Estradiol coupling to endothelial nitric oxide stimulates gonadotropin-releasing hormone release from rat median eminence via a membrane receptor.

The median eminence (ME), which is the common termination field for adenohypophysiotropic systems, has been shown to produce nitric oxide (NO), a signaling molecule involved in neuroendocrine secretion. Using an ex vivo technique, 17beta-estradiol exposure to ME fragments, including vascular tissues, stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol or testosterone had no effect. 17Beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA. Furthermore, estradiol-stimulated NO stimulates GnRH release. This was demonstrated by hemoglobin (a NO scavenger), N(omega)-nitro-L-arginine methyl ester, and L-N5-(1-iminoethyl)ornithine (nitric oxide synthase inhibitors) inhibition of estradiol stimulated NO and GnRH release. In this regard, L-N5-(1-iminoethyl)ornithine, specific for endotheliol constitutive nitric oxide synthase, was significantly more potent, suggesting that the estradiol-stimulated NO release arose from vascular endothelial cells. Additionally, the NO-stimulated GnRH release occurs via guanylyl cyclase activation in GnRH nerve terminals, as ODQ, a potent and selective inhibitor of NO-sensitive guanylyl cyclase, abolished the estradiol-stimulated GnRH release. The results suggest that at physiological concentrations, 17beta-estradiol may have immediate actions on ME endothelial cells via nongenomic signaling pathways leading to NO-stimulated GnRH release.

Mu3 opiate receptor expression in lung and lung carcinoma: ligand binding and coupling to nitric oxide release.

The mu3 opiate receptor subtype is expressed in human surgical specimens of both normal lung and non-small-cell lung carcinoma. Nitric oxide (NO) release is mediated through the mu3 receptor, and in lung carcinoma, morphine-stimulated NO release is significantly higher and prolonged than in normal lung. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis we show that specific mu opioid receptor transcripts are present in lung carcinoma and other cells with the mu3 profile. Our findings identify a unique role for the mu3 opiate receptor in opiate-mediated NO release and suggest that endogenous opiates, through their release of NO, may play a role in cancer progression.

Functional assessment of disease-free saphenous vein grafts at redo coronary artery bypass grafting.

BACKGROUND: Reoperations for coronary artery bypass grafting are on the rise. The general rule of replacing all saphenous vein grafts (SVGs) older than 5 years of age at the time of reoperation has recently been challenged on clinical grounds. This study provides functional data of endothelial behavior in long-term vein grafts. METHODS: Previously placed SVGs were removed at the time of redo operations. Nitric oxide (NO) measurements in real time were carried out before and after stimulation with morphine. The measurements were compared to the angiographic appearance of the grafts obtained prior to operation. Grafts were categorized into 3 groups: disease-free, moderately diseased, and severely diseased. RESULTS: Sixteen grafts were analyzed. Five were angiographically disease-free, 4 had moderate, and 7 severe disease. In the disease-free group, peak NO production after 10(-6) mol/L morphine stimulation was 35 mol/L, equivalent to the production of native saphenous vein. The severely diseased group did not demonstrate an increase in NO production, and the moderately diseased group produced a small rise in production. CONCLUSIONS: Measurement of NO release of old SVGs, when angiographically pristine, equals that of native saphenous vein. These findings support the recent clinical observations that long-term angiographically disease-free vein grafts are biologically privileged.

Anandamide amidase inhibition enhances anandamide-stimulated nitric oxide release in invertebrate neural tissues.

Anandamide, an endogenous cannabinoid signaling molecule, in a concentration dependent manner, initiates the release of nitric oxide (NO) from leech and mussel ganglia. SR 141716A, a cannabinoid antagonist, blocks the anandamide stimulated release of NO from these tissues. Methyl arachidonyl fluorophosphonate (MAFP), a specific anandamide amidase inhibitor, when administered to either ganglia with anandamide (10-6 M) did not increase the peak level of NO release but did significantly extend NO release from 12 to 18 min (P<0.05). Lower levels of anandamide (10-8 and 10-7 M) do not stimulate the release of significant amounts of NO from these tissues. However, in the presence of MAFP (2.5 nM), the lower anandamide concentrations were able to release significant peak amounts of NO. In mussel neural tissues, the peak NO release increased from 2.2+/-1.3 nM to 8.6+/-2.1 nM. Taken together, the results indirectly demonstrate the presence of anandamide amidase in these tissues, suggesting that the enzyme may serve as an endogenous regulator of anandamide action.

Morphine and anandamide coupling to nitric oxide stimulates GnRH and CRF release from rat median eminence: neurovascular regulation.

Nitric oxide (NO) is involved in neurohormonal secretion from median eminence neuroendocrine nerve terminals. We report that stimulation of NO release from median eminence fragments including vascular tissues occurs by mu3 receptor activation by morphine, or by cannabinoid type 1 receptor activation by anandamide. The released levels of NO are lower after anandamide than after morphine stimulation. These processes can be blocked by L-NAME, a specific nitric oxide synthase inhibitor, by naloxone for the morphine-stimulated NO release, or SR 141716A, a specific CB1 receptor inhibitor, for the anandamide-stimulated NO release. Furthermore, morphine and anandamide, by this NO dependent process, influences neurohormonal release from median eminence nerve terminals within 10 min. Via this NO dependent process, morphine stimulates both GnRH and CRF release, whereas anandamide selectively stimulates GnRH release. These observations together with previous data suggest that morphine and the anandamide-stimulated NO originates from the vascular endothelium of the portal plexus. These results indicate that endothelial cells of the median eminence may be involved in the release of neurohormones.

Morphine- and anandamide-stimulated nitric oxide production inhibits presynaptic dopamine release.

Morphine and anandamide stimulate the release of nitric oxide (NO) in diverse tissues. The present study examines the consequences of this action on neurotransmitter release in ganglia from two invertebrates: ventral chain ganglia from the leech Hirudo medicinalis and the pedal ganglion from the mussel Mytilus edulis. In these ganglia, preloaded serotonin (5-HT) and dopamine (DA) can be released by 50 mM KCl. Anandamide, an endogenous cannabinoid substance, suppresses the potassium-stimulated release of [3H]DA (80%), but not 5-HT, in a concentration-dependent manner, from the neural tissues of both. The effect of anandamide can be antagonized by pre-exposing the neural tissues of both animals to SR 141716A, a potent cannabinoid receptor antagonist. Prior treatment of the ganglia with N-omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, significantly diminishes the inhibitory effect of anandamide. Morphine also inhibits [3H]DA release in a naloxone- and L-NAME-sensitive manner. Anandamide and morphine act through separate mechanisms since the respective antagonists show no cross-reactivity. The NO donor, SNAP, depressed the potassium-stimulated release of preloaded [3H]DA, but not 5-HT, in the neural tissues of both animals. D-Ala2-Met5 enkephalinamide (DAMA) also inhibited the potassium-stimulated release of [3H]DA in a naloxone-sensitive process. However, the effect of DAMA was seen in the presence of L-NAME (10(-4) M), indicating that the opioid peptide inhibition of the presynaptic release of DA is not coupled to NO. We postulate that cannabinoids and their endogenous effectors play a prominent role in the regulation of catecholamine release in invertebrates via NO release as is the case for opiate alkaloids.

Dopamine and morphine stimulate nitric oxide release in human endometrial glandular epithelial cells.

OBJECTIVE: Previous studies have shown that human endometrial glandular epithelial cells contain endothelial nitric oxide synthase indicating that the endometrium might produce nitric oxide (NO). We conducted this study to identify stimuli that can activate a transient NO release from endometrial glandular epithelial cells because NO is an important intracellular and intercellular signal transduction pathway in reproductive cycle. METHODS: Endometrial glandular epithelial cells, free of endothelial cells, were isolated from human endometrial specimens and maintained viable in RPMI 1640 medium with 2% fetal bovine serum for 2-4 days. Nitric oxide release from the glandular cells in response to stimuli was monitored continuously amperometrically. RESULTS: Among the substances examined, we found that dopamine and morphine stimulated a transient surge of NO production that was dose-dependent, whereas estrogen, progesterone, or relaxin (RLX) had no short-term effect on NO release. Cells treated with RLX or dopamine for 4 days enhanced the dopamine-induced NO release fourfold to sixfold, with the peak of the NO surge shifting from 35 to 15 seconds. CONCLUSION: Endometrial glandular cells were capable of producing NO. Dopamine and morphine were potent stimuli for a transient surge of NO release from endometrial glandular cells. Furthermore, prolonged exposure to dopamine or RLX enhanced the sensitivity of NO release in endometrial glands.

Macrophage behavior associated with acute and chronic exposure to HIV GP120, morphine and anandamide: endothelial implications.

We demonstrate that immediate exposure to gp120 (5 min; 0.1 microg/ml) results in a significant shift of the macrophage population to an amoeboid and motile category (P<0.01; 91.7+/-5.5 vs. a control value of 42.4+/-4.2) and prior exposure with anti-gp120 antagonizes this shift. Acute exposure of the macrophages to morphine (10(-6) M) or anandamide (10(-6) M) resulted in the cells rounding up (shape factors of 0.84 and 0.87 respectively) and becoming non-motile. The action is blocked by prior treatment with the specific antagonists naloxone and SR 141716A. Chronic exposure (6 h) of the cells to all three agents resulted in a random migration pattern. Further, all agents blocked chemotaxis induced by DAMA and IL-1. Observation of the cells behavior during chronic exposure revealed a sporadic activity pattern with gp120 whereas morphine and anandamide first induced a period of inactivity which is followed by a period of activity (chemokinesis). Recent work from our laboratory has demonstrated that both morphine and anandamide acutely stimulate constitutive macrophage nitric oxide (NO) release, which then induces macrophage rounding and inactivity. It was therefore of interest to examine their behavior by exposing macrophages to the NO-donor SNAP. In a concentration dependent manner SNAP exhibited the same behavioral actions as both substances of abuse. Given this, we next determined if macrophages exposed to gp120 would release NO. We demonstrated that NO was released only when exposed to morphine and anandamide not gp120. Thus. the chemokinetic inducing activities of these agents may be the basis for excitotoxin liberation in neural tissues and/or a higher viral load in various organ systems since cellular adherence and random migration are stimulated.

Nociceptin, endomorphin-1 and -2 do not interact with invertebrate immune and neural mu 3 opiate receptor.

AIM: To determine if endomorphin-1, -2 and nociceptin (orphanin FQ) bind to the mu 3 opiate receptor subtype or release nitric oxide as mu 3 selective ligands do. METHODS: These opioid peptides were examined for their ability to displace [3H]dihydromorphine (DHM) binding from the invertebrate (immunocytes and pedal ganglia) mu 3 opiate receptor in membrane homogenates. The ligands were also tested for their ability to release nitric oxide from the same intact tissues utilizing an amperometric probe that measures nitric oxide in real-time. RESULTS: Endomorphin-1, -2 and nociceptin do not displace [3H]DHM binding from immunocyte or pedal ganglia membrane homogenates nor do they release nitric oxide from these tissues. CONCLUSION: Since these newly discovered opioid peptides do not interact with the mu 3 opiate receptor subtype, endogenous morphine's significance is enhanced because it appears to be the only naturally occurring opiate ligand for the receptor. Furthermore, since this study involves invertebrate tissues, this signal system had to evolve early during evolution.

Endomorphin-1 and -2 inhibit human vascular sympathetic norepinephrine release: lack of interaction with mu 3 opiate receptor subtype.

AIM: To determine if endomorphin-1 (End-1) and -2 (End-2) interact with mu 3 opiate receptor subtype and in this way cause vascular hypotension. METHODS: Amperometric nitric oxide (NO) determinations associated with opiate binding displacement analysis and preloaded [3H]norepinephrine KCl stimulated release in human vascular tissues from sympathetic nerve fibers in vitro. RESULTS: The endomorphins did not release NO from human monocytes, granulocytes, saphenous vein, and internal thoracic artery endothelium and did not displace opiate alkaloid binding to mu 3 receptor. However, they did inhibit KCl-stimulated [3H]norpinephrine release from vascular nerves. CONCLUSION: The data strongly suggested that End-1 and -2 caused hypotension by blocking sympathetic vascular sympathetic activity.

2-arachidonyl-glycerol stimulates nitric oxide release from human immune and vascular tissues and invertebrate immunocytes by cannabinoid receptor 1.

The pharmacological physiological effects of the endogenous cannabinomimetic (endocannabinoid) anandamide have been well characterized. Another endocannabinoid, 2-arachidonoyl-glycerol (2-AG), has been less-widely studied. 2-AG occurs in vertebrate and invertebrate tissues and binds to both cannabinoid receptors (CB1 and CB2). In the current study, 2-AG was found to cause human monocytes and immunocytes from Mytilus edulis to become round and immobile, which may correlate with decreased production of cytokines and adhesion molecules, i.e. an immunosuppressive response. In addition, exposure of these cells to 2-AG results in nitric oxide (NO) release, which is blocked by the nitric oxide synthase inhibitor, l-NAME and a CB1 antagonist, but not by a CB2 antagonist. The results obtained in the human vascular system were similar to those obtained in immune cells. Treatment of human saphenous veins and atria with 2-AG stimulated basal NO release, which was antagonized by l-NAME and a CB1 antagonist. Taken together these results indicate that 2-AG exerts immune and vascular actions similar to those observed with anandamide.

Nitric oxide mediates laminin-induced neurite outgrowth in PC12 cells.

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.

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.

Leech immunocytes contain proopiomelanocortin: nitric oxide mediates hemolymph proopiomelanocortin processing.

This report establishes the presence of mammalian-like proopiomelanocotropic hormone (POMC), and six of its peptides, including adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (MSH), in the immune tissues of the leech Theromyzon tessulatum. The 25.4-kDa protein was purified by high pressure gel permeation chromatography, anti-ACTH-affinity column, and reverse-phase HPLC. Its characterization was performed by Edman degradation, enzymatic treatments, and electrospray mass spectrometry. Leech POMC exhibits considerable amino acid sequence similarity to mammalian POMC. Of the six peptides, three showed high sequence similarity to their vertebrate counterparts met-enkephalin, alpha-MSH, and ACTH: 100, 84.6, and 70%, respectively; whereas gamma-MSH, beta-endorphin, and gamma-lipotropin hormone exhibited only 45, 20, and 10% sequence identity, respectively. No dibasic amino acid residues were found at the C terminus of the gamma- and beta-MSH peptides. In contrast, the leech alpha-MSH was flanked at its C-terminal by the Gly-Arg-Lys amidation signal. ACTH and corticotropin-like intermediary pituitary peptide were also C-terminally flanked by dibasic amino acid residues. The coding region of leech POMC was obtained by reverse transcription-PCR using degenerated oligonucleotide primers. Circulating levels of ACTH and MSH were 10 and 1 fmol/microl hemolymph, respectively. Morphine, in a dose-dependent manner, increased the levels of both peptides threefold; this effect was blocked by naloxone treatment. Similar results were found with the anandamide. Leech ACTH was processed to MSH by the enzymes neutral endopeptidase (24.11) and angiotensin-converting enzyme. Leech alpha-MSH had the same activity as authentic alpha-MSH in two bioasssay systems. Taken together, the study demonstrates that POMC is present in invertebrates and its immunoregulatory actions have been conserved during evolution.