(Stroke. 1999;30:153-159.)
© 1999 American Heart Association, Inc.
Original Contributions |
Superoxide Generation Links Protein Kinase C Activation to Impaired ATP-Sensitive K+ Channel Function After Brain Injury
From the Departments of Anesthesia and Pharmacology, University of Pennsylvania and The Children's Hospital of Philadelphia, Philadelphia, Pa.
Correspondence to William M. Armstead, PhD, Department of Anesthesia, The Children's Hospital of Philadelphia, 34th and Civic Center Blvd, Philadelphia, PA 19104. Email armsteaw{at}mail.med.upenn.edu
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Background and Purpose—Endothelin-1, in concentrations similar to that present in cerebrospinal fluid after fluid percussion brain injury (FPI), increases superoxide anion (O2-) production. Endothelin-1 also contributes to altered cerebral hemodynamics after FPI through impairment of ATP-sensitive K+ (KATP) channel function through protein kinase C (PKC) activation. Generation of O2- additionally occurs after FPI. Nitric oxide and cGMP elicit pial artery dilation through KATP channel activation. The present study was designed to determine whether PKC activation generates O2-, which, in turn, could link such activation to impaired KATP channel function after FPI.
Methods—Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase–inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O2- generation.
Results—Phorbol 12,13-dibutyrate (10-6 mol/L), a PKC activator, increased superoxide dismutase-inhibitable NBT reduction from 1±1 to 37±5 pmol/mm2. Staurosporine (10-7 mol/L), a PKC antagonist, blocked the NBT reduction after phorbol 12,13-dibutyrate and blunted the NBT reduction observed after FPI (1±1 to 15±2 versus 1±1 to 5±1 pmol/mm2 after FPI in the absence versus presence of staurosporine). Exposure of the cerebral cortex to a xanthine oxidase O2--generating system increased NBT reduction in a manner similar to FPI and blunted pial artery dilation to the KATP channel agonists cromakalim and calcitonin gene–related peptide, the nitric oxide releasers sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and the cGMP analogue 8-bromo-cGMP (10±1% and 21±1% versus 4±1% and 9±1% for 10-8 and 10-6 mol/L cromakalim before and after activated oxygen-generating system exposure).
Conclusions—These data show that PKC activation increases O2- production and contributes to such production observed after FPI. These data also show that an activated system that generates an amount of O2- similar to that observed with FPI blunted pial artery dilation to KATP channel agonists and nitric oxide/cGMP. These data suggest, therefore, that O2- generation links PKC activation to impaired KATP channel function after FPI.
Key Words: cerebral circulation • newborn • nitric oxide
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Introduction |
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Traumatic brain injury is a leading cause of morbidity and mortality in children.1 Decreased cerebral blood flow has been described in children after brain injury and may contribute to the severity of sequelae.2 Fluid percussion injury (FPI) in animals has been suggested to model human concussive trauma.3 In the newborn pig, FPI results in pial artery vasoconstriction and reductions in cerebral blood flow within 10 minutes of injury.4 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) cGMP in response to several nitric oxide (NO)–dependent stimuli were attenuated after FPI in piglets.5 6 Dilation to the NO releaser sodium nitroprusside (SNP) and the cGMP analogue 8-bromo-cGMP appears dependent on activation of the ATP-sensitive K+ (KATP) channel,7 an important contributor to the regulation of vascular tone.8 Recently, it has been observed that responses to SNP, 8-bromo-cGMP, and the KATP channel agonist cromakalim were blunted after FPI, suggesting that impaired function of mechanisms distal to NO synthase contributes to altered cerebral hemodynamics after FPI.9 Because an antagonist for the peptide endothelin-1 (ET-1), BQ 123, and the protein kinase C (PKC) inhibitor staurosporine partially restored decreased dilation to KATP agonists, NO releasers, and a cGMP analogue after FPI, ET-1 appears to contribute to altered cerebral hemodynamics after FPI through impairment of KATP channel function via activation of PKC.10 In separate experiments under non–brain injury conditions, it was also observed that coadministration of ET-1 with KATP channel agonists blunted dilation to such stimuli, suggesting that this peptide directly interacts with and impairs the KATP channel.10 However, pathways involved in linking PKC activation to KATP channel impairment are uncertain.
Superoxide anion (O2-) production is thought to antagonize NO function and to contribute to altered cerebral hemodynamics after FPI because O2- scavengers partially restored decreased NO-dependent dilator responses after FPI.11 Moreover, ET-1, in concentrations present in CSF after FPI, has been observed to release O2-.12 Therefore, ET-1 may link O2- generation to altered NO-dependent dilation after FPI.12 Interestingly, ET-1 is known to activate PKC,13 while PKC activation may lead to the generation of O2-.14 However, the effect of O2- on cerebrovascular K+ channel function is uncertain.
Therefore, the present study was designed to determine whether PKC activation generates O2-, which, in turn, could link such activation to impaired KATP channel function after FPI. Three major types of experiments were performed to address this hypothesis. First, the ability of PKC activation to generate O2- was explored. Second, the role of PKC activation in O2- generation after FPI was investigated. Third, the effects of an exogenous activated oxygen-generating system applied to the cerebral cortical surface on vascular responses to KATP channel agonists were explored.
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Materials and Methods |
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Newborn pigs (1 to 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 to 50 mg/kg, supplemented with 5 mg/kg per hour 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. The trachea was cannulated, and the animals
were mechanically ventilated with room air. A heating pad was used to
maintain the animals at 37°C to 38°C. One or 2 cranial windows were placed in the parietal skull of these anesthetized animals. The window consists of 3 parts: a stainless steel ring, a circular glass coverslip, and 3 ports consisting of 17-gauge hypodermic needles attached to 3 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 space under the window was filled with artificial CSF of the following composition (in mg/L): KCl 220, MgCl2 132, CaCl2 221, NaCl 7710, urea 402, dextrose 665, and NaHCO3 2066. This artificial CSF had the following chemistry: pH 7.33, PCO2 46 mm Hg, and PO2 43 mm Hg, which was similar to endogenous CSF. Pial arterial vessels were observed with a dissecting microscope, a television camera mounted on the microscope, and a video monitor. Vascular diameter was measured with a video microscaler (model VPA 550, For-A-Corp).
Methods for brain FPI have been described previously.15 A device designed by the Medical College of Virginia was used. A small opening in the parietal skull contralateral to the cranial window(s) 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, while 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 (37°C). The percussion device was supported by 2 brackets mounted on a metal platform. FPI was induced by striking the piston with a 4.8-kg pendulum. The intensity of the blow (usually 1.9 to 2.3 atm with a constant duration of 19 to 23 milliseconds) 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 2 types of pial
arterial vessels—small arteries (baseline diameter, 120 to
160 µm) and arterioles (baseline diameter, 50 to 70
µm)—were examined to determine whether segmental differences in the
actions of O2- could be
identified. Pial arterial vessel diameter was determined
every minute for a 10-minute exposure period after infusion onto the
exposed parietal cortex of artificial CSF containing no drug and after
infusion of artificial CSF containing a drug.
Ten major types of experiments were performed: (1) generation of O2- with phorbol 12,13-dibutyrate (n=7); (2) generation of O2- with phorbol 12,13-dibutyrate in the presence of staurosporine (n=7); (3) generation of O2- with an activated oxygen-generating system (n=7); (4) generation of O2- with an inactivated oxygen-generating system (n=5); (5) generation of O2- with FPI (n=7); (6) generation of O2- with FPI in the presence of staurosporine (n=7); (7) vascular response to agonists before and after generation of O2- with an activated oxygen-generating system (n=7); (8) vascular response to agonists before and after generation of O2- with an inactivated oxygen-generating system (n=7); (9) time control for agonist responses (n=5); and (10) sham control for O2- generation (n=7).
In the vascular response experiments, responses of arterial vessels to the synthetic KATP channel agonist (-) cromakalim (10-8 and 10-6 mol/L, SmithKline Beecham), the endogenous KATP channel activator calcitonin gene–related peptide (CGRP) (10-8 and 10-6 mol/L, Sigma Chemical), the cGMP analogue 8-bromo-cGMP (10-8 and 10-6 mol/L, Research Biochemical International), and the NO donors SNP (10-8 and 10-6 mol/L, Sigma) and S-nitroso-N-acetylpenicillamine (SNAP) (10-8 and 10-6 mol/L, Research Biochemical International) were obtained before and 20 minutes after exposure to the active or inactive oxygen-generating system for 20 minutes. The active oxygen-generating system consisted of 0.2 U/mL of xanthine oxidase, 0.6 mmol/L hypoxanthine, and 0.02 mmol/L FeCl3 administered repeatedly at 5 minutes intervals over a 20-minute period. Piglets treated with the inactivated oxygen-generating system were initially treated with oxypurinol (50 mg/kg 30 minutes before experimentation) to inhibit endogenous xanthine oxidase. They were treated as above, but the xanthine oxidase in the system was replaced with xanthine oxidase that had been boiled for 30 minutes to inactivate the enzyme. Each of the agonists was applied in a randomized ascending concentration manner. There was a period of 20 minutes after the highest concentration of one agonist was washed off before a different agonist was infused. The percent changes in artery diameter values were calculated on the basis of the diameter measured in the control period for each agonist to obtain pre–oxygen-generating system (control) values; the diameter in the control period before agonist administration after treatment with the oxygen-generating system was used for post–oxygen-generating system exposure values. Time control experiments were conducted in a separate series of animals and were designed to obtain responses to agonists initially and then 20 minutes later.
In the first 2 series of experiments designed to investigate generation of O2-, phorbol 12,13-dibutyrate (10-6 mol/L, Sigma) was applied to the cerebral cortex for 20 minutes in either the absence or presence of staurosporine (10-7 mol/L, Calbiochem). In the second set of such series of experiments, O2- generation in the presence of either an active or inactive oxygen-generating system was investigated. In the final pair of such experiments, generation of O2- with FPI was investigated in the absence and presence of staurosporine (10-7 mol/L) pretreatment. In these experiments, staurosporine was administered 20 minutes before FPI and then was kept in continued contact with the cerebral cortex for the duration of the experiment. An additional series of animals (sham control) were used to investigate the O2- generation that occurs under control conditions.
The stock staurosporine (10-4 mol/L) and phorbol 12,13-dibutyrate (10-3 mol/L) solutions were made by dissolving these agents in a small amount of dimethyl sulfoxide (200 µL), followed by ethanol. This vehicle was then diluted 1:1000 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.
O2- Analysis
Superoxide dismutase (SOD)–inhibitable nitroblue tetrazolium
(NBT) reduction was determined as an index of
O2- generation, as previously
described.11 16 Such reduction was determined by placing
NBT (Sigma, 2.4 mmol/L) dissolved in artificial CSF under one
window and NBT (2.4 mmol/L) and SOD (Sigma, 60 U/mL) in artificial
CSF under the other window 1 hour after FPI. Two windows were placed
contralateral to the adapter for induction of FPI for these
experiments.
NBT is water soluble and forms a yellow solution that is converted to nitroblue formazan, an insoluble purple precipitate, in the presence of reducing agents, eg, O2-. The SOD-inhibitable NBT reduction was determined by the difference in the quantities of nitroblue formazan precipitated on the brain surface under the 2 windows. Although NBT can be reduced by a variety of agents, SOD provides specificity for the assay. Slices of the brain surface 1 mm thick under each cranial window were obtained. The slices were minced and homogenized in 1N NaOH and 0.1% sodium dodecyl sulfate solution. The homogenate was centrifuged at 20 000g for 20 minutes. The supernatant was discarded, and the pellet was resuspended in 3 mL of pyridine. The formazan was dissolved in the pyridine during heating at 80°C for 1 hour. Particulate matter was removed by a second centrifugation at 10 000g for 10 minutes. The concentration of nitroblue formazan in the supernatant was then determined spectrophotometrically at 515 nm. The nitroblue formazan on the side with NBT alone was analyzed against the background of the SOD-treated side. Freshly prepared calibration solutions were used with each set of samples and treated identically to the samples. Recovery of NBT averaged 88±4%.
Statistical Analysis
Pial artery diameter, systemic arterial pressure,
and amount of NBT reduced were analyzed with ANOVA for repeated
measures or t test when appropriate. 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 1 vessel in each animal. Values are represented as
mean±SEM of absolute values or as percentages of change from control
values. Data presented as percent change were compared by
nonparametric means with the Wilcoxon signed rank
test.
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Role of PKC Activation in O2- Generation During Non–Brain Injury and Brain Injury Conditions
Topical application of phorbol 12,13-dibutyrate (10-6 mol/L), a PKC activator, to the cerebral cortical surface of non–brain injured animals increased SOD-inhibitable NBT reduction (Figure 1
).
Such NBT reduction by this PKC activator was blocked by
topical coadministration of staurosporine
(10-7 mol/L), a PKC antagonist
(Figure 1). Under brain injured conditions, SOD-inhibitable NBT
reduction was increased 60 minutes after FPI. Such enhanced NBT
reduction after FPI was blunted by staurosporine
pretreatment before FPI (Figure 1).
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Influence of a Xanthine Oxidase
O2--Generating System on
O2- Production and Pial Artery
Dilation to Vasoactive Stimuli
Exposure of the cerebral cortex to an active xanthine oxidase
O2--generating system increased
NBT reduction in a manner similar to FPI (Figure 1
). However,
similar exposure of the cerebral cortex to an inactive
oxygen-generating system did not change NBT reduction compared with
control (Figure 1). Cromakalim and CGRP
(10-8, 10-6 mol/L),
synthetic and endogenous KATP channel
agonists, respectively, elicited reproducible pial small-artery (120 to
160 µm) and arteriole (50 to 70 µm) vasodilation (data
not shown). These increases in vessel diameter were attenuated after
exposure of the cerebral cortical surface to the active
oxygen-generating system (Figure 2). The
inactive oxygen-generating system, however, had no effect on pial
artery dilation to the KATP channel agonists
(Figure 2). Similarly, the NO releasers, SNP and SNAP, and the
cGMP analogue 8-bromo-cGMP elicited reproducible pial artery
dilation. As with the KATP channel
agonists, the active oxygen-generating system attenuated pial dilation
to SNP, SNAP, and 8-bromo-cGMP, while the inactive oxygen-generating
system had no effect on these responses (Figures 3 and 4).
Treatment with the active oxygen-generating system increased pial
small-artery diameter from 136±5 to 166±8 µm, while pial
arteriole diameter was increased from 61±1 to 84±2 µm (n=7).
Such increases in diameter were rapid in onset, peaked at 
10 minutes
of active oxygen-generating exposure, but were reversed (returned to
control diameter) within 20 minutes of the end of the exposure. The
inactive oxygen-generating system exposure had no effect on pial artery
diameter.
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The arterial blood gas and pH values for the piglets at the beginning, during O2- generation, and at the end of the experiment were no different between all the experimental groups (eg, 7.43±0.01, 33±1, and 93±4 versus 7.44±0.01, 34±2, and 95±5 versus 7.43±0.01, 34±1, and 92±5 mm Hg for pH, PCO2, and PO2 for the beginning, during active oxygen-generating exposure, and at the end of the experiment, respectively; n=7).
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Results of the present study show that topical administration of phorbol 12,13-dibutyrate, a PKC activator, results in increased SOD-inhibitable NBT reduction by newborn pig brains, indicating that O2- was generated. Since staurosporine, a PKC inhibitor, coadministered with the phorbol 12,13-dibutyrate blocked such elevation in SOD-inhibitable NBT reduction as well as the phorbol-induced pial artery vasoconstriction,10 these data indicate that PKC activation generates O2- in a selective manner. Moreover, this antagonist also attenuated brain injury–induced elevated SOD-inhibitable NBT reduction. Earlier observations that staurosporine blocked piglet pial artery vasoconstriction induced by phorbol 12,13-dibutyrate are supportive of the specificity of staurosporine for inhibition of PKC.10 Previously, FPI has been observed to be associated with generation of O2- on the cerebral cortical surface, and ET-1 released into CSF by FPI appears to be at least one mechanism whereby brain injury generates such radicals.12 Interestingly, ET-1 is known to activate PKC.13 Taken together, then, these data suggest that ET-1 released into CSF after FPI contributes to the generation of O2- after injury through activation of PKC. It should be cautioned, however, that FPI also increases CSF levels of a number of other vasoactive substances, which, in turn, may also contribute to impaired reactivity of cerebral arteries. Additionally, it should be cautioned that concerns related to the accuracy of the NBT assay have recently been raised.17
The cerebrovascular consequences of free radical production are not fully understood. However, there is a significant amount of evidence that supports a role of oxygen radicals in brain injury. For example, brain injury in cats has been reported to cause the generation of superoxide for at least 1 hour after injury.18 In that study, the sustained dilation and abnormal responsiveness of pial arterioles observed after injury could be reversed by treatment with the free radical scavengers (SOD) and catalase.18 Oxygen radicals also have been shown to increase blood-brain barrier permeability,19 produce ultrastructural changes in pial vessel endothelium,19 and cause abnormal arteriolar reactivity.20 In addition, oxygen radical scavengers have been shown to improve vascular function and blood flow during focal ischemia in rats, which may account for the observed reductions in infarct size.21 Recently, a trial with SOD in humans with severe head injuries showed that death and vegetative state were increased in patients receiving a placebo compared with those receiving polyethylene glycol and SOD.22 Intracellular generation of superoxide or other species could alter structures and/or production of nucleotides, second messengers, receptors, and membranes, and the movement of superoxide out of the cell through anion channels could result in high concentrations of activated oxygen species at cell surfaces, including endothelium. Such oxygen species are thought to antagonize NO function and to contribute to altered cerebral hemodynamics after FPI in the piglet because free radical scavengers partially restored decreased CSF cGMP concentration and decreased responses to NO-dependent dilator stimuli such as opioids.11
The role of the systemic presser response after FPI in altered adult cerebral hemodynamics has been investigated. For example, it was hypothesized that acute elevations of blood pressure after injury in the adult result in the release and metabolism of arachidonic acid, which would generate oxygen free radicals, causing cerebral functional abnormalities.15 18 19 23 However, in contrast to studies performed in adult and juvenile animals, there was no acute elevation in blood pressure after FPI in the newborn pig.4 Since the elevation in systemic blood pressure was thought to be an absolute requirement for cerebral generation of free radicals after injury,15,18,19,23 the observed decrease in blood pressure was initially perplexing. More recent studies, however, have shown that the peptide ET-1 is released after FPI in the piglet.12 Topical administration of ET-1 in the same concentration observed after FPI resulted in the generation of substantial amounts of superoxide on the cerebral cortical surface.12 These results, therefore, link the cerebral release of this peptide to superoxide generation after FPI in the piglet. Interestingly, decreased opioid-induced dilation and associated CSF cGMP release after FPI were partially restored in animals pretreated with the ET-1 antagonist BQ123.12 These data, then, suggest that ET-1 contributes to altered cerebral hemodynamics after FPI, at least in part, through elevated superoxide production.
Although the aforementioned studies indicate that generation of
superoxide contributes to altered cerebral hemodynamics
after FPI, the role of more distal signal transduction mechanisms, such
as impaired K+ channel function, was not
considered. For example, the membrane potential of vascular muscle is a
major determinant of vascular tone, and activity of
K+ channels is a major regulator of membrane
potential.8 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.8 Because opioids elicit dilation through
KATP channel activation,24 altered
dilation to such stimuli after FPI6 could relate to
impaired K+ channel function. Interestingly,
global cerebral ischemia has been observed to impair pial
artery responses to the KATP channel opener
aprikalim in piglets.25 Similarly, another study found
that FPI blunted pial artery dilation elicited by the
KATP channel agonists CGRP and
cromakalim.9 Additionally, although CGRP has been linked
to cAMP-dependent dilator mechanisms by others,8 results
of that study did not support this idea since pial responses were not
associated with changes in cortical periarachnoid CSF cAMP and were
also not altered by Rp 8-bromo-cAMP, a cAMP
antagonist.9 Since glibenclamide blocked
dilation to CGRP and cromakalim while responses were unchanged in the
presence of iberiotoxin,9 these data indicate that these
agents are selective endogenous and synthetic
activators of the KATP channel,
respectively, consistent with previous
studies.7 26 Similarly, dilator responses to the NO
releasers SNP, SNAP, and the cGMP analogue 8-bromo-cGMP were also
blunted after FPI.9 However, responses to brain
natriuretic peptide, an activator of
particulate guanylate cyclase, and the nonselective dilator
papaverine were unchanged. These changes in responsiveness of pial
arteries after FPI were observed in the presence of moderate
vasoconstriction (17% reduction in vessel diameter). Similar
reductions in pial artery diameter in the presence of coadministered
thromboxane mimic (U46619) did not alter responses to
cromakalim, CGRP, SNP, SNAP, and 8-bromo-cGMP. Thus, there were marked
alterations in dilator mechanisms that were not the result of indirect
effects of increased vascular tone. Therefore, these data indicate that
KATP channel function was selectively impaired
after brain injury. Activation of KATP channels
has been observed to contribute to the dilation of pial arteries in the
newborn pig in response to the NO releaser SNP.7 However,
others do not ascribe such a role for KATP
channels in NO dilation since pial responses to SNP were unchanged by
glibenclamide.27 28 While the reasons for such differences
are uncertain, such observations could result from differences in
species, age, or experimental conditions. Nonetheless, impaired
KATP channel function could serve as a common
mechanism for altered cerebral hemodynamics after FPI.
Because the ET-1 antagonist BQ123 and the PKC
inhibitor staurosporine partially restored
decreased dilation to KATP agonists, NO
releasers, and a cGMP analogue after FPI, ET-1 appears to contribute to
altered cerebral hemodynamics after FPI through
impairment of KATP channel function via
activation of PKC.10 In separate experiments under
non–brain injury conditions, it was observed that coadministration of
ET-1 with KATP channel agonists blunted dilation
to such stimuli, suggesting that this peptide directly interacts with
and impairs the KATP channel,10
consistent with other studies.29 30 The mechanism
by which this impairment occurs is currently uncertain.
Because PKC activation contributes to KATP channel impairment after FPI, as described above, and also results in O2- generation, as observed in the present study, it was hypothesized that such superoxide generation could link PKC to impaired KATP channel function after brain injury. New data in the present study are the first to show that generation of oxygen free radicals through an activated oxygen-generating system results in attenuated pial artery dilation to KATP channel agonists, NO releasers, and a cGMP analogue. These data suggest, therefore, that ET-1 impairs KATP channel function after FPI through superoxide generation due to PKC activation. These studies extend previous observations that PKC activation inhibits the K+ current in isolated feline cerebral vascular smooth muscle cells31 to the intact animal and also demonstrate the physiological relevance of such observations in the context of impaired cerebral artery reactivity after FPI. The activated oxygen-generating system had been observed in a previous study to result in pial vessels that were ultrastructurally abnormal.20 Lesions consisted of increased numbers of vascular cytoplasmic inclusions, more numerous surface pits, and mitochondrial injury.20 Although pial artery diameter returned to the pretreatment diameter after removal of the activated oxygen-generating system, pial artery responsiveness was altered. In the earlier study, pial artery dilation in response to hypercapnia and hypotension was reduced, while that to isoproterenol or constriction to norepinephrine was unchanged after activated oxygen-generating system treatment,20 observations similar to those obtained with a piglet model of global cerebral ischemia.32 33 After FPI, cerebral blood flow and cerebral oxygenation are reduced,4 suggesting that ischemia may occur after such injury in the piglet as well. Results of the present study show that the activated oxygen-generating system produced a reduction of NBT similar to that observed after FPI, indicating that approximately the same amount of superoxide is generated with either intervention. The inactive oxygen-generating system, however, did not cause the reduction of NBT, nor did it alter vascular responses to KATP channel agonists, NO releasers, or a cGMP analogue, thereby giving specificity to the conclusions related to the actions of oxygen free radicals. By repeated application of the activated oxygen-generating system for 20 minutes in the present study, superoxide was generated continuously over the application period. However, the effect of topical application of the activated oxygen-generating system on endothelial cells may be attenuated by intervening tissue, although ultrastructural endothelial alternations appear considerable.20 Intracellular generation of superoxide or other species after FPI could result in higher concentrations of more active species at cell surfaces, including endothelium.
In conclusion, results of the present study show that PKC activation increases O2- production and contributes to such production observed after FPI. These data also show that an activated oxygen-generating system that generates an amount of O2- similar to that observed with FPI attenuated pial artery dilation to KATP channel agonists, NO releasers, and a cGMP analogue. These data, therefore, suggest that O2- generation links PKC activation to impaired KATP channel function after FPI.
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health and the American Heart Association. Dr Armstead is an Established Investigator of the American Heart Association. The author thanks Joseph Quinn for technical assistance in the performance of the experiments.
Received July 23, 1998; revision received September 22, 1998; accepted October 14, 1998.
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Editorial Comment
Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska
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Many investigators have suggested that the formation of oxygen radicals, during a variety of conditions, produces adverse effects on the cerebral circulation. For example, acute increases in arterial blood pressure damage cerebrovascular endothelium through the production of oxygen radicals,1 impaired responses of cerebral blood vessels during brain injury can be restored by scavengers of oxygen radicals,2 and oxygen radicals increase the permeability of the blood-brain barrier.3 In addition, recent studies by Kasemsri and Armstead4 5 have shown that topical application of ET-1, at a level seen during FPI, produces an increase in oxygen radical formation and impairs KATP channel function of cerebral blood vessels through activation of PKC. The purpose of the present study was to determine whether activation of PKC generates oxygen radicals and thus accounts for impaired cerebrovascular reactivity during brain injury.
In the present study, the investigators measured oxygen radical formation during activation of PKC and FPI. In addition, the investigators measured in vivo responses of piglet cerebral arterioles to activation of KATP channels, NO donors, and activators of cGMP before and after generation of oxygen radicals. The authors report that activation of PKC and FPI increased oxygen radical formation, which could be attenuated by pretreatment with staurosporine. Furthermore, the authors report that dilation of cerebral blood vessels to the agonists was inhibited by oxygen radical formation.
Thus, on the basis of the findings of the present study, the authors suggest that activation of PKC during brain injury produces an increase in oxygen radical formation and accounts for impaired responses of cerebral blood vessels to activators of KATP channels, NO donors, and activators of cGMP. These findings may have important implications regarding therapeutic approaches used in the treatment of brain injury in newborns.
Received July 23, 1998; revision received September 22, 1998; accepted October 14, 1998.
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injury. Circ Res.. 1985;57:508–516.
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- Kasemsri T, Armstead WM. Endothelin production links superoxide generation to altered opioid-induced pial artery vasodilation after brain injury in pigs. Stroke.. 1997;28:190–197.
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