Introduction

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The study of vascular force generation by isolated small arteries is often performed using a wire myograph, which enables the measurement of isometric tension and relaxation responses. Most myographs that are used are variations of the original system described by Bevan and Osher (1972) and modified by Mulvany and Halpern (1977). The procedure consists of inserting thin wires through the lumen of the vessel and using these wires to attach the segment to "feet" which are in turn connected to a force transducer and a translation stage micrometer that permit measurement of tension and stretching of the segment, respectively.

Over the past several years, our laboratory has used a wire myograph system to demonstrate that cumulative addition of extracellular Ca²⁺ to preconstricted mesenteric branch arteries causes dose-dependent, sensory-nerve-dependent relaxation (Bukoski et al., 1997). The sensitivity of this relaxation event for extracellular Ca²⁺, as reflected by the ED₅₀ value for Ca²⁺, lies between 1.5 and 2.0 mM (Mupanomunda et al., 1999). Over the course of ongoing work, we found that both the magnitude and the Ca²⁺ sensitivity of the Ca²⁺-induced relaxation response became greatly reduced. We therefore initiated a series of experiments to determine the underlying cause. The results indicate that a surface oxidation product on the tungsten wires that are used to mount the vessels on the myograph significantly impairs relaxation of isolated arterial segments.

	Experimental Procedures

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Animals. All procedures involving animals were performed in accordance with approval of the Institutional Animal Care and Use Committee at both universities. Male Wistar rats (8-10 weeks of age) were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and housed in the North Carolina Central University animal care facility in colony rooms with fixed light/dark cycles and constant temperature and humidity and provided with Purina rodent chow and water ad libitum (Purina, St. Louis, MO). Mesenteric tissue was isolated while the rats were anesthetized with a mixture of ketamine and xylazine (100:5, mg/kg). Male golden Syrian hamsters (9-12 weeks of age) were obtained from Charles Rivers Laboratories (Wilmington, MA) and maintained at Western Michigan University under conditions similar to the rats. Hamsters were killed by CO₂ asphyxiation followed by cervical dislocation, their cremaster muscles removed and second and third order arterioles isolated by hand dissection as described previously (Jackson, 2000).

Isolation of Vascular Smooth Muscle Cells. Vascular smooth muscle cells were isolated enzymatically from hamster cremasteric arterioles as described previously (Jackson, 2000). Arteriolar segments were placed into 1 ml of dissociation solution (DS, in mmol/l: 140 NaCl, 5 KCl, 1 MgCl₂, 0.1 mM CaCl₂, 10 HEPES, 10 glucose, 1 mg/ml bovine serum albumin, 10 µM sodium nitroprusside, and 10 µM diltiazem, pH 7.4, with NaOH, 295-300 mOsm) at room temperature. After 10 min of incubation, most of this solution was removed and replaced with 1 ml of DS containing 1.5 mg/ml papain and 1 mg/ml dithioerythritol and the arteriolar segments incubated at 37°C for 35 min. The papain solution was then removed and replaced with 1 ml of DS containing 1.5 mg/ml collagenase, 1 mg/ml elastase, and 1 mg/ml soybean trypsin inhibitor, and the segments were incubated for an additional 16 to 19 min at 37°C. The enzyme-containing solution was then replaced with 4 ml of DS at room temperature and allowed to settle for approximately 10 min. This solution was then removed and replaced with 1 ml of fresh DS not containing sodium nitroprusside or diltiazem. Cells were released from the segments by gentle trituration (1-4 strokes) using a 100 to 1000 µl Eppendorf style pipettor and stored in this solution for up to 4 h at room temperature.

Biophysical Measurements. Isometric force generation by rat mesenteric branch arteries was measured using previously described methods (Bukoski et al., 1997). The small intestine and the attending mesenterium was pinned to a dissecting dish filled with ice-cold physiologic salt solution (PSS) of the following composition (in mmol/l) 150 NaCl, 5.4 KCl, 1.17 MgSO₄7H₂O, 1.18 NaH₂PO₄, 6.0 NaHCO₃, 1.0 CaCl₂, 20 HEPES, and 5.5 glucose, pH 7.4. Branch II and III segments were isolated by microdissection taking special care to leave a portion of the omental membrane attached to the adventitial surface of the blood vessel. Following these preparative procedures, vessels were mounted on a wire myograph (Kent Scientific, Litchfield, CT) using the indicated wires and immersed in PSS warmed to 37°C and gassed with 95% air/5% CO₂. After a 15-min equilibration period, the segments were stretched to a predetermined length that was equivalent to an internal diameter of 200 to 225 µm and allowed to equilibrate for an additional 15 min. After the equilibration period, the vessels were contracted with 5 µM phenylephrine until reproducible contractile responses were obtained (three to four times).

Laser Raman Spectral Analysis. Raman spectra of tungsten wire samples was performed by NAMAR Scientific (Russellville, AR) and collected using a maximum power of 2 mW of 514.5-nm radiation from an Omnichrome model 150 argon-ion laser for Raman excitation. Radiation from the sample was collected in a 180° back-scattering geometry by a model BHSM Olympus microscope and a 50× magnification objective (Olympus, Tokyo, Japan). The scattered radiation was directed into a Renishaw System 1000 Raman spectrometer where the laser radiation was filtered using a series of holographic notch filters and the Raman signal dispersed by high resolution grating (1800 grooves/mm) onto a thermo-electrically cooled CCD detector (70°C) (Renishaw, Gloucestershire, UK). The Raman spectra were collected and stored using Renishaw Raman software operated on a Pentium-based PC. The combined spectral resolution and reproducibility were experimentally determined to be better than 3 cm¹. Spectral calibration was performed using the atomic emission lines of a neon arc lamp.

UV/VIS Spectral Analysis. In solution with a neutral pH, the metatungstate and paratungstate anions are in equilibrium with the tungstate anion (Pope, 1983). We therefore performed UV/VIS spectral analysis of tungstate compounds to provide information about the spectral characteristics of dilute solutions of these salts and their stability over time. Salts were dissolved as described under the materials section (see below) to a final concentration of 5 µM, and spectral scans from 190 to 320 nm were collected using a Beckman Coulter, Inc. (Fullerton, CA) DU 640 scanning spectrophotometer.

Electrophysiology. The perforated-patch technique was used to assess the effects of tungsten compounds on macroscopic K⁺ currents using voltage clamp protocols as described previously (Jackson et al., 1997). An aliquot of cell-containing solution was placed on the cover-slipped bottom of a 1-ml flow-through chamber, allowed to settle for 5 to 10 min, and then superfused with HEPES-buffered PSS (in mmol/l): 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, 10 Glucose, pH 7.4, with NaOH at room temp (295-300 mOsm). Patch-pipettes were constructed from 1-mm i.d. × 1.5-mm o.d. Corning 7052 glass tubes (Garner Glass Company, Claremount, CA), fire polished, their tips filled with pipette solution (see below), and then back-filled with the same solution containing 240 µg/ml amphotericin B as described previously. Electrical access was gained in 10 to 15 min and was maximal in 15 to 30 min. Pipette solution contained (in mmol/liter) 100 K-aspartate, 43 KCl, 1 MgCl₂, 10 HEPES, 0.5 EGTA, pH 7 to 7.2 adjusted with NaOH, 295 to 300 mOsm adjusted with sucrose. Pipettes had tip resistances of 3 to 4 M when filled with this solution. Seals were made on vascular smooth muscle cells by gently touching the fire-polished tip of the pipette to a cell and then applying gentle suction by mouth. Seal resistances were all greater than 10 G for the studies presented, and no attempt was made to correct for leakage currents.

Materials. The tungsten and gold wires were obtained from Wirenetics (Sunnyvale, CA); phenylephrine was obtained from ACROS Organics (Morris Plains, New Jersey); sodium tungstate (Na₂WO₄) and sodium metatungstate [3Na₂WO₄ · 9WO₃ (also written as Na₆W₁₂O₃₉)] were obtained from the Aldrich Chemical Co (Milwaukee, WI); ammonium paratungstate [(NH₄)₁₀W₁₂O₄₁] was obtained from CERAC (Milwaukee, WI). All other chemicals were of reagent grade or better and obtained from the Sigma Chemical Co or Invitrogen (Carlsbad, CA). Phenylephrine was dissolved in PSS containing 100 µM ascorbic acid, and the tungstate compounds were dissolved in acidified water that was then buffered with HEPES (0.1 M final concentration), and the pH was adjusted to 7.4 with NaOH.

Statistical Analysis. The agonist concentration eliciting 50% of the maximal relaxation (EC₅₀) was determined from plots of the percentage of initial tension versus the concentration of agonist. All data are presented as mean ± S.E.M., and statistical analysis was performed using the SYSTAT software package (SPSS Science, Chicago, IL). Comparisons among groups were performed using ANOVA with a repeated measures design when appropriate. A value of p < 0.05 was taken to indicate a statistically significant difference.

	Results

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In experiments carried out previously, we found that mesenteric branch arteries mounted on 28-µm tungsten wires and precontracted with 5-µM PE, responded to the cumulative addition of extracellular Ca²⁺ with a dose-dependent relaxation to 8.1 ± 1.46% of the initial tension response to PE and with an ED₅₀ for Ca²⁺ of 1.65 ± 0.07 mM (n = 11). These results were similar to those reported previously for arteries precontracted with methoxamine (Mupanomunda et al., 1999). In more recent studies, we found that the response of isolated mesenteric branch arteries was significantly attenuated, with an obvious reduction in the maximal relaxation response (54.8 ± 4.5% of initial PE tension, n = 6, p < 0.001) and in the ED₅₀ value for Ca²⁺ (4.58 ± 0.16 mM, n = 6, p < 0.001). Multiple experimental parameters could have contributed to the reduced relaxation. These include the purity of the water used to prepare the physiologic salt solution, the colony or genotype of the rats used as the tissue source, the purity of the gas mixture used to aerate the buffer, and the amount of sunlight present in the room. Each of these was tested as a contributing factor, but none was found to affect the impaired dilator response.

During this same time period, we initiated experiments to study relaxation responses of murine arteries and obtained 14-µm diameter tungsten wire to accommodate the smaller lumen of these vessels. We subsequently mounted rat arteries on this smaller wire to test the hypothesis that it was the 28-µm diameter tungsten wire that was attenuating the relaxation response. In these experiments, adjacent vessel segments were mounted on either 14- or 28-µm tungsten wire and the relaxation response to either the cumulative addition of Ca²⁺ or acetylcholine was assessed. Both the magnitude (83.5 ± 3.9%, n = 6) and the Ca²⁺ sensitivity (ED₅₀ = 2.35 ± 0.20 mM, n = 6) of the Ca²⁺-induced relaxation response of vessels mounted on the 14-µm tungsten wire were significantly enhanced compared with those mounted on 28-µm wire (p < 0.001) (Fig. 1A).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Response of PE-contracted mesenteric branch arteries to cumulative addition of extracellular Ca2+ (A) or acetylcholine (B) when mounted on aged 28-µm tungsten wires, 14-µm tungsten wires, or 28-µm tungsten wire after removal of surface oxidation by abrasion with fine sandpaper. Values are mean ± S.E.M., n = 6 per group; *, significant difference at p < 0.05.

To learn whether the attenuated response seen when vessels were mounted on 28-µm wire was limited to the relaxation induced by extracellular Ca²⁺, or whether it extended to another vasodilator pathway, we assessed the response of arteries to the cumulative addition of acetylcholine. Both the maximal relaxation to acetylcholine (66.0 ± 5.3% on 28-µm wire versus 97.4 ± 0.8% on 14-µm wire, n = 5-7, p < 0.001) and the ED₅₀ values for acetylcholine (1.03 ± 0.44 µM on 28-µm wire versus 0.04 ± 0.005 µM on 14-µm wire, p = 0.002) were significantly reduced on the 28-µm wire (Fig. 1B).

These findings caused us to consider the possibility that the 28-µm wire was contaminated with a vasotoxic substance. We therefore performed several experiments in which the tungsten wires were soaked in ethanol to remove possible contaminating organic substances. No improvement in the responses was observed (data not shown). We next considered the possibility that the surface of the 28-µm wire had become oxidized and the oxidation product was inhibiting the relaxation. As a test, we compared the responses of vessels mounted on the 28-µm tungsten wires with those of segments mounted on 28-µm wire taken from the same stock, but which had been cleaned by abrading the surface with fine sandpaper. Sanding the 28-µm wire restored the relaxation response of the artery to both the cumulative addition of extracellular Ca²⁺ and to acetylcholine so that differences in the maximal response and ED₅₀ values for each dilator were no longer detected (Fig. 1, A and B).

To learn whether minor surface oxidation might also be present on the 14-µm tungsten wire and acting to cause more subtle inhibition of the responses, we compared relaxation responses of arteries mounted on 14-µm tungsten wire with those of adjacent segments mounted on 14-µm wire made from gold. Vessels mounted on 14-µm gold wire had significantly enhanced sensitivity to the vasodilator effect of extracellular Ca²⁺ (ED₅₀ tungsten = 1.83 ± 0.06 mM versus ED₅₀ gold = 1.45 ± 0.02 mM; n = 6, p = 0.001) while no difference in the maximal response (92.5 ± 1.1% for tungsten versus 93.7 ± 2.6% for tungsten; n = 6, p = 0.686) was detected (Fig. 2A). In contrast, no differences in the vasodilator response to acetylcholine were detected (ED₅₀ tungsten = 0.010 ± 0.002 versus ED₅₀ gold = 0.010 ± 0.002 µM; n = 6, p = 0.981) (maximal response for gold 89 ± 2.5% versus 89.3 ± 7.2% for tungsten; n = 5-7, p = 0.890) (Fig. 2B). These results are consistent with the hypothesis that the more recently purchased 14-µm tungsten wire was causing a slight but significant inhibition of the response to Ca²⁺, but not the response to acetylcholine.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of mounting vessels on 14-µm tungsten wires or 14-µm gold wires on ligand-induced relaxation. A, response of PE-contracted mesenteric branch arteries to cumulative addition of extracellular Ca2+, n = 6 per group; repeated measures ANOVA gave an overall p value of 0.015; *, significant difference at a p value of at least <0.05. B, response to acetylcholine, n = 8 for tungsten wire and 5 for gold wire, repeated measures ANOVA gave an overall p value of 0.177; no significant differences were detected.

Working on the assumption that surface oxidation was responsible for the reduced response of arteries mounted on the 28-µm tungsten wire, laser Raman spectral analysis was performed on a segment of the corroded 28-µm tungsten wire and of an adjacent segment that had its surface cleaned with fine sandpaper. The results of the spectral analysis indicated that the surface of the corroded wire was in an oxidized state and that the oxidation product could be removed by sanding (Fig. 3). The Raman spectra for the tungsten oxide coating determined at two different places on the wire were compared with reference spectra in the literature (Griffith and Lesniak, 1969). Analysis of the oxide in one region most closely matched that of Na₁₂[W₁₂O₄₂]¹² although the lack of lattice vibrations in the low-frequency region indicated a microcrystalline form. The oxide in the second region most closely matched that of the paratungstate [W₁₂O₄₂]¹² anion, indicating an amorphous tungstate structure (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Laser Raman spectra of corroded 28-µm tungsten wire and a segment of the same wire that was cleaned with fine sandpaper.

In view of these results, we tested the effect of ammonium paratungstate [(NH₄)₁₀W₁₂O₄₁] and two structurally dissimilar tungsten oxide formulations, sodium tungstate (Na₂WO₄) and sodium metatungstate (3Na₂WO₄ · 9WO₃) on the relaxation responses of isolated arteries. These salts were dissolved in acidified water, then buffered to pH 7.0 to 7.4. Because the paratungstate and metatungstate anions are in slow equilibrium with the tungstate anion (Pope, 1983), UV/VIS spectral scans of dilute (5 µM) solutions of ammonium paratungstate, sodium metatungstate, and sodium tungstate were collected to determine whether spectral characteristics of these compounds vary and might provide information about stability of the salts in solution (Souchay et al., 1972). Excitation from 190 to 320 nm resulted in the absorption spectra illustrated in Fig. 4. Sodium metatungstate showed the most complicated spectrum absorbing from 190 to 290 nm with a broad peak centered at around 258 nm. Ammonium paratungstate showed a lower level of absorbance from 190 to 290 nm and lacked the broad peak at 258 nm characteristic of sodium metatungstate. In contrast, sodium tungstate was characterized by a much lower level of absorbance at 190 nm, which was completely absent at 212 nm and higher. These spectra did not change over a 2-h period, indicating that appreciable conversion of meta- and paratungstate to tungstate was not occurring during the time interval between which the solution was prepared and used in the experiment.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   UV/VIS spectra of 5 µmol/l solutions of ammonium paratungstate, sodium metatungstate, and sodium tungstate. Spectra were collected as described under Experimental Procedures using freshly prepared solutions and after the solutions were incubated at 37°C for 2 h. Abs, absorbance units relative to water.

Pretreatment of arteries with 1 µM ammonium paratungstate significantly inhibited Ca²⁺-induced relaxation (Fig. 5A) such that there was a reduction in both the maximal response (control = 84.4 ± 1.8% versus ammonium paratungstate = 50.7 ± 4.2%; n = 6, p < 0.001) and the ED₅₀ value (ED₅₀ control = 1.91 ± 0.15 versus ammonium paratungstate = 4.33 ± 0.31 µM; n = 6, p < 0.001). Similarly, when the effect of the paratungstate anion on the acetylcholine-induced relaxation response was examined, there was a significant reduction in both the maximal response (control = 87.3 ± 2.4% versus ammonium paratungstate = 68.8 ± 8.0%; n = 6, p < 0.05) (Fig. 5B) and sensitivity (ED₅₀ control = 6.9 ± 1.9 versus ammonium paratungstate = 22.6 ± 0.9.6 nM; n = 6, p < 0.037).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of 1 µmol/l ammonium paratungstate on the relaxation response of PE-contracted arteries mounted on 14-µm tungsten wires to the cumulative addition of extracellular Ca2+ (A) or acetylcholine (B). Values are mean ± S.E.M., n = 6 per group; *, significant difference at p < 0.05.

In contrast, 100 µM sodium tungstate was without effect on the relaxation response of the arteries to the cumulative addition of extracellular Ca²⁺ (Fig. 6A), whereas 50 µM sodium metatungstate caused a slight but significant increase in the relaxation response to the lowest concentrations of extracellular Ca²⁺ (Fig. 6B). To test whether the ammonium cation might be responsible for the attenuated relaxation responses, we assessed the effect of 10 µM ammonium chloride (NH₄Cl) on Ca²⁺-induced relaxation in two preparations and found that it was without effect (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of 100 µmol/l sodium tungstate (A) or 50 µmol/l sodium metatungstate (B) on the relaxation response of PE-contracted arteries mounted on 14-µm tungsten wires to the cumulative addition of extracellular Ca2+. Values are mean ± S.E.M., n = 5 per group; *, significant difference at p < 0.05.

Previously, we showed that Ca²⁺-induced relaxation can be inhibited by blockers of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels such as tetraethylammonium (TEA) and iberiotoxin (Bian and Bukoski, 1995; Ishioka and Bukoski, 1999). Therefore, we tested the hypothesis that paratungstate anion might inhibit Ca²⁺-induced relaxation by blocking BK_Ca channels. We found that 1 µM paratungstate anion had no effect (p > 0.05) on macroscopic K⁺ currents in vascular smooth muscle cells (Fig. 7A). Likewise, in six cells from three animals, 1 µM sodium metatungstate was without effect (data not shown). As a positive control, we examined the effects of 1 mM TEA, which, at this concentration, selectively blocks vascular smooth muscle BK_Ca channels (Nelson and Quayle, 1995). In contrast to the lack of effect of the tungsten compounds, this quaternary ammonium BK_Ca blocker significantly inhibited currents at positive membrane potentials (Fig. 7B), consistent with other studies in the literature (Nelson and Quayle, 1995).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   A, lack of effect of paratungstate anions on whole-cell K+ currents in vascular smooth muscle cells. Shown are current densities in the absence (control) and presence of 1 µM ammonium paratungstate. Data are mean ± S.E.M., n = 6; factorial analysis of variance indicated a significant effect of voltage (p < 0.05), but no significant paratungstate or interaction (voltage × paratungstate) effects (p > 0.05). B, inhibition by TEA of whole-cell K+ currents in vascular smooth muscle cells. Shown are current densities in the absence (control) and presence of 1 mM TEA. Data are mean ± S.E.M., n = 6; factorial analysis of variance indicated significant effects of voltage and TEA, as well as a significant interaction (voltage × TEA) between these two treatments (p < 0.05).

Since it is well accepted that oxygen free radicals can diminish relaxation responses to certain vasodilators, we assessed the effect of the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (4-OH-TEMPO) on Ca²⁺-induced relaxation in the presence and absence of 1 µM ammonium paratungstate. Pretreatment with this compound did not affect Ca²⁺-induced relaxation or the inhibitory effect of paratungstate on Ca²⁺-induced relaxation (Fig. 8), indicating that neither the superoxide anion nor downstream reactive oxygen species are involved.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Effect of 1 µmol/l ammonium paratungstate on Ca2+-induced relaxation in the presence and absence of 1 µmol/l of the superoxide dismutase mimetic 4-OH-TEMPO. Values are mean ± S.E.M., n = 4 per group; no effect of 4-OH-TEMPO was detected.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We have performed experiments to determine the mechanism(s) underlying the unexpected decrease in the Ca²⁺-induced relaxation response of isolated mesenteric branch arteries associated with aged tungsten wire. The new findings of this study include the demonstration that 1) spontaneous oxidation of tungsten wire significantly inhibits vasorelaxation responses to both Ca²⁺ and acetylcholine; 2) the major tungsten oxidation product is similar to the paratungstate [W₁₂O₄₂]¹² anion; 3) low (1 µmol/l) concentrations of ammonium paratungstate significantly attenuate Ca²⁺ and acetylcholine-induced relaxation; and 4) substitution of tungsten wire with gold wire significantly enhances the Ca²⁺ sensitivity of the Ca²⁺-induced relaxation event.

The wire myograph technique originally described by Bevan and Osher (1972) and modified by Mulvany and Halpern (1977) has permitted a large number of studies of vascular physiology and pharmacology on isolated arteries as small as 100 µm in diameter. The basic approach of the method is to thread thin wires into the lumen of a vessel segment and to use these wires to affix the vessel by means of screws to two metal or Plexiglas feet, with one affixed to a translation micrometer and the other affixed to a force transducer. Although the original method used platinum wires for mounting vessel segments (Bevan and Osher, 1972), at present most laboratories use either tungsten (Steeds et al., 1997; Liu et al., 1998; Thorin et al., 1998) or stainless steel wire (Buss et al., 1999; Scotland et al., 1999) presumably because their tensile strength is greater than that of platinum.

The present report indicates that the tungsten wire that our laboratory has routinely used for wire myography undergoes spontaneous surface oxidation. Surface oxidation is a common property of the transition metals (Griffith and Lesniak, 1969). We used laser Raman spectroscopy to analyze the state of the tungsten wire because, to our knowledge, it is the only method that is available for detecting oxidation on the surface of very small metal samples. The results showed that the surface of the wire was in an oxidized state and indicated that the oxidation product was similar to the paratungstate anion. Our findings that the biological effects of the oxidized wire are mimicked by low concentrations of ammonium paratungstate, and that two other structurally unrelated compounds (sodium tungstate and sodium metatungstate; Fig. 9) do not inhibit vascular relaxation, have led to the conclusion that the surface oxidation product is a paratungstate-like compound.

View larger version (61K): [in this window] [in a new window]   Fig. 9.   Bond and polyhedral representations of tungstate anions. Bond (A) and polyhedral (B) representations of the oxygen coordination of the tungstate WO42 anion; C, polyhedral representation of the paratungstate anion; D, polyhedral representation of the metatungstate anion based on the Keggin structure for tetrahedral heteroatoms. Adapted from Pope (1983) with permission.

The paratungstate and metatungstate anions are complex structures, which in solution are in very slow equilibrium (on the order of days) with one another. This relationship is illustrated by Scheme 1 (Pope, 1983).

View larger version (10K): [in this window] [in a new window]   Scheme 1.

We therefore thought it important to determine whether the polytungstates that we tested spontaneously dissociate to form the tungstate anion at neutral pH. The approach that we used was to analyze the UV/VIS spectra of diluted solutions of these compounds (Souchay et al., 1972). The absorption spectra of the meta- and paratungstate anions were different from the spectrum of the tungstate anion and did not vary over the 2-h period that was examined, indicating that they were chemically stable during the course of our experiments.

Tungsten and sodium tungstate have been studied extensively in the past with special emphasis on the ability of dietary tungsten and tungstate to inhibit xanthine oxidase and H₂O₂ production (Owen and Dundas, 1969; Smith et al., 1987; Swei et al., 1999) and of tungstate to mimic the serum glucose clearing action of insulin, presumable by stimulating glucose 6-phosphatase activity (Barbera et al., 1997; Foster et al., 1998). To our knowledge, however, there is no published information about the vascular effects of ammonium paratungstate. We therefore believe that our data are the first to demonstrate that ammonium paratungstate significantly impairs both Ca²⁺ and acetylcholine-induced relaxation.

A question of primary concern is the mechanism by which the paratungstate anion suppresses the vasodilator effect of extracellular Ca²⁺. Early studies from Bohr (1962) were interpreted to indicate that relaxation induced by very high concentrations of extracellular Ca²⁺ was the result of either a membrane-stabilizing effect of the Ca²⁺ cation or an effect of the cation to stimulate the smooth muscle cell Na⁺ pump and induce hyperpolarization (Webb and Bohr, 1978). In contrast with these earlier reports, collective evidence from our laboratory supports the following pathway for the high sensitivity Ca²⁺-induced relaxation of the isolated mesenteric branch artery. Extracellular Ca²⁺ binds to a Ca²⁺-sensing receptor located on perivascular sensory nerves; activation of this receptor results in the production and/or release of a nerve-derived hyperpolarizing vasodilator factor; this hyperpolarizing factor binds to a receptor on adjacent smooth muscle cells; and activation of the receptor is coupled with opening of an iberiotoxin-sensitive K_Ca channel, which leads to hyperpolarizing vasodilation (Bukoski, 1998, 2001).

The finding that the paratungstate anion blocks both Ca²⁺-induced relaxation and attenuates acetylcholine-induced relaxation makes it unlikely that the compound is acting by inhibiting a sensory nerve Ca²⁺ receptor, since this receptor is not believed to be active in acetylcholine-induced relaxation. Moreover, the electrophysiological studies reported here failed to demonstrate inhibition of BK_Ca channels by the paratungstate anion. Additional evidence obtained using 4-OH-TEMPO rule out the possibility that paratungstate-generated superoxide anions are chemically inactivating a nerve-derived vasodilator. These data indicate that paratungstate must be acting at some other point along the signaling cascade, either at the receptor for the nerve-derived hyperpolarizing factor, at a site intermediate to activation of the receptor and opening of the K_Ca channel, or at a site distal to K_Ca channel opening. Further research will be required to determine the precise mechanism of action.

Our results surrounding the finding that surface oxidation of tungsten wire impairs arterial relaxation are of interest for several reasons. One is that it would seem prudent to stop the use of tungsten wire for mounting arterial segments on wire myographs. In place of tungsten, we recommend that another nonreactive wire be used such as gold or tungsten-free stainless steel. A second point of interest is that our analysis has shown that the paratungstate anion is a potent inhibitor of Ca²⁺-induced relaxation. This compound may find utility as a tool in the study of mechanisms of Ca²⁺-induced relaxation. The third deals with the finding that very low, physiologic concentrations of extracellular Ca²⁺ are able to elicit nearly complete relaxation of isolated arteries when they are mounted on gold wire. The finding that the ED₅₀ for Ca²⁺ is less than 1.5 mM when studied using gold wire compares well with our recent demonstration that interstitial Ca²⁺ in the duodenal submucosa (Mupanomunda et al., 1999) and the renal cortex (Mupanomunda et al., 2000) ranges from 1 to slightly more than 2 mM and suggests that a Ca²⁺-activated dilator system may be functional in select vascular beds under physiologic conditions.