INTRODUCTION

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IN RESPONSE TO HYPOXIA, cerebral vascular contractile and relaxation responses are markedly altered. In general, acute hypoxia is associated with vasodilation of systemic blood vessels, with increased blood flow and oxygen delivery. (In contrast, in the pulmonary vascular bed, hypoxia-induced vasoconstriction serves to direct blood away from poorly oxygenated alveoli.) In long-term hypoxia (LTH), organ blood flows may be increased, decreased, or little changed from normoxic control values depending on the species, organ, degree of hypoxia, and other factors. On going to high altitude, cerebral blood flow initially increases and then with acclimatization returns to near normal values (8, 32).

Studies of acute hypoxia-mediated vascular smooth muscle (VSM) responses suggest these to be mediated, in part, by K⁺ channel activation, which, in turn, plays a major role in modulating vessel contractility (1, 27). Although we and others have described a number of effects of LTH on cerebral vascular reactivity (16, 17, 34, 38), less well defined are the relative roles of K⁺ channels in these responses. In vascular smooth muscle, K⁺ channel activation plays a major role in modulating membrane potential, voltage-gated L-type Ca²⁺ channel activity, intracellular Ca²⁺ concentration ([Ca²⁺]_i), and myogenic tone (22). Four types of K⁺ channels have been identified in cerebral and other arteries: ATP sensitive (K_ATP), Ca²⁺ activated (K_Ca), voltage dependent (K_v), and inward rectifier (K_IR). Recently, we reported on some aspects of the role of these several channels in modulating the NE-induced [Ca²⁺]_i and contractile responses in normoxic adult and fetal cerebral vessels (14). LTH-mediated cerebrovascular responses are less well defined, and few studies have examined the relative roles of K⁺ channels in association with chronic hypoxia.

In the present studies, we tested the hypothesis that, in part, the LTH-associated depressed or otherwise altered tension responses seen in cerebral arteries were secondary to altered plasma membrane K⁺ channel function. We also tested the hypothesis that in the fetus the responses of cerebral K⁺ channels to LTH differed from those responses in the adult.

	METHODS

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Tissue preparation. We obtained main branch middle cerebral arteries (MCA) from 29 near-term fetuses (~140 days gestation) and 30 nonpregnant adult sheep (age 18-24 mo) that had been maintained near sea level [300 m; arterial PO₂ (Pa_O2) = 95 ± 5 and 25 ± 2 Torr for ewe and fetus, respectively]. We also obtained MCA from 28 fetuses and 30 adult ewes that had been acclimatized to high altitude (3,820 m, 12,470 ft, Pa_O2 = 60 ± 5 and 19 ± 3 Torr for ewe and fetus, respectively; Barcroft Laboratory, White Mountain Research Station, Bishop, CA) for 110+ days. (Note: to optimize animal use, other tissues from these animals were shared with other investigators in our research group.) Both nonpregnant and pregnant ewes were obtained from Nebeker Ranch (Lancaster, CA), as previously described (16, 19). The animals from high altitude were transported to our laboratory just before the studies. Shortly after arrival, we placed a tracheal catheter in the ewe, through which N₂ flowed at a rate to maintain Pa_O2 at ~60 Torr (9), and this was maintained until the time of the experimental study. All hypoxemic fetuses were singletons, and when normoxic twin fetuses were obtained, we used only one fetus of the pair. The ewes were anesthetized and euthanized with 100 mg/kg intravenous pentobarbital sodium. We removed the fetal or adult brain, placed it in iced saline, and dissected out and cleaned the cerebral arteries. Our laboratory has shown that this method of death has no significant effect on vessel reactivity compared with use of other anesthetic agents (18). To avoid the complication of endothelium-mediated effects, we removed the endothelium by carefully inserting a small wire three times (18). To confirm endothelium removal, we contracted the vessel with 10⁵ M 5-hydroxytryptamine and at the plateau added 10⁶ M ADP. Vessels that relaxed >20% after this treatment were rejected for further study. Cerebral arteries [diameter of 450 ± 25 µm for adult and 350 ± 25 µm for fetus (15), with no difference in sea level vs. high altitude] were used within a few minutes of being obtained for simultaneous measurements of tension and [Ca²⁺]_i (15). Unless otherwise noted, all chemical compounds were purchased from Sigma Chemical (St. Louis, MO).

Contractility and intracellular Ca²+ measurements. We cut the middle cerebral arteries into rings 2 mm in length and mounted them on two tungsten wires (0.13 mm diameter; A-M Systems, Carlsborg, WA). We attached one wire to an isometric force transducer (Kent Scientific, Litchfield, CT) and the other to a post attached to a micrometer that was used to vary resting tension in a 5-ml tissue bath mounted on Jasco CAF-110 intracellular Ca²⁺ analyzer (Jasco, Easton, MD). We then measured vascular tension, as previously described (13, 15). This, with vessel inside diameter measurements in combination with measurements of vessel wall thickness, length, and potassium-induced force, enabled calculation of force per unit cross-sectional area, as previously described. MCA rings were equilibrated under 0.3-g tension at 25°C for 40 min before being loaded with the acetoxymethyl ester of fura 2 (fura 2-AM; Molecular Probes, Eugene, OR), a fluorescent Ca²⁺ indicator that is a measure of mean cytoplasmic [Ca²⁺]_i (7). Fura 2 fluorescence and force were measured simultaneously at 38°C, as previously described (15). As we have noted, although some investigators may prefer the transformation of fluorescence to [Ca²⁺]_i, in tissues such as cerebral arteries the presentation of the ratio is less ambiguous (15). During all contractility experiments, we continuously digitized, normalized, and recorded contractile tensions and the fluorescence ratio (F_340/380) using an on-line computer. For all vessels, we evaluated the contractile response for tension and fluorescence ratio by measuring the maximum peak height and expressing it as percent of maximal tension achieved to 120 mM K⁺ (K_max; a measure of "efficacy") and calculated pD₂ [the negative logarithm of the 50% effective concentration, or half-maximal concentration for norepinephrine (NE), and an index of tissue "sensitivity" or "potency"] (15).

Relative roles of the several K⁺ channels with hypoxia. To determine the potential role of the several types of K⁺ channels in modulating NE-induced changes in [Ca²⁺]_i and vascular tension in the two oxygenation states, we quantified these variables in the presence of selective K⁺ channel activators or blockers. For all studies, after initial K⁺ (120 mM) depolarization to determine K_max, we performed NE dose-response curves to establish the maximum contraction, percent K_max, and pD₂.

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Statistical analysis. All values were calculated as means ± SE. In all cases, n refers to the number of vessel segments (which corresponds to the number of animals) studied. The n values for the different experiments are given in Table 1. Because of the nature of these studies, several statistical tests were used to test for significant differences. For testing differences between two groups, we used a simple unpaired Student's t-test. For multiple comparisons, one-way and two-way ANOVA (age, oxygen status) coupled with Duncan's multiple-range test was used. When appropriate, we used ANOVA with repeated measures. A P value of <0.05 was considered significant.

	RESULTS

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Contractile and [Ca²+]_i responses to K⁺ and NE. Figure 1 shows the NE-induced responses of vascular tension and the fura 2 fluorescence ratio (F_340/380), a measure of [Ca²⁺]_i, for adult and fetal middle cerebral arteries under conditions of normoxia and LTH. Maximum K⁺ (120 mM)-induced tension values (g) for normoxic and LTH adult MCA were 1.6 ± 0.1 and 1.4 ± 0.1, respectively, whereas the maximum K⁺-induced fluorescence ratios in normoxic and hypoxic adult MCA were 0.15 ± 0.02 and 0.11 ± 0.01, respectively (for n values, see Table 1). As shown in Fig. 1A, the maximum NE-induced tensions were 1.7 ± 0.1 and 1.4 ± 0.2, respectively, whereas the pD₂ values were similar. In turn, the NE-induced fluorescence ratios for the two vessel groups were 0.14 ± 0.02 and 0.11 ± 0.01 (Fig. 1B; Table 1). For normoxic and hypoxic fetal MCA, the maximum tension responses to K⁺ depolarization were 1.0 ± 0.1 and 1.1 ± 0.1, respectively (Table 1). As seen in Fig. 1, C and D, for fetal MCA the NE-induced tension values, fluorescence ratios, and pD₂ values were not significantly different between the two oxygen-exposure groups (Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Norepinephrine (NE) dose-response relationships for adult and fetal main branch middle cerebral arteries (MCA) under normoxic control conditions and in response to long-term hypoxia. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, we induced subsequent contractions using cumulative doses of NE added in half-log increments (see METHODS for details). Points shown are means ± SE. A: vascular tensions (g) for normoxic () and long-term hypoxic () adult MCA. B: fluorescence ratios (F340/380) for normoxic and hypoxic adult MCA. C: vascular tensions (g) for normoxic and long-term hypoxic fetal MCA. D: F340/380 for normoxic and hypoxic fetal MCA.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Long-term hypoxia is associated with an increase in Ca2+ sensitivity in adult, but not fetal, cerebral arteries. A: NE-induced vascular tension (g) as a function of F340/380 for normoxic control adult MCA. Slope = 7.3 ± 0.6. B: NE-induced tension (g) as a function of F340/380 for long-term hypoxic adult MCA. Slope = 15.5 ± 1.3 (P < 0.01 different from normoxic control). C: NE-induced vascular tension (g) as a function of F340/380 for normoxic control fetal MCA. Slope = 4.1 ± 0.4. D: NE-induced tension (g) as a function of F340/380 for long-term hypoxic fetal MCA. Slope = 3.7 ± 0.6.

Role of K_ATP in vascular [Ca²+]_i and tension. To examine the role of ATP-sensitive K⁺ channel activation on adult and fetal MCA tension and fluorescence ratio, we first contracted the vessels with 10⁵ M NE and then on the plateau of the contractile response administered pinacidil in increasing half-log dose increments (10⁷ to 10³ M). As shown in Fig. 3A for adult MCA, at concentrations above 10⁶ M, pinacidil inhibited tension with little difference between normoxic and LTH vessels [see Table 1 for half-maximal inhibitory concentration (pIC₅₀) values]. In contrast, whereas the fluorescence ratio showed essentially no change in normoxic adult vessels (14), long-term hypoxemic vessels displayed ~40% inhibition (Fig. 3B). As shown in Fig. 3C, fetal cerebral vessels from the LTH animals were much less sensitive to increasing pinacidil concentrations (see Table 1 for pIC₅₀ values). In addition, hypoxic fetal vessels also showed less sensitivity to pinacidil-induced inhibition of [Ca²⁺]_i (Fig. 3D, Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Long-term hypoxia is associated with decreased sensitivity of both tension and intracellular Ca2+ concentration to inhibition by the ATP-sensitive K+ channel activator pinacidil in fetal cerebral arteries. Points shown are means ± SE. A: percent inhibition of NE-induced tension in normoxic () and long-term hypoxic () adult MCA in response to pinacidil (n = 4 each). Vessels were first contracted with 105 M NE and then at the plateau of contraction the channel agonist was given in half-log increments (see METHODS for details). B: percent inhibition by pinacidil of NE-induced F340/380 (a measure of intracellular Ca2+ concentration) in normoxic and hypoxic adult MCA. C: percent inhibition of NE-induced tension in normoxic and long-term hypoxic fetal MCA in response to pinacidil (n = 3 each). Vessels were first contracted with 105 M NE, and then at the plateau of contraction the channel agonist was given in half-log increments (see METHODS for details). D: percent inhibition by pinacidil of NE-induced fluorescence in normoxic and hypoxic fetal MCA.

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Role of K_Ca in vascular [Ca²+]_i and tension. Figure 4 shows the effect of the K_Ca channel opener NS-1619 (10⁹ to 10⁶ M) on hypoxic and normoxic precontracted adult and fetal MCA. In the adult LTH vessel, tension was only modestly inhibited at NS-1619 concentrations above 3 × 10⁸ M, 27 ± 8% at 10⁶ M, significantly less than the 42 ± 9% inhibition seen in the normoxic vessel (14) (Fig. 4A; Table 1). In contrast, in hypoxic adult artery the fluorescence ratio did not change significantly in response to NS-1619 (Fig. 4B). In comparison, Fig. 4C shows the effect of K_Ca channel activation (at concentrations above 3 × 10⁸ M NS-1619) in inhibiting vascular tension only 41 ± 5% at 10⁶ M in the hypoxic fetal MCA. This significantly smaller inhibition contrasts with essentially 100% inhibition seen in the normoxic fetal vessel (14). As shown in Fig. 4D, the fluorescence ratio was essentially unchanged in the hypoxic fetal MCA in response to NS-1619 (Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Long-term hypoxia is associated with decreased inhibition of tension by the KCa channel activator NS-1619 in fetal cerebral arteries. Points shown are means ± SE. A: percent inhibition of NE-induced tension in normoxic () and hypoxic () adult main branch MCA in response to NS-1619 (n = 4 each). Vessels were first contracted with 105 M NE, and then at the plateau of contraction the channel agonist was given in half-log increments (see METHODS for details). B: lack of effect of NS-1619 in inhibiting NE-induced F340/380 response in either normoxic or hypoxic adult MCA. C: percent inhibition of NE-induced tension in normoxic and hypoxic fetal main branch MCA in response to NS-1619 (n = 4 each). Vessels were first contracted with 105 M NE, and then at the plateau of contraction the channel agonist was given in half-log increments (see METHODS for details). Points shown are means ± SE. D: relative lack of effect of NS-1619 in inhibiting NE-induced F340/380 response in either normoxic or hypoxic fetal MCA.

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Role of K_v in [Ca²+]_i and tension. We also examined the role of inhibition of K_v channels on tension and fluorescence ratio of hypoxic adult cerebral arteries. As in the other studies, we measured these variables after the administration of 10⁴ M 4-AP followed in 15 min by a NE dose response. In LTH adult MCA, 4-AP pretreatment was associated with 30-40% decreases in NE-induced tension and [Ca²⁺]_i, which were only slightly greater inhibition than that of normoxic controls (14) (Table 1). For both LTH and normoxic fetal MCA, administration of 10⁴ M 4-AP had no significant effect on either the NE dose-response of tension or fluorescence ratio (Table 1).

Role of K_IR in [Ca²⁺]_i and tension. To explore the possible effect of hypoxia in modulating K_IR channel activity, we administered 10⁵ M BaCl₂ to inhibit these channels, after which we performed a NE dose response. As we have reported (14), in normoxic adult MCA maximum vascular tension was not significantly altered (Fig. 5A), despite a modest decrease in fluorescence ratio (Fig. 5B). In contrast, in vessels from ewes subjected to LTH, BaCl₂ resulted in significant 50% reduction of maximum NE-induced tension (Fig. 5C) and fluorescence ratio (Fig. 5D) (Table 1). In the hypoxic fetal MCA, 10⁵ M BaCl₂ significantly inhibited both the NE-induced tension and fluorescence ratio, but these responses were not significantly different from those seen in normoxic fetal vessels (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   NE dose-response relationships for normoxic and hypoxic adult MCA in presence or absence of BaCl2. Points shown are means ± SE. A: vascular tension (g) for normoxic adult MCA in response to NE under control conditions (). Also shown is the NE dose-response after administration of 105 M BaCl2 (). B: F340/380 for normoxic adult MCA in response to NE under control conditions and after administration of 105 M BaCl2. C: vascular tension (g) for hypoxic adult MCA under control conditions. Also shown is the NE dose-response after administration of 105 M BaCl2. D: F340/380 for hypoxic adult MCA under control conditions and after administration of BaCl2.

	DISCUSSION

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Cerebral arteries, like most VSM, are exquisitely sensitive to K⁺ channel activation in modulating membrane potential and voltage-gated L-type Ca²⁺ channel activity. The present studies offer several important observations on the K⁺ channel responses of adult and fetal cerebral arteries exposed to long-term, high-altitude hypoxia. First, in response to K_Ca channel activation by NS-1619, LTH fetal MCA showed significantly less inhibition of NE-induced tension and [Ca²⁺]_i than normoxic controls (Fig. 4, C and D). By comparison, in LTH adult vessels, K_Ca channel activation showed modest inhibition of tension compared with normoxic control, with no significant change in [Ca²⁺]_i (Fig. 4, A and B). Second, in response to pinacidil-induced K_ATP channel activation, LTH fetal MCA showed significantly decreased sensitivity (e.g., a lower pIC₅₀ value) of both tension and [Ca²⁺]_i, compared with normoxic controls (Fig. 3, C and D). In contrast, in hypoxic adult MCA the effect of activation of K_ATP channel on tension was little different from that of normoxic controls, whereas the significantly greater inhibition of [Ca²⁺]_i again suggests somewhat greater Ca²⁺ sensitivity (Fig. 3, A and B). Third, in adult MCA, hypoxia was associated with significant inhibition by BaCl₂ of NE-induced tension and to a lesser extent [Ca²⁺]_i, again demonstrating the greater sensitivity to Ca²⁺ under conditions of chronic hypoxia (Fig. 5). In hypoxic fetal MCA, the BaCl₂-induced inhibition was not significantly different from that observed in the normoxic vessels. Fourth, notable in these studies was the fact that both the adult and fetal hypoxic cerebral arteries showed little alteration in NE-induced tension or [Ca²⁺]_i responses after activation or blockade of K_ATP, K_Ca, or K_v channels compared with normoxic controls. This suggests that, in terms of overall regulation of cerebral artery responses to LTH, K⁺ channel activity and its linkage to myogenic tone is probably of greater importance than adrenergic-mediated mechanisms per se. Fifth, in adult, but not fetal, MCA, LTH was associated with a decrease in both maximal NE-induced tension and fluorescence ratio (e.g., decreased efficacy) (Fig. 1). Because this decrease in tension was less than the decrease in [Ca²⁺]_i, LTH was associated with a significant increase in Ca²⁺ sensitivity in adult vessels (Fig. 2). Sixth, overall in response to LTH, adult cerebral arteries demonstrated increased Ca²⁺ sensitivity (Figs. 2, A and B; 3, A and B; 5, C and D). In contrast, LTH fetal cerebral arteries showed relatively little change in Ca²⁺ sensitivity (Fig. 2, C and D), with only a slight decrease when K_Ca channels were activated (Fig. 4, C and D). That is, in fetal MCA, exposure to LTH significantly decreased the inhibition of vascular tone produced by activation of K_Ca but not K_ATP channels. This was associated with a tendency for the fetal cerebral arteries to function in a manner similar to more "mature" adult vessels and was particularly evident in terms of the effect of the K_Ca channel opener NS-1619 (Fig. 4, C and D; Table 1). In addition, the differing patterns of response to activation of K_ATP and K_Ca channels suggests differential regulation of these channels by LTH. Overall, the present studies support the hypothesis that LTH is associated with significantly altered K_ATP and K_Ca channel function in cerebral arteries, especially in those of the fetus.

Developmental differences in contractile responses in long-term hypoxemia. Previously, we and others have reported significant differences in cerebral artery signal transduction mechanisms with development from fetus to adult (13, 15, 19, 26, 38), and this includes the function of K_ATP and K_Ca channels (14).

Hypoxic modulation of K+ channel function in cerebral arteries. In previous studies, our laboratory has reported for cerebral arteries of both adult and fetus that LTH is associated with a significant decrease or downregulation of ₁-adrenergic-receptor density and NE-induced Ins(1,4,5)P₃ responses (34), Ins(1,4,5)P₃-receptor density (38), and other signal transduction components (16, 17). Under normoxic conditions, vessels in the cerebral microcirculation exist in a partially contracted state and constrict further or dilate depending on the tissue requirements for blood and/or O₂. This tone is an important determinant of vascular resistance and blood pressure and to a great extent is regulated by the VSM membrane potential, which, in turn, is regulated by the plasma membrane K⁺ channels (22). Little is known of the regulation of these channels in chronic hypoxia, however. As noted earlier, the main branch MCA employed in the present studies is not the major site of vascular resistance; however, because the K⁺ channels in the smaller arterioles cannot be examined in the manner of the present studies, we trust that the insights gained from these data may also apply to the smaller vessels. Although the four distinct types of K⁺ channels are fairly well defined structurally, and selective pharmacological blockers exist for each channel type, selective activators have been described for only the K_ATP and K_Ca channels.

Role of K_ATP channels. The sensitivity of these channels to changes in cellular metabolic state (opening in response to a decrease in intracellular ATP concentration) suggests that they would be activated by hypoxia. Although some studies suggest that acute moderate hypoxia may not result in significant ATP decrease, the extent to which this changes with chronic hypoxia is unknown. In rat cerebral arteries, the K_ATP channel inhibitor glibenclamide reduced hypoxia-induced vasodilation (33). In the present study, in hypoxic adult MCA, the K_ATP channel opener pinacidil inhibited NE-induced elevation in [Ca²⁺]_i, compared with its minimal effect in the normoxic vessel (Fig. 3B). The mechanistic basis of this hypoxia-associated increase in Ca²⁺ sensitivity is not clear. LTH-associated changes in RhoA-Rho kinase, or other enzymes, by increasing phosphorylation of myosin light chain phosphatase, may increase Ca²⁺ sensitivity; however, this remains to be determined. In hypoxic fetal MCA, the reduction in pIC₅₀ values of pinacidil-induced inhibition of both tension and [Ca²⁺]_i of about one-half log (i.e., a threefold change) (Fig. 3, C and D) may be related to a similar, or other, mechanism. Again in hypoxic fetal MCA, administration of pinacidil lowered maximum NE-induced tension and [Ca²⁺]_i (Table 1). As an aside, the pinacidil concentrations required for half-maximal inhibition of ~10⁵ M is an order of magnitude higher than that observed in anesthetized cat pial arteries in vivo (12). This raises the issue of relative insensitivity of these in vitro endothelium-denuded vessel rings, compared with vessels in vivo.

Role of K_Ca channels. Although large-conductance K⁺ channels activated by membrane depolarization and an increase in [Ca²⁺]_i are an important feature of essentially all smooth muscle cells (21), the physiological regulation of K_Ca channel activity is largely unknown (22). Hypoxia activates K_Ca channels in pulmonary artery (35) and portal vein (20) VSM cells, and such activation is associated with Ca²⁺ release from intracellular stores. With development from fetus to adult, ovine pulmonary arteries demonstrate a maturational change from K_Ca to K_v channels (30), but whether such change occurs in other vessels is unknown.

Role of K_v channels. These channels (also called delayed-rectifier channels) open to allow K⁺ efflux and membrane repolarization when the membrane is depolarized (22). In pulmonary artery VSM cells, K_v channels can be regulated by cellular O₂ tension (1, 2, 27), and hypoxia-induced inhibition of K_v channels is an important mechanism of pulmonary hypoxic vasoconstriction (23, 36). A novel delayed-rectifier K_v channel in pulmonary arterial smooth muscle cells, activated by ATP, may be the channel inhibited by hypoxia (24, 25). Nonetheless, in the present study LTH showed little influence on 4-AP modulation of NE-induced contraction (Table 1).

Role of K_IR channels. In contrast to K_Ca and K_v channels, which are activated by membrane depolarization, the K_IR channels are activated by membrane hyperpolarization and may play a role in maintaining resting membrane potential, although this is poorly understood (6, 22). As noted, the effect of BaCl₂ in both LTH and normoxic fetal MCA may have been secondary to Ba²⁺ blockade of L-type Ca²⁺ channels rather than to inhibition of K_IR channels per se (Table 1), and the mechanisms of the significant inhibition of NE-induced contraction by BaCl₂ in hypoxic adult vessels (Fig. 5C) are unclear.

Perspectives. The role of cerebral artery plasma membrane K⁺ channels, and their sensitivity to various agents, may differ greatly as a function of species and developmental age. Here we show that particularly the K_Ca and K_ATP channels are affected by LTH. By quantifying simultaneously [Ca²⁺]_i and tension, the present studies are the first to demonstrate in fetal and adult cerebral arteries the effect of high-altitude LTH on the function of several K⁺ channels. The dependence of fetal cerebral arteries on extracellular Ca²⁺ (15) is associated with these vessels being exquisitely sensitive to K_ATP and K_Ca channel activation, compared with adult, and these channels play an important, albeit differing, role in the regulation of vascular tone in both fetal and adult cerebral arteries (14). As evidenced in the present report, in adult cerebral arteries LTH significantly decreased NE-induced contractility (Fig. 1) and increased [Ca²⁺]_i sensitivity (Fig. 2). In addition, although hypoxemic adult cerebral arteries were only moderately affected by the K_ATP channel opener pinacidil or the K_Ca channel opener NS-1619, their sensitivity to Ca²⁺ was significantly increased. In contrast, LTH fetal cerebral arteries showed significantly decreased inhibition of NE-induced contraction by the K_Ca channel opener NS-1619 and to a lesser extent by the K_ATP channel opener pinacidil. The differing pattern of these responses strongly supports the idea of differential regulation of these channels by hypoxia. In some respects (e.g., less inhibition by NS-1619), LTH was associated with the fetal cerebral arteries functioning more like mature adult than fetal normoxic vessels. In both adult and fetal vessels, LTH appeared to affect K_ATP and K_Ca function per se, without significantly altering the coupling of these channels to adrenergic-mediated contraction. Because cerebral artery vascular tone appears to be determined primarily by myogenic regulation, and because both K_ATP and K_Ca channels play a key role in regulating this tone (21, 22, 28), the present findings that chronic hypoxia suppressed or otherwise altered K_ATP and K_Ca channel activities in a developmental age-dependent manner supports the hypotheses that stimulated the study. This may have significant implications in understanding the regulation of cerebral blood flow in both the chronically hypoxic fetus and adult. For instance, in the hypoxic fetus the cerebral arteries may be near-maximally dilated so that K⁺ channel activation with hyperpolarization can result in little further inhibition of tone. Obviously, many questions remain as to the mechanisms by which LTH alters K⁺ channel activity and Ca²⁺ sensitivity.