Vasopressin induced cyclooxygenase dependent superoxide generation contributes to K+ channel function impairment after brain injury

Brain Research 910 (2001) 19–28 www.elsevier.com / locate / bres

Research report

Vasopressin induced cyclooxygenase dependent superoxide generation contributes to K 1 channel function impairment after brain injury William M. Armstead* Departments of Anesthesia and Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA Accepted 12 June 2001

Abstract This study determined if vasopressin generates superoxide anion (O 2 2 ) in a cyclooxygenase dependent manner and if such production contributes to impairment of dilation to activators of ATP sensitive K 1 (KATP ) and calcium sensitive K 1 (K ca ) channels following fluid percussion brain injury (FPI) in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD) inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O 2 2 generation. Under non-brain injury conditions, topical vasopressin (40 pg / ml, the concentration present in CSF following FPI) increased SOD inhibitable NBT reduction from 161 to 2564 pmol / mm 2 . Indomethacin, a cyclooxygenase inhibitor, blunted such NBT reduction (161 to 561 pmol / mm 2 ), while the vasopressin antagonist, l-(b-mercapto-b b-cyclopentamethylene propionic acid) 2-(o-methyl)-Tyr-AVP (MEAVP) blocked NBT reduction. MEAVP and indomethacin also blunted the NBT reduction observed after FPI. Under non-brain injury conditions, vasopressin (40 pg / ml) coadministered with the KATP and K ca channel agonists, cromakalim and NS1619 (10 28 , 10 26 M) diminished dilation to these K 1 channel agonists while indomethacin partially prevented such impairment (1361 and 2361 vs. 461 and 1062 vs. 861 and 1961% for cromakalim in untreated, vasopressin, and vasopressin plus indomethacin treated piglets, respectively). Cromakalim and NS1619 induced pial artery dilation was attenuated following FPI, while indomethacin or MEAVP preadministration partially prevented such impairment (1361 and 2361, sham control; 161 and 461, FPI; 861 and 1663%, FPI–indomethacin pretreated for responses to cromakalim 10 28 , 10 26 M, respectively). These data show that vasopressin increased O 22 production in a cyclooxygenase dependent manner and contributed to this production after FPI. These data also show that vasopressin blunted KATP and K ca channel mediated cerebrovasodilation in a cyclooxygenase dependent manner. These data suggest that vasopressin induced cyclooxygenase dependent O 22 generation contributes to KATP and K ca channel function impairment after FPI.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Newborn; Cerebral circulation; Oxygen free radical; KATP and K ca channel

1. Introduction Relaxation of blood vessels can be mediated by several mechanisms, including cGMP, cAMP, and K 1 channels [20]. 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 [28]. Activation

*Present address: Department of Anesthesia, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA. Tel.: 11-215573-3674; fax: 11-215-349-5078. E-mail address: [email protected] (W.M. Armstead).

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 [27,28]. 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 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 [20]. While several recent studies have characterized the role of K 1 channels in cerebrovascular control under physiological

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02716-0

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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 [29,33]. Although the effects of traumatic brain injury have been well described for adult animal models [15,25,26,37], 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 [25,26]. 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 regional cerebral blood flow decreased and remained depressed longer, in newborns compared to juveniles [10]. Vasopressin contributes to the regulation of cerebral hemodynamics in the piglet [11–13]. Vasopressin is released into CSF by FPI and also contributes to impaired pial artery dilation to opioids such as dynorphin following brain injury in the piglet [9]. Dynorphin elicits pial vasodilation via activation of KATP and K ca channels [6,32]. Since KATP and K ca channel function is impaired after FPI [4,5], altered dilation to this opioid could relate to such impaired K 1 channel function. Interestingly, vasopressin has been observed to block the KATP channel in porcine coronary artery smooth muscle cells [34]. However, vasopressin did not appear to have any direct effect on the K ca channel in the above studies [34]. More recent studies, though, have shown that vasopressin does modulate both KATP and K ca channel agonist induced pial artery dilation and contributes to impairment of both K 1 channel subtypes following FPI in the newborn pig [1,31]. However, the mechanism whereby vasopressin might contribute to KATP and K ca channel function impairment following FPI is uncertain. For example, while activation of protein kinase C (PKC) by vasopressin has been observed to generate superoxide anion (O 2 2 ), which, in turn, contributes to KATP channel function impairment following FPI [1,3], such a role for this mechanism has not been similarly observed for K ca channel impairment following this insult [1]. While cyclooxygenase activation has been observed to contribute to O 2 2 generation following FPI [21], the role of such a mechanism in vasopressin induced impairment of KATP and K ca channel mediated vasodilation following FPI is uncertain. Therefore, this study was designed to characterize mechanisms involved in impaired cerebrovascular control contributory to secondary ischemia following traumatic brain injury. Specifically, this study was designed to determine whether vasopressin generates O 2 2 in a cyclooxygenase dependent manner, which could link vasopressin release to impaired KATP and K ca channel induced pial artery dilation after FPI.

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 hour, 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 37–398C. 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 378C and had the following chemistry: pH 7.33, pCO 2 46 mmHg, and pPO 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 [37]. 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 photoelectri-

W.M. Armstead / Brain Research 910 (2001) 19 – 28

cally 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 14 major types of experiments were performed: (1) generation of O 2 2 with vasopressin (n57); (2) generation of O 2 2 with vasopressin in the presence of indomethacin, a cyclooxygenase inhibitor (n57); (3) generation of O 2 2 with vasopressin in the presence of l-(bmercapto-b b-cyclopentamethylene propionic acid) 2-(omethyl)-Tyr-AVP (MEAVP), a vasopressin receptor antagonist (n57); (4) generation of O 2 2 with FPI (n57); (5) generation of O 22 with FPI in indomethacin pretreated animals (n57); (6) generation of O 2 2 with FPI in MEAVP pretreated animals (n57); (7) vascular responses to agonists in non-injured animals (n56); (8) vascular responses to agonists coadministered with vasopressin (n56); (9) vascular responses to agonists in indomethacin pretreated animals (n56); (10) vascular responses to agonists in MEAVP pretreated animals (n56); (11) vascular responses to agonists in the absence of FPI (sham control) (n56); (12) vascular responses to agonists after FPI (n56); (13) vascular responseS to agonists after FPI in indomethacin pretreated animals (n56); and (14) vascular responses to agonists after FPI in MEAVP pretreated animals (n56). In the first three series of experiments designed to investigate the generation of O 2 2 , lysine vasopressin, the vasopressin isoform present in the pig (40 mg / ml, Sigma), was applied to the cerebral cortex for 20 min in either the absence or presence of indomethacin (5 mg / kg, i.v.) or MEAVP (5 mg / kg, i.v.) (both Sigma). In the next three series of experiments, generation of O 2 2 1 h after FPI was investigated in the absence and presence of indomethacin or MEAVP. Because the technique for measurement of O 2 2 generation (see below) involves placement of detection solutions on the cerebral cortex for 20 min, such measurement in fact reflects O 2 2 generation during the first 20-min period 1 h after FPI. In the vasopressin experiments, the responses of arterial vessels to the synthetic KATP channel agonist (2) cromakalim (10 28 , 10 26 M, SmithKline 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) were obtained in the absence of vasopressin, in the presence of vasopressin (40 pg / ml, the concentration

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observed in CSF after FPI), in the presence of vasopressin and indomethacin, and in the presence of vasopressin and MEAVP. Because vasopressin is a vasodilator, U46619 (0.3 ng / ml), a vasoconstrictor, was coadministered with vasopressin to make certain that pial artery diameter was equivalent in the absence and presence of vasopressin. In the FPI experiments, responses of arterial vessels to cromakalim, CGRP, and NS1619 were obtained before and 60 min after brain injury in the absence and presence of pretreatment 30 min prior to injury with MEAVP (5 mg / kg, i.v.) or indomethacin (5 mg / kg, i.v.). Sham control experiments were designed such that responses were obtained initially and then again 60 min later. Each of the drugs was applied in an ascending concentration manner. There was a period of 20 min after the highest concentration of one drug was washed off before a different drug was infused. The percent change in artery diameter values was 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. O 2 2 analysis SOD-inhibitable NBT reduction was determined as an index of O 2 generation, as previously described 2 [2,3,19,21]. 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 h after FPI. Because such solutions remained on the surface for 20 min, data are quantified as pmol NBT reduced for 20 min. 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, e.g. O 2 2 . The SOD-inhibitable NBT reduction was determined by the difference in the quantities of nitroblue formazan precipitated on the brain surface under the two 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 1 N NaOH and 0.1% sodium dodecyl sulfate solution. The supernatant was discarded, and the pellet was resuspended in 3 ml of pyridine. The formazan was dissolved in the pyridine during heating at 808C for 1 h. Particulate matter was removed by a second centrifugation at 10,0003g for 10 min. 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

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used with each set of samples and treated identically to the samples.

3.2. Role of cyclooxygenase activation in vasopressin modulation of KATP and Kca channel agonist induced pial artery dilation under non-injury conditions

2.3. Statistical analysis Pial arteriolar diameter, systemic arterial pressure, and amount of NBT reduced 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 alpha 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.

3. Results

3.1. Role of cyclooxygenase activation in vasopressin induced O 2 2 generation during non-brain injury conditions Topical vasopressin (40 pg / ml, the concentration present in CSF after FPI) to the cerebral cortical surface of non-brain injured animals increased SOD-inhibitable NBT reduction (Fig. 1A). Such NBT reduction by vasopressin was blunted by indomethacin (5 mg / kg, i.v.) and blocked by the vasopressin antagonist MEAVP (5 mg / kg, i.v.) (Fig. 1A). Under brain injury conditions, SOD inhibitable NBT reduction was increased 1 h after FPI (Fig. 1B). Such enhanced NBT reduction after FPI was blunted by both indomethacin and MEAVP (Fig. 1B).

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). Vasopressin (40 pg / ml) increased pial small artery diameter from 14166 to 15967 mm, n56. During coadministration of U46619 (0.3 ng / ml) with vasopressin (40 pg / ml), there was no net change in pial artery diameter (142610 vs. 151610 mm). Under such conditions of equivalent baseline diameter, vasopressin / U46619 coadministered with cromakalim, CGRP or NS1619, attenuated pial small artery dilation to these K 1 channel agonists (Figs. 2 and 3). Attenuated responses were partially restored when these agonists were coadministered with vasopressin in indomethacin pretreated animals (Figs. 2 and 3). However, in MEAVP pretreated animals, vasopressin coadministered with cromakalim, CGRP, or NS1619 did not attenuate vascular responses to the latter agonists (Figs. 2 and 3). Similar effects were observed in pial arterioles.

3.3. Role of cyclooxygenase activation in vasopressin impairment of KATP and Kca channel agonist induced pial artery dilation following FPI Cromakalim, CGRP, and NS1619 induced pial small artery dilation was attenuated within 1 h post FPI (Figs. 4 and 5). In animals pretreated with the vasopressin antagonist MEAVP or the cyclooxygenase inhibitor in-

Fig. 1. (A) Determination of SOD-inhibitable NBT reduction in newborn pig brain before (control) and after topical vasopressin (LVP, 40 pg / ml), as well as after indomethacin (5 mg / kg, i.v., Indo) or MEAVP (5 mg / kg, i.v.). (B) Determination of SOD-inhibitable NBT reduction in newborn pig brain before (control) and after FPI, as well as after FPI plus indomethacin (Indo) or FPI plus MEAVP, n57. *P,0.05 versus control; 1 P,0.05 versus absence of indomethacin or MEAVP.

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Fig. 2. Influence of coadministered vasopressin (40 pg / ml), vasopressin and the cyclooxygenase inhibitor indomethacin (5 mg / kg, i.v.), or vasopressin and the vasopressin antagonist MEAVP (5 mg / kg, i.v.) on pial small artery dilation to cromakalim and CGRP (10 28 , 10 26 M) under conditions of equivalent baseline diameter, n56. *P,0.05 versus control; 1 P,0.05 versus absence of indomethacin.

domethacin, such impaired vasodilation was partially prevented, though responses were still attenuated compared to control (Figs. 4 and 5). Similar effects were observed in pial arterioles.

3.4. Confirmation of effective receptor blockade Vascular responses to vasopressin were blocked by MEAVP (5 mg / kg, i.v.) (1061 vs. 161% for vasopressin

Fig. 3. Influence of coadministered vasopressin (40 pg / ml), vasopressin and the cyclooxygenase inhibitor indomethacin (5 mg / kg, i.v.) or vasopressin and the vasopressin antagonist MEAVP (5 mg / kg, i.v.) on pial small artery dilation to NS1619 (10 28 , 10 26 M) under conditions of equivalent baseline diameter, n56. *P,0.05 versus control; 1 P,0.05 versus absence of indomethacin.

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Fig. 4. Influence of cromakalim and CGRP (10 28 , 10 26 M) or pial small artery diameter before (control), after FPI, after FPI in indomethacin (5 mg / kg, i.v.) pretreated animals, and after FPI in MEAVP (5 mg / kg, i.v.) pretreated animals, n56. *P,0.05 versus control; 1 P,0.05 versus absence of indomethacin or MEAVP.

40 pg / ml before and after MEAVP, respectively, n56). MEAVP did not have any significant effect on pial artery diameter.

3.5. Blood chemistry and intensity of injury Blood chemistry values were obtained at the beginning

Fig. 5. Influence of NS1619 (10 28 , 10 26 M) on pial small artery diameter before (control), after FPI, after FPI in indomethacin (5 mg / kg, i.v.) pretreated animals, and after FPI in MEAVP (5 mg / kg, i.v.) pretreated animals, n56. *P,0.05 versus control; 1 P,0.05 versus absence of indomethacin or MEAVP.

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and the end of all experiments. These values were 7.4460.01, 3664 and 9767 mmHg versus 7.4360.02, 3564, and 9467 mmHg for pH, pCO 2 , and pPO 2 , respectively, before and after injury. Administration of MEAVP did not significantly effect blood chemistry values. The amplitude of the pressure pulse used as an index of injury intensity was 2.060.1 atm.

4. Discussion Results of the present study show that under non-brain injury conditions, topical administration of vasopressin, in a concentration observed in CSF after FPI [9], resulted in increased SOD-inhibitable NBT reduction by newborn pig brain, indicating that O 2 was generated. Because in2 domethacin blunted such elevation in SOD-inhibitable NBT reduction by vasopressin, these data indicate that activation of cyclooxygenase contributes to O 2 2 generation by vasopressin. Moreover, the vasopressin antagonist MEAVP blocked such NBT reduction, indicating that vasopressin generates O 2 2 in a selective manner. Additionally, both indomethacin and MEAVP blunted brain injuryinduced elevated SOD-inhibitable NBT reduction. Previously, FPI has been observed to be associated with generation of O 2 2 on the piglet cerebral cortical surface [3,19]. In those studies, it was also observed that FPI caused the release of endothelin-1 (ET-1) into CSF, which in turn contributed to the generation of O 2 2 after injury through activation of PKC [3,19]. More recent studies have shown that vasopressin contributes to O 2 generation 2 following FPI in a PKC dependent manner [1]. Additionally, other studies observed that the opioid NOC / oFQ contributes to O 2 2 generation following this insult in a mechanism dependent on the activation of cyclooxygenase [21]. Results of the present study extend the latter observations to indicate that several vasoactive substances released into CSF following brain injury have the ability to contribute to O 2 2 generation after this insult via separate signal transduction pathways. Previously, the dose of indomethacin used in this study was shown to reduce cortical periarachnoid CSF prostaglandin concentrations to non-detectable concentrations and inhibit the conversion of exogenous arachidonic acid to prostaglandins on the cerebral surface by 90% [22]. A byproduct of such metabolism is the generation of O 2 2 . However, it should be noted that concerns related to the accuracy of the NBT assay have been raised [17]. Such concerns, related in large part to the specificity of NBT reduction as an index of O 2 2 , have been limited by recent piglet studies supportive of such specificity [2]. The cerebrovascular consequences of free radical production are not fully understood. It has been suggested that O2 2 could be involved in irreversible vascular damage, delayed hypoperfusion, and edema produced by cerebral ischemia / reperfusion [35]. Topical application of a xan-

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thine / xanthine oxidase activated oxygen generating system, severe hypertension, topical application of arachidonic acid, and fluid percussion brain injury cause morphological, functional, and biochemical cerebral artery abnormalities, which include reduced responsiveness to vasoconstrictor and vasodilator stimuli [19,23,36,37,39]. Superoxide anion and species derived from it, such as hydrogen peroxide and hydroxyl radical, appear to mediate these abnormalities [19,39]. Intracellular generation of O 2 2 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 the endothelium. More importantly, current concepts point toward the significant contribution to damage by the reaction of superoxide with nitric oxide to form the highly reactive pro-oxidant, peroxynitrite [14,24] The latter species and not O 2 2 is currently thought to be the more direct mediator of damage. Results of the present study also show that coadministration of vasopressin with cromakalim, CGRP, or NS1619 attenuated vasodilation to these K 1 channel agonists in a non-injury state similar to recent observations [1,31]. Because vasopressin had a vascular effect of its own, U46619 was coadministered with vasopressin in a concentration that resulted in a no net change in pial artery diameter. Therefore, vasopressin, in effect, was coadministered with K 1 channel activators under conditions of equivalent baseline diameter. Because previous studies have shown that U46619, in concentrations higher than that used in the present study (1 vs. 0.3 ng / ml), did not have any effect on pial dilation to cromakalim and CGRP [5], vasopressin alone appears to exert these inhibitory effects. Similar unpublished studies have observed that U46619 (1 ng / ml) did not affect NS1619 induced pial artery dilation. Because MEAVP blocked the ability of vasopressin to inhibit vasodilator responses to KATP and K ca channel agonists, such inhibition was not non-specific. Although the precise concentration at the receptor level is uncertain, the concentration investigated (e.g. 40 pg / ml) is approximately the one observed for vasopressin in cortical periarachnoid CSF 1 h after FPI [9]. Since CSF concentrations reflect but are not equivalent to changes in substance concentration at the receptor level (where the effective concentration presumably is higher), these data support the functional significance of the interaction between vasopressin and K 1 channel activators. Additional results of the present study show that the cyclooxygenase inhibitor indomethacin partially restored decremented pial artery dilator responses to both KATP and K ca channel agonists observed in the presence of coadministered vasopressin under non-brain injury conditions. These data suggest that activation of this signal transduction pathway by vasopressin contributes to such impaired K 1 channel vascular responsiveness. These data are,

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however, in contrast to recent observations indicating that vasopressin induced PKC activation contributed to only KATP but not K ca channel vascular impairment [1]. Results of the present study, therefore, extend those of recent studies and indicate that different mechanisms subserve the ability of vasopressin to modulate KATP and K ca channel dependent vascular activity. Another series of experiments was designed to further investigate the functional significance of the above described modulatory role of vasopressin in K 1 channel mediated vasodilation. In particular, cromakalim, CGRP, and NS1619 induced pial artery dilation was attenuated within 1 h of FPI, consistent with previous studies [4,5]. MEAVP partially prevented such diminished K 1 channel agonist vasodilation post insult, similar to recent observations [1,31]. These data suggest that vasopressin contributes to KATP and K ca channel function impairment following FPI. Previous studies showing that MEAVP attenuated pial artery vasoconstriction induced by FPI [9] indicate that vasopressin contributes to impaired cerebral hemodynamics following brain injury. In that systemic MEAVP blocked the vascular action of topical vasopressin without affecting the response to other substances [12]; these data indicate that this antagonist was selective for vasopressin and that it crosses the blood brain barrier in sufficient quantity. New data in the present study show that indomethacin partially prevented cromakalim, CGRP, and NS1619 dilator impairment following FPI. These data suggest that vasopressin contributes to KATP and K ca channel function impairment following FPI via activation of cyclooxygenase. Previous studies have shown that cyclooxygenase activation generates O 2 and that O 2 2 2 generation can impair KATP and K ca channel mediated vasodilation [1,3]. Taken together, then, these data suggest that vasopressin activates cyclooxygenase, which, in turn, generates O 2 2 to impair KATP and K ca channel function following FPI. On the other hand, the lack of protection for NS1619 induced pial artery dilation following FPI with the PKC inhibitor chelerythrine [1] could relate to a mechanism by which vasopressin generates O 2 2 to impair K ca channel function independent of PKC activation [1]. Experiments in the present study showing that indomethacin partially protected responses to both KATP and K ca channel agonists following FPI, therefore, suggest that there are fundamental differences in the mechanism whereby O 2 2 interacts with and impairs KATP and K ca channel function. Mechanistic pathways whereby O 22 so produced might impair both types of K 1 channels, however, are presently uncertain. It should be noted, though, that others have observed that O 22 induced cerebrovasodilation is mediated by activation of K ca channels [38]. While reasons for such differences are uncertain, it is speculated that low concentrations of O 2 2 might activate while higher concentrations inhibit K ca channel function. Cellular sites of origin for vasopressin detected in CSF in previous studies [9] include neurons, glia, vascular

smooth muscle, and endothelial cells. The present experimental design, however, does not allow any conclusion to be drawn with respect to cellular sites of origin for vasopressin. For a similar reason, no conclusions can also be drawn with respect to the cellular site of origin for the O2 generated by either vasopressin or FPI. Because 2 MEAVP had no significant effect on baseline pial artery diameter, vasopressin probably has minimal contribution to cerebrovascular tone during physiologic conditions. In that pial small arteries exhibited about the same percentage decrease in responsiveness to K 1 channel activators following FPI as that observed with pial arterioles, these data also suggest that there are probably minimal regional segmental vascular differences in altered K 1 channel agonist activity following FPI. Previous studies have investigated the selectivity of the agents used as probes for KATP and K ca channel activation induced pial artery dilation. Cromakalim induced pial artery dilation has been observed to be blocked by glibenclamide and unchanged by iberiotoxin, KATP and K ca channel antagonists, respectively [7]. Conversely, NS1619 induced pial artery dilation was blocked by iberiotoxin and unchanged by glibenclamide [4,6,7]. These data suggest that cromakalim and NS1619 are selective KATP and K ca channel agonists in the piglet cerebral circulation. Pial arteries have been shown to be innervated by CGRP containing nerve fibers [16]. CGRP produces hyperpolarization of cerebral vascular muscle in vitro [30], and cross selectivity experiments have similarly been performed supportive of its selectivity for the KATP channel in the piglet [7]. Inclusion of data for CGRP in the present study, therefore, lends physiologic functional perspective to results indicative of vasopressin’s modulatory role in KATP channel vascular function. 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 [18]. 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 [6]. 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. Previous studies have observed that vasopressin is released into CSF and contributes to altered dilation to the opioid dynorphin following FPI in the newborn pig [9]. Results of the present study extend the latter observations to indicate that vasopressin also modulates KATP and K ca channel agonist mediated vascular activity following FPI, suggestive of more distal signal transduction impairment post insult. Results of this study also suggest that O 22 generation via cyclooxygenase activation contributes to KATP and K ca channel function impairment following FPI. Alternatively, vasopressin modulation of KATP and K ca channel function following FPI could relate to a physio-

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logic antagonism of K 1 channel induced vasodilation. Specifically, vasopressin reverses from a dilator to a vasoconstrictor following FPI [8]. Such vasoconstriction could, therefore, oppose the ability of KATP and K ca channel agonists to vasodilate. Additionally, brain injury could alter the number or binding of K 1 channels available for activation, the degree of hyperpolarization that subsequently occurs or the ultimate response to hyperpolarization itself. In conclusion, results of the present study show that vasopressin increased O 2 2 production in a cyclooxygenase dependent manner and contributed to this production after FPI. These data also show that vasopressin blunted KATP and K ca channel mediated cerebrovasodilation in a cyclooxygenase dependent manner. These data suggest that vasopressin induced cyclooxygenase dependent O 2 2 generation contributes to KATP and K ca channel function impairment after FPI.

[12]

[13]

[14]

[15]

[16]

[17] [18]

Acknowledgements

[19]

This research was supported by grants from the National Institutes of Health and the University of Pennsylvania. The author thanks John Ross for technical assistance in the performance of the experiments.

[20]

References

[21]

[22] [23]

[1] W.M. Armstead, Vasopressin induced PKC dependent superoxide generation contributes to KATP but not K ca channel function impairment after brain injury, Stroke 32 (2001) 1408–1414. [2] W.M. Armstead, NOC / oFQ PKC-dependent superoxide generation contributes to hypoxic–ischemic impairment of NMDA cerebrovasodilation, Am. J. Physiol. 279 (2000) H2678–H2684. [3] W.M. Armstead, Superoxide generation links protein kinase C activation to impaired ATP-sensitive K 1 channel function after brain injury, Stroke 30 (1999) 153–159. [4] W.M. Armstead, Role of impaired cAMP and calcium sensitive K 1 channel function in altered cerebral hemodynamics following brain injury, Brain Res. 768 (1997) 177–184. [5] W.M. Armstead, Brain injury impairs ATP-sensitive K 1 channel function in piglet cerebral arteries, Stroke 28 (1997) 2273–2280. [6] W.M. Armstead, Role of activation of calcium-sensitive K 1 channels and cAMP in opioid-induced pial artery dilation, Brain Res. 747 (1997) 252–258. [7] W.M. Armstead, Role of ATP-sensitive K 1 channels in cGMPmediate pial artery vasodilation, Am. J. Physiol. 270 (1996) H423– H426. [8] W.M. Armstead, Influence of brain injury on vasopressin-induced pial artery vasodilation: role of superoxide anion, Am. J. Physiol. 270 (1996) H1272–H1278. [9] W.M. Armstead, Role of vasopressin in altered pial artery responses to dynorphin and b endorphin following brain injury, J. Neurotrauma 13 (1996) 115–123. [10] W.M. Armstead, C.D. Kurth, Different cerebral hemodynamic responses following fluid percussion brain injury in the newborn and juvenile pig, J. Neurotrauma 11 (1994) 487–497. [11] W.M. Armstead, C.W. Leffler, D.W. Busija, R. Mirro, Vasopressin

[24]

[25]

[26]

[27]

[28]

[29] [30]

[31] [32]

[33]

27

and prostanoid mechanisms in control of cerebral blood flow in hypotensive newborn pigs, Am. J. Physiol. 258 (1990) H408–H413. W.M. Armstead, R. Mirro, D.W. Busija, C.W. Leffler, Vascular responses to vasopressin are tone dependent in the cerebral circulation of the newborn pig, Circ. Res. 64 (1989) 136–144. W.M. Armstead, R. Mirro, S.L. Zuckerman, C.W. Leffler, Vasopressin modulates cerebrovascular responses to opioids in newborn pigs, J. Pharmacol. Exp. Ther. 260 (1992) 1107–1112. J.S. Beckman, Peroxynitrite versus hydroxyl radical: the role of nitric oxide in superoxide-dependent cerebral injury, Ann. NY Acad. Sci. 738 (1994) 69–75. D.S. DeWitt, D.C. Prough, C.L. Taylor, M. Whitley, Reduced cerebral blood flow, oxygen delivery, and electroencephalographic activity after traumatic brain injury and mild hemorrhage in cats, J. Neurosurg. 76 (1992) 812–821. L. Edvinsson, R. Ekman, I. Jansen, J. McCulloch, R. Uddman, Calcitonin gene related peptide and cerebral blood vessels: distribution and vasomotor effects, J. Cereb. Blood Flow Metab. 7 (1987) 720–728. I. Fridovich, Superoxide anion radical (O 2 2 ) superoxide dismutase, and related matters, J. Biol. Chem. 272 (1997) 18515–18517. M. Holland, P.D. Langton, N.B. Standen, J.P. Boyle, Effects of the BK ca channel activator NS1619, on rat cerebral artery smooth muscle, Br. J. Pharmacol. 117 (1996) 119–129. T. Kasemsri, W.M. Armstead, Endothelin production links superoxide generation to altered opioid-induced pial artery vasodilation after brain injury in pigs, Stroke 28 (1997) 190–197. T. Kitazono, D.D. Heistad, F.M. Faraci, Role of ATP-sensitive K 1 channels in CGRP-induced dilation of basilar artery in vivo, Am. J. Physiol. 265 (1993) H581–H585. M. Kulkarni, W.M. Armstead, Superoxide generation links nociceptin / orphanin FQ (NOC / oFQ) release to impaired N-methyl-D-aspartate cerebrovasodilation after brain injury, Stroke 31 (2000) 1990– 1996. C.W. Leffler, D.W. Busija, Arachidonate metabolism on the cerebral surface of newborn pigs, Prostaglandins 39 (1985) 811–818. C.W. Leffler, D.W. Busija, W.M. Armstead, Activated oxygen and arachidonate effects on newborn cerebral arterioles, Am. J. Physiol. 259 (1990) H1230–H1238. S.A. Lipton, Y.B. Choi, Z.H. Pan, A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso compounds, Nature 364 (1993) 626–632. T.K. McIntosh, R.L. Hayes, W.I. Dewey, V. Agura, A. Faden, Endogenous opioids may mediate secondary damage after experimental brain injury, Am. J. Physiol. 253 (1987) E565–E574. T.K. McIntosh, R. Vink, L. Noble, I. Yamakami, S. Fernyak, H. Soares, A.I. Faden, Traumatic brain injury in the rat: characterization of a lateral fluid percussion model, Neuroscience 28 (1989) 233–244. M.T. Nelson, J.B. Potlak, J.F. Worley, N.B. Standen, Calcium channels, potassium channels and voltage dependence of arterial smooth muscle tone, Am. J. Physiol. 259 (1990) C3–C18. M.T. Nelson, J.M. Quayle, Physiological roles and properties of potassium channels in arterial smooth muscle, Am. J. Physiol. 268 (1995) C799–C822. J.C. Rodriguez, S.T. Brown, Childhood injuries in the United States, Am. J. Dis. Child. 144 (1990) 626–636. A. Saito, T. Makaki, Y. Uchiyama, T.J.F. Lee, K. Goto, Calcitonin gene related peptide and vasodilator nerves in large cerebral arteries of cats, J. Pharmacol. Exp. Ther. 248 (1989) 462–466. A. Salvucci, W.M. Armstead, Vasopressin impairs KATP and K ca channel function after brain injury, Brain Res. 887 (2000) 406–412. V. Shankar, W.M. Armstead, Opioids contribute to hypoxia-induced pial artery dilation through activation of ATP-sensitive K 1 channels, Am. J. Physiol. 269 (1995) H997–H1002. J.J. Tepas, M.L. Dokler, Critical care of the injured child, Semin. Pediatr. Surg. 4 (1995) 120–127.

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W.M. Armstead / Brain Research 910 (2001) 19 – 28

[34] T. Wakatsuki, Y. Nakaya, I. Inoue, Vasopressin modulates K 1 channel activities of cultured smooth muscle cells from porcine coronary artery, Am. J. Physiol. 263 (1992) H491–H496. [35] D.B. Watson, R. Busto, W.J. Goldberg, M. Santiso, S. Yoshida, M.D. Ginsberg, Lipid peroxidation in vivo induced by reversible global ischemia in rat brain, J. Neurochem. 41 (1984) 268–274. [36] E.P. Wei, W. Christman, H.A. Kontos, J.T. Povlishock, Effects of oxygen radicals on cerebral arterioles, Am. J. Physiol. 248 (1985) H157–H167. [37] E.P. Wei, W.D. Dietrich, J.T. Povlishock, R.M. Navari, H.A. Kontos,

Functional, morphological, and metabolic abnormalities of the cerebral microcirculation after concussive brain injury in cats, Circ. Res. 46 (1980) 37–47. [38] E.P. Wei, H.A. Kontos, J.S. Beckman, Mechanisms of cerebral vasodilation by superoxide, hydrogen peroxide, and peroxynitrite, Am. J. Physiol. 271 (1996) H1262–H1266. [39] E.P. Wei, H.A. Kontos, V.C. Christman, D.S. DeWitt, J.T. Povlishock, Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilation after acute hypertension, Circ. Res. 57 (1985) 781–787.