Caterina Fimiani

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.

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 morphines and anandamides 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.

T-cell homeostasis alteration in HIV-1 infected subjects with low CD4 T-cell count despite undetectable virus load during HAART.

Objective:To investigate the pathogenesis of low CD4 T-cell count in subjects who are immunological non responders (InR) to HAART. Design:Thirty-five HIV-positive subjects on HAART for at least 1 year, all with undetectable HIV-1 RNA, were studied. Patients were defined as InR according to a CD4 cell increase < 20% from CD4 cell baseline or CD4 cell count < 200/μl; subjects with a CD4 T-cell increase > 20% from baseline and a CD4 cell count > 200/μl were defined as immunological responders (IR). We performed a comprehensive study to characterize the immune response of InR. Methods:The immunological phenotype of peripheral blood mononuclear cells, thymic naive T cells, T-cell receptor Vβ repertoire, serum concentration of interleukin (IL)-7, the expression of IL-7Rα on naive and memory CD4 and CD8 T cells, and regulatory T cells (Treg) were studied. Results:In InR a significant reduction (P < 0.0001) of naive and thymic naive CD4 T cells was associated with a reduced expression of IL-7Rα in both cell subsets, with an increased serum concentration of IL-7 was observed. Furthermore, an increased immune activation with a reduced Treg frequency and increased number of expansions of Vβ families was observed. Conclusions:The reduced expression of IL-7Rα associated with the persistent immune activation and the alteration of Treg frequencies in part explains the low level of CD4 T cells observed in InR.

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.

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.

Altered Clonogenic Capability and Stromal Cell Function Characterize Bone Marrow of HIV-Infected Subjects with Low CD4 + T Cell Counts Despite Viral Suppression during HAART

BACKGROUND Inflammatory cytokines in bone marrow may impair hematolymphopoiesis in human immunodeficiency virus (HIV)-infected subjects who do not experience reconstitution of CD4(+) T cells despite suppression of virus replication while receiving highly active antiretroviral therapy (HAART) (immunological nonresponders). METHODS Bone marrow samples from 12 immunological nonresponders receiving HAART were studied and compared with samples from 11 immunological responders. The mean CD4(+) T cell count (+/- standard deviation) was 174 +/- 68 cells/mm(3) and plasma HIV RNA levels had been <50 copies/mL for at least 1 year for individuals enrolled in the study. The clonogenic capability of bone marrow samples was evaluated using the colony forming cell assay and the long-term culture-initiating cell assay. CD34(+) cells from the colony forming cell assay were pooled for real-time polymerase chain reaction analysis of Fas and Fas ligand. Bone marrow cytokine production (interleukin-2 and tumor necrosis factor-alpha) and stromal interleukin-7 levels were analyzed by enzyme-linked immunosorbent assay in both groups. Flow cytometric analysis of CD4(+) and CD8(+) T cell subsets was performed. RESULTS A reduced clonogenic capability and a decrease in the level of more primitive progenitor cells were observed in parallel with lower production of interleukin-2 and increased tumor necrosis factor-alpha levels. A significant upregulation of Fas and Fas ligand on CD34(+) cells and a higher stromal interleukin-7 production were observed. Impairment of the naive T cell compartment and persistent T cell activation were observed in peripheral blood. CONCLUSIONS Samples from immunological nonresponders show reduced growth of in vitro colonies and an altered cytokine production in bone marrow. The cytokine pattern observed and the altered Fas and Fas ligand pathway may determine stem cell apoptosis and low CD4(+) cell recovery. These features, which are similar to those observed in HIV-infected subjects before starting therapy, persist despite treatment.

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.

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.

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 morphines ability to release NO. Taken together, the data demonstrate that cannabinoid signalling is involved with the regulation of the microvascular environment.

μ3 Opiate receptor expression in lung and lung carcinoma : ligand binding and coupling to nitric oxide release

The μ3 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 μ3 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 μ opioid receptor transcripts are present in lung carcinoma and other cells with the μ3 profile. Our findings identify a unique role for the μ3 opiate receptor in opiate-mediated NO release and suggest that endogenous opiates, through their release of NO, may play a role in cancer progression.