Cyclic gmp is a measure of physiologic stress.

This study was undertaken to determine whether patients who were critically ill evidenced elevated levels of blood cyclic guanosine monophosphate (cGMP). Cyclic guanosine monophosphate levels correlated with severity of illness as measured by the APACHE II severity of illness scoring system (p < 0.01). Cyclic guanosine monophosphate also correlated with the level of carboxyhemoglobin (HbCO) (p < 0.001). The correlation between cGMP and creatinine was p < 0.0001. Patients with end-stage disease (renal or liver) tended to have elevated levels of cGMP (p < 0.0001). We conclude that the induction of these two molecules may be linked in patients with increasing severity of illness.

Metallothionein induction in islets of Langerhans and insulinoma cells.

Isolated pancreatic islets from rat and mouse and the insulinoma cell lines, betaHC9 and RINm5F, were investigated to determine the regulation of metallothionein (MT). Dexamethasone (DEX) increased rat and mouse islet and insulinoma cell MT levels in a time- and concentration-dependent manner. Rat islet MT expression was increased with interleukin-1beta (IL-1beta), but not tumor necrosis factor-alpha (TNF). However, MT induction by IL-1beta and TNF was synergistic with DEX in rat islets and insulinoma cells. Mouse islet MT failed to respond to IL-1beta alone, although IL-1beta and TNF were synergistic. IL-1beta and TNF did not synergize with DEX for mouse islet MT induction. Zinc sulfate induced MT in rat islets but not mouse islets. MT messenger RNA levels were significantly increased in rat islets in response to DEX and IL-1beta plus DEX. The inducible nitric oxide synthase inhibitors N(G)-monomethyl-L-arginine and aminoguanidine failed to inhibit IL-1beta induced MT levels in insulinoma cells, and the nitric oxide generating agent sodium nitroprusside failed to significantly affect MT levels. Phorbol dibutyrate increased MT levels in rat islets and betaHC9 cells, but phorbol dibutyrate and IL-1beta effects were not additive. Transgenic MT-null and wild-type mouse islets had similar insulin contents, but basal and glucose-stimulated insulin release from MT-null islets were significantly lower than in wild-type islets. Blood glucose levels in MT-null mice were, however, slightly lower than those in wild-type mice. Thus, MT induction in pancreatic islets and beta-cells is regulated by cytokines and DEX, and protein kinase C activation may play a role. However, regulation of MT induction in mouse and rat islets differs. MT also appears to modulate insulin release from pancreatic islets.

Metallothionein in human disease.

Evidence concerning a role for metallothionein (MT) in human disease is reviewed. Current knowledge of MT is juxtaposed with our understanding of the pathogenesis of disease. MT is known to modulate three fundamental processes: 1) the release of gaseous mediators such as hydroxyl radical or nitric oxide; 2) apoptosis, and 3) the binding and exchange of heavy metals such as zinc, cadmium or copper. The capability to specifically manipulate MT levels in cells and in mice is beginning to provide answers regarding how MT could impact complex disease scenarios. Associations among MT and several diseases, including cancer, circulatory and septic shock, coronary artery disease, and Alzheimer's disease have been made. Strong evidence exists that MT modulates the immune system. The primary function of MT remains unknown.

Measurement of self-esteem in repeat assault victims.

Background characterization of assault-related injuries have demonstrated that lifestyle, substance abuse, education, employment, mental illness, and high-risk behavior contribute to low self-esteem in repeat assault victims. Recurrent-assault patients have never been studied with respect to self-esteem. This study evaluated self-esteem and assault-related injury in 28 consecutive male assault patients (11 first-assault and 17 recurrent-assault patients) and 19 controls with no previous assault history. Study participants were administered the Walmyr Assessment Scales Index of Self-Esteem (WASISE) as part of a three-item survey to determine the relationship between self-esteem and assault-related injury. No demographic differences were found between the groups. The mean (+/- standard error) WASISE score for recurrent-assault patients (34.9 +/- 3.4) was significantly higher than mean scores for no-assault and first-time assault-injured patients (14.7 +/- 1.4 and 15.0 +/- 2.3, respectively). The recurrent-assault patients had a lower mean education level than both no-assault and first-time assault-injured patients (2.1 +/- 0.26 and 1.9 +/- 0.57, respectively). Eighteen (2%) first-time assault-injured and 11 (7%) recurrent-assault patients were employed. These data suggest that self-esteem, education, and employment history need to be considered when evaluating and developing interventions for repeat-assault patients.

Opposite effects of metallothionein I and spermine on mitochondrial function.

Previously, we reported that the stress-induced protein metallothionein I (MT) modulated the oxygen consumption (VO2) of isolated rat liver mitochondria [Life Sci. 55 221-226, 1994]. We now present confirmation of this finding, and the additional observations that in rat liver mitochondria, MT caused swelling and depolarization. These actions of MT were inhibited by the aliphatic polyamine, spermine. Our findings suggest that mitochondrial function could be influenced by the balance between MT and spermine.

Beta-endorphin modulates calcium channel activity in human neutrophils.

10(-6) M n-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated Ca2+ flux in human neutrophils is characterized by a profile composed of two peaks of different amplitude and breadth. beta-Endorphin inhibited the magnitude and modulated the kinetics of the second peak in a manner which was dose-dependent and could reflect either negative cooperativity or heterogeneity of binding sites. The second peak arises from calcium channel activity since in the presence of nifedipine or EGTA it was not evident while the first peak was reduced about 24%. Similarly, at 15 degrees C, where we were unable to detect any channel activity, the first peak was diminished by 35% and beta-endorphin had no detectable effect on this peak. These results led us to conclude that the first peak is chiefly composed of Ca2+ recruited from cytosolic stores which are relatively insensitive to the above treatments and a smaller fraction of calcium originating in calcium channel activity. Hence, we reason that beta-endorphin modulates only the calcium ion flux arising from calcium channel function.

Effect of metallothionein I on mitochondrial oxygen consumption.

The effect of the stress-induced, cysteine-rich protein, metallothionein I (MT), on oxygen consumption by rat liver mitochondria was studied. Using a Clark-type oxygen electrode we found that electron transport from succinate to oxygen was enhanced by MT whereas ADP-initiated oxygen consumption was inhibited by MT. The MT effect was concentration dependent. No evidence was found for the provision by MT of an alternate pathway to oxygen.

Reduction of cytochrome C by fragments of heat shock protein 70.

Using absorption spectroscopy, we have found that two highly conserved fragments of heat shock protein 70 reduce cytochrome c. The fragments are SCV and YSCVGVF. YSCVGVF was more potent than SCV at low concentrations (10(-6)-10(-4)M). At high concentrations (5 x 10(-4)-10(-3)M) SCV was more potent than YSCVGVF. Reduction of CC by YSCVGVF was reversed by cytochrome c oxidase. These findings show that heat shock protein 70 and/or small intracellular peptides could carry electrons to cytochromes.

Prostaglandin D2 modulates human neutrophil intracellular calcium flux and inhibits superoxide release via its ring carbonyl.

We compared the effects of prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF2) and various ketones on superoxide (OX) release by human neutrophils, which had been stimulated by N-formyl methionyl leucyl phenylalanine (FMLP). Our data suggested that the ring carbonyl of PGD2 is essential to its inhibitory effect on OX release, but the carbonyl group as a ketone, alone is not sufficient. Using the fluorescent Ca2+ probe, Fura-2AM, we found that PGD2 increased the rate of decline of FMLP stimulated intracellular free Ca2+ (Ca)i, but that PGF2 had no effect. cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. Furthermore, the ring carbonyl of PGD2 is crucial to its effect on OX as well as on (Ca)i.

Human erythrocytes have binding sites for beta-endorphin.

Monoiodinated human beta-endorphin was found to bind specifically to human erythrocytes. Unlabeled beta-endorphin and beta-endorphin inhibited binding, but (-)naloxone, [D-Ala2, D-Leu5]-enkephalin, and leu- and met-enkephalin did not. Immunoelectron microscopy, using rabbit anti-beta-endorphin antibody, an antirabbit IgG secondary antibody, and complexed horseradish peroxidase, revealed that at low concentrations beta-endorphin binds to the cell surface. Electron spin resonance spectroscopy showed no effect of beta-endorphin on membrane fluidity. This receptor does not appear to conform to the characteristics of an opiate receptor.

Phospholipase C activity in human polymorphonuclear leukocytes: partial characterization and effect of indomethacin.

Several hormones act at the cellular level to increase diacylglycerol via increased catabolism of phosphatidylinositol by phospholipase C. Diacylglycerol stimulates protein kinase C, leading to protein phosphorylation and hormone action. Since phospholipase C activity has not been well studied in man, we have established an assay for phospholipase C in human neutrophils. In this assay sonicates of neutrophils were incubated with L-3-phosphatidyl-[U 14C]-inositol and the incubation mixture extracted with chloroform/methanol. Following the additions of 2 mol/l KCl and chloroform, phospholipase C activity was determined by counting [14C] in the aqueous phase. The phospholipase C activity was linear with respect to time and the quantity of added enzyme. Optimum substrate concentration and pH were 2 mmol/l and 7.0, respectively. Optimal activity was dependent on Ca2+ (2 mmol/l) and deoxycholate (2 mmol/l). Naloxone, and PGD2, which affect various aspects of leucocyte function, had no significant effects on neutrophil PLC activity. The effects of various compounds with phospholipase A2 inhibitory activity were also tested on this enzyme. Of these, mepacrine, lidocaine and indomethacin inhibited the enzyme activity. The inhibition by indomethacin was of the noncompetitive type with an apparent Km of 0.17 X 10(-6) mol/l and apparent Ki of 3.6 X 10(-6) mol/l. From these data we conclude that indomethacin is capable of inhibiting phospholipase C activity in neutrophils at clinically significant levels and that this may be relevant in the therapeutic action of this drug.

Macrophage-conditioned medium and interleukin 1 suppress vascular contractility.

Isolated rat aortas, after incubation in medium conditioned by endotoxin-stimulated peritoneal macrophages, exhibited diminished contraction to norepinephrine (maximal contraction: control medium = 713 +/- 37 (SE) mg tension/mg tissue; medium conditioned by macrophages = 437 +/- 38, P less than .001). Medium containing endotoxin alone or medium conditioned by nonstimulated macrophages had no effect on aortic tissue response to norepinephrine. Stimulation of peritoneal macrophages in vivo by sterile silica particles also induced diminished contractile responses to norepinephrine by subsequently isolated aortas. Incubation of rat aortas with human monocyte-derived interleukin 1 or recombinant human tumor necrosis factor resulted in diminished aortic contraction and sensitivity to norepinephrine, and gel filtration of medium conditioned by endotoxin-stimulated macrophages yielded suppressive activity at a molecular weight equivalent to interleukin 1 and tumor necrosis factor. The data suggest that mononuclear phagocytes may contribute to altered vascular function in sepsis via the release and vascular modulatory effects of interleukin 1 and tumor necrosis factor.

Beta-endorphin and lipopolysaccharide interactions with human neutrophils.

The binding of the Escherichia coli peptide, N-formyl methionyl leucyl phenylalanine (FMLP), to human neutrophils was found to be reduced by E coli lipopolysaccharide (LPS). This reduction is reversed by human ß-endorphin 1-31. ß-Endorphin (BE) also increased the binding of FMLP in the absence of LPS. Structural analogs of BE, namely BE 1-27 and N-acetyl BE 1-31, were equal to BE in potency. BE 6-31, however, was less potent than BE. These effects may be mediated by a neutrophil binding site for BE, which was found to have a K(D) of 4.1 × 10(7) and 315,930 sites per cell. These findings provide an explanation for the authors' previous observation that BE enhances the chemotaxis of neutrophils toward FMLP. Furthermore, these data suggest that there may be a role for BE in the modulation of neutrophilic function in the septic state.

The effect of enkephalins and prostaglandins on O-2 release by neutrophils.

Derivatives of superoxide (O-2), produced by phagocytic cells, are thought to play a role in the adult respiratory distress syndrome (ARDS) and other disease states. Control of the release of O-2 may prove beneficial. Using human neutrophils as a source of O-2, and an assay for O-2 based upon the reduction of cytochrome C, we found that prostaglandin D2 (PGD2), leucine enkephalin (LE), and methionine enkephalin (ME) inhibited O-2 release. The Escherichia coli product, N-formyl methionyl leucyl phenylalanine (FMLP), was employed to stimulate O-2 release. PGD2 was most potent while there was no significant difference between LE and ME. Another peptide, thyrotropin releasing hormone (TRH), had no effect on O-2 release. There was no correlation between the potency of the inhibitory effect on O-2 release and the effect of these agents on the binding of [3H] FMLP to human neutrophils. Comparison of different but structurally related prostaglandins (PGD2, PGE2, and PGF2 alpha) revealed that PGD2 was more potent than PGE2 in inhibiting O-2 and that PGF2 alpha had no effect. This result suggested that the presence and position of the carbonyl group was an important determinant of the magnitude of inhibition.

Inhibition by naloxone of neutrophil superoxide release: a potentially useful antiinflammatory effect.

The working hypothesis of many studies of shock has been that naloxone acts by blocking centrally and/or peripherally located opioid receptors. At plasma concentrations used to treat experimental shock (10(-6) M and above), naloxone inhibited the in vitro release of superoxide (O2-) by human neutrophils that were stimulated by the E. coli peptide N-formyl methionyl leucyl phenylalanine (FMLP). Superoxide release stimulated by phorbol 12,13-dibutyrate (PDB) was also inhibited by naloxone. Naloxone had no effect on the FMLP-stimulated release of beta-glucuronidase or lysozyme. Naloxone had no effect on 3H FMLP receptor binding. Studies utilizing 3H naloxone revealed the presence of a ligand-specific naloxone binding site on human neutrophils with a Kd of 1.2 X 10(-5) M, which is close to the ID50 of the inhibitory effect upon O2- release (1.8 X 10(-5). Thyrotropin releasing factor (TRF) had no effect upon 3H naloxone binding or on O2- release. Verapamil, a calcium channel blocker, inhibited 3H naloxone binding, and O2- release while nifedipine, another calcium channel blocker had no effect on either assay except at 10(-4) M, at which concentration 3H naloxone binding as well as the release of O2- were increased. These experiments suggest that the inhibitory effect of naloxone upon O2- release is mediated via a specific binding site.

Naloxone inhibits superoxide release from human neutrophils.

Using the superoxide dismutase inhibitable reduction of cytochrome c assay, we studied, the effect of (-) naloxone on N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated superoxide (O2-) release from human neutrophils. Neutrophils were pre-incubated with the range of concentrations of (-) naloxone that is administered in models of experimental sepsis (10(-6) - 10(-4.5) M). (-) Naloxone inhibited O2- release in a dose dependent manner. 02- produced by a cell-free xanthine-xanthine oxidase system was not inhibited by (-) naloxone, indicating that (-) naloxone was not scavanging O2-. There was no difference between the effect of (-) and (+) naloxone suggesting that the inhibition of O2- was not specific for an opiate receptor. Another opiate antagonist, nalorphine, as well as the opiate agonist, morphine, also inhibited O2- release in the same concentration range. There was no difference between the effect of naloxone and morphine.

Human neutrophil migration is enhanced by beta-endorphin.

Using a serum-free chemotaxis-under-agarose assay, we measured the effect of beta-endorphin on directed migration of human neutrophils toward 10(-7) M N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neutrophils were pre-incubated with a range of beta-endorphin concentrations. beta-endorphin enhanced migration of neutrophils toward FMLP. This effect was maximal at 10(-9) M beta-endorphin. Naloxone inhibited the beta-endorphin effect, suggesting that enhanced migration is mediated via an opiate receptor.