Introduction

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The L-arginine/nitric oxide (NO) pathway (Moncada et al., 1991) has been implicated in the control of several biological processes in the cardiovascular and nervous systems. In these systems, NO is released by the action of constitutive Ca²⁺-dependent NO synthases (NOS; Moncada et al., 1991; Snyder and Bredt, 1992). Another isoform of this enzyme, the inducible Ca²⁺-independent NOS (iNOS), is expressed in phagocytic and other cell types after activation by endotoxin (bacterial lipopolysaccharide; LPS) and/or cytokines. Much larger amounts of NO are produced by this enzyme, accounting for the cytotoxicity of macrophages toward parasites and tumor cells and for the progressive hypotension present in pathological conditions such as septic shock (Moncada et al., 1991).

Excessive NO production has been shown to play a pivotal role in septic shock. Current knowledge suggests that high and continuous NO production during septic shock is the major cause of the vascular hyporesponsiveness to vasoconstrictors (Gray et al., 1990, 1991; Julou-Shaeffer et al., 1990; Mulder et al., 1994). For instance, inhibition of iNOS attenuates the circulatory changes and multiple organ failure caused by LPS in the rat (Wu et al., 1996). Moreover, guanylate cyclase inhibition reverses the hyporesponsiveness to vasoconstrictors (Fleming et al., 1991).

Besides NO, endothelial cells produce other chemical species able to induce vasodilatation, among them prostacyclin (Moncada et al., 1976) and endothelium-derived hyperpolarizing factor (EDHF; Chen et al., 1988; Taylor and Weston, 1988). Changes in membrane potential leading to repolarization/hyperpolarization have been associated with the relaxation induced by some endothelium-dependent vasodilatory substances. These membrane-potential changes initially were associated with the release of EDHF, whose chemical identity still is unknown (for reviews, see Garland et al., 1995; Mombouli and Vanhoutte, 1997; and Edwards and Weston, 1998). Although it has been shown that NO is also able to induce hyperpolarization in arterial smooth muscle (Tare et al., 1990), there are evidences indicating that relaxant response to endothelial-derived NO could be blocked independently of the accompanying smooth muscle hyperpolarization (for a review, see Garland et al., 1995), suggesting that the concomitant release of NO and EDHF may underlie the relaxant effect of some vasodilatory substances. Both NO and EDHF seem to induce K⁺ channel opening, thus explaining the hyperpolarization. For instance, NO activates voltage-dependent K⁺ channels, causing hyperpolarization and relaxation of pulmonary arterial smooth muscle (Yuan, 1996; Zhao et al., 1997). Similarly, EDHF-induced relaxation of vascular smooth muscle seems to involve opening of voltage-dependent K⁺ channels (Petersson et al., 1997). Finally, the mechanisms of potassium channel activation induced by NO is still a matter of controversy. For instance, the NO-induced opening of calcium-dependent K⁺ channels can be mediated through a cGMP-dependent protein kinase (Archer et al., 1994) or directly by NO itself (Bolotina et al., 1994; Mistry and Garland, 1998). In addition, NO seems to activate potassium channels in pathological conditions such as septic shock (Hall et al., 1996; Price et al., 1997; Wu et al., 1998).

In the current report, we have attempted to study the effects of the exposure of the rat vascular system to NO and its consequences regarding the responses to vasoconstrictors and to vasodilators. The classical approach of increasing NO production by endotoxin (LPS) did not seem adequate for this, because LPS is known to release a vast array of mediators, several of them with important actions in the vascular system (for a review, see Brandtzaeg, 1996). Therefore, we have developed a model for exposing the vascular system of the rat to NO, namely the infusion of NO donors. As our results will show, a brief exposure to NO is enough to render the rat vascular system hyporesponsive to vasoconstrictors, which resembles remarkably the hemodynamic changes seen in septic shock. This NO effect is long-lasting, persisting for at least 24 h. In addition, simultaneously to the hyporesponsivity to vasoconstrictors, NO donor infusion potentiated the vascular responses to vasodilators. Finally, opening of K⁺ channels seems to be involved in NO effects.

	Materials and Methods

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Surgical Procedures. All procedures were approved by our Institutional Ethics Committee and are in accordance with National Institutes of Health Animal Care Guidelines. Both male and female Wistar rats (3-4 months old; weighing 200-300 g) were used in this study. Animals were kept at a 12-h light/12-h dark cycle and had free access to food and water. They were anesthetized with ketamine/xylazine (90/15 mg/kg i.m., supplemented at 45- to 60-min intervals), as suggested by Gratton et al. (1995). The left femoral vein and right carotid artery were isolated, and heparinized polyethylene catheters (PE 20 and PE 50) were inserted for drug administration and recording of MAP and heart rate (HR) and blood withdrawal, respectively. Immediately after artery cannulation, heparin diluted (30 IU/ml) in sterile Dulbecco's PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM NaHPO₄, pH 7.4) was injected to prevent clotting. When necessary, another catheter was inserted into the bladder for urine withdrawal. Animals were allowed to breathe spontaneously via a tracheal cannula. Body temperature was monitored by a rectal thermometer and maintained at 36 ± 1°C. Data were recorded (at a 10-s sampling rate) with a Digi-Med Blood Pressure Analyzer system (model 190) connected to a Digi-Med System Integrator (model 200; Micro-Med, Louisville, KY) software, running under Windows 95 (Microsoft Corporation, Redmond, WA). All MAP and HR values shown are those recorded at the peak of the effect produced by a given compound. Results are expressed as a mean either of changes in MAP (mm Hg) or changes in HR (beats per minute; bpm) in relation to basal values. All animals were sacrificed by an overdose of pentobarbitone immediately after ending the experiment.

NO Donor Infusions. Figure 1 shows a typical experiment using phenylephrine and sodium nitroprusside (SNP). The same basic protocol was followed throughout the study. After surgery, a stabilization period of 30 min was allowed for. Then, increasing doses of vasoconstrictors (3, 10, and 30 nmol/kg phenylephrine; 3, 10, and 30 pmol/kg angiotensin I and angiotensin II) or vasodilators (3, 10, and 30 nmol/kg acetylcholine, bradykinin, and SNP; 1, 3, and 10 nmol/kg iloprost) were injected as boluses in a volume of 50 µl followed by a catheter flush with 150 µl of sterile PBS. Control values were obtained by injection of 200 µl of sterile PBS. Changes in MAP began immediately after injection and lasted for 2 to 3 min, the peak being observed in the first minute for all vasoactive compounds tested. The next dose was only injected once the changes in MAP induced by the previous one had subsided fully (usually within 10 min of injection). After obtaining a control dose-response curve to a given agonist and once MAP had fully returned to baseline levels, infusion of SNP (250 nmol/kg/min), S-nitroso-N-acetyl-DL-penicillamine (SNAP; 85 nmol/kg/min), or N-acetyl-DL-penicillamine (NAP; 85 nmol/kg/min) was started and maintained for a 30-min period. MAP fell to about 40 mm Hg for SNP and SNAP infusions and remained around this value throughout the infusion period. A recovery period of 30 min, starting with the end of the infusion, then was allowed. MAP returned to baseline levels within 5 to 15 min, and, by the end of the recovery period, it was around preinfusion levels. At given times (30, 60, and 120 min) after the end of recovery period, successive dose-response curves to the same vasoconstrictor or vasodilator compound were obtained. Only one such compound was studied in any given animal. Control groups were subjected to a similar protocol, in which the animals were infused with PBS alone during 30 min (20 µl/min/30 min). Next, we studied the effect of NO donor infusion on vascular responses to selected compounds 24 h after the end of the infusion. Because maintaining animals anesthetized for long time periods is difficult, this part of the study was conducted by using a somewhat different protocol. Groups of naive animals were anesthetized with tribromoethanol (125 mg/kg, i.p.) to allow insertion and fixation of a butterfly catheter into the caudal vein for the 30-min infusion of SNP, SNAP, or PBS as described above. After the infusion, the caudal catheter was removed and the animal was accommodated in warmed surroundings until recovery of anesthesia (1 h). Twenty-four hours later, the animals were anesthetized with ketamine/xylazine and dose-response curves to phenylephrine or to bradykinin were performed as described above.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Trace recording of a typical experiment showing the decrease in phenylephrine vasoconstrictor effect on MAP after SNP infusion in an anesthetized rat. Increasing bolus doses (3, 10, and 30 nmol/kg) of phenylephrine were injected consecutively before and 30 min after a 30-min infusion with SNP (250 nmol/kg/min). Each dose was administered after the effect of the preceding one had disappeared. Note that MAP fell to about 40 mm Hg and remained around this value throughout the infusion period (30 min), but promptly returned to basal levels after ending infusion.

Treatment with K⁺ Channel Inhibitors. For these experiments we used two protocols. In the first, dose-response curves to phenylephrine or to bradykinin were obtained as described above. Then, nonspecific K⁺ channel blocker tetraethylammonium (TEA; 360 µmol/kg), the ATP-dependent K⁺ channel blocker glibenclamide (40 µmol/kg), or the voltage-dependent K⁺ channel blocker 4-aminopyridine (4-AP; 1 µmol/kg) was slowly (during 3-5 min) injected i.v., and, after stabilization of the MAP (usually 5-10 min), infusion with SNP was initiated as described. Thirty minutes after the end of the infusion, new dose-response curves to phenylephrine and to bradykinin were obtained as described. The effects of K⁺ channel inhibitors alone were evaluated in animals in which the infusion of SNP was substituted by PBS. The second protocol was similar, except that K⁺ channel blockers were injected after the infusion of SNP. In this case, dose-response curves to phenylephrine or to bradykinin were obtained before and 30 min after the end of SNP infusion, K⁺ channel blockers were injected, and new dose-responses curves to phenylephrine or to bradykinin were obtained. Finally, for studying TEA effects on the long-lasting effect of NO donor infusion, it also was injected 24 h after SNP infusion and dose-response curves to phenylephrine were made.

NO_x Assay. Briefly, plasma (deproteinized by zinc sulfate and diluted 1:1 with Milli-Q water) and urine (nondiluted) were subjected to nitrate conversion, as described by Granger et al. (1990). Nitrate was converted to nitrite by using Escherichia coli nitrate reductase for 2 h at 37°C. Samples were centrifuged for bacteria removal, and 100 µl of each sample was mixed with Griess reagent (1% sulfanilamide in 10% phosphoric acid/0.1% naphthyl-ethylenediamine in Milli-Q water) in a 96-well plate and read at 540 nm in a plate reader. Standard curves of nitrite and nitrate (0-150 µM) were run simultaneously. Because under these conditions nitrate conversion was always greater than 90%, no corrections were made. Values are expressed as µM NO_x (nitrate + nitrite).

Drugs. The following drugs and reagents were used in this study: phenylephrine, angiotensin I, angiotensin II, acetylcholine, bradykinin, SNP, NAP, sulfanilamide, -naphtyl-ethylenediamine, sodium nitrate, sodium nitrite, and glibenclamide (all purchased from Sigma Chemical Co., St. Louis, MO); ketamine (obtained from Parke-Davis, Sao Paulo, SP, Brazil); xylazine (Ronpum; kindly donated by Bayer, Sao Paulo, SP, Brazil); iloprost (a kind gift from Dr. E. Antunes, Universidade de Campinas, SP, Brazil); TEA (Sigma) and 4-AP (Research Biochemicals, Natick, MA), which were donated kindly by Dr. M. C. O. Salgado (Faculty of Medicine, Ribeirao Preto, USP, Brazil); and S-nitroso-N-acetyl-DL-penicillamine (synthesized in house by the method of Field et al., 1978). All compounds were diluted in sterile PBS except glibenclamide, which was prepared as a concentrated stock solution in dimethyl sulfoxide.

Statistical Analysis. Results are expressed as the mean ± S.E.M. (n = 4-7 for each group). Statistical significance was analyzed by either Student's t test for paired or unpaired samples or one-way ANOVA followed by Bonferroni's post hoc t test, when applicable. A value of P < .05 was considered statistically significant.

	Results

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Effects on MAP. A typical experiment using phenylephrine before and after SNP infusion is shown in Fig. 1. For the sake of brevity, only the dose-response curve obtained 30 min after the end of NO donor infusion is depicted. All vasoconstrictors increased MAP dose-dependently (Fig. 2, circles). The responses evoked by angiotensin II (data not shown) were identical with those caused by angiotensin I. To allow for a better analysis, we performed time-matched dose-response curves in control animals, which were infused with sterile PBS only. Similarly, the hypotensive effects of bradykinin and acetylcholine also were dose-dependent (Fig. 3, circles).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effects of SNP and SNAP infusions on vasoconstrictive effects of phenylephrine and angiotensin I on MAP of anesthetized rats. Dose-response curves for phenylephrine (left) and angiotensin I (right) were obtained at 30 (A), 60 (B), and 120 (C) min after a 30-min recovery period after infusion with either PBS (20 µl/min; ), SNP (250 nmol/kg/min; ), or SNAP (85 nmol/kg/min; ). D, responses to phenylephrine 24 h after infusion of either PBS (20 µl/min; circles) or SNP (250 nmol/kg/min, triangles) during 30 min. Each point represents mean ± S.E.M. of four to seven animals. Statistical analyses were performed by ANOVA followed by Bonferroni's post hoc t test in A-C or Student's t test for unpaired samples in D. *P < .05, comparing effects between SNP and SNAP groups in relation to PBS group, and #P < .05, comparing effects between PBS- and SNAP-infused animals. Where there is no error bar, it was covered by the symbol. After infusion and recovery, MAP values were 95.7 ± 7.9, 94.9 ± 8.6, and 107.6 ± 4.1 mm Hg for animals that received PBS, SNP, and SNAP, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of SNP and SNAP infusions on vasodilatory effect of bradykinin and acetylcholine on MAP of anesthetized rats. Dose-response curves for bradykinin (left) and acetylcholine (right) were performed 30 (A), 60 (B), and 120 (C) min after a 30-min recovery period after infusion with either PBS (20 µl/min; ), SNP (250 nmol/kg/min; ), or SNAP (85 nmol/kg/min; ). D, responses to bradykinin 24 h after a 30-min infusion of either PBS (20 µl/min; ) or SNP (250 nmol/kg/min, ). Each point represents the mean ± S.E.M. of four to seven animals. Differences between SNP and SNAP groups in relation to PBS group were all significant (*P < .05, ANOVA followed by Bonferroni's t test in A-C or Student's t test for unpaired samples in D). Where there is no error bar, it was covered by the symbol. After infusion and recovery, MAP values were 90.7 ± 7.9, 101.9 ± 3.7, and 105.2 ± 3.9 mm Hg for animals that received PBS, SNP, or SNAP, respectively.

Effects of NO Donor Infusion. Both SNP and SNAP infusions caused reductions in normal MAP (90 ± 1.5 mm Hg; n = 83) of around 40 to 60 mm Hg (for a typical recording, see Fig. 1). On the other hand, HR was not changed by SNP or SNAP infusions. For instance, the average HR of control animals during sterile PBS infusion was 226 ± 8 bpm, whereas values observed in SNP and SNAP groups were 208 ± 15 bpm and 235 ± 10 bpm (n = 10 each), respectively. None of these parameters was changed by infusion of NAP, the non-nitrosylated parent compound of SNAP. Although we did test the influence of SNP at higher concentrations (up to 1000 nmol/kg/min) and for longer periods of infusion (up to 120 min; data not shown), we selected the 250-nmol/kg/min regimen because the hypotension was reproducible and fully reversible, indicating that animals were not developing hemodynamic failure. For SNAP we selected the 85-nmol/kg/min regimen because its effects on MAP were identical with those of SNP.

Effects of K⁺ Channel Blockers. Next, we sought to study the involvement of K⁺ channels in responses induced by NO donor infusion. Some control experiments were made initially. Glibenclamide (40 µmol/kg, at dose able to fully inhibit the vasodilatory effect of cromakalim, an ATP-dependent K⁺ channel opener; data not shown) caused an initial MAP reduction of 23.4 ± 4.0 mm Hg (n = 16) followed by a sustained increase of 45.8 ± 3.4 mm Hg (n = 16), which was accompanied by a heart rate fall of 51 ± 5 bpm. Both TEA (360 µmol/kg; n = 15) and 4-AP (1 µmol/kg; n = 14) increased MAP transiently by 43.4 ± 3.6 and 26.6 ± 6.6 mm Hg, respectively, without altering the heart rate. All of these effects subsided in the next 10 to 20 min. Neither TEA nor 4-AP affected the hypotension caused by SNP infusion, but glibenclamide attenuated it by 30 to 40% (data not shown). At the doses used, none of K⁺ channel blockers induced any changes in the effects of phenylephrine or bradykinin in control animals (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effects of potassium channels blockers injected before infusion with SNP in vascular responsiveness to phenylephrine and to bradykinin in anesthetized rats. After surgery, vessel cannulation, and equilibration period, dose-response curves to phenylephrine (left) or to bradykinin (right) were performed (). Potassium channel blockers then were injected i.v. (40 µmol/kg glibenclamide, A; 1 µmol/kg 4-AP, B; or 360 µmol/kg tetraethylammonium, C). After MAP had returned to normal levels, an infusion of SNP (250 nmol/kg/min) for 30 min was started. After a recovery period, new dose-response curves to phenylephrine and bradykinin were performed (). Each point represents the mean ± S.E.M. of four to eight experiments. Statistical comparison was performed in relation to the corresponding effect obtained before potassium channel blocker injection and SNP infusion (*P < .05, Student's t test for paired samples).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effects of potassium channels blockers injected after infusion with SNP in vascular responsiveness to phenylephrine and to bradykinin in anesthetized rats. After surgery, vessel cannulation, and equilibration period, dose-response curves to phenylephrine (left) or to bradykinin (right) were performed (). After MAP had returned to normal levels, an infusion of SNP (250 nmol/kg/min) for 30 min was started. To confirm that the SNP effects were present, new dose-response curves to phenylephrine and to bradykinin () were made. Finally, potassium channel blockers were injected i.v. (40 µmol/kg glibenclamide, A; 1 µmol/kg 4-AP, B; or 360 µmol/kg tetraethylammonium, C). After MAP normalization, new dose-response curves to phenylephrine and bradykinin were obtained (). In D, the protocol was identical except that SNP was infused 24 h before TEA injection. Each point represents the mean ± S.E.M. of four to eight experiments. Statistical analyses were performed by ANOVA followed by Bonferroni's post hoc t test. *P < .05, comparing effects between PBS- () and SNP-infused () animals, and #P < 0.05, comparing effects between PBS- and SNP-infused K+ channel blocker-treated animals ().

	Discussion

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The main findings of the present report can be summarized as follows: 1) infusion of NO donors induced a state of profound hyporesponsiveness to vasoconstrictors (phenylephrine and angiotensins I and II) in the rat vascular system, which resembles the pattern seen in septic shock; 2) exposure to NO donors also potentiated endothelium-dependent vasodilator responses to bradykinin and to acetylcholine; 3) these NO-induced modifications in vascular responsiveness persisted for at least 24 h after NO donor infusion; 4) blockade of K⁺ channels with TEA or, more specifically, of voltage-dependent K⁺ channels with 4-AP before NO donor infusion practically abolished the changes in responses to phenylephrine and bradykinin; and 5) 4-AP, when administered after NO donor infusion, failed to normalize reduced phenylephrine and increased bradykinin responses, whereas TEA reversed the hyporesponsiveness to phenylephrine (even 24 h after NO donor infusion) but was without effect on bradykinin responses.

The effects of NO donor infusion can be ascribed to NO because NAP, the non-nitrosylated parent compound of SNAP, was completely devoid of any effect. In addition, MAP returned rapidly to basal levels once the infusion was terminated. We did not find increased NO_x nor changes in nitrosothiol levels in plasma during or after NO donor infusion (data not shown). These findings indicate that the effective amount of NO released during infusions was rather small. Katsuki et al. (1977) showed diminished pressor responses to vasoconstrictors during SNP infusion. Perhaps the most important contribution of the current study is that a relatively brief exposure to NO donors (and, consequently, to small amounts of NO) is effective in changing rat vascular responses for at least 24 h after NO infusion, when MAP has had returned to normal levels.

Activation of K⁺ channels causes membrane hyperpolarization, reduction in Ca²⁺ influx, and vascular relaxation (Okabe et al., 1987). Using patch-clamp techniques, it was shown that NO modulates directly the activity of calcium-activated K⁺ channels (Bolotina et al., 1994; Mistry and Garland, 1998). Our results indicate that the mechanism by which NO infusion affects vessel responsiveness involves, at least in part, K⁺ channels.

When TEA and 4-AP were injected before NO donor infusion, they completely abolished NO effects on phenylephrine- and bradykinin-induced responses (Fig. 4). Based on these findings, one could conclude that voltage-dependent K⁺ channels would account for these NO effects. However, closer inspection of Fig. 5 suggests that this may not be the case. When injected after NO donor infusion, 4-AP failed to alter the decreased responses to phenylephrine. This finding suggests that voltage-dependent K⁺ channel appears to be important for the installation of decreased phenylephrine responses, but it is not essential for its maintenance. On the other hand, TEA-sensitive potassium channels are important for the onset and maintenance of the decreased response to phenylephrine. Importantly, even 24 h after ending NO donor infusion, TEA administration fully reverses the diminished phenylephrine pressor responses. This result points out that a TEA-sensitive, but 4-AP-insensitive, subpopulation of K⁺ channels is important for the maintenance of this long-lasting effect of NO donors on phenylephrine responses.

Concerning bradykinin, activation of K⁺ channels seems to be important only in the onset of the hyper-responsiveness. Although NO effects were blocked by TEA and by 4-AP when given before NO infusion, administration of these compounds after NO donor infusion failed to block the hyperresponsiveness to bradykinin, except when very small doses of the peptide were used. This may indicate that, after exposure to NO, responses triggered by low-bradykinin doses are more dependent on K⁺ channel opening, whereas those induced by higher doses should rely on some other mechanism.

Another important aspect concerns the potentiation of endothelium-dependent (bradykinin and acetylcholine), but not endothelium-independent (iloprost and SNP), responses. At present, we do not have a clear explanation for this difference. The only hypothesis that we can offer at present would be that events causing an increase in bradykinin and acetylcholine responses seem to be occurring at the endothelial level. It may be that, for example, ion channels (or other transduction mechanisms) are affected differentially by NO in endothelial and smooth muscle cells. This possibility warrants further investigation.

Data presented here do not offer explanations on why a short exposure to NO may cause long-term changes in vessel sensitivity. However, considering that the presence of cysteinyl sulfhydryl groups on regulatory domains of K⁺ channels is critical for their activity (Rusppersberg et al., 1991; Islam et al., 1993; Wang et al., 1997) and that NO is highly reactive toward sulfhydryl groups, it is conceivable that reaction of NO with -SH groups may affect K⁺ channel activity. Indeed, nitrothiosylation of cytoplasmic domain of potassium channels increases their open probability (Abderrahmane et al., 1998).

Another explanation for the long-term effect of NO infusion in vessel sensitivity is related to the formation of intracellular nitrosothiols (RSNO), formed by the rapid reaction of NO with intracellular thiols (such as glutathione). These compounds can undergo homolytic cleavage of the S---N bond to give NO and thiyl radical (for a review, see Stamler, 1994). Recently, NO release from intracellular S-nitrosothiol pools has been shown to play a direct role in the decrease of rat blood pressure induced by acetylcholine and bradykinin (Davisson et al., 1996). Therefore, if NO donor infusion replenishes S-nitrosothiol pools in endothelial cells and these pools release NO back it would explain, at least in part, 1) why only endothelium-dependent vasodilators had their effects potentiated by NO donor infusion and 2) the long-term hyposensitivity to vasoconstrictors and hypersensitivity to vasodilators. Thus, an increased, continuous NO release from S-nitrosothiol pools would, in turn, increase the open probability of K⁺ channels, leading to hyperpolarization. Alternatively, our data could be explained by an increase in EDHF release caused by NO donor infusion. This suggestion is based on reports showing that NO inhibits endothelial NO synthase (Buga et al., 1993) and also damps EDHF production (Bauersachs et al., 1996). Therefore, the impaired NO synthesis and the consequent increase in EDHF release would explain the observed changes in vascular response induced by NO donor infusion. Although our data do not allow for exclusion of this possibility, it seems unlikely because a long-term inhibition on NO synthesis would have some effect on the blood pressure. These possibilities are now being investigated in our laboratory.

We sought to investigate the involvement of soluble guanylate cyclase on NO-induced changes on vascular responsiveness, but guanylate cyclase inhibitors, methylene blue and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1, failed to change the vascular responses to SNP, SNAP, acetylcholine, bradykinin, and phenylephrine in either PBS- or NO donor-infused animals (data not shown). One possible explanation for these findings would be that NO donor effects are not related to guanylate cyclase activation. For instance, NO donors open calcium-activated K⁺ channels in cell-free membrane patches (Bolotina et al., 1994; Mistry and Garland, 1998). However, several reports show that in other preparations (such as pulmonary artery rings) NO effects on the same type of K⁺ channels seem to be mediated by a cGMP-dependent protein kinase (for example, Archer et al., 1994), indicating that this still is an unresolved issue. Alternatively, guanylate cyclase inhibitors may have failed to inhibit the enzyme activity in vivo because of pharmacokinetic aspects or the inherent complexity of blood pressure as the experimental preparation.

In accordance with the present results, i.v. injection of E. coli also induces hyporesponsiveness to vasoconstrictors and hyperresponsiveness to vasodilators, which persist for at least 24 h after septic shock onset (manuscript in preparation). The involvement of potassium channels in this model currently is being evaluated in our laboratory.

In conclusion, our results indicate, for the first time, that NO donor infusion reproduces the changes in vascular responsiveness to vasoconstrictors and vasodilators seen in septic shock. In addition, our results suggest that the role of bradykinin in the septic shock hypotension may be more important than described previously. Another important piece of information provided by our in vivo study is that the onset of these vascular changes induced by NO appear to need activation of K⁺ channels, mainly of the voltage-dependent type, but not of the ATP-sensitive type. The long-lasting effects of NO on phenylephrine responses, however, are likely to depend on TEA-sensitive K⁺ channel population, whereas NO should rely on some other mechanism when responses to bradykinin are considered. Finally, our data demonstrate that the NO-induced changes in vascular responsiveness are much more profound, long-lasting, and important than anticipated previously. If applicable to septic shock, our data may help to understand the hyporesponsiveness to vasoconstrictors and point out the putative importance of the hyper-responsiveness to vasodilators in this condition. Further studies directed to understanding the relationship between NO, K⁺ channel activity, and altered responses to vasoconstrictors and vasodilators ultimately may lead to a better management of septic shock.