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Departments of Anesthesia and Pharmacology, University of Pennsylvania, and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In piglets, pial arteries constrict,
ATP-sensitive K+
(KATP) channel function is
impaired, and cerebrospinal fluid endothelin-1 (ET-1) increases to
10
10 M after brain injury
[fluid percussion injury (FPI)]. Nitric oxide (NO) elicits
dilation via guanosine 3',5'-cyclic monophosphate (cGMP)
and KATP channel activation. This
study was designed to characterize the relationship between ET-1 and
impaired function of KATP channels
after FPI. Injury was produced via the lateral FPI technique in piglets
equipped with a closed cranial window. Cromakalim, a
KATP agonist, produced dilation
that was attenuated by FPI and partially restored by BQ-123, an ET-1
antagonist (11 ± 1 and 23 ± 2 vs. 2 ± 1 and 4 ± 1 vs. 8 ± 1 and 17 ± 2% for responses to
108 and
106 M cromakalim before
FPI, after FPI, and after FPI with BQ-123, respectively). Because ET-1
constriction may antagonize dilation, separate experiments were
conducted under conditions of equivalent baseline diameter in the
absence and presence of ET-1
(1010 M). Cromakalim
dilation was attenuated by ET-1 and partially restored by the protein
kinase C (PKC) inhibitor staurosporine (12 ± 1 and 28 ± 1 vs. 2 ± 1 and 21 ± 3 vs. 9 ± 1 and 29 ± 2% for
108 and
106 M cromakalim,
cromakalim with ET-1, and cromakalim with ET-1+staurosporine, respectively). Similar interactions were observed with
calcitonin gene-related peptide, 8-bromoguanosine
3',5'-cyclic monophosphate, and the NO releasers sodium
nitroprusside and
S-nitroso-N-acetylpenicillamine. These data show that ET-1 blunts
KATP channel-, NO-, and
cGMP-mediated dilation. These data suggest that ET-1 contributes to
altered cerebral hemodynamics after FPI through impairment of
KATP channel function via PKC
activation.
newborn; cerebral circulation; nitric oxide; cyclic nucleotides
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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TRAUMATIC BRAIN INJURY is a leading cause of morbity and mortality in children (23). Decreased cerebral blood flow has been described in children after brain injury and may contribute to the severity of sequelae (27). Fluid percussion injury (FPI) in animals has been suggested to model human concussive trauma (14). In the newborn pig, FPI results in pial artery vasoconstriction and reductions in cerebral blood flow within 10 min of injury (7). Additionally, neurohumoral control of the cerebral circulation is altered after brain injury. For example, pial dilation and associated elevations in cortical periarachnoid cerebrospinal fluid (CSF) guanosine 3',5'-cyclic monophosphate (cGMP) in response to several nitric oxide (NO)-dependent stimuli were attenuated after FPI in piglets (3, 38). Dilation to the NO releaser sodium nitroprusside (SNP) and the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) appears dependent on activation of the ATP-sensitive K+ (KATP) channel (4), an important contributor to the regulation of vascular tone (19). Recently, it has been observed that responses to SNP, 8-BrcGMP, and the KATP-channel agonist cromakalim were blunted after FPI, suggesting that impaired function of mechanisms distal to NO synthase (NOS) contributes to altered cerebrohemodynamics after FPI (1). However, pathways involved in eliciting such alterations are uncertain.
Endothelin (ET) is a 21-amino acid peptide with potent vasoconstrictor properties (43). There are three pharmacologically distinct isoforms of ET, ET-1, ET-2, and ET-3, with ET-1 having the most potent vasoconstrictive effect (17). ET-1 and its receptor ETA are prevalent in the central nervous system (15, 16). In the piglet, ET-1 elicits potent pial artery constriction, whereas cortical periarachnoid CSF ET-1 concentration is increased after FPI and contributes to altered dilator response to several NO-dependent stimuli (5, 18). For example, ET-1, in concentrations present in CSF after injury, releases oxygen free radicals, which, in turn, destabilize NO and contribute to altered cerebral hemodynamics after FPI (18). In those studies, however, the role of ET-1 in altered mechanisms distal to NOS was not considered. Interestingly, ET-1 has been reported to block the KATP channel in porcine coronary artery smooth muscle and guinea pig ventricular cells (28, 34, 40). The interaction of ET-1 with the KATP channel, however, is unknown in the cerebral circulation, and the role of this relationship in altered cerebral hemodynamics after FPI is uncertain.
The present study, therefore, was designed to characterize the relationship between ET-1 and impaired function of KATP channels after FPI. In addition, ET-1 is thought to elicit vasoconstriction, at least in part, through activation of protein kinase C (PKC) (29), whereas PKC activation contributes to altered responses of several stimuli after FPI (6). The role of this second messenger system in the interaction between ET and the KATP channel was, therefore, studied as well.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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One hundred twelve newborn pigs (1-5 days old) of either sex were
used in these experiments. All protocols were approved by the
Institutional Animal Care and Use Committee. Animals were anesthetized
with ketamine hydrochloride (33 mg/kg) and acepromazine (3.3 mg)
intramuscularly. Anesthesia was maintained with 
-chloralose (30-50 mg/kg, supplemented with 5 mg · kg1 · h1
iv). A catheter was inserted into a femoral artery to monitor blood
pressure and to sample for blood-gas tensions and pH. Drugs to maintain
anesthesia were administered through a second catheter placed in a
femoral vein. Blood was withdrawn through this catheter as well when
indicated by the protocol. The trachea was cannulated, and the animals
were mechanically ventilated with room air. A heating pad was used to
maintain the animals at 37-39°C.
A cranial window was placed in the parietal skull of these anesthetized animals. This window consisted of three parts: a stainless steel ring, a circular glass coverslip, and three ports consisting of 17-gauge hypodermic needles attached to three precut holes in the stainless steel ring. For placement, the dura was cut and retracted over the cut bone edge. The cranial window was placed in the opening and cemented in place with dental acrylic. The volume under the window was filled with a solution similar to CSF of the following composition (in mg/l): 220 KCl, 132 MgCl2, 221 CaCl2, 7,710 NaCl, 402 urea, 665 dextrose, and 2,066 NaHCO3. This artificial CSF had the following chemistry: pH 7.33, PCO2 46 mmHg, and PO2 43 mmHg; this was similar to endogenous CSF. Pial arterial vessels were observed with a dissecting microscope, a television camera mounted on the microscope, and a video-output screen. Vascular diameter was measured with a video microscaler.
Methods for brain FPI have been described previously (41). A device designed by the Medical College of Virginia was used. A small opening in the parietal skull contralateral to the cranial window was made. A metal shaft was sealed into the opening on top of intact dura. This shaft was connected to transducer housing, which was, in turn, connected to the fluid percussion device. The device itself consisted of an acrylic plastic cylindrical reservoir 60 cm long, 4.5 cm in diameter, and 0.5 cm thick. One end of the device was connected to the transducer housing, whereas the other end had an acrylic plastic piston mounted on O-rings. The exposed end of the piston was covered with a rubber pad. The entire system was filled with 0.9% saline. The percussion device was supported by two brackets mounted on a platform. FPI was induced by striking the piston with a 4.8-kg pendulum. The intensity of the blow (usually 1.9-2.3 atm with a constant duration of 19-23 ms) was controlled by varying the height from which the pendulum was allowed to fall. The pressure pulse of the blow was recorded on a storage oscilloscope triggered photoelectrically by the fall of the pendulum. The amplitude of the pressure pulse was used to determine the intensity of the injury.
Protocol. Drug effects on the diameter of two types of pial arterial vessels, small arteries (resting diameter, 120-160 µm) and arterioles (resting diameter, 50-70 µm), were examined to determine whether segmental differences in the actions of ET-1 could be identified. Pial arterial vessel diameter was determined every minute for a 10-min exposure period after infusion onto the exposed parietal cortex of artificial CSF containing no drug and after infusion of artificial CSF containing a drug. Typically, 2-3 ml of CSF were flushed through the window over a 30-s period, and excess CSF was allowed to run off through one of the needle ports.
We performed seven types of experiments for each drug: 1) FPI without BQ-123 or staurosporine pretreatment (n = 7); 2) FPI with BQ-123 pretreatment (n = 7); 3) FPI with staurosporine pretreatment (n = 7); 4) ET-1 with and without BQ-123 coadministration (n = 8); 5) ET-1 with and without staurosporine coadministration (n = 7 or 8); 6) BQ-123 or staurosporine coadministration (n = 5); and 7) time controls (n = 5). In the FPI experiments, responses of arterial vessels to the synthetic KATP-channel agonist (
)-cromakalim (108
and 106 M, SmithKline
Beecham), the endogenous
KATP-channel activator calcitonin
gene-related peptide (CGRP;
108 and
106 M, Sigma Chemical), the
cGMP analog 8-BrcGMP
[108 and
106 M, Research
Biochemicals International (RBI)], and the NO donors sodium
nitroprusside (SNP) (108
and 106 M, Sigma Chemical)
and
S-nitroso-N-acetylpenicillamine
(SNAP) (108 and
106 M, RBI) were obtained
before and 1 h after FPI. In animals that were pretreated with BQ-123
(106 M, RBI) or
staurosporine (107 M,
Calbiochem), a PKC inhibitor, antagonists were applied 20 min before
FPI. Each of the drugs was applied in an ascending concentration
manner. There was a period of 20 min after the highest concentration of
one drug was washed off before a different drug was infused. The
percent change in artery diameter values were calculated on the basis
of the diameter measured in the control period for each drug before
injury for preinjury (control) values, whereas the diameter present in
the control period before the drug administration after injury was used
for brain injury values.
In the ET-1 experiments, the response of arterial vessels to
cromakalim, CGRP, 8-BrcGMP, SNP, and SNAP
(108 and
106 M) were obtained in the
absence of ET-1, in the presence of ET-1 (1010 M), a concentration
observed in CSF after FPI (4), and in the presence of ET-1 and BQ-123
(106 M). Because ET-1
(1010 M) elicits
constriction, blood was withdrawn from the animals as necessary after
ET-1 administration to dilate pial arteries and return the vessel
diameters to resting values observed before ET-1 administration.
Agonists were then coadministered with ET-1, and the mean arterial
blood pressure maintained constant through titrated blood withdrawal or
saline infusion. There was a 20-min period after one drug was washed
off before the next drug was administered. Each of the drugs was
applied in an ascending concentration manner. For determination of
specificity, responses to ET-1
(1010 M) were obtained in
the absence and presence of BQ-123, whereas responses to papaverine
(108 and
106 M, Sigma Chemical) were
obtained in the absence and presence of ET-1. To determine the role of
PKC in the action of ET-1 on KATP
channel activation, staurosporine was coadministered with ET-1 instead
of BQ-123 in the above protocol in a separate series of animals. In
both of these series, the percent change in artery diameter values were
calculated on the basis of the diameter present in the resting period
(control) for each animal before administration of drugs. Specificity
of staurosporine action was determined by obtaining responses to
phorbol 12,13-dibutyrate
(108 and
106 M; Sigma Chemical), an
activator of PKC, in the absence and presence of staurosporine.
Responses to CGRP, cromakalim, SNP, SNAP, and 8-BrcGMP were also
obtained in the absence and presence of either BQ-123 or staurosporine
without superimposed FPI or coadministered ET-1. Time-control
experiments were conducted in a separate series of animals and were
designed to obtain responses to drugs initially and then 1 h later
(designated as time 1 and
time 2 in Table
1).
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4 M) and phorbol
12,13-dibutyrate (103 M)
solutions were made by dissolving these agents in a small amount of
dimethyl sulfoxide (200 µl), followed by ethanol. This vehicle was
then diluted 1:1,000 in CSF to make the working solution. Appropriate
aliquots of the vehicle for all other agents (0.9% saline) were added
to CSF infused under the window. These CSF vehicles had no effect on
pial artery diameter. All drugs were made fresh on the day of use.
Statistical analysis. Pial artery diameter and systemic arterial pressure values were analyzed with analysis of variance for repeated measures. If the value was significant, Fisher's exact test was performed. A value of P < 0.05 was considered significant. The n values reflect data for one vessel in each animal. Values are represented as means ± SE of absolute values or as percentages of change from control values. Data presented as percent change were compared by nonparametric means with the Wilcoxin signed-rank test.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Influence of BQ-123 on KATP channel-,
NO-, and cGMP-induced pial artery dilation after FPI.
Cromakalim and CGRP (108
and 106 M), synthetic and
endogenous KATP channel agonists,
respectively, elicited reproducible pial small artery (120-160
µm) and arteriole (50-70 µm) vasodilation (Table 1). These
increases in vessel diameter were attenuated after FPI and partially
restored by BQ-123 pretreatment (Fig. 1).
The NO releasers SNP and SNAP
(108 and
106 M) produced similar
reproducible pial artery dilation (Table 1). As with the
KATP-channel agonists, FPI
attenuated dilator responses to SNP and SNAP, whereas BQ-123
pretreatment partially restored the responses after FPI (Fig.
2). The pial vessels showed a similar
response profile to the cGMP analog 8-BrcGMP
(108 and
106 M) before (Table 1) and
after FPI with and without pretreatment with BQ-123 (Fig.
3).
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Influence of staurosporine on KATP
channel-, NO-, and cGMP-induced pial artery dilation after FPI.
Similar to the finding above, FPI attenuated pial artery responses to
cromakalim, CGRP, SNP, SNAP, and 8-BrcGMP (Table
2). However, pretreatment with
staurosporine (107 M)
partially restored decremented pial artery responses to the above
agonists after FPI (Table 2).
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Influence of ET-1 on KATP channel-, NO-,
and cGMP-induced pial artery dilation.
ET-1 (1010 M) decreased
pial small artery diameter from 137 ± 2 to 128 ± 3 µm,
whereas arteriole diameter was decreased from 65 ± 1 to 59 ± 1 µm (n = 5). Blood withdrawal
(4-10 ml iv) during ET-1 administration resulted in no change in
pial small artery or arteriole diameter (143 ± 4 vs. 140 ± 4 µm and 68 ± 2 vs. 67 ± 2 µm, respectively,
n = 46). Similarly, there was no
change in mean arterial blood pressure (66 ± 1 vs. 65 ± 2 mmHg,
n = 46).
8 and
106 M) was attenuated by
coadministration with ET-1
(1010 M) under conditions
of equivalent baseline diameter in the absence and presence of ET-1 as
described above (Fig. 4). Attenuated
responses were fully restored when these agonists were coadministered
with ET-1 and BQ-123 (106
M) (Fig. 4). Similarly, pial artery dilator responses to SNP, SNAP, and
8-BrcGMP were attenuated by ET-1 and restored by BQ-123 (Figs.
5 and 6).
However, responses to papaverine
(108 and
106 M) were unchanged by
ET-1 (9 ± 1 and 18 ± 1 vs. 9 ± 1 and 17 ± 1% in the
absence and presence of ET-1, respectively,
n = 5).
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Influence of ET-1 on KATP channel-, NO-,
and cGMP-induced pial artery dilation: Role of PKC.
Under conditions of equivalent baseline diameter before and after ET-1,
as described above, pial arterial vasodilation in response to
cromakalim and CGRP (108
and 106 M) was attenuated
by coadministration with ET-1
(1010 M) and partially
restored by staurosporine, a PKC inhibitor, coadministered with ET-1
(Fig. 7). Staurosporine had similar actions on vasodilator responses to SNP and SNAP
(108 and
106 M) when coadministered
with ET-1 (1010 M) (Fig.
8). In addition, in the presence of ET-1,
staurosporine was able to partially restore dilator responses to
8-BrcGMP as well (Fig. 9).
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Influence of BQ-123 and staurosporine on KATP channel-, NO-, and cGMP-induced pial artery dilation in the absence of FPI or ET-1. Responses to cromakalim, CGRP, SNP, SNAP, and 8-BrcGMP were obtained in the absence of brain injury or local application of ET-1. These data show that pial artery responses to the above agonists were unchanged when coadministered with either BQ-123 or staurosporine (Table 3).
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Influence of BQ-123 and staurosporine on baseline pial artery
diameter and responses to ET-1 and phorbol 12,13-dibutyrate.
BQ-123 (106 M) had no
effect on pial small artery or arteriole diameter (140 ± 4 vs. 142 ± 4 µm and 65 ± 3 vs. 67 ± 3 µm,
n = 24). Similarly, staurosporine
(107 M) had no effect on
pial vessel diameters (148 ± 5 vs. 149 ± 5 µm and 69 ± 2 vs. 70 ± 2 µm, n = 22). However,
BQ-123 blocked pial constriction to ET-1
(1010 M; 8 ± 1 vs. 1 ± 1%, n = 5).
Similarly, staurosporine blunted pial constriction to phorbol 12,- 13-dibutyrate (108 and
106 M; 9 ± 1 and
23 ± 2 vs. 3 ± 1 and 8 ± 1%,
n = 5).
Blood chemistry. Blood chemistry values were obtained at the beginning and end of all experiments. In the FPI experiments, these values were 7.45 ± 0.01, 32 ± 1 mmHg, and 98 ± 2 mmHg vs. 7.44 ± 0.01, 33 ± 1 mmHg, and 95 ± 2 mmHg for pH, PCO2, and PO2 before FPI and after FPI, respectively (n = 28). In the non-FPI experiments, these values were 7.43 ± 0.01, 35 ± 1 mmHg, and 92 ± 2 mmHg vs. 7.44 ± 0.01, 36 ± 1 mmHg, and 91 ± 2 mmHg for pH, PCO2, and PO2 at the beginning and end of experiments, respectively (n = 67). Mean arterial blood pressure decreased from 65 ± 1 to 53 ± 2 mmHg within 60 min of FPI (n = 28), whereas it remained stable in the non-FPI experiments (66 ± 1 vs. 65 ± 1 mmHg for the beginning and end of experiments, respectively, n = 67). The mean level of intensity of FPI was 2.1 ± 0.1 atm.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Results of the present study show that FPI blunted pial artery dilation
elicited by the KATP-channel
agonists cromakalim and CGRP, the NO releasers SNP and SNAP, and the
cGMP analog 8-BrcGMP, consistent with recent observations (1). Because
similar blunted responsiveness was observed in both pial small arteries
and arterioles, the effects of FPI do not appear to reflect regional
vascular differences. When animals were pretreated with the
ETA-receptor antagonist BQ-123 or
the PKC inhibitor staurosporine, responses to these dilator stimuli
were partially restored after FPI. In separate experiments, ET-1
(1010 M) coadministration
also blunted pial artery responses to these dilators, whereas the
addition of BQ-123 fully restored decremented agonist-induced dilation.
NO and cGMP elicit pial artery dilation via opening of the
KATP and not the calcium-dependent
K+ channel in the newborn pig (2,
4). In previous studies, it had also been observed that FPI resulted in
elevation of ET-1 concentration in cortical periarachnoid CSF to
1010 M in the newborn
pig (5, 18). Results of the present experiments, therefore, indicate
that such elevated CSF ET-1 concentrations contribute to altered
cerebral responses to KATP
channel-dependent dilator stimuli. There are at least two possible
explanations for these observations. First, because ET-1
(1010 M) elicits pial
constriction in the piglet (8), decremented agonist dilation could
relate to physiological opposition to stimuli-induced increased pial
artery diameter. Alternatively, there could be a direct interaction of
ET-1 with the KATP channel, as
observed in porcine coronary artery smooth muscle and guinea pig
ventricular cells (28, 34, 40). To investigate the latter possibility, experiments were designed so as to maintain baseline pial artery diameter at equivalent values in the absence and presence of ET-1. Under such conditions, coadministration of ET-1 with dilator stimuli resulted in similar decremented agonist responses, suggesting that ET-1
contributes to impairment of KATP
channel function after FPI. Because BQ-123 fully restored decremented
responses observed with ET-1 under equivalent baseline conditions and
blocked pial constriction to topical ET-1, these data demonstrate the
specificity of action for ET-1 impairment of
KATP channel function. Because responses to papaverine were unchanged in the presence of
coadministered ET-1, these data indicate that ET-1 impairment of
dilator responsiveness is not an epiphenomenon. Responses to papaverine
have previously been observed to be unchanged after FPI (1), consistent
with the inability of a CSF concentration of ET-1 reached after FPI to
inhibit responses to this dilator. Additionally, responses to
cromakalim, CGRP, SNP, SNAP, and 8-BrcGMP were unchanged when coadministered with BQ-123 in the absence of FPI or local
administration of ET-1, further indicating specificity of action with
BQ-123. Finally, experiments were designed to characterize at least one mechanism by which ET-1 contributes to altered
KATP channel function after FPI.
Because ET-1 is thought to elicit cerebral vasoconstriction, at least
in part, via activation of PKC (29), experiments were designed to
obtain responses to the same dilator stimuli and ET-1 coadministered
with the PKC inhibitor staurosporine, under conditions of equivalent
baseline diameter. These results show that staurosporine partially
restored decremented dilator responses, indicating that PKC activation
contributes to ET-1 impairment of
KATP channel function. Specificity
of staurosporine action was demonstrated by observing that
staurosporine blunted responses to the PKC activator phorbol
12,13-dibutyrate but did not alter responses to
KATP channel, NO, and cGMP
agonists in the absence of FPI or locally administered ET-1. However,
mechanisms whereby PKC activation couples to the KATP channel are uncertain, while
the role of other biochemical pathways in impaired
KATP channel function is equally
unknown.
Membrane potential of vascular muscle is a major determinant of vascular tone, and activity of K+ channels is a major regulator of membrane potential (32). Activation or opening of these channels increases K+ efflux, thereby producing hyperpolarization of vascular muscle. Membrane hyperpolarization closes voltage-dependent calcium channels and thereby causes relaxation of vascular muscle (31, 32). Direct measurements of membrane potential and K+ current in vitro indicate that several different types of K+ channels are present in cerebral blood vessels. In addition, a number of pharmacological studies using activators and inhibitors have provided functional evidence that K+ channels, especially KATP channels, regulate tone of cerebral blood vessels in vitro and in vivo (19). For example, an outward K+ current has been reported to produce dilation of dispersed cerebral arterial smooth muscle cells (11), whereas KATP channel openers have been observed to cause dilation of isolated basilar and middle cerebral arteries (30). Moreover, in vivo studies support the idea that KATP channels are present in cerebral arterioles (26). On the other hand, it has also been reported that KATP channel openers such as cromakalim do not dilate cerebral arteries (25).
Several substances have been suggested to be endogenous activators of KATP channels. In the cerebral circulation, pial arteries have been shown to be innervated by CGRP-containing nerve fibers (12). CGRP produces hyperpolarization of cerebral vascular muscle in vitro (33), whereas dilator responses of cerebral arteries are inhibited by glibenclamide, a KATP-channel antagonist, indicating that dilation is mediated by opening of this K+ channel (20).
Previous studies in the newborn pig have observed that SNP and 8-BrcGMP elicit dilation via activation of KATP channels (4). However, others have also observed that responses to SNP were unchanged by glibenclamide (9, 22). Although the reasons for such differences are uncertain, such observations could result from differences in species, age, or experimental conditions. Additionally, in vivo approaches to the study of ion channels are limited in that pharmacological probes can only serve as an indirect index of ion channel contribution to vascular responsiveness. It has also been observed that responses to several NO-dependent dilator stimuli, including opioids and vasopressin, were blunted after FPI and partially restored by the preadministration of oxygen free radical scavengers (3, 39), indicating that impaired NO function contributes to altered cerebral hemodynamics after FPI. Because responses to SNP and 8-BrcGMP were also blunted after FPI in the present study, these data suggest that impaired function of mechanisms distal to NOS also contribute to altered hemodynamics after FPI (1). Because many different substances, including opioids (35), are thought to elicit K+ channel-dependent dilation, such observations, therefore, suggest that impaired K+ channel function could serve as a common mechanism for altered cerebral hemodynamics after FPI (1). However, impaired functionality after brain injury is not nonspecific because responses to brain natriuretic peptide and the nonselective dilator papaverine were unchanged (1).
Although several other types of cerebral injury models have observed similar impairment of KATP channel function, others have not. For example, dilation of pial arteries in response to RP-52891, a KATP channel activator, was observed to be impaired in diabetic rats (26), while basilar artery dilation to aprikalim, another KATP agonist, was blunted in stroke-prone spontaneously hypertensive rats (21). Additionally, global ischemia has been observed to impair pial artery responses to CGRP and aprikalim in piglets, indicating that impairment of KATP channel function can be achieved by an acute stimulus (10, 24). In contrast, responses to aprikalim and CGRP were augmented in a rat model of subarachnoid hemorrhage, whereas responses to SNP were decreased and those to 8-BrcGMP were unchanged, indicating that KATP channel function was preserved but production of cGMP, and not its action, was blunted in this injury model (36). Although mechanisms for impaired KATP channel function after FPI are uncertain, data from the present study are the first to suggest that FPI-associated release of ET-1 (5, 18) could contribute to such impairment. Alternatively, brain injury could alter the number or binding of K+ channels available for activation, the degree of hyperpolarization that subsequently occurs or the ultimate response to hyperpolarization itself.
PKC has been shown to mediate vasoconstrictive effects of ET-1 in
isolated preparations of rat aorta (42), in afferent arterioles of
isolated perfused hydronephrotic kidney (37), and in the bovine
cerebral artery (13). In the present study, staurosporine, an inhibitor
of PKC, was able to partially restore pial arterial vasodilation by
both KATP-dependent and
NO-dependent stimuli in the presence of a brain injury concentration of
ET-1. Therefore, it appears that ET-1 activates PKC, resulting in
impairment of KATP channel
function. Alternatively, ET-1 may cause production of other activators
of PKC that are present in CSF after FPI such as the opioids, dynorphin
and 
-endorphin (6).
Although many studies have characterized the hemodynamic effects of brain injury in adult animal models, few have done so in the newborn-to-infant time period. Results of recent studies show that developmental changes result in markedly different effects of brain injury on cerebral hemodynamics in the newborn and juvenile pig (7). For example, the following were observed: 1) pial vessels constricted more and cerebral blood flow falls and remained depressed longer in newborns vs. juveniles; 2) there are marked increases in intracranial pressure in the newborn but modest increases in intracranial pressure in the juvenile; and 3) there were differences in cerebral oxygenation, an index of metabolism; increased saturation followed by prolonged desaturation of hemoglobin for oxygen in the newborn; and modest increases in saturation followed by mild desaturation in the juvenile (7). Furthermore, systemic arterial pressure has been observed to increase in the adult studies (41) and in juvenile pigs (7), whereas systemic arterial pressure decreases after brain injury in the newborn pig (7). These data suggest that cerebral and systemic hemodynamic responses after brain injury are age dependent. In fact, it has been suggested previously that the newborn is exquisitely sensitive to brain injury (7). Because both newborn pigs and children <1 yr of age have skulls with unfused sutures, the age period of pigs chosen in these studies may approximate the newborn-to-infant time period in humans.
In conclusion, results of the present study show that ET-1 blunts KATP channel-, NO-, and cGMP-mediated dilation. These data suggest that ET-1 contributes to altered cerebral hemodynamics after FPI through impairment of KATP channel function via PKC activation.
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ACKNOWLEDGEMENTS |
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The authors thank Joseph Quinn for technical assistance in the performance of the experiments.
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FOOTNOTES |
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This research was supported by grants from the National Institutes of Health and the American Heart Association (AHA). W. M. Armstead is an Established Investigator of the AHA.
Address for reprint requests: W. M. Armstead, Dept. of Anesthesiology, The Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104.
Received 19 May 1997; accepted in final form 20 August 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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1.
Armstead, W. M. Brain injury impairs ATP-sensitive
K+ channel function in piglet
cerebral arteries. Stroke. In
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Role of activation of calcium-sensitive K+ channels in NO- and hypoxia-induced pial artery vasodilation.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H1785-H1790,
1997
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Armstead, W. M.
Influence of brain injury on vasopressin- induced pial artery vasodilation: role of superoxide anion.
Am. J. Physiol.
270 (Heart Circ. Physiol. 39):
H1272-H1278,
1996
4.
Armstead, W. M.
Role of ATP-sensitive K+ channels in cGMP-mediated pial artery vasodilation.
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