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Protein tyrosine kinase and mitogen-activated protein kinase activation contribute to KATP and Kca channel impairment after brain injury
Brain Research 943 (2002) 276–282 www.elsevier.com / locate / bres
Research report
Protein tyrosine kinase and mitogen-activated protein kinase activation contribute to KATP and K ca channel impairment after brain injury William M. Armstead* Departments of Anesthesia and Pharmacology, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA Accepted 9 January 2002
Abstract Previous studies have observed that pial artery dilation to activators of the ATP sensitive K (KATP ) and calcium sensitive K (K ca ) channel was blunted following fluid percussion brain injury (FPI) in the piglet. In recent studies in the rat, protein tyrosine kinase (PTK) activation was observed to contribute to KATP channel impairment after FPI, but such a role in K ca channel impairment was unclear. This study investigated the role of PTK and mitogen activated protein kinase (MAPK) activation in blunted pial dilation to KATP and K ca channel agonists following FPI in piglets equipped with a closed cranial window. Cromakalim and NS1619 (10 28 , 10 26 M) induced pial artery dilation was blunted after FPI, but partially restored by the PTK inhibitors genistein (10 26 M) and tyrphostin A23 (10 25 M) (1061 and 1961%, sham control; 261 and 461%, FPI; and 761 and 1161% FPI-genistein pretreated for NS1619 10 28 , 10 26 M, respectively). Cromakalim- and NS1619-induced pial dilation was also partially restored after FPI by pretreatment with the MAPK inhibitors U0126 (10 26 M) and PD98059 (10 25 M) (1261 and 2161%, sham control; 261 and 461%, FPI; and 661 and 1062%, FPI-U0126 pretreated for NS1619 10 28 , 10 26 M, respectively). These data suggest that PTK and MAPK activation contribute to KATP and K ca channel impairment following FPI. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Newborn; Cerebral circulation; K 1 channel; Signal transduction
1. Introduction Relaxation of blood vessels can be mediated by several mechanisms, including cGMP, cAMP, and K 1 channels [21]. Membrane potential of vascular muscle is a major determinant of vascular tone, and activity of K 1 channels is a major regulator of membrane potential [27]. Activation or opening of these channels increases K 1 efflux, thereby producing hyperpolarization of vascular muscle. Membrane hyperpolarization closes voltage-dependent calcium channels and thereby causes relaxation of vascular muscle [26,27]. Direct measurements of membrane potential and K 1 current in vitro indicate that several different types of K 1 channels are present in cerebral blood vessels. In addition, a number of pharmacological studies using activators and inhibitors have provided functional evidence *Tel.: 11-215-573-3674; fax: 11-215-349-5078. E-mail address: [email protected] (W.M. Armstead).
that K 1 channels, especially ATP sensitive K 1 (KATP ) and calcium sensitive K 1 (K ca ) channels, regulate tone of cerebral blood vessels in vitro and in vivo [21]. While several recent studies have characterized the role of K 1 channels in cerebrovascular control under physiological conditions, less is known concerning their contributions under pathological conditions. Traumatic brain injury is one of the major causes of morbidity, mortality, and pediatric intensive care unit admissions of children today [29,30]. Although the effects of traumatic brain injury have been well described for adult animal models [14,23,24,31], few have investigated these effects in the newborn. To reproduce some of the biomechanical aspects of closed head injury, fluid percussion brain injury (FPI) has been used in the adults of several species [23,24]. Earlier studies have compared the cerebral hemodynamic effects of FPI in newborn (1–5 days old) and juvenile (3–4 weeks old) pigs. For example, it was observed that pial vessels constricted more, and that
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02703-8
W.M. Armstead / Brain Research 943 (2002) 276 – 282
regional cerebral blood flow decreased and remained depressed longer, in newborns than in juveniles [13]. Activation of protein kinase C (PKC) is thought to contribute to the cerebral vasospasm associated with pathologic conditions such as subarachnoid hemorrhage [22]. Activation of PKC, in turn, promotes interaction with other more distal signaling pathways, such as protein tyrosine kinase (PTK) and its substrate, mitogen-activated protein kinase (MAPK), also thought to contribute to cerebral vasospasm [22]. Previous studies in the piglet have observed a role for PKC activation in the impairment of pial artery dilation to activators of the KATP but not the K ca channel after FPI [3]. More recent studies noted the contribution of PTK activation to KATP channel pial artery dilation impairment following FPI in the adult rat, but such a role in K ca channel impairment was less clear [18]. Additionally, the role of the more distal signal transducer, MAPK, in such impairment following FPI was not considered. Therefore, this study investigated the role of PTK and MAPK activation in blunted pial artery dilation to KATP and K ca channel agonists following FPI in piglets.
2. Methods Newborn (1–5 days old) 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 per 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 (measured rectally). A cranial window was placed 0.5 cm from bregma and the mid sagittal line 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 CaCl 2 , 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO 3 . This artificial CSF was warmed to 37 8C and 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 micro-
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scope, and a video output screen. Vascular diameter was measured with a video microscaler. Methods for brain FPI have been described previously [31]. 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, also 0.5 cm from bregma. 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 insult (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 insult 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.
2.1. Protocol 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. The following eight major types of experiments were carried out: (1) vascular responses to agonists in the absence of FPI (sham control), (2) vascular responses to agonists after FPI, (3) vascular responses to agonists after FPI in genistein-pretreated animals, (4) vascular responses to agonists after FPI in tyrphostin A23-pretreated animals, (5) vascular responses to agonists after FPI in U0126pretreated animals, (6) vascular responses to agonists after FPI in PD98059-pretreated animals (all n56), (7) vascular responses to agonists in sham genistein-pretreated animals, and (8) vascular responses to agonists in sham U0126pretreated animals. In the first series of experiments (sham control), responses were obtained initially and then again 1 h later. These responses included pial artery dilation to the synthetic KATP channel agonist (2)cromakalim (10 28 , 10 26 M) (Smith Kline Beecham), the endogenous KATP channel activator calcitonin gene related peptide (CGRP, 10 28 , 10 26 M) and the synthetic K ca channel activator NS1619 (10 28 , 10 26 M) (both Sigma). In the FPI experiments, responses of arterial vessels to cromakalim, CGRP, and
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NS1619 were obtained before and 1 h after FPI in the absence and presence of pretreatment 30 min prior to injury with genistein (10 26 M), tyrphostin A23 (10 25 M), U0126 (10 26 M), or PD98059 (10 25 M). In the last two series of experiments, responses to agonists were obtained before and after administration of genistein or U0126 in the absence of FPI. Each of the drugs were 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 changes 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.
2.2. Statistical analysis Pial artery diameter and systemic arterial blood pressure values were analyzed using ANOVA for repeated measures. If the value was significant, the data were then analyzed by Fisher’s protected least significant difference test. An a -level of P,0.05 was considered significant in all statistical tests. Values are represented as means6S.E. of the absolute values or percent changes from control values.
3764, and 9666 mmHg versus 7.4560.03, 3665, and 8767 mmHg for pH, PCO 2 , and PO 2 , respectively before and after injury. Administration of PTK or MAPK inhibitors did not significantly affect blood chemistry values. The amplitude of the pressure pulse used as an index of injury intensity was 2.160.1 atm.
3.2. Role of PTK activation in impaired KATP and Kca channel agonist-induced pial artery dilation following FPI Cromakalim, CGRP, and NS1619 (10 28 , 10 26 M) elicited reproducible pial small artery (120–160 mm) and arteriole (50–70 mm) vasodilation (data not shown). Pial small artery dilation to all three K 1 channel agonists was blunted within 1 h post FPI (Figs. 1 and 2). In animals pretreated with PTK inhibitors genistein (10 26 M) or tyrphostin A23 (10 25 M), such impaired vasodilation was partially prevented, though responses were still attenuated compared to control (Figs. 1 and 2). Similar effects were observed in pial arterioles. Genistein had no effect on vascular responses to K 1 channel agonists in sham control animals (1361 and 2461 vs. 1461 and 2562% for cromakalim in the absence and presence of genistein, respectively).
3.3. Role of MAPK activation in impaired KATP and Kca channel agonist-induced pial artery dilation following FPI
3. Results
3.1. Blood chemistry and intensity of injury Blood chemistry values were obtained at the beginning and end of all experiments. These values were 7.4660.03,
Pial small artery dilation to cromakalim, CGRP, and NS1619 was blunted following FPI, as described above (Figs. 1–4). In animals pretreated with the MAPK inhibitors U0126 (10 26 M) or PD98059 (10 25 M), however, such impaired vasodilation was partially prevented, though
Fig. 1. Influence of cromakalim and CGRP (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in genistein (10 26 M) pretreated animals, and after FPI in tyrphostin A23 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of genistein or tyrphostin A23.
W.M. Armstead / Brain Research 943 (2002) 276 – 282
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Fig. 2. Influence of NS1619 (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in genistein (10 26 M) pretreated animals, and after FPI in tyrphostin A23 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of genistein or tyrphostin A23.
responses were still attenuated compared to control (Figs. 3 and 4). Similar effects were observed in pial arterioles. U0126 had no effect on vascular responses to K 1 channel agonists in sham control animals.
inhibitors U0126 and PD98059 also had no significant effect on pial artery diameter.
4. Discussion
3.4. Influence of PTK and MAPK inhibitors on pial artery diameter The PTK inhibitors genistein and tyrphostin had minimal effects on pial small artery diameter (11866 vs. 12066 mm for genistein, n56). Similarly, the MAPK
Results of the present study show that pial artery dilation induced by activators of the KATP and K ca channel was blunted within 1 h of FPI, consistent with previous piglet studies [1–4,6,8,9,19]. New data show that pretreatment with the PTK inhibitors genistein or tyrphostin A23
Fig. 3. Influence of cromakalim and CGRP (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in U0126 (10 26 M) pretreated animals, and after FPI in PD98059 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of U0126 or PD98059.
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W.M. Armstead / Brain Research 943 (2002) 276 – 282
Fig. 4. Influence of NS1619 (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in U0126 (10 26 M) pretreated animals, and after FPI in PD98059 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of U0126 or PD98059.
partially protected dilation to cromakalim and CGRP following FPI. Because pial arteries are innervated by CGRP containing nerve fibers [15], inclusion of such data lends physiologic functional perspective to this study. These data confirm those observed in the adult rat in which genistein pretreatment partially protected dilation to cromakalim and CGRP following FPI [18]. Data from the present study strengthen conclusions for the role of PTK activation in such K 1 channel-induced dilator impairment in that a second structurally unrelated inhibitor of this pathway yielded similar results. However, the above study in the adult rat [18] did not rigorously address the concept that both KATP and K ca channel impairment post FPI might result from PTK activation. Specifically, use of a selective K ca channel agonist was not employed. Instead, because these authors observed that the CGRP-induced dilation restored by genistein was blunted by the K ca channel antagonist iberiotoxin but unchanged by glibenclamide following FPI, it was concluded that both subtypes of K 1 channel were impaired by PTK activation following FPI [18]. However, these data contradict that of Kitazono et al. [21] who observed that CGRP elicited dilation of the rat basilar artery via KATP channel activation. Additionally, these data are inconsistent with those observed in the piglet where CGRP similarly elicited pial dilation via KATP but not K ca channel activation [9,11]. A potential explanation for the above divergent conclusions regarding the K 1 channel subtype which mediates dilation to CGRP could relate to a change induced by brain injury in the relative importance of subtype involvement in mediation of vascular activity. Such an explanation is limited by the observation that FPI did not change such K 1 channel coupling to CGRP in the piglet [3]. While species, age, and / or regional vascular differences could also have contributed, the
salient point of all of the above discussion is that while PTK activation has a role in KATP channel impairment following FPI, such a role in K ca channel impairment was less clear. Therefore, experiments in the present study were designed to investigate the contribution of PTK to impaired K ca channel mediated dilation following FPI through the use of the synthetic selective channel agonist, NS1619. Results of these studies show that genistein and tyrphostin A23 pretreatment partially protected responses to NS1619 post FPI. Considering that NS1619-induced pial artery dilation has been observed to be blocked by the K ca channel antagonist iberiotoxin but unchanged by the KATP antagonist glibenclamide in the piglet both before and after FPI [3,8,10], these data more clearly indicate that PTK activation contributes to K ca channel impairment after FPI. However, it has also been observed that NS1619 may additionally possess calcium channel antagonistic activity and, therefore, may not be useful as a probe for K ca channel activation [17]. In contrast, recent observations in the piglet show that vasoconstrictor responses to the calcium channel agonist Bay K8644 were unchanged in the presence of NS1619 [10]. These results suggest that NS1619 has no calcium channel blocking activity and, therefore, may be considered to be selective for activation of K ca channels in the newborn pig. A final series of experiments was designed to characterize the role of MAPK activation in KATP and K ca channel impairment after FPI. PTK is a substrate for MAPK [22]. Results of the present study show that pretreatment with U0126 and PD98059, MAPK inhibitors [16], partially protected vascular responses to cromakalim, CGRP, and NS1619 following FPI. These results are the first to show a role for MAPK activation in impaired KATP or K ca channel
W.M. Armstead / Brain Research 943 (2002) 276 – 282
function following FPI. Mechanistically in this scenario, then, endothelin and vasopressin released into CSF following FPI [5,12,19], would activate PKC [3,6] to impair KATP channel-mediated pial artery dilation [3,19] via sequential activation of PTK and MAPK. However, the mechanism for K ca channel impairment following FPI remains somewhat less clear in that PKC activation, which subsequently activates PTK, does not contribute to K ca channel impairment following FPI in the piglet [3]. Since cyclooxygenase activation does contribute to K ca channel impairment after FPI [1,2], these data suggest that an as yet unidentified mechanism links cyclooxygenase to PTK and MAPK activation. Interestingly, PTK activation has been observed to subserve a permissive role in the action of at least one cyclooxygenase metabolism product [28]. The choice of concentration for the PTK and MAPK probes used in this study was based on in vitro selectivity assay data [16,20,25]. Such probes had no significant effect on baseline pial artery diameter indicating little role for PTK and MAPK activation in the tonic tone of the piglet pial vasculature. Previous studies have observed that KATP and K ca channel-induced pial artery dilation was impaired to a greater extent and for a longer period of time in newborn versus juvenile pigs [4]. The functional significance of such impairment has been observed in the age-related impairment of hypotensive cerebrovascular autoregulation [5], a KATP and K ca channel-dependent dilator stimulus [7], following FPI. Interestingly, cerebrovascular dysregulation during hypotension following FPI in the rat was partially protected by genistein pretreatment [18]. The relative agedependent contribution of PTK and MAPK activation to impairment of such a physiologic stimulus post FPI is uncertain. In conclusion, therefore, results of the present study show that PTK and MAPK activation contribute to KATP and K ca channel impairment following FPI.
[4] [5]
[6]
[7] [8]
[9] [10]
[11]
[12]
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[16]
[17]
[18]
Acknowledgements
[19]
The author thanks John Ross for 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.
[20]
[21]
[22]
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Research report
Protein tyrosine kinase and mitogen-activated protein kinase activation contribute to KATP and K ca channel impairment after brain injury William M. Armstead* Departments of Anesthesia and Pharmacology, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA Accepted 9 January 2002
Abstract Previous studies have observed that pial artery dilation to activators of the ATP sensitive K (KATP ) and calcium sensitive K (K ca ) channel was blunted following fluid percussion brain injury (FPI) in the piglet. In recent studies in the rat, protein tyrosine kinase (PTK) activation was observed to contribute to KATP channel impairment after FPI, but such a role in K ca channel impairment was unclear. This study investigated the role of PTK and mitogen activated protein kinase (MAPK) activation in blunted pial dilation to KATP and K ca channel agonists following FPI in piglets equipped with a closed cranial window. Cromakalim and NS1619 (10 28 , 10 26 M) induced pial artery dilation was blunted after FPI, but partially restored by the PTK inhibitors genistein (10 26 M) and tyrphostin A23 (10 25 M) (1061 and 1961%, sham control; 261 and 461%, FPI; and 761 and 1161% FPI-genistein pretreated for NS1619 10 28 , 10 26 M, respectively). Cromakalim- and NS1619-induced pial dilation was also partially restored after FPI by pretreatment with the MAPK inhibitors U0126 (10 26 M) and PD98059 (10 25 M) (1261 and 2161%, sham control; 261 and 461%, FPI; and 661 and 1062%, FPI-U0126 pretreated for NS1619 10 28 , 10 26 M, respectively). These data suggest that PTK and MAPK activation contribute to KATP and K ca channel impairment following FPI. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Newborn; Cerebral circulation; K 1 channel; Signal transduction
1. Introduction Relaxation of blood vessels can be mediated by several mechanisms, including cGMP, cAMP, and K 1 channels [21]. Membrane potential of vascular muscle is a major determinant of vascular tone, and activity of K 1 channels is a major regulator of membrane potential [27]. Activation or opening of these channels increases K 1 efflux, thereby producing hyperpolarization of vascular muscle. Membrane hyperpolarization closes voltage-dependent calcium channels and thereby causes relaxation of vascular muscle [26,27]. Direct measurements of membrane potential and K 1 current in vitro indicate that several different types of K 1 channels are present in cerebral blood vessels. In addition, a number of pharmacological studies using activators and inhibitors have provided functional evidence *Tel.: 11-215-573-3674; fax: 11-215-349-5078. E-mail address: [email protected] (W.M. Armstead).
that K 1 channels, especially ATP sensitive K 1 (KATP ) and calcium sensitive K 1 (K ca ) channels, regulate tone of cerebral blood vessels in vitro and in vivo [21]. While several recent studies have characterized the role of K 1 channels in cerebrovascular control under physiological conditions, less is known concerning their contributions under pathological conditions. Traumatic brain injury is one of the major causes of morbidity, mortality, and pediatric intensive care unit admissions of children today [29,30]. Although the effects of traumatic brain injury have been well described for adult animal models [14,23,24,31], few have investigated these effects in the newborn. To reproduce some of the biomechanical aspects of closed head injury, fluid percussion brain injury (FPI) has been used in the adults of several species [23,24]. Earlier studies have compared the cerebral hemodynamic effects of FPI in newborn (1–5 days old) and juvenile (3–4 weeks old) pigs. For example, it was observed that pial vessels constricted more, and that
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02703-8
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regional cerebral blood flow decreased and remained depressed longer, in newborns than in juveniles [13]. Activation of protein kinase C (PKC) is thought to contribute to the cerebral vasospasm associated with pathologic conditions such as subarachnoid hemorrhage [22]. Activation of PKC, in turn, promotes interaction with other more distal signaling pathways, such as protein tyrosine kinase (PTK) and its substrate, mitogen-activated protein kinase (MAPK), also thought to contribute to cerebral vasospasm [22]. Previous studies in the piglet have observed a role for PKC activation in the impairment of pial artery dilation to activators of the KATP but not the K ca channel after FPI [3]. More recent studies noted the contribution of PTK activation to KATP channel pial artery dilation impairment following FPI in the adult rat, but such a role in K ca channel impairment was less clear [18]. Additionally, the role of the more distal signal transducer, MAPK, in such impairment following FPI was not considered. Therefore, this study investigated the role of PTK and MAPK activation in blunted pial artery dilation to KATP and K ca channel agonists following FPI in piglets.
2. Methods Newborn (1–5 days old) 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 per 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 (measured rectally). A cranial window was placed 0.5 cm from bregma and the mid sagittal line 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 CaCl 2 , 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO 3 . This artificial CSF was warmed to 37 8C and 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 micro-
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scope, and a video output screen. Vascular diameter was measured with a video microscaler. Methods for brain FPI have been described previously [31]. 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, also 0.5 cm from bregma. 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 insult (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 insult 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.
2.1. Protocol 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. The following eight major types of experiments were carried out: (1) vascular responses to agonists in the absence of FPI (sham control), (2) vascular responses to agonists after FPI, (3) vascular responses to agonists after FPI in genistein-pretreated animals, (4) vascular responses to agonists after FPI in tyrphostin A23-pretreated animals, (5) vascular responses to agonists after FPI in U0126pretreated animals, (6) vascular responses to agonists after FPI in PD98059-pretreated animals (all n56), (7) vascular responses to agonists in sham genistein-pretreated animals, and (8) vascular responses to agonists in sham U0126pretreated animals. In the first series of experiments (sham control), responses were obtained initially and then again 1 h later. These responses included pial artery dilation to the synthetic KATP channel agonist (2)cromakalim (10 28 , 10 26 M) (Smith Kline Beecham), the endogenous KATP channel activator calcitonin gene related peptide (CGRP, 10 28 , 10 26 M) and the synthetic K ca channel activator NS1619 (10 28 , 10 26 M) (both Sigma). In the FPI experiments, responses of arterial vessels to cromakalim, CGRP, and
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NS1619 were obtained before and 1 h after FPI in the absence and presence of pretreatment 30 min prior to injury with genistein (10 26 M), tyrphostin A23 (10 25 M), U0126 (10 26 M), or PD98059 (10 25 M). In the last two series of experiments, responses to agonists were obtained before and after administration of genistein or U0126 in the absence of FPI. Each of the drugs were 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 changes 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.
2.2. Statistical analysis Pial artery diameter and systemic arterial blood pressure values were analyzed using ANOVA for repeated measures. If the value was significant, the data were then analyzed by Fisher’s protected least significant difference test. An a -level of P,0.05 was considered significant in all statistical tests. Values are represented as means6S.E. of the absolute values or percent changes from control values.
3764, and 9666 mmHg versus 7.4560.03, 3665, and 8767 mmHg for pH, PCO 2 , and PO 2 , respectively before and after injury. Administration of PTK or MAPK inhibitors did not significantly affect blood chemistry values. The amplitude of the pressure pulse used as an index of injury intensity was 2.160.1 atm.
3.2. Role of PTK activation in impaired KATP and Kca channel agonist-induced pial artery dilation following FPI Cromakalim, CGRP, and NS1619 (10 28 , 10 26 M) elicited reproducible pial small artery (120–160 mm) and arteriole (50–70 mm) vasodilation (data not shown). Pial small artery dilation to all three K 1 channel agonists was blunted within 1 h post FPI (Figs. 1 and 2). In animals pretreated with PTK inhibitors genistein (10 26 M) or tyrphostin A23 (10 25 M), such impaired vasodilation was partially prevented, though responses were still attenuated compared to control (Figs. 1 and 2). Similar effects were observed in pial arterioles. Genistein had no effect on vascular responses to K 1 channel agonists in sham control animals (1361 and 2461 vs. 1461 and 2562% for cromakalim in the absence and presence of genistein, respectively).
3.3. Role of MAPK activation in impaired KATP and Kca channel agonist-induced pial artery dilation following FPI
3. Results
3.1. Blood chemistry and intensity of injury Blood chemistry values were obtained at the beginning and end of all experiments. These values were 7.4660.03,
Pial small artery dilation to cromakalim, CGRP, and NS1619 was blunted following FPI, as described above (Figs. 1–4). In animals pretreated with the MAPK inhibitors U0126 (10 26 M) or PD98059 (10 25 M), however, such impaired vasodilation was partially prevented, though
Fig. 1. Influence of cromakalim and CGRP (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in genistein (10 26 M) pretreated animals, and after FPI in tyrphostin A23 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of genistein or tyrphostin A23.
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Fig. 2. Influence of NS1619 (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in genistein (10 26 M) pretreated animals, and after FPI in tyrphostin A23 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of genistein or tyrphostin A23.
responses were still attenuated compared to control (Figs. 3 and 4). Similar effects were observed in pial arterioles. U0126 had no effect on vascular responses to K 1 channel agonists in sham control animals.
inhibitors U0126 and PD98059 also had no significant effect on pial artery diameter.
4. Discussion
3.4. Influence of PTK and MAPK inhibitors on pial artery diameter The PTK inhibitors genistein and tyrphostin had minimal effects on pial small artery diameter (11866 vs. 12066 mm for genistein, n56). Similarly, the MAPK
Results of the present study show that pial artery dilation induced by activators of the KATP and K ca channel was blunted within 1 h of FPI, consistent with previous piglet studies [1–4,6,8,9,19]. New data show that pretreatment with the PTK inhibitors genistein or tyrphostin A23
Fig. 3. Influence of cromakalim and CGRP (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in U0126 (10 26 M) pretreated animals, and after FPI in PD98059 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of U0126 or PD98059.
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Fig. 4. Influence of NS1619 (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in U0126 (10 26 M) pretreated animals, and after FPI in PD98059 (10 25 M) pretreated animals, n56. *P,0.05 versus control, 1 P,0.05 versus absence of U0126 or PD98059.
partially protected dilation to cromakalim and CGRP following FPI. Because pial arteries are innervated by CGRP containing nerve fibers [15], inclusion of such data lends physiologic functional perspective to this study. These data confirm those observed in the adult rat in which genistein pretreatment partially protected dilation to cromakalim and CGRP following FPI [18]. Data from the present study strengthen conclusions for the role of PTK activation in such K 1 channel-induced dilator impairment in that a second structurally unrelated inhibitor of this pathway yielded similar results. However, the above study in the adult rat [18] did not rigorously address the concept that both KATP and K ca channel impairment post FPI might result from PTK activation. Specifically, use of a selective K ca channel agonist was not employed. Instead, because these authors observed that the CGRP-induced dilation restored by genistein was blunted by the K ca channel antagonist iberiotoxin but unchanged by glibenclamide following FPI, it was concluded that both subtypes of K 1 channel were impaired by PTK activation following FPI [18]. However, these data contradict that of Kitazono et al. [21] who observed that CGRP elicited dilation of the rat basilar artery via KATP channel activation. Additionally, these data are inconsistent with those observed in the piglet where CGRP similarly elicited pial dilation via KATP but not K ca channel activation [9,11]. A potential explanation for the above divergent conclusions regarding the K 1 channel subtype which mediates dilation to CGRP could relate to a change induced by brain injury in the relative importance of subtype involvement in mediation of vascular activity. Such an explanation is limited by the observation that FPI did not change such K 1 channel coupling to CGRP in the piglet [3]. While species, age, and / or regional vascular differences could also have contributed, the
salient point of all of the above discussion is that while PTK activation has a role in KATP channel impairment following FPI, such a role in K ca channel impairment was less clear. Therefore, experiments in the present study were designed to investigate the contribution of PTK to impaired K ca channel mediated dilation following FPI through the use of the synthetic selective channel agonist, NS1619. Results of these studies show that genistein and tyrphostin A23 pretreatment partially protected responses to NS1619 post FPI. Considering that NS1619-induced pial artery dilation has been observed to be blocked by the K ca channel antagonist iberiotoxin but unchanged by the KATP antagonist glibenclamide in the piglet both before and after FPI [3,8,10], these data more clearly indicate that PTK activation contributes to K ca channel impairment after FPI. However, it has also been observed that NS1619 may additionally possess calcium channel antagonistic activity and, therefore, may not be useful as a probe for K ca channel activation [17]. In contrast, recent observations in the piglet show that vasoconstrictor responses to the calcium channel agonist Bay K8644 were unchanged in the presence of NS1619 [10]. These results suggest that NS1619 has no calcium channel blocking activity and, therefore, may be considered to be selective for activation of K ca channels in the newborn pig. A final series of experiments was designed to characterize the role of MAPK activation in KATP and K ca channel impairment after FPI. PTK is a substrate for MAPK [22]. Results of the present study show that pretreatment with U0126 and PD98059, MAPK inhibitors [16], partially protected vascular responses to cromakalim, CGRP, and NS1619 following FPI. These results are the first to show a role for MAPK activation in impaired KATP or K ca channel
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function following FPI. Mechanistically in this scenario, then, endothelin and vasopressin released into CSF following FPI [5,12,19], would activate PKC [3,6] to impair KATP channel-mediated pial artery dilation [3,19] via sequential activation of PTK and MAPK. However, the mechanism for K ca channel impairment following FPI remains somewhat less clear in that PKC activation, which subsequently activates PTK, does not contribute to K ca channel impairment following FPI in the piglet [3]. Since cyclooxygenase activation does contribute to K ca channel impairment after FPI [1,2], these data suggest that an as yet unidentified mechanism links cyclooxygenase to PTK and MAPK activation. Interestingly, PTK activation has been observed to subserve a permissive role in the action of at least one cyclooxygenase metabolism product [28]. The choice of concentration for the PTK and MAPK probes used in this study was based on in vitro selectivity assay data [16,20,25]. Such probes had no significant effect on baseline pial artery diameter indicating little role for PTK and MAPK activation in the tonic tone of the piglet pial vasculature. Previous studies have observed that KATP and K ca channel-induced pial artery dilation was impaired to a greater extent and for a longer period of time in newborn versus juvenile pigs [4]. The functional significance of such impairment has been observed in the age-related impairment of hypotensive cerebrovascular autoregulation [5], a KATP and K ca channel-dependent dilator stimulus [7], following FPI. Interestingly, cerebrovascular dysregulation during hypotension following FPI in the rat was partially protected by genistein pretreatment [18]. The relative agedependent contribution of PTK and MAPK activation to impairment of such a physiologic stimulus post FPI is uncertain. In conclusion, therefore, results of the present study show that PTK and MAPK activation contribute to KATP and K ca channel impairment following FPI.
[4] [5]
[6]
[7] [8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements
[19]
The author thanks John Ross for 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.
[20]
[21]
[22]
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