Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-D-L-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2ß. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Intracorporeal Videoprobe (IVP).

The objective of the work in the EC project IVP is the development and evaluation of two prototypes of video systems:- a small wired videoprobe with a CMOS image sensorand- an autonomous video-capsule with a telemetric link for image data transfer to an external PC-based system.

Rapid S-nitrosothiol metabolism by platelets and megakaryocytes.

RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs. Exposure of platelets to GSNO (S-nitrosoglutathione) for as little as 5 s inhibited thrombin-induced platelet aggregation by >95%; however, AlbSNO (S-nitrosoalbumin) was much less effective over these short time periods. Incubation of 1 microM GSNO or AlbSNO with platelets and megakaryocytes resulted in a 25-34% loss of RSNO recoverable from the supernatant (P <0.02) within 30 s. This rapid cell-mediated RSNO decay did not progress further over 5 min, and could not be accounted for by release of free NO. The gamma-glutamyl transpeptidase inhibitor acivicin (100 microM) partially decreased GSNO decay, whereas the membrane-impermeable thiol-blocking agent 5,5'-dithiobis-(2-nitrobenzoic acid) (100 microM) completely blocked cell-mediated GSNO decay and partially blocked AlbSNO decay. Our results highlight differences between high- and low-molecular-mass RSNOs with regard to their rapid metabolism/uptake and subsequent cellular responses, and indicate a critical role for extracellular thiols in RSNO metabolism by platelets and megakaryocytes.

Substance P is involved in terminating the hypothalamo- pituitary-adrenal axis response to acute stress through centrally located neurokinin-1 receptors.

The neurokinin substance P (SP) has been previously shown to inhibit basal hypothalamo-pituitary-adrenal (HPA) axis activity. This study was designed to investigate the effects of central injection of the specific neurokinin-1 receptor antagonist RP67580 on the HPA axis response to acute restraint stress. In non-restrained rats injected with RP67580, plasma ACTH and corticosterone levels were elevated at 30 and 60 min compared to rats injected with vehicle, but there were no differences between vehicle and RP67580 groups at 4h. In restrained rats injected with vehicle, plasma ACTH and corticosterone levels were significantly elevated at 30 min and 60 min following initiation of the stress but had returned to basal levels at 4h. In restrained rats injected icv with RP67580, plasma corticosterone and ACTH levels were significantly elevated at 30 min and 60 min, with no significant differences compared to the restraint stressed vehicle-injected group. However, in the RP67580-injected group, corticosterone and ACTH levels remained significantly elevated at 4h following onset of restraint compared to those in the restraint stressed vehicle-injected group. Corticotrophin-releasing factor mRNA levels in the parvocellular subdivision of the paraventricular nucleus of the hypothalamus and POMC mRNA levels in the anterior pituitary were significantly increased in the stressed group 4h following injection with RP67580 compared to the stressed group injected with vehicle alone. These data show that endogenous SP does not inhibit the initial magnitude of the HPA axis response to restraint stress, but does act through neurokinin-1 receptors at a central level to reduce the duration of the response to stress. This suggests that SP may be an important central agent controlling the transition between acute and chronic stress.

Expression and protective effects of urocortin in cardiac myocytes.

Reverse transcription PCR showed that mRNA encoding the CRH-like molecule, urocortin, is expressed in a rat cardiac myocyte cell line and in primary cultures of cardiac myocytes. Identity of the amplified with the published sequence was established by restriction mapping and direct sequencing. Expression of urocortin mRNA was increased 12-18 h after thermal injury. Urocortin peptide protected cardiac myocytes from cell death induced by hypoxia. The data suggest that urocortin is an endogenous cardiac myocyte peptide which modulates the cellular response to stress.

C/EBP activates the human corticotropin-releasing hormone gene promoter.

The purpose of these studies was to identify whether transcription factors, associated with cytokine signalling, affected promoter activity of the corticotropin releasing hormone (CRH) gene. Fragments of a 3.6 kb sequence of the human CRH gene promoter were amplified by PCR and ligated upstream of a CAT reporter. These constructs were transfected into a variety of cell lines, either alone or together, with transcription factor expression vectors. Basal activity of a 3070 bp CRH promoter fragment was only seen in neuronal and lymphoblastoid cell lines. Promoter activity was increased by the transcription factors C/EBPbeta (NF-IL6) and more strongly, by C/EBPdelta (NF-IL6beta). Increased CRH promoter activity following phorbol ester treatment was inhibited by a dominant negative NF-IL6 mutant, showing that the effects of phorbol ester were principally mediated by C/EBP. Moreover, the inverse changes in the expression of CRH in the hypothalamus and spleens of arthritic rats were paralleled by similar inverse changes in NF-IL6beta expression in these organs. These data show that some transcription factors associated with cytokine signalling can also activate the CRH promoter.

The role of endogenous serotonin in adjuvant-induced arthritis in the rat.

We have investigated the effects of serotonin depletion on the progress and severity of adjuvant-induced arthritis in the Piebald-Viral-Glaxo (PVG) strain of rat. Total body depletion of serotonin was achieved using p-chlorophenylalanine given i.p. Two paradigms were investigated. First we depleted serotonin at the time of injection of the adjuvant to determine whether serotonin was involved in the initial induction phase. Secondly, we depleted serotonin at the time of onset of the inflammation. Serotonin levels in the hypothalamic paraventricular nucleus (PVN) were reduced by > 95%. Depletion at the time of induction had no effect on the severity of the disease (determined by the increase (determined by the increase in hind paw volume) 14 days after injection of the adjuvant. In contrast, depletion at the time of onset of the disease resulted in a significant reduction in severity at day 14, suggesting a pro-inflammatory role for serotonin in this model. The decrease in corticotrophin-releasing factor (CRF) mRNA in the PVN associated with the development of adjuvant arthritis in PVG rat was reversed in the serotonin-depleted animals. Central serotonin could be one of the factors responsible for the reduced expression of CRF mRNA in response to adjuvant-induced arthritis in this rat strain. These data suggest that serotonin antagonists may be efficacious in reducing the severity of acute inflammatory episodes.

Inhibition of hypothalamic nitric oxide synthase gene expression in the rat paraventricular nucleus by food deprivation is independent of serotonin depletion.

We have investigated the effects of food deprivation on nitric oxide synthase (NOS) transcript levels in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Food deprivation for 48 h significantly and consistently reduced NOS transcript prevalence by approximately 50% in both sites. Since there is considerable evidence for an important role of 5-HT in feeding behaviour, we then examined the effect of food deprivation on NOS gene expression in the PVN following para-chlorophenylalanine (PCPA)-induced hypothalamic 5-HT depletion. As starvation causes central down-regulation of the thyroid axis, changes in thyrotropin-releasing hormone (TRH) and pituitary thyrotrophin (TSH) transcript prevalence were used as internal controls. PCPA pretreatment (200 mg/kg body weight as a single daily dose ip for 2 days) had no significant effect on basal levels of NOS, TRH or TSH transcripts, or on the effect of a subsequent 48 h fast, which significantly reduced all three. These results show for the first time, that food deprivation for 48 h significantly reduces NOS gene expression in the rat PVN and SON. Secondly, that basal levels and the fasting-induced reductions in the prevalence of NOS, TRH and TSH transcripts were not affected by PCPA-induced hypothalamic 5-HT depletion. Therefore, at least under the experimental conditions used here, 5-HT does not appear to be involved in setting baseline levels- or in the starvation-induced inhibition of NOS or thyroid axis gene expression in the PVN.

Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis.

1. Adjuvant-induced arthritis (AA) is an experimental inflammation of the joints that results in chronic activation of the hypothalamo-pituitary-adrenal (HPA) axis. 2. In this study the role of hypothalamic corticotrophin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of the HPA axis in this condition both in Sprague-Dawley (SD), and Piebald-Viral-Glaxo (PVG) rats has been further characterized. 3. The increase in AVP peptide content of portal blood (as early as day 11), just prior to the onset of arthritis is confirmed and further increases, peaking at day 16 are shown, coincident with the progression of inflammation in the PVG rats. 4. The increase in AVP is associated with a significant increase in the expression of AVP but not CRF mRNAs in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN) of arthritic SD rats. 5. In the presence of maximal inflammation of SD rats there was a significant decrease in the maximum binding of [125I]-Tyr-oCRF to anterior pituitary membranes, whereas AVP receptor concentration in anterior pituitary membranes from both PVG and SD rats showed a significant increase with respect to controls. 6. The basal adrenocorticotrophin (ACTH) secretion in vitro was similar in both control and arthritic SD rats but that from arthritic PVG rat pituitaries was significantly greater than the respective controls (436 +/- 91 v 167 +/- 23 pg/tube). The ACTH response of pituitaries of arthritic PVG rats to CRF or the combination of CRF and AVP was significantly higher compared with the controls, although the ACTH response of arthritic SD rat pituitaries was unchanged. 7. The results are consistent with the view that activation of the parvocellular vasopressin system has an important role in the adaptation of the HPA axis to experimentally-induced chronic stress of arthritis.

Hypothalamic nitric oxide synthase gene expression is regulated by thyroid hormones.

We investigated the effects of thyroid status on nitric oxide synthase (NOS) gene expression in the rat hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). Propylthiouracil (PTU)-induced hypothyroidism in male rats produced a highly significant reduction in NOS gene transcripts in the PVN and SON, as assessed by quantitative in situ hybridization histochemistry with a specific oligodeoxynucleotide probe. The addition of T3 (40 micrograms/kg) to the PTU-containing diet completely prevented the reduction in NOS transcripts. Hyperthyroidism, induced by adding 160 micrograms/kg T3 to the food, more than doubled the prevalence of NOS transcripts in the PVN and SON after a similar time. Up-regulation of NOS gene transcripts induced by the osmotic stimulus of chronic salt loading was markedly attenuated by PTU-induced hypothyroidism. These results demonstrate a major effect of thyroid status on regulation of NOS gene expression in the hypothalamus.

Water deprivation in the rat induces nitric oxide synthase (NOS) gene expression in the hypothalamic paraventricular and supraoptic nuclei.

We examined the effects of water deprivation on nitric oxide synthase (NOS) gene expression in the rat hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. Dehydration caused a significant increase in NOS gene transcripts in the PVN and SON but not in the subfornical organ (SFO). The results suggest that dehydration has a major effect on the NOS gene expression in the PVN and SON.

S-100 antigen-positive folliculostellate cells are not the source of IL-6 gene expression in human pituitary adenomas.

We have investigated the expression of IL-6 in a random selection of 27 human pituitary adenomas, comprising 8 somatotroph, 5 corticotroph, 3 mammotroph and 11 endocrinologically inactive adenomas, using a 35S-labelled 1.1kb riboprobe complementary to human IL-6. Positive and negative IL-6 transcript controls were generated from the IL-6-secreting human bladder carcinoma cell line T24/83. Tissue from a malignant melanoma was used as a positive S-100 immunocytochemical control tissue. Of the 27 human pituitary adenomas examined by in situ hybridization, 7 (26%) contained IL-6 transcripts: these were 3 of 5 corticotroph adenomas, 2 of 8 somatotrophinomas and 2 of 11 endocrinologically inactive adenomas. In each case, IL-6 transcript-positive cells constituted less than 1% of the total pituitary tissue mass examined. Alternate wax embedded 3 microns thick sections from 5 of the 7 IL-6 transcript positive tumours were examined immunocytochemically for S-100 antigen, or by in situ hybridization for IL-6 transcripts. Immunocytochemistry for S-100 antigen was completely negative in 3 of the 5 tumours and in the remaining 2, there was no evidence of IL-6 transcripts and S-100 antigen co-localization in any of the sections examined. This suggests that in pituitary adenomas, cells other than classical folliculostellate cells are responsible for IL-6 production.

Vasopressin is located within lymphocytes in the rat spleen.

Using dual antigen immunocytochemical staining with a specific antiserum for arginine vasopressin (AVP), we have detected AVP immunoreactivity in clusters of large immunoglobulin (Ig) G-containing cells, probably plasma cells, within the rat spleen, and in smaller cells which are IgG-negative. Vasopressin-positive cells were detected principally throughout the white pulp areas in the subcortical region of the spleen. IgG staining could only be detected within the cells and not on the cell surface, demonstrating that the antiserum is recognising genuine intracellular IgG and not cell surface antigens. Reversed-phase HPLC of spleen tissue extract revealed a single peak of AVP immunoreactivity which co-eluted with the standard. This is the first evidence that AVP is found within lymphocytes of the immune system and provides further information about the important interaction between the endocrine and immune systems.

Endogenous substance P inhibits the expression of corticotropin-releasing hormone during a chronic inflammatory stress.

We have investigated the effects of a chronic inflammatory stress on substance P (SP) levels in the hypothalami of rats given adjuvant-induced arthritis (AA). Fourteen days after injection of Mycobacterium butyricum, substance P concentrations in the paraventricular nucleus (PVN) and median eminence/arcuate nucleus were significantly increased. In AA rats injected intraperitoneally with the specific neurokinin-1 receptor antagonist RP67580, plasma ACTH and corticosterone concentrations were significantly elevated, and corticotropin-releasing hormone (CRH) mRNA in the PVN was increased compared to the AA group which received saline alone. The increases in hypothalamic SP in AA, together with the data demonstrating that HPA axis activity is enhanced in AA following injection of a SP antagonist, are consistent with the hypothesis that SP is acting as an inhibitor of CRH expression in this model of chronic inflammatory stress.

The diurnal expression of genes encoding vasopressin and vasoactive intestinal peptide within the rat suprachiasmatic nucleus is influenced by circulating glucocorticoids.

The mammalian suprachiasmatic nucleus (SCN) is the endogenous pacemaker generating the diurnal rhythm of the stress hormones ACTH and glucocorticoid secretion. In the present study, we have employed male rats entrained to a 12:12 h (light:dark) photoperiod to investigate the effects of chronic and acute administration of exogenous glucocorticoids upon the diurnal expression of vasopressin and vasoactive intestinal peptide (VIP) mRNA in the SCN by semiquantitative in situ hybridization histochemistry. Chronic administration of exogenous glucocorticoids significantly enhanced vasopressin mRNA expression only at zeitgeber time (ZT) 5, while the otherwise rhythmic expression of vasopressin mRNA was unaffected at ZT11, ZT17 and ZT23. In contrast, the same treatment abolished the rhythmic expression of VIP mRNA resulting in constantly elevated mRNA levels. In adrenalectomized rats given an overnight supplement of dexamethasone in their drinking water, the expression of both vasopressin and VIP mRNA in the SCN was elevated the following morning at ZT6 when compared to adrenalectomised rats kept on 0.9% saline. These results suggest that glucocorticoids influence the expression of vasopressin during a narrow window of time in the diurnal cycle coinciding with the time where entrainment of the circadian pacemaker with non-photic cues is possible. Constantly elevated levels of glucocorticoids may also interfere with the suprachiasmatic expression of VIP mRNA which is thought to be driven by photic cues.

The effects of restraint or hypertonic saline stress on corticotrophin-releasing factor, arginine vasopressin, and proenkephalin A mRNAs in the CFY, Sprague-Dawley and Wistar strains of rat.

It is generally assumed that the stress response of different strains of rat will be identical following exposure to acute stress. In the present study we have examined the activation of the hypothalamo-pituitary-adrenal axis in the Wistar, Sprague-Dawley and CFY strains of rat following exposure to either the predominantly psychological stress of restraint or the physical stress of i.p. hypertonic saline injection. We have investigated the hypothalamic activation of corticotrophin-releasing factor (CRF) and proenkephalin A (PEA) mRNAs in the parvocellular cells of the paraventricular nucleus (PVN) and arginine vasopressin (AVP) in both the magnocellular and parvocellular regions in the PVN following acute stress. In addition we have measured corticosterone as an index of end-point activation. Circulating corticosterone and CRF mRNA were increased in all three strains following either stress. AVP and PEA mRNAs were increased following hypertonic saline but only in the CFY strain following restraint. Overall the relative increase in the parameters measured was greater in the CFY strain of rat than the other strains. These data demonstrate marked differences in response to acute stress in the three strains of rat examined. These varying responses must be taken into consideration when designing or interpreting any study investigating the stress response.

Contents of corticotropin-releasing hormone and arginine vasopressin immunoreactivity in the spleen and thymus during a chronic inflammatory stress.

We have previously found that proopiomelanocortin (POMC) mRNA and levels of adrenocorticotropin (ACTH) and beta-endorphin peptides are increased in the spleen and thymus of rats with adjuvant-induced arthritis (AA), and immunologically mediated inflammatory disease. To determine whether alterations in immune tissue POMC during AA are also accompanied by changes in immune tissue corticotropin-releasing hormone immunoreactivity (ir-CHR) and arginine vasopressin (AVP), we measured ir-CRH and AVP by radioimmunoassays in spleen and thymic extracts 14 days following injection of adjuvant. Ir-CRH was detectable in all extracts of spleen and thymus. Total contents of ir-CRH in the spleen and thymus were not altered following arthritis, although a significant decrease was observed in splenic extracts from arthritis rats (40.0 +/- 4.2 fmol/g tissue) compared to controls (69.5 +/- 8.4 fmol/g tissue) when contents were expressed as amount per weight of tissue. Low levels of AVP were also detected in immune tissues, with contents significantly increased in spleens from arthritis animals (17.4 +/- 1.6 fmol/g tissue) compared to controls (10.6 +/- 1.9 fmol/g) but thymic contents of AVP were not altered by arthritis (10.6 +/- 1.3 fmol/g) compared to controls (9.2 +/- 0.7 fmol/g). Control levels of AVP were significantly higher in spleens and thymuses from female rats (53 +/- 5 and 25 +/- 4 fmol/g tissue, respectively) compared to males. G-50 chromatography revealed that the principal form of splenic ir-CRH is CRH(1-41), although in non-arthritic animals some ir-CHR eluted in a position indicating a slightly larger form.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal monosodium glutamate treatment alters both the activity and the sensitivity of the rat hypothalamo-pituitary-adrenocortical axis.

We have investigated the effects of monosodium glutamate (MSG) lesioning of the arcuate nucleus on both central and peripheral components of the hypothalamo-pituitary-adrenocortical (HPA) axis under basal conditions and under acute and chronic stress. Plasma ACTH levels were lower in MSG-lesioned rats (27 +/- 7 pg/ml) compared with controls (71 +/- 18 pg/ml) while corticosterone levels were elevated (523 +/- 84 ng/ml compared with 176 +/- 34 ng/ml). Quantitative in situ hybridization histochemistry revealed that corticotrophin-releasing factor mRNA levels in the medial parvocellular part of the hypothalamic paraventricular nucleus were significantly lower in MSG-treated rats. MSG lesioning resulted in an enhanced response of corticosterone to restraint stress (1309 +/- 92 ng/ml compared with 628 +/- 125 ng/ml in sham-lesioned animals), while ACTH responses to restraint stress in MSG-lesioned and sham-MSG groups were not significantly different (160 +/- 24 pg/ml and 167 +/- 24 pg/ml respectively). These data suggest that MSG-lesioned rats have an increased adrenocortical sensitivity. In rats subjected to the chronic osmotic stimulus of drinking 2% saline for 12 days, plasma ACTH levels were significantly reduced (15 +/- 5 pg/ml) and the ACTH and corticosterone responses to restraint stress were eliminated. ACTH levels were also reduced in MSG-treated animals given 2% saline and the ACTH response to acute stress remained absent in these animals. However, a robust corticosterone response to restraint stress was observed in saline-treated MSG-lesioned rats.(ABSTRACT TRUNCATED AT 250 WORDS)