Pulmonary and systemic vascular responsiveness to TNF-alpha in conscious rats

Endotoxin decreases pulmonary vascular reactivity. Because tumor necrosis factor-alpha (TNF-alpha) is a primary mediator of endotoxemia, we tested whether TNF-alpha altered pulmonary vascular reactivity in conscious adult female rats. Osmotic pumps were implanted intraperitoneally, and low-dose TNF-alpha (62 micrograms, TNF62; n = 7), high-dose TNF-alpha (> or = 250 micrograms, TNF250; n = 5), or saline (n = 5) was administered for 2 wk. Pulmonary pressor responses to 14% O2 and angiotensin II (ANG II, 0.0206 micrograms/min for 10 min) were measured without (day 13) or after (day 14) administration of nitro-L-arginine (4.4 mg/kg iv), an inhibitor of endothelium-derived relaxing factor (EDRF). TNF-alpha administration slightly decreased (P < or = 0.08) baseline pulmonary arterial pressure in TNF250 rats and decreased (P < or = 0.05) hypoxia- and ANG II-induced constrictions in TNF62 and TNF250 rats. Whereas nitro-L-arginine potentiated (P < or = 0.05) pressure responses in control rats, it had no effect on hypoxic responses in TNF-alpha-treated rats. Nitro-L-arginine increased (P < or = 0.05) ANG II-induced vasoconstriction in TNF-alpha-treated rats, but the pulmonary arterial pressure response was still lower (P < or = 0.05) in TNF250 than in control and TNF62 rats. These results suggest that chronic TNF-alpha decreases 1) pulmonary vascular reactivity in the intact rat, 2) hypoxic pulmonary vasoconstriction by a mechanism that is independent of EDRF, and 3) ANG II-induced constriction by a mechanism that is partly EDRF dependent.     

Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12

We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-alpha and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-alpha and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naive macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-gamma dose dependently and differentially regulated subsequent LPS-induced production of TNF-alpha, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-alpha and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-alpha responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-alpha response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-alpha production. We also found that the initial LPS priming of naive macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-gamma in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-alpha and NO responses in LPS-primed macrophages.     

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes

We studied the pathways of macrophage response to lipopolysaccharide (LPS). When mouse macrophages pre-exposed to LPS were restimulated with this agent, reduced tumour necrosis factor-alpha (TNF-alpha) responses (desensitization/endotoxin tolerance) were accompanied by increased (priming) nitric oxide (NO) responses. Priming was also inducible with recombinant interferon-beta (IFN-beta). The requirement of TNF-alpha biosynthesis in the LPS-induced priming was also suggested by the observation that both anti-TNF-alpha serum and pentoxifylline inhibited this effect. However, addition of mouse recombinant TNF-alpha (mrTNF-alpha) did not enhance the priming induced by LPS or IFN-beta, and preincubation with mrTNF-alpha alone, or in association with other cytokines produced by macrophages (interleukin-1 beta, interleukin-6, or leukaemia inhibitory factor), did not induce a priming effect. We found however, that pentoxifylline, which blocked the priming, also decreased the level of membrane-bound TNF-alpha. Furthermore, exposure to compound BB-3103 (a metalloproteinase inhibitor that blocks the processing of membrane-bound TNF-alpha yielding to the secreted cytokine) enhanced the priming effect, the expression of membrane TNF-alpha and the specific binding of LPS. These observations suggest that the membrane form of TNF-alpha is involved in the interaction of LPS with a receptor required for LPS-induced priming.     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway

IL-1 plays an important role in the pathophysiologic responses to infection and inflammation, in part by mediating its own production and that of other proinflammatory cytokines. However, the relative contribution of IL-1 alpha and IL-1 beta to the inflammatory response has not been well clarified. Using IL-1 beta-deficient (IL-1 beta -/-) mice, we investigated the specific role of IL-1 beta in the in vivo and in vitro response to LPS. No differences between IL-1 beta +/+ and IL-1 beta -/- mice were observed in circulating levels for IL-1 alpha, IL-6, or TNF-alpha after the systemic administration of either a low (5 micrograms/kg) or high (5 mg/kg) dose of LPS. IL-1 beta -/- mice also had a normal response to LPS in terms of activation of the hypothalamus-pituitary-adrenal axis, hypoglycemia, serum amyloid A production, and anorexia. IL-1 beta -/- mice were normally sensitive to the lethal effect of LPS and were protected against LPS toxicity when pretreated with low-dose LPS. However, in vitro, peritoneal macrophages from IL-1 beta -/- mice stimulated with LPS produced significantly less IL-1 alpha than macrophages from IL-1 beta +/+ mice (p < 0.05). No differences in IL-6 or TNF-alpha synthesis were observed between macrophages from IL-1 beta +/+ and IL-1 beta -/- mice. In summary, our results suggest that either IL-1 beta is not essential for the in vivo systemic response to LPS or that its role can be fulfilled by other cytokines with overlapping activities.     

Production of inflammatory mediators by human macrophages obtained from ascites

Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.     

Endothelin 1 mediates ex vivo coronary vasoconstriction caused by exogenous and endogenous cytokines

Treatment of rats with cytokines has been associated with an increase in the circulating levels of endothelin 1 (ET-1). Here we show that administration of tumor necrosis factor alpha (TNF-alpha; 4 micrograms.kg-1) to anesthetized rats caused within 15 min a strong elevation in the circulating levels of ET-1. This was associated with a striking coronary vasoconstriction in hearts from these animals when they were removed and perfused in vitro by the Langendorff technique. This vasoconstriction was largely overcome by treatment with either the endothelin type A (ETA) receptor antagonist FR 139317 or antibody against ET-1. Furthermore, it was mimicked by in vivo exposure to exogenous ET-1. Endogenously produced TNF-alpha may also cause such a coronary vasoconstriction, for treatment with interleukin 2 (600 micrograms.kg-1) produced an increase in coronary perfusion pressure that correlated with the increases in circulating TNF-alpha. This coronary vasoconstriction was substantially reversed by treatment either with antibody against TNF-alpha or with FR 139317. We suggest, therefore, that cytokine-driven changes in the production of ET-1 are key events in the development of vascular pathologies.     

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-alpha, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.     

Tumour necrosis factor-alpha as a target of melanocortins in haemorrhagic shock, in the anaesthetized rat

The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved (mostly through the activation of inducible nitric oxide synthase) in the pathogenesis of circulatory shock. On the other hand, melanocortin peptides are potent and effective in reversing haemorrhagic shock, both in animals (rat, dog) and in humans. This prompted us to study the influence of the melanocortin peptide ACTH-(1-24) on the blood levels of TNF-alpha in haemorrhage-shocked rats and on the in vitro production of TNF-alpha by lipopolysaccharide (LPS)-activated macrophages. Plasma levels of TNF-alpha were undetectable before starting bleeding and greatly increased 20 min after bleeding termination in saline-treated rats. In rats treated with ACTH-(1-24) the almost complete restoration of cardiovascular function was associated with markedly reduced levels of TNF-alpha 20 min after bleeding termination. On the other hand, ACTH-(1-24) did not influence TNF-alpha plasma levels in sham-operated, unbled rats. In vitro, ACTH-(1-24) (25-100 nM) dose-dependently reduced the LPS-stimulated production of TNF-alpha by peritoneal macrophages harvested from untreated, unbled rats. These results indicate that inhibition of TNF-alpha overproduction may be an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock.     

Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor-alpha in mice with acute graft-versus-host disease

Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35-fold. Activated GVHD splenocytes secreted large amounts of TNF-alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF-alpha antibody (Ab) both abrogated NO production and restored LPS-induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl-arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin co-stimulates staphylococcal enterotoxin beta (SEB) cytokine production and phenotypic cell cycling in Long-Evans rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that is considered to be a potent immunotoxicant. In the present study, we examined the effect of 25 micrograms/kg TCDD on cytokine production and T lymphocyte phenotype, cell cycling and receptor activity in female Long-Evans rats that had been injected with 50 micrograms of Staphylococcal Enterotoxin B (SEB). In the SEB-injected rats, TCDD increased the serum levels of interleukin-2 (IL-2) but did not affect the serum levels of interleukin-1 (IL-1), interleukin-6 (IL-6) or tumor necrosis factor (TNF). The ability of spleen cells and peritoneal cells to produce cytokines in response to SEB restimulation in vitro was also evaluated. TCDD exposure significantly enhanced IL-2 production by spleen cells from SEB-primed rats after 6 h or 24 h in cultures co-stimulated with SEB in vitro. However, TCDD treatment did not alter the production of IL-6 and TNF in these cultures. Although TCDD did not influence the production of IL-6 and TNF in peritoneal cells from SEB-primed rats with SEB restimultion in vitro, IL-1 production was significantly increased at 2 h. Both the kinetics and extent of SEB-induced IL-2 receptor (IL-2R) and T-cell receptor (TCR) expression on CD4+ cells was unaffected by TCDD. TCDD did not significantly alter the percentage or the total numbers of CD4+ and CD8+ subpopulations at various times after SEB injection. However, flow cytometric analysis showed that TCDD exposure increased the percentage of both CD4+ and CD8+ cells cycling in the S and G2M phase. TCDD, in the absence of SEB priming, did not affect any of the immune parameters tested. Nevertheless, collectively these results showed that TCDD can enhance the production of IL-2 and the percentage of CD4+ and CD8+ cells cycling in SEB-exposed Long-Evans rats. Histopatholgically, there were not observable effects of SEB on lymphoid organs while thymic atrophy and diffuse hepatocellular hypertrophy was observed in the TCDD-treated animals.     

Ursodeoxycholic acid modifies gut-derived endotoxemia in neonatal rats

We developed a model for the translocation of intraluminal endotoxin in the neonatal animal and used it to examine the capacity of a nonhepatotoxic bile acid, ursodeoxycholic acid (UDCA), to modify endotoxin translocation and cytokine response. Three-d-old Sprague-Dawley rats were randomized to receive enterally either no drug, lipopolysaccharide (LPS, 1 mg/animal), or UDCA (400 micrograms/animal) alone, or UDCA followed by LPS 1 h later. One h after LPS administration, the rats were killed and plasma endotoxin and tumor necrosis factor (TNF) were measured. Control animals had low circulating endotoxin (21.2 +/- 7.6 endotoxin units) and TNF (0.06 +/- 0.02 ng/mL). Enteral administration of LPS 1 h before the rats were killed resulted in significant elevation of endotoxin (249.5 +/- 71.3, p = 0.008) and TNF (3.6 +/- 1.3, p = 0.019). UDCA alone did not alter endotoxin levels (8.7 +/- 2.1). UDCA 1 h before LPS prevented the rise in endotoxin (38.9 +/- 11.2 endotoxin units) and TNF (0.2 +/- 0.05) significantly. Chenodeoxycholic acid was studied in a similar group of experiments and prevented neither the translocation of LPS nor the development of increased TNF levels in animals receiving LPS. In conclusion, LPS can cross the intestinal barrier in the normal neonatal rat. UDCA, administered before LPS, can decrease the translocation of LPS and prevent the cytokine response as measured by TNF levels. We speculate that UDCA, administered prophylactically, might reduce morbidity in clinical conditions leading to gut-derived endotoxemia.     

Different receptor mechanisms mediate the effects of endotoxin and interleukin-1 on female sexual behavior

Activation of the immune system by lipopolysaccharide (LPS) produces physiological, neuroendocrine and behavioral effects, some of which are mediated by cytokine production. We have previously shown that the cytokine interleukin-1 (IL-1) inhibits sexual behavior in female, but not male rats, while producing a comparable suppression of locomotion in both sexes. The present study examined the effects of LPS on sexual behavior and locomotion of male and female rats, and the involvement of IL-1 receptors in mediating the effects of IL-1 and LPS on females' behavior. Peripheral (i.p.) administration of LPS (50 or 250 microg/kg) significantly decreased sexual behavior in females, up to 6 h after administration, while it had no effect on male sexual behavior. However, locomotor activity, measured in the open-field test, was similarly reduced by LPS in both males and females. Pretreatment with the IL-1 receptor antagonist (IL-1ra) either i.p. (10 mg/kg) or intracerebroventricularly (i.c.v.) (50 microg/rat) did not prevent the inhibition of female sexual behavior and locomotion induced by either i.p. (50 microg/kg) or i.c.v. (200 or 400 ng/rat) administration of LPS, respectively. However, identical doses of IL-1ra significantly reversed the effects of IL-1beta, administered either i.p. (5 microg/kg) or i.c.v. (50 ng/rat), respectively. These results demonstrate that both LPS and IL-1beta produce marked inhibition of sexual behavior in female, but not in male rats. However, IL-1 receptors are not required for the effects of LPS on sexual behavior in female rats.     

Distinct tumor necrosis factor-alpha responses in alveolar and peritoneal macrophages are associated with local levels of endotoxin

Tumor necrosis factor alpha (TNF-alpha) responses of alveolar macrophages (AMs) and peritoneal macrophages (PMs) were studied in rats after intravenous injection of lipopolysaccharide (LPS). High levels of plasma TNF-alpha, increased pulmonary myeloperoxidase activity, and leukopenia occurred within 2 h after LPS injection. Alveolar spaces exhibited a strict compartment property, as manifested by only slightly increased LPS and TNF-alpha levels in alveolar lavage fluid and an unchanged capacity of AMs to produce TNF-alpha. By contrast, the peritoneal cavity had greatly increased local LPS and TNF-alpha levels and a diminished PMs TNF-alpha response to LPS. The amount of LPS in the alveolar spaces was less than 0.2% of the level in peritoneal fluid. These results indicate that activation of resident macrophages is dependent on the amounts of local LPS and, in addition, suggest that resident AMs neither participate in the plasma TNF-alpha response nor contribute to neutrophil sequestration in the lung during the early stages of endotoxemia.     

16 beta-Methyl-17 alpha,21-diesterified glucocorticoids as partial agonists of glucocorticoid in rat endotoxin-induced inflammation

OBJECTIVE AND DESIGN: We investigated the anti-inflammatory effects of 16 beta-methyl-17 alpha,21-diesterified glucocorticoids which are well known as potent topical glucocorticoids in man on endotoxin-induced uveitis (EIU) in rats. MATERIAL: Female Lewis rats were used. TREATMENT: Glucocorticoids were instilled (0.01%-1.0%) or subcutaneously injected (0.1-10 mg/kg) to rats. METHODS: To elicit EIU, LPS (500 micrograms/kg) was injected into the footpad of rats. Twelve hours after LPS injection, cell number in aqueous humor was counted by flow cytometry. Endotoxin-induced in vivo tumor necrosis factor-alpha (TNF-alpha) production was also examined. RESULTS: 16 beta-methyl-17 alpha,21-diesterified glucocorticoids showed no effects or some enhancement of cell infiltration into the aqueous humor in EIU by topical instillation. Systemic injection of these glucocorticoids showed only weak inhibition of cell infiltration and TNF-alpha production. On the other hand, betamethasone phosphate strongly inhibited the cell infiltration and TNF-alpha production. Combined systemic injection of 16 beta-methyl-17 alpha,21-diesterified glucocorticoids and betamethasone phosphate reduced the inhibitory effects of the latter. CONCLUSIONS: These results suggest that 16 beta-methyl-17 alpha,21-diesterified glucocorticoids might act as partial agonists of glucocorticoid in rats.     

Different roles of brain interleukin 1 in the adrenocorticotropin response to central versus peripheral administration of lipopolysaccharide in the rat

Although it is well established that peripheral administration of endotoxin activates the hypothalamic-pituitary-adrenal (HPA) axis, information is very limited regarding whether central administration of endotoxin can similarly stimulate the endocrine axis. Moreover, it is also unknown whether a difference exists in the mode of involvement of brain-derived cytokines in determining the HPA response to peripheral vs central administration of endotoxin. In the present study, the authors attempted to gain more knowledge on these issues focusing on interleukin (IL) 1 in the brain, one of key pro-inflammatory cytokines mediating the immuno-endocrine network. In male rats, both intravenous (i.v., 100 micrograms/kg body weight) and intracerebroventricular [i.c.v. (the 3rd ventricle), 10 micrograms] injections of Escherichia coli lipopolysaccharide (LPS) caused a significant elevation of adrenocorticotropin (ACTH) levels in plasma, even though peaked ACTH responses occurred earlier after the i.v. (60 min post-injection) than the i.c.v. (120 min post-injection) LPS. Although the ACTH response to i.c.v. LPS was significantly suppressed by a prior (5 min) i.c.v. administration of IL-1 receptor antagonist (IL-1Ra, 1 microgram), the hormonal response to i.v. LPS was not. That this dose of IL-1Ra was not biologically a small dose was indicated by another experiment that the same dose of i.c.v. IL-1Ra was able to significantly suppress the ACTH response to an i.c.v. injection of recombinant human IL-1 beta (50 ng). These results suggest that i.c.v. LPS, as i.v. LPS, can stimulate ACTH secretion in the rat, and this hormonal response may, at least in part, be mediated by brain-derived IL-1. Although there is one previous study reporting an important role of central IL-1 in mediating the HPA response to systemic LPS treatment, our present data suggest that such a mechanism may not operate before and during an early, peak phase of ACTH secretion after i.v. LPS.     

Interactions between bacterial pyrogen and proteolipid extracted from the cerebrum (I)

As proteolipid of the myelin sheath and its parent glial membrane may possible interact with bacterial pyrogen (lipopolysaccharide, LPS) during penetration into the brain, we investigated the interaction of LPS with proteolipid derived from the cerebrum of rabbits, rats and chickens. Intravenous administration of LPS (1 microgram/kg) produced a febrile response in rabbits, but not in rats and chickens. Marked hyperthermia was observed in these three species after intracisternal administration of LPS (0.01-0.1 microgram/kg). Dinitrophenol (30 mg/kg s.c.) induced a high fever in these three species tested, particularly in the chickens. The pyrogenicity of LPS given intravenously to rabbits was inactivated by incubation of LPS with proteolipid in vitro. Inactivation effects of proteolipid extracted from the three species was in the order of: chickens, rats and rabbits. In rats, the inactivation effects of proteolipid from the adult animal were more potent than in the case of newborn animals. The febrile response induced by dinitrophenol and leucocytic pyrogen in rabbits, however, was not suppressed by incubation with proteolipid extracted from the rabbit brain. These results suggest that proteolipids do play an important role in the mechanism of penetration of LPS into the brain.     

The effects of treatment with lipopolysaccharide on responsiveness of rat blood vessels

Effects of treatment with lipopolysaccharide (LPS) both in vivo (intraperitoneal administration of 20 mg.kg-1 LPS for 6 hours) and in vitro (incubation with 1 microgram.kg-1 LPS for 6 hours) on the responsiveness of the rat thoracic aorta, carotid, renal, femoral, mesenteric, pulmonary arteries, and the femoral and mesenteric veins were examined. Intraperitoneal administration of LPS did not change the blood pressure of rats, but increased the heart rate significantly. The same amplitude of relaxation was produced by L-arginine in the aortic strips treated by LPS in vivo and in vitro, and the responses were inhibited by NG-nitro-L-arginine (L-NOARG). The contractile responses to phenylephrine in the aortic strips were reduced by LPS-treatment in vivo or in vitro, but the extent of inhibition was greater in vivo than in vitro. Further, the attenuation of contractile responses to phenylephrine was completely reversed by L-NOARG in the strips treated with LPS in vitro, whereas the reversal by L-NOARG was incomplete in the strips treated with LPS in vivo. Different amplitudes of relaxations were also produced by L-arginine or SNP in the blood vessels treated by LPS in vivo or in vitro. However, the tail artery treated with LPS in vivo or in vitro did not relax in response to L-arginine but did produce a relaxation to SNP. These results suggest that the hyporesponsiveness of rat blood vessels after treatment with LPS in vivo or in vitro may be related to an enhanced NO production in the smooth muscle cells and that there is a possible heterogeneity of regional induction or activation of L-arginine/NO pathway by LPS in rat blood vessels.     

Longtime administration of growth hormone-releasing hormone (GHRH) does not restore the reduced efficiency of GHRH on sleep endocrine activity in 2 old-aged subjects--a preliminary study

Aging results in a more shallow sleep accompanied by a blunted growth hormone (GH) secretion. In young male normal controls repetitive administration of GH-releasing hormone (GHRH) at the beginning of the night results in an increased secretion of GH, a blunting of cortisol and a stimulation of slow-wave sleep (SWS). In healthy elderly men and women, however, GHRH exerts only weak effects on sleep-endocrine activity. In a previous report continuous treatment of healthy elderly males by repetitive administration of GHRH (during 12 days administration with 100 micrograms GHRH i.v. at 9.00 h every second day, "priming") enhanced GHRH stimulated GH secretion at daytime markedly. We tested if priming with GHRH results in a more distinct modulation of the nocturnal hormone secretion and of the sleep EEG than acute administration of the peptide. Two elderly male controls spent first three consecutive nights in the sleep laboratory, the first of which served for adaptation to laboratory conditions. During the two other nights (at days 1 and 2) sleep EEG was recorded and blood was sampled for determining the secretion of GH, cortisol and ACTH. In one of the nights the subjects received 50 micrograms GHRH hourly between 22.00 h and 1.00 h (4 x 50 micrograms) or placebo. The next examination followed after the priming period at day 14 and the last was performed two weeks after treatment at day 28. After the baseline administration of 4 x 50 micrograms GHRH before priming no clear changes of sleep EEG towards improved sleep were detectable, whereas GH secretion was increased. After priming sleep period time and SWS time were lower compared to the baseline night with GHRH administration, whereas REM time duration increased. GHRH induced GH secretion was not enhanced after priming. ACTH secretion was markedly enhanced compared to baseline stimulation. We conclude that priming with GHRH has no sleep improving effect and does not change hormone secretion in elderly normal subjects. Hence in the elderly priming with GHRH is not capable to induce a rejuvenation of sleep endocrine activity.