INTRODUCTION

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NITRIC OXIDE (NO) and nitrovasodilators cause vasodilation by activating guanylate cyclase and increasing cGMP in vascular smooth muscle (9). Although the mechanism by which cGMP reduces vascular tone is uncertain, changes in the membrane potential are thought to be involved. The membrane potential of vascular muscle is a major determinant of vascular tone, and the activity of K⁺ channels is a major regulator of membrane potential (10, 17). 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 (16). Several types of K⁺ channels, including ATP sensitive (K_ATP), calcium sensitive (K_Ca), delayed rectifier, and inward rectifier, have been identified. Activation of K_ATP but not K_Ca channels has been observed to contribute to the dilation of pial arteries in the newborn pig in response to the NO releaser sodium nitroprusside (SNP) (3, 4). However, others do not ascribe such a role to K_ATP channels in NO dilation, since pial vessel responses to SNP were unchanged by glibenclamide (6, 12). Although the reasons for such differences are uncertain, such observations could result from differences in species, age, or experimental conditions.

Several mechanisms have been proposed to account for hypoxia-induced cerebral vasodilation. These possibilities include adenosine, prostaglandins, and NO (7, 18, 26). In the newborn pig, NO, cGMP, and the opioids methionine enkephalin and leucine enkephalin have been observed to contribute separately to hypoxic pial artery dilation (1, 2, 22, 23). Alternatively, these second messengers can also modulate opioid contributions to hypoxic dilation. For example, NO releasers and cGMP analogs elevate cortical periarachnoid cerebrospinal fluid (CSF) opioid concentration, whereas an NO synthase inhibitor attenuates hypoxic release of opioids (22). These data suggest that NO also contributes to hypoxic dilation, at least in part, via the formation of cGMP and the subsequent release of opioids. Opioid and hypoxic pial artery dilation were also associated with activation of the K_ATP channel (20). Furthermore, recent data suggest that the neuronal isoform of NO synthase contributes to hypoxic opioid release, whereas the endothelial isoform contributes to dilation to exogenously administered opioids (23, 25). With respect to hypoxia, however, it is uncertain whether the release of NO and cGMP and activation of K_ATP channels are the cause or the result of opioid release. The role of the K_ATP channel in the previously observed ability of N-nitro-L-arginine (L-NNA) to attenuate hypoxic pial artery dilation in the piglet is equally uncertain. Arginine analogs such as L-NNA have been observed to block pial artery dilation to K_ATP channel agonists in the adult cat and rat (12). On the other hand, it has also been observed that dilation in response to activation of the K_ATP channel is not mediated by NO in the adult rat (12).

This study, therefore, was designed to characterize the relationship among NO, opioids, and K_ATP channel activation in hypoxic pial artery dilation.

	METHODS

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All experiments were approved by the Institutional Animal Care and Use Committee. Pigs (1-5 days old) of either gender were first anesthetized with ketamine hydrochloride-acepromazine (33 mg/kg im and 3.3 mg/kg im, respectively). Anesthesia was maintained with -chloralose (30-50 mg/kg initially, supplemented with 5 mg/kg iv). A catheter was inserted into the femoral artery to record blood pressure and to sample for blood gases and pH. Another catheter was placed in a femoral vein for injection of drugs. The trachea was cannulated, and the animals were ventilated with room air. The body temperature was maintained at 37-38°C with a heating pad.

For insertion of the cranial window, the scalp was removed and an opening was made in the skull over the parietal cortex. The dura was cut and retracted over the cut bone edge. The cranial window was placed in the hole and cemented in place with dental acrylic. The space under the window was filled with artificial CSF of the following composition (in mM): 3.0 KCl, 1.5 MgCl₂, 1.5 CaCl₂, 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO₃ (pH 7.30-7.36, PCO₂ 42-49 mmHg, PO₂ 40-50 mmHg).

Pial arterioles were observed with a dissecting microscope, a television camera mounted on the microscope, and a video monitor. Vascular diameter was measured with a video microscaler.

Protocol. Pial artery diameter (small artery 120-160 µm, arteriole 50-70 µm) was determined at 1-min intervals for a 10-min exposure period after injection under the window of artificial CSF containing no drug and CSF containing a drug. Diameters were also measured 10-15 min after the highest concentration of a drug was flushed off the cerebral cortical surface with CSF containing no drug. Typically 1-2 ml of CSF were flushed through the window over a 30-s period. Needles incorporated into the side of the window allowed for the injection of CSF under the window and the runoff of excess CSF. The peak responses were measured, and a CSF sample for vasoactive metabolite analysis was collected at the end of the 10-min exposure period. The cerebral cortical periarachnoid CSF (300 µl) was collected slowly by infusing artificial CSF into one side of the window and allowing the CSF under the window to drip freely into a collection tube on the opposite side.

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NO synthase activity analysis. NO synthase activity was determined by quantification of [¹⁴C]citrulline converted from [¹⁴C]arginine using a method recently described (14). Briefly, the left brain cortex was exposed to L-NNA for 10 min, and it and the vehicle (CSF)-treated right brain cortex were frozen for later analysis. This assay involved dissolving the tissue in a 50 mM Tris-2 mM EDTA buffer, pH 7.4, sonication, and centrifugation at 10,000 g. The tissue supernatant was added to a mixture containing 1 µM L-[¹⁴C]arginine, 1 mM NADPH, and 1 mM CaCl₂, and the reaction was stopped with a buffer containing 30 mM HEPES and 3 mM EDTA. [¹⁴C]citrulline was quantified by scintillation spectroscopy, and the protein concentration in the tissue supernatant was determined by the use of the Bradford method.

Opioid analysis. The CSF samples were acidified with 1 N acetic acid to prevent protein degradation and stored at 20°C. RIA kits for methionine enkephalin and leucine enkephalin are commercially available (IncStar, Stillwater, MN; Peninsula Laboratory, Belmont, CA). The RIA used simultaneous addition of the sample, antiopioid antibody, and the ¹²⁵I derivative of the opioid. After an overnight incubation at 4°C, the free opioid was separated from the opioid bound to the antibody by the addition of saturated ammonium sulfate in the presence of rabbit carrier -globulin. After centrifugation at 760 g for 10 min, the supernate was decanted and the pellet was counted using a gamma scintillation counter. All samples and standards were assayed in duplicate. Data were calculated as percent B/B_o vs. concentration, where B/B_o is [(average cpm of sample  average cpm of nonspecific binding tube)/average cpm of total binding tube × average cpm of nonspecific binding tube] × 100.

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. All measures were analyzed using ANOVA for repeated measures. If the values were significant, the Fishers test was performed. An -level of P < 0.05 was considered significant in all statistical tests. The n values reflect data for one vessel in each animal. Values are means ± SE of absolute values or as percentages of change from control values. Data presented as percent change were compared by nonparametric means using the Wilcoxon signed rank test and Bonferroni correction.

	RESULTS

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Role of NO and cGMP in K_ATP channel agonist-induced pial artery dilation. Cromakalim and CGRP (10⁸ and 10⁶ M) elicited reproducible pial small artery (120-160 µm) and arteriole (50-70 µm) dilation (Table 1). Pial vessel dilation in response to both of these K_ATP agonists was unchanged in the presence of 10⁶ or 10³ M L-NNA, an NO synthase inhibitor (Fig. 1). However, 10⁶ M L-NNA decreased pial small artery and arteriole diameters by 8 ± 1 and 15 ± 1%, whereas 10³ M L-NNA decreased these diameters by 15 ± 1 and 20 ± 2%, respectively (n = 8). L-NNA at 10⁶ M blocked substance P dilation, whereas responses to SNP were unchanged (Fig. 2). L-NNA at 10³ M had similar effects on substance P and SNP pial vessel dilation. NO synthase activity in the cerebral cortex was decreased by 79% in the side treated with 10⁶ M L-NNA and by 89% in the side treated with 10³ M L-NNA (20.3 ± 3.8 vs. 4.4 ± 0.9 vs. 2.3 ± 0.5 pmol · mg protein¹ · min¹, n = 4).

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Influence of 106 and 103 M N-nitro-L-arginine (L-NNA) on pial small artery and arteriole responses to cromakalim and calcitonin gene-related peptide (CGRP; 108 and 106 M). Values are means ± SE; n = 8.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Influence of 106 M L-NNA on pial small artery and arteriole responses to substance P and sodium nitroprusside (SNP; 108 and 106 M). Values are means ± SE; n = 8. * P < 0.05 compared with corresponding control value.

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View larger version (18K): [in this window] [in a new window]   Fig. 3.   A: influence of cromakalim and CGRP (108 and 106 M) on cerebrospinal fluid cGMP. Values are means ± SE; n = 8. C, control. B: influence of Rp diastereomer of 8-bromoguanosine 3',5'-cyclic monophosphothioate (Rp-8-BrcGMPS 105 M) on pial small artery and arteriole responses to cromakalim and CGRP. Values are means ± SE; n = 8.

Role of K_ATP channel blockade in the attenuation of hypoxic pial artery dilation by L-NNA. Moderate and severe hypoxia (arterial PO₂ 35 and 25 mmHg, respectively) elicited pial artery dilation when induced on three separate occasions (Table 2). Glibenclamide (10⁶ M), a K_ATP antagonist, attenuated hypoxic pial artery dilation but had no effect on hypoxia-associated elevation in cortical periarachnoid CSF cGMP concentration (Fig. 4). Coadministration of L-NNA with glibenclamide had no further effect on the already diminished dilator response to hypoxia but blocked the hypoxia-associated rise in CSF cGMP (Fig. 4). Glibenclamide had no effect on resting CSF CGMP concentration, whereas L-NNA significantly decreased control CSF cGMP (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   A: influence of moderate and severe hypoxia on cortical periarachnoid cerebrospinal fluid cGMP in absence (vehicle) and presence of glibenclamide (106 M) or glibenclamide + 106 M L-NNA. Values are means ± SE; n = 8. B: influence of moderate and severe hypoxia on pial artery diameter in absence (control) and presence of glibenclamide or glibenclamide + L-NNA. Values are means ± SE; n = 8. * P < 0.05 compared with corresponding control; + P < 0.05 compared with corresponding glibenclamide alone.

Role of the K_ATP channel in opioid release. Neither cromakalim nor CGRP affected CSF methionine enkephalin or leucine enkephalin concentration (Fig. 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Influence of cromakalim and CGRP (108 and 106 M) on cortical periarachnoid cerebrospinal fluid methionine enkephalin (A) and leucine enkephalin (B) concentrations. Values are means ± SE; n = 6. C, control.

Blood chemistry and mean arterial blood pressure. Blood chemistry and mean arterial blood pressure values were obtained at the beginning and end of all normoxia experiments as well as during normoxia and hypoxia in experiments designed to characterize the role of K_ATP channels and NO in hypoxia-induced pial artery dilation. Hypoxia decreased arterial PO₂ as expected, whereas the pH, arterial PCO₂, and mean arterial blood pressure were unchanged (Table 3).

	DISCUSSION

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Results of the present study show that pial artery dilation elicited by cromakalim and CGRP was unchanged by the NO synthase inhibitor L-NNA. Additional data show that dilation by these agents was not associated with elevated CSF cGMP concentration and unchanged by Rp-8-BrcGMPS, a protein kinase G inhibitor thought to be a cGMP antagonist (14). In the piglet, Rp-8-BrcGMPS has been observed to block cGMP pial artery dilation while cAMP-induced dilation was unchanged (22, 24). Because the K_ATP channel antagonist glibenclamide has been observed to block dilation to cromakalim and CGRP while responses were unchanged in the presence of the K_Ca antagonist iberiotoxin, it has been suggested that these agents are selective synthetic and endogenous activators of the K_ATP channel, respectively (5). Additionally, although CGRP has been linked to a cAMP-dependent dilator mechanism by others (10), recent results in the piglet do not support this idea, since pial artery responses were not associated with changes in CSF cAMP and were also not altered by the Rp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphothioate, a cAMP antagonist (5). Taken together, then, data from the present study show that K_ATP channel activation is not associated with the release of NO or cGMP.

Arginine analogs such as L-NNA have been observed to block pial artery dilation to K_ATP channel agonists in the adult cat (12), but not in the adult rat (21). A partial explanation for this discrepancy is that the effectiveness of K_ATP channel blockade may be arginine analog choice, dose, and species dependent (12). For example, in the cat, N-monomethyl-L-arginine is a more effective blocker of the K_ATP channel than L-NNA (12). However, L-NNA, in the adult cat, can be used to distinguish between the traditional NO-dependent activation of guanylate cyclase and mediation of effects via K_ATP channels. In this species, there is a substantial difference between the dose required to block the production of endothelium-derived relaxing factor by ACh (20 µM) and the dose required for effective blockade of K_ATP channels (250 µM) (12). Although the precise action by which arginine analogs block K_ATP channels is uncertain, it has been suggested that the K_ATP channel has an arginine site and that the arginine analogs act directly on the channel without participation of NO (12). In the present study, two L-NNA concentrations (10⁶ and 10³ M) were similarly used to characterize the potential interaction of an arginine analog with an arginine site on the K_ATP channel. These data show that dilation to cromakalim and CGRP was unchanged in the presence of either L-NNA concentration. However, both L-NNA concentrations decreased pial artery diameter and resting CSF cGMP concentration. Furthermore, both L-NNA concentrations blocked substance P dilation, whereas responses to SNP were unchanged. Taken together, these data show that L-NNA, in a concentration that blocked responses to substance P, an endothelium-derived relaxing factor-dependent dilator, had no effect on K_ATP channel agonist-induced dilation. This concentration of L-NNA also blunted the tonic dilatory influence of NO on the cerebral circulation, since pial vessels constricted and basal levels of cGMP were decreased. Because a higher L-NNA concentration (10³ M) similarly had no effect on cromakalim or CGRP dilation, these data further suggest that the K_ATP channel does not have an arginine site in the newborn pig cerebral circulation. However, systemically administered N-nitro-L-arginine methyl ester has also been observed to partially block the pial vessel dilation to aprikalim, another K_ATP agonist, in the newborn pig, suggesting that a part of the dilation with activation of K_ATP channels could involve NO (6). Reasons for such differences are uncertain but could relate to route of administration or choice of NO synthase inhibitor.

Several substances have been suggested to be endogenous activators of K_ATP channels. In the cerebral circulation, pial arteries have been shown to be innervated by CGRP-containing nerve fibers (8). CGRP produces hyperpolarization of cerebral vascular muscle in vitro (19), whereas dilator responses of cerebral arteries are inhibited by glibenclamide, a K_ATP channel antagonist, indicating that dilation is mediated by opening of this K⁺ channel (11).

Additional experiments were designed to further investigate the relationship between NO and the K_ATP channel during hypoxia. Glibenclamide attenuated hypoxic pial artery dilation but had no effect on hypoxia-associated elevation in cortical periarachnoid CSF cGMP concentration. Coadministration of L-NNA with glibenclamide had no further effect on the already diminished dilator response to hypoxia but blocked the hypoxia-associated rise in CSF cGMP. Glibenclamide had no effect on resting CSF cGMP concentration, whereas L-NNA significantly decreased control CSF cGMP. These data show that NO and activation of K_ATP channels contribute to hypoxic pial artery dilation, consistent with previous studies (1, 20, 22). The attenuation of hypoxic pial vessel dilation by glibenclamide in the absence of any effect on the ability of hypoxia to release CSF cGMP suggests that NO is released by this stimulus independent of opening the K_ATP channel. The observation that L-NNA coadministration with glibenclamide did not further decrement the already attenuated hypoxic pial vessel dilatory response but blocked the release of CSF cGMP during hypoxia suggests that NO and K_ATP channel contributions to hypoxic dilation must be occurring at a common site. In fact, these data are consistent with the suggestion that NO activates the K_ATP channel to contribute to hypoxic pial vessel dilation. Therefore, K_ATP channels are involved in the mediation of NO dilation, but not its release. Reasons for differences between our data, which indicate a role for NO and K_ATP channel activation in hypoxic pial vessel dilation, and those of Leffler et al. (13), which do not, are uncertain but could relate to a more prolonged hypoxic exposure in our study (10 min) than in the study of Leffler et al. (5 min). It is not inconceivable, therefore, that a more robust hypoxic exposure could activate mechanisms not recruited during a shorter stimulation period.

Finally, experiments were designed to determine the ability of K_ATP channel activation to increase CSF opioid concentration. Results show that neither cromakalim nor CGRP had an effect on CSF methionine enkephalin or leucine enkephalin concentrations. Previous studies have observed that both of these opioids and activation of the K_ATP channel contributed to hypoxic pial artery dilation (20). Additionally, these opioids, at least in part, elicit dilation via K_ATP channel activation (20). Furthermore, NO and cGMP contribute to hypoxia-associated opioid release (22). Results of the present study, therefore, clarify this relationship and suggest that opioid release during hypoxia is the cause and not the result of K_ATP channel activation.

In conclusion, results of the present study show that K_ATP channel agonists do not elicit dilation via NO/cGMP and do not release opioids. NO release during hypoxia also is independent of K_ATP channel activation. These data suggest that hypoxic dilation results, at least in part, from the sequential release of NO, cGMP, and opioids, which in turn activate the K_ATP channel. Additionally, NO and opioids may also act in parallel to activate the K_ATP channel to elicit dilation. This proposed mechanism is summarized as a schematic diagram in Fig. 6. The data that resulted in this mechanism are novel, since data in previous publications could only indicate that NO, opioids, and K_ATP channel activation were involved in hypoxic pial vessel dilation. Such studies could not, however, discern the relationship between the above (e.g., NO releases opioids, which activate the K_ATP channel, or K_ATP channel activation releases NO, which then releases opioids). Because the response to hypoxia was not ablated with combined NO synthase and K_ATP channel blockade, data in the present study also suggest that additional mechanisms contribute to the vascular response as well. Furthermore, it is speculated that one or more of the other multiple redundant pathways involved in hypoxic cerebrovasodilation are upregulated in a compensatory manner when an initial pathway for dilation is blocked.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Schematic diagram illustrating relationship among NO, cAMP, opioids, and ATP-sensitive K+ (KATP) channel in hypoxic pial artery dilation.