Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation J. L. DINERMAN, D. L. LAWSON, AND J. L. MEHTA Department of Medicine, University of Florida College of Medicine, and Veterans Affairs Medical Center, Gainesville, Florida 32610
DINERMAN, J. L., D. L. LAWSON, AND J. L. MEHTA. Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H698-H701, 1991.-To evaluate the role of endothelium in nitroglycerin (NTG) -mediated vascular relaxation, epinephrine-contracted rat thoracic aortic segments with and without intact endothelium were exposed to NTG (lOvl' to 10m5 M). Aortic segments with intact (endo+, n = 15) and denuded endothelium (endo-, n = 9) exhibited typical NTGinduced relaxation. However, the mean effective concentration of NTG was lower for endo- than for endo+ segments (P < 0.001). To determine if this phenomenon related to nitric oxide (NO) generation by endothelium, six endo+ segments were treated with N”-monomethyl-L-arginine (L-NMMA), an inhibitor of NO production. These endo+ segments exhibited greater (P < 0.001) relaxation in response to NTG than the untreated endo+ segments. Oxyhemoglobin, an inhibitor of guanylate cyclase activation, greatly diminished NTG-mediated relaxation of all aortic segments. To determine if the enhanced NTGmediated relaxation of endosegments was unique to the guanosine 3’,5’-cyclic monophosphate-dependent vasodilator NTG, other endo+ and endo- segments were exposed to adenosine 3’,5’-cyclic monophosphate-dependent vasodilator papaverine (10S8 to low4 M), and no difference in EC0 was noted between endo+ and endo- segments. Thus endothelium attenuates NTG-mediated vasorelaxation, and this attenuation is abolished by inhibition of endothelial NO production with LNMMA. These observations indicate that endothelium is a dynamic modulator of vascular smooth muscle relaxant effects of NTG. This modulation appears to result from a competitive interaction between endothelial NO and NTG.
endothelium-derived
relaxing
factor; nitric
oxide
SERVES as an important modulator of vascular smooth muscle tone through the formation of several vasoactive species (28). Endothelium is a source of potent vasodilatory compounds such as prostacyclin and endothelium-derived relaxing factor (EDRF), which has been identified as nitric oxide (NO; 12). Endothelium also generates vasoconstrictor species, collectively known as endothelium-derived constricting factors (EDCFs), for which the biological activity may be accounted for, at least in part, by endothelium (16) and superoxide anions (7, 15). The relative contributions of these opposing factors may be of importance in the determination of vascular tone. Nitroglycerin (NTG) is generally thought to be an endothelium-independent smooth muscle relaxant. It is known that through formation of intracellular nitrosoENDOTHELIUM
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$1.50 Copyright
thiols, subsequent activation of guanylate cyclase, and accumulation of guanosine 3’,5’-cyclic monophosphate (cGMP), NTG shares with EDRF a final common pathway leading to vascular smooth muscle relaxation (5). Endothelial formation of EDCFs tends to oppose the actions of these factors and may thereby diminish the effect of vasodilatory influences. Production of the peptide known to account for the activity of endothelin-1 from its messenger RNA is inhibited as intracellular cGMP rises (1). We postulated that the presence of intact endothelium would alter NTG-mediated vasorelaxation, because both EDRF and NTG share a common pathway in the process of accumulation of cGMP in smooth muscle. These two relatively similar agents could either have cumulative or synergistic effects or compete with each other for activation of processes leading to smooth muscle relaxation. This was examined in rat thoracic aortic segments with and without intact endothelium. MATERIALS
AND
METHODS
Preparation of rat aortic segments. Thoracic aortas of Male Sprague-Dawley rats (Charles River, Portage, MI) weighing 150-200 g were harvested after exsanguination under light halothane anesthesia, carefully cleaned of fat and connective tissue, and cut into 3- or 4mm segments. The endothelium of a subset of vessels was removed by gentle rubbing. The segments were washed, mounted onto wire stirrups, and suspended in organ baths at 37°C containing 5 ml of aerated Krebs-Ringer buffer of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaC12, 1.2 KH2P04, 1.2 MgC1,, 12.5 NaHC03, 0.01 NaEDTA, and 11.1 dextrose. The segments were connected to myograph transducers (Grass Instruments, Quincy, MA) to record changes in isometric force (4, lo), stretched to a preload force of 5 g, and allowed to equilibrate for -2 h. During the equilibration period, the Krebs-Ringer buffer in the organ baths was replaced every 15 min. Endothelial integrity was determined in each experiment by characteristic relaxation after treatment with acetylcholine (ACh). These vascular segments will be referred to as endo+. In deendothelialized segments (subsequently referred to as endo-), ACh did not cause relaxation. Fresh buffer was then added to the baths, and the responses to various agonists and antagonists were recorded. Study protocols. Parallel series of aortic rings with and without endothelium were stimulated with epinephrine
0 1991 the American
Physiological
Society
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ENDOTHELIUM
AND NITROGLYCERIN
to achieve -3-5 g (80% of maximum) of stable tension above baseline. In some experiments norepinephrine was used as the agonist. Once stable contraction had been obtained, increasing concentrations of NTG (lOsl' to 10e5 M) were added to the organ bath, and changes in force were recorded. In several experiments, vessels were treated with oxyhemoglobin (10 PM) or NG-monomethyl+arginine (L-NMMA, 50 PM) before precontraction with epinephrine. In later experiments a variety of precontracted aortic segments were exposed to a non-nitroso vasodilator papaverine (lOwl' to lo-* M). Reagents. NTG was obtained from Parke-Davis, Morristown, NJ. L-NMMA was a gift from Dr. S. Moncada, and indomethacin was a gift from Merck Sharpe & Dohme, West Point, PA. Oxyhemoglobin was prepared by adding 10 M excess of Na2S204 to a 1 mM solution of type 1 bovine hemoglobin (Sigma). Na2S204was removed by dialysis against 100 vol of distilled Hz0 for 2 h at 4°C. All other reagents used were obtained from Sigma Chemical, St. Louis, MO. All reagents and buffers were prepared fresh daily and were kept on ice during the experiments. Statistical analysis. Force generated by aortic segments was divided by the wet weight of the segments to obtain force in grams per milligram of tissue. All data are expressed as geometric means t SE. Magnitude of relaxation was expressed as percent decrease from stable contraction. Linear regression of the straight portion of dose-response curves from each experiment was used to obtain mean effective concentration (EC&), which was expressed in moles per liter. Statistical analysis of data was performed using Student’s t test for paired and unpaired data and a Bonferroni correction was applied for multiple comparisons, where appropriate. A P value co.05 was considered significant. RESULTS
Effect of endothelium on vascular contraction. Addition of epinephrine to organ baths was associated with a concentration-dependent increase in contraction of both endo+ and endo- aortic segments. The threshold concentration of epinephrine required to initiate contraction was less (P < 0.03) for endo- than for endo+ segments (Fig. 1, Table 1). Similar data were obtained with norepinephrine as the agonist. Increased sensitivity of vascular smooth muscle in endo- segments is due to loss of EDRF in keeping with previous reports (3). Effect of L-NMMA and oxyhemoglobin on epinephrinemediated contraction. L-NMMA as well as oxyhemoglobin caused small contractions in endo+ segments (Fig. lC), probably because of inhibition of endogenous EDRF and its binding, respectively. The concentration of epinephrine required to initiate further contraction was less (P C 0.05) for vessels pretreated with L-NMMA or oxyhemoglobin than for untreated endo+ vessels (Table 1). Similar results were obtained when norepinephrine was used as the agonist (data not shown). Presence of endothelium and NTG-mediated vasorelaxation. Addition of NTG to organ baths resulted in a concentration-dependent decrease in the force generated by aortic segments. The magnitude of relaxation was greater for endo- aortic segments than for endo+ aortic
H699
2 mkn
endothelium
-./
-6
4 -5 (-log M)
4
EPI -8
0
L -/
4 EPI
4
-9
-7 (-log M)
L-NMMA
4 L-iJMMA
+ endothelium
4 -5
EbI
intact
in tact
-7 (-log M)
-9
FIG. 1. A: representative experiment showing nitroglycerin (NTG)mediated relaxation of an epinephrine (Epi)-precontracted rat thoracic aortic segment with intact endothelium. B: vascular relaxation is much more pronounced at all concentrations of NTG in another segment from the same aorta that had been deendothelialized. C: enhanced NTG-mediated relaxation of another segment with intact endothelium that had been pretreated with NC’-monomethyl-L-arginine (L-NMMA) before contraction with epinephrine.
TABLE 1. Effects of epinephrine and NTG on rat aortic segments Segments Endo+ EndoL-NMMA + endo+ HbOZ + endo+
n
15 9
Epinephrine Threshold
2.40t1.11 4.64kl.32
x lo-’ x lo-lo*
6 1.47k1.57 x lo-' 3 1.00tl.70 x 1o-g
EC&
NTG
2.50t0.77 x 1O-7 2.50t0.72 2.38t0.69 4.60t0.94
x lo-‘“f x lo-‘-/x 10-6j-
Data from multiple experiments in geometric means t SE; n = no. refer to segments with intact and of segments. Endo+ and endodenuded endothelium, respectively. L-NMMA, p-monomethyl-L-arginine; Hb02, oxyhemoglobin; NTG, nitroglycerin. * P < 0.05 vs. epinephrine threshold concentration in endo+ segments; j- P < 0.001 vs. ECso of NTG in endo + segments.
segments. This was observed at each concentration in all experiments. A representative experiment is shown in Fig. 1, and data from multiple experiments are summarized in Fig. 2. The mean EC50 of NTG was 10 times lower for endo- than for endo+ segments (P < 0.001). Effect of L-NMMA and oxyhemoglobin on NTG-mediated vasorelaxation. To determine if the enhanced relaxation noted for endo- vessels was related to mechanical properties of, or generation of nitric oxide by endothelium, endo+ aortic segments were pretreated with LNMMA and this resulted in significantly greater relaxation of vascular smooth muscle when these segments were subsequently exposed to NTG (Table 1). The EC50 of NTG was lower (P < 0.001) than that for the untreated endo+ vascular segments but was similar to that for endo- segments. Pretreatment of endo+ segments with oxyhemoglobin, on the other hand, resulted in significant attenuation of NTG-mediated vasorelaxation (P c
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H700
ENDOTHELIUM
AND
NITROGLYCERIN
Previous studies have shown that endothelium modulates the smooth muscle relaxant effects of ADP, histamine, and LTD,; agents that exert their effects in part via release of EDRF (3). The augmented NTG-mediated relaxation of vascular segments denuded of endothelium compared with segments with intact endothelium in the present study reflects the importance of endothelium in modulating the vascular response to NTG. It is notewor60thy that Nakagawa et al., like us, observed enhanced NTG-mediated vasorelaxation in deendothelialized ca80nine coronary arteries; they also observed greater accu90mulation of cGMP vessels in these segments than in 00 endo+ segments in response to NTG (11). I I 0 0 T v7 The major mechanisms by which endothelium may b b -0 5 5 -X attenuate the response to vascular smooth muscle to m NTG are through its function as a “mechanical barrier” Nitroglycerin (M) and by production of EDCFs. It is possible that disrupFIG. 2. Augmented nitroglycerin-mediated relaxation of rat thoracic tion of the endothelial integrity contributes to the augaortic segments with denuded endothelium (0, n = 9) compared with mented vasoreactivity by allowing enhanced availability those with intact endothelium (m, n = 15). of NTG in the proximate milieu of the vascular smooth 0.001) compared with the untreated endo+ segments. muscle. Once access to vascular smooth muscle is obThe presence of oxyhemoglobin also attenuated the tained, NTG stimulates guanylate cyclase and causes marked NTG-mediated relaxation of L-NMMA-preintense relaxation. This hypothesis is supported by pretreated segments (mean E& 1.52 x 10m6 M). vious observations of enhanced smooth muscle relaxaEffect of indomethacin and superoxide dismutase (SOD) tion in response to neutrophil-derived NO in deendoon NTG-mediated vasorelaxation. To determine if the thelialized vascular segments (10). However, endotheenhanced NTG-mediated relaxation of endo+ vessels lium serving as a simple mechanical barrier is an unlikely pretreated with L-NMMA related to generation of pros- scenario in vascular ring preparations suspended in an taglandins or superoxide anions, segments were pre- organ bath. treated with the cyclooxygenase inhibitor indomethacin To further examine the concept of endothelium serving or the superoxide anion scavenger SOD. Neither of these as a potential chemical modulator, we pretreated endo+ agents modified the relaxation of L-NMMA-pretreated segments with L-NMMA, which resulted in a modest endo+ aortic segments (mean E& of NTG 3.48 x 10B8 contraction and augmented sensitivity to epinephrine M for indomethacin-exposed segments and 5.39 x 10v8 (Fig. 1C). L-NMMA is a stereospecific, competitive anM for SOD-exposed segments). tagonist of L-arginine-mediated NO production (13). It Effect of intact endothelium and L-NMMA on papavis possible that in L-NMMA-treated endo+ segments, erine-mediated vasorelaxation. To investigate whether baseline smooth muscle tone was influenced by inhibithe enhanced relaxation of endo- or L-NMMA-treated tion of EDRF and by the release of one or more EDCFs. endo+ aortic segments was a unique response to NTG, Indeed, Boulanger and Luscher (1) have recently dempretreated endo+ aortic onstrated L-NMMA increases the formation of endotheendo+, endo- and L-NMMA segments were exposed to papaverine, a non-nitroso lin-1 in porcine aortic segments with intact endothelium. vasodilator. Although characteristic relaxation was noted We expected attenuation of NTG-mediated relaxation for all aortic segments, the E&s of papaverine for endo+ in the presence of the constrictor influence of endothelinsegments. However, we observed (4.11 t 2.73 X 10m5,n = 3), endo- (4.96 t 1.77 X 10B5,n 1 in L-NMMA-treated = 4), and L-NMMA-pretreated endo+ aortic segments markedly enhanced NTG-induced relaxation in these segments, similar to that in endo- segments. L-NMMA (4.65 t 0.50 X 10v5, n = 4) were similar. is unlikely to physically disrupt the endothelial integrity; as such, the concept of endothelium serving as a meDISCUSSION chanical barrier is not tenable in these experiments. Endothelium serves an important role in vascular ho- Thus it appears that the basal release of EDRF from meostasis by modulating vasomotor tone through the intact endothelium affects the vasorelaxation caused by production of constrictor and dilator substances. It has NTG. Accordingly, it is likely that the enhanced NTGbeen proposed that in states of health, endotheliummediated relaxation occurs in the absence of endothelium derived substances that exhibit smooth muscle relaxant because competition between EDRF and NTG for actiproperties predominate (8). In states of hypoxia or endo- vation of guanylate cyclase is absent. Supporting this thelial dysfunction, vasoconstrictor influences may be concept is the close similarity of the mechanism of action left relatively unopposed. Vascular segments with dis- of EDRF and NTG, both of which cause vasorelaxation rupted or dysfunctional endothelium may also exhibit by causing an increase in vascular cGMP accumulation increased contraction in response to smooth muscle ag- (5). In this regard, our experiments with papaverine, a onists (3, 7). Our present observation that a lower con- vasodilator not related to EDRF, are significant. Unlike centration of epinephrine or norepinephrine is required NTG, papaverine, which increases vascular adenosine to initiate contraction of deendothelialized vascular seg- 3’,5’-cyclic monophosphate, caused a similar degree of relaxation in endo+ and endo- segments. Treatment of ments is in keeping with this concept. Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 2, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
ENDOTHELIUM
AND
endo+ segments with L-NMMA also did not influence papaverine-mediated relaxation. Based on these observations, papaverine may be considered a true “EDRFindependent” vasodilator. Our studies showing the reduction of NTG-mediated vasorelaxation in the presence of oxyhemoglobin are in keeping with previous reports (9). Indomethacin did not modify NT@-mediated vasorelaxation, indicating that prostaglandins are not involved in the actions of NTG on smooth muscle tone (14). Although SOD prevents degradation of EDRF by scavenging superoxide anions (7), the presence of SOD did not potentiate the vasorelaxant potential of NTG. In contrast, inhibition of EDRF synthesis by L-NMMA pretreatment resulted in marked potentiation of NTG-mediated vasorelaxation. It is noteworthy that like L-NMMA, oxyhemoglobin has been shown to stimulate production of endothelin-1 (1). This stimulation of endothelin-1 is also associated with diminished cellular cGMP accumulation as a result of inhibition of EDRF activity. In keeping with this concept, oxyhemoglobin pretreatment contracted the vascular segments and augmented their sensitivity to agonists in our experiments (Table 1). However, NTGmediated vasorelaxation was attenuated in the presence of oxyhemoglobin in all endo+ and endo- segments as well as in others pretreated with L-NMMA. The most likely mechanism for this is binding of NTG as well as NO to oxyhemoglobin, thereby inhibiting smooth muscle guanylate cyclase stimulation (9). Two recent studies on endothelial modulation of the EDRF donor SIN-l support our observations that endogenous EDRF inhibits the effects of cGMP-dependent vasodilators. Busse et al. (2) demonstrated that the ability of SIN-l to elicit vasodilation in the rabbit femoral artery was markedly enhanced in vessels denuded of endothelium, and they concluded that this effect was mediated by EDRF or another endothelium-derived substance that alters the action or metabolism of cGMP (3). Similar findings were noted in canine coronary arteries by Flavahan and Vanhoutte (4); these investigators, however, found that the pretreatment of endo+ segments with SOD and catalase enhanced SIN-l-mediated vasodilation (4). Although we did not note similar augmentation of NTG-mediated vasorelaxation by SOD, these differences may relate to differences in superoxide radical production between canine and rat vascular endothelium. Nevertheless, both these studies support our observations and suggest inhibitory interactions between cGMPdependent vasodilators and EDRF. In summary we have demonstrated enhanced NTGmediated vasorelaxation in rat thoracic aortic segments denuded of endothelium compared with those with intact endothelium. These results indicate the important role of vascular endothelium as both a dynamic barrier and a modulator of smooth muscle response to vasodilator stimuli, particularly those that share the same mechanism of vasorelaxation as EDRF. These findings may have clinical relevance to the pharmacotherapy of diseases in which dysfunction of vascular endothelium exists. Based on the observations described herein, NTG
H701
NITROGLYCERIN
may be considered lator.
an endothelium-interactive
vasodi-
This study was supported by the Medical Research Service of the Department of Veterans Affairs and by a grant from the American Heart Association, Florida Affiliate. J. L. Mehta is a Clinical Investigator of the Department of Veterans Affairs, Central Office. Address for reprint requests: J. L. Mehta, Dept. of Medicine, Univ. of Florida, Box J-277, JHMHC, Gainesville, FL 32610. Received
6 August
1990; accepted
in final
form
6 November
1990.
REFERENCES 1. BOULANGER, C., AND T. F. LUSCHER. Release of endothelin from porcine aorta: inhibition by endothelium-derived nitric oxide. J. CZin. Invest. 85: 587-590, 1990. 2. BUSSE, R., U. POHL, A. MOLSCH, AND E. BASSENGE. Modulation of the vasodilator action of SIN-l by the endothelium. J. Cardiovast. Pharmacol. 14, Suppl. II: 581-585, 1989. 3. DINERMAN, J. L., AND J. L. MEHTA. Endothelial, platelet and leukocyte interactions in ischemic heart disease: insights into potential mechanisms and their clinical relevance. J. Am. CoLl. Cardiol. 116: 207-222, 1990. 4. FLAVAHAN, N. A., AND P. M. VANHOUTTE. Mechanisms underlying the inhibitory interaction between the nitrovasodilator SIN- 1 and the endothelium. J. Cardiovasc. Pharmacol. 14, Suppl. II: 586-590, 1989. 5. IGNARRO, L. J. Endothelium-derived nitric oxide: pharmacology and relationship to the actions of organic nitrate esters. Pharm. Res. 6: 651-659, 1989. 6. LAWSON, D. L., C. SMITH, J. L. MEHTA, P. MEHTA, AND W. W. NICHOLS. Leukotriene D, potentiates the contractile effects of epinephrine and norepinephrine on rat aortic rings. J. PharmacoZ. Exp. Ther. 116: 1201-1206, 1988. 7. LAWSON, D. L., W. W. NICHOLS, P. MEHTA, AND W. H. DONNELLY. Superoxide radical-mediated endothelial injury and vasoconstriction of rat thoracic aortic rings. J. Lab. CZin. Med. 115: 541-548,199O. 8. LUSCHER, T. F. Endothelium-derived relaxing and contracting factors: potential role in coronary artery disease. Eur. Heart J. 10: 847-857,1989. 9. MARTIN, W., G. M. VILLANI, D. JOTHIANANDAN, AND R. F. FURCHGOTT. Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J. Pharmacol. Exp. Ther. 232: 708-716, 1985. 10. MEHTA, J. L., D. L. LAWSON, W. W. NICHOLS, AND P. MEHTA. Modulation of vascular tone by neutrophils. Dependence on endothelial integrity. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1315-H1320,1989. 11. NAKAGAWA, H., K. OKUMURA, H. HASHIMOTO, T. ITO, K. OGAWA, AND T. SATAKE. Effects of atria1 natriuretic peptide and organic nitrates on levels of relaxation and cyclic nucleotide of canine coronary artery with and without endothelial injury. Hearts VesseZs 4: 19-25, 1988. 12. PALMER, R. M., A. G. FERRIGE, AND S. MONCADA. Nitric oxide release accounts for the biologic activity of endothelium-derived relaxation factor. Nature Lond. 327: 524-526, 1987. 13. REES, D. D., R. M. J. PALMER, H. F. HODSON, AND S. MONCADA. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxations. Br. J. PharmacoZ. 96: 418-424, 1989. 14. TER RIET, M., W. W. NICHOLS, L. THOMPSON, D. LAWSON, AND J. L. MEHTA. Role of prostaglandins in coronary flow response to nitroglycerin in dogs subjected to temporary coronary occlusion. J. Vast. Med. Biol. 1: 272-277, 1989. 15. VANHOUTTE, P. M., AND Z. S. KATUSIC. Endothelium-derived contracting factor-endothelin and/or superoxide anion? Trends Pharmacol. Sci. 9: 229-230, 1988. 16. YANAGISAWA, M., H. KURIHARA, S. KIMURA, Y. TOMOBE, M. KOBAYASHI, Y. MITSUI, Y. YAZAKI, K. GOTO, AND T. MASAKI. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lond. 332: 411-415, 1988.
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DINERMAN, J. L., D. L. LAWSON, AND J. L. MEHTA. Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H698-H701, 1991.-To evaluate the role of endothelium in nitroglycerin (NTG) -mediated vascular relaxation, epinephrine-contracted rat thoracic aortic segments with and without intact endothelium were exposed to NTG (lOvl' to 10m5 M). Aortic segments with intact (endo+, n = 15) and denuded endothelium (endo-, n = 9) exhibited typical NTGinduced relaxation. However, the mean effective concentration of NTG was lower for endo- than for endo+ segments (P < 0.001). To determine if this phenomenon related to nitric oxide (NO) generation by endothelium, six endo+ segments were treated with N”-monomethyl-L-arginine (L-NMMA), an inhibitor of NO production. These endo+ segments exhibited greater (P < 0.001) relaxation in response to NTG than the untreated endo+ segments. Oxyhemoglobin, an inhibitor of guanylate cyclase activation, greatly diminished NTG-mediated relaxation of all aortic segments. To determine if the enhanced NTGmediated relaxation of endosegments was unique to the guanosine 3’,5’-cyclic monophosphate-dependent vasodilator NTG, other endo+ and endo- segments were exposed to adenosine 3’,5’-cyclic monophosphate-dependent vasodilator papaverine (10S8 to low4 M), and no difference in EC0 was noted between endo+ and endo- segments. Thus endothelium attenuates NTG-mediated vasorelaxation, and this attenuation is abolished by inhibition of endothelial NO production with LNMMA. These observations indicate that endothelium is a dynamic modulator of vascular smooth muscle relaxant effects of NTG. This modulation appears to result from a competitive interaction between endothelial NO and NTG.
endothelium-derived
relaxing
factor; nitric
oxide
SERVES as an important modulator of vascular smooth muscle tone through the formation of several vasoactive species (28). Endothelium is a source of potent vasodilatory compounds such as prostacyclin and endothelium-derived relaxing factor (EDRF), which has been identified as nitric oxide (NO; 12). Endothelium also generates vasoconstrictor species, collectively known as endothelium-derived constricting factors (EDCFs), for which the biological activity may be accounted for, at least in part, by endothelium (16) and superoxide anions (7, 15). The relative contributions of these opposing factors may be of importance in the determination of vascular tone. Nitroglycerin (NTG) is generally thought to be an endothelium-independent smooth muscle relaxant. It is known that through formation of intracellular nitrosoENDOTHELIUM
H698
0363-6135/91
$1.50 Copyright
thiols, subsequent activation of guanylate cyclase, and accumulation of guanosine 3’,5’-cyclic monophosphate (cGMP), NTG shares with EDRF a final common pathway leading to vascular smooth muscle relaxation (5). Endothelial formation of EDCFs tends to oppose the actions of these factors and may thereby diminish the effect of vasodilatory influences. Production of the peptide known to account for the activity of endothelin-1 from its messenger RNA is inhibited as intracellular cGMP rises (1). We postulated that the presence of intact endothelium would alter NTG-mediated vasorelaxation, because both EDRF and NTG share a common pathway in the process of accumulation of cGMP in smooth muscle. These two relatively similar agents could either have cumulative or synergistic effects or compete with each other for activation of processes leading to smooth muscle relaxation. This was examined in rat thoracic aortic segments with and without intact endothelium. MATERIALS
AND
METHODS
Preparation of rat aortic segments. Thoracic aortas of Male Sprague-Dawley rats (Charles River, Portage, MI) weighing 150-200 g were harvested after exsanguination under light halothane anesthesia, carefully cleaned of fat and connective tissue, and cut into 3- or 4mm segments. The endothelium of a subset of vessels was removed by gentle rubbing. The segments were washed, mounted onto wire stirrups, and suspended in organ baths at 37°C containing 5 ml of aerated Krebs-Ringer buffer of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaC12, 1.2 KH2P04, 1.2 MgC1,, 12.5 NaHC03, 0.01 NaEDTA, and 11.1 dextrose. The segments were connected to myograph transducers (Grass Instruments, Quincy, MA) to record changes in isometric force (4, lo), stretched to a preload force of 5 g, and allowed to equilibrate for -2 h. During the equilibration period, the Krebs-Ringer buffer in the organ baths was replaced every 15 min. Endothelial integrity was determined in each experiment by characteristic relaxation after treatment with acetylcholine (ACh). These vascular segments will be referred to as endo+. In deendothelialized segments (subsequently referred to as endo-), ACh did not cause relaxation. Fresh buffer was then added to the baths, and the responses to various agonists and antagonists were recorded. Study protocols. Parallel series of aortic rings with and without endothelium were stimulated with epinephrine
0 1991 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 2, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
ENDOTHELIUM
AND NITROGLYCERIN
to achieve -3-5 g (80% of maximum) of stable tension above baseline. In some experiments norepinephrine was used as the agonist. Once stable contraction had been obtained, increasing concentrations of NTG (lOsl' to 10e5 M) were added to the organ bath, and changes in force were recorded. In several experiments, vessels were treated with oxyhemoglobin (10 PM) or NG-monomethyl+arginine (L-NMMA, 50 PM) before precontraction with epinephrine. In later experiments a variety of precontracted aortic segments were exposed to a non-nitroso vasodilator papaverine (lOwl' to lo-* M). Reagents. NTG was obtained from Parke-Davis, Morristown, NJ. L-NMMA was a gift from Dr. S. Moncada, and indomethacin was a gift from Merck Sharpe & Dohme, West Point, PA. Oxyhemoglobin was prepared by adding 10 M excess of Na2S204 to a 1 mM solution of type 1 bovine hemoglobin (Sigma). Na2S204was removed by dialysis against 100 vol of distilled Hz0 for 2 h at 4°C. All other reagents used were obtained from Sigma Chemical, St. Louis, MO. All reagents and buffers were prepared fresh daily and were kept on ice during the experiments. Statistical analysis. Force generated by aortic segments was divided by the wet weight of the segments to obtain force in grams per milligram of tissue. All data are expressed as geometric means t SE. Magnitude of relaxation was expressed as percent decrease from stable contraction. Linear regression of the straight portion of dose-response curves from each experiment was used to obtain mean effective concentration (EC&), which was expressed in moles per liter. Statistical analysis of data was performed using Student’s t test for paired and unpaired data and a Bonferroni correction was applied for multiple comparisons, where appropriate. A P value co.05 was considered significant. RESULTS
Effect of endothelium on vascular contraction. Addition of epinephrine to organ baths was associated with a concentration-dependent increase in contraction of both endo+ and endo- aortic segments. The threshold concentration of epinephrine required to initiate contraction was less (P < 0.03) for endo- than for endo+ segments (Fig. 1, Table 1). Similar data were obtained with norepinephrine as the agonist. Increased sensitivity of vascular smooth muscle in endo- segments is due to loss of EDRF in keeping with previous reports (3). Effect of L-NMMA and oxyhemoglobin on epinephrinemediated contraction. L-NMMA as well as oxyhemoglobin caused small contractions in endo+ segments (Fig. lC), probably because of inhibition of endogenous EDRF and its binding, respectively. The concentration of epinephrine required to initiate further contraction was less (P C 0.05) for vessels pretreated with L-NMMA or oxyhemoglobin than for untreated endo+ vessels (Table 1). Similar results were obtained when norepinephrine was used as the agonist (data not shown). Presence of endothelium and NTG-mediated vasorelaxation. Addition of NTG to organ baths resulted in a concentration-dependent decrease in the force generated by aortic segments. The magnitude of relaxation was greater for endo- aortic segments than for endo+ aortic
H699
2 mkn
endothelium
-./
-6
4 -5 (-log M)
4
EPI -8
0
L -/
4 EPI
4
-9
-7 (-log M)
L-NMMA
4 L-iJMMA
+ endothelium
4 -5
EbI
intact
in tact
-7 (-log M)
-9
FIG. 1. A: representative experiment showing nitroglycerin (NTG)mediated relaxation of an epinephrine (Epi)-precontracted rat thoracic aortic segment with intact endothelium. B: vascular relaxation is much more pronounced at all concentrations of NTG in another segment from the same aorta that had been deendothelialized. C: enhanced NTG-mediated relaxation of another segment with intact endothelium that had been pretreated with NC’-monomethyl-L-arginine (L-NMMA) before contraction with epinephrine.
TABLE 1. Effects of epinephrine and NTG on rat aortic segments Segments Endo+ EndoL-NMMA + endo+ HbOZ + endo+
n
15 9
Epinephrine Threshold
2.40t1.11 4.64kl.32
x lo-’ x lo-lo*
6 1.47k1.57 x lo-' 3 1.00tl.70 x 1o-g
EC&
NTG
2.50t0.77 x 1O-7 2.50t0.72 2.38t0.69 4.60t0.94
x lo-‘“f x lo-‘-/x 10-6j-
Data from multiple experiments in geometric means t SE; n = no. refer to segments with intact and of segments. Endo+ and endodenuded endothelium, respectively. L-NMMA, p-monomethyl-L-arginine; Hb02, oxyhemoglobin; NTG, nitroglycerin. * P < 0.05 vs. epinephrine threshold concentration in endo+ segments; j- P < 0.001 vs. ECso of NTG in endo + segments.
segments. This was observed at each concentration in all experiments. A representative experiment is shown in Fig. 1, and data from multiple experiments are summarized in Fig. 2. The mean EC50 of NTG was 10 times lower for endo- than for endo+ segments (P < 0.001). Effect of L-NMMA and oxyhemoglobin on NTG-mediated vasorelaxation. To determine if the enhanced relaxation noted for endo- vessels was related to mechanical properties of, or generation of nitric oxide by endothelium, endo+ aortic segments were pretreated with LNMMA and this resulted in significantly greater relaxation of vascular smooth muscle when these segments were subsequently exposed to NTG (Table 1). The EC50 of NTG was lower (P < 0.001) than that for the untreated endo+ vascular segments but was similar to that for endo- segments. Pretreatment of endo+ segments with oxyhemoglobin, on the other hand, resulted in significant attenuation of NTG-mediated vasorelaxation (P c
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H700
ENDOTHELIUM
AND
NITROGLYCERIN
Previous studies have shown that endothelium modulates the smooth muscle relaxant effects of ADP, histamine, and LTD,; agents that exert their effects in part via release of EDRF (3). The augmented NTG-mediated relaxation of vascular segments denuded of endothelium compared with segments with intact endothelium in the present study reflects the importance of endothelium in modulating the vascular response to NTG. It is notewor60thy that Nakagawa et al., like us, observed enhanced NTG-mediated vasorelaxation in deendothelialized ca80nine coronary arteries; they also observed greater accu90mulation of cGMP vessels in these segments than in 00 endo+ segments in response to NTG (11). I I 0 0 T v7 The major mechanisms by which endothelium may b b -0 5 5 -X attenuate the response to vascular smooth muscle to m NTG are through its function as a “mechanical barrier” Nitroglycerin (M) and by production of EDCFs. It is possible that disrupFIG. 2. Augmented nitroglycerin-mediated relaxation of rat thoracic tion of the endothelial integrity contributes to the augaortic segments with denuded endothelium (0, n = 9) compared with mented vasoreactivity by allowing enhanced availability those with intact endothelium (m, n = 15). of NTG in the proximate milieu of the vascular smooth 0.001) compared with the untreated endo+ segments. muscle. Once access to vascular smooth muscle is obThe presence of oxyhemoglobin also attenuated the tained, NTG stimulates guanylate cyclase and causes marked NTG-mediated relaxation of L-NMMA-preintense relaxation. This hypothesis is supported by pretreated segments (mean E& 1.52 x 10m6 M). vious observations of enhanced smooth muscle relaxaEffect of indomethacin and superoxide dismutase (SOD) tion in response to neutrophil-derived NO in deendoon NTG-mediated vasorelaxation. To determine if the thelialized vascular segments (10). However, endotheenhanced NTG-mediated relaxation of endo+ vessels lium serving as a simple mechanical barrier is an unlikely pretreated with L-NMMA related to generation of pros- scenario in vascular ring preparations suspended in an taglandins or superoxide anions, segments were pre- organ bath. treated with the cyclooxygenase inhibitor indomethacin To further examine the concept of endothelium serving or the superoxide anion scavenger SOD. Neither of these as a potential chemical modulator, we pretreated endo+ agents modified the relaxation of L-NMMA-pretreated segments with L-NMMA, which resulted in a modest endo+ aortic segments (mean E& of NTG 3.48 x 10B8 contraction and augmented sensitivity to epinephrine M for indomethacin-exposed segments and 5.39 x 10v8 (Fig. 1C). L-NMMA is a stereospecific, competitive anM for SOD-exposed segments). tagonist of L-arginine-mediated NO production (13). It Effect of intact endothelium and L-NMMA on papavis possible that in L-NMMA-treated endo+ segments, erine-mediated vasorelaxation. To investigate whether baseline smooth muscle tone was influenced by inhibithe enhanced relaxation of endo- or L-NMMA-treated tion of EDRF and by the release of one or more EDCFs. endo+ aortic segments was a unique response to NTG, Indeed, Boulanger and Luscher (1) have recently dempretreated endo+ aortic onstrated L-NMMA increases the formation of endotheendo+, endo- and L-NMMA segments were exposed to papaverine, a non-nitroso lin-1 in porcine aortic segments with intact endothelium. vasodilator. Although characteristic relaxation was noted We expected attenuation of NTG-mediated relaxation for all aortic segments, the E&s of papaverine for endo+ in the presence of the constrictor influence of endothelinsegments. However, we observed (4.11 t 2.73 X 10m5,n = 3), endo- (4.96 t 1.77 X 10B5,n 1 in L-NMMA-treated = 4), and L-NMMA-pretreated endo+ aortic segments markedly enhanced NTG-induced relaxation in these segments, similar to that in endo- segments. L-NMMA (4.65 t 0.50 X 10v5, n = 4) were similar. is unlikely to physically disrupt the endothelial integrity; as such, the concept of endothelium serving as a meDISCUSSION chanical barrier is not tenable in these experiments. Endothelium serves an important role in vascular ho- Thus it appears that the basal release of EDRF from meostasis by modulating vasomotor tone through the intact endothelium affects the vasorelaxation caused by production of constrictor and dilator substances. It has NTG. Accordingly, it is likely that the enhanced NTGbeen proposed that in states of health, endotheliummediated relaxation occurs in the absence of endothelium derived substances that exhibit smooth muscle relaxant because competition between EDRF and NTG for actiproperties predominate (8). In states of hypoxia or endo- vation of guanylate cyclase is absent. Supporting this thelial dysfunction, vasoconstrictor influences may be concept is the close similarity of the mechanism of action left relatively unopposed. Vascular segments with dis- of EDRF and NTG, both of which cause vasorelaxation rupted or dysfunctional endothelium may also exhibit by causing an increase in vascular cGMP accumulation increased contraction in response to smooth muscle ag- (5). In this regard, our experiments with papaverine, a onists (3, 7). Our present observation that a lower con- vasodilator not related to EDRF, are significant. Unlike centration of epinephrine or norepinephrine is required NTG, papaverine, which increases vascular adenosine to initiate contraction of deendothelialized vascular seg- 3’,5’-cyclic monophosphate, caused a similar degree of relaxation in endo+ and endo- segments. Treatment of ments is in keeping with this concept. Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 2, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
ENDOTHELIUM
AND
endo+ segments with L-NMMA also did not influence papaverine-mediated relaxation. Based on these observations, papaverine may be considered a true “EDRFindependent” vasodilator. Our studies showing the reduction of NTG-mediated vasorelaxation in the presence of oxyhemoglobin are in keeping with previous reports (9). Indomethacin did not modify NT@-mediated vasorelaxation, indicating that prostaglandins are not involved in the actions of NTG on smooth muscle tone (14). Although SOD prevents degradation of EDRF by scavenging superoxide anions (7), the presence of SOD did not potentiate the vasorelaxant potential of NTG. In contrast, inhibition of EDRF synthesis by L-NMMA pretreatment resulted in marked potentiation of NTG-mediated vasorelaxation. It is noteworthy that like L-NMMA, oxyhemoglobin has been shown to stimulate production of endothelin-1 (1). This stimulation of endothelin-1 is also associated with diminished cellular cGMP accumulation as a result of inhibition of EDRF activity. In keeping with this concept, oxyhemoglobin pretreatment contracted the vascular segments and augmented their sensitivity to agonists in our experiments (Table 1). However, NTGmediated vasorelaxation was attenuated in the presence of oxyhemoglobin in all endo+ and endo- segments as well as in others pretreated with L-NMMA. The most likely mechanism for this is binding of NTG as well as NO to oxyhemoglobin, thereby inhibiting smooth muscle guanylate cyclase stimulation (9). Two recent studies on endothelial modulation of the EDRF donor SIN-l support our observations that endogenous EDRF inhibits the effects of cGMP-dependent vasodilators. Busse et al. (2) demonstrated that the ability of SIN-l to elicit vasodilation in the rabbit femoral artery was markedly enhanced in vessels denuded of endothelium, and they concluded that this effect was mediated by EDRF or another endothelium-derived substance that alters the action or metabolism of cGMP (3). Similar findings were noted in canine coronary arteries by Flavahan and Vanhoutte (4); these investigators, however, found that the pretreatment of endo+ segments with SOD and catalase enhanced SIN-l-mediated vasodilation (4). Although we did not note similar augmentation of NTG-mediated vasorelaxation by SOD, these differences may relate to differences in superoxide radical production between canine and rat vascular endothelium. Nevertheless, both these studies support our observations and suggest inhibitory interactions between cGMPdependent vasodilators and EDRF. In summary we have demonstrated enhanced NTGmediated vasorelaxation in rat thoracic aortic segments denuded of endothelium compared with those with intact endothelium. These results indicate the important role of vascular endothelium as both a dynamic barrier and a modulator of smooth muscle response to vasodilator stimuli, particularly those that share the same mechanism of vasorelaxation as EDRF. These findings may have clinical relevance to the pharmacotherapy of diseases in which dysfunction of vascular endothelium exists. Based on the observations described herein, NTG
H701
NITROGLYCERIN
may be considered lator.
an endothelium-interactive
vasodi-
This study was supported by the Medical Research Service of the Department of Veterans Affairs and by a grant from the American Heart Association, Florida Affiliate. J. L. Mehta is a Clinical Investigator of the Department of Veterans Affairs, Central Office. Address for reprint requests: J. L. Mehta, Dept. of Medicine, Univ. of Florida, Box J-277, JHMHC, Gainesville, FL 32610. Received
6 August
1990; accepted
in final
form
6 November
1990.
REFERENCES 1. BOULANGER, C., AND T. F. LUSCHER. Release of endothelin from porcine aorta: inhibition by endothelium-derived nitric oxide. J. CZin. Invest. 85: 587-590, 1990. 2. BUSSE, R., U. POHL, A. MOLSCH, AND E. BASSENGE. Modulation of the vasodilator action of SIN-l by the endothelium. J. Cardiovast. Pharmacol. 14, Suppl. II: 581-585, 1989. 3. DINERMAN, J. L., AND J. L. MEHTA. Endothelial, platelet and leukocyte interactions in ischemic heart disease: insights into potential mechanisms and their clinical relevance. J. Am. CoLl. Cardiol. 116: 207-222, 1990. 4. FLAVAHAN, N. A., AND P. M. VANHOUTTE. Mechanisms underlying the inhibitory interaction between the nitrovasodilator SIN- 1 and the endothelium. J. Cardiovasc. Pharmacol. 14, Suppl. II: 586-590, 1989. 5. IGNARRO, L. J. Endothelium-derived nitric oxide: pharmacology and relationship to the actions of organic nitrate esters. Pharm. Res. 6: 651-659, 1989. 6. LAWSON, D. L., C. SMITH, J. L. MEHTA, P. MEHTA, AND W. W. NICHOLS. Leukotriene D, potentiates the contractile effects of epinephrine and norepinephrine on rat aortic rings. J. PharmacoZ. Exp. Ther. 116: 1201-1206, 1988. 7. LAWSON, D. L., W. W. NICHOLS, P. MEHTA, AND W. H. DONNELLY. Superoxide radical-mediated endothelial injury and vasoconstriction of rat thoracic aortic rings. J. Lab. CZin. Med. 115: 541-548,199O. 8. LUSCHER, T. F. Endothelium-derived relaxing and contracting factors: potential role in coronary artery disease. Eur. Heart J. 10: 847-857,1989. 9. MARTIN, W., G. M. VILLANI, D. JOTHIANANDAN, AND R. F. FURCHGOTT. Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J. Pharmacol. Exp. Ther. 232: 708-716, 1985. 10. MEHTA, J. L., D. L. LAWSON, W. W. NICHOLS, AND P. MEHTA. Modulation of vascular tone by neutrophils. Dependence on endothelial integrity. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1315-H1320,1989. 11. NAKAGAWA, H., K. OKUMURA, H. HASHIMOTO, T. ITO, K. OGAWA, AND T. SATAKE. Effects of atria1 natriuretic peptide and organic nitrates on levels of relaxation and cyclic nucleotide of canine coronary artery with and without endothelial injury. Hearts VesseZs 4: 19-25, 1988. 12. PALMER, R. M., A. G. FERRIGE, AND S. MONCADA. Nitric oxide release accounts for the biologic activity of endothelium-derived relaxation factor. Nature Lond. 327: 524-526, 1987. 13. REES, D. D., R. M. J. PALMER, H. F. HODSON, AND S. MONCADA. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxations. Br. J. PharmacoZ. 96: 418-424, 1989. 14. TER RIET, M., W. W. NICHOLS, L. THOMPSON, D. LAWSON, AND J. L. MEHTA. Role of prostaglandins in coronary flow response to nitroglycerin in dogs subjected to temporary coronary occlusion. J. Vast. Med. Biol. 1: 272-277, 1989. 15. VANHOUTTE, P. M., AND Z. S. KATUSIC. Endothelium-derived contracting factor-endothelin and/or superoxide anion? Trends Pharmacol. Sci. 9: 229-230, 1988. 16. YANAGISAWA, M., H. KURIHARA, S. KIMURA, Y. TOMOBE, M. KOBAYASHI, Y. MITSUI, Y. YAZAKI, K. GOTO, AND T. MASAKI. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lond. 332: 411-415, 1988.
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