Introduction ischemia results in reductions in pial artery diameter and

Brain Research 884 2000 51–58 www.elsevier.com locate bres Research report 1 Role of cAMP and K channel-dependent mechanisms in piglet hypoxic ischemic impaired nociceptin orphanin FQ-induced cerebrovasodilation Galia Ben-Haim, William M. Armstead Departments of Anesthesia and Pharmacology , University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA Accepted 22 August 2000 Abstract 1 This study was designed to determine the role of altered cAMP and K channel-dependent mechanisms in impaired pial artery dilation to the newly described opioid, nociceptin orphanin FQ NOC oFQ following hypoxia ischemia in newborn pigs equipped with a closed cranial window. Recent studies have observed that NOC oFQ elicits pial dilation via release of cAMP, which, in turn, activates the 1 calcium sensitive K and the ATP-dependent K K channel. Global cerebral ischemia 20 min was induced via elevation of ca ATP 28 26 intracranial pressure, while hypoxia 10 min decreased pO to 3563 mmHg with unchanged pCO . Topical NOC oFQ 10 , 10 M 2 2 induced vasodilation was attenuated by ischemia reperfusion I1R and reversed to vasoconstriction by hypoxia ischemia reperfusion H1I1R at 1 h of reperfusion control, 961 and 1661; I1R, 361 and 661; H1I1R, 2761 and 21261. Such altered dilation returned to control values within 4 h in I1R animals and within 12 h in H1I1R animals. NOC oFQ dilation was associated with elevated CSF cAMP in control animals but such biochemical changes were attenuated in I1R animals and reversed to decreases in cAMP concentration in H1I1R animals control, 1037658 and 19196209 fmol ml; I1R, 1068633 and 1289630 fmol ml; H1I1R, 976636 26 28 26 and 772627 fmol ml for absence and presence of NOC oFQ 10 M, respectively. Topical 8-Bromo cAMP 10 , 10 M pial dilation was unchanged by I1R but blunted by H1I1R control, 1061 and 2061; I1R, 1161 and 2062; H1I1R, 061 and 062. Pituitary adenylate cyclase activating polypeptide and cromakalim, adenylate cyclase and K channel activators, respectively, ATP elicited dilation that was blunted by both I1R and H1I1R while NS1619, a K channel activator, elicited dilation that was unchanged ca by I1R but blunted by H1I1R. These data indicate that impaired NOC oFQ dilation following I1R results form altered adenylate cyclase and K channel-dependent mechanisms. These data further indicate that impaired NOC oFQ dilation following H1I1R results ATP not only from altered adenylate cyclase and K channel but also from altered cAMP and K channel-dependent mechanisms.  2000 ATP ca Elsevier Science B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Ischemia 1 Keywords : Newborn; Cerebral circulation; Opioid; K channel; Cyclic nucleotide

1. Introduction ischemia results in reductions in pial artery diameter and

cerebral blood flow as well as impaired cerebrovascular Episodes of inadequate oxygen supply to the brain can control during hypotension and hypercapnia in a newborn result in significant neurological sequelae. Babies are pig model [17–19]. Less, however, is known about the frequently exposed to hypoxic ischemic insults during the cerebrovascular consequences of combined hypoxia is- perinatal period. One contributor to neurological damage is chemia. thought to be cerebrovascular dysfunction. Global cerebral The membrane potential of vascular smooth muscle is a major determinant of vascular tone and activity of potas- 1 sium K channels is a major regulator of membrane Corresponding author. Tel.: 11-215-573-3674; fax: 11-215-349- potential [23]. Activation or opening of these channels 5078. E-mail address : armsteawmail.med.upenn.edu W.M. Armstead. increases potassium efflux, thereby producing hyperpolari- 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 8 2 - 1 52 G zation of vascular muscle. Membrane hyperpolarization oFQ-induced pial dilation following hypoxia ischemia is closes voltage-dependent calcium channels, causing relaxa- unknown. Interestingly, it has been observed that K ATP 1 tion of vascular muscle [22]. Several types of K channels, channel function is impaired following global cerebral including ATP-sensitive K , calcium-sensitive K , ischemia in the piglet [9]. Such studies did not observe any ATP ca 1 delayed rectifier and inward rectifier K channels have effect of that insult on K or cAMP agonist mediated ca been identified. Pharmacological studies using activators dilation [8]. However, those studies did not investigate the and inhibitors have additionally provided functional evi- effects of combined hypoxia ischemia on cAMP, K and ATP 1 dence that K channels, especially K and K channels, K channel function. ATP ca ca regulate tone of cerebral blood vessels in vitro and in vivo Therefore, the present study was designed to determine 1 [10,16,22,23]. In the piglet, cAMP elicits pial artery the role of altered cAMP and K channel-dependent dilation predominantly via activation of the K channel mechanisms in impaired pial artery dilation to NOC oFQ ca with a more minor K channel contributing component observed following hypoxia ischemia. ATP [3,4]. Opioids have been observed to be important in the control of the cerebral circulation of the piglet during 2. Methods physiological and pathological conditions [5]. During the last 5 years, several groups have isolated and cloned a new Newborn 1–5-day-old, 1.3–2.1 kg pigs of either sex G-protein-coupled receptor that showed high homology were used in these experiments. All protocols were ap- with opioid receptors. This opioid-like receptor, however, proved by the Institutional Animal Care and Use Commit- displayed no affinity for opioid ligands and remained an tee. Piglets were initially anesthetized with isoflurane 1–2 ‘orphan’ until late 1995 [15]. At that time, two indepen- MAC. Anesthesia was maintained with a-chloralose 30– dent groups [21,24] identified a 17-amino acid peptide that 50 mg kg, supplemented with 5 mg kg per h i.v.. A did not bind to the classical opioid receptors m, d, k but catheter was inserted into a femoral artery to monitor activated the orphan receptor in a nanomolar concentration blood pressure and to sample for blood gas tensions and range and could therefore be considered the endogenous pH. Drugs to maintain anesthesia were administered ligand for the orphan receptor. This peptide was named through a second catheter placed in a femoral vein. The orphanin FQ by Reinscheid et al. [24] because its sequence trachea was cannulated, and the animals were mechanical- begins with phenylalanine F and ends with a glutamine ly ventilated with room air. A heating pad was used to Q. The same peptide was called nociceptin by Meunier et maintain the animals at 37–398C. al. [21] because it increased the reactivity to pain in A cranial window was placed in the parietal skull of animals in contrast with the analgesic effects of opioid these anesthetized animals. This window consisted of three drugs. The orphan receptor therefore will be referred to as parts: a stainless steel ring, a circular glass coverslip, and ORL-1 for opioid receptor-like 1 and its endogenous three ports consisting of 17-gauge hypodermic needles ligand, NOC oFQ for nociceptin orphanin FQ. In situ attached to three precut holes in the stainless steel ring. For hybridization studies have demonstrated localization of placement, the dura was cut and retracted over the cut bone ORL-1 in several regions of the central nervous system edge. The cranial window was placed in the opening and including the cerebral cortex, thalamus, and hypothalamus cemented in place with dental acrylic. The volume under [11–13]. A similar distribution has been observed for the window was filled with a solution, similar to CSF, of NOC oFQ. It has therefore been suggested that this opioid the following composition in mM: 3.0 KCl, 1.5 MgC1 , 2 system may play a role in memory, nociception, learning, 1.5 CaCl , 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 2 and emotion [20]. Recently, NOC oFQ has been observed NaHCO . This artificial CSF was warmed to 378C and had 3 to elicit pial artery vasodilation in the newborn pig [2]. The the following chemistry: pH 7.33, pCO 546 mmHg, and 2 mechanism for such pial dilation involved the release of pO 543 mmHg, which was similar to that of endogenous 2 cAMP, and subsequent activation of the K and K CSF. Pial arterial vessels were observed with a dissecting ATP ca channel [2]. However, little is known about the role of microscope, a television camera mounted on the micro- NOC oFQ in the physiological or pathophysiological scope, and a video output screen. Vascular diameter was control of cerebral hemodynamics. measured with a video microscaler. For production of CSF NOC oFQ concentration has been recently ob- cerebral ischemia, a hollow stainless steel bolt was im- served to increase following I1R in the piglet [1]. planted in a small 2 mm hole in the skull. Additionally, NOC oFQ-induced pial artery vasodilation is attenuated following I1R and reversed to vasoconstriction 2.1. Protocol following combined hypoxia ischemia reperfusion H1 I1R [1]. Such impaired NOC oFQ-mediated pial dilation Two types of pial arterial vessels, small arteries resting appears to contribute to reduced cerebral blood flow and diameter 120–160 mm and arterioles resting diameter pial artery vasoconstriction observed following hypoxia 50–70 mm, were examined to determine whether segmen- ischemia [1]. However, the mechanism for impaired NOC tal differences in the effects of hypoxia ischemia could be G . Ben-Haim, W.M. Armstead Brain Research 884 2000 51 –58 53 identified. Pial arterial vessel diameter was determined assay determines cyclic nucleotide concentration for bind- every minute for a 10-min exposure period after infusion ing to an antiserum that has a high specificity for the cyclic onto the exposed parietal cortex of artificial CSF prior to nucleotide. The antibody-bound cyclic nucleotide is then drug application and after infusion of artificial CSF reacted with an anti-rabbit second antibody bound to containing a drug. Typically, 2–3 ml of CSF were flushed fluoromicrospheres. Labeled cyclic nucleotide bound to the through the window over a 30-s period, and excess CSF primary rabbit antibody can then be measured by determin- was allowed to run off through one of the needle ports. For ing the amount of light emitted by the fluoromicrospheres. sample collection, 300 ml of the total cranial window All unknowns were assayed at two dilutions, with the volume of 500 ml was collected by slowly infusing CSF lower limit of detection being 100 fmol ml. The con- into one side of the window and allowing the CSF to drip centration of the unlabeled cyclic nucleotides is calculated freely into a collection tube on the opposite side. from the standard curve via linear regression analysis. Techniques for induction of total cerebral ischemia in the piglet have been well documented [17,19]. Briefly, 2.3. Statistical analysis total cerebral ischemia was accomplished by infusing artificial CSF into a hollow bolt in the cranium to maintain Pial artery diameter, systemic arterial pressure, and an intracranial pressure 15 mmHg greater than the numeri- cyclic nucleotide values were analyzed using repeated cal mean of systolic and diastolic arterial blood [19]. measures of analysis or t-tests where appropriate. If the F Intracranial pressure was monitored via a sidearm of the value was significant, the Fisher test was performed on all cranial window. Blood flow in pial arterioles, viewed with data analyzed by repeated measures. An a level of P,0.05 a microscope and video monitor, stopped completely on was considered significant in all statistical tests. The n elevation of intracranial pressure and did not resume until values reflect data for one vessel in each animal. Values are the pressure was lowered [19]. To prevent the arterial represented as means6S.E.M. of absolute values or as pressure from rising inordinately Cushing response, percentages of change from control values. venous blood was withdrawn as necessary to maintain mean arterial pressure no greater than 100 mmHg. As the cerebral ischemic response subsided, the shed blood was

3. Results