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Biomedical subjects

G B Stefano

Publications and source records attributed to G B Stefano.

At least 91 records · Page 5Linked to original sources
PubMed1998

Naltrexone suppresses the rejection of cardiac tissue transplantation.

The present study demonstrates the following: 1. Transplantation of cardiac tissue induces an inflammatory response that ultimately leads to the rejection of the tissue by the host within 9 days; 2. Treatment with the opiate antagonist, naltrexone, significantly increased the survival of the transplanted cardiac tissue to 13 days, suggesting the involvement of opioid signaling molecules in tissue rejection; 3. In further experiments it was demonstrated that in mixed lymphocyte populations from different mice, the DNA synthesis inhibitor, mitomycin C, reduced the lymphocyte proliferative response as did naltrexone; 4. Mice injected with naltrexone for 10 days and given concanavalin A exhibited a suppressed spleen lymphocyte proliferative response compared to controls. Taken together, these data suggest that endogenous opioid signals not only activate immunocytes, but also stimulate DNA synthesis.

Analysis of Variance↗
PubMed1998

Aspirin inhibits granulocyte and monocyte adherence to saphenous vein endothelia in a process not mediated by nitric oxide.

We examined the effects of aspirin on human immunocyte adherence to human saphenous vein preparations in vitro. Monocytes and granulocytes were analyzed by phase-contrast microscopy in conjunction with a time-lapse video recording system for cell counting in the medium. Saphenous vein samples taken from five volunteer subjects during their elective coronary artery bypass grafting procedures were used for an in vitro experiment in which each subject's immune cells were added to his/her respective endothelial tissues. In addition, aspirin or morphine was added to the preparations. After 30 min the endothelium and the medium were examined for cell numbers using feature color detection software. The endothelium exposed to a 10 min preincubation with 10 mmol/l aspirin was found to have significantly fewer monocytes and granulocytes attached. The saphenous vein preparations were examined for nitric oxide (NO) release using an NO-selective amperometric microprobe. Aspirin at its immunocyte inhibitory concentrations did not induce NO release from the endothelium, whereas morphine, which is known to stimulate NO production by endothelium, did so. These findings demonstrate that the administration of aspirin in high doses can diminish human granulocyte and monocyte adherence to saphenous vein endothelium in vitro through an NO-independent mechanism.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Delta2 opioid receptor subtype on human vascular endothelium uncouples morphine stimulated nitric oxide release.

We demonstrate the presence of both delta and mu opioid receptors on the endothelium of human saphenous vein and internal thoracic artery. Displacement analysis revealed that a variety of opioid peptides were found to be ineffective in displacing specifically bound 3H dihydromorphine and only delta2 ligands were effective in regard to 3H Ala2-met5 enkephalinamide (DAMA), indicating the presence of mu3 and delta2 opioid receptor sites, respectively. Confirming the presence of both mu and delta sites we demonstrated positive immunostaining with anti-delta and anti-mu receptor antibodies. Exposure of these vessels to DAMA significantly enhances granulocyte adherence (P<0.01) even in vessels 5 min later exposed to 10(-6) M morphine. Unlike morphine, DAMA did not stimulate nitric oxide from either blood vessel and human granulocytes. Additionally, DAMA preadministered before morphine exposure to the endothelium or granulocytes, inhibited the morphine-stimulated release of NO in a dose-dependent manner. The data indicate that opioid peptides and opiate alkaloids regulate endothelial function in an antagonistic manner thereby influencing the microvascular environment.

Cell Adhesion↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Anesthetics, Intravenous↗
PubMed1998

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.

Animals↗
PubMed1998

Isolation and characterization of a leech neuropeptide in rat brains: coupling to nitric oxide release in leech, rat and human tissues.

The osmoregulator peptide (leech osmoregulatory factor, LORF; IPEPYVWD) was first found in the leech central nervous system (CNS). Given the fact that certain peptides can be found in mammals and invertebrates, e.g., opioid, we examined rat brains to determine if LORF was present. This peptide was found and isolated by successive reversed-phase HPLC purification steps and characterized by electrospray mass spectrometry measurement. It was sequenced by Edman degradation and quantified in different tissues by ELISA. Our results demonstrate the presence of LORF in the hypothalamus, thalamus, and striatum (6 pmol/mg of protein extract) and in other brain areas at lower levels. This octapeptide is also present in the rat duodenum and liver (10 to 14 pmol/mg) and at lower levels in heart, lung, pancreas and caudal spinal cord (< 5 pmol/mg). The testes, adrenals and kidneys have the lowest levels of all the tissues examined (ca. 0.5 pmol/mg of protein). Furthermore, we also demonstrate that LORF is coupled to nitric oxide (NO) release in leech CNS, rat hypothalamus and human saphenous vein in a manner which is inhibited by a nitric oxide synthase inhibitor as well as an antibody directed toward LORF. The study demonstrates that LORF, and its function in relation to NO release, has been conserved over more than 400 million years of evolution.

Animals↗
PubMed1998

Autoimmunovascular regulation: morphine and anandamide and ancondamide stimulated nitric oxide release.

The hypothesis concerning morphine as an endogenous signal molecule has been strengthened with the recent discovery of a new opiate receptor subtype, designated mu3. This opiate receptor is opiate alkaloid sensitive and opioid peptide insensitive, including peptides previously shown to have affinities for mu opiate receptors. This receptor is coupled to nitric oxide release in human endothelial cells, granulocytes and monocytes and in invertebrate immunocytes and microglia. In relation to the endothelium, it has also been coupled to vasodilation via nitric oxide. Given the known influence of nitric oxide in downregulating cell adhesion, the role of this compound has now been investigated in also diminishing endothelial-immunocyte interaction. Morphine, via nitric oxide, has the potential to diminish adhesion molecule expression and in so doing calm an inflammatory process between immunocytes and the endothelial surface. In this regard, the potential for abuse is also present.

Arachidonic Acids↗
PubMed1998

Mytilus edulis hemolymph contain prodynorphin.

We have characterized a prodynorphin (prodyn) molecule in hemocytes and hemolymph of the bivalve mollusk Mytilus edulis. The ca. 16-kDa protein was purified by cut-off filtration prepurification, anti-leucine-enkephalin affinity column separation followed by reversed-phase HPLC. Its primary sequence was determined by Edman degradation, endoproteinase Glu-C digestion and CNBr treatment. Mytilus prodyn contains, alpha-neo-endorphin, dynorphin-A and dynorphin-B at the C-terminus, exhibiting 100, 70.5 and 85% of identity with the rat prodyn-derived counterparts, respectively. The number of leucine-enkephalins in this precursor is identical to that found in vertebrates. Mytilus prodyn is distinguished from that found in leeches in that the N-terminus is longer. Additionally, by sequence comparison, the presence of an orphanin FQ-like peptide, exhibiting 50% sequence similarity with that found in mammals, is demonstrated. This report constitutes the first complete biochemical characterization of a prodyn in a non-parasitic invertebrate and mollusk.

Amino Acid Sequence↗
PubMed1998

Antagonism of LPS and IFN-gamma 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.

Adenylyl Cyclase Inhibitors↗
PubMed1998

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.

Arachidonic Acids↗
PubMed1998

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-alpha (TNFalpha), 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. METHODS: Sixteen patients submitted to elective coronary artery bypass grafting (CABG) surgery were randomized to either low-dose opioid anaesthesia consisting of thoracic epidural analgesia combined with inhalational anaesthesia (group I) or high-dose fentanyl anaesthesia (group II). From each patient 18 blood samples were taken perioperatively. Cytokine analyses were performed with ELISA, CRP and SAP mere measured with rocket immunoelectrophoresis (RIE). RESULTS: Surgery and CPB elicited a marked, transient and almost simultaneous proinflammatory and anti-inflammatory cytokine response with no differences between the groups. The cytokine levels returned to preoperative levels 1-3 d after operation. Anaesthesia and surgery did not affect SAP plasma levels while patients showed a major increase in CRP concentrations preceding the cytokine responses. CONCLUSION: CABG performed during two different anaesthetic techniques, high-dose fentanyl versus low-dose opioid anaesthesia, elicited a well-defined cytokine response with minor variation in the time course of each cytokine. The cytokine production was not modified by type of anaesthesia. Finally, SAP is not an acute-phase protein in men.

Acute-Phase Proteins↗
PubMed1998

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.

Analgesics, Opioid↗
PubMed1997

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.

Adult↗
PubMed1997

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.

Adrenocorticotropic Hormone↗
PubMed1997

Nitric oxide inhibits the dopamine-induced K+ current via guanylate cyclase in Aplysia neurons.

Nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS) and has been implicated in inter- and intracellular communication in the nervous system. The present study was undertaken to assess the effects of sodium nitroprusside (SNP) and hydroxylamine (HOA), NO donors, on a dopamine (DA)-induced K+ current in identified Aplysia neurons using voltage-clamp and pressure ejection techniques. Bath-applied SNP (10-25 microM) reduced the DA-induced K+ current without affecting the resting membrane conductance and holding current. The DA-induced K+ current also was inhibited by the focal application of 200 microM HOA to the neuron somata. The DA-induced K+ current suppressing effects of SNP and HOA are completely reversible. Pretreatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a specific inhibitor of NO-stimulated guanylate cyclase, and hemoglobin (50 microM), a nitric oxide scavenger, decreased the SNP-induced inhibition of the DA-induced current. In contrast, intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) or bath-applied 3-isobutyl-1-methylxanthine (IBMX; 50 microM), a non-specific phosphodiesterase inhibitor, inhibited the DA-induced current, mimicking the effect of the NO donors. These results demonstrate that SNP and HOA inhibit the DA-induced K+ current and that the mechanism of NO inhibition of the DA-induced current involves cGMP-dependent protein kinase.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Amino-acid-sequence determination and biological activity of cytin, a naturally occurring specific chymotrypsin inhibitor from the leech Theromyzon tessulatum.

We purified a chymotrypsin inhibitor, designated cytin, from the rhynchobdellid leech Theromyzon tessulatum. This 7.4-kDa peptide was purified to apparent homogeneity by gel-permeation and anion-exchange chromatographies, followed by reverse-phase HPLC. The structure of cytin was determined by reduction, S-beta-pyridilethylation, automated Edman degradation, and electrospray mass spectrometry. Cytin is formed by the association of two protein chains, which are linked by a disulfide bridge. Chain A consists of 43 and chain B of 22 amino acid residues. Chain B exhibits 40-63% sequence similarity with the N-terminal sequences of subtilisin/chymotrypsin inhibitors isolated from barley seeds. Cytin inhibited chymotrypsin (Ki 600 pM) and weakly inhibited trypsin (Ki 350 nM). This chymotrypsin inhibitor, in contrast to others isolated from leeches, does not inhibit elastase or cathepsin G. Furthermore, cytin (10 microM) significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharide (1 U/ml) in a manner similar to that of aprotinin. These data indicate that this chymotrypsin inhibitor may be biomedically significant.

Amino Acid Sequence↗