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Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
eNOS-/-
nNOS-/-
iNOS-/-
CONCLUDING REMARKS
REFERENCES
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Nitric oxide (NO) plays an essential
role in the maintenance of cardiovascular and renal homeostasis.
Endogenous NO is produced by three different NO synthase (NOS)
isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal
NOS (nNOS). To investigate which NOS is responsible for NO production
in different tissues, NOS knockout (
/) mice have been generated for
the three isoforms. This review focuses on the regulation of
cardiovascular and renal function in relation to blood pressure
homeostasis in the different NOS/ mice. Although regulation of
vascular tone and cardiac function in eNOS/ has been extensively
studied, far less is known about renal function in these mice.
eNOS/ mice are hypertensive, but the mechanism responsible for
their high blood pressure is still not clear. Less is known about
cardiovascular and renal control in nNOS/ mice, probably because
their blood pressure is normal. Recent data suggest that nNOS plays
important roles in cardiac function, renal homeostasis, and regulation
of vascular tone under certain conditions, but these are only now
beginning to be studied. Inasmuch as iNOS is absent from the
cardiovascular system under physiological conditions, it may become
important to blood pressure regulation only during pathological
conditions related to inflammatory processes. However, iNOS is
constitutively expressed in the kidney, where its function is largely
unknown. Overall, the study of NOS knockout mice has been very useful
and produced many answers, but it has also raised new questions. The appearance of compensatory mechanisms suggests the importance of the
different isoforms to specific processes, but it also complicates interpretation of the data. In addition, deletion of a single gene may
have physiologically significant effects in addition to those being
studied. Thus the presence or absence of a specific phenotype may not
reflect the most important physiological function of the absent gene.
endothelial nitric oxide synthase; neuronal nitric oxide synthase; inducible nitric oxide synthase; knockout mice; blood pressure
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
eNOS-/-
nNOS-/-
iNOS-/-
CONCLUDING REMARKS
REFERENCES
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NITRIC OXIDE (NO) plays an important role in the maintenance of cardiovascular and renal homeostasis. In the cardiovascular system, it is involved in regulation of vascular tone (22), cardiac contractility (67), cell growth (76), vascular remodeling (49), and baroreflex function (7, 48, 111). In the kidney, NO regulates salt and fluid reabsorption (63, 89), hemodynamics (44), renin secretion (46), and tubuloglomerular feedback (TGF; 73, 106, 107). Most of these processes are important in both short- and long-term regulation of arterial blood pressure and have been extensively studied in the last 10 years.
Endogenous NO is enzymatically produced from conversion of the amino
acid L-arginine to L-citrulline, a reaction
catalyzed by the enzyme NO synthase (NOS). Three different NOS isoforms have been cloned and characterized: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). The three isoforms are
differentially expressed throughout the cardiovascular system and the
kidney, and one or more isoforms can be expressed in the same cell
type. To answer important questions regarding which isoforms produce the NO that regulates physiological processes, mice with disruption of
each of the NOS genes have been generated. Studies of the different NOS
knockout (/) mice have provided many answers but have also raised
new questions regarding the role of the various NOS isoforms. Many
reviews have concentrated on different pathophysiological aspects
studied in NOS/ mice (12, 25, 34, 35, 50, 78, 93).
This review will focus on recent data concerning the regulation of
cardiovascular and renal function in relation to blood pressure
homeostasis in the different NOS/ mice.
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eNOS / |
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TOP
ABSTRACT
INTRODUCTION
eNOS-/-
nNOS-/-
iNOS-/-
CONCLUDING REMARKS
REFERENCES
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Blood pressure.
Given the location of e, n, and iNOS expression in the cardiovascular
and renal systems, NO produced by each isoform has the potential to
alter blood pressure. Most studies have shown that eNOS/ mice have
higher blood pressure than wild-type mice, although the magnitude of
hypertension reported by different laboratories varies. Differences in
systolic arterial pressure in conscious eNOS/ compared with
wild-type mice range from 20 (45) to 50 mmHg (85,
86), while differences in mean blood pressure in anesthetized
mice range from 14 (5) to 37 mmHg (101). The
varying magnitude of the hypertension observed in eNOS/ may be due
to the use of different methods for measuring blood pressure or the genetic backgrounds of the strains used. However, even with these differences, eNOS/ mice were found to be hypertensive in all cases.
/ is caused by the lack of endothelium-derived
NO and the resulting increase in arterial tone and peripheral
resistance. However, this simple explanation is not fully supported by
the data, as discussed below.
Vascular function.
The amount of eNOS expressed in the vascular endothelium, together with
the data showing that NO is an important endothelium-derived relaxing
factor, indicates that eNOS-derived NO is essential to regulation of
vascular tone (22). Thus the hypertension observed in
eNOS/ can be partly attributed to increased vascular resistance caused by the lack of endothelial NO. Several investigators have studied the response of eNOS/ arteries to vasodilator stimuli. It
was first reported that aortic rings isolated from eNOS/ do not
relax in response to ACh (36). Lamping and Faraci
(47) observed a complete lack of ACh-induced relaxation in
carotid artery rings, with no difference between male and female mice. To verify that eNOS mediates ACh-induced relaxation in carotid arteries, Scotland et al. (80) transfected the endothelium
of eNOS/ mouse carotid arteries with an adenoviral vector carrying the gene for eNOS and in this way restored ACh-induced relaxation. These data indicate that 1) in large vessels, such as the
aorta or carotid artery, eNOS mediates ACh-induced vasodilatation and 2) there is no compensatory vasodilator mechanism for ACh.
/. These responses could be prevented by
K+ channel blockers in eNOS/ but not in wild-type mice,
suggesting that they are mediated by an endothelium-derived
hyperpolarizing factor (EDHF) (18, 79). Sun et al.
(91) reported that in gracilis muscle arterioles of male
eNOS/, flow-induced dilatation was completely blocked by
indomethacin, whereas this drug only inhibited 50% of the response in
wild-type mice. Interestingly, the same group reported that in female
eNOS/, a different vasodilator mechanism compensates for the lack
of eNOS. In females, flow-induced vasodilatation was not prevented by
indomethacin but was completely blocked by a Ca2+-activated
K+ channel blocker or a cytochrome P450 inhibitor
(31). A gender difference in the relative contribution of
NO to endothelium-dependent vasodilatation has been reported in
wild-type mice and in rats (31, 32, 55, 91). Thus a
different compensatory mechanism could be activated in the absence of
eNOS in male or female mice. However, the precise explanation for these
differences is still not known.
On the basis of these data, it is possible to conclude that
eNOS-derived NO is an important mediator of vasodilator stimuli that
affect vascular tone in most arteries studied. In large arteries the
lack of endothelial NO in eNOS/ impairs the relaxant effect of
vasodilators, and this is apparently not compensated for by other
endothelial vasodilators. In resistance arteries, the lack of eNOS is
sometimes compensated for by EDHF, a cyclooxygenase product, or by
nNOS-derived NO, as in the brain pial arterioles and the coronary
microcirculation (33, 56) (Fig.
1). It is important to note that in some
cases such as the pulmonary circulation, the lack of eNOS is not
compensated for at all (20). Thus most data indicate that
the mechanisms that compensate for the lack of eNOS-derived NO are
specific to different vascular beds, and one or more mechanisms may be
activated to regulate arterial tone in the absence of eNOS. Although
the absence of eNOS appears to be compensated for in most resistance
arteries, eNOS/ mice exhibit higher blood pressure and it is still
not known whether they have increased total peripheral resistance. To
us, this raises the questions of how much of the hypertension observed
in eNOS/ is caused by an increase in vascular tone and whether
other mechanisms such as increased cardiac output and increased salt
and fluid absorption by the kidney may be involved in the hypertension
exhibited by these mice.
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Renal function. Extracellular fluid volume is regulated by the kidney, where eNOS is expressed in the endothelium of the renal vasculature, including the afferent and efferent arterioles and vasa recta. eNOS is also present in proximal tubules, thick ascending limbs, and collecting ducts (44, 96). However, despite the important role of NO in the regulation of various physiological processes in the kidney, little is known about the contribution of the three NOS isoforms to these mechanisms.
NO is known to be involved in the regulation of renin secretion. However, the data are still contradictory, with some reports concluding that endothelial NO stimulates renin secretion, whereas others found the opposite (46). In eNOS/, a decrease in total kidney renin mRNA was observed, whereas plasma renin levels were elevated by 50% compared with wild-type mice despite the high blood
pressure (81). These data suggest that eNOS-derived NO tonically inhibits renin release and that regulation of renin release
by renal perfusion pressure is impaired because renin levels would be
low due to the high blood pressure. Wagner et al. (101)
found a decrease in renin mRNA levels in total kidney homogenates but
also reported lower levels of renin activity in the eNOS/ kidney.
Although this latter result contradicts the increase in plasma renin
observed by others, total renin activity does not necessarily reflect
plasma levels or rate of renin secretion. In a recent study,
Beierwaltes et al. (5) found no difference in plasma renin
content between eNOS/ and wild-type mice. They also found that in
anesthetized mice, renin secretion in response to reduced perfusion
pressure was normal in eNOS/, whereas acute inhibition of NOS
completely prevented pressure-dependent renin release in wild-type
mice. The authors concluded that pressure-dependent renin release
is completely compensated for in eNOS/. Inasmuch as NO derived from
macula densa nNOS regulates renin secretion, it is possible that this
pathway is upregulated in eNOS/. It is important to note that
plasma renin levels have been found to be either equal or increased in
eNOS/ in the context of increased blood pressure (5,
81), suggesting that this mechanism may contribute to
hypertension. The precise role of NO in the regulation of renin
secretion is still unknown, as are the molecular mechanisms by which NO
acts in renin-secreting cells.
Previous in vivo and in vitro data support a role for
endothelium-derived NO in the regulation of renal vascular tone
(44). In eNOS/, basal renal perfusion pressure was
increased, but renal blood flow was similar to wild-type mice
(5). Acute infusion of the NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME) decreased renal blood flow in wild-type mice but
had no effect in eNOS/. These data suggest that in the renal
vasculature compensatory mechanisms are activated in the absence of
eNOS to maintain normal renal blood flow. However, the mechanism by
which renal blood flow is maintained is not known. In rabbit afferent
arterioles, NOS inhibition reduced basal arteriolar diameter (38,
43). In contrast, Patzak et al. (66) reported that
in isolated perfused afferent arterioles from wild-type mice, neither
L-arginine nor L-NAME changed basal arteriole
diameter, suggesting that basal NO activity is negligible in this
preparation. However, angiotensin II-induced contractions were greatly
enhanced in afferent arterioles of eNOS/, suggesting that
eNOS-derived NO decreases the constrictor response to angiotensin II.
Although NO has been shown to modulate the vasoconstrictor response to
other hormones, the role of eNOS-derived NO in these responses has not
been studied to our knowledge.
NO is an important regulator of the renal medullary microcirculation
(53, 65). Medullary infusion of the nonspecific NOS inhibitor L-NAME decreases medullary blood flow and
increases sodium retention and blood pressure (54).
Conflicting results have been published regarding a role for nNOS in
this process. It was first shown that infusion of anti-sense
oligonucleotides for nNOS into the medullary interstitium caused
salt-sensitive hypertension in rats (51, 52), suggesting a
role for nNOS in blood flow regulation. However, infusion of selective
nNOS inhibitors into the renal medulla did not affect medullary blood flow, although it decreased NO levels (41), suggesting
that nNOS does not play a role in medullary blood flow regulation. Thus
the particular NOS isoform responsible for regulation of medullary
blood flow or the cell type where the NO that affects blood flow is
produced is unknown. In the renal medulla, NO could be produced by the
thick ascending limb (62), descending vasa recta
(75), or medullary collecting duct (11, 57).
Despite the important role of NO in the medullary microcirculation, NOS knockout mice have not yet been used to study this physiological process.
NO has been shown to inhibit NaCl and fluid reabsorption along the
nephron and promote renal sodium and water excretion; however, very
little is known about the role of endogenous NO and the contribution of
the different NOS isoforms to this process (63). We
reported that in the rat thick ascending limb, L-arginine
stimulates endogenous NO production and decreases NaCl and
NaHCO3 reabsorption (60-62, 70). To
investigate which NOS isoform mediates this response, we studied the
effect of L-arginine on thick ascending limbs from eNOS/, iNOS/, and nNOS/ mice. We found that in thick limbs from wild-type, nNOS/, and iNOS/ mice, L-arginine
inhibited NaCl absorption, whereas it had no effect in eNOS/. A NO
donor was able to inhibit NaCl transport in eNOS/, indicating that the second messenger cascade for NO was intact (69). These
data suggest that eNOS-derived NO inhibits NaCl absorption in this nephron segment and that the lack of eNOS is not compensated for by
other NOS isoforms.
Although the proximal tubule and collecting duct also express eNOS,
little is known about basal or stimulated reabsorption rates in these
tubular segments from eNOS/. In proximal tubules microperfused in
vivo, Wang (103) reported no differences in basal fluid
and bicarbonate reabsorption rates in eNOS/ compared with wild-type
mice. In contrast, Adler et al. (1) reported that basal oxygen
consumption in cortical renal slices was higher in eNOS/ than in
wild-type mice. The renal cortex is composed mostly of proximal tubules
and a minor fraction of vascular cells, distal tubules, and cortical
thick ascending limbs. Because the basal metabolic rate of epithelial
cells is much higher than that of vascular cells, and because NO has
been shown to inhibit proximal tubule sodium reabsorption
(63), the higher rate of oxygen consumption found in
eNOS/ is likely due to increased basal rates of sodium reabsorption
by the proximal tubule, caused by the lack of eNOS-derived NO. However,
the precise role of eNOS in the proximal tubule is currently unknown.
Thus it is possible that a lack of eNOS-derived NO may chronically
increase reabsorption of NaCl by the nephron and increase TGF
responses, contributing to the hypertension observed in eNOS/ (Fig.
2).
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Cardiac function. Elimination of NO produced by eNOS in the heart may also enhance cardiac output and contractility. Recently, a large number of publications have centered on the role of NO in regulation of cardiac function. In the heart, eNOS is expressed not only in the endothelium of the coronary vessels but also in cardiac myocytes. In addition to eNOS, cardiac myocytes have also been shown to express nNOS in the mitochondria and sarcoplasmic reticulum (42, 110), suggesting that NO plays an important role in myocyte physiology.
Most investigators have reported that the basal heart rate in conscious eNOS/ mice is significantly lower than in wild-type mice (23,
45, 81, 85, 110). However, others have reported that heart rates
measured in anesthetized animals were no different between eNOS/
and wild-type mice (27, 36). An acute increase in blood
pressure decreases the heart rate via a baroreflex mechanism. However,
during long-term increases in blood pressure, the baroreceptor resets
to the new pressure and the heart rate returns to baseline. In
eNOS/, the increase in blood pressure is chronic and thus the heart
rate would be expected to be normal. Although there is still no
explanation for the decreased heart rate observed in conscious
eNOS/, it is possible that eNOS-derived NO can affect baroreflex
resetting or be involved in establishing the baroreceptor setpoint
(30, 48, 84). In addition, the fact that the heart rate is
not different in anesthetized mice may reflect the loss of baroreflex
influence caused by the anesthesia. Overall, it is still not clear why
eNOS/ have a lower heart rate than wild-type mice.
In eNOS/ mice, basal parameters of cardiac function in vivo appear
to be normal. Assessment of cardiac function by echocardiography showed
no significant differences in left ventricular shortening fraction,
ejection fraction, and cardiac output between eNOS/ and wild-type
mice (110). Basal systolic contractility, reflected by the
maximum rate of pressure development (dP/dtmax), was no different from wild-type mice as measured with an intraventricular pressure catheter (27). An increase in left ventricular
mass and posterior wall thickness together with an increase in myocyte size indicative of cardiac hypertrophy have been observed in eNOS/ (110). Although these changes are most likely due to
hypertension, a role for NO in cardiac myocyte growth cannot be ruled out.
Basal cardiac contractility was found to be similar between eNOS/
and wild-type mice. However, data obtained from perfused whole heart
(Langendorff) preparations or in vivo suggest that during
-adrenergic stimulation cardiac contractility is influenced by
eNOS-derived NO. Gyurko et al. (27) found that in an
isolated perfused whole heart preparation, isoproterenol-stimulated
cardiac contractility was enhanced in hearts from eNOS/, whereas
-adrenergic receptor density was no different between eNOS/ and
wild-type mice. In vivo assessment of cardiac function showed that
isoproterenol-stimulated cardiac contractility was enhanced in
eNOS/ compared with wild-type mice. The effect of isoproterenol in
eNOS/ was similar to the response observed in wild-type mice
treated with the NOS inhibitor L-NNA, supporting a role for
eNOS in mediating this effect (27). Another group of
investigators also reported that isoproterenol-stimulated cardiac
contractility was enhanced in hearts from eNOS/ mice (100). These data indicate that in the intact animal,
eNOS-derived NO modulates the systolic response to -adrenergic stimulation.
The mechanism by which eNOS modulates -adrenergic-stimulated cardiac
contractility appears to involve activation of
3-adrenergic receptors. It has been shown that in
3-adrenergic receptor knockout mice
(3/), the contractile response to isoproterenol is
enhanced to the same extent as in eNOS/ (100). In
addition, NOS inhibitors enhance the -adrenergic inotropic response
in wild-type mice but not in 3/. These data suggest
that binding of isoproterenol to the 1- and
2-adrenergic receptors stimulates cardiac contractility, whereas simultaneous binding to the 3 receptor modulates
the positive inotropic effect by activating eNOS. A recent report confirms the latter hypothesis by showing that a selective
3-receptor agonist decreased intracellular calcium and
sarcomere length in wild-type cardiac myocytes but failed to produce
this effect in myocytes from eNOS/ (4). However, the
mechanism for the increase in intracellular calcium observed in
eNOS/ mice remains unclear.
Whereas most data support a role for eNOS in decreasing -adrenergic
effects on contractility, in vitro studies using isolated myocytes are
more controversial. Han et al. (28) first reported that in
isolated cardiac myocytes from eNOS/, the muscarinic agonist
carbachol failed to reverse isoproterenol-stimulated contractions. These authors showed that carbachol failed to decrease
isoproterenol-stimulated L-type Ca2+ channel
activation in eNOS/ mice, whereas it completely abolished the
effect of isoproterenol in myocytes from wild-type mice. Although these
data are consistent with eNOS-derived NO blunting -adrenergic stimulation of contractility, others have failed to reproduce these
results. Vandecasteele et al. (99) found no difference in
-adrenergic-induced contractility of isolated papillary muscles between eNOS/ and wild-type mice. They also reported that carbachol blocks isoproterenol-stimulated L-type Ca2+
channel currents to the same extent in isolated eNOS/ and wild-type myocytes. In agreement with the last report, two other groups of
investigators found no differences in isoproterenol-stimulated L-type Ca2+ channel currents or the inhibitory
effect of muscarinic receptor agonists between isolated eNOS/ and
wild-type myocytes (6, 24). Consistent with a role for
eNOS in decreasing the -adrenergic increase in contractility,
Barouch et al. (4) found that the isoproterenol-stimulated
increase in intracellular calcium was enhanced in myocytes from
eNOS/. Contractility data obtained from isolated myocytes are still
unresolved. The different results observed in isolated cell
preparations may be attributable to blockade or activation of second
messenger cascades caused by physical disruption of the tissue or the
different protocols used to obtain it. Although the data suggest that
eNOS-derived NO affects intracellular calcium balance, the mechanism
involved is still unclear.
In summary, in vivo and in vitro data indicate that, under basal
conditions, cardiac function is normal in eNOS/, suggesting that
the lack of eNOS is compensated for by other mechanisms or that eNOS
only plays a minor role in basal cardiac function. However, in vivo
data indicate that when sympathetic output and adrenergic discharge are
increased, eNOS mediates the negative inotropic effect caused by
stimulation of the 3-adrenergic receptor. Thus eNOS-derived NO appears to be an important physiological modulator of
cardiac contractility.
Finally, genetic deletion of eNOS may disrupt the function of other
important regulators of blood pressure by affecting central nervous
system activity. For example, Stauss et al. (85, 87) found
that blood pressure is more variable in eNOS/ compared with
wild-type mice, suggesting that baroreflex responses are blunted in the
former. Still, little is known about other central nervous system
effects of deleting eNOS, in particular regarding blood pressure control.
Studies in eNOS/ mice have clearly demonstrated that NO produced by
eNOS plays an important role in the regulation of blood pressure.
Although one might expect that eliminating eNOS from the vasculature
would play a predominant role in the hypertension seen in eNOS/
mice, other vasodilators partially compensate for the loss of eNOS in
resistance vessels. Thus the hypertension observed in these mice can
also be attributed to the lack of eNOS-derived NO in the kidney and heart.
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nNOS/ |
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TOP
ABSTRACT
INTRODUCTION
eNOS-/-
nNOS-/-
iNOS-/-
CONCLUDING REMARKS
REFERENCES
|
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Blood pressure.
In contrast to the findings observed in eNOS/, blood pressure in
nNOS/ mice has generally been shown to be similar to that of
wild-type controls (4, 40, 59, 98). These data suggest
that genetic deletion of nNOS is compensated for, in terms of blood
pressure regulation. Alternatively, the hypotensive actions of nNOS may
be counterbalanced by its hypertensive effects. Acute administration of
the nNOS inhibitor 7-nitroindazole (7-NI) to eNOS/ mice
significantly reduced blood pressure (45), suggesting that
nNOS contributes to their hypertension. However, Barouch et al.
(4) reported that systolic blood pressure in mice
deficient in both eNOS and nNOS (e-nNOS/) was higher than in
wild-type mice and similar to eNOS/. Although nNOS may be
antihypertensive in some cases (74, 106), in others it
appears to be prohypertensive (4, 45). Thus the role of
nNOS in the global regulation of blood pressure is still not well
defined, and more work is needed in this area.
Vascular function.
The data showing that blood pressure in nNOS/ is similar to that of
wild-type mice suggest that nNOS does not play an important role in the
regulation of basal vascular tone. However, it has been shown that nNOS
is expressed in vascular smooth muscle cells (10, 33) and
also in cardiac myocytes (110). Therefore, while vascular
tone is regulated by eNOS under basal conditions, it is possible that
when eNOS-dependent vasodilatation is impaired, nNOS-derived NO could
modulate vascular tone. For example, in brain pial arterioles of
eNOS/, ACh-induced dilatation was found to be reduced by only 25%
compared with wild-type controls. In the presence of the nNOS inhibitor
7-NI, ACh-induced dilatation was reduced by 50% in eNOS/ but
normal in wild-type mice, suggesting that nNOS-derived NO compensates
for the lack of eNOS (56). This is supported by Huang's
study (33), showing that in isolated perfused coronary
arterioles of eNOS/, which exhibited normal flow-induced
dilatation, 7-NI blunted flow-induced dilatation by 40% but had no
effect in wild-type arterioles. In addition, these authors found
upregulation of nNOS expression in the endothelium and smooth muscle of
coronary arteries. Brandes et al. (9) observed increased
sensitivity of soluble guanylate cyclase in aortic rings of eNOS/.
These data suggest that despite the small amount of nNOS in blood
vessels, low levels of nNOS-derived NO could compensate for the lack of
eNOS in these mice. To date, compensation by nNOS has only been evident
in eNOS/ arterioles, but it could also be important in other
conditions where endothelium-dependent vasodilatation is impaired, such
as hypercholesterolemia (8, 90) and diabetes
(16).
Renal function.
In the kidney, nNOS is expressed in macula densa cells, collecting
ducts (77, 105, 107), and in thick ascending limbs (Garvin, unpublished observations). nNOS expression is significantly higher in the macula densa compared with other tubular cells, suggesting an important role for this isoform in modulating the function of the macula densa and juxtaglomerular apparatus
(106). In fact, studies using pharmacological inhibitors
of NOS have shown that nNOS-derived NO produced in the macula densa
blunts TGF responses in rats, rabbits, and mice (74, 95,
98). In wild-type mice, inhibition of macula densa nNOS with
7-NI increases the magnitude of the TGF response, seen as greater
constriction of the afferent arteriole (74). Vallon et al.
(98) studied TGF responses in nNOS/ in vivo by
monitoring changes in proximal stop-flow pressure while increasing
luminal perfusion rates to the distal nephron. They found no difference
in TGF responses between nNOS/ and wild-type mice; however,
blocking NOS with L-NNA increased TGF in wild-type mice but
had no effect in nNOS/. In agreement with this report, we found no
difference in TGF responses between nNOS/ and wild-type mice.
However, 7-NI potentiated TGF responses in wild-type mice, whereas it
did not affect TGF in nNOS/ (74). Taken together,
these data suggest that in wild-type mice, nNOS-derived NO produced in
the macula densa blunts TGF. However, chronic deletion of nNOS is
compensated for by some mechanism that helps maintain glomerular
hemodynamics. Because we have shown that NO produced by the thick
ascending limb (presumably by eNOS) can also blunt TGF responses
(102), it could be that this mechanism is upregulated in
nNOS/ to maintain normal TGF.
/ exhibited
higher fluid and chloride absorption rates compared with proximal
tubules of normal mice, suggesting that endogenously produced NO
inhibits proximal tubule transport. In contrast to the study of Vallon et al. (98), Wang et al. (104) reported that
nNOS knockout mice exhibited lower fluid and bicarbonate absorption
rates than proximal tubules from wild-type mice, suggesting that NO
produced by nNOS stimulates rather than inhibits transport in the
proximal tubule.
The explanation for the disparate results regarding the role of
nNOS-derived NO in proximal tubule transport is unclear. However, in
nNOS/ mice, nNOS is genetically deleted from all tissues, not just
the proximal tubule. Thus the difference between wild-type and
nNOS/ may be due to the effect of deleting nNOS from other organs
or a change in the control of neural innervation of the proximal
tubule. In fact, it has been shown that the effects of NOS inhibition
in proximal tubule transport are modulated by different degrees of
neural activity (63, 108).
Cardiac function.
In the heart, nNOS is found in the sarcoplasmic reticulum
(109) and mitochondria of cardiac myocytes
(42), cholinergic and nonadrenergic/noncholinergic nerve
terminals, and in sympathetic nerve terminals, where it has been
postulated to play a role in catecholamine release and reuptake
(14, 15, 40). Although blood pressure is normal in
nNOS/, studies have shown that this NOS isoform is important for
maintenance of normal cardiac function. Basal heart rates have been
found to be slightly increased in nNOS/ (14, 40). It
has also been reported that heart rate variability is decreased in
nNOS/, suggesting that the increased heart rate may be due to
reduced parasympathetic tone (40). In addition, atropine,
a muscarinic antagonist, increased the heart rate in wild-type mice but
had no effect in nNOS/, suggesting that muscarinic tone was already
blunted (40). In agreement with nNOS controlling heart
rate, Choate et al. (14) reported that vagal nerve
stimulation caused a much slower decrease in nNOS/ heart rate
compared with wild-type mice, but the magnitude of the response was
similar in both strains. The heart rate decrease caused by a muscarinic
agonist was similar in both strains. These data suggest that vagal
control of bradycardia is modulated by nNOS-derived NO via a
presynaptic mechanism. The exact mechanism by which nNOS-derived NO
modulates parasympathetic tone has not been fully studied to our knowledge.
/ myocytes during electrical stimulation. In addition, the contractile response to isoproterenol was enhanced in nNOS/ myocytes.
Similarly, Barouch et al. (4) found that in vivo basal
cardiac contractility was enhanced in nNOS/ as shown by increased
dP/dtmax. However, when they studied the
-adrenergic response, they found decreased isoproterenol-stimulated
contractility in nNOS/ compared with wild-type mice. These authors
also reported that isoproterenol-stimulated increases in intracellular
calcium and contractility in isolated cardiac myocytes were almost
completely abolished in nNOS/. These data suggest that nNOS-derived
NO increases intracellular calcium and cardiac contractility, whereas
eNOS-derived NO has the opposite effect. Interestingly, in double
e-nNOS/ mice basal cardiac contractility in vivo was even higher
than in nNOS/, eNOS/, and wild-type hearts and
isoproterenol-stimulated contractility was almost normal
(4). Overall, the data suggest that under basal conditions
both eNOS and nNOS decrease contractility; however, during
-adrenergic stimulation, eNOS decreases contractility while nNOS
increases it (Fig. 3). As with other
intracellular signaling molecules (e.g., cAMP), the effect of the
second messenger is regulated by strictly controlling the site of
production and the intracellular location of the target protein
complexes (21, 39, 83). For NO signaling, these data
suggest a new level of regulation that has not yet been studied.
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iNOS/ |
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TOP
ABSTRACT
INTRODUCTION
eNOS-/-
nNOS-/-
iNOS-/-
CONCLUDING REMARKS
REFERENCES
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Blood pressure.
The gene locus coding for iNOS has been shown to cosegregate with blood
pressure in the Dahl salt-sensitive rat (17). Whereas in
most tissues iNOS is only induced by proinflammatory factors, it is
expressed constitutively in the renal medulla (44).
Despite the implication that iNOS may be involved in the development of salt-sensitive hypertension, there have been very few studies testing
this hypothesis or questioning the role of iNOS in blood pressure
regulation. Recently, Ihrig et al. (37) reported that basal systolic blood pressure was elevated by 10 mmHg in iNOS/ at 3 mo of age, but was no different from wild-type mice at 9 or 12 mo.
Feeding iNOS/ a high-salt diet for 8 wk did not increase blood
pressure further by 3 mo and had no effect at 9 to 12 mo. Interestingly, and similar to reports in eNOS/ mice, iNOS/ had
higher plasma cholesterol levels than the wild type (19, 37). Ullrich et al. (97) also observed no
differences in mean arterial pressure in 2- to 5-mo old iNOS/. We
could find no other studies regarding blood pressure regulation in
iNOS/ under physiological conditions, nor of its relationship to
age, salt sensitivity, and other vasoactive hormones. Thus more work in this area is needed.
Vascular function.
iNOS expression has not been found in the vasculature under
physiological conditions and thus is not likely to play a role in basal
regulation of vascular tone. Only after its induction by inflammatory
factors such as lipopolysaccharides, tumor necrosis factor, or
interleukin-1 has iNOS-derived NO been shown to affect vascular tone
(13, 88, 97). Given that iNOS induction appears to occur
only under pathological conditions (i.e., septic shock) and there are
few studies on its role in vascular tone regulation in iNOS/ mice,
it will not be discussed further here.
Renal function.
In contrast to findings in other organs, iNOS is constitutively
expressed in the renal medulla and proximal tubule (2, 44). However, the role of iNOS in the regulation of nephron transport has not been studied extensively. It was first reported that
in proximal tubules stimulated with lipopolysaccharides, L-arginine decreased Na+-K+-ATPase
activity, consistent with data showing that exogenous NO inhibits both
apical Na+ entry and basolateral Na+ pump
activity in this segment (26). In contrast to previous results, Wang (103) recently reported that in proximal
tubules of iNOS/ perfused in vivo, basal fluid and bicarbonate
reabsorption were lower than in wild-type mice. They concluded that
under basal conditions NO produced by iNOS in the proximal tubule
stimulates solute and fluid reabsorption. There is currently no
explanation for these disparate results, and more data are needed to
resolve this issue.
Cardiac function.
We know of no data regarding iNOS expression in mouse hearts under
basal conditions. Ullrich et al. (97) reported that
iNOS/ mice have a heart rate similar to that of wild-type mice as
well as normal blood pressure. It has been proposed that iNOS induction in the heart during chronic inflammation may lead to heart failure and
other deleterious effects. However, it is still not defined whether
iNOS induction is important to the development of heart failure or
whether it is a consequence of the inflammatory response. Conflicting
results have been obtained from experiments in which iNOS was
overexpressed in cardiac myocytes. Heger et al. (29) found
that iNOS overexpression caused a small decrease in heart rate and
cardiac output but no other abnormalities in cardiac function,
histology or anatomy. However, Mungrue et al. (58) found
increased ventricular size, abnormal conduction, and increased mortality in these mice. Although the different results may be due to
different levels of iNOS expression in these mice, the precise role of
iNOS in the heart is still unclear.
| |
CONCLUDING REMARKS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
eNOS-/-
nNOS-/-
iNOS-/-
CONCLUDING REMARKS
REFERENCES
|
|---|
Genetic deletion of the various NOS isoforms has greatly aided our understanding of how NO and the three NOS isoforms regulate blood pressure and cardiovascular/renal function. However, many questions remain that cannot be answered with these models. Further clarification will require the development of inducible tissue-specific knockout of e, i, and nNOS. Additionally, the completion of the Human Genome Project has fundamentally changed the way we describe a gene and its function. It is now apparent that the concept of "one gene, one protein" was naive. Genetic deletion of a single gene may have physiologically significant effects in addition to, or more important than, those being studied. Thus the set of parameters we choose to study in knockout mice may not in fact reflect the most important physiological function of the absent gene.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. O. H. Cingolani for valuable scientific discussion during preparation of this manuscript.
| |
FOOTNOTES |
|---|
This work was supported in part by a grant from the National Heart, Lung, and Blood Institute (HL-28982) to J. L. Garvin.
Address for reprint requests and other correspondence: P. Ortiz, Division of Hypertension and Vascular Research, Dept. of Internal Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202 (E-mail: portiz1{at}hfhs.org).
10.1152/ajpregu.00401.2002
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
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iNOS-/-
CONCLUDING REMARKS
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