Reginald Matejec

Release of β-endorphin immunoreactive material under perioperative conditions into blood or cerebrospinal fluid: Significance for postoperative pain?

The function of beta-endorphin immunoreactive material (IRM) released under perioperative conditions remains to be clarified. In 17 patients undergoing orthopedic surgery, we determined beta-endorphin IRM in venous blood plasma and in cerebrospinal fluid (CSF) before surgery (t(A)); after termination of surgery and general anesthesia, but still under spinal anesthesia (t(B)); on occurrence of postoperative pain (t(C)); and 1 day after the operation (t(D)). Pain severity was rated by the patients by using a visual analog scale. Patients felt postoperative pain (t(C)), but they felt no pain at times t(A), t(B), and t(D). beta-Endorphin IRM plasma levels before surgery (t(A)) or with postoperative pain (t(C)) proved to be significantly higher than levels determined just after surgery, but still under spinal anesthesia (t(B)), or those determined 1 day after the operation (t(D)); beta-endorphin IRM plasma levels at times t(A) and t(C) correlated positively with postoperative pain severity (t(C)). beta-Endorphin IRM CSF levels after surgery, but still under spinal anesthesia (t(B)), were significantly higher than levels determined at times t(A), t(C), or t(D). No correlation was found between beta-endorphin IRM CSF levels and pain severity. In conclusion, postoperative pain severity appears to be related to beta-endorphin IRM levels in plasma before surgery as well as with postoperative pain; the analgesic significance of this material remains to be elucidated.

Interleukin-6 Contributes to the Paracrine Effects of Cardiospheres Cultured from Human, Murine and Rat Hearts

Cardiosphere‐derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned‐medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor’s heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin‑6. CDCs were also generated from Interleukin‑6−/−‐mice and their wildtype littermates. The supernatant of the cultured Interleukin‑6−/−‐CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin‑6+/+‐CDCs showed a positive effect. To confirm the hypothesis that Interleukin‑6 contributes to the paracrine effects, CMCs were incubated with Interleukin‑6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin‑6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin‑6 is involved in this phenomenon. J. Cell. Physiol. 229: 1681–1689, 2014.

Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and α-keto acid homeostasis or immunocompetence

Summary.We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, β-alanine and DFMO on neutrophil amino and α-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of •NO-synthase [L-NAME], an •NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [β-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.

Corticotropin-Releasing Hormone Reduces Pressure Pain Sensitivity in Humans without Involvement of β-Endorphin(1–31), but Does Not Reduce Heat Pain Sensitivity

In the present study the effects of intravenously administered corticotropin-releasing hormone (CRH) on the release of proopiomelanocortin (POMC) derivatives such as adrenocorticotropic hormone (ACTH), β-lipotropin (β-LPH) and β-endorphin (β-END) as well as direct effects of CRH on pain sensitivity were examined. In 16 healthy volunteers we studied the effects of 100 µg intravenously administered CRH in absence or presence of 12 mg naloxone on heat or pressure pain sensitivity, using a double-blind, cross-over and placebo-controlled design. To evaluate analgesic effects of CRH via release of POMC derivatives, we determined plasma concentrations of β-END-immunoreactive material (IRM), authentic β-END (β-END(1–31)) and β-LPH IRM, in parallel with heat and pressure pain tolerance thresholds before and 15 and 30 min after treatment with CRH (or placebo), and 5 min after naloxone (or placebo) administration which was administered 40 min after CRH (or placebo) injection. CRH increased levels of β-END IRM, β-END(1–31) and β-LPH IRM. As compared to β-END IRM levels measured by a commercial RIA kit, the β-END(1–31) levels determined by a highly specific two-site RIA, proved to be remarkably small. Furthermore, CRH did not induce increases of heat pain tolerance thresholds, but of pressure pain tolerance thresholds, which, however, were not reversible by naloxone. Neither β-END nor β-LPH IRM nor β-END(1–31) levels correlated with heat or pressure pain tolerance thresholds. We conclude that CRH does not modulate heat, but pressure pain; POMC derivatives like β-END IRM, β-END(1–31) or β-LPH do not mediate this effect.

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine.

Summary.The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O2−) generation, hydrogen peroxide (H2O2) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H2O2 generation. Formation of O2− on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O2− and H2O2 formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.

Release of melanotroph- and corticotroph-type proopiomelanocortin derivatives into blood after administration of corticotropin-releasing hormone in patients with septic shock without adrenocortical insufficiency.

The aim of the study was to assess the adequacy of pituitary function by determining the plasma concentrations of corticotroph-type (corticotropin, &bgr;-endorphin immunoreactive material [&bgr;-END IRM], authentic &bgr;-END, and &bgr;-lipotropin IRM) as well as melanotroph-type (&agr;-melanocyte-stimulating hormone [&agr;-MSH] and N-acetyl-&bgr;-END [Nac-&bgr;-END] IRM) proopiomelanocortin (POMC) derivatives in patients under septic shock upon administration of corticotropin-releasing hormone (CRH). The objectives were to assess whether an insufficient release of corticotroph- or melanotroph-type POMC derivatives from the pituitary into the cardiovascular compartment correlates with the 28-day mortality rate. Seventeen patients with septic shock but without adrenocortical insufficiency and 16 healthy volunteers were enrolled in the study, and CRH stimulation tests were performed with an i.v. bolus injection of 100 &mgr;g human CRH. After treatment with CRH, plasma concentrations of corticotroph-type POMC derivatives increased in survivors and nonsurvivors, melanotroph-type POMC derivatives such as &agr;-MSH or Nac-&bgr;-END IRM increased only in survivors in contrast to nonsurvivors. The release of &agr;-MSH and Nac-&bgr;-END IRM was suppressed by dexamethasone in survivors but not in nonsurvivors. In patients with septic shock, the response of the pituitary to CRH stimulation in terms of &agr;-MSH or Nac-&bgr;-END IRM release was impaired in nonsurvivors compared with survivors or controls. Reduced responses of &agr;-MSH or Nac-&bgr;-END IRM to CRH and the invalid suppression by dexamethasone reflect a state of dysfunction of the melanotroph-type POMC system in nonsurvivors. Considering anticytokine and anti-inflammatory effects of &agr;-MSH, this dysfunction may increase the risk of death in patients with septic shock.

Benzodiazepine receptor-dependent modulation of neutrophil (PMN) free amino- and α-keto acid profiles or immune functions

Summary.We have examined the effects of midazolam, Ro 5-4864 (agonist for “peripheral” [p] benzodiazepine receptors [BR]), PK 11195 (antagonist for pBR), flumazenil (antagonist for “central” BR), naloxone (antagonist for opiate receptors) and the combination of midazolam and Ro 5-4864, PK 11195, flumazenil or naloxone on intracellular amino- and α-keto acids and the immune function markers superoxide anion (O2−), hydrogen peroxide (H2O2) and released myeloperoxidase (MPO) activity in neutrophils (PMN). Only midazolam and Ro 5-4864 led to significant changes in the dynamic PMN free amino- and α-keto acid pools. Concerning PMN immune function markers, midazolam and Ro 5-4864 significantly decreased O2− and H2O2 formation and released MPO. When midazolam and Ro 5-4864 were applied together they appeared to act additively. Pre-incubation with PK 11195 partially neutralized the midazolam effects whereas flumazenil or naloxone showed no effects. We therefore believe that pBR are involved in the signal transmission of anesthetic-induced cellular metabolic changes in PMN.

Intracellular alpha-keto acid quantification by fluorescence-HPLC

Procedures for the analysis of free α-keto acids in human fluids (i.e. plasma, cerebrospinal fluid, urine, etc.) as well as for studying the dynamic free α-keto acid pools in differentiated tissues and organ cells have been the subject of growing clinical interest in the study of metabolic regulatory and pathophysiological phenomena. Due to the high instability and polarity of the α-keto acids being examined, the development of a quantitative and reproducible analysis of metabolically relevant intracellular α-keto acids still presents a substantial methodological challenge. The aim of small sample size, rapid, non-damaging and “metabolism-neutral” cell isolation, careful sample preparation and stability, as well as reproducible analytics technology is not often achieved. Only few of the methods described can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular α-keto acid analysis.

Nitric oxide and polyamine pathway-dependent modulation of neutrophil free amino- and α-keto acid profiles or host defense capability

Summary.We have examined the effects of Nω-nitro-L-arginine-methylester-hydrochloride [L-NAME; inhibitor of nitric oxide synthase], S-nitroso-N-acetyl-penicillamine [SNAP; nitric oxide donor], α-difluoro-methyl-ornithine [DFMO; inhibitor of ornithine decarboxylase] arginine or ornithine as well as the combination of arginine or ornithine with L-NAME, SNAP or DFMO on intracellular free amino- and α-keto acid profiles and the immune function markers superoxide anion and hydrogen peroxide generation as well as released myeloperoxidase activity in neutrophils (PMN). Although the underlying mechanisms still remain unclear, we believe from our results that nitric oxide as well as polyamine-dependent pathways are involved in the signal transmission of free radical molecule, beneficial nutritional therapy or maleficient pharmacological stress-induced alterations in PMN nutrient composition. Relevant changes in intragranulocyte free amino- and α-keto acid homeostasis and metabolism, especially, may be one of the determinants in PMN nutrition that positively or negatively influences and modulate neutrophil host defence capability and immunocompetence.

Effects of ornithine on neutrophil (PMN) free amino acid and α-keto acid profiles and immune functions in vitro

Summary.The objective of this study was to determine the effects of ornithine on polymorphonuclear leucocyte (PMN) free amino- and α-keto acid profiles, superoxide anion (O2−) generation, hydrogen peroxide (H2O2) formation and released myeloperoxidase acitivity (MPO). Exogenous ornithine significantly increased PMN asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, α-ketoglutarate and pyruvate as intracellular ornithine increased. Concerning PMN immune function markers ornithine increased H2O2-generation and MPO acitivity while O2−-formation was decreased. We believe therefore that ornithine is important for affecting PMN “susceptible free amino- and α-keto acid pool” although the mechanisms are not yet clear. This may be one of the determinants in PMN nutrition considerably influencing and modulating PMN host defense capability.