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Indigo Carmine Inhibits Endothelium-Dependent and -Independent Vasodilation

Originally published1 Feb 1996https://doi.org/10.1161/01.HYP.27.2.228Hypertension. 1996;27:228–234

    Abstract

    Abstract To investigate the potential mechanisms by which indigo carmine produces hypertension, we tested the hypothesis that indigo carmine inhibits endothelium-dependent vasodilation and determined the possible site of the inhibition (endothelium versus smooth muscle). Using isolated rat thoracic aortic rings that were precontracted with phenylephrine, we examined vasodilatory responses to acetylcholine, histamine, and Ca2+ ionophore A23187 (in endothelium-intact rings) and sodium nitroprusside and isoproterenol (in endothelium-denuded rings) in the presence and absence of indigo carmine. In addition, the effects of methylene blue on the acetylcholine- and sodium nitroprusside–induced vasodilation were compared with those of indigo carmine. Indigo carmine (10−6, 10−5, and 10−4 mol/L) significantly inhibited receptor- and non–receptor-mediated endothelium-dependent vasorelaxation. Indigo carmine (10−4 mol/L) also inhibited endothelium-independent vasorelaxation induced by sodium nitroprusside (an activator of vascular smooth muscle soluble guanylyl cyclase), although to a lesser extent than vasodilation from acetylcholine, histamine, and Ca2+ ionophore A23187. In contrast, indigo carmine (10−4 mol/L) had no effect on the vasodilation induced by isoproterenol (an activator of adenylyl cyclase), indicating that indigo carmine selectively inhibits nitric oxide–mediated responses. Methylene blue, a known inhibitor of soluble guanylyl cyclase, inhibited both acetylcholine- and sodium nitroprusside–induced vasorelaxation. The inhibition was also greater in the acetylcholine- than the sodium nitroprusside–induced vasodilation. These results suggest that indigo carmine, like methylene blue, may inhibit endothelium-dependent relaxation by a mechanism that involves two levels. The major action of indigo carmine appears to be at the level of nitric oxide generation and/or release from the endothelial cell. In addition, indigo carmine appears to inhibit vascular smooth muscle guanylyl cyclase. Thus, indigo carmine may elevate blood pressure by interfering with these nitric oxide–mediated vasodilatory mechanisms.

    Indigo carmine (sodium indigotin disulfonate), a blue dye that is often administered intravenously to determine patency of the urinary collecting system, has been reported to cause mild to severe hypertension in some patients.1234 The hypertension associated with IC was attributed to an increase in total peripheral resistance either due to direct activation of α-adrenergic receptors or indirectly through the release of catecholamines.14 In addition, IC may have serotonin-like effects as a result of its structural similarity to serotonin.3

    Vascular endothelium plays an important role in the regulation of vascular smooth muscle tone by releasing vasodilatory and/or vasoconstricting substances.56 One of the most powerful substances released from endothelium is EDRF, now identified as NO.78 NO plays a major role in maintaining resting vascular tone, and lack of NO production has been implicated as a cause of vasospasm and hypertension.6 NO is released under basal conditions and in response to a wide range of vasodilators.910 Inhibition of NO synthesis increases blood pressure.11 Thus, IC may elevate blood pressure by interfering with these NO-mediated vasodilatory mechanisms. NO is produced in endothelial cells from the amino acid l-arginine by a constitutive enzyme, NO synthase, which is Ca2+-, calmodulin-, and NADPH-dependent.12 Intracellular free Ca2+ concentration is probably the major factor involved in the activation of NO synthase in endothelial cells.1314 Intracellular Ca2+ can be increased in the endothelial cells by agonist-receptor interaction through both extracellular Ca2+ influx and Ca2+ release from intracellular stores (eg, ACh, histamine) or by nonreceptor mechanisms that involve direct Ca2+ transport through the cell membrane (eg, Ca2+ ionophore A23187).15161718 NO thus formed in endothelial cells causes vasodilation by stimulating vascular smooth muscle soluble guanylyl cyclase and elevating cGMP levels.19 Inhibition of any site in this l-arginine–NO–guanylyl cyclase pathway can cause an impairment of endothelium-dependent vasodilation and hypertension. The goals of the present study were (1) to examine whether IC inhibits endothelium-dependent vasodilation and (2) to determine whether the site of the inhibition is at the endothelium or at the vascular smooth muscle soluble guanylyl cyclase level. We attempted to answer these questions by using endothelium-dependent (ACh, histamine, A23187) and endothelium-independent (SNP) vasodilators. Further, by using different receptor- and non-receptor–activating agents, we tested whether the inhibition involves specific endothelial receptors or is distal to receptor sites.

    Methods

    The protocol was approved by the Institutional Animal Care and Use Committee of the Oregon Health Sciences University. The method used for the in vitro study of rat thoracic aortic rings has been described in detail.20 Briefly, thoracic aorta was removed from male Sprague-Dawley rats (body weight, 250 to 300 g; Simonsen Laboratories, Gilroy, Calif) that were anesthetized with isoflurane. Rings approximately 3 mm wide were then cut, mounted between two stainless steel wires, and placed in a 20-mL organ bath containing a modified K-H solution of the following composition (mmol/L): KCl 4.75, KH2PO4 1.19, MgSO4 1.19, CaCl2 1.25, NaCl 119, NaHCO3 25, and glucose 11, pH 7.4. The solution was continuously aerated with a gas mixture of 95% O2/5% CO2 and maintained at 37°C. Rings were equilibrated for 60 to 90 minutes with resting tension of 2 g. Preliminary length-tension experiments suggested that 2 g was the optimal resting tension for these vessels. Isometric tensions were recorded with force-displacement transducers (model FT 03, Grass Instruments) and recorded on a polygraph (model 7, Grass Instruments). Some of the rings were denuded of endothelium according to the protocol. The endothelium was removed by insertion of a small forceps into the lumen of the ring and gentle rolling of the ring for a few seconds. Three to four rings from the same aorta were studied simultaneously. Since for each experimental condition only one ring was used from each animal, the numbers of experiments (n) and the numbers of animals used are the same. A total of 75 animals were used.

    Concentration Responses of Endothelium-Dependent and -Independent Vasodilators in the Presence of IC

    After 60 to 90 minutes of equilibration, active tone was induced with EC50 of phenylephrine as determined previously (10−7 mol/L for endothelium-intact rings and 5×10−8 mol/L for endothelium-denuded rings).2122 The functional integrity of endothelium was confirmed by observation of a minimum of 40% relaxation in response to ACh 10−5 mol/L. Lack of relaxation in response to ACh in the denuded preparation was taken to indicate effective functional removal of endothelium. After ACh testing, the bathing solution was changed several times until tension returned to the baseline 2-g resting tension.

    To determine the site(s) of IC inhibition in the NO-dependent vasodilatory cascade, the relaxation responses to various endothelium-dependent and endothelium-independent vasodilators were tested in the presence and absence of IC.

    Endothelium-dependent vasodilation. Rings with intact endothelium were contracted submaximally as before with EC50 of phenylephrine. ACh (10−8 to 10−4 mol/L) or histamine (10−7 to 10−3 mol/L) was then added to the bath to obtain control cumulative concentration-relaxation response curves. After control curves for these vasodilators in the individual rings were obtained, the rings were treated with either a single concentration of IC (10−6, 10−5, or 10−4 mol/L) or an equal volume of added distilled water (time control) for 10 minutes. The experiment was repeated to obtain second concentration-response curves for these vasodilators in the presence of IC or vehicle. The rings were then washed several times with normal K-H solution for 60 to 90 minutes until the tension returned to the baseline 2-g resting tension. The third concentration-relaxation response curves were obtained to determine whether the preparation had recovered from the exposure of IC. In each experiment, one ring was designated as a time control throughout all the experimental protocol, thus confirming the stability of vasodilatory responses to ACh or histamine and contractile responses to phenylephrine. In the case of A23187 (10−8 to 3×10−6 mol/L), our preliminary experiments demonstrated that the preparation did not fully recover from A23187 treatment even with extensive washings (up to 2 hours). Therefore, the first control dose response for A23187 before IC treatment was not obtained. From the beginning, one ring was used as control and other rings were treated with various concentrations of IC (10−6, 10−5, and 10−4 mol/L). In separate experiments, to rule out possible involvement of prostacyclin and other prostanoid-mediated responses by some of the vasodilators used (eg, ACh), aortic rings with intact endothelium were studied in K-H solution containing indomethacin 10−5 mol/L, an inhibitor of cyclooxygenase.23

    Endothelium-independent vasodilation. The same protocol described for ACh and histamine as above were used to study responses in endothelium-denuded rings. In this experiment, the vasodilatory responses to SNP (10−9 to 10−7 mol/L) and isoproterenol (10−8 to 10−5 mol/L) were studied with and without IC.

    Effect of methylene blue on endothelium-dependent and endothelium-independent vasodilation. To compare the effects of IC with those of methylene blue, rings with and without endothelium were incubated with methylene blue (10−6 and 10−5 mol/L) for 10 minutes. The rest of the protocol was the same as above.

    Drugs

    The following drugs were used: ACh chloride, l-phenylephrine hydrochloride, indomethacin, histamine, (−)isoproterenol, IC, methylene blue, SNP, Ca2+ ionophore A23187, and DMSO. All drugs were purchased from Sigma Chemical Co and prepared in distilled water just before use except for indomethacin and A23187, which were dissolved in absolute ethanol and DMSO, respectively. The stock solutions of indomethacin and A23187 in ethanol and in DMSO were diluted with distilled water. Final bath concentrations of vehicles (ethanol and DMSO), at concentrations of 0.0001% to 0.01%, did not produce a measurable response during preliminary testing in aortic rings with and without endothelium.

    Data Analysis

    Relaxation responses produced with increasing concentrations of various vasodilators were expressed as the percentage relaxation from the contractile state elicited by EC50 of phenylephrine. All data are expressed as mean±SEM. Statistical analysis of the data was performed with one-way ANOVA between experimental groups (eg, comparison between responses from IC–treated rings and those of appropriate time control). When three or more groups of data were compared, significant differences were determined by the Scheffé F test. Data analysis within groups (eg, comparison between before and after IC treatment within each group) were performed with ANOVA for repeated measures. P<.05 was considered statistically significant.

    All statistical analyses were performed with StatView 11 software (Abacus Concepts Inc).

    Results

    Tension Evoked by Phenylephrine and IC

    Because the response to phenylephrine was increased (without a change in maximal contraction) in endothelium-denuded rings relative to endothelium-intact rings,2122 EC50 of phenylephrine was chosen to precontract the preparations (10−7 mol/L for endothelium-intact rings and 5×10−8 mol/L for endothelium-denuded rings) (Table 1). Thus, a similar baseline tension was obtained with phenylephrine in all groups of rings before IC treatment whether endothelium was intact or removed (Table 1). Also, there was no significant difference in the phenylephrine-evoked baseline tone within each experimental group of rings before and after treatment with IC except in rings with intact endothelium that were treated with 10−4 mol/L (Table 1). Under the latter conditions, the phenylephrine response was significantly enhanced. In this case, the concentration of phenylephrine was decreased to 3×10−8 mol/L to match a tension developed by phenylephrine before IC treatment (1450±145 mg). IC itself caused small but significant contraction both in endothelium-intact and endothelium-denuded rings (≈3% to 6% of 1.2- to 1.3-g tension evoked by EC50 of phenylephrine in endothelium-intact rings), which was greater in the endothelium-intact rings (Table 2). The contraction occurred at 10−5 mol/L in endothelium-intact rings and at 10−4 mol/L in endothelium-denuded rings.

    Endothelium-Dependent Vasodilation

    In aortic rings with intact endothelium precontracted with phenylephrine, ACh and histamine produced concentration-dependent relaxations (Figs 1 and 2) that were similar among all experimental groups of rings before IC treatment (time-control rings and rings to be treated) (Figs 1A and 2A).

    IC 10−5 and 10−4 mol/L significantly inhibited vasodilation induced by ACh and histamine with a shift of dose-response curves to the right and downward (Figs 1B and 2B). IC 10−6 mol/L inhibited vasodilation induced by low concentrations (3×10−7 and 10−6 mol/L for ACh and 10−5 and 10−4 mol/L for histamine) without affecting responses to high concentrations of these agents (Figs 1B and 2B). Responses to ACh and histamine completely recovered from IC after the washout procedures (Figs 1C and 2C). Time-control responses to ACh and histamine (first, second, and third times) were stable (Figs 1D and 2D). Since time-control responses to ACh or histamine during IC exposure were not different from pre–IC responses to these agents, comparisons were made between appropriate time-control and IC–treated groups unless otherwise stated.

    Vasodilation induced by the non–receptor-mediated endothelium-dependent agent A23187 (10−8 to 3×10−6 mol/L) was also significantly inhibited by IC at concentrations of 10−5 and 10−4 mol/L, with lesser inhibition with 10−6 mol/L (Fig 3A). Once the preparation was exposed to A23187, responses were diminished to 30% to 40% of the original response despite extensive washings. Nevertheless, complete recovery from the IC effect still can be seen (Fig 3B).

    Indomethacin had no effect on the inhibition of ACh-induced vasodilation by IC (data not shown).

    Endothelium-Independent Vasodilation

    SNP produced a dose-dependent vasodilation that was similar among the experimental groups of rings before IC treatment (Fig 4A). IC inhibited the SNP-induced vasodilation to a lesser extent than vasodilation to ACh, histamine, or A23187 (Fig 4B). Whereas low concentrations of IC (10−6 and 10−5 mol/L) had no effect on the SNP-induced vasodilation, high concentrations (10−4 mol/L) caused significant inhibition. SNP responses completely recovered from IC exposure after the washout (Fig 4C). Time-control responses to SNP were stable (Fig 4D). IC had no effect on the isoproterenol-induced endothelium-independent vasodilation (Fig 5).

    Effect of Methylene Blue on ACh- and SNP-Induced Vasodilation

    Methylene blue inhibited both vasodilation induced by ACh and that induced by SNP (Fig 6A and 6B). Methylene blue had much less effect on SNP-induced than on ACh-induced vasodilation.

    Discussion

    The principal finding of this study is that IC inhibits NO-mediated endothelium-dependent and -independent vasodilation in rat aortic vascular smooth muscle. IC inhibited both receptor- and non–receptor-mediated endothelium-dependent vasodilation induced by ACh, histamine, and A23187 and endothelium-independent vasodilation induced by SNP. Agents like ACh, histamine, and A23187 produce vasodilation through the endothelial synthesis and release of NO, which activates vascular smooth muscle soluble guanylyl cyclase to produce an increase of cGMP concentration.151619 SNP produces vasodilation by activation of soluble guanylyl cyclase after its breakdown to NO.24 Thus, the site of action for NO is soluble guanylyl cyclase, whether it is released endogenously from endothelial cell activation by receptor-mediated (ACh, histamine) or non–receptor-mediated (A23187) agents or is produced intracellularly in the smooth muscle cells from metabolism of the nitrovasodilator SNP.24 Our findings that IC had less influence on SNP than on the endothelium-mediated agents ACh, histamine, and A23187 suggest that IC may have a predominantly inhibitory action on the NO-generating and/or NO-releasing mechanisms in the endothelium, with a minor action at the level of vascular smooth muscle guanylyl cyclase. Although our present results suggest the major site of action on the endothelium, the mechanisms by which IC elicits such an effect in the endothelium are unclear at present. On the basis of the known mechanism of NO synthesis and release, possibilities include (1) an interference with receptor activation and signal transduction, (2) a reduction of intracellular calcium availability, (3) direct inhibition of NO synthase, and (4) interactions with NO itself. NO synthesis in vascular endothelium results from an activation of constitutive NO synthase that depends primarily on the intracellular Ca2+ concentration.1314 An increase in intracellular Ca2+ in the endothelial cells can be achieved by extracellular Ca2+ influx and/or Ca2+ release from intracellular stores. Drugs such as ACh and histamine, which act via endothelial membrane receptors, increase the intracellular Ca2+ concentration through extracellular Ca2+ influx and Ca2+ release from intracellular stores.1718 A23187 bypasses receptor-mediated processes by directly transporting Ca2+ across the cell membrane.25 Our present results suggest that it is unlikely that IC inhibits endothelium-dependent vasodilation by interacting with specific endothelial receptors (muscarinic or histaminergic receptors), on the basis of two observations. First, the vasodilation induced by activation of two entirely different receptor systems was inhibited to a similar extent by IC. Second, the vasodilation from A23187 that bypasses receptor effects was also inhibited to a degree similar to that from the receptor-mediated agents, consistent with an inhibition distal to the receptors and intracellular Ca2+ availability. Since the SNP response was inhibited less than the ACh, histamine, and A23187 responses, the inhibition may involve sites distal to the Ca2+ availability in the endothelium and proximal to guanylyl cyclase, including NO synthase.

    The inhibition by IC was similar to that of methylene blue, a known inhibitor of soluble guanylyl cyclase. Methylene blue inhibits both ACh- and nitrovasodilator-induced relaxation.262728 IC may have a mechanism of action similar to that of methylene blue. Although methylene blue is known to inhibit a soluble guanylyl cyclase, it has been reported that methylene blue has more potent inhibitory activity on endothelium-dependent vasodilation than on vasodilation induced by nitrovasodilators.262728 The latter observations are consistent with the results of our present study (Fig 6). Methylene blue can inactivate NO via superoxide generation29 and also acts as a direct inhibitor of NO synthase.3031 These additional actions of methylene blue on the inhibition of NO synthesis and NO destruction may explain the greater inhibitory action on ACh-induced vasodilation than on SNP-induced vasodilation. Methylene blue can affect iron-containing enzymes,32 easily oxidizing ferrous hemoprotein to the ferric form.33 The mechanism of inhibition of guanylyl cyclase, an iron-containing enzyme, by methylene blue may involve oxidation of the hemoprotein linked to guanylyl cyclase.34 Recently, it was reported that NO synthase is a cytochrome P-450–type hemoprotein.35 Thus, NO synthase may also be inhibited by methylene blue oxidation of hemoprotein. IC, as an electron mediator, can oxidize or reduce iron-containing enzymes.36 Thus, it is possible that IC, like methylene blue, may act on the hemoprotein of soluble guanylyl cyclase and NO synthase, resulting in an inactivation of both enzymes. It is unknown, however, whether IC, like methylene blue, can generate superoxide radicals that destroy NO. On the basis of its known redox properties,36 IC, like methylene blue, may generate superoxide radicals during its auto-oxidation. Our observations that the inhibition by IC was promptly reversed suggests a direct chemical interaction with NO as a major mechanism rather than an enzyme inhibition, which would be expected to last longer.

    Although the endothelium releases two major vasodilators, NO and prostacyclin, our present ACh results indicate that IC does not affect the prostacyclin-mediated vasodilation, since the inhibition of ACh-induced vasodilation by IC was still present after the cyclooxygenase activity was blocked by indomethacin. This view was further supported by the observation that the concentration of IC (10−4 mol/L) that inhibited the vasodilation induced by SNP, an activator of soluble guanylyl cyclase, had no effect on the vasodilation induced by isoproterenol, an activator of adenylyl cyclase. This suggests that IC selectively inhibits NO-mediated vasodilation.

    IC itself caused small but significant contractions, which were greater with an intact endothelium (Table 2). This may be due in part to inhibition of the effects of spontaneously released NO by IC. In addition, IC augmented phenylephrine-evoked tone in aortic rings containing endothelium (Table 1). This supports the view that IC inhibits basal release of NO from endothelium in addition to the release of NO by receptor- and non–receptor-mediated endothelium-dependent vasodilators. Basal release of NO modulates the contractile responses to many vasoconstrictors, including phenylephrine.10 Although IC caused a greater contraction in rings containing endothelium, it also caused a slight contraction in rings of aorta lacking endothelial cells. This may be due to direct α-adrenergic receptor stimulation or indirect release of catecholamines, as was previously suggested for IC,4 and/or to blockade of basal unstimulated guanylyl cyclase activity if IC can enter cells. In the present study, methylene blue, like IC, produced a contraction that was greater in endothelium-containing rings than in endothelium-absent rings, consistent with observations by others.2737

    Low concentrations of IC that inhibit the endothelium-dependent relaxation by ACh, histamine, and A23187 are within the range clinically used (10−6 and 10−5 mol/L; 0.46 and 4.6 μg/mL). Methylene blue, which inhibits both soluble guanylyl cyclase and NO synthase, has been shown to cause a short-lived hypertension in a dose-dependent manner in the rat model.38 Inhibition of NO synthase with NG-monomethyl-l-arginine also causes an increase in blood pressure.11 Although we cannot extrapolate in vitro data directly to in vivo conditions, hypertension associated with intravenous administration of IC is consistent with inhibitory actions on NO-generating mechanisms. High concentrations of IC (10−4 mol/L; 46 μg/mL) also inhibit SNP-induced relaxation. However, such high concentrations are not likely during the clinical administration of IC.

    In summary, IC inhibits endothelium-dependent vasorelaxation induced by ACh, histamine, and A23187 and endothelium-independent vasorelaxation induced by SNP in rings of rat aorta. The inhibition was selective for agents that produce vasorelaxation in association with NO production and a rise in cGMP. Cyclooxygenase activity does not appear to contribute. IC has significantly less inhibitory effect with SNP vasodilation than with endothelium-dependent vasodilation by ACh, histamine, and A23187, indicating that IC may inhibit the NO-generating mechanism in the endothelium more than soluble guanylyl cyclase activity. The site of the inhibitory effect on endothelial NO production is probably distal to plasma membrane receptors and cytosolic Ca2+ availability. Therefore, the most likely site of action is at the NO synthase and/or NO stability level. The possibility that IC produces its inhibitory effects on endothelium-dependent vasorelaxation by interfering with NO synthase (and/or NO destruction) and soluble guanylyl cyclase warrants further investigation.

    Selected Abbreviations and Acronyms

    ACh=acetylcholine
    EDRF=endothelium-derived relaxing factor
    IC=indigo carmine
    K-H=Krebs-Henseleit
    NO=nitric oxide
    SNP=sodium nitroprusside

    Figure 1.

    Figure 1. Effect of IC on endothelium-dependent relaxation induced by ACh in isolated rat thoracic aortic rings containing endothelium before IC exposure (A), during exposure to various concentrations of IC (B), and after several washings with K-H solution during 60 to 90 minutes after exposure to IC (C). Time-control responses to ACh before, during, and after IC are shown in D. Data are expressed as percent relaxation of contractions elicited by EC50 of phenylephrine (10−7 mol/L) before addition of ACh in nearly all groups of rings. For the IC 10−4 mol/L treatment group, the phenylephrine concentration was lowered to 3×10−8 mol/L. Tensions (in milligrams) elicited by phenylephrine before IC treatment and after the washout of IC were 1281±139 and 1394±153, respectively. For IC treatment groups, tensions developed were 1336±193, 1474±172, 1573±203, and 1430±142 for time-control rings and rings treated with IC 10−6, 10−5, and 10−4 mol/L, respectively. Each data point represents mean±SEM of 5 to 14 preparations from 14 animals. *P<.05 compared with time control during IC exposure.

    Figure 2.

    Figure 2. Effect of IC on endothelium-dependent relaxation induced by histamine in isolated rat thoracic aortic rings containing endothelium before IC exposure (A), during exposure to various concentrations of IC (B), and after several washings with K-H solution during 60 to 90 minutes after the exposure to IC (C). Time-control responses to histamine before, during, and after IC are shown in D. Data are expressed as percent relaxation of contractions elicited by EC50 of phenylephrine (10−7 mol/L) before addition of histamine in nearly all groups of rings. For the IC 10−4 mol/L treatment group, the phenylephrine concentration was lowered to 3×10−8 mol/L. Tensions (in milligrams) elicited by phenylephrine before IC treatment and after the washout of IC were 1237±160 and 1306±138, respectively. For IC treatment groups, tensions developed were 1221±159, 1332±165, 1507±147, and 1390±117 for time-control rings and rings treated with IC 10−6, 10−5, and 10−4 mol/L, respectively. Each data point represents mean±SEM of 4 to 9 preparations from 9 animals. *P<.05 compared with time control during IC exposure.

    Figure 3.

    Figure 3. Effect of IC on endothelium-dependent relaxation induced by Ca2+ ionophore A23187 in isolated rat thoracic aortic rings containing endothelium in the presence of various concentrations of IC or isovolumetric water (Control) (A), and partial recovery of control response to A23187 and comparable recovery responses after several washings with K-H solution during 60 to 90 minutes after exposure to IC (B). Data are expressed as percent relaxation of contractions elicited by EC50 of phenylephrine (10−7 mol/L) before addition of A23187 in nearly all groups of rings. For the IC 10−4 mol/L treatment group, the phenylephrine concentration was lowered to 3×10−8 mol/L. Tensions (in milligrams) elicited by phenylephrine in control rings and rings treated with IC 10−6, 10−5, and 10−4 mol/L were 1447±217, 1493±133, 1639±154, and 1580±176, respectively. Tension developed after the washout of IC was 1470±135. Each data point represents mean±SEM of 5 to 14 preparations from 14 animals. *P<.05 compared with control.

    Figure 4.

    Figure 4. Effect of IC on endothelium-independent relaxation induced by SNP in isolated rat thoracic aortic rings without endothelium before IC exposure (A), during exposure to various concentrations of IC (B), and after several washings with K-H solution during 60 to 90 minutes after exposure to IC (C). Time-control responses to SNP before, during, and after IC are shown in D. Data are expressed as percent relaxation of contractions elicited by EC50 of phenylephrine (5×10−8 mol/L) before addition of SNP. Tensions (in milligrams) elicited by phenylephrine before IC treatment and after the washout of IC were 1516±169 and 1378±143, respectively. For the IC treatment groups, tensions developed were 1478±157, 1409±103, 1501±158, and 1387±160 for time-control rings and rings treated with IC 10−6, 10−5, and 10−4 mol/L, respectively. Each data point represents mean±SEM of 5 to 14 preparations from 14 animals. *P<.05 compared with time control during IC exposure.

    Figure 5.

    Figure 5. Effect of IC on endothelium-independent relaxation induced by isoproterenol in isolated rat thoracic aortic rings without endothelium during exposure to various concentrations of IC or isovolumetric water (Control). Data are expressed as percent relaxation of contractions elicited by EC50 of phenylephrine (5×10−8 mol/L) before addition of isoproterenol. Tensions (in milligrams) elicited by phenylephrine in control rings and rings treated with IC 10−6, 10−5, and 10−4 mol/L were 1514±20, 1365±104, 1455±178, and 1301±198, respectively. Each data point represents mean±SEM of 6 to 11 preparations from 11 animals. *P<.05 compared with time control during IC exposure.

    Figure 6.

    Figure 6. Effect of methylene blue (MB) on endothelium-dependent relaxation induced by ACh (A) and endothelium-independent relaxation induced by SNP (B) during exposure to methylene blue 10−6 or 10−5 mol/L or isovolumetric water (Control). ACh responses were studied in aortic rings containing endothelium and SNP responses in those without endothelium. Data are expressed as percent relaxation of contraction elicited by EC50 of phenylephrine 10−7 mol/L in endothelium-containing rings or 5×10−8 mol/L in endothelium-denuded rings before addition of ACh or SNP, respectively. When the endothelium-containing rings were treated with methylene blue 10−5 mol/L, the phenylephrine concentration was lowered to 3×10−8 mol/L. Tensions (in milligrams) elicited by phenylephrine before addition of ACh were 1287±152, 1371±203, and 1401±140 in time-control rings and rings treated with methylene blue 10−6 and 10−5 mol/L, respectively. Tensions developed before addition of SNP in endothelium-denuded rings were 1224±141, 1521±256, and 1503±154 in time-control rings and rings treated with methylene blue 10−6 and 10−5 mol/L, respectively. Each data point represents mean±SEM of 3 to 4 preparations from 8 animals. *P<.05 compared with time control during IC exposure.

    Table 1. Baseline Active Tension Developed With PE50 in Aortic Rings With and Without Endothelium Before and After Indigo Carmine Treatment

    Experimental GroupIntact ED (PE50, 10−7 mol/L)Denuded ED (PE50, 5×10−8 mol/L)
    Before ICAfter ICBefore ICAfter IC
    Control1259±1501335 ±190 (42)1480±1841496±179 (25)
    IC 10−6 mol/L1324±1521433±157 (18)1398±981432 ±103 (11)
    IC 10−5 mol/L1296±1741573 ±168 (42)1445±1891478±168 (25)
    IC 10−4 mol/L1345±1622208±216 (38)121456±1701344 ±179 (16)

    ED indicates endothelium; PE50, phenylephrine concentration that produces 50% of maximal tension. Data are milligrams, mean±SEM. Parentheses indicate the number of preparations studied.

    1P<.05 compared with before IC treatment.

    2P<.05 compared with time control.

    Table 2. Active Tension Developed With Indigo Carmine

    Experimental GroupEndothelium
    IntactDenuded
    Control2.0±2.0 (42)3.2±0.5 (25)
    IC 10−6 mol/L7.8±7.8 (18)6.7±6.7 (11)
    IC 10−5 mol/L47.8±14.1 (42)125.9±12.1 (25)
    IC 10−4 mol/L85.1±9.5 (38)151.2 ±12.2 (16)1

    Data are milligrams, mean±SEM. Parentheses indicate the number of preparations studied.

    1P<.05 compared with respective controls.

    This study was supported in part by the American Heart Association, Oregon Affiliate, and by the Medical Research Foundation of Oregon. We thank John Hromco for his technical assistance and Becki Stephenson for her secretarial assistance.

    Footnotes

    Correspondence to Kyoung S.K. Chang, MD, PhD, Department of Anesthesiology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201. E-mail [email protected]

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