, the
thiorphan-induced ventilatory response to hypoxia is similar to that
observed in the NEP -/- mice exposed to 8% hypoxia. Because work by
Hachisu et al. (8) in the rat revealed that thiorphan crosses the
blood-brain barrier, thiorphan may exert its effects on both central
and peripheral chemoreceptors.
Our results clearly demonstrate an enhancement in the expression of the
hypoxic ventilatory response in the NEP -/- mice. This enhancement in
the ventilatory response to acute hypoxia may involve peripheral and/or
central chemoreceptors. With regard to the possible role of peripheral
chemoreceptors, the ventilatory response in the knockout mice resembles
the enhanced hypoxic response of cat carotid bodies after inhibition of
NEP activity (12). The greater increase in ventilatory response to
acute hypoxia demonstrated in the NEP -/- mice is consistent with the
results of in vivo data by Kumar et al. (12). In this study, they
investigated NEP activity of the cat carotid body and assessed its
significance in chemoreception. To assess the significance of NEP in
chemoreception, close carotid body administration of phosphoramidon, a
NEP inhibitor, significantly potentiated the carotid body response to
hypoxia but not to hypercapnia (12). The results of their study are important, because they demonstrated for the first time that inhibition of NEP activity augments the hypoxic response of the carotid body, a
finding similar to that observed in our NEP -/- mice under conditions of acute hypoxia. With regard to the possible role of NEP in degrading SP in the central nervous system and influencing the respiratory control apparatus, Matsas et al. (17) have demonstrated that a
metalloendopeptidase that appeared to be similar to endopeptidase 24.11 is the principal enzyme hydrolyzing SP in various areas of the human
brain and that the enzymatic activity was inhibited by phosphoramidon.
Immunohistochemical staining in a later study by Back and Gorenstein
(1) demonstrated significant NEP-like immunoreactivity throughout the
medulla, a region where chemoreceptors are known to have their synaptic
connections. The possibility that centrally located NEP may play a role
in chemoreception is also bolstered by the findings in another study in
which local application of NEP inhibitors increased the responses of
tracheal tone and phrenic nerve activity to both hypercapnia and
hypoxia in cats (10). Accordingly, because the findings of our study cannot distinguish whether the absence of NEP is exerting its effects
peripherally and/or centrally, future studies will need to investigate
the contribution of these two chemoreceptor foci in the NEP -/- mice.
The contribution of SP in hypoxia-induced ventilatory responses has
been the focus of considerable interest. For example, the importance of
SP as a chemotransducer of hypoxia has been clearly demonstrated in a
study in which the administration of a SP antagonist in vivo has been
shown to block the response to infused SP and attenuate or completely
abolish the carotid body excitation to hypoxia (24). An inhibition of
SP degradation by NEP inhibitors (12) presumably increases the levels
of this neuropeptide, both in the peripheral chemoreceptors and
centrally in areas such as the nucleus tractus solitarius, a region
rich in SP-like immunoreactivity (26) and an area demonstrated to receive afferent inputs from chemoreceptors (20). The importance of
afferent nerve fibers in mediating the increase in the ventilatory response to hypoxia has been clearly demonstrated by Ledlie et al.
(15), who demonstrated that the decrease in
TE and increase in f with
hypoxia were significantly less after carotid sinus denervation in
anesthetized dogs. Furthermore, our laboratory has previously
demonstrated that ablation of neuropeptide-containing C fibers by
neonatal treatment with capsaicin significantly reduces the tachypnic
component of the ventilatory response to hypoxia (6).
The contribution of SP to the ventilatory response to hypoxia has also
been investigated in the respiratory centers of the brain.
Microinjections of SP in the area of the nucleus tractus solitarius has
been shown to produce an increase in f in parallel with an increase in
VT (3). As this is an area known
to receive chemoreceptor afferents, it is possible that an increase in
SP levels may be responsible in part for the increase in f in parallel with the increase in VT so noted
in the NEP -/- mice but not in the Wt mice. The increased ventilatory
response in the NEP -/- mice may also be due to an increase in the
levels of SP in the peripheral carotid body chemoreceptors and/or
central chemoreceptors of the NEP -/- mice, possibly because of greater
increases in the sensory discharge of these chemoreceptor foci. In
support of this hypothesis, several studies have shown that local
administration of SP increases the sensory discharge of carotid body
chemoreceptors (18, 19). It is important to note that SP may elicit
both an inhibitory and excitatory effect on chemoreceptor activity. McQueen (18) demonstrated in the cat that the effect of SP on carotid
chemoreceptor activity appeared biphasic, where SP initially caused a
slight inhibition of activity followed by a dose-dependent increase in
discharge rate. In another study by Monti-Bloch and Eyzaguirre (19), SP
either excited or depressed the carotid body discharge rate in the cat
in a dose-dependent manner. Thus, whereas several studies have
demonstrated an enhancement of chemoreceptor discharge rate with SP,
the neuropeptide may also exert an inhibitory effect under certain circumstances.
In summary, the alteration in the ventilatory response to hypoxia in
the NEP -/- mice suggests that NEP plays a critical role in regulating
the expression of the ventilatory response to acute hypoxia via the
peripheral and/or central chemoreceptors. Neurophysiological studies
will be needed to delineate the contribution of NEP at these sites of
chemoreception. The mechanism by which this increased ventilatory
response occurs may be explained in part by an increase in the
availability of SP, which in turn would cause an increase in the
chemoreceptor discharge rate. This increase in afferent discharge would
lead to an enhancement of the ventilatory response as observed in the
NEP -/- and C57BL/6J mice treated with the NEP inhibitor. This
mechanism is supported by a recent study by Kumar et al. (12), who
demonstrated that close carotid body administration of a NEP inhibitor
significantly potentiated the carotid body response to hypoxia in the
cat. The lack of NEP or the inhibition of this enzyme leads to an
enhanced ventilatory response to acute hypoxia, largely through an
effect on the tachypnic response to hypoxia. Further work is required
to evaluate the effects of NEP on the levels of neuroactive peptides,
both peripherally and in the respiratory centers of the brain.
| |
ACKNOWLEDGEMENTS |
This study was funded by a grant from the Plum foundation (to
G. T. De Sanctis). G. T. De Sanctis was supported by a
Partner's Nesson Award; N. P. Gerard was supported by National Heart,
Lung, and Blood Institute Grant HL-41587; and H. Grasemann was
supported by a grant from the Deutsche Forschungsgemeinschaft.
| |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. T. De
Sanctis, Division of Pulmonary and Critical Care Medicine, Brigham and
Women's Hospital and Harvard Medical School, 75 Francis St., Boston,
MA 02115 (E-mail: gdesanctis{at}rics.bwh.harvard.edu).
Received 17 February 1999; accepted in final form 8 June 1999.
| |
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