INTRODUCTION

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OPENING OF CHLORIDE (Cl) ion channels depolarizes vascular smooth muscle (VSM). This is because Cl is transported into VSM cells against its electrochemical gradient, resulting in an intracellular Cl concentration that is much higher than that predicted by passive distribution (8). Cl currents are activated by both vasoconstrictors (3, 20, 38) and stretch (28). Agonist-induced Cl current contributes functionally to the VSM response to catecholamines because when Cl channels are blocked or Cl transport is interrupted contraction is inhibited (7, 23). Endothelial disruption greatly augments the Cl dependence of norepinephrine (NE)-induced contraction, suggesting that the Cl current activated by -adrenergic receptors is regulated by an endothelial factor (24).

Two distinct Cl conductances have been identified in VSM. A calcium-activated Cl current (I_Cl,Ca) has been well characterized (26). This current is activated by both spontaneous (40) and agonist-induced (3, 31) release of intracellular calcium stores at calcium concentrations ranging from 180 to 600 nM (30). These channels have a very low conductance of 1-2 pS (19) and an anion selectivity of iodide (I) > Cl (40). I_Cl,Ca is inhibited by a number of pharmacological agents, including DIDS and niflumic acid (25). VSM cells also possess a typical "volume-activated" Cl current (I_Cl,vol) (29, 41). These channels are activated by hypotonic conditions associated with cell swelling. I_Cl,vol is outwardly rectifying, inactivates at positive potentials (more than approximately +60 mV) and, like I_Cl,Ca, has an ion selectivity of I > Cl. The channel is blocked by DIDS and by the antiestrogen agent tamoxifen. It has been proposed that I_Cl,vol is encoded by the ClC-3 gene (11), which is the most highly expressed member of the ClC gene family in VSM (22).

In previous experiments we used anion substitution as a tool to study the contribution of Cl currents to vasoconstrictor responses of the rat aorta (23). Replacement of Cl with methanesulfonate at the same time that NE was applied resulted in immediate potentiation of contractile responses. Substituting bromide (Br) or I for Cl initially depressed contractile responses of endothelium-intact rings to NE but subsequently potentiated them. In the current study, when anion substitution was performed without the concomitant addition of NE, a small, immediate relaxation to I or Br was the only response seen. However, when the same experiment was performed in endothelium-denuded rings, this initial relaxation was followed 10-15 min later by a very large contractile response. The current experiments were designed to investigate the mechanism underlying these responses to anion substitution.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Adult male Sprague-Dawley rats (250-300 g) were obtained from Harlan Sprague Dawley. The animals were killed by exposure to 100% CO₂ for 5 min followed by cervical dislocation. Thoracic aortas were removed, cleaned of adherent connective tissue, and cut into 4- to 5-mm rings. The rings were prepared for recording isometric force as previously described (23). In some preparations the endothelium was removed by gentle rubbing with the edge of a fine, serrated forceps. Passive stretch was set at 2.5 g, and the rings were allowed to equilibrate in physiological salt solution (PSS) at 37°C for 120 min before the start of experimentation. Normal PSS contained (in mM) 130 NaCl, 4.7 KCl, 1.18 KH₂PO₄, 1.17 MgSO₄ · 7H₂O, 14.9 NaHCO₃, 1.6 CaCl₂ · H₂O, 5.5 dextrose, and 0.03 CaNa₂-EDTA (pH 7.30). NaI and NaBr PSS were prepared by equimolar substitution of these salts for NaCl. The total concentration of Cl from sources other than NaCl was 7.9 mM. HEPES-buffered PSS was prepared by substituting NaHCO₃ with 10 mM HEPES and titrating the pH to 7.3.

For the concentration-response curve to I (see Fig. 4), mixtures of 130 mM NaCl, 130 mM NaI, and 130 mM Na-methanesulfonate PSS were used. The 130 mM Na-methanesulfonate buffer was prepared by substituting NaCl with 130 mM NaOH and titrating the pH of the buffer to 7.30 with methanesulfonic acid while the solution was being aerated with 95% O₂-5% CO₂. The measured osmolality of this buffer was 292 compared with 293 mosmol/kgH₂O for normal PSS (5500 vapor pressure osmometer). The various I concentrations were achieved by mixing either normal 130 mM NaCl PSS or 130 mM Na-methanesulfonate buffer with 130 mM NaI buffer in fixed ratios. In one set of experiments, Cl was simply replaced with I and NaCl plus NaI to equal 130 mM. In a second set of studies, Cl was present only at the constant 7.9 mM (4.7 mM KCl + 1.6 mM CaCl₂) as in all experiments involving I or Br replacement. In these experiments NaI plus Na-methanesulfonate equals 130 mM.

Prazosin, yohimbine, and [NOC-18,DETA/NO,(Z)-1-[2- (2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2- diolate] (detaNONOate) stock solutions were prepared in water. NE stock was prepared in water containing 10 mM ascorbic acid as a preservative. DIDS and hemoglobin (rat) were dissolved directly into PSS at the desired concentration. N-nitro-L-arginine (L-NNA) was dissolved in 1 N HCl. Bumetanide and nifedipine were dissolved in ethanol, whereas tamoxifen, niflumic acid, indomethacin, 17-octadecynoic acid (ODYA), and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) were dissolved in DMSO. Solvents were added to the buffer at no greater than a 1,000:1 dilution yielding a final solvent concentration of 0.1%. Indomethacin treatment consisted of a 1-h exposure at 10⁶ M during the 2-h equilibration period, which preceded all experiments. Nitric oxide synthesis was inhibited by incubation in L-NNA (10⁴ M) for 20 min before eliciting a response. No individual ring was used for more than a single pharmacological intervention (drug or combination of drugs) to avoid potential effects of incomplete washout. All drugs and all salts for the preparation of PSS were obtained from Sigma Chemical with the exception of ODQ and detaNONOate, which were obtained from Alexis Pharmaceuticals, and ODYA, which was obtained from Biomol.

At the beginning of each experiment a contractile response to 90 mM KCl was recorded. KCl was added directly to the buffer from a 1 M stock without adjusting for changes in tonicity. Many of the data are reported as a percentage of the response of each individual ring to this initial, maximal response to KCl. Traces of typical responses are drawn accompanied by a solid, vertical bar representing the magnitude of this response. The figures show mean responses ± SE, and the n values represent the number of tissues from different rats that underwent a given intervention. Differences between groups were assessed using Student's t-tests. Linear regression was performed using Microsoft Excel software and significance assessed using ANOVA. P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Anion substitution has complex effects on NE-induced contractile responses of intact rings of rat aorta (Fig. 1). The concentration of NE was titrated for each ring to achieve an approximately ED₂₅ response in normal PSS (n = 10). The average concentration of NE was 1.0 ± 0.23 × 10⁷ M. The replacement of NaCl with either NaBr or NaI at the same instant that NE is added (no time for transport of the alternative anion before agonist binding) produced a diminution of initial tension development compared with control responses obtained in normal PSS. Br causes less suppression of the initial response (Br: control 26.0 ± 2.3% of the response to 90 mM KCl, treated 14.3 ± 4.6% vs. I: control 31.2 ± 3.8%, treated 6.7 ± 4.3%, P < 0.05 for percentage suppression) and an earlier onset of potentiation than does I. We have previously reported the early inhibitory effects of anion substitution (23) and speculated that this effect was related to inward movement of the substituted anion down its instantaneously infinite concentration gradient, resulting in membrane hyperpolarization. As time passes the initial suppressive effect of anion substitution changes to a large augmentation of the contractile response. NE-induced contractions in the presence of NaBr or NaI were significantly larger at both the 10- and 20-min time points, but there was no statistically significant difference between the magnitude of the potentiating effects of I and Br.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A: anion substitution alters contractile response to norepinephrine (NE) in endothelium-intact aortic rings. Approximately ED25 contractile responses to NE were obtained in normal physiological salt solution (PSS) and then repeated in the presence of either 130 mM NaBr (solid bars, n = 5) or 130 mM NaI PSS (hatched bars, n = 5). Anion substitution was concurrent with addition of NE. Both Br and I suppressed initial peak response to NE (I > Br), but at 10- and 20-min time points contractile responses were significantly larger than control. B: typical experimental recordings are displayed with control (gray) and anion-substituted (black) responses superimposed to better illustrate changes observed. Solid bar on left represents magnitude of response of each ring to 90 mM KCl. * P < 0.05 compared with Cl.

To define the direct effects of alternative anions in the absence of agonist stimulation, we exposed both endothelium-intact and -denuded (rubbed) rings to 130 mM I alone (Fig. 2). Both intact and rubbed rings underwent an immediate relaxation from basal tension. This relaxation occurred despite the absence of identifiable active contractile tone. This relaxation was not significantly different between intact (215 ± 9 mg, n = 27 rings from 9 rats) and rubbed (234 ± 6 mg, n = 67 rings from 18 rats) rings. Over a 40-min period intact rings slowly recovered from this relaxation and some developed small contractile responses. At 40 min average tension was 4.2 ± 1.2% of the response to 90 mM KCl. Tensions below baseline at 40 min (7 of 27) were also calculated as a percentage of the response to 90 mM KCl and averaged in as negative values. Rubbed rings contracted vigorously to I and average tension at 40 min was 93.7 ± 1.9% (n = 67 rings from 18 rats). I-induced contraction was not inhibited by -adrenergic receptor blockade with prazosin (10⁶ M) plus yohimbine (10⁶ M; control 92.7 ± 11.2%, treated 91.1 ± 6.2%, n = 3). This combination completely blocked contractions of comparable size induced by NE (2 × 10⁸ M; control 93.7 ± 9.3%, treated 0%, n = 3). These data demonstrate that I-induced contraction is inhibited by an intact endothelial layer and is not caused by release of endogenous catecholamines from adrenergic nerve endings within the vascular wall.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Both intact (C) and endothelium-denuded (rubbed, D) rings undergo initial small relaxation on exposure to 130 mM I. Magnitude of this relaxation response was not significantly different between intact and rubbed rings (bar graph, A). After significant delay, rubbed rings () began to contract and after 40 min contractile response to I closely approximated response elicited by 90 mM KCl (scatter plot, B). At this same time point, tension in intact rings was often still below baseline (). Solid black bars to left of typical responses represent magnitude of each ring's response to 90 mM KCl.

Figure 2 includes a scatterplot of the standardized force-generating capacity of each ring (response to 90 mM KCl) plotted against the response of each ring to I. There is a significant difference in the maximal force-generating capacity between intact (3.69 ± 0.13 g) and rubbed (2.65 ± 0.06 g) rings. However, there is significant overlap between the two groups with regard to the magnitude of the KCl response but no overlap with respect to the magnitude of the I response. Within groups there is no significant correlation between the size of the KCl response and the size of the response to I [intact, r² = 0.023, not significant (ns); rubbed, r² = 0.034, ns]. These results suggest that the response to I is all or none and is related to the removal of some endothelial influence rather than to varying degrees of VSM cell damage caused during the rubbing process.

I produced both larger initial relaxations (Br: 126 ± 18 mg, I: 308 ± 24 mg, n = 5) and larger contractions (Br: 45.7 ± 27.3%, I: 105 ± 3.3%) than Br (Fig. 3). The average time to onset of contraction was not significantly different but tended to be longer with I than with Br (Br 12 min 51 s ± 2 min 4 s, I 14 min 40 s ± 53 s). Br-induced contractions were highly variable and frequently transient with large fluctuations in tone (see typical responses, Fig. 3). Both the relaxation and the contraction induced by I were concentration dependent (Fig. 4) and did not peak until the I concentration reached 130 mM. This suggested that high intracellular I concentrations were required for the response. To test this hypothesis we repeated the dose response to I using Na-methanesulfonate as a substitute for the NaCl component of the buffer. Methanesulfonate has been employed frequently as an extracellular Cl substitute because it permeates anion channels very poorly (33) and, unlike many other large anions, has a negligible effect on the ionized calcium concentration of physiological buffers (18). In addition, methanesulfonate is a poor substrate for the Na-K-2Cl cotransporter (6). On the basis of the selectivity of the Cl/HCO₃ exchanger for Cl over other large anions (32), it is likely that inward methanesulfonate transport by this mechanism is also minimal. We hypothesized that using methanesulfonate would limit the extracellular anions available for transport and force I loading of the VSM cells at lower extracellular concentrations of I. Under these conditions the concentration response relationship for I was shifted remarkably to the left. With simple I substitution for Cl, 98 mM I was required to produce a contractile response of 59 ± 5.4% of the response to 90 mM KCl. In contrast, when Na-methanesulfonate is substituted for the NaCl portion of the buffer, a response of similar magnitude (56 ± 12.9%) is obtained in only 16 mM I. We have shown previously that replacement of extracellular Cl with methanesulfonate causes a small contraction of variable size (average ~20% of maximal response to KCl) in rubbed rings (24). This finding is confirmed in these experiments by the response seen in 0 mM I (130 mM methanesulfonate, 18.7 ± 2.9%).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A: typical responses of three different tissues to Br (left) or I (right). Br substitution (solid bars, B, n = 5) for Cl produces both smaller initial relaxations and smaller subsequent contractions than does I substitution (hatched bars, B, n = 5). Time delay to onset of contraction tended to be longer with I but was not statistically different. Br-induced contractions can be transient and may consist of bursts of phasic responses with early peaks that are not maintained. Solid black bars to left of typical responses (A) represent magnitude of each ring's response to 90 mM KCl. * P < 0.05 comparing I to Br.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves to I using either Cl (, n = 4) or methanesulfonate (MS, , n = 3) as alternative extracellular anion. When Cl was simply replaced with I (NaCl + NaI = 130 mM) the threshold for significant relaxation response (A) was 16 mM and the largest responses occurred only after all 130 mM NaCl had been replaced with NaI. Contractile responses to I (B) occurred at similar but slightly higher concentrations and peak effects again required 130 mM I. , Magnitude of contractile response when NaI + Na-MS = 130 mM and no NaCl was added to PSS (see METHODS). Lack of significant extracellular Cl as substrate for transport forces use of I at lower extracellular concentrations of I. This results in a leftward shift in the concentration-response curve to I. No relaxation data are shown under these conditions because Na-MS substitution alone produces immediate small contractile responses in rubbed rings (24).

Under normal conditions, contractile responses to NE in the endothelium-intact rat aorta are about 75% dependent on calcium entry through nifedipine-sensitive, voltage-dependent calcium channels (24). To determine the source of calcium responsible for I-induced contraction, rings were treated with nifedipine (10⁶ M) either 10 min before changing to NaI buffer or after the contraction to I reached a plateau at 40 min (Fig. 5). Pretreatment with nifedipine had no effect on the relaxation response to I but completely inhibited the contraction (0%, n = 6). These data suggest that although the contractile response is a completely voltage-dependent phenomenon, the relaxation (and presumably the initial suppression of NE-induced contraction) is not mediated by hyperpolarization as previously suggested (23).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   I-induced contractions are completely inhibited by nifedipine (106 M). Typical responses of intact (A) or rubbed (B) aortic rings demonstrate that initial relaxation responses to I were unaltered by nifedipine, whereas contractile response of the rubbed ring was completely inhibited. Treatment with nifedipine (n = 6) after full response to I had already developed resulted in 100% relaxation of all active tension (C). Solid black bars to left of typical responses represent magnitude of each ring's response to 90 mM KCl.

The complete inhibition of I-induced contraction by nifedipine suggested that the contractile response to I was due to either direct activation of voltage-sensitive calcium channels or membrane depolarization. To discern between these two possibilities, inhibitors of Cl transport and Cl channel blockers were employed. Control contractile responses to 130 mM I were obtained in rubbed rings. The tissues were then incubated for 20 min in one of the following: 1:1,000 dilutions of DMSO or ethanol, 10 mM HEPES buffer (0 HCO₃), 10⁵ M bumetanide, 10⁵ or 10⁴ M niflumic acid, 10⁴ M DIDS, or 10⁵ M tamoxifen. The rings were then reexposed to 130 mM NaI in the continued presence of the pharmacological intervention. Figure 6 demonstrates that the contractile response to I could be repeated three times without a significant change in the response. The contraction was also not altered by the solvents used to dilute the drugs employed in these experiments. Neither time nor solvents produced any change in the initial relaxation response (data not shown). As shown in Fig. 7, HEPES buffer, which inhibits Cl/HCO₃ exchange by being HCO₃ free, had no effect on the relaxation response to I (control 245 ± 17 mg, treated 254 ± 32 mg) but almost completely blocked the contractile response to I (control 101.4 ± 6.1%, treated 11.2 ± 7.8%). Bumetanide, which inhibits Na-K-2Cl cotransport, did not significantly alter either the relaxation (control 245 ± 34 mg, treated 250 ± 35 mg) or the contraction (control 95.2 ± 4.8%, treated 80.5 ± 15.2%) to I. We have previously demonstrated that HEPES buffer inhibits the contractile response to NE but does not affect responses to KCl in any way. The effect of HEPES buffer on responses to NE is absent when both HEPES and HCO₃ are present, suggesting that the effect is due to the absence of HCO₃ not the presence of HEPES (23). The results obtained with HEPES and bumetanide suggest that I is taken up by VSM cells via anion exchange and that the ability of I to cause contraction is dependent on I getting into VSM cells.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   A: time controls demonstrate reproducibility of contractile response to I. There was no significant difference between first and third response to I (n = 5). B: vehicle controls demonstrate that contractile response to I (open bars) was unaffected by either of 2 vehicles (hatched bars; DMSO, n = 3 and ethanol, n = 3), which were used to administer drugs in subsequent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Effects of altering Cl transport or blocking Cl channels (hatched bars) on control responses to I (open bars). Substitution of HEPES for NaHCO3 results in inhibition of Cl/HCO3 exchange. A 20-min incubation in HEPES-buffered PSS followed by substitution of I for Cl in HEPES buffer almost completely inhibited contractile response to I (n = 8). Inhibition of Na-K-2Cl cotransport with bumetanide (Bumet; 105 M, n = 6) did not alter response to I. Cl channel blocking agents niflumic acid (105 M, n = 3, and 104 M, n = 4), DIDS (104 M, n = 5), and tamoxifen (Tamox; 105 M, n = 5) all inhibited contractile responses to I. Negative values reported for niflumic acid and DIDS reflect the fact that in the presence of these drugs tension remained below baseline throughout 40-min period. The degree to which tension was below baseline was normalized to the maximum response to KCl to facilitate display of data. * P < 0.05 vs. control.

Three different Cl channel blockers significantly inhibited contractile responses to 130 mM I but none of them inhibited the initial relaxation (Fig. 7). Niflumic acid at 10⁵ M (control contraction 90.7 ± 9.7%, treated contraction 28.0 ± 20.8%, control relaxation 383 ± 31.8 mg, treated relaxation 367 ± 21.6 mg) and tamoxifen (control contraction 98.9 ± 8.7%, treated contraction 47.4 ± 13.3%, control relaxation 238 ± 21 mg, treated relaxation 220 ± 20 mg) partially inhibited contraction to I. Niflumic acid at 10⁴ M (control contraction 101.6 ± 17.0%, treated contraction 9.3 ± 1.4%, control relaxation 208 ± 33 mg, treated relaxation 287 ± 28 mg) and 10⁴ M DIDS (control contraction 98.1 ± 15.7%, treated contraction 12.9 ± 1.1%, control relaxation 347 ± 45.5 mg, treated relaxation 417 ± 20.4 mg) inhibited contraction completely. We have demonstrated previously that 10³ M DIDS and 10⁵ M tamoxifen do not alter KCl-induced contraction of rat aortas (23), whereas 10⁴ M niflumic acid inhibits the response by ~50%. We therefore used niflumic acid at both 10⁵ and 10⁴ M. Niflumic acid does not inhibit calcium current directly at 10⁵ M (25). These data suggest that the contractile effect of I is not likely to be due to direct activation of calcium channels and must require depolarization. This depolarization appears to be Cl-channel dependent.

The requirement for endothelium removal to induce contractile responses to I suggested that the anion channels responsible for the depolarizing response to I were under the regulatory control of the endothelium. We used inhibitors of known pathways for the production of endothelium-dependent vasodilators in an attempt to mimic the effect of rubbing and to determine what endothelial factor is involved. Intact rings were treated with indomethacin (10⁶ M), an inhibitor of cyclooxygenase and therefore prostacyclin production, ODYA (10⁵ M), an inhibitor of cytochrome P-450 metabolism and putative inhibitor of endothelium-derived hyperpolarizing factor production, or L-NNA (10⁴ M), an inhibitor of nitric oxide synthase. These compounds were used both alone or in combination (n = 3 for all groups). None of these interventions duplicated the effect of rubbing on the contractile phase of the response to I (Fig. 8). Contractions tended to be larger whenever L-NNA was used, and when the L-NNA results were pooled from all combinations that included L-NNA (n = 9 rings from 3 rats) a significant effect can be demonstrated (control 7.0 ± 1.9%, L-NNA treated 15.5 ± 2.1%, P < 0.05). Although this result reaches statistical significance, the response to 130 mM I in the presence of L-NNA is not nearly as large as that obtained after rubbing.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   A: intact rings were treated with indomethacin (Indo, 106 M), 17-octadecynoic acid (ODYA, 105 M), or N-nitro-L-arginine (L-NNA; 104 M), alone or in combination (n = 3 for all groups). Only pretreatment with L-NNA had any effect on contractile response to I. Effect was small but significant (* P < 0.05) when all rings receiving L-NNA (alone or in combination with Indo or ODYA) were grouped and compared with their control responses (n = 9 rings from 3 animals). Inhibition of soluble guanylate cyclase with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 106 M) in intact rings did not reproduce effect of L-NNA. B: typical response demonstrates that this concentration of ODQ almost completely reverses vasodilator effects of detaNONOate in rat aortic rings after constriction with NE. * P < 0.05 compared with control.

The small magnitude of the effect of L-NNA suggested that rather than specifically regulating the ion channels responsible for I-mediated depolarization, inhibition of resting nitric oxide production might be nonspecifically increasing vascular reactivity. This effect could be mediated by releasing a tonic inhibitory effect on voltage-dependent calcium (34) or potassium (1) channels. To evaluate the role of nitric oxide in another way, ODQ was used to inhibit NO-mediated activation of soluble guanylate cyclase (Fig. 8). Pretreatment with ODQ (10⁶ M) did not alter the contractile response of aortic rings to NaI (control 3.3 ± 3.4%, treated 2.7 ± 1.1%). This concentration of ODQ was very effective at reversing the vasodilator effect of nitric oxide (detaNONOate, 10⁶-10⁴ M). On the basis of these results the ability of nitric oxide synthase inhibition to induce small contractile responses to I in intact rings is not due to an ability to reduce cGMP levels. Finally, to eliminate the possibility that a stored form of nitric oxide was involved in endothelial suppression of responses to I, 10⁵ M hemoglobin was used as an nitric oxide scavenger. Three intact rings underwent a control exposure to I followed by a 10-min incubation in hemoglobin. The rings were then reexposed to I. No significant contractile response to I was seen in the presence of hemoglobin (control 2.1 ± 1.2%, treated 4.2 ± 2.4%).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The response of isolated blood vessels to anion substitution occurs in two phases. An initial relaxation response is endothelium independent and related to both the anion used [I > Br, not seen with methanesulfonate (24)] and its concentration. This relaxation is not voltage-sensitive calcium channel or Cl channel dependent as it was not altered by nifedipine or by Cl channel blockers. The contractile phase of the response to I is also concentration dependent and elicited by I > Br. This contraction is not induced by an endothelial factor and is in fact prevented by the presence of an intact endothelium. I-induced contraction can be inhibited by calcium channel (nifedipine) or Cl channel (niflumic acid, tamoxifen, DIDS) blockers and by interruption of Cl/HCO₃ exchange. The induction of a contractile response to I by mechanical endothelial disruption cannot be mimicked by drugs that inhibit the production of known endothelial vasodilators (NO, prostaglandins, P-450 metabolites). These results are best explained by the hypothesis that VSM cells take up I via an anion exchanger and subsequent I efflux through anion channels produces depolarization, which is sufficient to activate voltage-sensitive calcium channels. It will be important to directly measure the effect of I substitution on membrane potential in future experiments.

Anion-induced relaxation occurs in rings having no evident myogenic or vasoconstrictor-induced tone. This relaxation was not seen when Cl was replaced with the impermeable anion methanesulfonate (23). A similar response was observed when thiocyanate anions were used as a Cl substitute by other investigators (42, 43). Neither calcium-free buffer nor nifedipine duplicate this effect of anion substitution. These data suggest that the ability of I or Br substitution to suppress the initial contractile response to NE is not due to membrane hyperpolarization as we previously hypothesized (23). I could either inhibit active but calcium-independent tone (35) or somehow alter the elastic properties of these isolated vessels. The immediate onset of relaxation and lack of inhibition by anion uptake blockade (HCO₃-free bumetanide) suggest that the initial site of action of the relaxant effect of I is extracellular.

The replacement of extracellular Cl with gluconate inhibits Cl/HCO₃ exchange and produces VSM cell alkalinization (4, 27). This effect is not seen in HEPES buffer and requires 10-20 min to complete. This time course is consistent with that of the onset of I-induced contraction; however, this pH change was not associated with contraction of human subcutaneous small arteries (4). If a pH change is directly responsible for the contractile effects of anion substitution, then contraction should be inversely proportional to the ability of the substitute anion to replace Cl as a substrate for the anion exchanger. The Na-independent anion exchangers expressed in rat aortic VSM cells are AE2 and AE3 (2). There are limited data regarding the anion selectivity of these proteins; however, AE2 expressed in Xenopus oocytes has a diminished ability to transport I compared with Cl (15). The same is true of the erythrocyte band 3 protein (AE1), and I is a worse substrate than Br (32). Although no data are available, one would predict that methanesulfonate, like gluconate, would be an extremely poor substrate for the anion exchanger, yet this anion causes only small and inconsistent late contractions (Fig. 4 and Ref. 24). Although a change in pH may occur with I substitution and may be important, it seems unlikely that alkalosis is the primary mechanism of the vasoconstrictor effect of I.

Contraction to I is likely to depend not only on the removal of extracellular Cl but also intracellular accumulation of the substitute anion. Nearly complete replacement of Cl (130 mM I, 7.9 mM Cl) is required to elicit a full contractile response to I. This suggests that a relatively high intracellular concentration of I is required to induce the response. This conclusion is supported by the large leftward shift in the concentration-response relationship that is observed when methanesulfonate is used to limit alternative anions for uptake. The ability of HEPES buffer to inhibit the contraction suggests that HCO₃-dependent anion exchange is important for I uptake. Decreased efficiency of anion exchange in the presence of I may account for the significant time delay before the onset of contraction as intracellular Cl is progressively replaced with I. The time required for I to induce contraction of resting tissues is much longer than the time required for I to potentiate NE-induced contraction. This may be due to the ability of catecholamines to accelerate inward anion transport (9). The lack of an effect of bumetanide on I-induced contraction suggests that this transporter does not play a major role in inward I transport in these tissues.

Once VSM cells are loaded with I or Br, efflux through anion channels will result in a depolarizing current. VSM has a significant resting Cl conductance, with estimates of relative permeability at rest for K and Cl (Cl/K) varying from 0.09 in the rat femoral artery (5) to 0.82 in rat portal vein (39). No data are available comparing anion permeability in endothelium-intact vs. denuded tissues. I_Cl,vol channels have a relative permeability for I compared with Cl (P_I/P_Cl) of 1.5-2 (29, 36, 40), whereas for I_Cl,Ca the ratio is 3.5 (26). Therefore if the anion gradients are equal, the depolarizing current produced under resting conditions by I efflux will exceed that produced by Cl efflux through the same number of open channels. If the total anion conductance at any given time produces sufficient depolarization to reach threshold for activation of voltage-sensitive calcium channels, contraction will occur. Replacement of Cl with thiocyanate (highly permeant anion) in unstimulated cells produced a significant outward current in rabbit pulmonary artery cells (13) and caused contraction of rat aortic rings (42). In resting, endothelium-intact aortic rings, anion conductance is apparently not high enough to cause contraction after I loading. However, NE activates a Cl conductance that contributes to depolarization (3) and contraction (23). Replacing Cl with a more permeable anion appears to augment this response. It seems remarkable, however, that rubbed rings contract so vigorously to anion substitution alone. Endothelium disruption may uncover a previously inactive VSM anion conductance. This conclusion is supported by previous experiments demonstrating that only rubbed rings contract when the outward Cl gradient is suddenly augmented by Cl replacement with methanesulfonate (24).

The two anion conductances that are likely to account for resting Cl permeability in VSM are I_Cl,vol and I_Cl,Ca. The ability of tamoxifen to partially inhibit I-induced contraction suggests that I_Cl,vol contributes significantly to resting anion conductance. This conclusion is supported by the ability of tamoxifen to decrease the holding current of isolated rabbit portal vein VSM cells held at 50 mV (12). It has been speculated that I_Cl,vol may be activated by mechanical stretch, allowing the passive tension placed on the rings to provide basal activation of this current (12, 28, 41). An I_Cl,vol has been recorded from VSM cells and may be encoded by the ClC-3 gene (11, 41). ClC-3 is the most highly expressed member of the ClC chloride channel gene family in human aortic VSM (22). The inability of tamoxifen to completely inhibit the response to I suggests that other anion channels are involved. I_Cl,Ca is selectively inhibited by 10⁵ M niflumic acid [IC₅₀ ~5 × 10⁶ M (Ref. 14)]. At a higher niflumic acid concentration (10⁴ M), I_Cl,vol is likely to also be partially inhibited [IC₅₀ ~1 × 10⁴ M (Ref. 12)]. The ability of niflumic acid to partially inhibit I-induced contraction at 10⁵ M and to completely block it at 10⁴ M suggests that in rubbed rings, both I_Cl,Ca and I_Cl,vol are active at rest. The activity of I_Cl,Ca may be accounted for by periodic activation of spontaneous transient inward Cl currents due to release of calcium from the sarcoplasmic reticulum (40). There is evidence to suggest that these events occur not only in isolated cells but also in whole tissue preparations (37). There is evidence that anion channel activity is regulated by the intracellular concentration of the permeant anion (10, 17), but there are no data available regarding the relative ability of alternative anions to induce these changes. In the current experiments the substitute anion may not only permeate anion channels more readily but could also contribute to an increase in anion conductance. Any pH change associated with anion replacement could also alter the probability of opening of VSM anion channels.

The ability of Cl channel blockers to inhibit the contractile response to I suggests that outward I movement (inward current) produces depolarization, activates voltage-dependent calcium channels, and induces contraction. Alternative explanations must be considered and include that these compounds 1) inhibit calcium channels directly, 2) interfere with some other mechanism of depolarization activated by I such as inhibition of potassium channels or activation of nonselective cation channels, or 3) inhibit anion exchange. DIDS and niflumic acid did not alter voltage-dependent calcium currents measured by patch-clamp recording from rabbit coronary artery myocytes (25). In addition, neither DIDS nor tamoxifen had any effect on contractile responses to submaximal (38 mM) potassium-induced depolarization of rat aortic rings (23). Niflumic acid also did not alter contractile responses of rat aortic rings to 25 mM KCl, which were completely relaxed by the potassium channel activator levcromakalim (7). These data suggest that these inhibitors do not alter VSM contraction by blocking calcium channels. Patch-clamp recording of rabbit portal vein myocytes demonstrated that niflumic acid did not alter either spontaneous calcium-activated potassium currents or catecholamine-induced nonselective cation currents (14). It is possible that the inhibitory effect of DIDS and niflumic acid on I-induced contraction are at least partially related to inhibition of anion exchange. Although DIDS has been demonstrated to inhibit anion exchange in VSM (8), the data for niflumic acid are more limited and are entirely based on interactions with AE1 (32). The relative ability of these agents to block Cl channels vs. inhibit anion exchange is difficult to quantify. Both actions may contribute to inhibition of I-induced contraction. We can find no evidence from the literature that tamoxifen inhibits anion exchange, but this possibility cannot be dismissed.

Endothelial disruption is required to induce a contractile response to I or Br. Rubbing may remove an endothelial factor that inhibits anion conductance. We have been unable to identify a critical factor by using inhibitors of endothelial vasodilator production (L-NNA, indomethacin, ODYA) on intact rings. Nitric oxide and prostaglandins do not appear to be involved. By definition such a factor would be an endothelium-derived hyperpolarizing factor in that it prevents I-induced depolarization; however, it does not appear to be a P-450 metabolite. It is possible that the response to I is induced by VSM damage related to the rubbing process. It is difficult to assess the significance of any injury to VSM cells induced by endothelial denuding. The mean maximal force-generating capacity of the denuded rings was slightly diminished, suggesting that some VSM damage occurred. However, there was no correlation between force-generating capacity (response to 90 mM KCl) and the size of the response to I either in the rubbed rings or in the intact rings, which are also at risk for partial endothelial and VSM injury during handling. The effect of rubbing could also be caused by loss of physical contact between the endothelium and the underlying VSM (16).

In summary, these data suggest that isolated rat aortic VSM has a significant resting anion current which is larger when the endothelium is absent. When the cells are loaded with I, this anion current can produce sufficient depolarization to result in a large contractile response. Both I_Cl,vol and I_Cl,Ca seem to contribute to this current. I-induced contractions may provide a useful bioassay for future study of the regulation of vascular anion channels. The mechanism by which endothelial cells control VSM anion current may be an important determinant of contractility. The withdrawal of this influence could play a critical role in the increase in vascular reactivity that accompanies endothelial damage.