Within-field damage and distribution patterns of the stalk borer, Eldana saccharina (Lepidoptera: Pyralidae), in sugarcane and a comparison with nematode damage.

The occurrence of Eldana saccharina (Lepidoptera: Pyralidae) was monitored in grids represented by plots in 12 nematicide trials in South African sugarcane fields. The trials encompassed a total of eight plant cane crops and 22 ratoon crops and were situated within commercial cane fields. Several measurements were made to characterize the damage caused by E. saccharina. These included the number of internodes per stalk, the percentage of internodes damaged and the percentage of stalks damaged. The mapping of E. saccharina infestation in plant crops of sugarcane showed that the borders of the trials were as infested as the centre, indicating invasion from outside the field plus internal spread within the field. Ratoon crops were less infested than plant crops. This could be explained by a shorter ratoon crop cycle and by the fields having areas that were more suitable for the borer than elsewhere. The location of these preferred areas could be predicted from one ratoon crop to the next but was not related to the distribution of the borer in the plant crop. This situation was thought to explain the apparent stabilization of E. saccharina infestation in ratoon cane. Because the borer was found at harvest only in stalks with more than 14 to 16 internodes, it appeared that the oldest shoots, or the shoots with the greatest growth potential, attracted the insect, possibly due to their higher nitrogen content, which would stimulate growth. All the trials were on sandy soil, and crop loss from nematodes was greater than that caused by E. saccharina.

Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

Cholinergic regulation of morphine release from human white blood cells: evidence for a novel nicotinic receptor via pharmacological and microarray analysis.

Recent work from our laboratory has demonstrated that human white blood cells make morphine and that substances of abuse, i.e. nicotine, alcohol and cocaine have the ability to release this endogenous substance, suggesting a common mechanism of action. We now demonstrate that the nicotinic process is more complex than formerly envisioned. The incorporation rate of 125I-labeled morphine into PMN and MN are 7.85+/-0.36%, 1.42+/-0.19%, respectfully, suggesting in MN this process is of low activity. Separate incubations of PMN with varying concentrations of nicotine or the nicotine agonist epibatidine resulted in a statistically significant enhancement of 125I-trace labeled morphine released into the extracellular medium. In order to ascertain the specificity of the nicotine stimulated morphine release the following experiments were performed. Co-incubation of hexamethonium dichloride (5 microg/ml and at 10 microg/ml), which preferentially blocks nicotinic receptors at autonomic ganglia, with nicotine, exerted a very weak inhibitory effect. Co-incubation of alpha-BuTx or atropine or chlorisondamine diiodide or dihydro-Beta-erythroidine hydrobromide, an alpha4Beta2 receptor antagonist, did not block nicotine induced morphine release alone or in combination, suggesting either the response was not specific or it was mediated by a novel nicotinic receptor. Human leukocyte total RNA isolated from whole blood were analyzed, using the Human Genome Survey microarray (Applied Biosystems), for cholinergic receptor expression. PMN nicotinic receptor gene expression was present and contained numerous variants (eight). The number of variants suggests that indeed a novel nicotinic receptor may be mediating this effect, while simultaneously demonstrating the significance of the cholinergic receptor expression in these immune cells.

Morphine inhibits AP-1 activity and CD14 expression in leukocytes by a nitric oxide and opioid receptor-dependent mechanism.

BACKGROUND: Activator protein 1 is a transcription factor involved in the regulation of proinflammatory mediators. Activation of phagocytes by lipopolysaccharide depends on the expression of CD14 on the cell surface. In this study, we investigated the effects of morphine and nitric oxide on CD14 expression and activator protein 1 activation in human blood monocytes and neutrophils as well as the leukocyte cell line HL-60. METHODS: Whole blood was incubated with morphine, the nitric oxide donor S-nitroso-N-acetyl-penicillamine, naloxone or nitric oxide synthase inhibitors Nomega-nitro-l-arginine and Nomega-nitro-l-arginine-methylester and stimulated with lipopolysaccharide. Activator protein 1 nuclear content was determined by flow cytometry in human blood neutrophils and monocytes. CD14 expression on neutrophils was measured after incubation with fluorescein isothiocyanate-labelled antibodies. Electric mobility shift assay served for evaluation of activator protein 1 nuclear binding in HL-60 cells. RESULTS: Incubation of whole blood with morphine and subsequent stimulation with lipopolysaccharide decreased activator protein 1 nuclear content. Exposure to naloxone before morphine treatment abolished morphine-induced inhibition of activator protein 1 activity in human blood monocytes and neutrophils. Nitric oxide synthase inhibitors also reversed morphine's effects. CD14 expression on neutrophils was reduced after morphine treatment. These effects were antagonized by nitric oxide synthase inhibitors and naloxone. CONCLUSION: Morphine inhibits activator protein 1 activation by a mu opioid receptor pathway coupled to nitric oxide as second messenger. The decrease in CD14 expression caused by morphine may play a role in inhibition of activator protein 1 activation following lipopolysaccharide treatment of phagocytes.

Morphine enhances nitric oxide release in the mammalian gastrointestinal tract via the micro(3) opiate receptor subtype: a hormonal role for endogenous morphine.

Studies from our laboratory have revealed a novel micro opiate receptor, micro(3), which is expressed in both human vascular tissues and leukocytes. The micro(3) receptor is selective for opiate alkaloids, insensitive to opioid peptides and is coupled to constitutive nitric oxide (cNO) release. We now identify the micro(3) receptor characteristics in mammalian gut tissues. It appears that the various regions of the mouse gut release low levels of NO (0.02 to 4.6 nM ) in a pulsatile manner. We demonstrate that morphine stimulates cNO release (peak level 17 nM) in the mouse stomach, small intestine and large intestine in a naloxone and L-NAME antagonizable manner. Opioid peptides do not exhibit cNO-stimulating capabilities in these tissues. Taken together, we surmise morphine acts as a hormone to limit gut activity via micro(3) coupled to NO release since micro opiate receptors are found in the gut and endogenous morphine is not but is found in blood.

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype.

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [3H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present.

Real-time RT-PCR measurement of the modulation of Mu opiate receptor expression by nitric oxide in human mononuclear cells.

BACKGROUND: In previous studies, we have attributed opiate alkaloid selectivity to a subtype of the neuronal mu receptor known as mu3, expressed on human blood cells. Opiate alkaloid activation of this receptor subtype leads to the release of constitutively derived nitric oxide. In this report, we show by real-time RT-PCR that the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) initiates the down regulation of mu receptor gene expression in human mononuclear cells after 30 minutes. Superoxide dismutase, a free radical scavenger, blocks the effect of SNAP. MATERIAL AND METHODS: Human mononuclear cells isolated from whole blood were treated with SNAP (100 microM), and also with SNAP plus superoxide dismutase (100 U/ml) at different time points. Real-time RT-PCR with total RNA extracted from the cells was used to analyze expression of the mu opiate receptor. RESULTS: Mu opiate receptor gene expression was significantly down regulated in cells treated with SNAP at 30 min, and superoxide dismutase blocked the effect of SNAP. At 2 and 6 hours, a rebound effect was observed as noted by an increase in mu receptor expression, and at 24 hours mu receptor expression returned to control levels in the SNAP-treated cells. CONCLUSIONS: This study confirms that human mononuclear cells express the mu opiate receptor transcript and demonstrates that nitric oxide is involved in regulation of its expression.

Ischemic preconditioning - an opiate constitutive nitric oxide molecular hypothesis.

Coronary artery disease is the number one cause of adult mortality due to a medical illness in the United States. Exciting new studies are looking at the role transient ischemia may play in preconditioning the myocardium to reduce the degree of infarction following a sustained ischemic insult. In this speculative review, we surmise ischemic preconditioning and the resulting protection afforded by it in response to abnormal insults arises from an already existing physiological process that may be associated with exercise. A brief ischemic episode mimics the cells response to normal dips in ATP levels caused by metabolic demand. In so doing, via constitutive nitric oxide synthase derived nitric oxide, it temporarily down regulates a cells excitatory state, thus protecting it from the next insult. Within this context, opiate and opioid actions can be incorporated into the protection scenario, as can other signal molecules since they may release nitric oxide. Instead of ischemia inducing nitric oxide release via a drop of ATP levels, various signal molecules, such as opiate alkaloids, have their cell surface receptors coupled to constitutive nitric oxide synthase thereby releasing nitric oxide, initiating associated cell activity dampening action. In conclusion, it appears as though endogenous nitric oxide stimulators offer their selective preconditioning protection by joining an already existing process that limits activation following normal physical exertion.

Morphine inhibits indolactam V-induced U937 cell adhesion and gelatinase secretion.

We demonstrate that indolactam V, a non-phorbol protein kinase C activator, promotes U937 cell attachment to fibronectin, type IV collagen and laminin. In the absence of indolactam V, 2-4% of U937 cells attach to all test substrates, however, in the presence of 100 nM indolactam V, 25, 16 and 11% of U937 cells attach to fibronectin, type IV collagen and laminin, respectively. When added concomitantly, 90 microM H-7, a protein kinase C inhibitor, reduces indolactam V-induced U937 cell adhesion to fibronectin by 91%. Monoclonal antibodies directed against both the beta1 and alpha 5 integrin subunits inhibit indolactam V-induced U937 cell adhesion to fibronectin by 62 and 52%, respectively. Indolactam V also promotes homotypic aggregation in U937 cells, which is blocked with either anti-ICAM or anti-LFA-1 antibodies. In addition, indolactam V promotes U937 cell secretion of a 92 kDa gelatinase as demonstrated by zymography. In the presence of low levels of morphine (10 nM-1.0 microM), the U937 cell attachment to matrix proteins was not significantly affected. However, in the presence of 10 microM morphine, the indolactam V treated cells exhibit a 71-74% reduction in cell adhesion to the matrix proteins. Further, 10 microM morphine also blocks indolactam V-induced homotypic aggregation and gelatinase secretion. The inhibitory effect of morphine on cell-matrix adhesion and gelatinase secretion was not inhibited by the opiate receptor antagonist naloxone (1 microM). While 10 microM naloxone did partially counteract the effect of 10 microM morphine on U937 cell attachment, this effect was likely non-specific since 10 microM naloxone alone increased cell adhesion. Supporting this conclusion, PCR analysis revealed that U937 cells do not express the mu high affinity morphine receptor. Also, indolactam V did not induce mu receptor expression, suggesting that morphine acts on U937 cells in a non-specific fashion.

Morphine reduces herpes simplex virus-1 pathogenesis in the murine flank.

Here we investigate the effect of morphine on herpes simplex virus-1 (HSV-1) pathogenesis using a murine flank scarification model. Murine flank scarification with HSV-1 results in primary lesions at the site of inoculation within three days and lesions at secondary sites within four days. The lesions are scored based on lesion size. Applying 0.1 mM morphine to the skin one-day post inoculation tested the effect of morphine on the formation of the herpes lesion. On days three through five, mice treated with morphine developed lesions with scores half of that observed in untreated animals, however, skin viral titers on these days were equivalent. Further, 1.0 microM morphine did not effect the replication rate of HSV-1 in Vero cells. Taken together, these data suggest the morphine reduced HSV-1 pathogenesis by modifying the host response to HSV-1 infection and not by reducing viral replication rates.

HIV gp120 and morphine alter mu opiate receptor expression in human vascular endothelium.

We find that chronic exposure of human saphenous vein, atria and internal thoracic artery endothelium to the human immunodeficiency virus surface glycoprotein gp120, results in an increase in endothelial mu opioid receptor expression (52%). gp120 acts, in this regard, as a proinflammatory cytokine (e.g. interleukin-1-alpha) by increasing endothelial mu opioid receptor expression. In contrast, morphine decreases mu opioid receptor expression by 90% in a dose dependent fashion. Pretreatment of these tissues with the respective antagonists e.g., naloxone and anti-gp120 blocks the opiate decrease and increase gp120 induced increase in mu expression, respectively. Further, pretreatment of these endothelia with morphine inhibits gp120-stimulated mu transcript expression. Therefore, the immune down-regulating action of morphine may prevent viral replication because this process requires immune activation that can, in part, be provided for by gp120 proinflammatory actions.

Vascular pulsations stimulating nitric oxide release during cyclic exercise may benefit health: a molecular approach (review).

It is widely assumed that all exercise, regardless of the degree of difficulty or strenuousness, is good (no pain-no gain). In this speculative review of the literature and our research findings we highlight the fact that strenuous exercise taken to the extreme initiates an immune and vascular proinflammatory situation. However, mild cyclic exercise appears to produce health benefits for an individual. In part, this is due to vascular cyclic pulsations, occurring in mild exercise, stimulating constitutive nitric oxide synthase derived nitric oxide release. This in turn down-regulates vascular endothelial cells and immunocytes, as well as their interaction and inhibits the disassociation of NF-kappaB, preventing the production of proinflammatory cytokines. The nitric oxide so generated may even scavenge excess free radicals, preventing tissue damage. Prolonged strenuous exercise appears to limit these positive phenomena because of the maintained and prolonged high blood pressure that reduces the cyclic pulsations, limiting nitric oxide production. We further note that pathological conditions, i.e., Parkinson's disorder, may benefit from mild exercise, i.e., cyclic nitric oxide production, since the inactivity associated with this disease may lead to compromised nitric oxide production, initiating a progressive deterioration of tissues, including peripheral adrenergic neurons, due to a lack of adequate basal nitric oxide levels required to maintain the vascular microenvironment in a mild state of inhibition. We conclude that mild exercise represents an alternate and economical therapy to preserve health and/or diminish the rate of decline of the normal physiological processes that may even be associated with aging.

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.

Identification of morphine in the adrenal medullary chromaffin PC-12 cell line.

Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.746+/-0.615 ng of morphine/million cells) were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived from the human neuronal mu 1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from this cell line. The transcript was absent. The study conclusively proves morphine, but not a mu opiate receptor, is constitutively expressed in the adrenal medulla chromaffin PC-12 cell line.

Endogenous morphine.

It is now well accepted that endogenous morphine is present in animals, both in invertebrates and vertebrates. It is a key signaling molecule that plays an important role in downregulating physiological responses, such as those in the immune system, including immune elements in the CNS. It has been demonstrated that a specific mu-opiate-receptor subtype, mu3, mediates these downregulatory effects through release of NO. This article examines morphine as an endogenous signaling molecule, in terms of its role in neural and immune regulation.

Human vascular and cardiac endothelia express mu opiate receptor transcripts.

Pharmacologic and immunologic evidence suggests that nitric oxide-coupled mu-subtype opiate receptors are expressed in human vascular endothelium. In this study, we present molecular evidence of mu opiate receptor expression. Using primers derived from the human neuronal mu1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from human endothelia. Sequence analysis of the RT-PCR products revealed 100% identity with the neuronal human mu1 receptor. We further show that pretreatment of human internal thoracic artery and cardiac atrial endothelium with the proinflammatory cytokines interleukin-1-alpha and -beta led to a significant increase in both the expression of the mu transcript and in morphine-stimulated nitric oxide release measured amperometrically. Taken together, these studies provide molecular evidence that mu-type opiate receptors are expressed in human vascular endothelia and that their expression can be upregulated by proinflammatory cytokines.

Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor.

We tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17beta-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17beta-estradiol conjugated to bovine serum albumin (E(2)-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17beta-estradiol and E(2)-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17beta-estradiol-stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca(2+)]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N', N'-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca(2+)]i chelator, reduced [Ca(2+)]i in response to E(2)-BSA, and depleting [Ca(2+)]i stores abolished the effect of 17beta-estradiol on NO release. Confocal photomicrographs using E(2)-BSA-FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase-polymerase chain reaction, human neutrophil granulocytes expressed ERalpha but not ERbeta, suggesting that ERalpha may be the membrane receptor for 17beta-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca(2+)]i. (Blood. 2000;95:3951-3958)

Morphine inhibits NF-kappaB nuclear binding in human neutrophils and monocytes by a nitric oxide-dependent mechanism.

BACKGROUND: The transcription factor NF-kappaB plays a pivotal role in gene expression of inflammatory mediators such as cytokines or adhesion molecules. NF-kappaB-mediated transcriptional activation of these genes is inhibited by nitric oxide (NO) in a variety of cells, including monocytes. Morphine mediates NO release in a naloxone antagonizable manner in monocytes and neutrophils. METHODS: The influence of morphine on NF-kappaB activation was investigated in a whole-blood flow cytometric assay. A specific antibody against the p65 subunit of NF-kappaB was used and detected by fluoresceine-isothiocyanate-labeled anti-immunoglobulin G. Nuclei were stained with propidium iodide. Leukocyte subpopulations were evaluated by gating on neutrophils and monocytes. The median fluorescence channel was determined. Different morphine concentrations (50 nm, 50 microm, 1 mm) and incubation intervals (10-150 min) were used. RESULTS: Morphine inhibits lipopolysaccharide-induced NF-kappaB nuclear binding in human blood neutrophils and monocytes in a time-, concentration-, and naloxone-sensitive-dependent manner. Similar effects were achieved with the NO donor S-nitroso-N-acetyl-pencillamine and the antioxidant N-acetyl-cysteine. The NO synthase inhibitors Nomega-nitro-l-arginine-methyl-esther and Nomega-nitro-l-arginine completely abolished the morphine-induced attenuation of NF-kappaB nuclear binding, demonstrating that the inhibitory action is mediated by NO release. CONCLUSION: Morphine causes immunosuppression, at least in part, via the NO-stimulated depression of NF-kappaB nuclear binding.

Ascaris suum, an intestinal parasite, produces morphine.

The parasitic worm Ascaris suum contains the opiate alkaloid morphine as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of this material is 1168 +/- 278 ng/g worm wet weight. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a naloxone-reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals' nerve chords. Furthermore, as determined by RT-PCR, Ascaris does not express the transcript of the neuronal mu receptor. Failure to demonstrate the expression of this opioid receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment.

Cell-surface estrogen receptors mediate calcium-dependent nitric oxide release in human endothelia.

BACKGROUND: Although estrogen replacement therapy has been associated with reduction of cardiovascular events in postmenopausal women, the mechanism for this benefit remains unclear. Because nitric oxide (NO) is considered an important endothelium-derived relaxing factor and may function to protect blood vessels against atherosclerotic development, we investigated the acute effects of physiological levels of estrogen on NO release from human internal thoracic artery endothelia and human arterial endothelia in culture. METHODS AND RESULTS: We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase activity in human endothelial cells by acting on a cell-surface receptor. NO release was measured in real time with an amperometric probe. 17beta-Estradiol exposure to internal thoracic artery endothelia and human arterial endothelia in culture stimulated NO release within seconds in a concentration-dependent manner. 17beta-Estradiol conjugated to bovine serum albumin also stimulated NO release, suggesting action through a cell-surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized this action. We further showed with the use of dual emission microfluorometry that 17beta-estradiol-stimulated release of endothelial NO was dependent on the initial stimulation of intracellular calcium transients. CONCLUSIONS: Physiological doses of estrogen immediately stimulate NO release from human endothelial cells through activation of a cell-surface estrogen receptor that is coupled to increases in intracellular calcium.