Biomedical subjects

G B Stefano

Publications and source records attributed to G B Stefano.

At least 19 recordsLinked to original sources

PubMed1997

Human monocyte adhesion is modulated by endothelin B receptor-coupled nitric oxide release.

Human monocytes have the capacity to produce both endothelin 1 (ET-1) and nitric oxide (NO), yet the roles of these mediators in monocyte function remain unclear. The relationship of ET-1 and NO release to monocyte adhesion was explored using peripheral blood monocytes (PBM) and the human monocytic cell lines THP-1 and U937. Specific binding of 125I-labeled ET-1 to THP-1 was abrogated by pretreatment with the endothelin B (ET(B)) receptor antagonist, BQ-788, but not by the endothelin A (ET(A)) receptor antagonist, BQ-123, consistent with predominant ET(B) receptor expression. Direct measurement of NO with an amperometric probe demonstrated the production of nanomolar concentrations of NO by PBM and THP-1 cells upon treatment with ET-1, which was abrogated by BQ-788, but not BQ-123, pretreatment, suggesting functional coupling of ET(B) receptors to NO release. Indeed, the presence of ET(B) receptor mRNA transcripts was detected in THP-1 and is consistent with previous reports that have demonstrated functional coupling of ET(B) receptors to constitutive NO synthase activation. In contrast, U937 cells did not release NO in response to ET-1 treatment, and mRNA transcripts were not detected in these cells, consistent their failure to bind 125I-labeled ET-1, as previously determined. Exposure of PBM to ET-1 markedly reduced the adhesion of these cells to human saphenous vein, whereas PBM adhesion in the presence of BQ-788 was restored to control levels. These data demonstrate that PBM interactions with the vascular wall can be reduced by autocrine production of NO and suggest that ET(B) receptors may attenuate monocyte activity at sites of inflammation.

Cell Adhesion↗

PubMed1997

A rapid and sensitive quantitation method of endogenous morphine in human plasma.

Endogenous morphine is found at very low levels (pg/ml) in human plasma. Without technical modification, the usual techniques, i.e., RIA or HPLC, can not directly detect its presence. As a result the initial purification process is very crucial. In this report we demonstrate this opiate alkaloids determination in 2 ml of human plasma. Following an acidified precipitation of proteins in plasma, a solid-phase extraction, Sep-pak collection, was employed to concentrate the endogenous morphine. The extract was dissolved in a pH 8.9 phosphate buffer and then extracted into an organic solvent (chloroform and isopropyl alcohol). The initially purified sample was separated by HPLC and detected with an electrochemical detector of 500 mV and 0.02 Hz. The results showed that the recovery of morphine by this technique was 86+5.7%, and the sensitivity was 50 pg/ml. Compared to the detection sensitivity of the HPLC (2 ng/ml), this method increased the detection limit by 40-fold. With this method the concentration of endogenous morphine in plasma was determined as 80 pg/ml.

Acetonitriles↗

PubMed1996

Inhibition of the Met-enkephalin-induced K+ current in B-cluster neurons of Aplysia by nitric oxide donor.

The effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on a methionine-enkephalin (Met-E)-induced K+ current recorded from B-cluster neurons in Aplysia cerebral ganglion were investigated with voltage-clamp and pressure ejection techniques. Bath-applied SNP (10-25 microM) reduced the Met-E-induced K+ current in the neurons without affecting the resting membrane conductance and holding current. The inhibitory effects of SNP were reversible. Pretreatment with methylene blue (10 microM), a non-specific inhibitor of guanylate cyclase, and hemoglobin (50 microM), a NO scavenger, decreased the SNP-induced inhibition of the Met-E-induced current. Intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) or bath-applied 3-isobutyl-1-methylxanthine (IBMX; 50 microM), a nonspecific phosphodiesterase inhibitor, inhibited the Met-E-induced current. Furthermore, 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), a more specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Met-E-induced current. These results suggest that SNP induces suppression of the Met-E-induced K+ current recorded from B-cluster neurons of Aplysia cerebral ganglion via stimulation of cGMP formation.

Animals↗

PubMed1996

Cannabinoid receptors are coupled to nitric oxide release in invertebrate immunocytes, microglia, and human monocytes.

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in invertebrate immunocytes and microglia. The anandamide-binding site is monophasic and of high affinity, exhibiting a Kd of 34.3 nM with a Bmax of 441 fmol/mg protein. These sites are highly selective, as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release in the invertebrate tissues examined as well as in human monocytes. Interestingly, the cannabinoid-stimulated release of nitric oxide initiates cell rounding. Thus, these cannabinoid actions resemble those of opiate alkaloids. In this regard, we demonstrate that these signaling systems use the same effector system, i.e. nitric oxide release, but separate receptors. Last, the presence of a cannabinoid receptor in selected evolutionary diverse organisms indicates that this signaling system has been conserved for more than 500 million years.

Animals↗

PubMed1996

Morphine-induced conformational changes in human monocytes, granulocytes, and endothelial cells and in invertebrate immunocytes and microglia are mediated by nitric oxide.

We evaluated the contribution of nitric oxide (NO) to morphine-induced rounding of spontaneously activated (mobile) ameboid human monocytes, granulocytes, or arterial endothelial cells and invertebrate immunocytes and microglia. Morphine induced significant rounding and inactivation of ameboid cells within 20 min except for arterial endothelial cells, which became rounded 24 h after morphine exposure. The effects of morphine on cell conformation were blocked in the presence of N-nitro-L-arginine, a nitric oxide synthase inhibitor. Treatment of cells with the NO donor, sodium nitroprusside, induced cell rounding similar to that observed following morphine exposure, suggesting that NO release may mediate morphine-induced changes in cell conformation. The contribution of NO release to morphine-induced cell rounding was determined by direct evaluation of NO concentration in real-time using a NO-specific amperometric probe. Significant increases in NO concentration were observed 2 min after morphine stimulation, whereas morphine-induced NO release was markedly impaired by pretreatment with N-nitro-L-arginine or the opiate alkaloid antagonist, naloxone. In contrast, opioid peptides failed to induce NO release, consistent with our previous observations that demonstrated the failure of opioid peptides to promote cell rounding. Taken together, these data suggest that morphine-induced NO release may be mediated by activation of the opiate alkaloid-selective, opioid peptide-insensitive micro3 receptor, and that functional coupling of morphine to NO production has been conserved during evolution and may modulate cellular activation.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Surgical anticipatory stress manifests itself in immunocyte desensitization: evidence for autoimmunoregulatory involvement.

The immunocyte behavior (conformational changes and locomotion in response to signal molecule challenge) in patients about to undergo elective cardiac surgery was studied to elucidate the effect of psychological anticipatory stress on the immune system. Granulocytes and monocytes from 10 patients and 35 non-surgical controls were examined. Computer-assisted microscopic image analysis, capable of measuring cellular conformational and velocity changes, was used to measure the responsiveness of these immunocytes to peptidergic and cytokine stimulation. Immunocyte desensitization would appear to account for the reduction in their abilities to respond to chemotaxic challenge associated with the pre-cardiac surgery state. Their abilities to respond to D-Ala2-Met-enkephalinamide (DAMA) were observed only at much higher concentrations than previously reported (10-11 M vs. 10-9 M prior to surgery). This finding, together with the observed decrease in adrenocorticotropin levels compared to non-surgical controls, suggests that neutral endopeptidase activity was elevated just prior to surgery. Indeed, neutral endopeptidase activity is statistically elevated in the pre-cardiac surgery state. Furthermore, glucocorticoid levels remained constant, within normal resting limits, in both groups. Thus, surgical anticipatory stress may manifest itself, in part, as a desensitization of various immunocytes. Thus, a psychological anticipatory stress response may be a precipitant of the desensitization. Although this desensitization seemed not to involve the entire hypothalamic-pituitary-adrenal axis, the data suggest that psychological anticipatory stress may initially involve and influence autoimmunoregulation.

Adrenocorticotropic Hormone↗

PubMed1996

Hyperstimulation of leukocytes by plasma from cardiopulmonary bypass patients is diminished by alpha-MSH pretreatment.

Cardiopulmonary bypass (CPB) results in a diffuse inflammatory response characterized in part by hyperstimulation of leukocytes. We have previously shown that this hyperstimulation appears to be due, in part, to an increase in the release of biological response modifiers (BRMs) such as cytokines. In the present study, we evaluated the ability of a naturally occurring immunocyte inhibitory substance, alpha-melanocyte-stimulating hormone (alpha-MSH), to prevent the hyperstimulation caused by CPB. Monocytes and granulocytes were pretreated with alpha-MSH (10(-6) M) before exposing the cells to plasma obtained from patients who had undergone CPB, as CPB plasma would stimulate native monocytes and granulocytes in a manner similar to that observed in CPB patients. Pretreatment of these cells with alpha-MSH significantly diminished the hyperstimulation induced by CPB plasma in a concentration-dependent manner. In contrast, when the cells were first or simultaneously exposed to CPB plasma and then to alpha-MSH, alpha-MSH had no effect. Furthermore, use of the specific neutral endopeptidase inhibitor, phosphoramidon, significantly increased the efficacy of alpha-MSH in inhibiting CPB-induced immunocyte activation. The data demonstrate that pretreatment of monocyte/macrophages and granulocytes with alpha-MSH effectively inhibits the immune hyperstimulation induced by CPB-plasma exposure. In addition, the data strongly suggest that preexposure to other naturally occurring immune inhibitory substances may diminish the hyperstimulation associated with CPB. The study also further confirms that this hyperstimulation may, in part, be due to BRMs released from immunocytes.

Cardiopulmonary Bypass↗

PubMed1996

Aprotinin diminishes inflammatory processes.

Many of the recent reports concerning cytokine levels in cardiopulmonary bypass have documented changes in the levels of these trauma indicators. In the present report, we also document their levels but in the presence of Aprotinin. Aprotinin is a protease inhibitor used not only to diminish bleeding, but also to diminish elements of the diffuse inflammatory response associated with this type of surgery. We report in plasma obtained from 20 patients that initially interleukin-8 (IL-8) levels (53.4 +/- 7 pg/ml) plasma to 185.5 +/- 30 pg/ml) increased 20 min from the start of surgery. This is followed by IL-6 (5.3 +/- 1.1 to 200 +/- 50 pg/ml) peaking 15 h post surgery. These levels return to normal by day 3 postop. IL-1 beta and tumour necrosis factor (TNF) levels remained at baseline for the observation period. Associated with these changes in cytokine levels is the activity state of immunocytes (granulocytes and monocytes) noted by conformational changes obtained from computer-assisted microscopy. The cells exhibited an ameboid conformation and became mobile (67%), peaking at 120 min after surgery began and returned to a more rounded conformation with only 6% exhibiting the ameboid conformation by day three. In in-vitro experiments, where immunocytes not exposed to cardiopulmonary bypass were exposed to plasma obtained from patients having undergone this surgery, their activity level rose to 65%. In the same experiment, when Aprotinin was added to the cell-plasma mixture, the level of activation dramatically dropped to 25%. Thus, aprotinin was found at high doses to lower cytokine and cellular activation associated with the acute inflammatory responses of cardiopulmonary bypass, suggesting that this may be initiated by hyperstimulated immunocytes.

Acute-Phase Reaction↗

PubMed1996

Evidence for morphine downregulating immunocytes during cardiopulmonary bypass in a porcine model.

Cardiopulmonary bypass is associated with both cellular immunosuppression and an inflammatory response. Previous studies have demonstrated that morphine, a naturally occurring substance, can downregulate granulocyte, monocyte and endothelial activity. It can even prevent the activation caused by exposing these cells to plasma obtained from patients undergoing cardiopulmonary bypass. The present study demonstrates that preadministering a high dose of morphine (3.3 mg/kg) to pigs prior to cardiopulmonary bypass also diminishes the activation levels of these cells. In animals not given morphine, monocyte activation levels were 45% compared to 14% exposed to the opiate. Granulocytes also exhibited the same statistically significant (P < 0.05) drop in cellular activation. Activation is determined by computer-assisted microscopic image analysis whereby cellular shape is indicative of the cells activity. Additionally, in animals pretreated with morphine, a twofold increase in the number of cells was obtained, indicating that the endothelium also was downregulated.

Animals↗

PubMed1996

The presence and effects of mammalian signal molecules in immunocytes of the insect Leucophaea maderae.

Opioid peptides activate immunocytes and opiate alkaloids inhibit this activation in the mussel, Mytilus edulis. Here we present evidence that cells of another invertebrate, Leucophaea maderae, can be influenced in a similar way by the Met-enkephalin analogue D-Ala2-Met5-enkephalin (DAMA) and morphine. Effects of different signal molecules on Leucophaea hemocytes were evaluated by computer-assisted image analysis of their conformational state. A small percentage of the untreated cells were found to display spontaneous conformational changes after 25 min of incubation without pharmacological agents which was noted as a decrease in both circularity factor and shape factor values. Activation caused the cells to become elliptical, a feature that appears to be characteristic of Leucophaea immunocytes. Administration of DAMA induced a similar activation of most of the cells. After 30 min these DAMA-activated cells started to display distinct locomotory activity not seen in the controls. alpha-Melanocyte-stimulating hormone (MSH, 10(-7)) added to the incubation medium after DAMA-activation caused the cells to return to their original "rounded" conformation. In addition, the presence of immunoreactive interleukin (IL-1), adrenocorticotropin (ACTH) and tumor necrosis factor (TNF) in the hemolymph was demonstrated. These data suggest an interaction between both vertebrate-type immunological signal molecules and neuropeptides in the regulation of immunological cells in Leucophaea.

Adrenocorticotropic Hormone↗

PubMed1996

The presence of the mu3 opiate receptor in invertebrate neural tissues.

A previous report demonstrated the presence of the newly discovered opiate alkaloid selective and opioid peptide insensitive mu3 receptor in ganglia of several invertebrate- and one vertebrate species as well as in microglial cells that had egressed from these ganglia after their maintenance in culture medium for several days. In the present study carried out in two representatives of invertebrates, the binding densities of this receptor determined in intact ganglia were compared with those in ganglia depleted of microglial cells. The aim was to ascertain whether the differences in binding capacity recorded in those two groups of ganglia might give an indication of the possible presence of this opiate receptor in nonmicroglial components of the nervous tissue, i.e., neurons. Within a period of 72 h of incubation, the gradual reduction in binding density had reached a plateau, in accordance with the termination of the egress of microglia. The fact that at least two thirds of the binding capacity of mu3 receptors were retained by the ganglia strongly suggests that part of this capacity may be attributed to neurons. This view is supported by additional data, in particular the demonstration of endogenous morphine in nervous tissue and its localization within distinct neurons.

Animals↗

PubMed1996

Neuroimmunologic implications in coronary artery disease.

In this review, the role of the macrophage in the pathophysiology of coronary artery disease (CAD) is examined. The central interaction of macrophage, endothelial cell and smooth muscle cell in the context of hyperlipidemia is considered. The macrophage appears to be at the beginning of a chain of events that starts with elevated low density lipoprotein (LDL). Stress, particularly in those with a core hostility, may be associated not only with higher catecholamine levels but also with higher serum lipid levels. These lipids will in turn be processed to oxidized LDL by macrophage and endothelial cells. Oxidized LDL molecules will contribute to atherosclerotic plaquing. A side effect of such plaque formation may be a diminished vasodilatory response to the nitric oxide (NO) produced by macrophages and endothelium. Indeed, paradoxical vasoconstriction occurs in atherosclerosis in response to neurotransmitters such as serotonin and acetylcholine, which under normal circumstances cause vasodilation. There also is evidence that both macrophages and endothelial cells can regulate NO production through a specific mu 3 morphine receptor, an effect that can be blocked by naloxone. The clinical effectiveness of morphine and nitroglycerin in CAD patients may relate to these mechanisms. More research will be needed to elucidate the neuroimmunologic basis for atherosclerosis with prospects for better treatment and management in future.

Animals↗

PubMed1996

Adrenocorticotropin--a central trigger in immune responsiveness: tonal inhibition of immune activation.

Adrenocorticotropin is known for its key role in mediating neuroendocrine responses, especially in response to stress. Recently, it has been recognized to have direct immunomodulatory actions, most of which are suppressive. This is a widely conserved action which occurs in invertebrates and vertebrates. This conservation illustrates the fundamental nature of adrenocorticotropin's immunomodulatory action. Such a mechanism of action helps explain why the immunocytes themselves can serve as a source of adrenocorticotropin. Regulation of adrenocorticotropin production and action is complex and the result is an integration of multiple mechanisms. Serum adrenocorticotropin levels fluctuate in response to stimulatory and inhibitory factors. Further, peptidases can specifically process adrenocorticotropin into smaller active fragments, inactive peptides or into peptides with different activities. These proteolytic enzymes have a differential tissue and cellular distribution. Immune stimulating factors such as interleukin-1 can overcome adrenocorticotropin inhibition and secondarily, block adrenocorticotropin production through the release of corticosteroids. With an endogenous presence and complex regulation it has been difficult to characterize adrenocorticotropin's role in the immune system. Here, we propose that adrenocorticotropin is a tonis regulator of immune response, i.e. it tonally inhibits immunocytes which undergo disinhibition as the result of exposure to stimulatory signals, e.g. cytokines, neuropeptides, etc. Thus, adrenocorticotropin appears to set the threshold for immunoactivation by controlling the degree of immunoexcitability.

Adjuvants, Immunologic↗

PubMed1996

A novel view of opiate tolerance.

Opiate substances occur as natural compounds in various invertebrate and vertebrate neural tissues. Recently we have discovered a novel opiate alkaloid-selective and opioid peptide-insensitive receptor, designated mu 3, that provides further evidence of the existence of separate morphine processes. Interestingly morphine biosynthesis appears to be linked to the dopamine pathway. Based on studies documenting the presence of morphine after stress, e.g., trauma, it is noted that this signal substance emerges after a timely delay. From this we speculate that this molecule can serve a specific effect to downregulate physiological processes after stress. We conclude that tolerance represents a natural process that terminates its action. In this regard a morphine hypothesis may be essential to a complete picture of motive circuitry. A speculative view of the psychiatric implications in schizophrenia, depression, and autism are presented with this in mind.

Alkaloids↗

PubMed1996

Opioid and opiate immunoregulatory processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel delta, mu, and kappa receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel mu3, opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Animals↗

PubMed1995

Effect of prolonged exposure to morphine on responsiveness of human and invertebrate immunocytes to stimulatory molecules.

This study deals with a novel role of morphine in the modulation of cellular responsiveness to immunostimulatory substances that, at first glance, appears to be in contrast to the well documented immunoinhibitory short-term effects of opiate alkaloids on cells simultaneously exposed to stimulatory molecules. Vertebrate and invertebrate immunocytes pre-exposed to morphine (10(-6) M) in vitro for at least 24 h prior to the administration of lipopolysaccharide (LPS; 1.0 micrograms/ml) or other immunoactivating substances have revealed a distinct enhancement of their responsiveness to these signals, e.g. monocytes exposed to LPS alone resulted in 21% activation, whereas the morphine pretreated level was at 40% (P < 0.01). Prolonged pretreatment with morphine of naive human monocytes had the same effect on their sensitivity to plasma from patients having undergone cardiopulmonary bypass (CPB) operations followed by a diffuse inflammatory response. These results suggest that endogenous opiates may participate, in more than one way, in re-establishing an organism's readiness to meet a new demand on its immune system. Additional support for the concept of a role of endogenous opiates in immunomodulation was obtained by the results of in vivo tests with experimentally induced stress in Mytilus. Following their stress-induced stimulation, these animals' immunocytes could be shown to become exposed for some time to a measurable rise in endogenous morphine-like material (9 pmol/ml increasing to 59). These immunocytes, like those preincubated with exogenous morphine, displayed a heightened sensitivity to stimulation by LPS (control 21.3 +/- 3.1% activation compared to 47.2 +/- 5.1) when the morphine levels dropped. The mechanism of this enhancement of responsiveness to immunostimulation following the prolonged exposure of immunocytes to morphine, and its relationship with the known short-term immunoinhibitory opiate effects on the immune system, remains to be ascertained.

Animals↗

PubMed1995

Presence of the mu3 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 mu3) 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 mu3 receptor is coupled to nitric oxide release and vasodilation.

Animals↗