Ursula Lang

Native and reconstituted HDL activate Stat3 in ventricular cardiomyocytes via ERK1/2: Role of sphingosine-1-phosphate

AIMS High-density lipoprotein (HDL) has been reported to have cardioprotective properties independent from its cholesterol transport activity. The influence of native HDL and reconstituted HDL (rHDL) on Stat3, the transcription factor playing an important role in myocardium adaptation to stress, was analysed in neonatal rat ventricular cardiomyocytes. We have investigated modulating the composition of rHDL as a means of expanding its function and potential cardioprotective effects. METHODS AND RESULTS Stat3 phosphorylation and activation were determined by western blotting and electrophoretic mobility shift assay (EMSA). In ventricular cardiomyocytes, HDL and the HDL constituent sphingosine-1-phosphate (S1P) induce a concentration- and time-dependent increase in Stat3 activation. They also enhance extracellular signal-regulated kinases (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. U0126, a specific inhibitor of MEK1/2, the upstream activator of ERK1/2, abolishes HDL- and S1P-induced Stat3 activation, whereas the p38 MAPK blocker SB203580 has no significant effect. Inhibition of the tyrosine kinase family Src (Src) caused a significant reduction of Stat3 activation, whereas inhibition of phosphatidylinositol 3-kinase (PI3K) had no effect. S1P and rHDL containing S1P have a similar strong stimulatory action on Stat3, ERK1/2, and p38 MAPK comparable to native HDL. S1P-free rHDL has a much weaker effect. Experiments with agonists and antagonists of the S1P receptor subtypes indicate that HDL and S1P activate Stat3 mainly through the S1P2 receptor. CONCLUSION In ventricular cardiomyocytes, addition of S1P to rHDL enhances its therapeutic potential by improving its capacity to activate Stat3. Activation of Stat3 occurs mainly via the S1P constituent and the lipid receptor S1P2 requiring stimulation of ERK1/2 and Src but not p38 MAPK or PI3K. The study underlines the therapeutic potential of tailoring rHDL to confront particular clinical situations.

Delayed sexual maturation induced by daily melatonin administration eliminates the LH response to naloxone despite normal responsiveness to GnRH in juvenile male rats

Daily administration of melatonin (MT) markedly delays sexual maturation in the male Wistar rat. In this study, we have evaluated pituitary responsiveness to GnRH and the level of tonic inhibition by endogenous opioids in normal juvenile male rats and in rats with delayed sexual development induced by daily afternoon MT injection (100 micrograms, s.c.) starting at 20 days of life. Plasma LH responses to repetitive intravenous GnRH administration (100 ng/100 g body weight), or to different doses of GnRH administered subcutaneously (5-100 ng/100 g body weight) were normal in MT-treated rats both at 30 and 40 days of life despite significantly lower number of pituitary GnRH receptors and decreased pituitary gonadotropin content. One naloxone (NAL) injection (2.5-5.0 mg/kg, s.c.) produced a significant increase of plasma LH in normal 40- and 55-day-old rats, which was not seen in MT-treated rats of the same age. In contrast, no increase of plasma LH was seen in 30-day-old control rats nor in MT-treated rats at this age. Pretreatment with morphine sulfate (10 mg/kg, s.c.), or with the potent Met-enkephalin analog FK 33-824 (1.0 mg/kg, s.c.) prevented the NAL-induced rise of plasma LH in control rats at day 40 of life. In all instances, plasma PRL levels were decreased after NAL both in untreated and in MT-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Native and reconstituted HDL protect cardiomyocytes from doxorubicin-induced apoptosis

AIMS We analysed the impact of native and reconstituted HDL on doxorubicin-induced cardiomyocyte apoptosis. While it is an effective anti-cancer agent, doxorubicin has serious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes, notably against oxidative stress. METHODS AND RESULTS Cultured neonatal rat ventricular cardiomyocytes were subjected to doxorubicin-induced stress, monitored as caspase3 activation, apoptotic DNA fragmentation and cell viability. The protective effects of HDL and sphingosine-1-phosphate (S1P) were investigated using native HDL, reconstituted HDL of varied composition and agonists and antagonists of S1P receptors. Anti-apoptotic signalling pathways were identified with specific inhibitors. Native and reconstituted HDL significantly decreased doxorubicin-induced cardiomyocyte apoptosis, essentially due to the S1P component of HDL. The latter was mediated by the S1P2 receptor, but not the S1P1 or S1P3 receptors. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) signalling pathway was required for the anti-apoptotic effects of HDL and S1P. The transcription factor Stat3 also played an important role, as inhibition of its activity compromised the protective effects of HDL and S1P on doxorubicin-induced apoptosis. CONCLUSION HDL and its sphingosine-1-phosphate component can protect cardiomyocytes against doxorubicin toxicity and may offer one means of reducing cardiotoxic side effects during doxorubicin therapy. The study identified anti-apoptotic pathways that could be exploited to improve cardiomyocyte survival.

Diurnal rhythm of melatonin action on sexual maturation of male rats.

Immature male Wistar rats with a 12:12 h light-dark cycle received daily s.c. melatonin injections of 100 micrograms from 20 to 40 days of age. Melatonin administration during the late photophase (9 h after the onset of light) or during the late scotophase (9 h after the onset of darkness) caused reduced weights of testes and seminal vesicles, lowered plasma levels of testosterone, LH and FSH, and decreased number of pituitary GnRH receptors, a series of observations which reflects delayed sexual maturation at 40 days. In contrast, no effect was observed when melatonin was injected during the early photophase of scotophase (5 h after the onset of light or darkness, respectively). In order to better investigate the night hours, melatonin action was studied in rats that were born and raised with a shifted 12:12 h light-dark cycle. When the light phase was shifted by 5 and 17 h 1 week before the animals were born, sensitivity to melatonin action was unchanged. The responsiveness of the neuroendocrine-reproductive axis to exogenous melatonin was then studied throughout the day-night cycle. The inhibitory influence of melatonin increased gradually during the late photophase and reached a peak just before the onset of darkness. No effect was observed during the first 7 h of the dark phase; however, melatonin injected 9 h after the onset of darkness again had an inhibitory influence equivalent to about 50% of that observed in the late photophase , whereas administration 2 h later remained without effect. These results demonstrate that the time of administration of melatonin within the day-night cycle is critical for melatonin action.(ABSTRACT TRUNCATED AT 250 WORDS)

The PGE2 – Stat3 interaction in doxorubicin-induced myocardial apoptosis

AIMS Both cyclooxygenase-2 (COX-2) and the transcription factor signal transducer and activator of transcription 3 (Stat3) are involved in adaptive growth and survival of cardiomyocytes. In ventricular cardiomyocytes, prostaglandin E(2) (PGE(2)), a major COX-2 product, leads to adaptive growth via Stat3 activation, but whether this transcription factor acts as a signalling molecule in PGE(2)-induced cell survival is unknown. Therefore, the purpose of this study was to determine whether PGE(2) counteracts cardiac apoptosis induced by doxorubicin (DOX), and if so, whether Stat3 plays a critical role in this cardioprotective effect. METHODS AND RESULTS Neonatal rat ventricular cardiomyocytes were incubated with DOX (0.5 microM) and/or PGE(2) (1 microM). Apoptosis was assessed by determining caspase3 activation and apoptotic DNA fragmentation. The role of Stat3 was evaluated in vitro and in vivo by transfecting cardiomyocytes with siRNA targeting rat Stat3 and by using cardiomyocyte-restricted Stat3 knockout (Stat3 KO) mice, respectively. Incubation of ventricular cardiomyocytes with PGE(2) led to a time-dependent decrease in the DOX-induced caspase3 activation, reaching a maximal inhibition of 70 +/- 5% after 4 h. Similarly, PGE(2) inhibited DOX-induced DNA fragmentation by 58 +/- 5% after 24 h. This antiapoptotic action of PGE(2) was strongly reduced by the ERK1/2 inhibitor, U0126, whereas the p38 MAP kinase inhibitor, SB203580, had no effect. Depleting Stat3 expression by 50-60% in isolated ventricular cardiomyocytes markedly reduced the protective effect of PGE(2) on DOX-induced caspase3 activation and DNA fragmentation. Likewise, the stable PGE(2) analogue, 16,16-dimethyl-PGE(2), was unable to counteract cardiac apoptosis induced by DOX in Stat3 KO mice. CONCLUSION Our results demonstrate that PGE(2) prevents myocardial apoptosis induced by DOX. This protection requires the activation of the prosurvival pathways of Stat3 and ERK1/2.

COX-2 expression in oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory disease of unknown cause, which possesses the potential for malignant transformation. Cyclooxygenases (COX) 1 and 2 are two enzymes known to convert arachidonic acid into prostaglandins. Recent studies have shown an overexpression of COX-2 in oral squamous cell carcinoma and its precursor lesions. The present study investigated the expression of the COX-2 protein in OLP by Western blot analysis. Thirty patients with different degrees of histologically confirmed disease activity participated in the study: 9 patients had a recent onset of active OLP, 12 patients had atrophic OLP with moderate or low activity, and 9 patients presented with atrophic OLP with complete loss of activity. The results showed a high expression of COX-2 in all OLP patients in comparison with the control group. The differences in COX-2 expression in the various stages of OLP were not statistically significant. In conclusion, our results suggest that COX-2 is present during the various clinical forms of OLP. The resulting sustained overexpression of COX-2 in the late stage of the disease could play a role in the malignant transformation of some OLP.

Location of melatonin receptors

We attempted to locate melatonin receptors in different organs of rats. Experiments with plasma membranes and cytosol from rat liver revealed binding activity in both cell fractions. In both cases maximal binding was achieved after 3 hours at 20°C and after 6 hours at 4°C. Both binding sites had an optimum pH at 7.5 and were inhibited by trypsin. Plasma membrane receptors seem to be more Ca++ dependent than cytosol binding sites. Dissociation constants were 8·10-9M for membrane receptors and 6·10-8M for cytosol binding sites. Receptor concentration of liver, lung, spleen and heart cytosol varied between 50-200 fmoles/mg protein; that of testes, kidney and eyes was 2-3 times as high; that of pituitary gland, hypothalamus, epididymis and adrenals was 6-8 times as high. Melatonin receptor concentrations of plasma membranes from liver and testes were about 10 fold lower than in the cytosol fraction whereas spleen and lung membranes showed no melatonin binding activity. The search of melatonin in different organs of normal rats or animals treated with melatonin have shown significant concentration or accumulation in organs which showed also high concentrations of melatonin binding sites: eyes, brain, testes, epididymis, adrenals and kidney. These organs with the exception of kidney are known to be presumably targets for melatonin action.

Solubilization of pituitary GnRH binding sites by means of a zwitterionic detergent

Solubilization in an active form of the pituitary plasma membrane protein carrying binding sites for gonadotropin-releasing hormone (GnRH) was possible by using as detergent CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate), which is a zwitterionic derivative of cholic acid. In contrast, the use of the non-ionic detergent Triton X-100 resulted in the loss of GnRH binding sites. CHAPS at a concentration of 5-10 mM allowed the solubilization of up to 80% of available sites, and remaining non-solubilized sites retained unaltered binding properties. Characterization of solubilized binding sites of GnRH was achieved by using as radioactive ligand and standard a stable GnRH analog. [D-Trp6,(NEt)Pro9,desGly10]-GnRH. Maximum binding of GnRH to solubilized binding sites was possible in the presence of a very low amount of CHAPS in the incubation medium. Scatchard analysis of classical saturation experiments indicated the presence of a single class of high affinity, low capacity binding sites. The mean value for the affinity constant KA was 0.36 +/- 0.07 X 10(10) M -1 (n = 5) for solubilized GnRH binding sites, when analysed in the presence of 2 mM CHAPS; when solubilized GnRH binding sites were analysed in the absence of CHAPS, significantly higher KA values were observed, ranging from 1.2 to 5.9 X 10(10) M -1. Parallel analysis of the plasma membranes prior to solubilization indicated a mean KA value of 0.53 +/- 0.13 X 10(10) M -1 (n = 5). The binding specificity of the pituitary GnRH binding sites as seen by evaluating cross-reactions with a panel of agonists and antagonists of GnRH was not altered by solubilization. Gel filtration on Sephadex G-25 of solubilized binding sites preincubated with radioiodinated GnRH analog demonstrated the presence of a large molecular weight complex. In conclusion, CHAPS appears to be quite suitable for the solubilization of the binding site moiety of the pituitary GnRH receptor with good retention of the binding characteristics, thus offering a new possibility for the purification and characterization of the GnRH receptor protein.

Nocturnal urinary melatonin excretion and plasma cortisol levels in children and adolescents after a single oral dose of dexamethasone

In the present study, the possible relationship between melatonin secretion as reflected by nocturnal melatonin excretion (2000 h‐0800 h) and the pituitary‐adrenocortical axis was investigated. Nocturnal urinary melatonin excretion and plasma cortisol levels were determined in 41 children with weight problems before and after a single oral dose of dexamethasone. A first group of 15 individuals with normal cortisol cycle (12·2 ± 1·4 at 0800 h and 3·1 ± 0·5 μg/100 ml at 1700 h), and levels below 1·0 μg/100 ml after dexamethasone, showed a highly significant increase in melatonin excretion during the night following dexamethasone treatment (63·5 ± 5·5 ng/12 h vs 33·6 ± 3·0 for the control night, P > 0·001). This increase was observed from prepuberty to young adulthood (pubertal stages PI—PV). In a second group of 16 subjects with mean cortisol levels similar at 0800 h and 1700 h (10·7 ± 1·3 and 9·3 ± 1·5 μg/100 ml respectively), but with a normal cortisol suppression after dexamethasone administration, nocturnal melatonin excretion increased from 21·2 ± 2·1 to 33·4 ± 3·0 ng/12 h (P > 0·01). A significant increase was found in prepubertal children (PI) whereas no change was observed at the end of pubertal development (stages PIV‐PV). A third group of 10 patients with both low amplitude cortisol cycles (16·2 ± 2·5 and 10·8 ± 2·4 μg/100 ml) and abnormal cortisol suppression after dexamethasone administration (9·1 ± 2·4 μg/100 ml), showed no increase in melatonin excretion (24·2 ± 2·7 and 24·9 ± 3·7 ng/12 h). However, one patient with delayed puberty had increased melatonin excretion after dexamethasone. No relationship was found between body weight and melatonin excretion. Six subjects with delayed puberty had high melatonin excretion before dexamethasone and showed a marked increase after dexamethasone treatment (76·1 ± 7·2 ng/12 h vs 38·6 ± 6·9 for the control night, P > 0·001), whatever the cortisol cycles and levels of suppression. Our results suggest a relationship between the patterns of cortisol and melatonin secretion. However, further studies are necessary to prove whether there exist interactions between the pituitary‐adrenal axis and the pineal gland and whether the response of melatonin to dexamethasone is also related to the stage of sexual maturation.

Pituitary receptor sites for LHRH: their relationship to regulation of gonadotropins secretion in male rats

Pituitary plasma membranes contain high affinity, low capacity binding sites for LHRH, that are likely to represent specific receptor sites for this hypothalamic hormone. The use of a highly potent analog of LHRH, DesGly10[DTrp6,(N-Et)Pro9]LHRH as radio-iodinated tracer allows a specific and sensitive measurement of these receptor sites (Biochem. Biophys. Res. Comm. 90:1249,1979). Castration of male rats produced a rapid and sustained increase of pituitary LHRH receptor content (LHRH-R) which parallelled the well-established augmentation of LH and FSH secretions and decrease of hypothalamic content of LHRH. Treatment during 7 days with either testosterone or estradiol allowed to fully restore, in a dose-dependent manner, normal levels of plasma gonadotropins, pituitary LHRH-R and hypothalamic LHRH content in acutely castrated (2 days) but not in chronically castrated (>28 days) rats. In the latter group, only plasma gonadotropins were normalized by sex steroid treatment. It is concluded that the rapid feedback action of sex steroids for the control of gonadotropins secretion is exercised mainly at the pituitary level rather than the hypothalamic level in chronically castrated rats.