Thomas V. Bilfinger

Presence of the μ3 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 μ3) 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 μ3 receptor is coupled to nitric oxide release and vasodilation.

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.

Comparison of survival after sublobar resections and ablative therapies for stage I non-small cell lung cancer.

BACKGROUND Lobectomy is the standard therapy for patients with stage I non-small cell lung cancer (NSCLC). Recently, sublobar resections (SLR), radiofrequency ablation (RFA), and percutaneous cryablation therapy (PCT) for high-risk patients unfit for standard resection have been reported. This study compares all 3 modalities in stage I NSCLC. STUDY DESIGN Patients with biopsied stage I NSCLC determined by PET/CT deemed medically unfit for standard resection were reviewed by a tumor board according to American College of Surgeons Oncology Group/NIH inoperability criteria before being offered SLR, RFA, or PCT under anesthesia. Patients were followed with CT scans alternating with PET scans. The primary end points were overall survival, cancer-specific survival, and cancer-free survival. Kaplan-Meier analysis and log-rank tests were used. RESULTS Sixty-four patients underwent SLR (n = 25; 11 men, 13 women; median age 66 years, range 49 to 85 years), RFA (n = 12; 8 men, 4 women; median age 74 years, range 62 to 83 years), and PCT (n = 27; 16 men, 11 women; median age 74 years; range 59 to 88 years). The probability of 3-year survival for the SLR, RFA, and PCT groups was 87.1%, 87.5%, and 77%, respectively (p > 0.05). The 3-year cancer-specific and cancer-free survival for SLR, RFA, and PCT groups was 90.6% and 60.8% versus 87.5% and 50% versus 90.2% and 45.6%, respectively. CONCLUSIONS This experience suggests comparable survival after sublobar resections and ablative therapies at 3 years. Ablative therapies appear to be a reasonable alternative in high-risk patients not fit for surgery. However, larger randomized studies with longer follow-up are needed to make recommendations for therapy.

Antagonism of LPS and IFN-γ 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.

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.

Endogenous morphine levels increase following cardiac surgery as part of the antiinflammatory response

Exogenous morphine downregulates the activity of immunocompetent cells such as lymphocytes, granulocytes and macrophages. Furthermore, morphine increases the secretion of CRH, ACTH and glucocorticoids, i.e. substances with inhibitory effects on the immune system. In the present study we tested the hypothesis that endogenous morphine production is increased as part of the antiinflammatory response to cardiac surgery. Sixteen patients submitted to elective coronary artery bypass grafting (CABG) surgery were randomized to either thoracic epidural analgesia combined with general anaesthesia (group I) or high-dose fentanyl anaesthesia (group II). Patients in group I did not receive morphine while patients in group II received systemic morphine for postoperative pain relief. From each patient 18 blood samples were taken perioperatively and tested for morphine. Furthermore, monocyte function with respect to motility and shape was determined by computer-assisted image analysis. A steep increase in plasma morphine concentrations was demonstrated on the first postoperative day in patients in group I (not given morphine). Plasma morphine levels remained significantly elevated during the following five postoperative days. Patients in group II given morphine as pain treatment showed a larger and earlier morphine peak related to the morphine administration. Computer-assisted image analysis of leukocyte behaviour revealed a biphasic increase in cell motility. In conclusion, we demonstrate for the first time that endogenous morphine levels increase after the trauma of surgery. We surmise that morphine is part of the antiinflammatory response to cardiac surgery.

Effects of anaesthesia based on high versus low doses of opioids on the cytokine and acute‐phase protein responses in patients undergoing cardiac surgery

Background: Cardiac surgery with cardiopulmonary bypass (CPB) evokes a systemic inflammatory response involving the proinflammatory cytokines tumor necrosis factor‐α (TNFα), interleukin (iL)‐1, IL‐6, IL‐8 and anti‐inflammatory cytokines such as IL‐10. Like IL‐10, opioids downregulate the immune responses in vivo and in vitro, including the activity of the cytokine‐producing monocytes and granulocytes. The proinflammatory cytokines are potent inducers of the hepatic acute‐phase protein synthesis. The aim of the present study was to investigate if choice of anaesthesia, based on high‐dose opioids (fentanyl) versus low‐dose opioids influenced the release of IL‐6, IL‐8, and IL‐10. Secondly, it was investigated whether serum amyloid P‐component (SAP) is an acute‐phase protein in man such as C‐reactive protein (CRP), with which it is physically and structurally related.

A case of tracheal injury after emergent endotracheal intubation : A review of the literature and causalities

IMPLICATIONS Tracheal lacerations are rare, but potentially fatal, complications of intubations. Diagnosis of such conditions is difficult, and thus a high level of suspicion must be kept. We present a case and review the literature for factors that indicate high-risk patients.

Long-term exposure of human blood vessels to HIV gp120, morphine, and anandamide increases endothelial adhesion of monocytes : Uncoupling of nitric oxide release

Acute exposure of human saphenous vein or internal thoracic artery endothelium to either morphine [27.4 +/- 3.7 and 35.4 +/- 4.1 nM nitric oxide (NO), respectively] or anandamide (18.3 +/- 2.2 and 24.3 +/- 3.0 nM, respectively) results in NO release, whereas exposure to the human immunodeficiency virus envelope protein gp120 does not. After the short-term exposure of the vessel endothelium, monocyte adherence is diminished with morphine and anandamide treatment (jointly by -80%), whereas it is enhanced with that of gp120 (approximately 40%), indicating that gp120 enhances the ability of the endothelium to adhere monocytes. Long-term or continuous exposure of the endothelia to all agents results in a significant enhancement of monocyte adherence (p < 0.05), which is further increased when exposed to either morphine and anandamide plus gp120. This is caused by a desensitization of the endothelium to further NO release after the initial exposure to either anandamide or morphine. The results serve to indicate that in individuals abusing opiates and or cannabinoids, a tissue [i.e., central nervous system (CNS)] viral load may be higher, and acquired immunodeficiency syndrome (AIDS) may progress more rapidly because monocyte adherence and mobility is significantly increased, indicating a higher level of transmembrane migration.