INTRODUCTION

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THE ARTERIOLAR ENDOTHELIUM has a pivotal role in mediation of certain agonist-induced dilations and in sensing and transmitting alterations of hemodynamic forces into vasomotor responses. On the basis of studies on large vessels and cultured endothelial cells, it is generally believed that endothelial synthesis/release of vasoactive mediators is controlled and preceded by changes in endothelial intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, recent studies revealed that in the endothelium of skeletal muscle arterioles, vessels that contribute importantly to the development of peripheral resistance, nitric oxide (NO) synthesis is activated by increases in shear stress without substantial increases in endothelial [Ca²⁺]_i, most likely by activation of Ca²⁺-independent tyrosine kinase pathways (35).

In contrast, arteriolar dilations to agonists that act on endothelial receptors, such as acetylcholine (ACh), are associated with significant increases in endothelial [Ca²⁺]_i (7, 23, 34). Interestingly, in the microcirculation, in contrast to large vessels, a major part of ACh-induced dilation is insensitive to the inhibition of NO synthase and cyclooxygenase. Pathways associated with NO- and prostaglandin-independent mediation involve smooth muscle hyperpolarization (3, 7, 14, 34); therefore, it is termed endothelium-derived hyperpolarizing factor (EDHF)-type dilation. Despite its profound effect on arteriolar myogenic tone, the intra- and intercellular pathways for EDHF-type signal transduction are not well understood.

Skeletal muscle arterioles are known to develop significant myogenic tone in response to intraluminal pressure, which depends on pressure-induced increases in smooth muscle [Ca²⁺]_i due to an influx of Ca²⁺ through voltage-operated Ca²⁺ channels (VOC) (33). Recently, we (34) demonstrated that EDHF-type arteriolar dilation to ACh is elicited by a decrease in smooth muscle [Ca²⁺]_i due to a hyperpolarization-dependent closure of VOC. However, the role of increases in endothelial [Ca²⁺]_i and the nature of coupling between endothelial activation and hyperpolarization-induced decreases in smooth muscle [Ca²⁺]_i in EDHF-type arteriolar dilation remained to be resolved.

From previous studies, we hypothesized that agonist-induced increases in arteriolar endothelial [Ca²⁺]_i elicit opening of endothelial Ca²⁺-activated K⁺ (K_Ca) channels, and the consequent hyperpolarization (11, 40) is conducted to the smooth muscle, likely via gap junctions (13, 14, 28, 40) eliciting decreases in smooth muscle [Ca²⁺]_i and arteriolar dilations.

To demonstrate that this hypothetic pathway is responsible for signal transduction of EDHF-type dilation of skeletal muscle arterioles, we measured simultaneous changes in endothelial [Ca²⁺]_i and diameter and characterized the role of endothelial K_Ca channels [by use of a combination of charybdotoxin (ChTX) and apamin to inhibit all K_Ca channels on endothelial cells] and gap junction-dependent endothelial-smooth muscle coupling. Also, we demonstrated on the arteriolar endothelium the presence of large conductance K_Ca (BK_Ca) channels, thought to be involved in arteriolar EDHF mediation (7), with immunohistochemistry.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Simultaneous measurement of endothelial [Ca²+]_i and diameter of isolated arterioles. Experiments were conducted on isolated gracilis muscle arterioles of 12-wk-old Wistar rats (n = 30) as previously described (34, 35). In brief, the arterioles were cannulated in an organ chamber containing physiological salt solution (mmol/l: 110 NaCl, 5.0 KCl, 2.5 CaCl₂, 1.0 MgSO₄, 1.0 KH₂PO₄, 5.0 glucose, and 24.0 NaHCO₃; equilibrated with 10% O₂-5% CO₂-85% N₂; pH = 7.4). Intraluminal pressure was kept constant at 80 mmHg by a pressure servocontrol system. The internal arteriolar diameter was measured by videomicroscopy using the automatic edge detection function (time resolution: 0.02 s) of Ionwizard software (Ionoptics; Milton, MA) (35). To assess changes in [Ca²⁺]_i, the arteriolar endothelium was selectively loaded with fura 2-AM (5 µmol/l, intraluminally for 10 min at 37°C) as previously described (35). Changes in background-corrected calcium fluorescence ratios (R_Ca), estimates of endothelial [Ca²⁺]_i, were measured by the ratiometric fluorescence method (time resolution: 0.02 s) using the Ionoptix Microfluorimeter System (Ionoptix) (35). The system was calibrated in vitro using prediluted buffered fura 2 solutions made to mimic intracellular conditions (Fura 2 Calcium Imaging Calibration Kit, Molecular Probes; Eugene, OR), and R_Ca was calculated using the following equation (16): R_Ca = K_d × (R  R_min)/(R_max  R)  (F_380 max/F_380 min), where K_d (dissociation constant) = 224 nmol/l, R is the ratio of 510-nm arteriolar emission intensity exciting at 340-510 nm arteriolar emission intensity exciting at 380 nm, R_max = 6.4 ± 0.1, R_min = 0.16 ± 0.02, F_380 max is the fluorescence intensity exciting at 380 nm for zero concentration of Ca²⁺, and F_380 min is the fluorescence intensity at a saturating concentration of Ca²⁺ (39.8 µmol/l) (7). To determine whether selective loading of the endothelium has been accomplished at the end of the experiments, responses to the VOC inhibitor nimodipine (10⁶ mol/l) were assessed, and then the endothelium was removed by intraluminal perfusion of air. Selective endothelial loading was considered successful if nimodipine dilated the vessels without affecting endothelium R_Ca and removal of the fura 2-loaded endothelium of arterioles eliminated 90% of the fluorescence signal (35). Only data obtained in arterioles that met these criteria were used. The protocol used produced selective and consistent loading of the endothelium in ~80% of arterioles.

Experimental protocols. Simultaneous changes in endothelial [Ca²⁺]_i and diameter of arterioles in response to ACh (10⁶ mol/l) were obtained in the presence of N-nitro-L-arginine methyl ester (L-NAME, 3 × 10⁴ mol/l) and indomethacin (10⁵ mol/l). The arterioles were then perfused intraluminally with apamin [10⁶ mol/l, an inhibitor of small conductance K_Ca (SK_Ca) channels] or ChTX [3 × 10⁷ mol/l, an inhibitor of BK_Ca and intermediate conductance K_Ca (IK_Ca) channels (24)] plus apamin and responses to ACh were reassessed.

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Measurement of smooth muscle [Ca²+]_i. In separate experiments, the arteriolar smooth muscle was loaded with fura 2 (2 µmol/l fura 2-AM in the superfusate; 30 min at 25°C) as previously described (33, 34). Simultaneous changes in smooth muscle [Ca²⁺]_i and arteriolar diameter in response to ACh and nimodipine (10⁶ mol/l) were assessed. In some experiments, responses were reassessed after intraluminal perfusion of ChTX and apamin.

Immunohistochemistry and Western blotting. Tissue samples from the gracilis muscle were embedded in OTC-4583 medium (Sakura, Finetek; Torrance, CA) and snap-frozen in liquid nitrogen, and immunolabeling was carried out according to the modified protocol of Segal et al. (30). In brief, tissue sections (4 µm thickness) were cut by using a cryostat and collected on Probe-on-Plus (Fisher Scientific) microscope slides. Sections were fixed with cold (4°C) acetone and exposed to 3% hydrogen peroxide (10 min, 25°C, in methanol) to quench endogenous peroxidase activity. Slides were washed three times for 5 min with phosphate-buffered saline (PBS). To block nonspecific binding of antibodies, sections were incubated overnight at 4°C with 10% normal horse serum, which was then aspirated from each section. Then sections were incubated for 1 h at room temperature with anti-BK_Ca channel antibody (directed against the intracellular COOH-terminus of the BK_Ca channel -subunit; Alomone Labs) (37) diluted 1:50 in PBS containing 0.1% bovine serum albumin and 0.2% Triton X-100. To label-bound primary antibody, sections were incubated for 45 min at 25°C with a 1:200 dilution of goat anti-rabbit secondary antibody conjugated to biotin (Vector Laboratories). Sections were then incubated for 45 min with avidin-biotinylated enzyme complex solution (Vectastain kit, Vector Laboratories). Between each step of labeling protocol, sections were rinsed in PBS solution. Sections were then exposed to diaminobenzidine tertrahydrochloride (DAB, Vector) solution for 2 min, rinsed in deionized water, permanently mounted with Vectamount, and covered with a coverslip. The specificity of the immunolabeling was evaluated by omitting the primary antibody or omitting both primary and secondary antibodies in control experiments. Further specificity controls were made by immunostaining sections after overnight incubation of the primary antibody with the homologous antigen for the BK_Ca channel (3 µg fusion protein per 1 µg antibody; Alomone Labs). Sections were visualized through an Olympus BX60 microscope connected to a CoolSnap CF CCD camera (Roper Scientific).

Materials. Fura 2-AM and BAPTA-AM were purchased from Molecular Probes (Eugene, OR); all other salts and chemicals were obtained from Sigma-Aldrich (St. Louis, MO). PA was dissolved in ethanol and stored under nitrogen at 80°C. After responses to each drug subsided, the system was flushed with physiological salt solution.

Data analysis. Arteriolar dilations were expressed as a percentage of the maximal dilation defined as the passive diameter at 80 mmHg intraluminal pressure. Changes in R_Ca are expressed as a percentage of the baseline value. Time kinetics of the responses were calculated with the transient analysis function of the Ionwizard software. All data are expressed as means ± SE. Statistical analyses were performed by analysis of variance followed by Tukey's post hoc test. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

ACh-induced changes in endothelial and smooth muscle [Ca²+]_i and EDHF-type arteriolar dilations. During the equilibration period, arterioles developed substantial myogenic tone (active diameter: 60 ± 4 µm, passive diameter: 122 ± 5 µm at 80 mmHg). In the presence of L-NAME and indomethacin, ACh elicited significant, rapid increases in endothelial [Ca²⁺]_i (101 ± 7%) that were followed by substantial dilations (73 ± 2%) with a coupling time of ~1.3 s (Fig. 1A shows an original tracing representative to 20 measurements). Nimodipine maximally dilated arterioles without affecting endothelial [Ca²⁺]_i. In arterioles with fura 2-loaded smooth muscle, ACh- or nimodipine-induced dilations were preceded by a significant decrease in smooth muscle [Ca²⁺]_i (Fig. 1B). ACh-induced increases in endothelial [Ca²⁺]_i or decreases in smooth muscle [Ca²⁺]_i had a similar time course, and the coupling times between onset of calcium and diameter responses in these experiments did not differ significantly (Fig. 1C).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A: simultaneous recordings (representative of 20 experiments) showing an increase in endothelial intracellular Ca2+ concentration ([Ca2+]i) (expressed as fluorescent ratio, ERCa) followed by arteriolar dilation after application of 106 mol/l acetylcholine (ACh). Coupling time (CT) between onset of responses is 1.3 s (inset). B: original trace from a separate experiment (representative of 10 experiments) shows ACh-induced decrease in smooth muscle [Ca2+]i (SM RCa) followed by arteriolar dilation. N-nitro-L-arginine methyl ester (L-NAME) and indomethacin were present in all experiments. C: summary data of coupling times between ACh-induced increases in endothelial [Ca2+]i and arteriolar diameter (left) and decreases in SM [Ca2+]i to ACh or the Ca2+ channel inhibitor nimodipine (Nimo, n = 5; not significant) (right). Data are means ± SE.

Role of endothelial [Ca²+]_i. ACh elicited biphasic increases in endothelial [Ca²⁺]_i with a rapid peak (maximum at ~10 s) that was followed by a plateau phase, whereas in the absence of Ca²⁺ in the perfusate solution, ACh elicited a monophasic peak rise that returned to baseline within ~40 s (Fig. 2A). Selective loading of the endothelium with BAPTA abolished ACh-induced changes in endothelial [Ca²⁺]_i and prevented dilations (Fig. 2, B and C), whereas it had no effect on either baseline diameter or nimodipine-induced dilations.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   A: original tracings showing ACh (106 mol/l)-induced changes in endothelial [Ca2+]i (expressed as ERCa) under control conditions and after intraluminal perfusion of Ca2+-free solution containing EGTA (left) or selective loading of the endothelium with the Ca2+-chelating agent BAPTA (right). B: summary data of ACh-induced peak changes in endothelial [Ca2+]i. C: arteriolar diameter before and after endothelial loading of BAPTA. L-NAME and indomethacin were present throughout the experiments. Data are means ± SE (n = 4). * P < 0.05.

Role of endothelial K_Ca channels. Intraluminal application of ChTX plus apamin had no effect on either baseline values or ACh-induced peak levels of endothelial [Ca²⁺]_i (Fig. 3A), whereas it abolished both ACh-induced decreases in smooth muscle [Ca²⁺]_i (Fig. 3B) and dilations (Fig. 3C). ACh-induced responses were not affected significantly by apamin alone. Intraluminal ChTX and apamin did not affect either baseline diameter or nimodipine-induced dilations, and their effects were reversible upon washout.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   ACh (106 mol/l)-induced peak changes in endothelial [Ca2+]i (expressed as ERCa) (A), smooth muscle (SM RCa) [Ca2+]i (B), and arteriolar diameter (C) in the absence and presence of charybdotoxin (ChTX, 3 × 107 mol/l) plus apamin (106 mol/l), inhibitors of Ca2+-activated K+ (KCa) channels in the vessel lumen. L-NAME and indomethacin were present throughout the experiments. Data are means ± SE (n = 4-5). * P < 0.05.

Immunohistochemistry and Western blotting. Samples from the gracilis muscle (n = 4) containing first-order arterioles were analyzed with immunohistochemistry. In the arterioles, strong immunostaining for BK_Ca channels was detected in endothelial cells (Fig. 4A). BK_Ca immunoreactivity was also present in smooth muscle cells (Fig. 4A). In control experiments on consecutive sections in the absence of the primary antibody (Fig. 4B) or both primary and secondary antibodies or after absorption of the primary antibody with the homologous antigen for BK_Ca (Fig. 4C), there was no evidence for nonspecific immunostaining.

View larger version (84K): [in this window] [in a new window]   Fig. 4.   A: light micrographs of cross sections of rat gracilis arterioles labeled for large conductance KCa (BKCa) channels. Strong immunostaining for BKCa was detected in endothelial cells (arrows). BKCa immunoreactivity was also present in smooth muscle cells (arrowhead). Consecutive sections served as negative control (B: primary antibody omitted, C: antibody preabsorption with homologous antigen). Hematoxyline counterstaining. Scale bars are 20 µm (A) and 8 µm (B and C), respectively. D: original Western blots of isolated aortic endothelial cell (lane 1) and arteriolar lysates (lanes 2 and 3) labeled with the anti-BKCa antibody. In control experiments, after absorption (abs) of the primary antibody with the homologous antigen for BKCa, there was no immunolabeling (lanes 4 and 5).

Role of gap junctions. The gap junction uncoupler PA significantly inhibited ACh-induced dilations in a concentration-dependent manner, whereas it had no effect on either baseline values or ACh-induced levels of endothelial [Ca²⁺]_i (Fig. 5A). Also, PA did not affect nimodipine-induced dilations (maximum: 98 ± 1%), and its effects were reversible upon washout. Glucose (60 mmol/l) elicited arteriolar dilations that were inhibited by removal of the endothelium (22), by KCl depolarization, and by administration of PA (Fig. 5B).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   A: inhibition by the gap junction uncoupler palmitoleic acid (PALM) of ACh (106 mol/l)-induced peak increases in endothelial [Ca2+]i and arteriolar dilations. * P < 0.05. B: arteriolar dilations to glucose (60 mmol/l) before and after removal of the endothelium or administration of KCl (80 mmol/l) or palmitoleic acid (105 mol/l). L-NAME and indomethacin were present throughout the experiments. Data are means ± SE (n = 4-6). * P < 0.05. C: proposed scheme for EDHF-type mediation of dilation in skeletal muscle arterioles. ACh induces an increase in endothelial [Ca2+]I-activating endothelial KCa channels; the hyperpolarization is conducted to the arteriolar SM via myoendothelial gap junctions, resulting in decreases in SM [Ca2+]i and arteriolar dilations. KATP, ATP-sensitive K+ channels; eNOS, endothelial nitric oxide synthase; Endo, endothelium.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The main findings of the present study are that in skeletal muscle arterioles, ACh-induced increases in endothelial [Ca²⁺]_i elicit a non-NO, non-prostaglandin, EDHF-type mediation of dilation that can be prevented by 1) selective loading of the endothelium with the Ca²⁺ chelator BAPTA; 2) intraluminal administration of the K_Ca channel inhibitors ChTX and apamin; and by 3) PA, an inhibitor of gap junctional communication. In addition, the presence of BK_Ca channels on the arteriolar endothelium was shown with immunohistochemistry.

In the present study to elucidate the nature of signaling pathways involved in non-NO, non-prostaglandin-mediated dilation of skeletal muscle microvessels, we selectively recorded changes in endothelial [Ca²⁺]_i in intact, pressurized arterioles simultaneously with changes in myogenic tone with high time resolution. Because many vasoconstrictors alter membrane potential and interfere with signaling mechanisms, it is important to note that we had not used any vasoactive agent to induce tone in arterioles.

Role of endothelial [Ca²+]_i In these conditions, we showed that ACh-induced, EDHF-type arteriolar dilation (in the absence of NO and prostaglandins) is preceded by a substantial, biphasic increase in arteriolar endothelial [Ca²⁺]_i (Fig. 1A), most likely due to an initial rapid Ca²⁺ release from intracellular stores followed by influx of extracellular Ca²⁺ (Fig. 2A) (35). EDHF mediation, likely due to a hyperpolarization-dependent inhibition of influx of extracellular Ca²⁺ via VOC in the smooth muscle (7, 34), significantly decreased smooth muscle [Ca²⁺]_i (Fig. 1B). The pivotal role for an increase in endothelial [Ca²⁺]_i in initiating the signal transduction leading to EDHF-type dilation is supported by the present finding that selective chelating of endothelial [Ca²⁺]_i with BAPTA (41) abolished ACh-induced responses (Fig. 2, A-C).

Role of endothelial K_Ca channels. Several Ca²⁺-sensitive mechanisms have been suggested to exist in the endothelium; however, the coupling between increases in endothelial [Ca²⁺]_i and EDHF-type dilation has not been well understood. In the present study, we were able to demonstrate that selective inhibition of endothelial K_Ca channels by intraluminal application of ChTX plus apamin (12) completely abolishes ACh-induced decreases in smooth muscle [Ca²⁺]_i (Fig. 3B) and arteriolar dilation (Fig. 3C) without affecting increases in endothelial [Ca²⁺]_i (Fig. 3A). The idea that in skeletal muscle microvessels increases in endothelial [Ca²⁺]_i primarily activate ChTX-sensitive K_Ca channels is supported by our previous findings that this EDHF-type arteriolar dilation in these vascular bed is insensitive to inhibitors of K_ATP and inward rectifying K⁺ (K_IR) channels and can be inhibited by ChTX and ChTX plus apamin to a similar extent (34). Membrane potential measurements also demonstrated that a major component of ACh-induced endothelial hyperpolarization in the rat aorta (21) and guinea pig mesenteric (40) and submucosal (11) arterioles is ChTX sensitive. It is believed that in many vascular beds, a ChTX-sensitive, Ca²⁺-induced increase in endothelial K⁺ conductance is due to the opening of BK_Ca channels, which was reported to have higher Ca²⁺ sensitivity than vascular SK_Ca channels (21).

Role of gap junction-dependent coupling. In the present study, simultaneous measurements of changes in [Ca²⁺]_i and diameter did not allow the measurement of changes in membrane potential as well, but results of simultaneous membrane potential measurements in endothelial and smooth muscle cells by Emerson and Segal (14) provide sufficient support for the idea that hyperpolarization of smooth muscle may result directly from instantaneous electronic conductance of hyperpolarization originating in endothelial cells (10, 14, 20, 40). Nevertheless, the finding that the coupling time between increases in endothelial [Ca²⁺]_i and dilations to ACh did not differ significantly from the electromechanical coupling time of the smooth muscle contractile apparatus, defined as the time shift between decreases in smooth muscle [Ca²⁺]_i and the mechanical response (Fig. 1C), suggests that Ca²⁺-induced opening of endothelial K_Ca channels (36) indeed results in instantaneous smooth muscle hyperpolarization-dependent decreases in smooth muscle [Ca²⁺]_i.