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(Circulation Research. 1996;79:256-262.)
© 1996 American Heart Association, Inc.

Articles

Hemodynamic Effects of L- and D-S-Nitrosocysteine in the Rat

Stereoselective S-Nitrosothiol Recognition Sites

Robin L. Davisson, Mark D. Travis, James N. Bates, Stephen J. Lewis

the Cardiovascular Center and the Departments of Pharmacology (R.L.D., M.D.T., S.J.L.) and Anesthesia (J.N.B.), The University of Iowa, Iowa City.

Correspondence to Stephen J. Lewis, PhD, Department of Pharmacology, 2-272, BSB, University of Iowa, Iowa City, IA 52242.

	Abstract

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The vasorelaxant effects of the endothelium-derived relaxing factor S-nitrosocysteine (SNC) may not be simply due to its decomposition to NO. The biological actions of SNC may also involve the transnitrosation of amino acids in the blood and in plasma membranes. The possibility that the SNC moiety possesses biological activity prompted us to examine whether the hemodynamic effects of this S-nitrosothiol involves the activation of stereoselective S-nitrosothiol receptors within the cardiovascular system. We examined (1) the hemodynamic effects produced by intravenous injections of the L and D isomers of SNC (L- and D-SNC, respectively; 100 to 800 nmol/kg), the L and D isomers of the parent thiols (L- and D-cysteine, respectively; 100 to 800 nmol/kg), the oxidized thiol L-cystine (100 to 800 nmol/kg), and the NO donor sodium nitroprusside (SNP, 1 to 36 µg/kg) in conscious freely moving rats, (2) the baroreceptor reflex–mediated changes in heart rate elicited in response to the falls in arterial pressure produced by L- and D-SNC and SNP in conscious rats, and (3) the relative decomposition of L- and D-SNC to NO upon addition to heparinized rat blood or upon direct application to cultured porcine aortic smooth muscle (PASM) cells. We now report that (1) L-SNC is a more potent hypotensive and vasodilator agent within the mesenteric bed and sympathetically intact and sympathetically denervated hindlimb beds of conscious rats than is D-SNC, (2) L- and D-SNC markedly inhibit baroreceptor reflex–mediated tachycardia in conscious rats and D-SNC is considerably more effective than L-SNC, (3) the intravenous injections of L- and D-cysteine or L-cystine do not affect arterial blood pressure or vascular resistances, and (4) L- and D-SNC decompose equally to NO upon application to rat blood or cultured PASM cells. These results suggest that the hemodynamic effects of endogenous SNC may involve its interaction with stereoselective S-nitrosothiol recognition sites within the vasculature and the baroreflex arc. These findings provide tentative evidence that membrane-bound S-nitrosothiol receptors may exist within the cardiovascular system.

Key Words: L- and D-S-nitrosocysteine • nitric oxide • vascular relaxation • rat

	Introduction

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S-Nitrosocysteine is an endothelium-derived relaxing factor.¹ It is generally assumed that the biological actions of this S-nitrosothiol are due to its decomposition to NO.² However, it is now evident that the vasodilator potencies of S-nitrosothiols are not obviously related to their decomposition to NO; ie, S-nitrosothiols do not necessarily have to enter VSM to produce vascular relaxation, and S-nitrosothiols decompose to NO only upon contact with viable cell membranes.^{3 4} Moreover, NO cannot be detected from lower vasorelaxant concentrations of SNC by either chemiluminescence,¹ agarose-hemoglobin trapping, or electron paramagnetic spectroscopy.⁵ These findings suggest that the S-nitrosothiol moieties possess biological activity that is not necessarily dependent upon their decomposition to NO. The biological activity of SNC may be related to its ability to nitrosate membrane proteins,^{6 7} including those within receptor-operated ion channels.⁸ Moreover, the transnitrosation of circulating amino acids may also be an important determinant of the hemodynamic actions of systemically injected SNC and other S-nitrosothiols.⁹

Endothelium-derived factors such as prostacyclin¹⁰ and endothelin¹¹ exert their biological effects via the stimulation of membrane-bound receptors. The possibility that endothelium-derived SNC¹ may exert its vasorelaxant effects via the activation of stereoselective S-nitrosothiol receptors on the VSM of resistance vessels has not been addressed. One approach to defining the possible existence of such S-nitrosothiol receptors is to examine the relative biological potencies of the L and D isomers of S-nitrosothiols. For most receptors, the stereoisomeric configuration of the agonist is a critical determinant of binding or agonist potency at the receptor.¹² Consequently, differences in the biological potencies of the L and D isomers would suggest the presence of stereoselective S-nitrosothiol recognition sites, especially if it could be established that the stereoisomers decompose equally to NO.

In the present study, we compared the dose-dependent effects produced by the intravenous injection of L-SNC and D-SNC and the NO donor SNP on MAP and vascular resistances in conscious normotensive rats. In particular, we examined the effects of L- and D-SNC and SNP on the vascular resistance of sympathetically intact and sympathetically denervated hindlimbs to more confidently assess the actions of these agents on the VSM of resistance arteries rather than nerve terminals in these beds. In order to determine whether stereoselective S-nitrosothiol recognition sites may exist outside the vasculature, we also examined the baroreceptor reflex–mediated increases in HR elicited in response to the falls in arterial blood pressure produced by L- and D-SNC and SNP in conscious rats. In order to determine whether any differences in the biological potencies of L- and D-SNC were due to their differential decomposition to NO, we also examined the relative breakdown of the stereoisomers of SNC to NO upon their addition to rat blood or cultured PASM cells.

The findings from these experiments provide evidence that the stereoisomeric configuration of SNC is an important factor in determining the biological activity of this S-nitrosothiol. This suggests that SNC may exert its effects by the interaction with stereoselective recognition sites within the cardiovascular system. These recognition sites may represent a family of stereoselective ligand-gated receptors for which S-nitrosothiols such as SNC are the unique ligand. Alternatively, the stereoisomeric configuration of SNC may be an important factor in its ability to nitrosate proteins within known receptors and/or ion channels within the vasculature.

	Materials and Methods

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Surgical Procedures and Experimental Protocols in Conscious Rats
The protocols described below were approved by the University of Iowa Animal Care and Use Committee. All conscious and anesthetized experiments were performed in male Sprague-Dawley rats (Harlan, Inc, Madison, Wis) weighing 300 to 350 g (n=34). Conscious rats were individually housed in Plexiglas cages and maintained on a daily 12-hour light/dark cycle with Purina rat chow and tap water available ad libitum.

The rats were anesthetized with acepromazine maleate (12 mg/kg IP) and ketamine (120 mg/kg IP). Catheters (PE-50) were implanted into the left carotid artery for the measurement of PP, MAP, and HR and into the right jugular vein for the administration of drugs. After catheterization, a midline laparotomy was performed, and the left lumbar sympathetic chain was isolated, cut, and removed caudally to the bifurcation of the left common iliac artery and vein. The right sympathetic chain was left intact. The sympathectomy was performed in order to more directly examine the effects of L- and D-SNC on the VSM of resistance vessels in the hindlimb. The sympathectomy eliminates the confounding influence of the baroreflex-mediated changes in sympathetic nerve input to the hindlimb vasculature that would occur in response to the L- and D-SNC–induced falls in arterial pressure and also eliminates any direct effects the stereoisomers may have on the release of neurotransmitters from the sympathetic terminals. The effectiveness of the denervation was determined by examining the effects on resistance produced by the ₁-adrenoceptor antagonist prazosin (100 µg/kg IV). Doppler flow probes were placed on the superior mesenteric and left and right iliac arteries for the measurement of mesenteric, innervated, and denervated hindlimb blood flows and the determination of MR, HLR_i, and HLR_d.

Other groups of rats were used to examine the hemodynamic effects of L- and D-cysteine and L-cystine. In these rats, the surgical sympathectomy was not performed. Doppler flow probes were placed on the superior mesenteric artery, left renal artery, and the lower abdominal aorta for the measurement of MR, RR, and HQR, respectively. The probes were sutured in place, and the leads and catheters were tunneled subcutaneously and exteriorized between the scapulas. The wounds were then sutured closed. To protect the probe wires and polyethylene tubing while allowing animals unrestricted movement during recovery and experimental testing, the free ends of the catheters and Doppler leads were led through a stainless steel skin button spring swivel assembly that was mounted to a ring-stand clamp and suspended above the cage. The skin button was attached to the skin incision in the scapular region using stainless steel sutures. Details of the Doppler technique, including construction of the probes, the reliability of the method for the estimation of flow velocity, and quantitative determination of percent changes in resistances, have been described previously.^{13 14 15} After a 7-day recovery period, animals were connected to a Beckman Dynograph coupled pressure transducer (Cobe Lab, Inc) and Doppler flowmeter (Bioengineering, University of Iowa) for recording HR, PP, and MAP and for blood flows, respectively. The effects of the intravenous injection of L- and D-SNC (100 to 800 nmol/kg, n=7), L- and D-cysteine (100 to 800 nmol/kg, n=5), cystine (100 to 800 nmol/kg IV, n=5), and SNP (1 to 10 µg/kg, n=9) on hemodynamic parameters were then determined.

The baroreceptor reflex–mediated increases in HR in response to the falls in MAP produced by L-SNC (50 to 800 nmol/kg IV), D-SNC (100 to 1200 nmol/kg IV), and SNP (1, 2.5, 5, 10, 24, and 36 µg/kg IV) were determined in another eight rats.

Cell Culture
PASM cells were grown to confluence in 12-well plates at 37°C in a humidified atmosphere containing 5% CO₂ (Corning Glass Works), as described previously.¹⁶ Cells were subcultured weekly by trypsinization. Cells underwent one change of DMEM (GIBCO) containing 10% FBS (Hyclone), MEM vitamin solution (GIBCO), 2 mmol/L L-glutamine (Sigma Chemical Co), 50 mmol/L gentamicin (Schering), and 15 mmol/L HEPES (Sigma).

Relative Decomposition of L- and D-SNC to NO
Sprague-Dawley rats (250 to 350 g) were anesthetized with urethane (1 g/kg IP), and catheters (PE-50) were inserted into the left lower abdominal aorta via the left femoral artery. One-milliliter volumes of blood were collected and mixed with 100 µL of heparin (100 U/L) to prevent clotting. A 400-µL aliquot of this mixture was added to one well of a 12-well plate. This well was sealed, and 100 µL of saline containing 0.01 to 50 nmol of L- or D-SNC was injected into the chamber. The NO released from L- and D-SNC was carried to the NO detector by a stream of nitrogen gas. The initial concentrations of L- and D-SNC in the wells were 0.02 to 100 µmol/L. These concentrations were chosen on the basis that the intravenous injection of 100 to 800 nmol/kg of L- or D-SNC to a 250- to 350-g rat (blood volume, 20 mL)¹⁷ would result in initial blood concentration of SNC of 5 to 40 µmol/L (see "Results" for further details).

Cultured PASM cells were grown to confluence on 12-well plates as described above. Twenty-four hours before experimentation, the DMEM was removed, and the cells were washed one time with serum-free medium and allowed to equilibrate overnight. PASM cells were then placed on a slide warmer at 37°C. The medium was removed from each well, and the cells in the sealed wells were purged of oxygen by a stream of nitrogen gas. At this time, 100-µL aliquots of L- and D-SNC (0.01 to 10 nmol) were injected into the sealed chamber onto the PASM cells. The initial concentration of L- and D-SNC in these solutions was 0.1 to 100 µmol/L. NO was measured using a NO analyzer (Dasibi, model 2108), as described previously.¹ The analyzer was initially calibrated using known concentrations of NO. The NO released by L- and D-SNC upon addition to blood or PASM cells was carried in a stream of nitrogen gas under vacuum to the chemiluminescence NO analyzer. In the above experiments, the headspace NO was monitored continuously until the NO levels returned to baseline levels. The initial rate of appearance and the total amounts of NO were monitored.

Drugs
L- and D-cysteine were obtained from Sigma. L- and D-cysteine are stereoisomerically pure in that they do not contain detectable amounts of the other stereoisomer (Sigma, personal communication, 1996). Sodium nitrite, acepromazine maleate, and cystine were also obtained from Sigma. SNP was obtained from Abbott Laboratories. Ketamine was obtained from Aveco Co. Stock solutions of the stereoisomers of SNC were prepared fresh just before use by reacting 1-mL solutions of 0.2 mol/L sodium nitrite (containing 100 µL of 1N HCl) and 0.2 mol/L of L- or D-cysteine, which resulted in a stable (pH 3) 0.1 mol/L stock solution of the respective isomers of SNC. The stock and test solutions of L- and D-SNC were routinely examined spectrophotometrically¹ to ensure that the concentrations of the stereoisomers were identical.

Statistical Analysis
The data are represented as mean±SEM. The single SEM values displayed on each of the dose-response curves in the figures were determined by the following formula: SEM=(EMS/n)^1/2, where EMS is the error mean square term from the ANOVA and n is the number of rats per group. The data were analyzed by repeated-measures ANOVA¹⁸ followed by Student's modified t test with the Bonferroni correction for multiple comparisons between means¹⁹ using the EMS terms from the ANOVA.¹⁸ The relationship between the baroreflex-mediated increase in HR in response to the nitrovasodilator-induced falls in MAP was determined by linear orthogonal decomposition of the sums of squares from the repeated-measures ANOVA.¹⁹

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemodynamic Studies in Conscious Rats
The resting hemodynamic variables of the four groups of conscious rats used in these studies are summarized in Tables 1 and 2. Each group received either L- and D-SNC (100 to 800 nmol/kg IV, n=7), SNP (1 to 10 µg/kg IV, n=9), L- and D-cysteine (100 to 800 nmol/kg IV, n=5), or cystine (100 to 800 nmol/kg IV, n=5). The resting resistances within the sympathetically denervated hindlimb beds were higher than those of the sympathetically innervated beds (Table 1). The mechanism responsible for this increase in baseline resistance will be addressed in "Discussion." At the end of the experiments to be described below, we injected the ₁-adrenoceptor antagonist prazosin (100 µg/kg IV) to check the effectiveness of the denervation procedure. For example, the injection of prazosin into the group of rats that had received L- and D-SNC (n=7) significantly lowered HLR_i (-39±5%, P<.05) but did not affect HLR_d (-4±3%, P>.05). This indicates that the surgical transection of the lumbar chain produced an effective sympathetic denervation of the ipsilateral hindlimb.

View this table: [in this window] [in a new window]   Table 1. Resting Hemodynamic Parameters in Two Groups of Conscious Rats

View this table: [in this window] [in a new window]   Table 2. Resting Hemodynamic Parameters in Two Groups of Conscious Rats

The effects of L- and D-SNC (100 to 800 nmol/kg IV) on MAP, MR, HLR_i, and HLR_d in conscious freely moving rats (n=7) are summarized in Fig 1. Although L- and D-SNC produced dose-dependent decreases in these hemodynamic variables, the L-isomer was a more potent hypotensive and vasodilator agent than the D-isomer. The finding that L-SNC was a more potent vasodilator than D-SNC in the sympathetically denervated hindlimb suggests that L-SNC is a more potent relaxant of VSM within the resistance vessels of the hindlimb. Although the arithmetic changes in hindlimb resistance produced by L- and D-SNC were greater in the denervated compared with the innervated hindlimb beds, the percent changes in resistance were similar in both beds. This is explained by the significantly higher resting resistances in the denervated hindlimbs (see Table 1). The injection of L-cysteine, D-cysteine, or L-cystine (100 to 800 nmol/kg IV, n=5) did not affect MAP or vascular resistances in the hindquarter, renal, or mesenteric beds (Fig 2).

View larger version (18K): [in this window] [in a new window]   Figure 1. Summary of the effects of L- and D-SNC (100 to 800 nmol/kg IV) on MAP, MR, HLRi, and HLRd in conscious rats (n=7). Each value represents the mean of the percent change from baseline. The 1 SEM value on each dose-response curve represents the SEM derived from the repeated-measures ANOVA (see "Materials and Methods"). *P<.05 vs D-SNC.

View larger version (18K): [in this window] [in a new window]   Figure 2. Summary of the effects of L- and D-cysteine (L- and D-cys, respectively) and cystine (100 to 800 nmol/kg IV) on MAP, HQR, RR, and MR in conscious rats (n=5). The 1 SEM value on each dose-response curve represents the SEM derived from the repeated-measures ANOVA (see "Materials and Methods"). Note that there were no significant responses at the P<.05 level.

The effects of the NO donor SNP (1 to 10 µg/kg IV) on MAP and vascular resistances of conscious rats (n=9) are summarized in Fig 3. SNP produced dose-dependent reductions in MAP and MR. In addition, SNP produced only a minor vasodilation in the sympathetically intact hindlimb bed but a pronounced vasodilation in the sympathetically denervated bed.

View larger version (11K): [in this window] [in a new window]   Figure 3. Summary of the effects of SNP (1 to 10 µg/kg IV) on MAP, MR, HLRi, and HLRd in conscious rats (n=9). Each value represents the mean of the percent change from baseline. The 1 SEM value on each dose-response curve represents the SEM derived from the repeated-measures ANOVA (see "Materials and Methods"). *P<.05 vs HLRi.

Baroreflex Studies
The arithmetic changes in HR in response to the arithmetic falls in MAP produced by L-SNC (50 to 800 nmol/kg IV), D-SNC (100 to 1200 nmol/kg IV), and the NO donor SNP (1 to 36 µg/kg IV) in a group of conscious rats (n=8) are summarized in Fig 4. Again, L-SNC was a more potent hypotensive agent than D-SNC in these conscious rats. The injection of SNP produced dose-dependent falls in MAP that were associated with baroreceptor reflex–mediated increases in HR. The magnitude of the reflex tachycardia in response to SNP was directly related to the magnitude of the fall in MAP. The injection of D-SNC produced dose-dependent falls in MAP that were associated with markedly smaller increases in HR compared with those produced by equidepressor doses of SNP. The injection of L-SNC also produced dose-dependent decreases in MAP that were associated with nonlinear responses in HR. The magnitude of the changes in HR in response to the falls in MAP produced by the 50, 100, and 200 nmol/kg doses of L-SNC was dependent upon the magnitude of the fall in MAP. However, the tachycardia was less in response to the falls in MAP produced by the 400 and 800 nmol/kg doses of L-SNC. The y intercepts (HR, in bpm) and the slope of the HR-MAP relationships (HR/MAP) are summarized in Table 3. The values for L-SNC are derived from the 50, 100, and 200 nmol/kg doses. The y intercepts of the HR-MAP relationship were identical for all three compounds. The slope of the HR-MAP relationship was greatest for SNP. The slopes for both D-SNC and L-SNC were significantly less than that for SNP. In addition, the slope for D-SNC was significantly less than that for L-SNC. For example, the 36 µg/kg dose of SNP, the 200 nmol/kg dose of L-SNC, and the 1200 nmol/kg dose of D-SNC produced falls in MAP of -21±3, -28±4, and -27±3 mm Hg, respectively. The tachycardia in response to these doses of SNP and L- and D-SNC were 98±8, 55±7, and 23±5 bpm, respectively (P<.05, SNP versus L- and D-SNC, L- versus D-SNC). Therefore, it appears that both stereoisomers of SNC inhibit baroreceptor reflex–mediated tachycardia and that D-SNC is more potent than L-SNC in this regard.

View larger version (13K): [in this window] [in a new window]   Figure 4. Summary of the baroreceptor reflex–mediated increases in HR in conscious rats in response to the falls in MAP produced by SNP (1, 2, 4, 8, 16, and 36 µg/kg IV), L-SNC (50 to 800 nmol/kg IV), or D-SNC (100 to 1200 nmol/kg IV) in a group of eight conscious rats. Each value represents the mean of the arithmetic changes in HR and MAP. The 2 SEM values on each dose-response curve represent the SEM values for HR and MAP derived from the repeated-measures ANOVA (see "Materials and Methods").

View this table: [in this window] [in a new window]   Table 3. Baroreceptor-Reflex Parameters in Conscious Rats

NO Measurements
The total amounts of NO (pmol) obtained by the addition of L- and D-SNC to rat blood are shown in Fig 5 (top panel). The volume of blood in 250- to 350-g rats is 20 mL.¹⁷ Assuming a rapid distribution of SNC into this volume, the 100 to 800 nmol/kg IV doses of L- and D-SNC would result in peak blood concentrations of 5 to 40 µmol/L. The addition of 100 µL solutions containing 0.01 to 50 nmol of L- or D-SNC to 400 µL of heparinized blood samples resulted in initial concentrations of 0.02 to 100 µmol/L of the S-nitrosothiol stereoisomers. The addition of L- and D-SNC to blood produced a concentration-dependent increase in the detectable amounts of extracellular NO. Concentrations of L- and D-SNC below 0.1 µmol/L did not generate detectable amounts of NO. Over this range of concentrations, there were no differences in the rate of appearance of NO or the total amounts of NO generated by L- and D-SNC. The NO was detectable within 1 to 2 seconds of application of L- or D-SNC to the blood. The majority of the headspace NO was detected over the first 1 to 3 minutes. It took 10 minutes for all the NO to be released from the higher concentrations of L- and D-SNC. Virtually identical results were obtained by the addition of L- and D-SNC to arterial blood collected from conscious rats (7 to 10 days after anesthesia). For example, the 1 µmol/L concentrations of L- and D-SNC yielded 32±4 and 35±3 pmol of NO, respectively (n=3), whereas the 10 µmol/L concentrations of L- and D-SNC yielded 177±16 and 169±15 pmol of NO, respectively (n=4). The ability to detect NO in our system is limited to some extent, since hemoglobin will trap the free radical, and as such, we cannot determine the true total amount of NO generated by L- and D-SNC. However, on the basis of our results, it would appear that L- and D-SNC decompose equally to NO in blood.

View larger version (18K): [in this window] [in a new window]   Figure 5. Top, Summary of the total amount of NO (pmol) generated by the addition of 10 to 50 000 pmol of L- or D-SNC to 500 µL rat whole blood (initial concentration, 0.02 to 100 µmol/L). Each value represents the mean±SEM of at least three determinations. Bottom, Summary of the amount of NO (pmol) generated by the addition of 100 µL solutions of 0.01 to 10 nmol of L- or D-SNC (initial concentration, 0.1 to 100 µmol/L) to cultured PASM cells. Each value represents the mean±SEM of at least four determinations.

We next examined the decomposition of L- and D-SNC to NO upon application to VSM cells. The total amounts of NO (pmol) obtained by the addition of 100 µL solutions of saline containing 0.01 to 10 nmol of L- or D-SNC (0.1 to 100 µmol/L) to cultured PASM cells are shown in Fig 5 (bottom panel). The addition of L- and D-SNC produced concentration-dependent increases in NO. The rate of appearance of NO (within 1 to 2 seconds) and the total amounts of NO generated from each concentration of the stereoisomers were equivalent. The large majority of the NO was detected over the first 1 to 3 minutes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that (1) L-SNC was a more potent hypotensive and vasodilator agent than D-SNC in conscious rats, (2) both stereoisomers inhibited baroreceptor reflex–mediated tachycardia, but D-SNC was more potent than L-SNC in this respect, (3) the stereoisomers of the parent thiols, L- and D-cysteine, did not affect hemodynamic parameters, and (4) the net decompositions of L- and D-SNC to NO were identical upon their addition to rat whole blood or PASM cells. Since L- and D-SNC decomposed equally to NO, it appears that the differences in the biological potencies of the stereoisomers may be related to the relative activity of the parent S-nitrosothiol. The lack of effects of bolus doses of L- and D-cysteine on MAP and vascular resistances suggests that under these conditions, the stereoisomers are not nitrosated efficiently enough by circulating S-nitrosothiols^{6 9} to produce biologically active compounds. The observation that the NO donor SNP was a weakly effective vasodilator in the sympathetically intact hindlimb suggests that NO is not the principal bioactive nitrosyl factor in this bed. This finding also suggests that the vasodilator effects of SNC in the hindlimb vasculature are not due to its decomposition to NO. These results support previous findings that the S-nitrosothiol moiety in itself is biologically active.^{3 4 5} Moreover, the present findings raise the possibility that the actions of SNC may be mediated by the interaction with stereoselective recognition sites within the cardiovascular system. In order to more convincingly determine that L- and D-SNC directly relax VSM, we examined the vasodilator actions of the stereoisomers in sympathetically denervated as well as sympathetically intact hindlimb beds. Our finding that L- and D-SNC produce a pronounced vasodilation in sympathetically denervated hindlimb beds suggests that the stereoisomers directly relaxed VSM within resistance vessels. The greater potency of L-SNC compared with D-SNC in the denervated hindlimb suggests that the stereoselective recognition sites for SNC may exist on the VSM of these vessels.

The resting resistance of the chronically sympathectomized hindlimb beds was considerably higher than that of the innervated beds. We have found that sham operation (in which all of the surgical procedures except the transection of the lumbar sympathetic chain are performed) does not cause an increase in ipsilateral hindlimb vascular resistance compared with resistance in the nonoperated limb, 7 days after surgery in conscious rats (R.L.D. and S.J.L., unpublished data, 1996). This suggests that the increase in hindquarter resistance produced by the transection of the lumbar sympathetic chain is not simply due to the trauma during surgery, the subsequent loss of distensibility, or scar formation in the area. The present study also demonstrated that whereas the injection of the selective ₁-adrenoceptor antagonist prazosin produced a pronounced vasodilation in the sympathetically intact hindlimb, it did not alter the resistance in the sympathetically denervated hindlimb. This latter finding clearly suggests that the increase in resting resistance in the denervated hindlimb is not due to the denervation-induced supersensitivity of ₁-adrenoceptors to circulating catecholamines. A change in the receptor density of ₂- or ß-adrenoceptors is unlikely to be responsible for this increase in resistance, since epinephrine, the endogenous agonist at these receptors, produces a vasodilation in the hindlimb.¹⁴ Our results are consistent with findings that neonatal treatment with the sympathetic neurotoxin resulted in an increase in hindlimb vascular resistance in adult rats.²⁰ The possible mechanisms for this increase in resistance are (1) the loss of postganglionic NO synthase–positive lumbar sympathetic vasodilator nerves¹⁴ and (2) the downregulation of endothelial NO synthase activity due to the loss of the trophic influence of the sympathetic nerves.¹⁵ Therefore, it would appear that the loss of the endothelial and neurogenic vasodilator systems outweighs the loss of the neurogenic vasoconstrictor input in the hindlimb bed.

The increases in HR in response to the L- and D-SNC–induced falls in MAP were markedly smaller than those observed in response to the hypotensive effects of the NO donor SNP. Moreover, a smaller tachycardia was observed with D-SNC than with L-SNC. These findings suggest that the SNC moiety inhibits baroreflex-mediated tachycardia and that D-SNC is more potent than L-SNC in this respect. The relative potencies of L- and D-SNC on baroreflex-mediated tachycardia are opposite their relative potencies on vascular resistance. To our knowledge, this is the first instance in which the relative potencies of the stereoisomers of a compound are opposite in different biological systems. The usual observation is that the L-isomer is more potent in each system under investigation.¹² Therefore, it would seem possible that different subtypes of stereoselective S-nitrosothiol recognition sites are expressed within the cardiovascular system. One subtype may be preferentially expressed in the vasculature and the other subtype preferentially expressed in the baroreflex arc.

These studies cannot define the mechanisms by which L- and D-SNC inhibit baroreflex-mediated increases in HR. The stereoisomers may inhibit the baroreflex-mediated tachycardia via inhibition of norepinephrine release from cardiac sympathetic terminals or by direct effects on the sinoatrial or atrioventricular nodes. L- and D-SNC equally inhibit the increase in carotid sinus baroreceptor afferent nerve activity in response to graded increases in carotid sinus pressure in anesthetized rabbits.²¹ The equal efficacy of L- and D-SNC may be related to the relatively prolonged exposure times of L- and D-SNC (10 minutes) to the carotid afferents, which allowed for the substantial decomposition of the stereoisomers to NO. Nonetheless, it is unlikely that the inhibition of baroreceptor afferent function contributes to baroreflex-mediated tachycardia in response to hypotensive episodes that unload the baroafferent terminals. The increases in HR in response to the falls in MAP produced by L-SNC were linear for doses up to 200 nmol/kg. The hypotensive effects of the 400 and 800 nmol/kg doses were associated with progressively smaller increases in HR. We have reported that L-SNC but not SNP activates vagal afferents in anesthetized rats.²² As such, the relatively smaller increases in HR produced by the 400 and 800 nmol/kg doses of L-SNC may involve the activation of cardiopulmonary afferents, which counterbalances the baroreflex-mediated tachycardia.

The stereoselective actions of SNC raise several interesting possibilities with regard to the signal transduction processes involved in mediating the hemodynamic effects of S-nitrosothiols. SNC is poorly lipophilic, ie, highly polar,³ and is therefore unlikely to enter cells readily enough or rapidly enough to be an important factor in exerting its hemodynamic effects. One possible explanation for our findings is that SNC stimulates membrane-spanning S-nitrosothiol receptors within the cardiovascular system. It is also possible that the stereoisomeric configuration of S-nitrosothiols plays a role in their S-nitrosation of functionally important thiols within membrane-spanning receptors such as the ₁-adrenoceptor²³ or ion channels such as the Ca²⁺-dependent K⁺ channel.²⁴ Another mechanism by which L- and D-SNC may exert their biological effects is via the transnitrosation of circulating amino acids.⁹ In particular, the ability of D-SNC to transnitrosate circulating L-amino acids may result in compounds that have more biological activity than D-SNC itself.

In summary, the present study provides evidence that ligand-gated S-nitrosothiol receptors may exist within the vasculature and baroreflex arc and that there may be more than one receptor subtype or that the receptor can exist in different conformational states. This possibility is further strengthened by our recent preliminary findings that the L-isomer of S-nitroso-ß,ß-dimethylcysteine is a much more potent vasodilator in conscious rats than the D-isomer.²⁵

	Selected Abbreviations and Acronyms

 

HLRi, HLRd = hindlimb vascular resistance, intact and denervated HQR = hindquarter vascular resistance HR = heart rate L-SNC, D-SNC = L and D isomers of SNC MAP = mean arterial blood pressure MR = mesenteric vascular resistance PASM = porcine aortic smooth muscle PP = pulsatile arterial blood pressure RR = renal vascular resistance SNC = S-nitrosocysteine SNP = sodium nitroprusside VSM = vascular smooth muscle

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-14388. We wish to acknowledge the expert assistance of Mike Burcham in preparation of the figures.

Received February 1, 1996; accepted April 24, 1996.

	References

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