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.

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.

NF-kappaB, nitric oxide and opiate signaling.

NF-kappaB, a DNA binding factor, has been implicated in inflammatory cytokine activation. NF-kappaB is activated by IkappaBalpha, its inhibitor, which is phosphorylated and proteolytically degraded. In this regard, NF-kappaB is also responsive to reactive oxygen intermediates and calcium. Reports also have emerged that demonstrate that nitric oxide inhibits NF-kappaB transcriptional activation in a variety of cells, including monocytes and endothelial cells. Recently, we have demonstrated that morphine, not opioid peptides, via the mu3 opiate receptor is coupled to constitutive nitric oxide release in these same cells. In this regard, we provide a scenario whereby morphine modulates NF-kappaB activation via nitric oxide. This pathway appears to be the key step in regulating inducible nitric oxide synthase expression, controlling the balance between constitutive nitric oxide synthase and the inducible form.

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.

Antagonism of LPS and IFN-gamma induced iNOS expression in human atrial endothelia by morphine, anandamide, and estrogen.

AIM: To determine whether inducible nitric oxide synthase (iNOS) stimulation of human atrial fragments can be diminished by the naturally occurring signal molecules, such as morphine, anandamide, and estrogen. The use of iNOS as an indicator is justified since it has been associated with initiation of various types of cellular damage either directly or indirectly. METHODS: Western blots were performed on control and drug-exposed atrial tissue before and after lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) exposure. RESULTS: Preincubation of the tissue with morphine, anandamide or estrogen prior to, but not after, the addition of LPS + IFN-gamma, blocked iNOS expression. The nitric oxide donor SNAP also blocked iNOS induction while preincubation of atrial fragments with an inhibitor of NOS, L-NAME, prior to morphine or anandamide exposure, restored LPS + IFN-gamma induction of iNOS. CONCLUSION: These data suggest a direct regulatory link at the transcriptional level between constitutive (c) NOS and iNOS in human atrial tissue.

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol 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, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

Opiate, cannabinoid, and eicosanoid signaling converges on common intracellular pathways nitric oxide coupling.

Scientific fields as they emerge initially appear to be unrelated to other projects even if they are in a similar area of interest. This is especially true in the case of opiate, cannabinoid, and eicosanoid signaling processes. In this limited speculative review, we attempt to examine aspects of their intracellular cascading signaling systems for their commonalities. We find intracellular calcium mobilization, nuclear factor kappa B involvement, adenylate cyclase activity, and, finally, constitutive nitric oxide release to be converging points for these signaling processes, occurring by separate and distinct receptor-mediated effector systems. Phosphokinase C, mitogen activated protein kinase, and cytosolic phospholipase A2 also represent points of common impact. In this regard, aspirin also appears to be involved in an aspect of this signaling convergence. We conclude that many of the physiological observations regarding the actions of these signaling molecules, for example, immunosuppression, neurotransmission, vasodilation, cellular adherence, and cytotoxicity, can now be understood by considering their converging biochemical cascades.

Morphine and anandamide stimulate intracellular calcium transients in human arterial endothelial cells: coupling to nitric oxide release.

Both morphine and anandamide significantly stimulated cultured endothelial intracellular calcium level increases in a concentration-dependent manner in cells pre-loaded with fura 2/AM. Morphine is more potent than anandamide (approximately 275 vs. 135 nM [Ca]i), and the [Ca]i for both ligands was blocked by prior exposure of the cells to their respective receptor antagonist, i.e., naloxone and SR 171416A. Various opioid peptides did not exhibit this ability, indicating a morphine-mu3-mediated process. In comparing the sequence of events concerning morphine's and anandamide's action in stimulating both [Ca]i and nitric oxide production in endothelial cells, we found that the first event precedes the second by 40+/-8 sec. The opiate and cannabinoid stimulation of [Ca]i was attenuated in cells leeched of calcium, strongly suggesting that intracellular calcium levels regulate cNOS activity.

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.

Pharmacological evidence for anandamide amidase in human cardiac and vascular tissues.

The present report demonstrates the presence of antianandamide and anticannabinoid receptor 1 immunopositive material on the saphenous vascular endothelium. The endogenous cannabinoid, anandamide, in a dose-dependent manner stimulated the release of nitric oxide (NO) from saphenous vein, internal thoracic artery and right atrium tissue segments in vitro. This process can be antagonized by the nitric oxide synthase (NOS) inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M; 3.4+/-0.9 nM NO; P<0.01 compared to anandamide alone), as well as by the cannabinoid receptor I antagonist SR 141716A (2.9+/-1.0 nM NO; P<0.01). Furthermore, in the presence of varying concentrations of methylarachidonylfluorophosphonate, an anandamide amidase inhibitor, 10(-8) M anandamide stimulates a higher peak level of NO that remains elevated for a longer period of time (P<0.05) compared to anandamide alone, demonstrating the presence of anandamide amidase in human vascular tissues. Morphine, as anandamide, can stimulate the release of NO from right atria. This process can be inhibited by the opiate receptor antagonist naloxone and the NOS inhibitor L-NAME. As expected SR 141716A (10(-6) M; 26+3.8 NO nM in the presence of 10(-7) M morphine) did not antagonize morphine's ability to release NO. Taken together, the data demonstrate that cannabinoid signalling is involved with the regulation of the microvascular environment.

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.

Methionine-enkephalin stimulates interleukin-6 mRNA expression: human plasma levels in coronary artery bypass grafting.

Opioid peptides have the ability to regulate immunocompetent cells. The present study extends this phenomenon to include an interaction with interleukin (IL)-6. We demonstrate that methionine-enkephalin (met-enkephalin) stimulates IL-6 mRNA expression in mice as determined by RNA isolation and hybridization with IL-6 cDNA. In mice, met-enkephalin [0.1 and 1 mg/kg intraperitoneal exposure for 6 days] also enhanced serum IL-6 levels. The in vivo data demonstrate that met-enkephalin upregulates IL-6 levels via an increase in transcriptional activity. Further, pre- and postoperative IL-6 and met-enkephalin levels were determined in the plasma of seven patients. First an increase in met-enkephalin plasma levels occurred (8.9+/-2.9 to 135+/-18 pg/ml; P<0.005), and then an increase in plasma IL-6 levels (2.1+/-1.5 to 237+/-50 pg/ml; P<0.001), suggesting that, as in mice, met-enkephalin induces IL-6 production. Taken together, the data strongly suggest that opioid peptides may be important as one of the chemical signaling molecules initiating the diffuse inflammatory response associated with coronary artery bypass grafting.

Morphine's immunoregulatory actions are not shared by fentanyl.

Fentanyl is a commonly used narcotic agent in anesthesia. It has strong analgesic properties which it shares with morphine. Unlike morphine, it does not possess the ability to bind to the mu3 receptor, and therefore does not have the ability to influence nitric oxide release as measured amperometrically. Cell adhesion also is not influenced. As a result, it also lacks the ability to downregulate the inflammatory response associated with surgery, especially cardiopulmonary bypass.

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.

Toxic shock syndrome due to group C streptococci. A case report.

BACKGROUND: Streptococcal toxic shock-like syndrome is a life-threatening illness which is on the increase. In early reports, only group A beta-hemolytic streptococcus was associated with the disease, but recent evidence indicates non-A streptococci groups are also involved. OBSERVATIONS: We describe the first reported case of streptococcal toxic shock-like syndrome caused by a group C strain in Italy. Prior to the disease, the patient, a 46-year-old man, had been in good health and had only a 3-day history of sore throat, low grade fever, vomiting, diarrhea, and myalgia before admission. Initially, diagnosis was based only on clinical evidence: shock, multiorgan failure, profound hypothermia, and no apparent signs of infection. Toxic cardiomyopathy was also present. RESULTS: Positive throat swab and blood culture confirmed a "definite case" following established criteria. Anamnesis showed a diagnosis of monoclonal gammopathy. Antibiotic treatment was begun immediately on admission of the patient, who was discharged 20 days later in good health. CONCLUSIONS: This case illustrates how an early diagnosis and prompt antibiotic therapy can determine a more favorable outcome.

Complications with fully implantable venous access systems in oncologic patients.

AIMS: To evaluate the complications caused by long-term central venous catheterization in patients with malignant hemopathies or solid tumors. METHODS: Retrospective study from June 1988 to June 1993 in 211 consecutive patients who required 223 venous access devices for long-term use. A consistent analysis was possible only in 161 of these patients. RESULTS: Fourteen catheter systems were removed for complications. Infections were the most common complications, with an overall incidence rate of 9.6%, i.e. 0.033/100 catheter days/patient. A significant difference was noted between the two groups of patients: 10 cases (24%) in malignant hemopathies, 6 cases (4.8%) in solid tumors (P = 0.0002). The main mechanical complication was thrombosis, with an incidence rate of 3%. CONCLUSIONS: Given the cost-benefit ratio, our study indicates that fully implantable venous access systems in oncologic patients are extremely useful.

[Thrombocytopenia and thrombophilic state in heparin therapy]. / Piastrinopenia e stato trombofilico in corso di terapia eparinica.

The authors report two clinical cases of thrombocytopenia and thrombosis which occurred during profilaxys and therapy with heparin. The mechanisms involved are reviewed and the possible therapeutic role of heparin-like drugs is discussed according to data presented in the international literature.

Infections by ampicillin-resistant enterococci: a case-control study.

We identified 17 (20%) of 83 consecutive enterococcal isolates from hospitalized patients with documented infection as high-level ampicillin-resistant enterococci (ARE). Of these, 16 isolates were identified as Enterococcus faecium and 1 isolate as Enterococcus raffinosus. A case-control study found no significant differences with respect to underlying diseases, central venous catheterization, nosocomial acquisition of the infection and sites of infection. Patients with ARE infection were older and had a higher inhospital fatality rate than those with ampicillin-susceptible Enterococcus (ASE) infection. Hospitalization in a surgery service (usually for an abdominal procedure), prolonged hospital stay, prior treatment with antibiotics (in particular imipenem and metronidazole), were also more frequent among patients with ARE infection. ARE isolates were more frequently resistant to imipenem, ciprofloxacin and streptomycin than ASE isolates.

High rate of oxacillin-resistant Staphylococcus aureus isolates in an Italian University Hospital.

We reviewed our routine clinical laboratory records from January 1990 to March 1993 to evaluate the rate of oxacillin-resistance among nosocomial isolates of Staphylococcus aureus. Of 265 clinically significant isolates, 174 (65%) were oxacillin-resistant S. aureus (ORSA). Most of these strains were obtained from surgery patients and/or were isolated from surgical wounds. The isolations of S. aureus increased during the study period: 45 in 1990, 50 in 1991, 130 in 1992 and 40 in the first trimester of 1993. The annual rates of ORSA among S. aureus isolated varied from 62 to 68% through these years. Most ORSA isolates proved resistant to ciprofloxacin, gentamicin and rifampicin, and susceptible to vancomycin, netilmicin and cotrimoxazole. Based on these results, the need for a stringent application of infection control measures is outlined.

Carbohydrate-deficient transferrin quantified by HPLC to determine heavy consumption of alcohol.

We developed a new fully automated ion-exchange chromatographic method for quantitating carbohydrate-deficient transferrin (CDT) on a Mono Q column. Quantitation relies on the selective absorbance of the iron-transferrin complex at 460 nm. Transferrin isoforms deficient in sialic acid, with pIs 5.7 and 5.9, can easily be separated and quantitated as a percentage of the total transferrin. This method has been applied to samples from teetotalers, occasional drinkers, patients with recent heavy alcohol consumption, and patients during detoxification. The sensitivity of the method was 55% in patients reporting 40-70 g daily ethanol consumption and nearly 100% in heavily intoxicated patients (70-500 g daily consumption). The half-life of the dominating pI 5.7 isoform in this group was 9.5 (+/- 1) days during detoxification. A CDT value > 0.8% is a highly specific marker for alcohol abuse and is greatly superior to other currently available biological markers.