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www.elsevier.com / locate / bres

Research report

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

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

calcium sensitive (K ) and the ATP-dependent K_ca (K_ATP) channel. Global cerebral ischemia (20 min) was induced via elevation of 28 26 intracranial pressure, while hypoxia (10 min) decreased pO to 352 63 mmHg with unchanged pCO . Topical NOC / oFQ (102 , 10 M) 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 and21261%). 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 KATPchannel activators, respectively, elicited dilation that was blunted by both I1R and H1I1R while NS1619, a K_cachannel activator, elicited dilation that was unchanged by I1R but blunted by H1I1R. These data indicate that impaired NOC / oFQ dilation following I1R results form altered adenylate cyclase and K_ATPchannel-dependent mechanisms. These data further indicate that impaired NOC / oFQ dilation following H1I1R results not only from altered adenylate cyclase and KATPchannel but also from altered cAMP and Kcachannel-dependent mechanisms.  2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Ischemia

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: 1

1-215-349-potential [23]. Activation or opening of these channels

5078.

E-mail address: [email protected] (W.M. Armstead). increases potassium efflux, thereby producing

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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 KATP

tion of vascular muscle [22]. Several types of K channels, channel function is impaired following global cerebral including ATP-sensitive (KATP), calcium-sensitive (K ),ca ischemia in the piglet [9]. Such studies did not observe any

delayed rectifier and inward rectifier K channels have effect of that insult on Kca or cAMP agonist mediated 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, KATP and

dence that K channels, especially KATPand Kcachannels, Kca channel function.

regulate tone of cerebral blood vessels in vitro and in vivo Therefore, the present study was designed to determine

[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 Kca channel mechanisms in impaired pial artery dilation to NOC / oFQ with a more minor KATP channel contributing component observed following hypoxia / ischemia.

[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 witha-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 24.62

and emotion [20]. Recently, NOC / oFQ has been observed NaHCO . This artificial CSF was warmed to 373 8C and had to elicit pial artery vasodilation in the newborn pig [2]. The the following chemistry: pH 7.33, pCO2546 mmHg, and mechanism for such pial dilation involved the release of pO2543 mmHg, which was similar to that of endogenous cAMP, and subsequent activation of the KATP and Kca CSF. Pial arterial vessels were observed with a dissecting 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 / 50–70mm), were examined to determine whether segmen-ischemia [1]. However, the mechanism for impaired NOC / tal differences in the effects of hypoxia / ischemia could be

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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. Ana 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

returned to the animal. Cerebral ischemia was maintained

for 20 min. In combined H1I1R animals, hypoxia ( pO25 3.1. Influence of I1R and H1I1R on NOC /oFQ pial

3563 mmHg) was produced for 10 min by decreasing the artery reactivity and release of cAMP

inspired O via inhalation of N , which was followed by2 2

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the total ischemia protocol as described above. Topical NOC / oFQ (10 , 10 M) elicited reproducible Three types of experiments were performed: (1) sham pial small artery (120–160mm) and arteriole (50–70mm) control (bolt inserted, but intracranial pressure not in- dilation over a 12-h period in sham control animals (data creased) (n514); (2) I1R (n514); and (3) H1I1R (n5 not shown). NOC / oFQ-induced pial small artery dilation

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was diminished within 1 h but returned to control value 14). NOC / oFQ (10 , 10 M, Phoenix) was topically

with 4 h of reperfusion in I1R animals (Fig. 1A). Similar applied before intervention (0 time) and at 1 and 4 h of

changes were observed in pial arterioles. Such NOC / oFQ-reperfusion in I1R animals or at 1, 4, 8, and 12 h of

induced vasodilation was associated with elevated cortical reperfusion in H1I1R animals. Responses at the same

periarachnoid CSF cAMP concentration (Fig. 2A). At 1 h intervals were obtained in sham control animals. In another

of reperfusion, this NOC / oFQ-induced increase in CSF series of animals, pial artery responses to 8-Bromo cAMP,

cAMP was attenuated, but such biochemical responses Sp 8-Bromo cAMPs, PACAP 1–27, cromakalim, and

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were restored to control (pre-ischemia) value within 4 h of NS1619 (10 , 10 M) (all RBI except for cromakalim

reperfusion (Fig. 2A). which was obtained from Smith Kline Beecham) were

In contrast, NOC / oFQ-induced vasodilation was re-obtained in sham control, I1R, and H1I1R animals at the

versed to pial artery vasoconstriction at both 1 and 4 h of 1-h time point only. Appropriate aliquots of the vehicle for

reperfusion after H1I1R (Fig. 1B). At 8 h of reperfusion all agents (0.9% saline) were added to CSF infused under

such vasoconstriction had returned to modest vasodilation, the window. This CSF vehicle had no effect on pial artery

whereas at 12 h of reperfusion NOC / oFQ dilation was not diameter.

different from that observed before the insult (Fig. 1B). Similar changes were observed in pial arterioles. NOC / 2.2. Cyclic nucleotide analysis oFQ associated elevation in CSF cAMP was blocked and, in fact, reversed to small stimulus-induced decreases in CSF sample collected after a 10-min exposure to an CSF cAMP at 1 and 4 h of reperfusion in such animals intervention were analyzed for cAMP concentration using (Fig. 2B). At 8 h of reperfusion, NOC / oFQ-induced scintillation proximity assay methods. Commercially avail- increases in CSF cAMP once again, though attenuated able kits for cAMP (Amersham) were used. Briefly, this compared to sham control. Finally, at 12 h of reperfusion,

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Fig. 1. (A) Influence of NOC / oFQ (10 , 10 M) on pial small artery diameter before (control) and at 1 and 4 h post I1R. (B) Influence of NOC / oFQ on pial small artery diameter before (control) and at 1, 4, 8, and 12 h post H1I1R, n57. *P,0.05 compared to corresponding control.

NOC / oFQ-induced elevated CSF cAMP was no different 8-Bromo cAMP and Sp 8-Bromo cAMPs was unchanged than that observed in sham control animals (Fig. 2B). (Figs. 3 and 4). However, pial small artery dilation Baseline CSF cAMP was unchanged by either I1R or induced by the adenylate cyclase activator PACAP and the H1I1R in the absence of NOC / oFQ administration (Figs. KATP channel activator cromakalim was attenuated within

2A,B). 1 h of reperfusion in I1R animals (Figs. 5 and 6).

NS1619-induced pial small artery dilation, though, was 3.2. Influence of I1R and H1I1R on pial artery unchanged in I1R animals (Fig. 7). Similar observations

vasodilation induced by 8-bromo cAMP, Sp 8-Bromo were made for agonist reactivities in pial arterioles.

cAMPs, PACAP, cromakalim, and NS1619 In contrast, pial small artery dilation induced by 8-Bromo cAMP and Sp 8-8-Bromo cAMPs was blocked in Topical 8-Bromo cAMP, Sp 8-Bromo cAMPs, PACAP, H1I1R animals within 1 h of reperfusion (Figs. 3 and 4).

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cromakalim, and NS1619 (10 , 10 M) elicited re- PACAP- and cromakalim-induced pial small artery dilation producible pial small artery and arteriole dilation (data not was modestly, though nonsignificantly, attenuated to a shown). After 1 h of reperfusion, in I1R animals, pial greater extent in H1I1R versus I1R animals at 1 h of small artery dilation induced by the cAMP analogues reperfusion (Figs. 5 and 6). Finally, NS1619-induced pial

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Fig. 2. (A) Influence of NOC / oFQ (10 , 10 M) on CSF cAMP (fmol / ml) in sham control animals and in I1R animals at 1 and 4 h of reperfusion. (B) Influence of NOC / oFQ on CSF cAMP in sham control and in H1I1R at 1, 4, 8, and 12 h of reperfusion, n57. *P,0.05 compared to corresponding 0 value; †P,0.05 compared to corresponding sham control value.

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Fig. 5. Influence of PACAP (10 , 10 M) on pial small artery and

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Fig. 3. Influence of 8-Bromo cAMP (10 , 10 M) on pial small artery arteriole diameter in sham control animals and in I1R and H1I1R and arteriole diameter in sham control animals and in I1R and H1I1R animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding control value.

control value.

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Fig. 4. Influence of Sp 8-Bromo cAMPs (10 , 10 M) on pial small Fig. 6. Influence of cromakalim (10 , 10 M) on pial small artery and artery and arteriole diameter in sham control animals and in I1R and arteriole diameter in sham control animals and in I1R and H1I1R H1I1R animals at 1 h of reperfusion, n57. *P,0.05 compared to animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding

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CSF cAMP concentration in sham control animals similar to previous observations [2], but new data in this study show that such stimulated cAMP release was attenuated at 1 h but returned to sham control induced release within 4 h of reperfusion. These data suggest that attenuated ability to elevate CSF cAMP contributes to impaired NOC / oFQ-induced pial artery dilation following ischemia / reperfu-sion. This insult, however, did not alter baseline CSF cAMP concentration.

In contrast, several differences in the observed parame-ters described above were noted when the effects of ischemia / reperfusion were compared to that of hypoxia / ischemia / reperfusion. For example, NOC / oFQ-induced vasodilation was reversed to pial artery vasoconstriction at both 1 and 4 h of reperfusion following hypoxia / ischemia / reperfusion. At 8 h of reperfusion such vasoconstriction was returned to modest vasodilation, whereas at 12 h of reperfusion NOC / oFQ dilation was no different from that observed before the insult. Concomitantly, stimulated CSF cAMP release by NOC / oFQ was blocked, if not reversed to modest decreases in CSF cAMP concentration at 1 and 4 h post hypoxia / ischemia / reperfusion. At 8 h post reperfusion, NOC / oFQ stimulated release of cAMP once

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Fig. 7. Influence of NS1619 (10 , 10 M) on pial small artery and

again, but such release was less than that in sham control

arteriole diameter in sham control animals and in I1R and H1I1R

animals. NOC / oFQ ability to stimulate cAMP release

animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding

comparable to that observed in the sham control animal

control value.

was not fully restored until 12 h of reperfusion. Similar to the ischemia / reperfusion insult, hypoxia / ischemia / re-perfusion did not alter baseline non-agonist-stimulated small artery dilation was attenuated at 1 h of reperfusion in CSF cAMP concentration. Taken together, these data H1I1R animals (Fig. 7). Similar observations were made suggest that the more profound impairment of NOC / oFQ-for reactivities of agonists in pial arterioles. induced pial artery dilation following hypoxia / ischemia versus that observed following ischemia could relate to the 3.3. Blood chemistry potentiated inability of this agonist to elevate CSF cAMP

concentration.

Blood chemistry and mean arterial blood pressure values In order to more fully determine potential contributory were obtained at the beginning and end of all experiments mechanisms for the observed decrement in NOC / oFQ-as well oFQ-as during hypoxia. Hypoxia decreoFQ-ased pO2 to induced pial vasodilation following hypoxia / ischemia, the 3563 mmHg, whereas the pH, pCO , and mean arterial2 effects of such insults on the ability of cAMP analogues, blood pressure values were unchanged. Values for pH, an adenylate cyclase activator, and activators of the KATP pCO , pO , and mean arterial blood pressure were2 2 and Kca channels to elicit vasodilation were explored. 7.4560.02, 3663, 9064, and 7065 mmHg at the start of Results of these studies show that pial artery dilation experiments versus 7.4460.02, 3763, 9165, and 6766 induced by the cAMP analogues, 8-Bromo cAMP and Sp mmHg, respectively, at the end of experiments. There were 8-Bromo cAMPs, was unchanged by ischemia / reperfusion, no group differences in either blood pressure or blood consistent with the observations of others who showed that chemistry values. the dilation to another analogue, dibutyryl cAMP, was similarly unchanged in a piglet global cerebral ischemia model [8]. In contrast, results of the present study show

4. Discussion that hypoxia / ischemia produces blunted pial dilation to

these same cAMP analogues. Such results extend those of Results of the present study show that NOC / oFQ- previous investigations [8] and indicate that while cAMP-induced pial artery dilation was diminished within 1 h of mediated dilation is resistant to influence by ischemia, reperfusion, but such dilation was not different from that such cyclic nucleotide vasodilation is susceptible to inhibi-observed before ischemia / reperfusion within 4 h of re- tion with a more robust insult like hypoxia / ischemia. perfusion similar to previous observations [1]. Such NOC / Additional results of the present study show that pial oFQ-induced vasodilation was accompanied by elevated artery dilation in response to topical PACAP, an activator

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of adenylate cyclase, were attenuated after both ischemic with hypoxia / ischemia presumably results in the more and hypoxia / ischemic insults. While uncertain as to the robust alteration of the vascular response with this insult. mechanism for diminished stimulated CSF concentrations The origin of the cAMP detected in CSF cannot be of cAMP with NOC / oFQ following ischemia or hypoxia / determined from the present experiments. Potential cellular ischemia, results of the latter studies suggest that an altered sites of origin include neurons, glia, vascular smooth activation of adenylate cyclase might contribute to such muscle, and endothelial cells.

diminished stimulated cAMP levels. These results are in Previous studies have investigated the selectivity of the contrast, however, to those observed for another adenylate agents used as probes for the role of KATPand Kca channel cyclase activator, forskolin, whose dilation was unchanged activation in impaired NOC / oFQ dilation post insult. following global cerebral ischemia in the piglet [8]. Cromakalim-induced pial artery dilation has been observed Reasons for such differences are uncertain but could relate to be blocked by glibenclamide and unchanged by to different pools / mechanisms for adenylate cyclase acti- iberiotoxin, KATPand Kca channel antagonists, respectively vation by these two substances. Alternatively, experimental [7]. Conversely, NS1619-induced pial artery dilation was differences related to duration of ischemia (20 min in the blocked by iberiotoxin and unchanged by glibenclamide present study, 10 min in the other) could account for such [4,6,7]. These data suggest that cromakalim and NS1619 a discrepancy. are selective KATP and Kca channel agonists in the piglet Moreover, other results of the present study show that cerebral circulation. However, it has also been observed cromakalim, a KATP channel activator, elicited pial artery that NS1619 may additionally possess calcium channel dilation that was blunted after both ischemia and hypoxia / antagonistic activity and, therefore, may not be useful as a ischemia. With respect to ischemia alone, these data are probe for Kca channel activation [14]. In contrast, recent consistent with those previously published [9]. Present observations in the piglet show that vasoconstrictor re-data, however, extend those previously published in that sponses to the calcium channel agonist Bay K8644 were the effects of combined hypoxia / ischemia on KATP chan- unchanged in the presence of NS1619 [4]. These results nel function had not been considered. suggest that NS1619 has no calcium channel-blocking The final series of experiments in this study investigated activity and, therefore, may be considered to be selective the effects of ischemia and hypoxia / ischemia on vasodila- for activation of Kca channels in the newborn pig. tion elicited by the Kcachannel activator, NS1619. Results Global cerebral ischemia in a piglet model has been of those studies show that such dilation was unchanged by previously observed to result in reductions in blood flow of ischemia, consistent with previous studies [8]. However, the cerebrum and altered pial artery dilation to stimuli such the observation that NS1619-induced pial vasodilation was as hemorrhagic hypotension and hypercapnia [17–19]. blunted following combined hypoxia / ischemia is novel in However, such ischemic effects are not nonselective in that that others had previously concluded that Kca channel although response to these stimuli were impaired, others mechanisms were resistant to impairment [8]. Reasons for (e.g., isoproterenol) were not [17,18].

such impairment with hypoxia / ischemia and not ischemia Opioids are important contributors to the regulation of alone are currently unknown. the newborn pig cerebral circulation during physiological With respect to an understanding of mechanisms in- and pathological conditions [5]. Because the present study volved in impairment of NOC / oFQ-induced vasodilation did not characterize responses to NOC / oFQ after ischemia following ischemia alone, then, such impairment appears or hypoxia / ischemia in the juvenile or adult, it is uncertain related to an attenuated ability to elevate CSF cAMP whether similar results could be expected in the adult. concentration, at least in part, due to impaired adenylate In conclusion, results of the present study show that cyclase activation, as well as to an impairment of KATP impaired NOC / oFQ dilation following ischemia / reperfu-channel function. Although cAMP elicits vasodilation via sion results from altered adenylate cyclase and KATP

KATP channel activation [2], such a signal transduction channel-dependent mechanisms. These data further indi-linkage cannot explain impaired NOC / oFQ vasodilation, cate that impaired NOC / oFQ dilation following hypoxia / since cAMP analogue dilation was intact after ischemia. ischemia / reperfusion results not only from altered Therefore, cAMP independent contribution of KATP chan- adenylate cyclase and KATP channel but also from altered nel activation to NOC / oFQ dilation must be involved in cAMP and Kca channel-dependent mechanisms.

the observed impairment following ischemia. Alternative-ly, a more marked inability to elevate CSF cAMP as well as impaired adenylate cyclase activation, cAMP analogue

dilation, KATP and Kca channel activation contribute to Acknowledgements

impaired NOC / oFQ-induced vasodilation following

hypo-xia / ischemia. NOC / oFQ-induced pial artery dilation is The authors thank Miriam Kulkarni for technical assis-dependent on cAMP, KATP and Kca channel-dependent tance in the performance of the experiments. This research mechanisms to elicit pial artery dilation [2], and interfer- was supported by grants from the National Institutes of ence with all of the above signal transduction pathways Health, the American Heart Association – PA, DE

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(1995) 799–822. Low, D.K. Grandy, Molecular cloning and tissue distribution of a

[24] R.K. Reinscheid, H.P. Nothacker, A. Bourson, A. Ardati, R.A. putative member of the rat opioid receptor gene family that is not a

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(1)

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

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 returned to the animal. Cerebral ischemia was maintained

for 20 min. In combined H1I1R animals, hypoxia ( pO25 3.1. Influence of I1R and H1I1R on NOC /oFQ pial 3563 mmHg) was produced for 10 min by decreasing the artery reactivity and release of cAMP

inspired O via inhalation of N , which was followed by2 2

28 26

was diminished within 1 h but returned to control value 14). NOC / oFQ (10 , 10 M, Phoenix) was topically

with 4 h of reperfusion in I1R animals (Fig. 1A). Similar applied before intervention (0 time) and at 1 and 4 h of

changes were observed in pial arterioles. Such NOC / oFQ-reperfusion in I1R animals or at 1, 4, 8, and 12 h of

induced vasodilation was associated with elevated cortical reperfusion in H1I1R animals. Responses at the same

periarachnoid CSF cAMP concentration (Fig. 2A). At 1 h intervals were obtained in sham control animals. In another

of reperfusion, this NOC / oFQ-induced increase in CSF series of animals, pial artery responses to 8-Bromo cAMP,

cAMP was attenuated, but such biochemical responses Sp 8-Bromo cAMPs, PACAP 1–27, cromakalim, and

28 26

were restored to control (pre-ischemia) value within 4 h of NS1619 (10 , 10 M) (all RBI except for cromakalim

reperfusion (Fig. 2A). which was obtained from Smith Kline Beecham) were

In contrast, NOC / oFQ-induced vasodilation was re-obtained in sham control, I1R, and H1I1R animals at the

versed to pial artery vasoconstriction at both 1 and 4 h of 1-h time point only. Appropriate aliquots of the vehicle for

reperfusion after H1I1R (Fig. 1B). At 8 h of reperfusion all agents (0.9% saline) were added to CSF infused under

such vasoconstriction had returned to modest vasodilation, the window. This CSF vehicle had no effect on pial artery

whereas at 12 h of reperfusion NOC / oFQ dilation was not diameter.

(2)

54 G. Ben-Haim, W.M. Armstead / Brain Research 884 (2000) 51 –58

28 26

2A,B). 1 h of reperfusion in I1R animals (Figs. 5 and 6).

NS1619-induced pial small artery dilation, though, was 3.2. Influence of I1R and H1I1R on pial artery unchanged in I1R animals (Fig. 7). Similar observations vasodilation induced by 8-bromo cAMP, Sp 8-Bromo were made for agonist reactivities in pial arterioles. cAMPs, PACAP, cromakalim, and NS1619 In contrast, pial small artery dilation induced by

8-Bromo cAMP and Sp 8-8-Bromo cAMPs was blocked in Topical 8-Bromo cAMP, Sp 8-Bromo cAMPs, PACAP, H1I1R animals within 1 h of reperfusion (Figs. 3 and 4).

28 26

Fig. 2. (A) Influence of NOC / oFQ (10 , 10 M) on CSF cAMP (fmol / ml) in sham control animals and in I1R animals at 1 and 4 h of reperfusion. (B) Influence of NOC / oFQ on CSF cAMP in sham control and in H1I1R at 1, 4, 8, and 12 h of reperfusion, n57. *P,0.05 compared to corresponding 0 value; †P,0.05 compared to corresponding sham control value.

(3)

28 26

Fig. 5. Influence of PACAP (10 , 10 M) on pial small artery and

28 26

Fig. 3. Influence of 8-Bromo cAMP (10 , 10 M) on pial small artery arteriole diameter in sham control animals and in I1R and H1I1R and arteriole diameter in sham control animals and in I1R and H1I1R animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding control value.

control value.

28 26 28 26

(4)

56 G. Ben-Haim, W.M. Armstead / Brain Research 884 (2000) 51 –58

28 26

Fig. 7. Influence of NS1619 (10 , 10 M) on pial small artery and

again, but such release was less than that in sham control

arteriole diameter in sham control animals and in I1R and H1I1R

animals. NOC / oFQ ability to stimulate cAMP release

animals at 1 h of reperfusion, n57. *P,0.05 compared to corresponding

comparable to that observed in the sham control animal

control value.

concentration.

pCO , pO , and mean arterial blood pressure were2 2 and Kca channels to elicit vasodilation were explored. 7.4560.02, 3663, 9064, and 7065 mmHg at the start of Results of these studies show that pial artery dilation experiments versus 7.4460.02, 3763, 9165, and 6766 induced by the cAMP analogues, 8-Bromo cAMP and Sp mmHg, respectively, at the end of experiments. There were 8-Bromo cAMPs, was unchanged by ischemia / reperfusion, no group differences in either blood pressure or blood consistent with the observations of others who showed that chemistry values. the dilation to another analogue, dibutyryl cAMP, was similarly unchanged in a piglet global cerebral ischemia model [8]. In contrast, results of the present study show 4. Discussion that hypoxia / ischemia produces blunted pial dilation to these same cAMP analogues. Such results extend those of Results of the present study show that NOC / oFQ- previous investigations [8] and indicate that while cAMP-induced pial artery dilation was diminished within 1 h of mediated dilation is resistant to influence by ischemia, reperfusion, but such dilation was not different from that such cyclic nucleotide vasodilation is susceptible to inhibi-observed before ischemia / reperfusion within 4 h of re- tion with a more robust insult like hypoxia / ischemia. perfusion similar to previous observations [1]. Such NOC / Additional results of the present study show that pial oFQ-induced vasodilation was accompanied by elevated artery dilation in response to topical PACAP, an activator

(5)

the observed impairment following ischemia. Alternative-ly, a more marked inability to elevate CSF cAMP as well as impaired adenylate cyclase activation, cAMP analogue

dilation, KATP and Kca channel activation contribute to Acknowledgements impaired NOC / oFQ-induced vasodilation following

(6)

Af-58 G. Ben-Haim, W.M. Armstead / Brain Research 884 (2000) 51 –58

Molecular cloning, tissue distribution and chromosomal localization

filiate, and the University of Pennsylvania Research

of a novel member of the opioid receptor gene family, FEBS Lett.

Foundation.

347 (1994) 279–283.

[13] K. Fukuda, S. Kato, K. Mori, M. Nishi, H. Takeshima, N. Iwabe, T. Miyata, T. Houtani, T. Sugimoto, DNA cloning and regional

References distribution of a novel member of the opioid receptor family, FEBS Lett. 343 (1994) 42–46.