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Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

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

Submitted 29 June 2004 ; accepted in final form 19 August 2004

	ABSTRACT

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NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na⁺-K⁺-2Cl^– cotransporter inhibitor) or amiloride (Na⁺/H⁺ exchange channel inhibitor). Suppressing amiloride-sensitive Na⁺/H⁺ exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na⁺-K⁺-2Cl^– channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na⁺/Ca²⁺ exchanger extrudes Na⁺ in exchange for Ca²⁺, thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na⁺/Ca²⁺ exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na⁺-K⁺-2Cl^– channels. The Na⁺/Ca²⁺ exchanger then extrudes Na⁺ and increases endothelial Ca²⁺. The increase in endothelial Ca²⁺ causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.

endothelial nitric oxide synthase; sodium/calcium exchanger; sodium-potassium-chloride cotransporter; sodium-hydrogen exchanger 1

The linkage of mucosal metabolism during absorption to decreased resistance of the major resistance vessels likely involves NaCl hyperosmolarity. Lundgren and colleagues (18, 19, 23) have shown that as nutrients are absorbed, there is a dramatic increase in sodium concentration in intestinal villi. By measuring venous blood and lymph osmolarity, Bohlen and Unthank (11) found that submucosal arterioles are exposed to these hypertonic conditions by the hypertonic lymph and venous blood flowing from the mucosa. Steenbergen and Bohlen (38) showed that a dose-dependent vasodilation of the submucosal vessels was caused by increasing NaCl osmolarity of the submucosal interstitial space by addition of NaCl. Another study, by Bohlen (8), showed that NaCl hyperosmolarity caused as large an increase in NO, measured with NO-sensitive microelectrodes, as equivalent hypertonicity during natural absorptive hyperemia.

The linkage of NaCl hyperosmolarity to an increase in NO generation potentially involves a number of intermediary steps. When cells are exposed to a hyperosmotic solution, a decrease in cell volume initially occurs because of water moving outside the cells via osmotic flow. This cell shrinkage stimulates a regulatory volume increase (RVI) characterized by activation of ion uptake systems and organic osmolyte transporters, allowing the cells to increase their volume toward original levels through the net uptake of Na⁺, Cl^–, K⁺, and water (32, 37). The most likely route for Na⁺ to enter endothelial cells during NaCl hyperosmolarity is through the major ion transport systems involved in cell volume regulation, such as the Na⁺/H⁺ exchanger and the Na⁺-K⁺-2Cl^– cotransporter (32). Loading a cell with Na⁺ would in turn stimulate accumulation of Ca²⁺ as Na⁺ is removed by the Na⁺/Ca²⁺ exchanger (NCX), which has been identified in endothelial cells (24, 25). The exchanger functions in forward mode when extruding Ca²⁺ from the cell and in reverse mode when bringing Ca²⁺ into the cell in exchange for removing excess intracellular Na⁺ (35).

We hypothesized that NaCl hyperosmolarity increases intracellular calcium concentration of endothelial cells, favoring the activation of nitric oxide synthase (NOS) and increased NO formation to initiate dilation. In addition, we determined the extent to which the Na⁺/H⁺ exchanger and the Na⁺-K⁺-2Cl^– cotransporter are involved in endothelial responses to NaCl hyperosmolarity. In these studies, physiological concentrations of NaCl comparable to those that naturally occur during nutrient absorption were applied to the entire exposed intestinal vasculature while arteriolar diameter, blood flow, and arteriolar wall NO concentration ([NO]) were measured. We used selective pharmacological blockers of the Na⁺/H⁺ exchanger, the Na⁺-K⁺-2Cl^– cotransporter, and the NCX to determine how each channel system affected intestinal vascular reactivity and NO generation to NaCl hyperosmolarity. The results indicate that even mild increases in NaCl hyperosmolarity predominately use the Na⁺-K⁺-2Cl^– cotransporter to admit sodium ions and that the NCX is crucial to the increase in vessel wall [NO] during NaCl hyperosmolarity.

	MATERIALS AND METHODS

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Animal and tissue preparation. All animal procedures were performed in accordance with the "Guiding Principles in the Care and Use of Animals" of the American Physiological Society. Male Sprague-Dawley rats between 250 and 350 g (Harlan, Indianapolis, IN) were anesthetized with thiopental sodium (200 mg/kg; Abbott, Chicago, IL) injected subcutaneously at three sites over both thighs and the lower back. If additional anesthesia was needed, one-fourth of the original dose was given intraperitoneally. Each rat was placed on a heating pad (35–36°C) to maintain a 37°C rectal temperature. To ensure a patent airway, the trachea was intubated with polyethylene tubing (PE-240). The left femoral artery was cannulated (PE-50) to measure arterial blood pressure. Normal saline (0.5 ml·100 g body wt^–1·h^–1) was given to the animal to compensate for fluid loss by urine formation and mechanical ventilation. All animals were ventilated with a Harvard Apparatus small-animal ventilator (model 683; Harvard Apparatus, South Natick, MA) at a rate of 70 breaths/min, the typical ventilation frequency for conscious adult rats. The tidal volume was based on the Harvard Apparatus nomogram; in addition, 0.25–0.5 ml was added per breath to compensate for the dead space of the cannula system. As long as the animals were ventilated and given fluid replacement every hour, the mean arterial pressure was virtually constant for the length of the experiments, and the percent saturation of hemoglobin with oxygen measured in the skin was 95% (Nonin Medical, Minneapolis, MN).

The small intestine was prepared for observation with an established technique (6). The abdominal midline was incised 1.5–2.0 cm. An 10-cm loop of the jejunal region of the small intestine was exteriorized from the abdominal cavity and placed into a heated chamber filled with warm (37°C) saline and covered with plastic wrap (Saran Wrap; Dow, Indianapolis, IN). The intestinal loop was slit longitudinally 2.5 cm along the antimesenteric border with a microcautery and wetted with additional saline to avoid drying of the tissue. The nerves and vascular supply to the small intestine are left intact with this technique. The intestinal debris was rinsed away. Small suture threads were tied to the edges of the intestinal incisions and used to stretch the intestine to physiological dimensions. The vasculature of the muscle-submucosal layers faced upward over a translucent pedestal for transillumination with a direct current-powered quartz-iodine 12-V light source. A fluid chamber was placed over the intestine and lowered into the heating chamber; the chamber housing clamps the sutures to the metal tissue support to secure the intestine in place. A 5 ml/min flow of bicarbonate-buffered physiological saline solution (in mM: 118 NaCl, 6 KCl, 25 Na₂HCO₃, and 3.5 CaCl₂) was passed through the chamber after being heated to 37.5 ± 0.5°C. The physiological saline solution was equilibrated with 90% nitrogen-5% carbon dioxide-5% oxygen. To prevent equilibration with the atmosphere, the fluid lines were protected until the fluid entered a stainless steel heating system. Isoproterenol and norepinephrine (both Sigma, St. Louis, MO) were added to the bathing fluid to partially suppress intestinal motility. The concentration of drugs used had minor effects on vascular resistance, and their concentration was in the range of 10–100 nM in the bathing medium. The use of both an - and a -adrenergic agonist had a synergistic effect to suppress bowel motility, a necessary condition for precise placement of a NO-sensitive microelectrode on the vessel wall.

Microvascular observation and flow velocity measurements. After surgery, the animal was transferred to the stage of an intravital microscope (model BHMJ; Olympus, Hyde Park, NY). Microvessels and small arteries were observed through the microscope with a x10 or x20 Nikon water-immersion lens. Images were recorded with 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). The dimensions of the vessels were measured with the virtual caliper of the image analysis system, and images were stored in digital format. The image analysis system was calibrated in the x- and y-directions with the image of a stage micrometer marked in 10- and 100-µm units. Red blood cell flow velocity was measured with an Optical Doppler Velocimeter (Microcirculation Research Institution, Texas A&M University, College Station, Texas). The linearity of the velocity versus signal output was evaluated with red blood cells on a rotating disk for velocities of 100–600 µm/s. Once both the mean red blood cell velocity and the arteriolar inner diameter were known, flow was calculated with the following equation: 3.14 x velocity x (diameter/2)². This equation assumes a circular diameter. The optical Doppler technique is limited by both very large and small diameters. Therefore, 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 (12). We did not use a correction factor for translating center line velocity to mean velocity because in calculation of percentage of control blood flow, the correction factor would be nulled out.

Perivascular NO measurements. Carbon fiber, recessed-tip glass microelectrodes were used with a polarographic technique to measure [NO] in the intestine. NO-sensitive microelectrodes have been used in past studies in our laboratory (8, 10, 27). The production and calibration of the NO-sensitive microelectrodes were based on our lab's experience and techniques developed by Buerk and colleagues (13) and Freidemann et al. (16). The sharpened outer tip diameter of the microelectrodes was 7–10 µm. A small tip recess of 2–3 µm was made by electrolytically removing carbon at +1.4 V in pH 10 NaHCO₃. Thereafter, the polarization voltage was decreased to +0.7 V to covalently bond Nafion (Aldrich Chemical, Milwaukee, WI) to the carbon. Nafion was used to fill the electrode recess, which diminished random electrical noise. The Nafion also eliminated interference with [NO] measurements caused by ascorbic acid, nitrate, norepinephrine, and tyrosine at physiological concentrations. Relative to a World Precision Instruments (Sarasota, FL) carbon fiber reference electrode or a simple silver-silver chloride electrode, the NO-sensitive microelectrodes were polarized at +0.7 or +0.9 V to measure [NO]. The currents generated were in the picoampere range and were measured with a Keithley model 6517A electrometer (Cleveland, OH). The rationale for using +0.7 or +0.9 V depended on the polarization voltage at which a given electrode was most sensitive to [NO].

On the morning of each experiment, a Diamond General (Ann Arbor, MI) gas tonometer system was used to establish a calibration curve by measurement of the microelectrode current at 0, 600, and 1,200 nM [NO] based on the composition of the NO-N₂ precision calibration gases (Matheson, Joliet, IL) in saline at 37.5°C. A linear current-[NO] relationship was found for each microelectrode used. Basal currents were typically 5–10 pA at 0 nM [NO]. A microelectrode was only used if it possessed sensitivity >1 pA/1,000 nM [NO], which roughly translated to a 1-mV increase in output voltage per each 4 nM increase in [NO]. For a typical periarteriolar [NO] measurement, this equaled 80–120 mV above baseline. Allowing for random noise and current drift, the working resolution of the microelectrodes was typically <10 nM. Also, the sensitivity of the microelectrodes to NO is retained when the osmolarity of the bathing fluid is altered as checked in past studies (8). In addition, the baseline fluid current of the electrode was monitored as the NaCl concentration was gradually increased, and no change in baseline, aside from the normal minor drift, was noted.

An interpolated baseline was calculated over time for each individual [NO] measurement to compensate for the slow electrical drift of the microelectrode. To obtain a baseline measurement, the microelectrode was placed 200 µm above the tissue, and the current generated was used as the 0 nM reference immediately before and after each tissue measurement. These data were used to calculate the rate of drift and an interpolated baseline current for any given time. The current during tissue measurements of [NO] had the baseline current for that time period subtracted to yield a value representing the current equivalent of the [NO].

For measurements of periarteriolar [NO], the sharpened microelectrode tip was pushed through the visceral muscle layers and placed as close to the arteriolar wall as possible. The ideal angle for the microelectrode was attained with the microelectrode shaft nearly parallel to the arteriole. The highest possible [NO] for a given arteriole was the goal for all measurements; therefore, when the vessel diameter changed or the intestine moved, a precision hydraulic micromanipulator (Narishige, Tokyo, Japan) was used to maintain close contact of the arteriole wall and the microelectrode tip.

Effect of Na⁺ transporter inhibitors amiloride and bumetanide on periarteriolar NO production and arteriolar blood flow during NaCl hyperosmolarity. In two separate series of experiments, the effects of amiloride or bumetanide on periarteriolar [NO], arteriolar diameter, and blood flow velocity were determined during NaCl hyperosmolarity. In both series of experiments, periarteriolar [NO], arteriolar diameter, and blood flow velocity were measured for an individual arteriole in the intestine while it was topically suffused with a bicarbonate solution at the control osmolarity of 300, 330, or 380 mosM before and after the addition of bumetanide (10 µM) or amiloride (10 µM). The concentration used for both amiloride and bumetanide was based on previously published reports directly related to this study (2, 17, 28–31, 33). Neither drug had access to the mucosal tissue layer by direct contact. The osmolarity of the bathing solution was changed by adding a 1 osM NaCl solution to the bicarbonate solution flow (5 ml/min) at varying rates from a syringe pump: 12.4 ml/h for 330 mosM and 38.3 ml/h for 380 mosM. Bumetanide or amiloride was added to the bicarbonate solution once for each experiment, and this solution was allowed to suffuse the intestine for 20 min before measurements were repeated with control solution, 330 mosM solution, and 380 mosM solution. All measurements, before and after the addition of bumetanide or amiloride, were made 10 min after the osmolarity of the bathing solution was changed, and both diameter and [NO] responses were stable.

Inhibition of NCX by KB-R7943 during NaCl hyperosmolarity. The effect of the NCX inhibitor KB-R7943 on endothelial NO production and arteriolar blood flow during NaCl hyperosmolarity was determined. The inhibitor did not have access to the villus mucosa epithelial cells. Periarteriolar [NO], arteriolar diameter, and blood flow velocity were measured for an individual arteriole in the intestine at the control osmolarity of 300 mosM and at 360 mosM before and after the administration of KB-R7943 (50 µM). The concentration of KB-R7943 used in this study was based on previously published reports (1, 14). The osmolarity of the bathing solution was changed to 360 mosM by adding a 1 osM NaCl solution to the bicarbonate solution flow (5 ml/min) at the rate of 28.125 ml/h. KB-R7943 was added to the bicarbonate solution once for each experiment, and this solution was allowed to suffuse the intestine for 20 min before measurements were made with control solution and with 360 mosM solution. All measurements, before and after the addition of KB-R7943, were made 10 min after the osmolarity of the bathing solution was changed and vascular behavior was stable.

Data and statistical analysis. All data are expressed as means ± SE. Statistical analysis was carried out with Statistica 6.0 software. To assess differences in each variable, comparisons were made with repeated-measures two-way ANOVA (rest vs. response; natural vs. pharmacological blockade) with post hoc analysis via the Fisher least significant difference procedure.

	RESULTS

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In these studies, under control conditions the mean arterial pressure was 128.4 ± 3.5 mmHg. If the mean arterial pressure was not relatively constant or fell below 90 mmHg for an extended period of time during any part of the experiment, the recorded measurements were not used. The baseline diameters of the arterioles used in these studies were 54.4 ± 3.7 µm. The red blood cell velocities measured under control conditions were 35.9 ± 3.4 mm/s, whereas the calculated baseline blood flows were 0.0987 ± 0.0175 mm³/s. Also, under control conditions the endothelial [NO] values were 552.7 ± 140.5 nM measured on the lateral flank of the arterioles.

Effect of Na⁺ transporter inhibitor amiloride on periarteriolar NO production and arteriolar blood flow during NaCl hyperosmolarity. A total of eight male Sprague-Dawley rats (319 ± 16.5 g) were used for these studies, and all values are percentage of control. As shown in Fig. 1 for arteriolar blood flow, at resting conditions of 300 mosM there was an insignificant change between the control value of natural blood flow and blood flow after amiloride was applied. When the bicarbonate suffusate was increased to 330 mosM, there was no significant difference between the elevated natural blood flow of 116.1 ± 6.3% and the elevated blood flow after amiloride was applied of 117.8 ± 29.4%. Also, at 380 mosM there was no significant change between elevated natural blood flow of 160.0 ± 18.1% and elevated blood flow at 380 mosM after administration of amiloride of 156.2 ± 32.0%.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effect of amiloride on intestinal arteriolar blood flow during NaCl hyperosmolarity. Blood flow increased significantly vs. control as the osmolarity of the bathing fluid was increased during natural conditions. During resting (300 mosM) and hyperosmotic (330 and 380 mosM) conditions, blood flow was not affected by the application of amiloride (10 µM). Data are means ± SE. *P < 0.05 vs. the control.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of amiloride on periarteriolar nitric oxide (NO) concentration ([NO]) during NaCl hyperosmolarity. During natural conditions, periarteriolar [NO] increased significantly vs. control as the bathing fluid osmolarity was increased. At 300 (rest) and 330 (hyperosmotic) mosM, amiloride (10 µM) had no affect on periarteriolar [NO]; however, at 380 mosM (hyperosmotic) there was a significant reduction in periarteriolar [NO] vs. the natural paired condition at 380 mosM after amiloride was applied. Data are means ± SE. *P < 0.05 vs. the control; #P < 0.05 vs. the natural paired condition.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effect of bumetanide on intestinal arteriolar blood flow during NaCl hyperosmolarity. Blood flow increased significantly vs. control when hyperosmotic conditions (330 and 380 mosM) were applied to the intestinal vasculature during natural conditions. At 330 and 380 mosM, bumetanide significantly reduced blood flow vs. the respective natural paired conditions. Data are means ± SE. *P < 0.05 vs. the control; #P < 0.05 vs. the natural paired condition.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effect of bumetanide on periarteriolar [NO] during NaCl hyperosmolarity. Periarteriolar [NO] increased significantly vs. control as the osmolarity of the bathing fluid was increased during natural conditions. At 330 and 380 mosM (hyperosmotic), there was a significant reduction in periarteriolar [NO] vs. the respective natural paired conditions after bumetanide was applied. Data are means ± SE. *P < 0.05 vs. the control; #P < 0.05 vs. the natural paired condition.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Effect of KB-R7943 on intestinal arteriolar blood flow during NaCl hyperosmolarity. During natural conditions, blood flow increased significantly vs. control as the osmolarity of the bathing fluid was increased. At 300 (rest) and 360 (hyperosmotic) mosM, there was a significant reduction in blood flow vs. control and vs. the natural paired condition after the application of KB-R7943 (50 µM). Data are means ± SE. *P < 0.05 vs. the control; #P < 0.05 vs. the natural paired condition.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effect of KB-R7943 on periarteriolar [NO] during NaCl hyperosmolarity. Periarteriolar [NO] increased significantly vs. control as the bathing fluid osmolarity was increased during natural conditions. At 300 mosM (rest), there was a significant decrease vs. control in periarteriolar [NO] after KB-R7943 (50 µM) was applied. At 360 mosM (hyperosmotic), application of KB-R7943 resulted in a significant reduction in periarteriolar [NO] vs. the natural paired condition. Data are means ± SE. *P < 0.05 vs. the control; #P < 0.05 vs. the natural paired condition.

	DISCUSSION

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In this study, arteriolar diameter, blood flow, and arteriolar wall [NO] were measured while the entire intestinal vasculature was exposed to physiological concentrations of NaCl comparable to those that naturally occur during nutrient absorptions (8, 11). Cell volume regulation in vascular endothelium is of critical importance in maintaining a blood-compatible selective permeability barrier to water and solutes (29). A decrease in cell volume initially occurs when cells are exposed to a hyperosmotic solution because of water moving outside the cells via osmotic flow. A RVI characterized by the activation of ion uptake systems and organic osmolyte transporters is stimulated by this decrease in cell volume (32, 37). Therefore, we first determined how sodium ions enter intestinal endothelial cells during NaCl hyperosmolarity by using selective pharmacological blockers of the major ion transport systems involved in cell volume regulation, the Na⁺/H⁺ exchanger and the Na⁺-K⁺-2Cl^– cotransporter (32). The Na⁺-K⁺-2Cl^– cotransporter is sensitive to the loop diuretic bumetanide, whereas the Na⁺/H⁺ exchanger is sensitive to the potassium-sparing diuretic amiloride. These pharmacological blockers are highly selective for their own channel system (20, 37), which allows their actions to be studied separately.

Suppressing the amiloride-sensitive Na⁺/H⁺ exchanger did not have any effect on intestinal blood flow at rest or during NaCl hyperosmolarity (Fig. 1) and only diminished the increase in vascular [NO] at 380 mosM during physiological NaCl hyperosmolarity (Fig. 2). These results suggest the Na⁺/H⁺ exchanger has a limited role in regulating vascular [NO] formation only at near-maximal NaCl hyperosmolarity but not intestinal blood flow at any level of NaCl hyperosmolarity. The blockade of Na⁺-K⁺-2Cl^– channels by bumetanide was much more efficient in blunting the effects of NaCl hyperosmolarity on blood flow (Fig. 3) and vascular [NO] (Fig. 4). The results with bumetanide were much more pronounced than when Na⁺/H⁺ exchanger channels were blocked with amiloride (Figs. 1 and 2). These results indicate that, as a part of the cellular response to NaCl hyperosmolarity, regulation of cell volume by Na⁺-K⁺-2Cl^– entry and to a lesser extent by Na⁺/H⁺ exchange somehow activates eNOS and the resultant NO generation increases intestinal blood flow. Bumetanide did lower both resting blood flow and [NO] (Figs. 3 and 4). We suspect this is because sodium entry into resting cells at normal osmolarity is a routine event and ultimately has effects on the intracellular calcium environment. However, from their new steady-state circumstances after bumetanide application, relative arteriolar responses to NaCl hyperosmolarity were nonetheless strongly attenuated.

Our in vivo observations are consistent with prior in vitro studies of endothelial volume regulation during hyperosmolarity. O'Donnell (29) measured the effects of bumetanide and amiloride on cell volume during hyperosmolarity in bovine aortic endothelial cells. Cells were transferred from hyposmotic to hyperosmotic medium, causing a decrease in cell volume to 73% of control. After 30 min, a regulatory volume response was observed in which cell volume increased to 86% of control. When bumetanide was added the RVI was inhibited by 75%, whereas the addition of amiloride blocked the RVI by 40%. We found, as shown in Figs. 1– 4, that bumetanide suppression of the Na⁺-K⁺-2Cl^– cotransporter had much greater effects on NO generation than did suppression of the Na⁺/H⁺ exchanger with amiloride.

Linking the accumulation of sodium ions in endothelial cells to increased NO formation, as shown in Figs. 2 and 4 under natural conditions, would require a number of intermediary steps. We believe the most important first step is calcium accumulation in the endothelial cells as excess sodium ions are removed. The activity of eNOS, which is a Ca²⁺/calmodulin-dependent, peripheral membrane-associated protein, is tightly controlled by its interaction with the scaffolding protein caveolin-1 localized within caveolar regions of endothelial cells (26). Prior studies suggested that local increases in caveolae endothelial subplasmalemmal Ca²⁺ concentration, instead of overall increases in intracellular Ca²⁺ concentration, may be sufficient for eNOS activation and subsequent NO production in endothelial cells (34, 40). This local distribution of Ca²⁺ in the subplasmalemmal region of caveolae in endothelial cells has been proposed to be regulated by the NCX (40). Furthermore, Teubl et al. (39) showed the presence of both NCX and eNOS in the membrane fraction positive for caveolin-1. They demonstrated that increasing intracellular sodium ion accumulation had the effect of facilitated Ca²⁺-dependent eNOS activation without detectable changes in whole cell intracellular Ca²⁺ concentration, and this event was blocked by NCX inhibition. In our studies, as shown in Figs. 5 and 6, suppression of NCX with KB-R7943 strongly suppressed the increased [NO] and intestinal blood flow associated with NaCl hyperosmolarity. In addition, suppression of the KB-R7943 had effects on resting blood flow and [NO]. This likely indicates that even at rest the NCX exchanger is actively removing sodium ions that enter intestinal endothelial cells through the Na⁺-K⁺-2Cl^– cotransporter, as our data support, as well as other means of sodium ion entry.

In these studies, we used NaCl hyperosmolarity because it is the physiological form of hyperosmolarity routinely developed in the small intestine and, of course, similar conditions exist in the kidney. There are a great many studies of hyperosmotic effects on vascular regulation in the literature that use agents other than NaCl to generate hyperosmolarity. However, the only two forms of hyperosmolarity that naturally occur in mammals are NaCl hyperosmolarity in the bowel and kidney due to transport phenomena and water deprivation to cause systemic NaCl hyperosmolarity and hyperglycemia associated with various forms of diabetes mellitus. Our finding that even a mild increase in NaCl hyperosmolarity to 330 mosM increased NO production and elevated intestinal blood flow by 16–31% (Figs. 1 and 2) has clinical implications. Systemic NaCl hyperosmolarity due to water deprivation is a routine clinical problem that both lowers cardiac output because of decreased cardiac filling and causes an unexplained drop in peripheral vascular resistance (21, 22). The decline in vascular resistance could be coupled to the Na⁺/Ca²⁺/NO mechanism this laboratory has documented (8, 15) during naturally occurring and exogenously produced NaCl hyperosmolarity. This natural mechanism is of great potential interest because the small intestine uses interstitial NaCl hyperosmolarity as a form of vascular signaling during food absorption to couple blood flow needs to the increased metabolism of absorption. These current studies show for the first time during NaCl hyperosmolarity a link, although indirect, of the Na⁺-K⁺-2Cl^– cotransporter allowing sodium ions to eventually influence eNOS regulation. A crucial step in this process is the reverse mode of the NCX to remove sodium ions from the cells in exchange for calcium entry, the first step in activation of eNOS to produce [NO].

	GRANTS

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

	ACKNOWLEDGMENTS

 
The authors thank Mary Ann Neil for technical assistance and Dr. Geoffrey P. Nase for comments on the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. G. Bohlen, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., MS 426, 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.

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