Nitric oxide inhibits norepinephrine stimulated contraction of human internal thoracic artery and rat aorta.

The effect of nitric oxide (NO) on norepinephrine-induced vascular contraction was evaluated using segments of rat aorta and human internal thoracic artery (ITA) and the NO donor, SNAP. NO levels were measured directly using an amperometric probe. Concentrations of NO greater than 2 nM were required to reduce vascular contraction induced by 100 nM norepinephrine (NE). Exposure of the aortic rings to SNAP prior to, or after NE addition, resulted in a similar attenuation of NE-induced contraction. In contrast, increased relaxation of ITA segments in response to SNAP was observed relative to that of rat aorta and significant development of contractile tone following NE addition was not observed. Evaluation of cytoskeletal actin demonstrated marked loss of F-actin content in smooth muscle cells following NO exposure, suggesting that NO may have direct and indirect effects on contractile tone. These data taken together suggest that vascular responsiveness to contractile agents may be significantly attenuated by prior or subsequent exposure to NO, and mechanisms in addition to vascular relaxation are likely to contribute to this effect.

Morphine stimulates iNOS expression via a rebound from inhibition in human macrophages: nitric oxide involvement.

We have previously demonstrated that exposure of human macrophages to morphine results in transient inhibition of cell migratory behavior and adoption of an inactive conformation followed by a return from inhibition resulting in a significant increase in migration velocity and number of activated cells. In the current report, we demonstrate that the return to activation is nitric oxide dependent and inhibited by prior exposure to the opiate antagonist, naloxone. Exposure of macrophages to morphine for 6 hours resulted in a marked inhibition of cell activity and shift of the cell confirmation from amoeboid to round. The inactivation period lasted approximately 2 hrs and was followed by a period of hyperactivity. Incubation of macrophages with naloxone, prior to addition of morphine, inhibited both inactivation and hyper activation phases whereas, naloxone administration just prior to the hyper activation phase did not affect subsequent hyper activation. Morphine acutely stimulates the transient release of nitric oxide (NO) resulting in subsequent macrophage rounding and inactivation. Prolonged observation of the cells revealed another phase of NO release 12 hours following initial morphine exposure that was characterized by prolonged NO production. These data are consistent with acute constitutive NO synthase activation and inducible NO synthase activation following prolonged morphine exposure. Release of NO and changes in cellular activation mediated by morphine was abrogated by NOS, or morphine inhibitors, added prior to morphine exposure. In contrast, NOS, or morphine inhibitors, added during the inhibitory phase had no impact on the subsequent hyper activation phase. It did, however, have an impact on the hyper activation phase when added prior to morphine. These data demonstrate that morphine is capable of induction of both cNOS and iNOS coupled NO release that regulates the macrophage activation state. This may provide insight into the functioning of morphine following periods of trauma or stress when the levels of the opiate increase and, subsequently, inflammatory function is markedly altered.

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.

Expression of functional delta opioid receptors in vascular smooth muscle.

We evaluated smooth muscle from human internal mammary artery and rat aorta for the presence of delta opioid receptors. Radioligand receptor competition studies using the delta-receptor selective agonist, [3H]-[D-Ala2, Met5] enkephalinamide (DAMA) suggested the expression of a high affinity binding site in rat and human blood vessels that was consistent with the delta-2 opioid receptor subtype. Using RT-PCR with primers to the cloned delta opioid receptor (DOR), a cDNA fragment identical to the known DOR sequence was obtained from the smooth muscle cell line, A-10. Stimulation of A-10 cells with DAMA resulted in a significant mobilization of intracellular calcium and membrane depolarization. Exposure of aortic rings denuded of endothelium to DAMA induced a naltrindole-senstive increase in contractile tone. These data demonstrate the presence of a functional DOR in vascular smooth muscle and a direct impact of opioids on vascular contractile tone.

Rebound from nitric oxide inhibition triggers enhanced monocyte activation and chemotaxis.

Exposure of human peripheral blood monocytes to the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) resulted in a rapid shift in cellular conformation of spontaneously activated cells from ameboid to round. The population of activated cells, approximately 7. 1 +/- 1.2%, was reduced 7-fold to 1.1 +/- 0.4% following 0.5 h exposure to SNAP. Observation of monocytes for 6 h demonstrated a gradual release from NO inhibition initiating at 2.5 h following SNAP treatment and a period of hyperactivity that was maximal at approximately 5 h following SNAP exposure. During the rebound from the NO inhibition phase, there was a significant increase in the population of activated monocytes and an increased responsiveness to chemotactic agents such as IL-1, IL-8, and fMLP relative to that of cells treated with the chemotactic agents alone. Conformational changes induced by SNAP were associated with a reduction in F-actin and loss of filopodial extension. The loss and recovery of F-actin staining paralleled changes in cell activity, suggesting that NO may alter cellular activity by modulation of cytoskeletal actin. These data taken together suggest that inhibition of monocyte activity by NO results in an excitatory phase observed subsequent to release from NO inhibition and increased sensitivity to chemotactic agents. We propose that this rebound from NO inhibition may provide increased immunosurveillance to rectify immunological problems that have been encountered during the period of inhibition.

Thrombin receptor activation inhibits monocyte spreading by induction of ET(B) receptor-coupled nitric oxide release.

The effect of thrombin receptor activation on monocyte conformation was evaluated using the human monocyte cell line, THP-1, and the thrombin mimetic peptide, Trap-14. Treatment of THP-1 cells with Trap-14 induced rapid rounding of ameboid cells adherent to fibronectin-coated slides, whereas cell rounding was abrogated in the presence of the nitric oxide synthase inhibitor, NG-nitro-L-arginine or the endothelin B receptor antagonist, BQ-788. Endothelin-1 (ET-1) levels in the culture supernatant increased markedly within minutes of Trap-14 exposure with a concomitant loss in cellular ET-1 immunoreactivity. Importantly, loss of ET-1 immunoreactivity was blocked by pretreatment with the vesicle translocation inhibitor, nocodazole. Trap-14 potently induced the release of NO from THP-1 cells, whereas NO release was ablated by preincubation with BQ-788. These data demonstrate that thrombin receptor activation may inhibit cellular spreading as a result of autocrine ET-1 release and subsequent endothelin B receptor-dependent NO production, and suggest that initial exposure of inflammatory cells to thrombin may limit cellular activation and recruitment.

Antagonism of LPS and IFN-gamma induction of iNOS in human saphenous vein endothelium by morphine and anandamide by nitric oxide inhibition of adenylate cyclase.

Nitric oxide (NO) production regulates vasodilation in many blood vessels. Additionally, constitutive NO release is being associated with positive biomedical phenomena, whereas inducible NO synthase (iNOS)-associated NO release with detrimental consequences in regard to endothelial inflammatory activities. As yet, an important link demonstrating why one is activated over the other is not available. Previous studies have demonstrated that morphine and anandamide effector processes are coupled to NO release in human endothelial cells (ECs). This study now extends this observation in that these endogenous signaling molecules may use NO directly to inhibit adenylate cyclase activity. Activation of human ECs, obtained from the saphenous vein, with morphine- or anandamide-stimulated NO release (35 nM and 28 nM, respectively) that peaked within 5 min and returned to basal levels within 10 min of agonist stimulation, consistent with constitutive NO synthase (cNOS) activation. Significant release of NO from ECs stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) occurred after 2 h after exposure and remained significantly increased over basal levels for 24-48 h (28 nM), consistent with iNOS activation. Preincubation of ECs with morphine or anandamide before, but not after, the addition of LPS + IFN, blocked iNOS activity. Exposure of ECs to the NO donor, SNAP, before the addition of LPS + IFN, blocked iNOS induction, whereas preincubation of ECs with inhibitors of NOS, before morphine or anandamide exposure, restored LPS + IFN induction of iNOS, suggesting a direct impact of NO on the regulation of iNOS activity. Morphine and anandamide stimulation of ECs did not stimulate cyclic adenosine monophosphate (cAMP) accumulation, whereas a marked increase in cAMP was observed in ECs treated with LPS + IFN (8.2 to 33 pmol/mg protein). Treatment of ECs with LPS + IFN did not induce cAMP accumulation in ECs treated with morphine, anandamide, or SNAP before LPS + IFN exposure. These data suggest that cAMP is required for the induction of iNOS in ECs and that NO may directly impair adenylate cyclase activity, preventing iNOS activation.

Cryopreserved veins in myocardial revascularization: possible mechanism for their increased failure.

BACKGROUND: Cryopreserved veins are used as conduits for myocardial revascularization. However, a high failure rate associated with their use has been reported anecdotally. METHODS: To find an explanation for the poor performance of cryopreserved vein grafts, we conducted a retrospective 5-year study on all patients at a single institution in whom cryopreserved vein grafts were used. We further performed in vitro studies measuring cell adhesion, nitric oxide production, and contractile capacity of saphenous vein, internal thoracic artery, and cryopreserved veins. RESULTS; Forty-one patients were identified in whom one or more cryopreserved veins were used as a last resort. Sixteen had events (death or recatheterization). Seven deaths occurred (17%). Event-free survival was 50% at 12 months. Activated granulocyte/monocyte endothelial adherence could be lowered in internal thoracic arteries and saphenous veins with morphine incubation (50% and 57%, respectively), but not in cryopreserved veins. Simultaneous increases in nitric oxide release were also found in internal thoracic arteries and saphenous veins, but not cryopreserved veins. In addition, cryopreserved veins showed a diminished contractile capacity under experimental conditions. CONCLUSIONS: In this highly select group of patients, cryopreserved veins had a high early failure rate, which may be partially due to the inability of the endothelium to participate in immunovascular processes.

Human monocyte adhesion is modulated by endothelin B receptor-coupled nitric oxide release.

Human monocytes have the capacity to produce both endothelin 1 (ET-1) and nitric oxide (NO), yet the roles of these mediators in monocyte function remain unclear. The relationship of ET-1 and NO release to monocyte adhesion was explored using peripheral blood monocytes (PBM) and the human monocytic cell lines THP-1 and U937. Specific binding of 125I-labeled ET-1 to THP-1 was abrogated by pretreatment with the endothelin B (ET(B)) receptor antagonist, BQ-788, but not by the endothelin A (ET(A)) receptor antagonist, BQ-123, consistent with predominant ET(B) receptor expression. Direct measurement of NO with an amperometric probe demonstrated the production of nanomolar concentrations of NO by PBM and THP-1 cells upon treatment with ET-1, which was abrogated by BQ-788, but not BQ-123, pretreatment, suggesting functional coupling of ET(B) receptors to NO release. Indeed, the presence of ET(B) receptor mRNA transcripts was detected in THP-1 and is consistent with previous reports that have demonstrated functional coupling of ET(B) receptors to constitutive NO synthase activation. In contrast, U937 cells did not release NO in response to ET-1 treatment, and mRNA transcripts were not detected in these cells, consistent their failure to bind 125I-labeled ET-1, as previously determined. Exposure of PBM to ET-1 markedly reduced the adhesion of these cells to human saphenous vein, whereas PBM adhesion in the presence of BQ-788 was restored to control levels. These data demonstrate that PBM interactions with the vascular wall can be reduced by autocrine production of NO and suggest that ET(B) receptors may attenuate monocyte activity at sites of inflammation.

Thrombin-induced vascular reactivity is modulated by ETB receptor-coupled nitric oxide release in rat aorta.

The role of endothelin (ET) receptors in thrombin-induced modulation of vascular tone was evaluated by direct measurement of ET-1 and ET receptor-coupled nitric oxide (NO) release and developed isometric tension in thrombin-treated aortic rings. Here we report that rapid release of ET-1 and subsequent ETB receptor activation are required for production of the potent vasodilator NO by thrombin-stimulated aorta. Thrombin-induced NO release is ablated by pretreatment with ETB receptor antagonists or after ET receptor desensitization by repeated stimulation with ET-1. Thrombin-induced relaxation of precontracted vessels was abrogated in the presence of ETB receptor antagonists and, in contrast, marked contraction to thrombin was observed. These data indicate that the endothelium-dependent vasodilator activity previously attributed to thrombin is indirect and requires ETB receptor-coupled NO release and suggest that ET receptor modulation of thrombin-induced vascular tone may contribute to the increased vasomotor tone observed in diseased and mechanically injured vessels.

Endothelin and nitric oxide release modulate aortic contraction to selected thrombin receptor agonists.

The contribution of endothelin-1 (ET-1) and nitric oxide (NO) release to vascular contraction induced by synthetic peptide agonists of the alpha-thrombin receptor, TRAP-14 and TRAP-6, was evaluated with the use of rings of rat aorta. TRAP-6 induced fourfold greater contraction than that induced by addition of TRAP-14. TRAP-14, but not TRAP-6, stimulation of aortic rings resulted in a rapid (in seconds) and dose-dependent increase in ET-1 levels detected in the vessel perfusate. Release of ET-1 in vessels denuded of endothelium was indistinguishable from that of intact vessels, suggesting that endothelial cells are not required for the observed ET-1 release. The contractile potency of TRAP-14 was reduced in the presence of BQ-123, a specific antagonist of the endothelin A subtype of ET receptors, whereas TRAP-14 potency was increased significantly by pretreatment with the NO synthetase inhibitor NG-nitro-L-arginine (L-NNA). The contractile potency of TRAP-6 was not altered in the presence of BQ-123 or L-NNA, suggesting that TRAP-14 but not TRAP-6 potency is modulated by ET-1 and NO release. These data indicate that TRAP-6 has limited function relative to TRAP-14 and that thrombin receptor activation is not sufficient to induce ET-1 and NO release from rat aorta.

Morphine-induced conformational changes in human monocytes, granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide.

We evaluated the contribution of nitric oxide (NO) to morphine-induced rounding of spontaneously activated (mobile) ameboid human monocytes, granulocytes, or arterial endothelial cells and invertebrate immunocytes and microglia. Morphine induced significant rounding and inactivation of ameboid cells within 20 min except for arterial endothelial cells, which became rounded 24 h after morphine exposure. The effects of morphine on cell conformation were blocked in the presence of N-nitro-L-arginine, a nitric oxide synthase inhibitor. Treatment of cells with the NO donor, sodium nitroprusside, induced cell rounding similar to that observed following morphine exposure, suggesting that NO release may mediate morphine-induced changes in cell conformation. The contribution of NO release to morphine-induced cell rounding was determined by direct evaluation of NO concentration in real-time using a NO-specific amperometric probe. Significant increases in NO concentration were observed 2 min after morphine stimulation, whereas morphine-induced NO release was markedly impaired by pretreatment with N-nitro-L-arginine or the opiate alkaloid antagonist, naloxone. In contrast, opioid peptides failed to induce NO release, consistent with our previous observations that demonstrated the failure of opioid peptides to promote cell rounding. Taken together, these data suggest that morphine-induced NO release may be mediated by activation of the opiate alkaloid-selective, opioid peptide-insensitive micro3 receptor, and that functional coupling of morphine to NO production has been conserved during evolution and may modulate cellular activation.

Morphine stimulates nitric oxide release from invertebrate microglia.

Morphine stimulates nitric oxide (NO) release in human endothelial cells. To determine whether this mechanism also occurs in invertebrates, the mussel Mytilus edulis was studied. Exposure of excised ganglia to morphine for 24 h resulted in a significant dose-dependent decrease in microglial egress that was naloxone sensitive. In coincubating the excised ganglia with morphine and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), an increase in microglial egress was observed, suggesting that morphine may stimulate microglia to release NO. Morphine exposure to these cells in vitro resulted in NO release (39.4 +/- 4.9 nM), a phenomenon found to be naloxone sensitive (10(-6) M; NO level = 5.9 +/- 2.6 nM) and L-NAME sensitive (10(-4) M; NO level = 2.8 +/- 1.8 nM). Opioid peptides did not stimulate NO release, indicating that the process was mediated by the opiate alkaloid selective mu 3 receptor. Coincubation of microglia with L-arginine or the superoxide scavenger, superoxide dismutase, resulted in significantly higher NO levels observed following morphine stimulation. Taken together, the data demonstrate that morphine can stimulate NO release in cells obtained from an invertebrate that represents an animal 500 million years divergent in evolution from man, underscoring the significance of this process and further substantiating the critical importance of morphine as a naturally occurring signal molecule.

Protease activated receptors modulate aortic vascular tone.

The effect of agonists of the known protease activated receptors (PAR), the thrombin and the PAR-2 receptors, on vasoactive mediator release and vascular tone were studied using rings of rat aorta. Stimulation of aortic rings with the thrombin receptor agonist, Trap-14, or the PAR-2 agonist, SLIGRL, resulted in a rapid release of nitric oxide. Trap-14 and SLIGRL-induced nitric oxide release was reduced by pre-treatment with BQ-788, an ETB endothelin receptor-specific antagonist. Consistent with a role for endothelin-1 receptor activation in Trap-14 and SLIGRL-induced nitric oxide release, endothelin-1 levels were increased significantly following 5 min treatment of aortic rings with Trap-14 or SLIGRL. Cumulative addition of Trap-14 to aortic rings denuded of endothelium resulted in dose-dependent contraction with an EC50 value of 23 +/- 5 microM, whereas SLIGRL addition failed to induce aortic contraction. These data suggest that the known protease activated receptors are functionally coupled to nitric oxide release. In addition, the thrombin receptor appears to modulate both vasodilator and contractile responses, whereas the PAR-2 receptor is linked only to vasodilation.

Opioid and opiate immunoregulatory processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel delta, mu, and kappa receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel mu3, opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Presence of the mu3 opiate receptor in endothelial cells. Coupling to nitric oxide production and vasodilation.

Initial confinement of opiate receptors to the nervous system has recently been broadened to several other cell types. Based on the well established hypotensive effect of morphine, we hypothesized that endothelial cells may represent a target for this opiate substance. Endothelial cells (human arterial and rat microvascular) contain a high affinity, saturable opiate binding site presumed to mediate the morphine effects that is stereoselectively and characteristically antagonized by naloxone. This opiate alkaloid-specific binding site is insensitive to opioid peptides. It is, therefore, considered to be the same subtype of opiate receptor (designated mu3) used in the mediation of morphine in other cell types exhibiting the same binding profile. Experiments with endothelial cultures and the aortic ring of rats cultured in vitro demonstrate that morphine exerts direct modulatory control over the activities of endothelial cells, which leads to vasodilation. It induces the production of nitric oxide, a process that is sensitive to naloxone antagonism and nitric oxide synthase inhibition. In contrast with that of opiates, the administration of opioid peptides does not induce nitric oxide production by endothelial cells. In conclusion, the data presented above reveal a novel site of morphine action, endothelial cells, where a mu3 receptor is coupled to nitric oxide release and vasodilation.

Detection of endothelial cell-derived nitric oxide: current trends and future directions.

The vascular endothelium is a significant site of NO release that inhibits cellular adhesion and maintains a non-thrombogenic surface. Use of newly described technology suggests for the first time that the maximal release of NO induced by cNOS and iNOS activation may be quite similar, implying that it is the duration of NO release and not the concentration of NO produced from stimulated endothelial cells that accounts for the different biological activities of the enzymes. The respective roles of cNOS and iNOS must be carefully evaluated since both enzymes may have potent biological effects at local sites of production.

Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase.

There is accumulating evidence that endothelial cells express a non-isopeptide-selective endothelin (ET) receptor, ETB, which may be responsible for ET-1-induced transient vasorelaxation. The purpose of the present study was to seek direct evidence for ETB receptor expression in human umbilical vein endothelial cells (HUVEC) and to characterize its functional role in HUVEC and in Chinese hamster ovary cells stably transfected with ETB receptor cDNA (CHO-ETB). Reverse polymerase chain reaction using HUVEC total RNA and ETB receptor-specific oligonucleotide primers firmly demonstrated the presence of an endogenous transcript of the appropriate molecular size. Next, a biotinylated ligand specifically recognizing the ETB receptor, IRL-1620, was synthesized, and immunocytochemical mapping of binding sites was performed in CHO-ETB cells. Specific binding of biotinylated IRL-1620 was evident in CHO-ETB cells, confirming appropriate cell surface receptor expression. Continuous nitric oxide (NO) monitoring with NO-selective electrode revealed a dose-dependent ET-1 stimulation of NO production by HUVEC. Stable transfection of CHO-ETB cells with endothelial nitric oxide synthase (NOS), but not mock-transfection, imparted responsiveness to ET-1 similar to that for HUVEC and was characterized by the immediate release of NO. Protein tyrosine kinase-dependent and calcium-calmodulin-dependent pathways were involved in ET-1-induced activation of the constitutive NOS in CHO-ETB/NOS cells, but coupling of the receptor to the enzyme in HUVEC appeared to be predominantly protein tyrosine kinase-dependent. Although sufficient, calcium/calmodulin system was not an obligatory prerequisite for the ET-1-induced activation of NOS in HUVEC. In conclusion, using two cell systems, we demonstrated that the ETB receptor is functionally coupled to NOS and coordinates the generation of NO via a tyrosine kinase-dependent and a calcium/calmodulin-dependent pathway.

Vascular contractile potency of endothelin-1 is increased in the presence of monocytes or macrophages.

Accumulation of inflammatory cells and altered responsiveness to vasoactive mediators are commonly observed events in atherosclerotic vessels. We studied the effect of monocytic cells on endothelin-1 (ET-1)-induced contraction of strips of guinea pig carotid artery. The vascular contractile potency of ET-1 was increased markedly in the presence of human peripheral blood monocytes, guinea pig alveolar macrophages (M phi), and the human monocytic cell line, THP-1. Specific binding of 125I-labeled ET-1 to these cells was detected, and Scatchard analysis indicated a dissociation constant value of approximately 1 nM. In contrast, the human monocytic cell line, U-937, failed to bind 125I-ET-1 and did not alter ET-1 potency, suggesting that the ability of monocytic cells to increase ET-1 potency requires expression of ET receptors. Selective inhibition of ET-1 binding to vascular smooth muscle with BQ-123, an ETA receptor antagonist that does not inhibit ET-1 binding to monocytes, resulted in complete inhibition of vascular contraction. These data indicate that ET-1-induced vasoconstriction may be increased by monocytic cells via stimulation of monocyte endothelin receptors.

Endothelin-receptor interactions. Role of a putative sulfhydryl on the endothelin receptor.

The mechanism of action of endothelin-receptor interactions was studied, using radioligand binding assays and SDS-PAGE, to investigate the possibility of disulfide interchange. Electrophoretic analysis suggested involvement of disulfide bond(s) in the receptor-ligand complex. Treatment of Et receptors with sulfhydryl-specific alkylating reagents (NEM or others) resulted in decreased ability to bind [125I]Et-1. [Dpr1-Asp15]Et-1, an antagonist homologous to Et but with an amide link replacing one of the disulfides, bound to Et receptors reversibly, but binding of Et-1 was less reversible. Preincubation of receptors with Et-1, but not with [Dpr1-Asp15]Et-1, protected receptors from alkylation with [14C]NEM. The data suggest that the Et receptor has a sulfhydryl group at or near the Et binding site. A model is proposed in which the role of the putative sulfhydryl group is discussed.