Protein kinase C activation generates superoxide and contributes to impairment of cerebrovasodilation induced by G protein activation after brain injury

Brain Research 971 (2003) 153–160 www.elsevier.com / locate / brainres

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Protein kinase C activation generates superoxide and contributes to impairment of cerebrovasodilation induced by G protein activation after brain injury William M. Armstead* Departments of Anesthesia and Pharmacology, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA Accepted 10 January 2003

Abstract Previous studies have observed that activation of protein kinase C (PKC) contributes to generation of superoxide anion (O 2 2 ) after fluid percussion brain injury (FPI). This study was designed to characterize the effects of FPI on the vascular activity of two activators of a pertussis toxin sensitive G protein, mastoparan and mastoparan-7, and the role of PKC dependent O 2 2 generation in such effects in newborn pigs equipped with a closed cranial window. Mastoparan (10 28 , 10 26 M) elicited pial artery dilation that was blunted by FPI and partially restored by the PKC inhibitor chelerythrine (10 27 M) or the O 2 2 free radical scavengers polyethylene glycol superoxide dismutase and catalase (SODCAT) (961 and 1661, sham control; 361 and 561, FPI; and 761 and 1161%, FPI SODCAT pretreated). Similar results were observed for mastoparan-7 but the inactive analogue mastoparan-17 had no effect on pial artery diameter. Exposure of the cerebral cortex to a xanthine oxidase O 2 2 generating system blunted mastoparan induced pial artery dilation similar to FPI (1061 and 1761 vs. 261 and 361%). Pertussis toxin (1 mg / ml) exposure blocked mastoparan and mastoparan-7 vasodilation. These data show that pertussis toxin sensitive G protein activation elicits cerebrovasodilation that is blunted following FPI in a PKC dependent manner. These data also show that O 2 2 generation similarly blunts G protein mediated cerebrovasodilation. These data suggest that PKC dependent O 22 generation contributes to impaired G protein mediated cerebrovasodilation after FPI.  2003 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators and receptors Topic: Signal transduction Keywords: Newborn; Cerebral circulation; Signal transduction; Oxygen free radical

1. Introduction Traumatic brain injury is a leading cause of morbidity 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 [23,24]. Fluid percussion injury (FPI) in animals has been suggested to model human concussive trauma [11]. In the newborn pig, FPI results in pial artery vasoconstriction and reductions in cerebral blood flow within 10 min of injury [8]. Additionally, neurohumoral control of the cerebral circulation is altered after brain injury. For example, pial dilation and associated elevations in cortical periarachnoid cerebrospi*Tel.: 11-215-573-3674; fax: 11-215-349-5078. E-mail address: [email protected] (W.M. Armstead).

nal fluid (CSF) cGMP in response to several nitric oxide (NO)-dependent stimuli were attenuated after FPI in piglets [7,27]. Dilation to the NO releaser sodium nitroprusside (SNP) and the cGMP analogue 8-bromo-cGMP appears dependent on activation of the ATP-sensitive K 1 (KATP ) channel [6], an important contributor to the regulation of vascular tone [14]. 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 [5]. Superoxide anion (O 2 2 ) production is thought to antagonize NO function and to contribute to altered cerebral hemodynamics after FPI because O 2 2 scavengers partially restored decreased NO-dependent dilator responses after FPI [26]. Moreover, O 2 2 generation contributes to KATP chan-

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02287-X

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nel agonist induced dilator impairment after FPI in a mechanism dependent on activation of protein kinase C (PKC) [3]. Receptors regulate the function of G proteins by catalyzing the release of bound GDP and the binding of GTP. In turn, GTP activates the G protein allowing it to activate effector proteins which subsequently transmit chemical signals to elicit a biological response. Mastoparan, an amphiphilic tetra decapeptide catalyzes nucleotide exchange on G proteins in a manner similar to that of receptors [12,15,16]. Like the effect of receptor agonists, pertussis toxin inhibits the effects of mastoparan on G i / Go proteins [12,15,16]. Because of its close mimicry of G protein receptor interaction, use of mastoparan may serve as a useful probe for the modeling of signal transduction to elicit a biological response. Interestingly, mastoparan has been observed to elicit coronary and pial artery vasodilation via activation of the KATP channel [1,17]. The present study sought to link the observations described above: (1) activation of PKC generates O 2 2 which contributes to KATP channel agonist induced dilator impairment after FPI, and (2) the G protein activator mastoparan elicits vasodilation via activation of the KATP channel. Therefore, the present study was designed to characterize the effects of FPI on the vascular activity of two activators of a pertussis toxin sensitive G protein, mastoparan and mastoparan-7, and the role of PKC dependent O 2 2 generation in such effects.

had the following chemistry: pH 7.33, pCO 2 46 mmHg, and pO 2 43 mmHg, which was similar to that of 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 [28]. A device designed by the Medical College of Virginia was used. A small opening was made in the parietal skull contralateral to the cranial window. A metal shaft was sealed into the opening on top of intact dura. This shaft was connected to the 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 injury (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 injury 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.

2. Methods

2.1. Protocol

Newborn (1–5 days old, 1.3–2.1 kg) pigs of either sex were used in these experiments. All protocols were approved by the Institutional Animal Care and Use Committee. Animals were sedated with isoflurane (1–2 MAC). Anesthesia was maintained with a-chloralose (30–50 mg / kg, supplemented with 5 mg / kg / h i.v.). 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 37–39 8C, monitored rectally. 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 mM): 3.0 KCl, 1.5 MgC1 2 , 1.5 CaCI 2 , 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO 3 . This artificial CSF was warmed to 37 8C and

Two types of pial arterial vessels, small arteries (resting diameter, 120–160 mm) and arterioles (resting diameter, 50–70 mm) were examined to determine whether segmental differences in the effects of FPI could be identified. 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. Seven types of experiments were performed (all n56): (1) sham control, (2) FPI, (3) FPI pretreated with chelerythrine, (4) FPI pretreated with SODCAT, (5) active OX, (6) inactive OX, and (7) pertussis toxin treated. In experiments designed to investigate the influence of FPI on vascular responses to G protein activators, mastoparan, mastoparan-7 and the inactive analogue mastoparan-17 (10 28 , 10 26 M) were topically applied before and 60 min after FPI. Chelerythrine (10 27 M) or SODCAT (1000 and 10 000 mg / kg i.v. of polyethylene glycol superoxide dismutase and catalase, respectively) were applied 30 min prior to FPI and response after FPI obtained in the continued administration of topical chelerythrine. Sham control experiments were designed to obtain responses to agonists initially and then again 60 min later. In the active and inactive OX experiments, responses were obtained before and 60 min after exposure to the active or inactive oxygen generating system for 20 min.

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The active oxygen generating system consisted of 0.2 U / ml of xanthine oxidase, 0.6 mM hypoxanthine, and 0.02 mM FeCl 3 administered repeatedly at 5-min intervals over a 20-min period. Piglets treated with the inactive oxygen generating system were initially treated with oxypurinol (50 mg / kg, i.v. 30 min 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 min to inactivate the enzyme. The vehicle for all agents was 0.9% saline, which had no effect on pial artery diameter. In pertussis toxin (PTX) animals, responses were obtained before and after PTX (1 mg / ml). The latter solution was administered topically for 1 h, washed off with artificial CSF and responses obtained 30 min after wash off.

2.2. Statistical analysis Pial artery diameter and systemic artery pressure values were analyzed using ANOVA for repeated measures. If the value was significant, the data were then analyzed by Fishers protected least significant difference test. An a level of P,0.05 was considered significant in all statistical tests. Values are represented as mean6S.E. of the absolute values or as percentage changes from control values.

3. Results

3.1. Influence of FPI on mastoparan induced pial artery dilation: role of PKC activation and O 2 2 generation Mastoparan and mastoparan-7 (10 28 , 10 26 M) elicited reproducible pial small artery (120–160 mm) and arteriole

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(50–70 mm) dilation while mastoparan-17 had no effect on pial artery diameter (14067 vs. 14168 mm for control and mastoparan-17 10 26 M). The onset of the vascular response was within 30 s and the duration of the dilation was 7–10 min, with a peak response observed between 2 and 3 min. Mastoparan and mastoparan-7 induced pial artery dilation was blunted by FPI (Figs. 1 and 2). Mastoparan-17 had no effect on pial artery diameter after FPI similar to that observed prior to injury (Fig. 3). In contrast, in animals pretreated with either the PKC inhibitor chelerythrine (10 27 M) or the O 2 2 free radical scavenger SODCAT, impaired vasodilator responses to mastoparan and mastoparan-7 observed post FPI were partially restored (Figs. 1 and 2). In fact, responses in pial small arteries at 10 28 M were fully restored back to control value, though not for pial arterioles. Chelerythrine and SODCAT had no effect on mastoparan-17 (Fig. 3). Further, the vehicle for these agents (0.9% saline) had no effect on pial artery diameter either before or after FPI or in the presence of chelerythrine or SODCAT (Fig. 3).

3.2. Influence of a xanthine oxidase O 2 2 generating system on pial artery dilation to mastoparan Mastoparan and mastoparan-7 induced pial small artery and arteriole dilation was attenuated after exposure of the cerebral cortical surface to the active oxygen generating system (Fig. 4). Treatment with the inactive oxygen generating system, however, had no effect on pial artery dilation to mastoparan and mastoparan-7. Neither the active nor the inactive system had any effect on mastoparan-17’s inability to influence pial artery diameter. Treatment with the active oxygen generating system

Fig. 1. Influence of mastoparan (10 28 , 10 26 M) on pial small artery and arteriole diameter before (control), after FPI, after FPI in chelerythrine (10 27 M) pretreated animals, and after FPI in SODCAT pretreated animals, n56. *, P,0.05 corresponding vs. control value. 1 , P,0.05 vs. corresponding nontreated FPI value.

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Fig. 2. Influence of mastoparan-7 (10 28 , 10 26 M) on pial small artery and arteriole diameter before (Control), after FPI, after FPI in chelerythrine (10 27 M) pretreated animals, and after FPI in SODCAT pretreated animals, n56. *, P,0.05 vs. corresponding control value. 1 , P,0.05 vs. corresponding nontreated FPI value.

increased pial artery diameter from 14267 to 17368 mm. Such increases in diameter were rapid in onset, peaked at 10 min, but were reversed (returned to control diameter) within 20 min of the end of the exposure. The inactive oxygen generating system had no effect on pial artery diameter.

3.3. Role of PTX in mastoparan induced pial artery dilation Mastoparan and mastoparan-7 induced pial small artery

and arteriole dilation was blocked after exposure of the cerebral cortical surface to PTX (1 mg / ml) (Fig. 5). PTX had no effect on mastoparan-17’s inability to affect pial artery diameter.

3.4. Influence of chelerythrine and SODCAT on pial artery diameter after FPI FPI produced pial small artery and arteriole vasoconstriction within 60 min of the insult (Fig. 6). The mag-

Fig. 3. Influence of mastoparan-17 (10 28 , 10 26 M) and vehicle (0.9% saline) on pial small artery and arteriole diameter before (control), after FPI, after FPI in chelerythrine (10 27 M) pretreated animals, and after FPI in SODCAT pretreated animals, n56.

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Fig. 4. Influence of mastoparan and mastoparan-7 (10 28 , 10 26 M) on pial small artery (SA) and arteriole diameters (A) before (control), after exposure to an activated oxygen-generating system (OX), and after exposure to an inactivated oxygen-generating system (inactive OX), n56. *, P,0.05 compared with corresponding control value.

nitude of such injury induced vasoconstriction was blunted by chelerythrine and SODCAT (Fig. 6).

3.5. Blood chemistry and injury intensity Values for pH, pCO 2 and pO 2 were obtained at the

beginning and end of all experiments. No statistical differences were observed in any of these parameters at the end vs. that observed at the beginning of the experiments. In addition, there were no group differences in any of these parameters. The amplitude of the pressure pulse, used as an index of injury intensity, was 1.960.1 atm.

Fig. 5. Influence of mastoparan and mastoparan-7 (10 28 , 10 26 M) on pial small (SA) and arteriole (A) diameters before (control) and after exposure to PTX (1 mg / ml), n56. *, P,0.05 compared with corresponding control value.

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Fig. 6. Influence of FPI on pial small artery diameter in the absence (control) and presence of chelerythrine (10 27 M) or SODCAT, n56. *, P,0.05 vs. control.

4. Discussion Results of the present study show that mastoparan and mastoparan-7 elicit pial artery dilation that was blunted by FPI. These data indicate that vasodilation induced by G protein activation was impaired by FPI. Because pial small arteries and arterioles were equally affected, these data suggest there are no regional vascular differences in brain injury effects on G protein function. Such interactions appear specific in that the inactive analogue, mastoparan17 [18], had no effect on pial artery diameter before or after FPI, thereby serving as a negative control. However, it should be acknowledged that mastoparan may also interact with other signaling systems and mitochondria. Additional results of the present study show that impaired responses to mastoparan and mastoparan-7 observed after FPI were partially restored in animals pretreated with either chelerythrine or SODCAT. These data indicate the involvement of O 2 in impaired G protein activation 2 mediated vasodilation after FPI. Since chelerythrine similarly afforded such protection of mastoparan induced vasodilation post insult, these data suggest the involvement of PKC activation in O 2 2 mediated impairment of such vasodilation post injury. Earlier observations that chelerythrine blocked O 2 2 generation and pial artery vasoconstriction by phorbol-12,13-dibutyrate, a PKC activator, are supportive of the efficacy and specificity of chelerythrine for inhibition of PKC [2]. Previously, it had been observed that FPI was associated with O 2 2 generation resulting from PKC activation [3]. Such O 2 2 production contributed to impairment of KATP channel induced pial artery dilation following FPI [3]. Since mastoparan has been observed to elicit dilation of the coronary artery via

KATP channel activation [17], taken together, these data therefore suggest that PKC dependent O 22 generation impairment of G protein mediated pial artery dilation contributes to the more distal KATP channel induced dilator impairment after FPI. There appear to be minimal regional vascular differences in the contribution of PKC activation and O 2 2 generation in G protein dilator impairment in that small arteries and arterioles were equally partially restored by chelerythrine and SODCAT except for small arteries at low concentration (10 28 M). Another series of experiments in the present study was designed to strengthen observations related to the role of O2 2 in impaired dilation to mastoparan. These data show that generation of O 2 through an activated oxygen 2 generating system results in blunted dilation to mastoparan and mastoparan-7 similar to that observed after FPI. Results from an earlier study showed that the concentration of agents used to generate O 2 2 in this system elicited the production of an amount of O 2 2 on the cerebral cortical surface similar to that observed 60 min after FPI [3]. This approach, then, biochemically mimicked the conditions of FPI with respect to the amount of O 2 2 generation. The inactive oxygen generating system does not cause the production of O 22 [3] and did not alter dilation to mastoparan or mastoparan-7 in the present study. Such data give specificity to the conclusion that O 22 generation is involved in such impairment. A final series of experiments showed that PTX administration blocked dilation to mastoparan and mastoparan-7. PTX catalyzes the ADP-ribosylation of a cysteine residue located close to the carboxy terminal of G protein subunits. The known PTX-sensitive G proteins are G a1 – 3 , G ao1,2 and G a11,2 [25]. These data, therefore, indicate that dilation to

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mastoparan involves coupling via a PTX sensitive G protein. The concentration of PTX used in the present study is one previously used in the piglet cerebral circulation [30]. Mastoparan is attractive as a cellular probe for the signaling activities of G proteins. The regulation of G proteins by mastoparan and by receptors appears to be similar in many ways [12,15,16]. While many physiologic stimuli, including hypoxia, hypotension and others elicit cerebrovasodilation via activation of KATP channels [4,9,14], it has been previously less well understood exactly how second messengers released in response to these stimuli ultimately resulted in altered K 1 channel activity. Recent data suggest that G proteins may serve to couple receptor simulation to KATP channel activation to result in vasodilation [17]. Although a previous study had observed that phorbol myristate acetate, a PKC activator, inhibited a pertussis toxin sensitive G protein in canine coronary arteries [10], the present data are the first to link PKC activation to O 2 2 generation in impaired G protein mediated cerebrovasodilation. It is speculated that the latter mechanism contributes to impairment of KATP channel mediated dilation after FPI. 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 h after injury [19]. 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 superoxide dismutase and catalase [19]. Oxygen radicals also have been shown to increase blood–brain barrier permeability [29], produce ultrastructural changes in pial vessel endothelium [29], and cause abnormal arteriolar reactivity [21]. 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 reduction in infarct size [13]. Intracellular generation of superoxide or other species could alter structure 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 [26]. The activated oxygen-generating system had been observed in a previous study to result in pial vessels that were ultrastructurally abnormal [21]. Lesions consisted of increased numbers of vascular cytoplasmic inclusions, more numerous surface pits, and mitochondrial injury [21].

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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 [21], observations similar to those obtained with a piglet model of global cerebral ischemia [20,22]. After FPI, cerebral blood flow and cerebral oxygenation are reduced [5], suggesting that ischemia may occur after such injury in the piglet as well. Results of a previous study show that the activated oxygen-generating system produced a reduction of nitroblue tetrazolium (NBT) similar to that observed after FPI, indicating that approximately the same amount of superoxide is generated with either intervention [3]. The inactive oxygen-generating system, however, did not cause the reduction of NBT [3], nor did it alter vascular responses to G protein activators, 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 min 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 alterations appear considerable [21]. 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 pertussis toxin sensitive G protein activation elicits cerebrovasodilation that is blunted following FPI in a PKC dependent manner. These data also show that O 2 2 generation similarly blunts G protein mediated cerebrovasodilation. These data suggest that PKC dependent O 2 2 generation contributes to impaired G protein mediated cerebrovasodilation after FPI.

Acknowledgements The author thanks John Ross for excellent technical assistance in the performance of the experiments. This research was supported by grants from the National Institutes of Health and the PA, DE Affiliate of the AHA.

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