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Transport of extracellular L-arginine via cationic amino acid transporter is required during in vivo endothelial nitric oxide production

Department of Cellular and Integrative Physiology, Indiana University Medical School, Indianapolis, Indiana

Submitted 7 December 2004 ; accepted in final form 8 April 2005

	ABSTRACT

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In cultured endothelial cells, 70–95% of extracellular L-arginine uptake has been attributed to the cationic amino acid transporter-1 protein (CAT-1). We tested the hypothesis that extracellular L-arginine entry into endothelial cells via CAT-1 plays a crucial role in endothelial nitric oxide (NO) production during in vivo conditions. Using L-lysine, the preferred amino acid transported by CAT-1, we competitively inhibited extracellular L-arginine transport into endothelial cells during conditions of NaCl hyperosmolarity, low oxygen, and flow increase. Our prior studies indicate that each of these perturbations causes NO-dependent vasodilation. The perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature. Suppression of extracellular L-arginine transport significantly and strongly inhibited increases in vascular [NO] and intestinal blood flow during NaCl hyperosmolarity, lowered oxygen tension, and increased flow. These results suggest that L-arginine from the extracellular space is accumulated by CAT-1. When CAT-1-mediated transport of extracellular L-arginine into endothelial cells was suppressed, the endothelial cell NO response to a wide range of physiological stimuli was strongly depressed.

intestine; blood flow; arterioles; L-lysine; oxygen; hyperosmolarity; shear

To what extent eNOS uses L-arginine from the extracellular space or from the intracellular stores has not been determined in the in vivo state. The half-saturating arginine concentration for eNOS is in the range of 1–10 µM (49, 52). Physiological L-arginine concentrations in the extracellular space are 50–200 µM (60). Within cultured endothelial cells, the L-arginine concentration range is 100–800 µM (4, 6, 30, 35) and even higher in freshly isolated endothelial cells (35). From the intracellular concentration, it would seem eNOS operates under a saturating intracellular arginine concentration and would not require any arginine from the extracellular space. However, basal NO production in vascular endothelial cells declines when the extracellular concentration of L-arginine is below typical plasma concentrations (33, 44). Our laboratory has shown that the in vivo NO concentration ([NO]) can be almost doubled with 1 mM L-arginine (12). The observation that extracellular L-arginine can enhance endothelial NO production, despite a saturating intracellular arginine concentration, has been termed the "arginine paradox" (41). A possible answer to the arginine paradox was proposed by McDonald et al. (45), who showed the existence of a caveolar complex between eNOS and CAT-1 in pulmonary artery endothelial cells. From these results it has been suggested that caveolar eNOS may be functionally isolated from the large internal store of L-arginine, potentially allowing the eNOS-CAT-1 complex to influence NO production through directed delivery of L-arginine to eNOS (1, 45). In this context, Closs et al. (23) found human endothelial cells have two L-arginine pools: pool I that is and pool II that is not freely exchangeable with the extracellular space via CAT-1.

In vivo and in vitro vascular studies (12, 26, 28, 38, 42, 53, 56) have shown that extracellular L-arginine administration above plasma concentrations improves either NO formation or vascular relaxation attributable to NO. This might be taken as evidence that the supply of L-arginine is somehow limiting to NO formation. Hardy and May (33), using cultured bovine aortic endothelial cells, found stimulation of eNOS with bradykinin and acetylcholine, along with other endothelium-dependent dilators, was associated with increased L-arginine uptake. In contrast, using the same cell source, Arnal et al. (2) showed that the presence of L-arginine in cell culture medium did not significantly increase bradykinin-stimulated NO release. In a similar context, MacKenzie and Wadsworth (44) found only a small increase in aortic relaxation to methacholine in the presence of 100 mM L-arginine vs. no L-arginine.

Even though the L-arginine sources for eNOS are controversial, there is enough in vitro and in vivo evidence to support the notion that extracellular L-arginine entry into endothelial cells via CAT-1 may have a role in endothelial NO production. However, the relevance of this system to in vivo conditions has not been determined by direct measurement of changes in vascular wall [NO] and blood flow as the CAT-1 transport of L-arginine is suppressed. Therefore, in these studies L-lysine, a competitive inhibitor of L-arginine entry via CAT-1 (21, 22, 33), was suffused over the rat intestinal microvasculature to suppress CAT-1 transport of L-arginine during conditions of NaCl hyperosmolarity, lowered oxygen availability, and increased blood flow. Prior studies from our laboratory (8, 12, 48) indicate that each of these physiological conditions results in NO-dependent vasodilation. For each perturbation, arteriolar diameter, blood flow, and arteriolar wall [NO] were measured before and after the suppression of CAT-1 L-arginine transport by L-lysine. For all conditions tested, the results suggest that CAT-1 transport of extracellular L-arginine into endothelial cells was essential for increased NO production with subsequent increases in arteriolar diameter and blood flow. Given the wide range of physiological stimuli we tested, CAT-1 transport of extracellular L-arginine into endothelial cells is a crucial component of in vivo vascular regulation.

	METHODS

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All animal procedures were submitted to, independently reviewed, and performed in accordance with the Institutional Animal Care and Use Committee Guidelines of Indiana University Medical School.

Animal and tissue preparation. Adult male Sprague-Dawley rats (250–350 g) (Harlan, Indianapolis, IN) were anesthetized with sodium thiopental (200 mg/kg, subcutaneous; Abbott, Chicago, IL). A rectal temperature of 37°C was maintained by placing each rat on a heating pad (35–36°C). The trachea was intubated with polyethylene tubing (PE-240) and mechanically ventilated (70 breaths/min) to have a percent saturation of hemoglobin with oxygen of 95%. The left femoral artery was cannulated to measure arterial blood pressure.

An established technique (7) was used to prepare the small intestine for observation. An 10-cm loop of the jejunal region was exteriorized and slit 2.5 cm with a microcautery along the antimesenteric border. The intestine was restored to physiological dimensions with small sutures tied to the edges of the intestinal incisions. After being heated to 37.5 ± 0.5°C, a 5 ml/min flow of bicarbonate-buffered physiological saline solution (118 mM NaCl, 6 mM KCl, 25 mM Na₂HCO₃, and 3.5 mM CaCl₂) was passed through the chamber. Equilibration of the physiological saline was done with 90% nitrogen, 5% carbon dioxide, and 5% oxygen. To partially suppress intestinal motility to allow micropipette placement, we added norepinephrine (Sigma Chemical, St. Louis, MO) to the bathing fluid. The concentration of norepinephrine was in the range of 10–100 nM and had minor effects on vascular resistance.

Microvascular observation and flow velocity measurements. Using a x10 or x20 Nikon water-immersion lens, we observed microvessels and small arteries through the microscope (model BHMJ; Olympus, Hyde Park, NY). A Video Scope camera (model CCD 200E; Videoscope International, Washington, DC) joined with a computerized digitizing and image analysis system (MetaMorph; Universal Imaging, Downingtown, PA) was used to record all images and measure vessel inner diameter. An Optical Doppler Velocimeter (Microcirculation Research Institution, Texas A&M, TX) was used to measure the red blood cell flow velocity. Red blood cells on a rotating disk for velocities of 100–600 µm/s were used to evaluate the linearity of the velocity versus signal output. The following equation, which assumes a circular diameter of arterioles, was used to calculate flow after both the mean blood cell velocity and the arteriolar inner diameter were known: 3.14 x velocity x (diameter/2)². For optimal signal-to-noise ratios, vessels with diameters greater than 30 µm and less than 80 µm were used at a magnification of x20 because the optical Doppler technique is limited by both very large and small diameters (16). In the calculation of percentage of control blood flow, we did not use a correction factor for translating centerline velocity to mean velocity because the correction factor would be nulled out.

Perivascular NO measurements. A polarographic technique, using carbon fiber, recessed-tip glass microelectrodes, was implemented to measure [NO] in the intestine. In past studies (8, 12, 48), NO-sensitive microelectrodes have been used by our laboratory. Our experience and techniques, developed by Buerk et al. (18) and Freidemann et al. (29), were the basis for the production and calibration of the NO-sensitive microelectrodes. The microelectrodes had a sharpened outer tip diameter of 7–10 µm. The NO-sensitive microelectrodes were polarized at +0.7 or +0.9 V relative to a World Precision Instruments carbon fiber reference electrode (Sarasota, FL) or a simple silver-silver chloride electrode. The polarization voltage at which a given electrode was most sensitive to [NO] was used, as measured with a Keithley model 6517A electrometer (Cleveland, OH). The currents generated were typically in the range of 0–20 pA. A calibration curve was established by measurement of the microelectrode current at [NO] of 0, 600, and 1,200 nM, based on the composition of the NO-N₂ precision calibration gases (Matheson, Joliet, IL) in saline at 37.5°C. The working resolution of the microelectrodes was typically <10 nM, allowing for random noise and current drift. As checked in past studies (8, 62), the sensitivity of the microelectrodes to NO is retained when the NaCl osmolarity of the bathing fluid is altered. The microelectrodes are completely insensitive to oxygen when positively polarized. The current generated when the microelectrode was placed 200 µm above the tissue was used as the 0 nM reference immediately before and after each tissue measurement to obtain a baseline measurement. The rate of drift and an interpolated baseline current for any given time was calculated from these data. The interpolated baseline current was subtracted from the current during the time period when tissue [NO] was measured to yield a value representing the current equivalent of the [NO].

Perivascular PO₂ measurements. Carbon fiber, recessed-tip glass microelectrodes with a sharpened outer tip diameter of 7–10 µm were used to measure perivascular PO₂. The electrodes were polarized at –0.7 V relative to the ground electrode, and current was measured with a Keithley 6517 electrometer. A tonometer was used to calibrate and test the electrodes for a linear current-PO₂ relationship at PO₂ of 0, 40, and 144 mmHg at 37°C.

Competitive inhibition of cationic amino acid transporter during NaCl hyperosmolarity. The effects of L-lysine, a competitive inhibitor of arginine transport through the cationic amino acid transporter (21), on endothelial NO production and arteriolar blood flow during NaCl hyperosmolarity were determined. The concentration of L-lysine used in these studies, 200 µM, was based on previously published reports (33, 44). L-Lysine did not have access to the epithelial cells of the mucosa to avoid absorptive hyperemia. At the control osmolarity of 300 mosM and at 360 mosM before and after the administration of L-lysine (200 µM), we measured the periarteriolar [NO], arteriolar diameter, and blood flow velocity for an individual arteriole in the intestine. L-Lysine was exposed to the tissue for 30 min to assure diffusion and bath turnover in this and all the other protocols.

Competitive inhibition of cationic amino acid transporter during low-oxygen conditions. During low-oxygen conditions, the effects of L-lysine competition on endothelial NO production and arteriole blood flow were determined. At the control condition (90% nitrogen, 5% carbon dioxide, and 5% oxygen) and low-oxygen condition (95% nitrogen and 5% carbon dioxide), we measured the periarteriolar [NO], arteriolar diameter, and blood flow velocity for an individual intestinal arteriole before and after L-lysine (200 µM) was applied. Before and after the addition of L-lysine, all measurements were made 5–10 min after the oxygen percentage in the bathing solution was changed. The typical bath partial pressure of oxygen is 40–45 mmHg with 5% oxygen and below 10 mmHg when 0% oxygen is used.

Competitive inhibition of Na⁺/Ca²⁺ exchanger during low-oxygen conditions. We found in a prior study (62) that the Na⁺/Ca²⁺ exchanger (NCX) is very active in in vivo endothelial cells. As sodium ions increase their leakage into endothelial cells at reduced oxygen tension (32, 61), this pump system might be activated to remove sodium ions in exchange for calcium ions. This would elevate intracellular calcium and possibly explain our prior findings that decreased vessel wall oxygen tension caused an increase in [NO] (9, 48).

The effects of KB-R7943, a selective inhibitor of the NCX, on periarteriolar PO₂ and arteriole blood flow were determined during low-oxygen conditions. On the basis of previously published reports (3, 24), 50 µM of KB-R7943 was used in these studies. We measured the periarteriolar PO₂, arteriolar diameter, and blood flow velocity for an individual intestinal arteriole before and after KB-R7943 was applied at the control condition of 90% nitrogen, 5% carbon dioxide, and 5% oxygen and at the low-oxygen condition of 95% nitrogen and 5% carbon dioxide. All measurements were made 5–10 min after the oxygen percentage in the bathing solution was changed.

The endothelium-dependent vasodilator bradykinin was used to determine whether the concentration of KB-R7943 used in our experiments had any nonspecific effect on arterial vasodilation. Known concentrations of bradykinin were suffused over the entire vasculature before and after KB-R7943 was applied, and the arteriole dilatory responses were directly compared.

Competitive inhibition of cationic amino acid transporter during collateral perfusion. To increase blood flow in a given vessel, we nontraumatically occluded either a large arteriole [first-order arteriole (1A)] or an intermediate-sized arteriole [second-order arteriole (2A)] in anatomical hemodynamic parallel using a glass micropipette, as previously reported (12). This procedure resulted in an increase in resting blood flow in 1As and 2As, because these vessels became collateral arterioles to perfuse the tissue normally perfused by the occluded vessels in the bowel wall. The blood flow in the selected vessels typically increased 50–100% during the occlusion of their parallel neighbor vessel. The venous drainage of the venule beside the occluded 1A or 2A was not impeded. Therefore, in flow-deficient areas of tissue, the normal venous outflow from that area precluded vasoactive materials in venous blood from causing vasodilation of the collateral perfusing 1A or 2A. The major stimulus to the collateral 1A or 2A should be flow-mediated vasodilation as the shear rate is increased. To study the effects of blocking CAT-1 transport of extracellular arginine, we measured periarteriolar [NO], arteriolar diameter, and blood flow velocity for an individual 1A or 2A providing collateral flow to an occluded arteriole before and after L-lysine was applied. After measurements were made with and without occlusion of a 1A or 2A, L-lysine (400 µM) was added to the bicarbonate solution, and this solution suffused the intestine for 30 min before measurements were made with and without the 1A or 2A occlusion. Responses to increased shear rate were not consistently suppressed by 200 µM L-lysine. However, 400 µM L-lysine was very effective in suppressing dilatory and [NO] responses. We believe this situation occurs because of the near doubling of blood flow involved to deliver plasma arginine to the tissue during the collateral perfusion protocol.

Data and statistical analysis. All data are expressed as means ± SE. Statistica 6.0 software was used for all statistical analysis. Comparisons were made using repeated-measures, two-way ANOVA (rest vs. response; natural vs. pharmacological blockade), with post hoc analysis via the Fishers least significant difference (LSD) procedure to assess differences in each variable.

	RESULTS

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For these studies, a total of 22 male Sprague-Dawley rats (315.1 ± 8.9 g) was used with a mean arterial pressure of 123.2 ± 2.5 mmHg. The recorded measurements were not used if during any part of the experiment the mean arterial pressure was not relatively constant or fell below 90 mmHg for an extended period of time. Under control conditions the arterioles used in this study had a baseline diameter of 53.1 ± 3.4 µm, whereas the measured red blood cell velocities were 35.5 ± 2.7 mM/s. The baseline blood flows were calculated to be 0.098 ± 0.021 mM³/s. Also, the average periarteriolar [NO], measured on the lateral flank of the arterioles, was 545.8 ± 144.7 nM under control conditions.

Figure 1A presents the averaged blood flow and NO responses to 200 µM L-lysine from experiments to be described in Figs. 2 and 4. After L-lysine was applied, the blood flow significantly decreased to 79.7 ± 5.7% of control and [NO] decreased to 90.8 ± 3.1% of control. We found a similar reduction in blood flow with 400 µM L-lysine (Fig. 3). We were requested to determine whether a component of the vasoconstriction during L-lysine exposure is independent of an NO mechanism. We compared resting blood flow to conditions during 0.5 mM N-nitro-L-arginine methyl ester (L-NAME) and the combination of L-NAME and 200 µM L-lysine. In addition, the ability of 200 nM bradykinin to increase intestinal blood flow after each type of suppression was evaluated. These data based on studies of four rats are shown in Fig. 1B. L-NAME lowered blood flow to 63.3 + 4.6% of control and strongly limited the vasodilation to bradykinin. The combination of L-NAME and L-lysine did not significantly alter the flow relative to that at rest or during bradykinin exposure with L-NAME alone. Therefore, for these conditions, L-lysine did not cause constriction over that induced by a strong suppression of eNOS with L-NAME. In addition, both 200 and 400 µM L-lysine reduced basal flow about one-half as much as did L-NAME, yet subsequent data in Figs. 2–5 show that these concentrations of L-lysine strongly suppressed endothelium-dependent vasodilation and generation of NO to a wide variety of mechanisms.

View larger version (25K): [in this window] [in a new window]   Fig. 1. A: average control values showing effects of 200 µM L-lysine on resting blood flow and nitric oxide concentration ([NO]) for experiments shown in Figs. 2 and 4. After the application of 200 µM L-lysine (or 400), both blood flow and [NO] were significantly decreased. B: to determine whether a component of the vasoconstriction caused by L-lysine was not endothelial nitric oxide synthase (eNOS) dependent, we measured effects of 200 µM L-lysine on intestinal blood flow after 0.5 mM N-nitro-L-arginine methyl ester (L-NAME) was used to suppress eNOS. The strong suppression of dilation to 200 nM bradykinin after L-NAME indicated that eNOS was inhibited. L-Lysine did not significantly alter resting blood flow or the response to bradykinin after L-NAME treatment. *P < 0.05 vs. control. #P < 0.05 vs. natural paired condition.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of L-lysine on intestinal arteriolar blood flow and periarteriolar [NO] during NaCl hyperosmolarity. A: during natural conditions, blood flow increased significantly compared with the control as the osmolarity of the bathing fluid was increased. At 300 mosM (rest) and at 360 mosM (hyperosmotic) there was a significant reduction in blood flow compared with the natural paired condition after the application of L-lysine (200 µM). B: periarteriolar [NO] increased significantly compared with the control as the bathing fluid osmolarity was increased during natural conditions. At 360 mosM (hyperosmotic), application of L-lysine (200 µM) resulted in a significant reduction in periarteriolar [NO] compared with the natural paired condition. Data are means ± SE. *P < 0.05 vs. the control. #P < 0.05 vs. natural paired condition.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effect of L-lysine on intestinal arteriolar blood flow and periarteriolar [NO] during low-oxygen conditions. A: blood flow increased significantly at 0% oxygen compared with the control at 5% oxygen during natural conditions. After L-lysine (200 µM) was applied, there was a significant decrease in blood flow compared with the natural paired condition at 0% and 5% oxygen. B: during natural conditions, periarteriolar [NO] increased significantly at 0% oxygen compared with the control at 5% oxygen. There was a significant reduction in periarteriolar [NO] at 0% oxygen after L-lysine (200 µM) was applied compared with the natural paired condition. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. natural paired condition.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Effect of L-lysine on intestinal arteriolar blood flow and periarteriolar [NO] during collateral perfusion. A: blood flow during natural conditions significantly increased compared with the control after the adjacent arteriole was occluded. There was a significantly diminished increase in blood flow compared with the natural paired condition during occlusion after L-lysine (400 µM) was applied. B: during natural conditions, periarteriolar [NO] significantly increased compared with the control after an adjacent arteriole was occluded. Compared with the natural paired condition, after L-lysine (400 µM) was applied there was a significantly diminished increase in periarteriolar [NO] after an adjacent arteriole was occluded. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. natural paired condition.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effect of KB-R7943 on intestinal arteriolar blood flow and periarteriolar PO2 during low-oxygen conditions. A: at 0% oxygen compared with the control at 5% oxygen during natural conditions blood flow increased significantly. After KB-R7943 (50 µM) was applied, there was a significant decrease in blood flow compared with the natural paired condition at 0% and 5% oxygen. B: there was a significant reduction in periarteriolar PO2 at 5% oxygen after KB-R7943 (50 µM) was applied compared with the control, as well as a significant reduction in periarteriolar PO2 at 0% oxygen compared with the control and the natural paired condition. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. natural paired condition.

For periarteriolar [NO], there was a consistent decline in the mean concentration with L-lysine that did not achieve a significant difference at the resting condition of 300 mosM due to variability between vessels (Fig. 2B). At 360 mosM, periarteriolar [NO] was increased significantly to 139.1 ± 10.2% during the natural state, but the periarteriolar [NO] did not increase (99.1 ± 6.5%) in response to NaCl hyperosmolarity in the presence of L-lysine. These results suggests that the cationic amino acid transporter may play an important role in regulating blood flow and periarteriolar [NO] during exposure of NaCl hyperosmolarity to the intestinal vasculature.

Effect of competitive inhibition of arginine transport via the cationic amino acid transporter by L-lysine during collateral perfusion. Five male Sprague-Dawley rats (282.2 ± 7.1 g) were used in these studies, and all values are percentages of control. During natural conditions, blood flow decreased significantly to 79.8 ± 3.6% compared with control after L-lysine was applied, as shown in Fig. 3A. After an adjacent 1A or 2A was occluded during natural conditions, blood flow in the arteriole significantly increased to 189.6 ± 22.9%. Compared with the natural occluded condition when flow was elevated, there was a significantly diminished increase in blood flow to 115.5 ± 13.2% during the occlusion after L-lysine was applied. Observation of both the larger arterioles used for NO measurements and the distal smaller arterioles indicated very limited vasodilation in the presence of L-lysine at a time blood flow should have increased during the occlusion protocol.

As shown in Fig. 3B, there was no significant difference compared with control in periarteriolar [NO] during natural conditions after 400 µM L-lysine was applied. The periarteriolar [NO] significantly increased to 161.1 ± 10.0% after an adjacent 1A was occluded during natural conditions. In the presence of L-lysine during the occlusion, there was a significantly diminished increase in [NO] of 114.1 ± 5.6%, or a response of about one-fourth of normal. These results suggest that during collateral perfusion, the interference with transport of extracellular arginine by the cationic amino acid transporter may play a significant role in regulating flow-mediated increases in periarteriolar NO production.

Effect of competitive inhibition of arginine transport via the cationic amino acid transporter by L-lysine during low-oxygen conditions. Seven male Sprague-Dawley rats (323.3 ± 9.2 g) were used in these studies, and all values are percentages of control. At the resting condition of 5% oxygen, there was a significant difference in blood flow to 82.7 ± 7.7% after L-lysine was applied compared with control, as shown in Fig. 4A. At 0% oxygen, there was no significant increase in blood flow (86.9 ± 7.2%) after the application of L-lysine, whereas the natural paired condition significantly increased flow to 125.3 ± 2.7% of control.

As shown in Fig. 4B, there was no significant difference in periarteriolar [NO] at 5% oxygen after the application of L-lysine compared with control, although the average [NO] was reduced to 90.1 ± 4.6% of normal. During natural conditions at 0% oxygen, periarteriolar [NO] was significantly increased to 132.7 ± 10.4%. After L-lysine was applied, the [NO] response (92.5 ± 5.4%) was absent compared with that during the natural condition at 0% oxygen. These results indicate the transport of arginine through the cationic amino acid transporter has a significant role in regulating blood flow and periarteriolar NO production when oxygen availability is decreased. As shown below, 0% oxygen in the bathing medium decreases the periarteriolar oxygen tension <10%. However, the increase in blood flow of 25% and increase in [NO] of 33% in normal conditions indicate the oxygen regulatory system of vessels has been activated to increase NO production and associated dilation.

Effect of NCX inhibition by KB-R7943 during low-oxygen conditions. In these studies, five male Sprague-Dawley rats (329.8 ± 7.8 g) were used and all values are percentage of control. There was a significant reduction in blood flow to 53.3 ± 4.7% after KB-R7943 was applied compared with control flow at 5% oxygen, as shown in Fig. 5A. At 0% oxygen, there was a significant increase in blood flow to 132.5 ± 8.23% compared with control under natural conditions. After KB-R7943 was administered, exposure to 0% oxygen media did significantly increase the blood flow to 65.6 ± 3.7% versus that of 53.3 ± 4.7% during exposure to 5% oxygen medium with KB-R7943. These data should be considered in the context of how the periarteriolar PO₂ is influenced when these flow events occur. There was a significant reduction in periarteriolar PO₂ at 5% oxygen after the application of KB-R7943 to 70.5 ± 5.2% compared with control, as shown in Fig. 5B. During natural conditions at 0% oxygen, periarteriolar PO₂ was diminished to 91.6 ± 3.4%. After KB-R7943 was applied, there was a significant PO₂ decrease to 61.2 ± 4.6% compared with control with KB-R7943 and the natural condition at 0% oxygen. Therefore, a much larger decrease in vessel wall oxygen tension occurred after suppression of the NCX at both oxygen tensions used. These results suggests that the transport of calcium ions into caveolar regions of endothelial cells via the NCX has a significant role in regulating blood flow and periarteriolar PO₂ levels during normal and low-oxygen conditions.

Because of the substantial constrictor effect KB-R7943 had on arteriole behavior, we were concerned that KB-R7943 may have nonspecific effects on the intestinal vasculature. We used the endothelium-dependent vasodilator bradykinin to determine whether the arteriole dilatory response was still intact. As shown in Fig. 6, KB-R7943 only significantly diminished the arteriole dilatory response at 20 nM bradykinin, which is a threshold dosage under normal conditions. However, KB-R7943 had no significant effect on the ability of arterioles to dilate in response to the application of 100 or 200 nM bradykinin. These results suggest that the observed responses to KB-R7943 are not a result of nonspecific effects on endothelium-dependent dilation but result from the specific inhibition of the NCX.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effect of KB-R7943 on arteriole dilation stimulated by bradykinin. At rest and at 20 nM bradykinin, there was a significant reduction in arteriole dilation compared with the control after KB-R7943 (50 µM) was applied to the intestinal vasculature. At 100 and 200 nM bradykinin, there was a significant increase in arteriole dilation compared with the control before and after KB-R7943 was administered. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. natural paired condition.

	DISCUSSION

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View larger version (29K): [in this window] [in a new window]   Fig. 7. Hypothetical scheme of NO production in vascular endothelial cells during different physiological stimuli. A: during NaCl hyperosmolarity, Na+ enters endothelial cells predominantly through the Na+-K+-2Cl– cotransporter. Na+ is then extruded for extracellular Ca2+ via the Na+/Ca2+ exchanger (NCX), which in turn increases the subplasmalemmal Ca2+ concentration. Ca2+ then complexes with calmodulin and eNOS, dissociating eNOS from the eNOS-inhibitory protein caveolin-1 (Cav-1). Some of the L-arginine that is oxidized by eNOS to form NO is transported into the cell by the cationic amino acid transporter CAT-1. B: acute shear increase stimulates an increase in intracellular Ca2+ through either the transport of extracellular Ca2+ or internal Ca2+ stores. Calmodulin and Ca2+ complex with and activate eNOS, which oxidizes L-arginine (some of which is transported into the cell by CAT-1) to form NO. C: low oxygen initiates a Na+ influx, possibly through the Na+-glucose cotransport and/or the Na+/H+ exchanger. Na+ is then exchanged for extracellular Ca2+ by the NCX, thereby increasing subplasmalemmal Ca2+, which then complexes with calmodulin to activate eNOS. Extracellular L-arginine transported into the caveolar region by CAT-1 is an important L-arginine source for NO production during low-oxygen conditions.

In the small intestine, the physiological form of hyperosmolarity routinely developed is NaCl hyperosmolarity during luminal nutrient absorption. In this study, the entire intestinal vasculature was exposed to a NaCl concentration comparable to concentrations that occur naturally in the submucosal layer during nutrient absorption (8, 13). Limiting the CAT-1 transport of extracellular L-arginine strongly suppressed the increase in intestinal blood flow and elevation in arteriolar wall [NO] during NaCl hyperosmolarity (Fig. 2). Our laboratory previously showed (62) that during NaCl hyperosmolarity, sodium ions enter intestinal endothelial cells mainly through Na⁺-K⁺-2Cl^– cotransporter channels, as shown in Fig. 7. The sodium ion is then extruded out of the cell in exchange for extracellular calcium ion into the cell by the NCX (62). The NCX transporter is localized in caveolae (57), along with eNOS and the CAT-1 transporter, as diagrammed in Fig. 7. During NaCl hyperosmolarity, the increase in eNOS activity is partly due to the NCX increasing endothelial calcium, which in concert with calmodulin binds to and activates eNOS by dissociating eNOS from caveolin-1 (46). The final step in the hyperosmolarity pathway is the transport of extracellular L-arginine into caveolar regions of intestinal endothelial cells via CAT-1 for increased NO production, as the data in Fig. 2 support.

Because they are in direct contact with moving blood, vascular endothelial cells constantly encounter shear stress (14). In both cultured cells (40) and our studies of in vivo vessels (9, 12), changes in shear stress have been shown to very quickly alter endothelial NO production. In our in vivo studies, the [NO] changes essentially as rapidly as the flow alteration, whether increased or decreased (9, 12). Multiple mechanisms have been implicated in increased eNOS activity during elevated shear, including endothelial calcium-activated potassium channels (55), and AKT activation of eNOS (15, 39). In vitro studies of vessels, such as those by Bryan et al. (17) and Jen et al. (37), report an increased endothelial calcium concentration as shear stress or flow is increased, although other in vitro studies do not find a shear-calcium relationship (58) or, in the case of Worthen and Nollert (59), a shear-calcium relationship depends on the presence of either thrombin or histamine. An intermediate position found by Muller et al. (47) for isolated coronary arterioles is both calcium-dependent and -independent components of shear-dependent vasodilation. An in vivo study by Duza and Sarelius (27) found that low flow is associated with a reduction in the frequency of endothelial calcium concentration transients. Given that a calcium-shear relationship is more probable than not, we included calcium and arginine transport as common components of the cellular shear mechanism in our overview model of endothelial function in Fig. 7. Our evidence of the arginine linkage is shown in Fig. 3. These data show that intestinal blood flow and arteriolar wall [NO] were significantly suppressed during elevated shear flow after the application of L-lysine to suppress the availability of L-arginine. During natural conditions, the forcing of increased flow in larger arterioles elevated flow to 190% of control and the [NO] increased to 161% of control. These are major responses compared with those during hyperosmolarity and reduced oxygen stresses (Figs. 2 and 4). During L-lysine exposure, the increase in flow was from the resting state of 80% of control to 116% during the increased flow stage, and the [NO] increased 17%. These are minor responses compared with those during the natural state. The limited blood flow response during forced collateral perfusion is the inability of arterioles to appropriately dilate, presumably because the NO mechanism is suppressed. We interpret these results to support a role for CAT-1-mediated transport of extracellular L-arginine in shear stress-induced endothelial NO production. What we do not know is whether the shear mechanism increases CAT-1 activity or whether a caveolar mechanism linked with eNOS activation increases the transport of L-arginine. It has been previously reported (25) that during the initial phase of increased NO production by shear stress, there appears to be a transient increase in intracellular free Ca²⁺. This phase lasts from seconds to roughly 30 min and is Ca²⁺/calmodulin dependent (14). Because our measurements were over this time frame, the Ca²⁺/calmodulin-dependent pathway seems a likely possibility to activate CAT-1 transport.

In many vascular beds, an important regulator of microvascular tone is oxygen. Previous studies have shown that NO release from endothelial cells, isolated vessels, and in vivo arterioles is increased during reduced oxygen availability (19, 48, 51, 51). Data from these types of studies indicate the possibility that endothelial cells are somehow an oxygen sensor and that when reduced oxygen is sensed, the NO mechanism is activated. This possibility is supported by our results in Fig. 4. When the intestinal vasculature is exposed to lower oxygen availability, there is a significant increase in arteriole [NO] and blood flow. These vascular responses to reduced oxygen availability are strongly suppressed when the transport of extracellular L-arginine by CAT-1 into arteriole endothelial cells is limited. These results suggest that during lowered oxygen conditions, the NO mechanism is activated and CAT-1 transport of extracellular L-arginine is required for the NO mechanism to properly function. In our studies, we did not cause hypoxia because lowering the bath oxygen tension only reduces perivascular oxygen tension by 8%, as shown in Fig. 5 and as previously reported in our earlier studies (8, 48). However, such seemingly small reductions in arteriolar wall oxygen tension can increase intestinal blood flow >25% and increase the periarteriolar [NO] by 33%, as shown in Fig. 4 and in our prior studies (8, 48). After L-lysine application, the ability of the vasculature to both increase blood flow and elevate NO was substantially suppressed.

We suspect that reduced oxygen tension initiates increased NO production by the entry of sodium ions into endothelial cells followed by exchange for extracellular calcium ions by NCX for Na⁺/Ca²⁺ exchange. During hypoxia, previous studies of cultured endothelial cells (34, 36) have implicated the activation of the Na⁺/H⁺ exchanger (NHE), causing an increase in intracellular sodium ions, which results in an increase in intracellular calcium ions via NCXs (43). In endothelial cells, localized increases in caveolae endothelial subplasmalemmal calcium concentration may be all that is required for eNOS activation and subsequent NO production (50, 60). This scenario is possible because Teubl et al. (57) have shown the presence of the NCX in endothelial caveolae and that calcium entry via reverse mode of the NCX is required for sodium-dependent facilitation of eNOS activation. A study on human umbilical vein endothelial cells suggested that under hypoxic conditions, glycolysis is activated, leading to the stimulation of sodium-glucose cotransport, which results in an influx of sodium. This sodium influx, in turn, activates the NCX causing a net influx of Ca²⁺ (5). There are also studies that reduced oxygen availability results in loading of sodium into endothelial cells (32, 61), with activation of the NCX thereafter. As shown in Fig. 5, after NCX blockade to suppress the exchange of intracellular sodium ions for extracellular calcium ions, the increase in blood flow to reduced oxygen tension was strongly suppressed. Furthermore, without a mechanism to allow normal vasodilation the vessel oxygen tension was lowered more by reduced oxygen availability than occurred during normal conditions. In effect, a greater oxygen deficit occurred because the vasculature was unable to use NO to defend itself. What we did not expect to happen was vasoconstriction at rest during NCX blockade with an attendant decrease in resting blood flow and vessel wall oxygen tension. We believe this indicates that NCX transporters in endothelial cells are removing sodium ions at rest and replacing them with calcium ions that influence resting NO generation. In times of reduced oxygen availability, this exchange process is even greater. The presumed effects on intracellular calcium concentration are to provide a higher rate of eNOS activity. This would explain the increase in [NO] we find during reduced oxygen tension during normal conditions in this (Fig. 4) and prior studies (9, 48). In reference to the role of CAT-1 in the endothelial response to decreased oxygen, as shown schematically in Fig. 7, the initiating event is likely in part an increase in sodium entry followed by Na⁺/Ca²⁺ exchange to activate eNOS, and as part of the overall activation of the cell, CAT-1 provides the L-arginine required for elevated NO production.

It is clear that in vascular endothelial cells, eNOS production of NO can result from a variety of mechanisms depending on the physiological stimulus. In this study we have shown that a common factor in the NO-eNOS mechanisms for NaCl hyperosmolarity, low oxygen, and increased flow shear is the transport of extracellular L-arginine into vascular endothelial cells via CAT-1. These studies further elucidate the mechanisms by which three physiologically relevant stimuli induce an increase in vascular endothelial NO production.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-20605 and HL-25824.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. G. Bohlen, Dept. of Cellular and Integrative Physiology, Indiana Univ. Medical School, 635 Barnhill Drive, Indianapolis, IN 46202 (e-mail: gbohlen{at}iupui.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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Anderson RG. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci USA 90: 10909–10913, 1993.[Abstract/Free Full Text]
2. Arnal JF, Munzel T, Venema RC, James NL, Bai CL, Mitch WE, and Harrison DG. Interactions between L-arginine and L-glutamine change endothelial NO production. An effect independent of NO synthase substrate availability. J Clin Invest 95: 2565–2572, 1995.[Web of Science][Medline]
3. Barron LA, Green GM, and Khalil RA. Gender differences in vascular smooth muscle reactivity to increases in extracellular sodium salt. Hypertension 39: 425–432, 2002.[Abstract/Free Full Text]
4. Baydoun AR, Emery PW, Pearson JD, and Mann GE. Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells. Biochem Biophys Res Commun 173: 940–948, 1990.[CrossRef][Web of Science][Medline]
5. Berna N, Arnould T, Remacle J, and Michiels C. Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na⁺-glucose cotransporter. J Cell Biochem 84: 115–131, 2001.[Web of Science][Medline]
6. Block ER, Herrera H, and Couch M. Hypoxia inhibits L-arginine uptake by pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 269: L574–L580, 1995.[Abstract/Free Full Text]
7. Bohlen HG. Determinants of resting and passive intestinal vascular pressures in rat and rabbit. Am J Physiol Gastrointest Liver Physiol 253: G587–G595, 1987.[Abstract/Free Full Text]
8. Bohlen HG. Mechanism of increased vessel wall nitric oxide concentrations during intestinal absorption. Am J Physiol Heart Circ Physiol 275: H542–H550, 1998.[Abstract/Free Full Text]
9. Bohlen HG. Protein kinase II in Zucker obese rats compromises oxygen and flow-mediated regulation of nitric oxide formation. Am J Physiol Heart Circ Physiol 286: H492–H497, 2004.[Abstract/Free Full Text]
10. Bohlen HG and Lash JM. Intestinal lymphatic vessels release endothelial-dependent vasodilators. Am J Physiol Heart Circ Physiol 262: H813–H819, 1992.[Abstract/Free Full Text]
11. Bohlen HG and Lash JM. Intestinal absorption of sodium and nitric oxide-dependent vasodilation interact to dominate resting vascular resistance. Circ Res 78: 231–237, 1996.[Abstract/Free Full Text]
12. Bohlen HG and Nase GP. Dependence of intestinal arteriolar regulation on flow-mediated nitric oxide formation. Am J Physiol Heart Circ Physiol 279: H2249–H2258, 2000.[Abstract/Free Full Text]
13. Bohlen HG and Unthank JL. Rat intestinal lymph osmolarity during glucose and oleic acid absorption. Am J Physiol Gastrointest Liver Physiol 257: G438–G446, 1989.[Abstract/Free Full Text]
14. Boo YC and Jo H. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases. Am J Physiol Cell Physiol 285: C499–C508, 2003.[Abstract/Free Full Text]
15. Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, and Jo H. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser¹¹⁷⁹ by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 277: 3388–3396, 2002.[Abstract/Free Full Text]
16. Borders JL and Granger HJ. An optical Doppler intravital velocimeter. Microvasc Res 27: 117–127, 1984.[CrossRef][Web of Science][Medline]
17. Bryan RM Jr, Steenberg ML, and Marrelli SP. Role of endothelium in shear stress-induced constrictions in rat middle cerebral artery. Stroke 32: 1394–1400, 2001.[Abstract/Free Full Text]
18. Buerk DG, Riva CE, and Cranstoun SD. Nitric oxide has a vasodilatory role in cat optic nerve head during flicker stimuli. Microvasc Res 52: 13–26, 1996.[CrossRef][Web of Science][Medline]
19. Busse R, Pohl U, Kellner C, and Klemm U. Endothelial cells are involved in the vasodilatory response to hypoxia. Pflügers Arch 397: 78–80, 1983.[CrossRef][Web of Science][Medline]
20. Closs EI. Expression, regulation and function of carrier proteins for cationic amino acids. Curr Opin Nephrol Hypertens 11: 99–107, 2002.[CrossRef][Web of Science][Medline]
21. Closs EI, Basha FZ, Habermeier A, and Forstermann U. Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B. Nitric Oxide 1: 65–73, 1997.[CrossRef][Web of Science][Medline]
22. Closs EI and Mann GE. Identification of carrier systems in plasma membranes of mammalian cells involved in transport of L-arginine. Methods Enzymol 301: 78–91, 1999.[Web of Science][Medline]
23. Closs EI, Scheld JS, Sharafi M, and Forstermann U. Substrate supply for nitric-oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters. Mol Pharmacol 57: 68–74, 2000.[Abstract/Free Full Text]
24. Coleman DA and Khalil RA. Physiologic increases in extracellular sodium salt enhance coronary vasoconstriction and Ca²⁺ entry. J Cardiovasc Pharmacol 40: 58–66, 2002.[CrossRef][Web of Science][Medline]
25. Corson MA, James NL, Latta SE, Nerem RM, Berk BC, and Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res 79: 984–991, 1996.[Abstract/Free Full Text]
26. Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ, and Cooke JP. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest 90: 1248–1253, 1992.[Web of Science][Medline]
27. Duza T and Sarelius IH. Localized transient increases in endothelial cell Ca²⁺ in arterioles in situ: implications for coordination of vascular function. Am J Physiol Heart Circ Physiol 286: H2322–H2331, 2004.[Abstract/Free Full Text]
28. Fike CD, Kaplowitz MR, Rehorst-Paea LA, and Nelin LD. L-Arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs. J Appl Physiol 88: 1797–1803, 2000.[Abstract/Free Full Text]
29. Friedemann MN, Robinson SW, and Gerhardt GA. o-Phenylenediamine-modified carbon fiber electrodes for the detection of nitric oxide. Anal Chem 68: 2621–2628, 1996.[Medline]
30. Gold ME, Bush PA, and Ignarro LJ. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun 164: 714–721, 1989.[CrossRef][Web of Science][Medline]
31. Greene B, Pacitti AJ, and Souba WW. Characterization of L-arginine transport by pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 264: L351–L356, 1993.[Abstract/Free Full Text]
32. Hammarstrom AK and Gage PW. Hypoxia and persistent sodium current. Eur Biophys J 31: 323–330, 2002.[CrossRef][Web of Science][Medline]
33. Hardy TA and May JM. Coordinate regulation of L-arginine uptake and nitric oxide synthase activity in cultured endothelial cells. Free Radic Biol Med 32: 122–131, 2002.[CrossRef][Web of Science][Medline]
34. Hattori R, Otani H, Moriguchi Y, Matsubara H, Yamamura T, Nakao Y, Omiya H, Osako M, and Imamura H. NHE and ICAM-1 expression in hypoxic/reoxygenated coronary microvascular endothelial cells. Am J Physiol Heart Circ Physiol 280: H2796–H2803, 2001.[Abstract/Free Full Text]
35. Hecker M, Sessa WC, Harris HJ, Anggard EE, and Vane JR. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle L-citrulline to L-arginine. Proc Natl Acad Sci USA 87: 8612–8616, 1990.[Abstract/Free Full Text]
36. Horikawa N, Nishioka M, Itoh N, Kuribayashi Y, Matsui K, and Ohashi N. The Na⁺/H⁺ exchanger SM-20220 attenuates ischemic injury in in vitro and in vivo models. Pharmacology 63: 76–81, 2001.[Web of Science][Medline]
37. Jen CJ, Jhiang SJ, and Chen HI. Invited review: effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo. J Appl Physiol 89: 1657–1662, 2000.[Abstract/Free Full Text]
38. Jiang J, Valen G, Tokuno S, Thoren P, and Pernow J. Endothelial dysfunction in atherosclerotic mice: improved relaxation by combined supplementation with L-arginine-tetrahydrobiopterin and enhanced vasoconstriction by endothelin. Br J Pharmacol 131: 1255–1261, 2000.[CrossRef][Web of Science][Medline]
39. Jin ZG, Wong C, Wu J, and Berk BC. Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate Akt and eNOS activation in endothelial cells. J Biol Chem 2005.
40. Kuchan MJ and Frangos JA. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am J Physiol Cell Physiol 266: C628–C636, 1994.[Abstract/Free Full Text]
41. Kurz S and Harrison DG. Insulin and the arginine paradox. J Clin Invest 99: 369–370, 1997.[Web of Science][Medline]
42. Lerman A, Burnett JC Jr, Higano ST, McKinley LJ, and Holmes DR Jr. Long-term L-arginine supplementation improves small-vessel coronary endothelial function in humans. Circulation 97: 2123–2128, 1998.[Abstract/Free Full Text]
43. Levitsky J, Gurell D, and Frishman WH. Sodium ion/hydrogen ion exchange inhibition: a new pharmacologic approach to myocardial ischemia and reperfusion injury. J Clin Pharmacol 38: 887–897, 1998.[Abstract]
44. MacKenzie A and Wadsworth RM. Extracellular L-arginine is required for optimal NO synthesis by eNOS and iNOS in the rat mesenteric artery wall. Br J Pharmacol 139: 1487–1497, 2003.[CrossRef][Web of Science][Medline]
45. McDonald KK, Zharikov S, Block ER, and Kilberg MS. A caveolar complex between the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the "arginine paradox." J Biol Chem 272: 31213–31216, 1997.[Abstract/Free Full Text]
46. Minshall RD, Sessa WC, Stan RV, Anderson RG, and Malik AB. Caveolin regulation of endothelial function. Am J Physiol Lung Cell Mol Physiol 285: L1179–L1183, 2003.[Abstract/Free Full Text]
47. Muller JM, Davis MJ, Kuo L, and Chilian WM. Changes in coronary endothelial cell Ca²⁺ concentration during shear stress- and agonist-induced vasodilation. Am J Physiol Heart Circ Physiol 276: H1706–H1714, 1999.[Abstract/Free Full Text]
48. Nase GP, Tuttle J, and Bohlen HG. Reduced perivascular PO₂ increases nitric oxide release from endothelial cells. Am J Physiol Heart Circ Physiol 285: H507–H515, 2003.[Abstract/Free Full Text]
49. Palmer RM and Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun 158: 348–352, 1989.[CrossRef][Web of Science][Medline]
50. Paltauf-Doburzynska J, Posch K, Paltauf G, and Graier WF. Stealth ryanodine-sensitive Ca²⁺ release contributes to activity of capacitative Ca²⁺ entry and nitric oxide synthase in bovine endothelial cells. J Physiol 513: 369–379, 1998.[Abstract/Free Full Text]
51. Pohl U and Busse R. Hypoxia stimulates release of endothelium-derived relaxant factor. Am J Physiol Heart Circ Physiol 256: H1595–H1600, 1989.[Abstract/Free Full Text]
52. Pollock JS, Forstermann U, Mitchell JA, Warner TD, Schmidt HH, Nakane M, and Murad F. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci USA 88: 10480–10484, 1991.[Abstract/Free Full Text]
53. Schini VB and Vanhoutte PM. L-Arginine evokes both endothelium-dependent and -independent relaxations in L-arginine-depleted aortas of the rat. Circ Res 68: 209–216, 1991.[Abstract/Free Full Text]
54. Steenbergen JM and Bohlen HG. Sodium hyperosmolarity of intestinal lymph causes arteriolar vasodilation in part mediated by EDRF. Am J Physiol Heart Circ Physiol 265: H323–H328, 1993.[Abstract/Free Full Text]
55. Sun D, Huang A, Koller A, and Kaley G. Endothelial K_Ca channels mediate flow-dependent dilation of arterioles of skeletal muscle and mesentery. Microvasc Res 61: 179–186, 2001.[CrossRef][Web of Science][Medline]
56. Sun D, Messina EJ, Koller A, Wolin MS, and Kaley G. Endothelium-dependent dilation to L-arginine in isolated rat skeletal muscle arterioles. Am J Physiol Heart Circ Physiol 262: H1211–H1216, 1992.[Abstract/Free Full Text]
57. Teubl M, Groschner K, Kohlwein SD, Mayer B, and Schmidt K. Na⁺/Ca²⁺ exchange facilitates Ca²⁺-dependent activation of endothelial nitric-oxide synthase. J Biol Chem 274: 29529–29535, 1999.[Abstract/Free Full Text]
58. Ungvari Z, Sun D, Huang A, Kaley G, and Koller A. Role of endothelial [Ca²⁺]_i in activation of eNOS in pressurized arterioles by agonists andwall shear stress. Am J Physiol Heart Circ Physiol 281: H606–H612, 2001.[Abstract/Free Full Text]
59. Worthen LM and Nollert MU. Intracellular calcium response of endothelial cells exposed to flow in the presence of thrombin or histamine. J Vasc Surg 32: 593–601, 2000.[CrossRef][Web of Science][Medline]
60. Wu G and Morris SM Jr. Arginine metabolism: nitric oxide and beyond. Biochem J 336: 1–17, 1998.[Web of Science][Medline]
61. Yamaji R, Fujita K, Takahashi S, Yoneda H, Nagao K, Masuda W, Naito M, Tsuruo T, Miyatake K, Inui H, and Nakano Y. Hypoxia up-regulates glyceraldehyde-3-phosphate dehydrogenase in mouse brain capillary endothelial cells: involvement of Na⁺/Ca²⁺ exchanger. Biochim Biophys Acta 1593: 269–276, 2003.[Medline]
62. Zani BG and Bohlen HG. Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production. Am J Physiol Heart Circ Physiol 288: H89–H95, 2005.[Abstract/Free Full Text]
63. Zharikov SI and Block ER. Characterization of L-arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells. Biochim Biophys Acta 1369: 173–183, 1998.[Medline]

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