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Am J Physiol Heart Circ Physiol 274: H1674-H1683, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 5, H1674-H1683, May 1998

ATP-dependent K+ channel activation reduces loss of opioid dilation after brain injury

William M. Armstead

Departments of Anesthesia and Pharmacology, University of Pennsylvania and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

ATP-dependent K+ (KATP) channel function is impaired after fluid percussion brain injury (FPI). Additionally, the nitric oxide (NO) releaser sodium nitroprusside and a cGMP analog elicit pial dilation via KATP channel activation, whereas opioids such as methionine enkephalin (Met) elicit pial dilation via NO and KATP channel activation. Decremented Met dilation contributes to reductions in pial artery diameter and altered cerebral hemodynamics after FPI. This study was designed to investigate the role of KATP channel activation before FPI in the loss of opioid dilation subsequent to FPI in newborn pigs equipped with a closed cranial window. FPI was produced by allowing a pendulum to strike a piston on a saline-filled cylinder that was fluid coupled to the brain via a hollow screw in the cranium. FPI blunted dilation to Met (7 ± 1, 11 ± 1, and 17 ± 1% before FPI vs. 1 ± 1, 4 ± 1, and 6 ± 1% after FPI for 10-10, 10-8, and 10-6 M Met, respectively). Met-associated elevation in cerebrospinal fluid (CSF) cGMP was similarly blunted (350 ± 12 and 636 ± 12 fmol/ml before FPI vs. 265 ± 5 and 312 ± 17 fmol/ml after FPI for control and 10-6 M Met, respectively). In piglets pretreated with cromakalim (10-10 M) 20 min before FPI, Met dilation was partially restored (7 ± 1, 10 ± 1, and 15 ± 1% before FPI vs. 4 ± 1, 7 ± 1, and 11 ± 1% after FPI for 10-10, 10-8, and 10-6 M Met, respectively). Met cGMP release was similarly partially restored (400 ± 9 and 665 ± 25 fmol/ml before FPI vs. 327 ± 11 and 564 ± 23 fmol/ml after FPI for control and 10-6 Met, respectively). Cromakalim (10-10 M) had no effect on pial diameter itself but prevented pial artery constriction by FPI (148 ± 5 to 124 ± 5 µm vs. 139 ± 4 to 141 ± 4 µm in the absence vs. presence of cromakalim pretreatment, respectively). In contrast, pretreatment with a subthreshold concentration of NS-1619, a calcium-dependent K+ channel agonist, did not restore vascular and biochemical parameters after FPI. These data indicate that prior KATP channel activation reduces the loss of opioid dilation after FPI.

newborn; cerebral circulation; nitric oxide; cyclic nucleotides; opioids

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

TRAUMATIC INJURY is the leading cause of death for infants and children (16, 19). Fluid percussion brain injury (FPI) is an experimental technique thought to model shaken impact syndrome (19, 20). Previous studies have shown that FPI resulted in pial arterial vasoconstriction and decreased cerebral blood flow within 10 min of injury in newborn pigs (10, 11). Additionally, responses to several nitric oxide (NO)-dependent dilator stimuli, including opioids, were blunted after FPI in piglets (3, 18, 36). Although blunted dilation to opioids such as methionine enkephalin and leucine enkephalin is thought to contribute to pial artery vasoconstriction after FPI (36, 37), little is currently known about the mechanism of control of the cerebral circulation in the newborn after traumatic brain injury.

The membrane potential of vascular smooth muscle is a major determinant of vascular tone, and activity of K+ channels is a major regulator of membrane potential (31). Activation or opening of these channels increases K+ efflux, thereby producing hyperpolarization of vascular muscle. Membrane hyperpolarization closes voltage-dependent calcium channels, causing relaxation of vascular muscle (30). Several types of K+ channels, including ATP-sensitive (KATP), calcium-sensitive (KCa2+), delayed rectifier, and inward rectifier K+ channels, have been identified. Pharmacological studies using activators and inhibitors have additionally provided functional evidence that K+ channels, especially KATP and KCa2+ channels, regulate the tone of cerebral blood vessels in vitro and in vivo (15, 23, 30, 31).

Cyclic nucleotides are recognized as second messengers mediating the actions of peptides and hormones on vascular smooth muscle. In the piglet, activation of KATP but not KCa2+ channels has recently been observed to contribute to cGMP-induced pial artery dilation (4, 6). Alternatively, cAMP elicits dilation via activation of the KCa2+ channel (34). Whereas opioids produce pial dilation in the piglet via both KATP and KCa2+ channel activation, the cGMP-KATP pathway appears to predominate (5, 32). Recently, it has been observed that both KATP and KCa2+ channel function is impaired after FPI in piglets (7, 8). For example, dilator responses to the KATP and KCa2+ channel agonists cromakalim and NS-1619, respectively, as well as to cGMP and cAMP analogs, were blunted after FPI (7, 8).

Preconditioning with brief periods of cardiac ischemia-reperfusion decreases the extent of cellular injury and reduces dysfunction to endothelium-dependent dilators after subsequent prolonged ischemia (17, 29). Although this phenomenon is a well-documented entity, the underlying mechanism of the protection is still not clear (25). One potential mechanism involves the KATP channel. For example, because the opening of the KATP channel before injury affords protection, and blockade of that channel eliminates that protection (12), the state of the KATP channel before the insult may determine the degree of injury after cardiac ischemia. Recently, it has been observed that prior KATP channel activation reduces infarct volume in an adult rat model of focal ischemia (35). However, little is known about the contribution of the K+ channel state before brain injury to altered cerebral hemodynamics observed after FPI in the newborn.

Therefore, the present study was designed to investigate the role of KATP channel activation before brain injury in the loss of opioid-induced pial artery dilation subsequent to FPI.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Newborn pigs (1-5 days old) of either sex were used in these experiments. All protocols were approved by the Institutional Animal Care and Use Committee. Animals were anesthetized with ketamine hydrochloride (33 mg/kg) and acepromazine (3.3 mg/kg) intramuscularly. Anesthesia was maintained with alpha -chloralose (30-50 mg/kg, supplemented with 5 mg · kg-1 · h-1 iv). 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°C.

A cranial window was placed in the parietal skull of these anesthetized animals. This window consisted of three parts: a stainless steel ring, a circular glass coverslip, and three ports consisting of 17-gauge hypodermic needles attached to three precut holes in the stainless steel ring. For placement, the dura was cut and retracted over the cut bone edge. The cranial window was placed in the opening and cemented in place with dental acrylic. The volume under the window was filled with a solution similar to cerebrospinal fluid (CSF) of the following composition (in mg/l): 220 KCl, 132 MgCl2, 221 CaCl2, 7,710 NaCl, 402 urea, 665 dextrose, and 2,066 NaHCO3. This artificial CSF had the following chemistry: pH 7.33, PCO2 46 mmHg, and PO2 43 mmHg, which was similar to 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 (38). A device designed by the Medical College of Virginia was used. Injury to the brain was produced contralaterally to where the cranial window was placed. The device itself consisted of an acrylic plastic cylindrical reservoir 60 cm long, 4.5 cm in diameter, and 0.5 cm thick. The entire system was filled with 0.9% saline. FPI was induced by striking the piston located on the end of the fluid-filled column with a 4.8-kg pendulum. The intensity of the blow (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 blow 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.

Protocol. Drug effects on the diameter of two types of pial arterial vessels, small arteries (resting diameter 120-160 µm) and arterioles (resting diameter 50-70 µm), were examined to determine whether segmental differences in the effects of the FPI could be identified. Pial arterial vessel diameter was determined every minute for a 10-min exposure period after infusion onto the exposed parietal cortex of artificial CSF containing no drug and after infusion of artificial CSF containing a drug. 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. A 300-µl CSF sample was collected at the end of the 10-min exposure period for later analysis of cGMP concentration.

Eight types of experiments were performed for each drug: 1) no FPI with cromakalim administration (n = 7); 2) FPI without K+ channel agonist pretreatment (n = 8); 3) FPI with cromakalim pretreatment (n = 8); 4) FPI with cromakalim and glibenclamide pretreatment (n = 8); 5) FPI with pinacidil pretreatment (n = 7); 6) FPI with NS-1619 pretreatment (n = 8); 7) FPI with cromakalim posttreatment (n = 7); and 8) time controls (n = 5).

In the FPI experiments, responses of arterial vessels to methionine enkephalin, leucine enkephalin, and dynorphin (each at 10-10, 10-8, and 10-6 M, Sigma Chemical) were obtained before and 1 h after FPI. In animals that were pretreated with a K+ channel agonist alone or with a K+ channel antagonist, the agents were applied 20 min before FPI. In animals posttreated with a K+ channel agonist, the agent was coadministered with opioids after FPI. The K+ channel agonists used were l-cromakalim (10-10 M, SmithKline Beecham), NS-1619 [10-10 M, Research Biochemicals International (RBI)], and pinacidil (10-10 M, RBI), whereas the antagonist was glibenclamide (10-6 M, Sigma). Each of the opioids was applied in three concentrations, in ascending order of concentration. 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. Post-FPI values for opioids in pretreated animals reflect values in the continued presence of the K+ channel agonist. Time-control experiments were conducted in a separate series of animals and were designed to obtain responses to drugs initially and then 1 h later (designated as time 1 and time 2, respectively, in Table 1). The stock glibenclamide solution (10-3 M) was made by initially dissolving this agent in a small amount of dimethyl sulfoxide (DMSO, 200 µl) and the balance in ethanol. This vehicle was then diluted 1:1,000 in CSF to make the working solution. This CSF-DMSO-ethanol vehicle had no effect on pial artery diameter. All other agents were dissolved in 0.9% saline.

                              
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  		Table 1.   Influence of methionine enkephalin, leucine enkephalin, and dynorphin on pial artery diameter

Cyclic nucleotide analysis. CSF samples collected after a 10-min exposure to a drug were analyzed for cGMP concentration using scintillation proximity assay methods. Commercially available kits for cGMP (Amersham, Arlington Heights, IL) were used. Briefly, this assay determines cyclic nucleotide concentration for binding to an antiserum that has a high specificity for the cyclic nucleotide. The antibody-bound cyclic nucleotide is then reacted with an anti-rabbit second antibody bound to fluoromicrospheres. Labeled cyclic nucleotide bound to the primary rabbit antibody can then be measured by determining the amount of light emitted by the fluoromicrospheres. All unknowns were assayed at two dilutions. The concentration of the unlabeled cyclic nucleotides is calculated from the standard curve via linear regression analysis.

Statistical analysis. Pial artery diameter, systemic arterial pressure, and cyclic nucleotide values were analyzed using analysis of variance for repeated measures. If the values were significant, the Fisher test was performed. An alpha  level of P < 0.05 was considered significant in all statistical tests. Then n values reflect data for one vessel in each animal. Values are represented as means ± SE of absolute values or as percent change from control values. Data presented as percent change were compared by nonparametric means using the Wilcoxon signed-rank test and Bonferroni correction.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Influence of cromakalim on opioid-induced pial artery dilation under non-brain-injury conditions. The opioids methionine enkephalin, leucine enkephalin, and dynorphin (each at 10-10, 10-8, and 10-6 M) elicited reproducible pial small artery (120-160 µm) and arteriole (50-70 µm) vasodilation (Table 1). When expressed on a percent basis, the coadministration of cromakalim (10-10 M) with each of these opioids had no effect on the resulting vasodilator responses compared with those observed in the absence of cromakalim (Table 2).

                              
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  		Table 2.   Influence of cromakalim on methionine enkephalin-, leucine enkephalin-, and dynorphin-induced pial artery dilation under non-brain-injury conditions

Influence of cromakalim pretreatment on opioid-induced pial artery dilation after FPI. Methionine enkephalin-induced increases in vessel diameter were attenuated after FPI and partially restored by cromakalim (10-10 M) pretreatment before FPI (Fig. 1). Pretreatment consisting of combined glibenclamide (10-6 M) and cromakalim administration, however, did not restore decremented methionine enkephalin dilation after FPI. For 10-10, 10-8, and 10-6 M methionine enkephalin these values were, respectively, 7 ± 1, 11 ± 1, and 16 ± 1% dilation during control conditions vs. 1 ± 1, 4 ± 1, and 6 ± 1% dilation after FPI vs. 4 ± 1, 7 ± 1, and 11 ± 1% dilation after FPI pretreated with cromakalim vs. 1 ± 1, 2 ± 1, and 3 ± 1% dilation after FPI pretreated with combined glibenclamide and cromakalim (n = 8 for each group). All groups were statistically different from control, but the cromakalim and glibenclamide pretreatment group was also statistically different from the group that received cromakalim alone. Methionine enkephalin produced dilation that was associated with increased cortical periarachnoid CSF cGMP, and these biochemical changes were blunted by FPI and partially restored by cromakalim (Fig. 1). Relative to a baseline CSF cGMP value of 1.0, CSF cGMP concentration increased in response to 10-10, 10-8, and 10-6 M methionine enkephalin, respectively, to 1.3 ± 0.1, 1.4 ± 0.1, and 1.8 ± 0.1 during control conditions vs. 1.1 ± 0.1, 1.1 ± 0.1, and 1.2 ± 0.1 after FPI vs. 1.2 ± 0.1, 1.4 ± 0.1, and 1.6 ± 0.1 after FPI pretreated with cromakalim. Leucine enkephalin (10-10, 10-8, and 10-6 M) also produced pial artery dilation (Table 1) that was similarly attenuated after FPI and partially restored by cromakalim pretreatment before FPI (Fig. 2). Glibenclamide coadministered with cromakalim pretreatment did not restore decremented leucine enkephalin dilation after FPI, similar to the results with methionine enkephalin. Leucine enkephalin-induced dilation was associated with an increase in CSF cGMP, which was attenuated after FPI and partially restored by cromakalim (Fig. 2). These values represent a change in CSF cGMP similar to that produced with methionine enkephalin.


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  		Fig. 1.   A: influence of methionine enkephalin (10-10, 10-8, and 10-6 M) on cGMP concentration in cortical periarachnoid cerebrospinal fluid (CSF) before fluid percussion brain injury (FPI) (control), after FPI (FPI), and after FPI in cromakalim (10-10 M)-pretreated animals (FPI-cromakalim) (n = 6). C, without methionine enkephalin. B: influence of methionine enkephalin on small pial artery and arteriole diameter before FPI (control), after FPI, and after FPI in cromakalim-pretreated animals (n = 8). * P < 0.05 compared with corresponding control value. + P < 0.05 compared with corresponding FPI value.


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  		Fig. 2.   A: influence of leucine enkephalin (10-10, 10-8, and 10-6 M) on cGMP concentration in cortical periarachnoid CSF before FPI (control), after FPI (FPI), and after FPI in cromakalim (10-10 M)-pretreated animals (FPI-cromakalim) (n = 6). C, without leucine enkephalin. B: influence of leucine enkephalin on small pial artery and arteriole diameter before FPI (control), after FPI, and after FPI in cromakalim-pretreated animals (n = 8). * P < 0.05 compared with corresponding control value. + P < 0.05 compared with corresponding FPI value.

In contrast, dynorphin (10-10, 10-8, and 10-6 M), which is a a vasodilator under resting conditions (Table 1), decreased pial artery diameter after FPI, and this opioid was restored to a vasodilator by cromakalim pretreatment before FPI (Fig. 3). Similar to results with methionine enkephalin and leucine enkephalin dilation, glibenclamide coadministered with cromakalim did not restore dynorphin dilation. Dynorphin produced dilation that was associated with a large increase in CSF cGMP. This biochemical change was blunted after FPI and partially restored by cromakalim (Fig. 3).


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  		Fig. 3.   A: influence of dynorphin (10-10, 10-8, and 10-6 M) on cGMP concentration in cortical periarachnoid CSF before FPI (control), after FPI (FPI), and after FPI in cromakalim (10-10 M)-pretreated animals (FPI-cromakalim) (n = 6). C, without dynorphin. B: influence of dynorphin on small pial artery and arteriole diameter before FPI (control), after FPI, and after FPI in cromakalim-pretreated animals (n = 8). * P < 0.05 compared with corresponding control value. + P < 0.05 compared with corresponding FPI value.

Influence of cromakalim posttreatment on opioid-induced pial artery dilation after FPI. In these experiments, cromakalim was coadministered with topically applied opioids only after FPI. In contrast to the ability of cromakalim pretreatment before FPI to partially restore decremented opioid dilation observed after FPI, this posttreatment of cromakalim had no effect on diminished opioid dilation after FPI (Table 3).

                              
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  		Table 3.   Influence of cromakalim posttreatment on opioid-induced pial artery dilation after FPI

Influence of pinacidil pretreatment on opioid-induced pial artery dilation after FPI. To determine if the ability of cromakalim pretreatment to restore opioid responsiveness after FPI was agent specific, we used another KATP channel opener, pinacidil, in these experiments. These data show that pinacidil (10-10 M) pretreatment partially restored decremented dilation to opioids observed after FPI, similarly to that observed with cromakalim pretreatment (Table 4).

                              
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  		Table 4.   Influence of pinacidil pretreatment on opioid-induced pial artery dilation after FPI

Influence of NS-1619 on opioid-induced pial artery dilation after FPI. In contrast to results obtained with cromakalim, NS-1619 (10-10 M) pretreatment did not restore decremented pial artery dilation to methionine enkephalin and leucine enkephalin after FPI (Figs. 4 and 5). Moreover, reversal of dynorphin from a dilator to a constrictor after FPI was not modified by NS-1619 pretreatment (Fig. 6). Similarly, opioid-associated elevations in CSF cGMP that were blunted by FPI were not restored by NS-1619 (Figs. 4-6).


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  		Fig. 4.   A: influence of methionine enkephalin (10-10, 10-8, and 10-6 M) on cGMP concentration in cortical periarachnoid CSF before FPI (control), after FPI (FPI), and after FPI in NS-1619 (10-10 M)-pretreated animals (FPI-NS-1619) (n = 6). C, without methionine enkephalin. B: influence of methionine enkephalin on small pial artery and arteriole diameter before FPI (control), after FPI, and after FPI in NS-1619-pretreated animals (n = 8). * P < 0.05 compared with corresponding control value.


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  		Fig. 5.   A: influence of leucine enkephalin (10-10, 10-8, and 10-6 M) on cGMP concentration in cortical periarachnoid CSF before FPI (control), after FPI (FPI), and after FPI in NS-1619 (10-10 M)-pretreated animals (FPI-NS-1619) (n = 6). C, without leucine enkephalin. B: influence of leucine enkephalin on small pial arteries and arterioles before FPI (control), after FPI, and after FPI in NS-1619-pretreated animals (n = 8). * P < 0.05 compared with corresponding control value.


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  		Fig. 6.   A: influence of dynorphin (10-10, 10-8, and 10-6 M) on cGMP concentration in cortical periarachnoid CSF before FPI (control), after FPI (FPI), and after FPI in NS-1619 (10-10 M)-pretreated animals (FPI-NS-1619) (n = 6). C, without dynorphin. B: influence of dynorphin on small pial arteries and arterioles before FPI (control), after FPI, and after FPI in NS-1619-pretreated animals (n = 8). * P < 0.05 compared with corresponding control value.

Influence of cromakalim, pinacidil, and NS-1619 on pial artery diameter and CSF cGMP before and after FPI. Cromakalim and pinacidil (10-10 M) had no effect on pial small artery or arteriole diameter, respectively, before FPI (e.g., 137 ± 4 vs. 139 ± 4 µm and 61 ± 4 vs. 62 ± 4 µm before and after cromakalim, respectively, n = 5). NS-1619 (10-10 M) similarly had no effect on pial diameters before FPI (133 ± 6 vs. 137 ± 6 µm and 60 ± 2 vs. 62 ± 2 µm, n = 5). After FPI, all three K+ channel agonists had similar nonsignificant effects on pial small artery and arteriole diameter (e.g., 130 ± 3 vs. 132 ± 4 µm and 57 ± 3 vs. 59 ± 3 µm for cromakalim, n = 5). However, a 10-fold higher concentration of either K+ channel opener had a significant dilator effect (e.g., 139 ± 4 vs. 148 ± 4 µm and 60 ± 3 vs. 67 ± 3 µm before and after 10-9 M cromakalim, respectively, n = 5).

In five piglets, FPI decreased pial small artery diameter from 148 ± 5 to 124 ± 5 µm and pial arterioles from 65 ± 3 to 53 ± 3 µm. In contrast, FPI did not decrease pial small artery or arteriole diameters, respectively, in cromakalim-pretreated animals (139 ± 4 vs. 141 ± 4 µm and 62 ± 2 vs. 61 ± 3 µm, n = 5). However, NS-1619 pretreatment did not prevent FPI-induced reductions in pial small artery or arteriole diameter (139 ± 2 vs. 114 ± 3 µm and 66 ± 2 vs. 56 ± 2 µm, n = 5).

Cromakalim and NS-1619 had no effect on baseline CSF cGMP concentration before injury. Cromakalim pretreatment partially restored the FPI-induced decrement in baseline CSF cGMP concentration (Figs. 1-3), whereas NS-1619 had no effect on baseline CSF cGMP concentration after FPI (Figs. 4-6).

Blood chemistry. Blood chemistry values were obtained at the beginning and end of all experiments. These pH, PCO2, and PO2 values were, respectively, 7.44 ± 0.01, 32 ± 1 mmHg, and 94 ± 2 mmHg before FPI vs. 7.43 ± 0.1, 33 ± 1 mmHg, and 93 ± 2 mmHg after FPI vs. 7.43 ± 0.01, 33 ± 1 mmHg, and 93 ± 2 mmHg at the end of the experiment (n = 46). Mean arterial blood pressure decreased from 66 ± 1 to 56 ± 2 mmHg within 60 min of FPI (n = 46). The mean level of FPI was 2.0 ± 0.1 atm.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Results of the present study show that methionine enkephalin and leucine enkephalin elicited pial artery dilation associated with increased CSF cGMP and that these vascular and biochemical changes were attenuated by brain injury, consistent with previous observations (36). Coadministration of cromakalim with these opioids under non-brain-injury conditions did not alter opioid vascular reactivity. In contrast, pretreatment with cromakalim before brain injury partially restored the decremented opioid-induced dilation and ability to increase CSF cGMP after brain injury. Moreover, dynorphin was reversed from a dilator to a constrictor after brain injury, and this response was also partially restored by cromakalim. Specificity of cromakalim interaction with the KATP channel was demonstrated because combined preadministration of glibenclamide with cromakalim did not restore decremented opioid dilation after FPI. Additional experiments show that the ability of KATP channel pretreatment to protect opioid responsiveness after FPI is not agonist dependent because similar data were obtained when cromakalim was replaced by pinacidil. The concentration of cromakalim or pinacidil used in these studies had no effect, by itself, on pial artery diameter or CSF cGMP concentration either before or after FPI. These data, therefore, suggest that such a subthreshold concentration for these vascular and biochemical activities must have still opened the KATP channel to cause some other action that resulted in partial protection of opioid-mediated responses. Alternatively, low levels of KATP channel function after FPI may be sufficient, when operating in conjunction with other mechanisms, to allow normal responsiveness to occur. For example, permissive effects have been suggested for NO and prostaglandins (1, 26). By extension, then, these data suggested that baseline KATP channel function was diminished after FPI, consistent with the observations that dilator responses to exogenously administered KATP channel openers were decremented after FPI (8). An additional explanation for these data relate to the potential for cromakalim preadministration to set (reset) the sensitivity of the KATP channel before FPI.

Additional results of this study show that cromakalim pretreatment also prevented reductions in baseline pial artery diameter and CSF cGMP concentration observed after FPI. However, posttreatment with cromakalim after FPI did not restore decremented opioid dilation after FPI. Because decremented opioid dilation is thought to contribute to FPI-associated reductions in pial artery caliber (36, 37), these data, taken together, suggest that KATP channel activation before the insult contributes to the degree of the alteration of cerebral hemodynamics after FPI.

In contrast, NS-1619 pretreatment did not restore opioid dilation or associated CSF cGMP release after FPI. Additionally, NS-1619 pretreatment did not prevent FPI-associated reductions in baseline pial artery diameter or CSF cGMP concentration. Therefore, these data suggest that the state of the KCa2+ channel before the insult does not contribute to the degree of alteration of cerebral hemodynamics after FPI. Because KCa2+ channel function is impaired after FPI (7), and opioids depend on both KATP and KCa2+ channel activation to fully elicit pial dilation (5, 32), these data further suggest that an enhanced KCa2+ channel contribution to baseline pial artery tone and opioid dilation could not have compensated for loss of KATP channel function after FPI. Related to the above, then, the openness of the KATP channel before the insult appears to convey unique properties to the cerebral circulation after FPI.

The K+ channel selectivity of cromakalim and NS-1619 has been described previously for the newborn pig cerebral circulation. For example, cromakalim pial dilation was blocked by glibenclamide and unchanged by iberiotoxin KATP and KCa2+ channel antagonists, respectively (4, 6). Conversely, NS-1619 pial dilation was blocked by iberiotoxin and unchanged by glibenclamide (6, 7). Additionally, whereas NS-1619 has been suggested to also block voltage-dependent calcium channels, this was not found to be the case for its actions on piglet pial arteries (6).

Previous studies in the newborn pig have also suggested that NO elicits dilation via activation of KATP channels because responses to sodium nitroprusside (SNP) and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) were blunted by the KATP channel antagonist glibenclamide (4). However, others have also observed that responses to SNP were unchanged by glibenclamide (13, 24). Although the reasons for such differences are uncertain, such observations could result from differences in species, age, or experimental conditions. Additionally, in vivo approaches to the study of ion channels are limited in that pharmacological probes can only serve as an indirect index of ion channel contribution to vascular responsiveness.

It has also been observed that responses to several NO-dependent dilator stimuli, including opioids and vasopressin, were blunted after FPI and partially restored by the preadministration of oxygen free radical scavengers (3, 37), indicating that impaired NO function contributes to altered cerebral hemodynamics after FPI. Because responses to cromakalim, SNP, and 8-BrcGMP have also been observed to be blunted after FPI (8), it has been suggested that impaired function of mechanisms distal to NO synthase also contributes to altered hemodynamics after FPI (8). Because many different substances, including opioids (5, 32), are thought to elicit K+ channel-dependent dilation, such observations, therefore, suggest that impaired K+ channel function could serve as a common mechanism for altered cerebral hemodynamics after FPI (8). However, impaired functionality after brain injury is not nonspecific, because responses to brain natriuretic peptide and the nonselective dilator papaverine were unchanged (8). Observations that KATP channel pretreatment partially restored resting as well as stimulated CSF cGMP concentrations indicate that impairment after FPI is not necessarily restricted exclusively to the KATP channel. As such, data in the present study support previous studies showing that impaired reactivity also relates to decremented NO production/function after brain injury (37). It is presently uncertain how KATP channel activation before FPI could partially restore decremented NO production subsequent to injury.

Although with several other types of cerebral injury models, similar impairment of KATP channel function has been observed, with other models it has not been observed. For example, dilation of pial arteries in response to RP-52891, a KATP channel activator, was observed to be impaired in diabetic rats (28), whereas basilar artery dilation to aprikalim, another KATP agonist, was blunted in stroke-prone spontaneously hypertensive rats (23). Additionally, global ischemia has been observed to impair pial artery responses to calcitonin gene-related peptide (CGRP) and aprikalim in piglets, indicating that impairment of KATP channel function can be achieved by an acute stimulus (14, 27). In contrast, responses to aprikalim and CGRP were augmented in a rat model of subarachnoid hemorrhage, whereas responses to SNP were decreased and 8-BrcGMP unchanged, indicating that KATP channel function was preserved but production of cGMP, and not its action, was blunted in this injury model (33). Although mechanisms for impaired KATP channel function after FPI are uncertain, data from a recent study suggest that FPI-associated release of endothelin (2, 22) could contribute to such impairment (21).

Although many studies have characterized the hemodynamic effects of brain injury in adult animal models, few have done so in the newborn-to-infant time period. Results of previous studies show that developmental changes result in markedly different effects of brain injury on cerebral hemodynamics in the newborn and juvenile pig (10). For example, the following were observed: 1) an increase in pial vessel constriction and a decrease in cerebral blood flow that remained depressed longer in newborn vs. juveniles; 2) marked increases in intracranial pressure in the newborn, but modest increases in intracranial pressure in the juvenile; 3) differences in cerebral oxygenation, an index of metabolism, with increased saturation followed by prolonged desaturation of hemoglobin for oxygen in the newborn and modest increases in saturation followed by mild desaturation in the juvenile (10). Furthermore, systemic arterial pressure has been observed to increase in the adult studies (38) and in juvenile pigs (10), whereas systemic arterial pressure decreases after brain injury in the newborn pig (10). These data suggest that cerebral and systemic hemodynamic responses after brain injury are age dependent. In fact, it has been suggested previously that the newborn is exquisitely sensitive to brain injury (10). Because both newborn pigs and children <1 yr of age have skulls with unfused sutures, the age period of pigs chosen in these studies may approximate the newborn-to-infant time period in the human.

Although opioids contribute to the regulation of piglet cerebral hemodynamics under resting conditions, their contributions become more robust during pathological situations (9). For example, methionine enkephalin and leucine enkephalin contribute to cerebrovascular dilation to hypoxia and hemorrhagic hypotension (9). Additionally, brain injury elevates CSF opioid concentrations in the piglet (11). Because naloxone attenuates pial artery constriction and reductions in cerebral blood flow after FPI, opioids contribute to altered cerebral hemodynamics after injury in the newborn pig (11). Such observations can be explained by the reversal of dynorphin from a dilator to a constrictor, as well as by enhanced vasoconstriction to beta -endorphin after FPI (9). Additionally, decremented dilation to methionine enkephalin and leucine enkephalin contributes to pial artery vasoconstriction after FPI (36, 37). Results of the present study therefore provide a mechanistic perspective to events involved in altered responses to an important regulator of cerebrohemodynamics.

In conclusion, results of the present study suggest that prior KATP channel activation reduces the alteration of cerebral hemodynamics after FPI.

    ACKNOWLEDGEMENTS

The author thanks Joseph Quinn for technical assistance in the performance of the experiments.

    FOOTNOTES

This research was supported by grants from the National Institutes of Health and the American Heart Association (AHA). W. M. Armstead is an Established Investigator of the AHA.

Address for reprint requests: W. M. Armstead, Dept. of Anesthesia, 34th & Civic Center Blvd., The Children's Hospital of Philadelphia, Philadelphia, PA 19104.

Received 11 November 1997; accepted in final form 4 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(5):H1674-H1683
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