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Role of endothelial intermediate conductance K_Ca channels in cerebral EDHF-mediated dilations

Department of Anesthesiology, Baylor College of Medicine, Houston, Texas 77030

Submitted 22 April 2003 ; accepted in final form 11 June 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The present study evaluated the role of endothelial intermediate conductance calcium-sensitive potassium channels (IK_Ca) in the mechanism of endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in pressurized cerebral arteries. Male rat middle cerebral arteries (MCA) were mounted in an isolated vessel chamber, pressurized (85 mmHg), and luminally perfused (100 µl/min). Artery diameter was measured simultaneously with either endothelial intracellular Ca²⁺ concentration ([Ca²⁺]_i; fura-2) or changes in endothelial membrane potential [4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS)]. Nitric oxide synthase and cyclooxygenase inhibitors were present throughout. Luminal application of UTP produced EDHF-mediated dilations that correlated with significant endothelial hyperpolarization. The dilation and endothelial hyperpolarization were virtually abolished by inhibitors of IK_Ca channels but not by selective inhibitors of small or large conductance K_Ca channels (apamin and iberiotoxin, respectively). Additionally, direct stimulation of endothelial IK_Ca channels with 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial hyperpolarization and vasodilatation that were blocked by inhibitors of IK_Ca channels. 1-EBIO hyperpolarized the endothelium but did not affect endothelial [Ca²⁺]_i. We conclude that the mechanism of EDHF-mediated dilations in cerebral arteries requires stimulation of endothelial IK_Ca channels to promote endothelial hyperpolarization and subsequent vasodilatation.

calcium; brain; purinergic; small conductance potassium channels; large conductance potassium channels; endothelium-derived hyperpolarizing factor

There are, however, a number of unifying hallmarks common among EDHF-mediated dilations. One in particular is the essential involvement of K_Ca [for review see Feletou and Vanhoutte (25)]. Although it was initially assumed that the K_Ca channels critical to EDHF-mediated dilations were large conductance K_Ca channels (BK_Ca) located on the smooth muscle (44), it is now generally believed that the K_Ca channels involved include intermediate conductance K_Ca (IK_Ca) alone or in combination with small conductance K_Ca (SK_Ca) located on the endothelium (3, 19, 20, 23, 30). The mechanism by which endothelial K_Ca channel activation translates into a smooth muscle hyperpolarization is still a matter of debate, but has been proposed to result from either 1) direct transfer of hyperpolarization from the endothelium via myoendothelial gap junctions (5, 6, 18, 20, 24, 45, 49) or 2) stimulation of smooth muscle inwardly rectifying K channels (K_IR) channels or Na⁺-K⁺-ATPase by modest elevations in extracellular K⁺ concentration ([K⁺]_o) in the intercellular space (20).

One additional role for endothelial K_Ca channels that is often assumed in intact arteries is to facilitate greater influx of extracellular Ca²⁺ into the endothelium. It is proposed that activated endothelial K_Ca channels hyperpolarize the endothelium, thus creating a greater electrical driving force for Ca²⁺ influx [for review, see Nilius and Droogmans (42)]. Given the essential roles for both Ca²⁺ and K_Ca channels in EDHF-mediated dilations, it is reasonable to speculate that endothelial K_Ca channels contribute to endothelial Ca²⁺ reaching or staying at the Ca²⁺ threshold required for EDHF-mediated dilations. However, it must be noted that, whereas a role for endothelial K channel activation on Ca²⁺ flux is widely supported from endothelial cell culture models (32, 35–37, 46), there are little data available regarding endothelial intracellular Ca²⁺ concentration ([Ca²⁺]_i) within the context of intact arteries. Furthermore, the data that exist from pressurized artery preparations appear to conflict regarding the relevance of membrane potential on endothelial Ca²⁺ handling (27, 33, 50).

In the present studies, experiments were designed to determine the role and subtype of endothelial K_Ca channels involved in EDHF-mediated dilations within pressurized cerebral arteries. Our use of an intact pressurized artery preparation is significant given that pressurized and nonpressurized vessel preparations differ in many respects, such as in resting membrane potential and [Ca²⁺]_i (29, 34). We evaluated the following three hypotheses regarding the mechanism of EDHF-mediated dilations in pressurized middle cerebral arteries (MCAs): 1) IK_Ca channel activation and endothelial hyperpolarization is critical to the EDHF-dependent mechanism, 2) IK_Ca channel activation and endothelial hyperpolarization is sufficient for EDHF-mediated vasodilatation, and 3) IK_Ca channel-mediated hyperpolarization of the endothelium is required for endothelial [Ca²⁺]_i to reach the threshold for initiating EDHF-mediated dilations.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
All experiments were approved by the Animal Protocol Review committee at Baylor College of Medicine. A total of 52 male Long-Evans rats (275–350 g) were anesthetized with isofluorane and decapitated. The brain was quickly harvested and placed in an ice-cold Krebs solution for the removal of the left and right MCAs. MCAs were kept in ice-cold Krebs until needed; all vessels were used within 4 h of being removed from the brain. Because the primary focus of these studies was to evaluate EDHF-mediated responses, inhibitors of NO synthase and cyclooxygenase [50 µM N^G-nitro-L-arginine methyl ester (L-NAME) and 10 µM indomethacin, respectively] were added to the luminal and abluminal Krebs buffer for all experiments.

Mounting of pressurized/perfused MCAs. MCAs were cannulated with two glass micropipettes as described in detail previously (39). In brief, MCAs were secured to the micropipettes with 12-0 nylon suture and verified to be free of leaks. Warmed (37°C) and gassed (20% O₂-5% CO₂-balance N₂) Krebs was circulated through the chamber (abluminally) and perfused through the lumen of the artery (100 µl/min). Mean intraluminal pressure was set to 85 mmHg and monitored by two inline pressure transducers. After mounting of the MCA, the vessel chamber was placed on the stage of an inverted fluorescence microscope equipped for the simultaneous measurement of artery diameter and either endothelial [Ca²⁺]_i or changes in endothelial membrane potential (V_m).

Simultaneous measurement of endothelial [Ca²⁺]_i and diameter in intact cerebral arteries. The method for selectively measuring endothelial [Ca²⁺]_i and diameter in pressurized MCA has recently been described in detail (39). Briefly, the endothelium was selectively loaded with fura-2 AM by adding it to the luminal perfusate. Fura-2 AM (0.67 µM) was coadministered with pluronic F-127 (0.02%) to ensure a more even dispersion of fura-2 AM. The artery was perfused luminally with the fura-2 solution for 4 min before a washout period of 15 min. This short incubation with a low concentration of fura-2 AM ensured that the endogenous endothelial esterases were not overwhelmed, thus ensuring that the fura-2 free acid was contained to the endothelium.

Measurement of endothelial [Ca²⁺]_i was performed by alternating between 340 and 380 nm bandpass excitation filters and measuring the fura-2 fluorescence at 510 nm (28, 39). The artery was transilluminated with a red light (>690 nm) for simultaneous measurement of diameter (Intracellular Imaging; Cincinnati, OH). Endothelial [Ca²⁺]_i was determined from a series of in situ calibration curves on the basis of the following equation [Ca²⁺]_i = [(R – R_min)/(R_max – R)] K_d, where R is the ratio of 340 to 380 fluorescence, R_min is the ratio in Ca²⁺-free conditions, R_max is the ratio in saturating Ca²⁺ conditions, is the ratio of the 380 fluorescence with Ca²⁺ unbound to bound, and K_d is the dissociation constant for fura-2 to Ca²⁺. An in situ calibration produced the following values: 1.52 (R_max), 0.18 (R_min), and 4.35 (). The K_d was determined to be 282 nM (34).

Measurement of endothelial membrane potential in intact cerebral arteries. Endothelial membrane potential (V_m) changes were measured in pressurized MCA by using a voltage-sensitive dye, 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS). This dye has previously been utilized to indicate changes in endothelial membrane potential in intact peripheral microvessels (1, 2, 10, 41). Selective endothelial loading of di-8-ANEPPS was achieved by delivering di-8-ANEPPS (10 µM/0.1% pluronic) luminally for 10 min followed by a 10-min washout period. During incubation, the dye becomes associated with the endothelial membrane. Confocal microscopy was utilized to ensure that the dye was confined to the endothelium (see Confocal microscopy of pressurized/perfused cerebral arteries). The ratio of the 560 and 620 emissions (excitation at 475 nm) changes with endothelial membrane potential such that a decrease in the ratio (560/620) indicates a hyperpolarization and an increase indicates a depolarization. The 560/620 ratios were acquired at a frequency of 12.5 Hz by using a fluorometer equipped with matched spinning excitation and emission filter wheels (C & L Instruments; Hummelstown, PA). Because changes in endothelial V_m result in relatively small changes in fluorescence intensity, we felt it was important to eliminate changes in fluorescence intensity due to vasodilatation. We therefore used an inhibitor of smooth muscle actin polymerization (10 µM cytochalasin D administered abluminally) (13) to dilate the arteries before administering the experimental drug. In some vessels, we used papaverine (300 µM, abluminally) as a vasodilator and obtained similar results as with cytochalasin D (not shown). Artery diameter was measured simultaneously with di-8-ANEPPS fluorescence by using an infrared light source and infrared-sensitive video camera. However, because the arteries were predilated, the reported diameter responses to the experimental drugs were obtained in a separate set of vessels.

Confocal microscopy of pressurized/perfused cerebral arteries. Nipkow-type confocal microscopy was used to confirm that the di-8-ANEPPS dye was confined to the endothelium. The vessel chamber containing pressurized/perfused arteries was mounted on the stage of a Nikon TE-2000 fluorescence microscope (Nikon Instruments) coupled with a CARV confocal module set up for detection of di-8-ANEPPS fluorescence (Atto Instruments, Rockville, MD). The di-8-ANEPPS dye was loaded as described above before imaging.

Delivery of pharmacological compounds to the artery. Luminal delivery of pharmacological compounds was achieved by adding the compound to one of the luminal reservoirs connected to the inflow pipette. A miniaturized manifold system was utilized to substantially reduce the dead volume in the connecting tubing and thus speed delivery of the compound to the luminal compartment of the perfused artery. Abluminal delivery of pharmacological compounds was achieved by adding the compound to the abluminal perfusate reservoir. Compounds reached the luminal or extraluminal surface of the artery with a <30-s lag time.

Chemicals and buffers. All drugs and chemicals were obtained from Sigma (St. Louis, MO) with the exceptions of fura-2 AM (TefLabs; Austin, TX), di-8-ANEPPS (Molecular Probes; Eugene, OR), 1-ethyl-2-benzimidazolinone (1-EBIO; Tocris; Ellisville, MO), and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM)-34 (a generous gift from Dr. Heike Wulff, University of California, Irvine). The standard Krebs buffer solution consisted of the following (in mM): 119 NaCl, 4.7 KCl, 21 NaHCO₃, 1.18 KH₂PO₄, 1.17 MgSO₄, 0.026 EDTA, 1.6 CaCl₂, and 5.5 glucose. The high K⁺ buffers consisted of either 60 mM [K⁺] or 90 mM [K⁺] (calibration of di-8-ANEPPS) with the appropriate reduction in Na⁺ concentration to maintain an isotonic buffer; the remaining ingredients remained the same as the standard buffer. The Krebs buffer was gassed with a mixture of 20% O₂-5% CO₂-75% N₂ to achieve a pH of 7.4 and a PCO₂ of 35 mmHg.

Iberiotoxin (IbTx; 100 nM) selectively blocks BK_Ca channels, whereas charybdotoxin (ChTx; 70 nM) is a nonselective BK_Ca/IK_Ca channel blocker. ChTx has also been shown to block the delayed rectifier (K_v) channels K_v1.2 and K_v1.3 (4). TRAM-34 (10 µM) and clotrimazole (CTZ; 10 µM) are IK_Ca channel blockers (54). TRAM-34 is a recently developed structural analog of CTZ that is a potent IK_Ca channel blocker and lacks the cP450 inhibitory activity of CTZ (54). Apamin (Apa; 100 nM) is a selective SK_Ca channel blocker. High K⁺ (60 mM) Krebs buffer was used to nonselectively block all K channels.

Calculations and statistical methods. All data are presented as means ± SE. Percent tone is defined as %tone = [(D_max – D_t)/D_max] x 100, where D_t is the vessel diameter after development of spontaneous tone and D_max is the maximum diameter of the artery corresponding to the Ca²⁺-free diameter. Percent diameter change is defined as %diameter change = [(D – D_base)/(D_max – D_base)] x 100, where D is the diameter in response to a given vasodilator and D_base is the diameter before the addition of the vasodilator. Percent maximum diameter is defined as %max diameter = (AUC_drug/AUC_max) x 100, where AUC_drug is the baseline corrected area under the curve for 200 s of the response and AUC_max is the maximum possible area under the curve for that time period [(maximum diameter – resting diameter) x 200 s].

For comparison of concentration-response curves, a two-way repeated-measures ANOVA was utilized followed by a Tukey's test for individual comparisons when appropriate. For comparison of multiple treatments within one group, a one-way repeated-measures ANOVA was used. Comparison of single treatments between two groups was performed by either paired or unpaired t-test. Significance was defined as P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Pressurized/perfused arteries developed spontaneous tone (constriction) within 20 to 40 min of mounting in the vessel chamber. The final tone developed by the artery was dependent on which combination of K⁺ channel inhibitors was used in addition to L-NAME and indomethacin and are summarized in Table 1.

The ability to selectively deliver di-8-ANEPPS to the endothelium was evaluated for this isolated vessel preparation by confocal microscopy. In three separate vessels studied, di-8-ANEPPS fluorescence appeared uniformly along the lumen of the vessel and was clearly confined to the endothelium (data not shown). In a fourth vessel, the dye leaked from an open side branch and resulted in dye loading of the smooth muscle. The smooth muscle loading was evident as bright circumferential bands perpendicular to the direction of flow and was clearly evident by confocal or standard fluorescence microscopy.

The di-8-ANEPPS ratio changes were calibrated by using valinomycin (5 µM) and various [K⁺]_o to voltage clamp endothelial V_m (52). Valinomycin is a K⁺ ionophore that serves to increase the conductance value for K⁺ and thus increase the contribution of inner/outer K⁺ concentrations to the overall membrane potential. Luminal valinomycin was delivered in the presence of both 5.9 and 90 mM [K⁺]_o. From the Nernst equation, these concentrations of [K⁺]_o should produce endothelial V_m values of –82 and –10 mV, respectively. The full range of the V_m change would be 72 mV. The measured corresponding range of the di-8-ANEPPS ratio change to 5.9 and 90 mM [K⁺]_o was 5.6 ± 0.5% (n = 3). Therefore, the calculated relationship between millivolt and ratio change is 12.9 mV for each 1% change in the 560/620 ratio. This relationship compares well with what others have reported from peripheral vessels (10–12 mV/1% ratio change) (2, 11).

Figure 1 shows a representative experiment in which endothelial V_m was determined with di-8-ANEPPS. Cytochalasin D (10 µM) administered abluminally produced a rapid and near-maximal dilation that was sustained throughout the experiment (Fig. 1A). Fluorescence intensities (F₅₆₀ and F₆₂₀) both decreased on artery dilation (Fig. 1B). The drop in fluorescence resulted from the near and far portions of the vessel (from the objective) going beyond the depth of field of the objective. However, as the drops in intensities were equivalent for each wavelength, the 560/620 ratio remained essentially the same (Fig. 1C). The luminal addition of UTP (10^–5 M) produced minimal further change in diameter, whereas the F₅₆₀ (dotted line) and F₆₂₀ (solid line) changed in opposite directions (F₅₆₀ decreased and F₆₂₀ increased; Fig. 1B). The 560/620 ratio decreased by 4%, thus indicating an endothelial hyperpolarization.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Representative traces depicting simultaneous measurement of middle cerebral artery diameter (A), di-8-ANEPPS fluorescence (F560 [dotted line] and F620 [solid line]) from the endothelium (B), and the ratio of F560/F620 (C). The ratio of F560/F620 reflects membrane potential changes in the endothelium in which hyperpolarization is reflected by a decrease in the ratio, and depolarization is reflected by an increase in the ratio. Cytochalasin D, an inhibitor of smooth muscle actin polymerization, was used to predilate the artery to eliminate any motion artifact associated with the response to UTP. Arrows indicate the addition of abluminal cytochalasin D (10 µM) followed by luminal UTP (10 µM). Note that UTP resulted in a decrease in the ratio due to opposite changes in F560 and F620. All experiments were performed in the presence of NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin.

Diameter and endothelial V_m responses to luminally delivered UTP (10^–5 M) were evaluated in the absence and presence of ChTx (70 nM). Diameter responses are reported from a separate group of vessels, because cytochalasin D was used to predilate the endothelial V_m group vessels. Diameter responses to UTP are reported as the %maximum achievable area under the curve over a 200-s time period (see MATERIALS AND METHODS). The diameter and V_m results are summarized in Fig. 2. Luminal UTP produced significant vasodilatation (88 ± 3%) and endothelial hyperpolarization (–42 ± 2 mV). The addition of ChTx to the bath virtually eliminated both the dilation (27 ± 14%) and endothelial hyperpolarization to UTP (–5 ± 13 mV). These results indicate that the dilation and endothelial hyperpolarization are mediated primarily by ChTx-sensitive K channels.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Summary of %maximum diameter (A) and change in endothelial membrane potential (Vm; B) in response to 10 µM UTP. Responses to luminal UTP were evaluated in the absence and presence of charybdotoxin (ChTx; 70 nM). The diameter and Vm responses were performed separately, because cytochalasin D was used to predilate the vessels in which Vm was measured (n = 4–9). All experiments were performed in the presence of L-NAME and indomethacin. *P < 0.05 compared with control group by unpaired t-test.

Because ChTx has been shown to inhibit both BK_Ca and IK_Ca channels as well as some voltage-sensitive K channels (K_v1.2 and K_v1.3) (4), we sought to determine which ChTx-sensitive channels were essential to the EDHF-mediated response. The %max diameter (Fig. 3) reflects the measured AUC in response to UTP as a percentage of theoretical maximum AUC over a 200-s period (see MATERIALS AND METHODS). Control responses to luminal 10 µM UTP produced significant artery vasodilatation (82 ± 2%) over the measurement period. Application of a selective BK_Ca channel blocker (IbTx 100 nM) had no significant effect on EDHF-mediated dilations to UTP (73 ± 11%). Similarly, application of a selective SK_Ca channel blocker (100 nM Apa) had no effect on dilations to UTP (88 ± 4%). However, application of a selective IK_Ca channel blocker (TRAM-34 10 µM) significantly reduced EDHF-mediated dilations to UTP (32 ± 4%). Combined inhibition of IK_Ca and K_v channels (TRAM-34 and 3 mM 4-aminopyridine) similarly reduced dilations to UTP (25 ± 6%), although the inhibition was not significantly different than with TRAM-34 alone (P = 0.84, two-way repeated-measures ANOVA). Thus it appears that IK_Ca channels by themselves contribute considerably to the EDHF-mediated dilation and likely reflect the critical K channel blocked by ChTx.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Summary of %maximum diameter responses to luminal UTP (10 µM) with iberiotoxin (IbTx), apamin (Apa), 1-[(2-chlorophenyl)-diphenylmethyl]-1H-pyrazole (TRAM)-34 alone, or TRAM-34 plus 4-aminopyridine (4-AP). The filled bar represents the pooled control response to UTP performed before the addition of IbTx (100 nM; n = 3), Apa (100 nM; n = 5), TRAM-34 (10 µM; n = 8), or TRAM-34 plus 4-AP (3 mM; n = 5). A washout period of at least 30 min was allowed between the two UTP responses. L-NAME and indomethacin were present throughout. *P < 0.05 compared with control group by paired t-test.

If activation of IK_Ca channels is the critical down-stream event from elevated endothelial [Ca²⁺]_i in the mechanism of EDHF-mediated vasodilatation, then direct activation of IK_Ca channels should produce a dilatation. We therefore utilized an IK_Ca channel opener (1-EBIO) to determine the effect of direct activation of endothelial IK_Ca channels on vessel diameter. Luminal administration of 1-EBIO (1 to 300 µM) produced concentration-dependent vasodilatation that was significantly inhibited by nonselective K channel inhibition with luminal 60 mM K⁺ (Fig. 4). Furthermore, when 1-EBIO was administered abluminally (smooth muscle side), responses were significantly attenuated compared with luminal administration.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Summary of %diameter change in response to 1-ethyl-2-benzimidazolinone (1-EBIO). 1-EBIO was delivered luminally (n = 7) as well as abluminally (n = 6). In addition, 1-EBIO was delivered luminally in the presence of 60 mM K+ containing Krebs (luminal w/high K+; n = 5). L-NAME and indomethacin were present throughout. Abluminal and luminal with high K+ responses were significantly different from the luminal response (2-way repeated measures ANOVA). *Individual differences compared with luminal response (P < 0.05, Tukey's test).

Although 1-EBIO has been demonstrated to be an effective activator of IK_Ca channels (15, 21, 31, 51), it was still incumbent on us to confirm that 1-EBIO acts specifically via stimulation of IK_Ca channels and produces the predicted hyperpolarization of the endothelium in this vessel preparation. Figure 5 summarizes diameter responses as a percentage of control dilations in response to luminal delivery of 1-EBIO (300 µM) with selected BK_Ca and IK_Ca channel blockers. Note that 1-EBIO produced a significant dilation that was unaffected by a selective BK_Ca channel blocker (IbTx 100 nM, n = 3) but was significantly attenuated by a BK_Ca/IK_Ca channel blocker (ChTx 70 nM, n = 3) or by an IK_Ca channel blocker (CTZ 10 µM, n = 4). In addition, Fig. 6 shows that luminal 1-EBIO resulted in significant hyperpolarization of the endothelium (–26 ± 4 mV) that was significantly attenuated in the presence of 100 nM ChTx (–9 ± 2 mV, P < 0.01). Taken as a whole, these data indicate that luminal 1-EBIO acts primarily by stimulating endothelial IK_Ca channels. Furthermore, the direct activation of endothelial IK_Ca channels is sufficient to produce significant endothelial hyperpolarization and subsequent vasodilatation.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Summary of diameter change (as %paired control response) in response to 300 µM 1-EBIO in the presence of either IbTx (n = 3), ChTx (n = 3), or clotrimazole (CTZ; n = 4). Data reflect the diameter change to 1-EBIO in the presence of the various KCa blockers as a percentage of the control response. A washout period of at least 30 min was allowed after the first 1-EBIO response. L-NAME and indomethacin were present throughout. *P < 0.05 compared with initial response by paired t-test.

View larger version (6K): [in this window] [in a new window]   Fig. 6. Summary of endothelial Vm in response to luminal 1-EBIO. 1-EBIO (300 µM) was administered in the absence and presence of ChTx (70 nM; n = 5 each). L-NAME and indomethacin were present throughout. *P < 0.01 compared with ChTx(-) response by unpaired t-test.

We then sought to determine whether 1-EBIO-mediated vasodilatation was a direct result of IK_Ca channel-mediated endothelial hyperpolarization or whether perhaps IK_Ca-mediated endothelial hyperpolarization was simply a means to increase endothelial [Ca²⁺]_i. For instance, as the endothelial membrane potential has been shown to modulate Ca²⁺ influx, a possible indirect mechanism for 1-EBIO-dependent dilations could be to promote endothelial Ca²⁺ influx secondary to endothelial hyperpolarization. In this latter scenario, 1-EBIO-mediated hyperpolarization would promote an influx of Ca²⁺, thus activating some other pathway or enzyme in the EDHF-dependent mechanism. To evaluate these possible mechanisms of 1-EBIO-mediated vasodilatation, endothelial [Ca²⁺]_i was measured in response to 1-EBIO. Endothelial [Ca²⁺]_i was determined fluorometrically by selectively loading the endothelium with fura-2 as demonstrated previously (39, 40). The effect of luminal 1-EBIO on endothelial [Ca²⁺]_i and diameter were determined simultaneously. Luminal UTP (10^–5 M) was subsequently administered to the same vessel as a positive control. Figure 7A summarizes artery diameter in response to 300 µM 1-EBIO. The dilation to UTP (10^–5 M) reflects a steady-state diameter equivalent to 99 ± 4% of maximum. Figure 7B summarizes the corresponding endothelial [Ca²⁺]_i in response to 1-EBIO and to the subsequent addition of UTP. Note that 1-EBIO did not alter endothelial [Ca²⁺]_i despite significant artery dilation. Importantly, the lack of increase in endothelial [Ca²⁺]_i to 1-EBIO was not due to an inability of the endothelium to increase [Ca²⁺]_i, as significant increases to luminal UTP were obtained from the same arteries. Thus it appears that 1-EBIO-mediated vasodilatation via stimulation of endothelial IK_Ca channels and endothelial hyperpolarization does not result from an increase in endothelial [Ca²⁺]_i. Rather, it appears that vasodilatation results directly from IK_Ca channel activation and subsequent endothelial hyperpolarization.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Summary of simultaneous measurements of diameter (A) and endothelial [Ca2+]i (B) in response to 300 µM 1-EBIO followed by 10 µM UTP (n = 4). 1-EBIO and UTP were each delivered luminally. Responses to UTP were performed as a control to verify that there was not generalized dysfunction in endothelial Ca2+ regulation. L-NAME and indomethacin were present throughout. *P < 0.05 compared with baseline values by 2-way repeated-measures ANOVA followed by Tukey's test for individual comparisons.

As EDHF-mediated dilations have been shown to critically depend on elevations in endothelial [Ca²⁺]_i, we also sought to determine whether the endothelial hyperpolarization was required for the production of sufficient increases in endothelial [Ca²⁺]_i to elicit EDHF-mediated dilations with an EDHF-dependent agonist. Artery diameter and endothelial [Ca²⁺]_i were measured simultaneously after the addition of luminal UTP (10^–7 to 10^–5 M). Figure 8 shows responses for control, high luminal K⁺, and ChTx/Apa. The addition of either luminal K⁺ (nonselective neutralization of all endothelial K⁺ channels) or ChTx/Apa (inhibition of all K_Ca) resulted in complete elimination of EDHF-mediated dilations (Fig. 8A). However, peak endothelial Ca²⁺ responses were not at all attenuated by the inhibition of either K_Ca channels or all K⁺ channels (Fig. 8B). Furthermore, plateau [Ca²⁺]_i (measured at 200 s from onset of Ca²⁺ increase) was not attenuated after K⁺ channel inhibition in either group (see Table 2). Thus in this intact artery preparation, neither peak [Ca²⁺]_i nor plateau [Ca²⁺_i] responses were sensitive to K channel activation. In fact, endothelial [Ca²⁺]_i responses in the high K⁺ and ChTx/Apa groups easily exceeded the [Ca²⁺]_i threshold (340 nM) normally needed to elicit EDHF-mediated dilations (40). These data demonstrate that endothelial hyperpolarization does not significantly affect increases in endothelial [Ca²⁺]_i in intact cerebral arteries. In addition, these data demonstrate that inhibition of EDHF-mediated dilations by K channel blockers is not due to a diminished capacity of the artery to elevate endothelial [Ca²⁺]_i.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Summary of simultaneous measurements of diameter (A) and endothelial [Ca2+]i (B) in response to UTP (10–7 to 10–5 M). All experiments were performed in the presence of L-NAME and indomethacin. Responses to UTP were performed in the presence of L-NAME/indomethacin alone (control; n = 5), with luminal 60 mM K+ (high K+; n = 5), and in the presence of ChTx/Apa (n = 4). Diameter responses for both high K+ and ChTx/Apa were significantly different compared with control (2-way repeated-measures ANOVA). *Individual differences (P < 0.05, Tukey's test).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The present studies provide new evidence for a critical role of endothelial IK_Ca channels in EDHF-mediated dilations in pressurized cerebral arteries. Herein, we evaluated the hypotheses that 1) IK_Ca channel activation and endothelial hyperpolarization is critical for agonist-mediated EDHF responses, and 2) selective activation of IK_Ca channels is sufficient to promote endothelial hyperpolarization and vasodilatation. These hypotheses were addressed in two ways. First, we used UTP to evaluate the role of IK_Ca channel activation on endothelial hyperpolarization and vasodilatation in response to an EDHF-dependent agonist. Second, we used an IK_Ca channel agonist (1-EBIO) to evaluate the effect of directly activating endothelial IK_Ca channels on endothelial membrane potential and vessel diameter. We also evaluated the hypothesis that IK_Ca channel-mediated hyperpolarization of the endothelium is required for endothelial [Ca²⁺]_i to reach the threshold for initiating EDHF-mediated dilations within the context of pressurized cerebral arteries. The hypotheses are addressed further below.

The first step in demonstrating that endothelial IK_Ca channel activation results in endothelial hyperpolarization and vasodilatation was to develop a method for measuring endothelial membrane potential in pressurized arteries. The gold standard for measuring cell membrane potential has traditionally been by microelectrode impalement. However, measurement of endothelial membrane potential in the pressurized MCA preparation is exceedingly difficult, as it would entail passing a microelectrode through two to three layers of smooth muscle and into a very thin endothelial cell. To our knowledge, measurement of endothelial membrane potential by microelectrode in a pressurized artery has only been reported by a single laboratory (24, 53). We therefore sought to apply a recently developed technique that utilizes a ratiometric voltage-sensitive fluorescent dye, di-8-ANEPPS, to measure changes in membrane potential. Di-8-ANEPPS has been used successfully for that purpose in a variety of tissues, including the endothelium from intact pressurized peripheral blood vessels (1, 2, 10, 11, 41). In the present study, we demonstrated that luminally applied di-8-ANEPPS could be confined to the endothelium and used to indicate changes in endothelial V_m in a pressurized cerebral artery preparation. We also performed an in vitro calibration of the dye and determined a relationship of 12.9 mV/1% ratio change. This relationship compares well with what others have reported from peripheral vessels (10–12 mV/1% ratio change) and thus appears to be a reasonable approximation of actual membrane potential change (2, 11).

Luminal application of UTP produced significant hyperpolarization of the endothelium ( = –43 mV) as well as maximal EDHF-dependent dilations. Furthermore, the endothelial hyperpolarization to UTP was virtually abolished after the administration of ChTx. These data demonstrated that EDHF-mediated dilations to UTP involve significant endothelial hyperpolarization and that ChTx-sensitive channels were the primary effectors of that hyperpolarization (Fig. 2). The extent to which UTP hyperpolarized the endothelium falls within the upper range of agonist-mediated endothelial hyperpolarization ( = –20 to –45 mV) reported from nonpressurized peripheral tissues (3, 8, 12, 20, 21, 30, 32, 38). To our knowledge, only one previous study (53) has reported endothelial membrane potential in an EDHF-mediated response from a pressurized vessel. In that study, Welsh and Segal (53) demonstrated endothelial hyperpolarizations to ACh (15–32 mV) within hamster cheek pouch arterioles. Interestingly, the resting endothelial membrane potential that they report (–30 mV) is notably depolarized from that of nonpressurized artery endothelium (–42 to –65 mV) (14, 21, 22, 26, 38). Thus a more depolarized resting endothelial membrane potential in the pressurized artery would permit a greater possible hyperpolarization and might contribute to the reason that our hyperpolarization values fall at the upper end of the nonpressurized range.

Luminal 1-EBIO was used to determine the effect of activating endothelial IK_Ca channels directly. Activation of endothelial IK_Ca channels with 1-EBIO resulted in vasodilatation and endothelial hyperpolarization ( = –26 mV). Furthermore, both vasodilatation and hyperpolarization were significantly attenuated after the application of ChTx. It should be noted that, whereas 1-EBIO has been shown to be a good activator of IK_Ca channels and to possess good selectivity of IK_Ca channels over BK_Ca channels (15, 21, 31, 51), it may have additional effects that should be keep in mind. For instance, 1-EBIO has been shown to activate certain SK_Ca channels (43) as well as to promote relaxation of the smooth muscle through a mechanism not involving hyperpolarization (51). In our studies, 1-EBIO produced concentration-dependent dilations when delivered luminally (to the endothelium). When delivered abluminally (to the smooth muscle), however, the dilations were significantly attenuated. The significantly greater potency of 1-EBIO when delivered luminally versus abluminally suggests that the primary site of action is on the endothelium. The vasodilatation to luminal 1-EBIO was unaffected by the presence of a selective BK_Ca channel blocker, IbTx, demonstrating that BK_Ca channels are not involved in the 1-EBIO response (Fig. 5). However, vasodilatation was significantly attenuated when 1-EBIO was administered in the presence of either ChTx, CTZ, or high luminal K⁺ (Figs. 4 and 5). Our data indicate that SK_Ca channels do not play a significant role in the vasodilatation to 1-EBIO in this particular preparation, because the inhibition with ChTx or CTZ was not significantly different from that with nonspecific K channel inhibition (high luminal K⁺). Together, these data indicate that luminal 1-EBIO acts primarily on endothelial IK_Ca channels in the pressurized MCA preparation. Furthermore, endothelial IK_Ca channels are capable of promoting significant endothelial hyperpolarization when directly activated.

Although arteries dilated subsequently to 1-EBIO-mediated endothelial hyperpolarization, it was not clear where endothelial hyperpolarization fit into the mechanism. Because endothelial membrane potential has been shown to modulate Ca²⁺ influx, a possible indirect mechanism for 1-EBIO-dependent dilations could be to promote endothelial Ca²⁺ influx secondary to endothelial hyperpolarization. Therefore, whereas 1-EBIO produced significant endothelium-dependent dilations by activating endothelial IK_Ca channels, it was still necessary to determine whether the dilation resulted directly from IK_Ca channel activation and endothelial hyperpolarization or whether the resulting endothelial hyperpolarization triggered another Ca²⁺-dependent mechanism by promoting endothelial Ca²⁺ influx. For instance, if 1-EBIO resulted in an increase in endothelial [Ca²⁺]_i, then the vasodilatation to 1-EBIO (or IK_Ca channel activation) could ultimately result from subsequent production of a Ca²⁺-dependent relaxing factor(s). However, the addition of 1-EBIO did not alter endothelial [Ca²⁺]_i from resting values despite the significant vasodilatory effect of 1-EBIO (Fig. 7). Taken as a whole, these data indicate that 1-EBIO-dependent vasodilatation of cerebral arteries is mediated primarily by direct activation of endothelial IK_Ca channels, without an involvement of elevated endothelial [Ca²⁺]_i. Furthermore, these data demonstrate that direct stimulation of endothelial IK_Ca channels alone is indeed sufficient to produce EDHF-like dilations. These findings are consistent with a model in which endothelial IK_Ca channels act downstream of endothelial Ca²⁺ as the critical effector of EDHF-mediated dilations.

From previous studies, we know that endothelial Ca²⁺ does play a critical role in EDHF-mediated dilations. For instance, an endothelial [Ca²⁺]_i threshold (340 nM) has been demonstrated for the initiation of EDHF-mediated dilations (40). Additionally, influx of extracellular Ca²⁺ has been shown to be essential to sustain EDHF-mediated dilations (9, 48). The specific function of endothelial [Ca²⁺]_i in EDHF-mediated dilations is still unclear, because elevated endothelial [Ca²⁺]_i could activate any number of enzymes or pathways, including a hypothetical EDHF synthase. However, in light of this and other recent studies, one clear role for endothelial [Ca²⁺]_i in the EDHF mechanism could simply be as the physiological activator of endothelial IK_Ca channels. In support of this conclusion, the reported EDHF-initiating [Ca²⁺]_i threshold falls right within the range of [Ca²⁺]_i required to produce significant activation of IK_Ca channels (250–500 nM Ca²⁺) (42).

Whereas the primary role for IK_Ca channel activation in the EDHF mechanism thus appears to be down-stream of calcium, a secondary role might be to augment agonist-mediated endothelial [Ca²⁺]_i responses. In cultured endothelial cells, Ca²⁺ influx has been shown to be dependent on membrane potential with hyperpolarization driving greater Ca²⁺ influx (32, 35, 46). Given the IK_Ca channel activation and endothelial hyperpolarization that results from EDHF-dependent agonists (Fig. 2), one would therefore presume that IK_Ca channel activation would augment agonist-mediated endothelial [Ca²⁺]_i responses. Conversely, one would expect that interrupting that positive feedback system by inhibiting IK_Ca channels would result in reduced [Ca²⁺]_i responses to EDHF-dependent agonists. Thus IK_Ca channel blockers might also be able to inhibit or modulate EDHF-mediated dilations by preventing endothelial [Ca²⁺]_i from reaching the EDHF-initiating threshold. It must be noted, however, that such a scenario is reasonable only as long as one assumes that the relationship between membrane potential and Ca²⁺ flux applies beyond cultured endothelial cells. Although that assumption is often made in the literature, there are very few studies that provide data regarding the role of membrane potential on endothelial Ca²⁺ flux in intact arteries. The issue is further complicated given that endothelial Ca²⁺ responses from intact arteries are more complex to interpret given the likely contribution of smooth muscle to endothelial [Ca²⁺]_i (17). In intact peripheral arteries, agonist-mediated Ca²⁺ flux has been shown to be reduced (27, 33) or unchanged (27, 50) in the presence of K channel blockers. Interestingly, the studies that reported reduced [Ca²⁺]_i responses used high [K⁺]_o to nonselectively block K channels (and presumably depolarize the endothelium and smooth muscle) (27, 33). In contrast, ChTx/Apa was without effect on endothelial [Ca²⁺]_i, despite effectively blocking the agonist-mediated dilator response (27, 50). In the present study, neither ChTx/Apa nor high [K⁺]_o reduced endothelial Ca²⁺ responses to UTP or prevented endothelial [Ca²⁺]_i from attaining the [Ca²⁺]_i threshold necessary for initiating EDHF-mediated dilations despite substantially reducing endothelial hyperpolarization (Figs. 2 and 8 and Table 2). It is not clear why these studies appear to conflict regarding the role of membrane potential in modulating endothelial [Ca²⁺]_i. One possible explanation is that the relative role of membrane potential is dependent on the contribution of other modulating factors such as intracellular Ca²⁺ stores or Ca²⁺ flux from coupled smooth muscle cells (17). However, irrespective of the reason, our data demonstrate that endothelial hyperpolarization does not significantly affect endothelial [Ca²⁺]_i in response to UTP in intact cerebral arteries. Furthermore, these data reinforce the conclusion that the critical role of endothelial IK_Ca channel activation is entirely down-stream of endothelial [Ca²⁺]_i in the EDHF-dependent mechanism.

Taken as a whole, these data demonstrate that EDHF-mediated dilations in the cerebral circulation require activation of endothelial IK_Ca channels. In addition, direct activation of endothelial IK_Ca channels (1-EBIO) produces endothelial hyperpolarization and vasodilatation that mimic the EDHF-mediated response. Because the vasodilatory effect of direct IK_Ca channel activation does not involve an increase in endothelial [Ca²⁺]_i, it appears that the critical role for endothelial IK_Ca channels is downstream of endothelial [Ca²⁺]_i in the EDHF mechanism. Thus the present data support a model in which increased endothelial [Ca²⁺]_i acts as the physiological activator of endothelial IK_Ca channels with IK_Ca channel activation and endothelial hyperpolarization being critical events in promoting the subsequent smooth muscle hyperpolarization. Further studies are required to determine the specific mechanism by which endothelial IK_Ca channel activation ultimately effects smooth muscle hyperpolarization and vasodilatation in cerebral arteries.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported by American Heart Association grants (Scientist Development Grant 0230353N and Bugher Foundation Award 0270110N) and National Institute of Neurological Disorders and Stroke Grants RO1-NS-37250 and PO1-NS-27616.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. P. Marrelli, Baylor College of Medicine, Dept. of Anesthesiology, One Baylor Plaza, Suite 434-D, Houston, TX 77030 (E-mail: marrelli{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

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