Involvement of the proteasome and caspase activation in hippocampal long-term depression induced by the serine protease subtilisin.

The serine protease subtilisin-A produces a long-term depression (LTD) of synaptic potentials in hippocampal slices which differs mechanistically from classical LTD. Since caspases have been implicated in hippocampal plasticity, this study examined a possible role for these enzymes in subtilisin-induced LTD. Subtilisin produced a concentration-dependent decrease in the size of field excitatory synaptic potentials (fEPSPs), which was not prevented or modified by the caspase inhibitors Z-VAD(OMe)-fmk and Z-DEVD-fmk. Similarly Z-VAD(OMe)-fmk did not modify the selective loss of protein expression produced by subtilisin. Subtilisin reduced the expression of procaspase-3 and caspase-9 but, while caspase-9 was converted to its conventionally activated form (39 kDa), caspase-3 was metabolised along a non-canonical pathway to a 29/30 kDa protein rather than the classical 17/19 kDa fragments. Both Z-VAD(OMe)-fmk and Z-DEVD-fmk were unable to prevent the reduced expression of Postsynaptic Density Protein-95, Vesicle-Associated Membrane Protein-1 and Unco-ordinated 5H3 proteins produced by subtilisin, although MG132 did produce partial recovery from subtilisin-induced depression of fEPSPs. When tested on long-term potentiation (LTP) induced by theta stimulation in the stratum radiatum, MG132 inhibited the immediate increase in fEPSP size but generated a higher plateau LTP. Twin LTP stimulation generated a further increase in LTP amplitude in control slices but not in slices exposed to MG132. The results indicate that subtilisin does produce caspase activation but that this does not contribute to its induction of LTD. However, activation of the proteasome does contribute to subtilisin-induced LTD and may also play a modulatory role in electrically induced LTP.

Tryptophan, adenosine, neurodegeneration and neuroprotection.

This review summarises the potential contributions of two groups of compounds to cerebral dysfunction and damage in metabolic disease. The kynurenines are oxidised metabolites of tryptophan, the kynurenine pathway being the major route for tryptophan catabolism in most tissues. The pathway includes quinolinic acid -- an agonist at N-methyl-D-aspartate (NMDA) receptors, kynurenic acid -- an antagonist at glutamate and nicotinic receptors, and other redox active compounds that are able to generate free radicals under many physiological and pathological conditions. The pathway is activated in immune-competent cells, including glia in the central nervous system, and may contribute substantially to delayed neuronal damage following an infarct or metabolic insult. Adenosine is an ubiquitous purine that can protect neurons by suppressing excitatory neurotransmitter release, reducing calcium fluxes and inhibiting NMDA receptors. The extent of brain injury is critically dependent on the balance between the two opposing forces of kynurenines and purines.

Purine metabolism and clinical status of patients with rheumatoid arthritis treated with dipyridamole.

The anti-inflammatory activities of methotrexate and sulphasalazine may be mediated by increases in endogenous adenosine levels. Since the vascular protective drug dipyridamole inhibits the uptake and metabolism of adenosine we have now tested this compound in patients with rheumatoid arthritis to assess its effects on their symptoms. Forty patients (aged 18-75 years) received dipyridamole 400 mg/day or placebo. The levels of adenosine and its major metabolites were determined by high performance liquid chromatography (HPLC) in blood samples taken at baseline and at monthly intervals during treatment for 6 months. After three months of treatment there was a significant reduction in the modified Health Assessment Questionnaire (mHAQ) score, but these effects were not maintained, and dipyridamole did not modify disease severity scores or the levels of adenosine and its metabolites. We conclude that the symptoms of rheumatoid arthritis were not modified by treatment with dipyridamole.

Modulation of cytokine release by purine receptors in patients with rheumatoid arthritis.

OBJECTIVE: Since adenosine receptors are known to modulate the release of some inflammatory mediators in control subjects, we have examined the effects of the mixed A1 and A2 adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on basal and lipopolysaccharide (LPS)-induced cytokine release in diluted whole blood cultures from rheumatoid arthritis (RA) patients and healthy volunteers. METHODS: Twenty-eight patients with rheumatoid arthritis aged 18-75 years gave their voluntary consent to participate and give a blood sample. Basal levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) were measured by ELISA, and whole blood cultures were prepared to assess the effects of LPS activation. RESULTS: Following a 40-hour incubation, activation of adenosine receptors by NECA, added to the cell cultures from rheumatoid arthritis patients, was found to suppress both the basal and LPS-induced release of TNF-alpha and IL-1beta, while causing an increase in the release of both basal and LPS-induced IL-6. In healthy volunteers basal cytokines were undetectable, but NECA alone induced the release of all three cytokines. Stimulated levels of TNF-alpha were more than double those in patients. In the control blood cultures, NECA suppressed LPS-induced release of TNF-alpha and IL-1beta, but increased IL-6 release. CONCLUSIONS: Adenosine receptor stimulation has a differential effect on the release of pro-inflammatory cytokines, and may induce cytokine release in normal subjects. Stimulated release of TNF-alpha is substantially lower in patients with rheumatoid arthritis than in control subjects, possibly indicating saturation, exhaustion or down-regulation of the release process.

Purine modulation of cytokine release during diuretic therapy of rheumatoid arthritis.

Since free radicals are implicated in rheumatoid arthritis (RA) and since uric acid is a free radical scavenger, we examined the effects of treating RA patients with with the diuretic bumetanide to try to improve their arthritic control. Seventy patients, aged 18-75 years, were randomised to receive bumetanide 4 mg/day or placebo. Uric acid levels increased, but not that of other purines, in the blood of drug-treated patients compared with placebo-treated controls. There were no significant changes in clinical measurements of disease activity or in ESR or CRP levels. There were no over all differences in the blood levels of the cytokines, nor in the basal or stimulated production of cytokines from the blood cultures. The adenosine receptor agonist 5'N-ethylcarboxamido-adenosine (NECA) used to modify cytokine release in cultures of whole blood taken from the patients, depressed the release of tumour necrosis factor-alpha (TNFalpha), but failed to depress the release of interleukin-1b (IL-1b) or interleukin-6 (IL-6), a difference from earlier studies of healthy control subjects and, thus, a difference which may contribute to the disease activity.

Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders.

The generation of reactive oxygen species (free radicals) is an important factor in the development and maintenance of rheumatoid arthritis in humans and animal models. One source of free radicals is nitric oxide produced within the synoviocytes and chondrocytes and giving rise to the highly toxic radical peroxynitrite. Several cytokines, including tumour necrosis factor-alpha (TNFalpha) are involved in the formation of free radicals, partly by increasing the activity of nitric oxide synthase. Indeed, nitric oxide may mediate some of the deleterious effects of cytokines on bone resorption. Aspirin, tetracyclines, steroids and methotrexate can suppress nitric oxide synthase. Dietary antioxidants include ascorbate and the tocopherols and beneficial effects of high doses have been reported especially in osteoarthritis. There is also evidence for beneficial effects of beta-carotene and selenium, the latter being a component of the antioxidant enzyme glutathione peroxidase. The polyunsaturated fatty acids (PUFA) include the n-3 compounds, some of which are precursors of eicosanoid synthesis, and the n-6 group which can increase formation of the pro-inflammatory cytokines TNFalpha and interleukin-6, and of reactive oxygen species. Some prostaglandins, however, suppress cytokine formation, so that n-3 PUFA often oppose the inflammatory effects of some n-6-PUFA. gamma-linolenic acid (GLA) is a precursor of prostaglandin E1, a fact which may account for its reported ability to ameliorate arthritic symptoms. Fish oil supplements, rich in n-3 PUFA such as eicosapentaenoic acid have been claimed as beneficial in rheumatoid arthritis, possibly by suppression of the immune system and its cytokine repertoire. Some other oils of marine origin (e.g. from the green-lipped mussel) and a range of vegetable oils (e.g. olive oil and evening primrose oil) have indirect anti-inflammatory actions, probably mediated via prostaglandin E1. Overall, there is a growing scientific rationale for the use of dietary supplements as adjuncts in the treatment of inflammatory disorders such as rheumatoid arthritis and osteoarthritis.

The role of kynurenines in the production of neuronal death, and the neuroprotective effect of purines.

The kynurenine metabolic pathway from tryptophan accounts for a large proportion of the metabolism of this amino acid in the brain. Although a major route for the generation of the essential co-factor nicotinamide adenine dinucleotide (NAD), two components of the pathway have marked effects on neurons. Quinolinic acid is an agonist at N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors, while kynurenic acid is an antagonist and, thus, a potential neuroprotectant. The levels of quinolinic acid are known to increase substantially following cerebral insults or infection, and has been most clearly implicated in the AIDS-dementia complex. The actions of quinolinic acid and NMDA show subtle differences, however, which suggest other factors contributing to cell damage. In this article we review the evidence that free radicals may be involved in the neurotoxic effects of quinolinic acid and consider the possibility that quinolinic acid might be involved in Alzheimer's disease. Finally, adenosine receptor ligands can modulate neuronal damage, supporting the view that they may represent suitable targets for the development of novel neuroprotectant drugs for the treatment of Alzheimer's and other neurodegenerative disorders.

Changes in urate metabolism after castration of patients suffering from carcinoma of the prostate.

13 patients with carcinoma of the prostate were investigated on a low purine diet before and after surgical castration, performed to slow progression of their disease. A control group of herniorrhaphy patients was similarly studied. The 2 groups were very similar pre-operatively except for urea which was higher pre-operatively in patients than controls (p0.004). Testosterone levels fell significantly in patients soon after surgery (W0.002). There was a significant fall in serum uric acid in the early post-operative phase in both patients (W0.004) and controls (W0.01) but the fall only remained significant one month after surgery in castrated patients (W0.007). Urinary uric acid levels in patients fell significantly soon after surgery (W0.04). This is a small study but results suggest that endogenous male hormones are at least partially responsible for serum uric acid levels since castration, with reduction in circulating testosterone, resulted in a fall in these levels.

Evaluation of popliteal cysts and painful calves with ultrasonography: comparison with arthrography.

Grey-scale ultrasonography will detect reliably the presence of clinically significant popliteal cysts, fluid collections which do not fill by arthrography, and will frequently demonstrate ruptured cysts and the soft tissue changes resulting from a recent leak. Forty-eight knees, in 25 patients with popliteal and/or calf pain were examined by ultrasonography followed by arthrography. Popliteal cysts were demonstrated in 40% (19/48) by ultrasound and in 46% (22/48) by arthrogram. For comparison between arthrography and ultrasonography chi2 = 8.58 and contingency coefficient, phi = 0.42 (p less than 0.01). Acute cyst rupture was shown in 2 patients (8%) by both arthrography and ultrasound. In a further study ultrasonography demonstrated popliteal cysts with a prevalence of 31% (22/72) in 36 patients with definite or classical rheumatoid arthritis compared with 4% (3/72) in controls closely matched for age and sex. This difference in prevalence between the rheumatoid patients and controls was highly significant chi2 = 17.48, p less than 0.001. Ultrasonography, therefore, will demonstrate noninvasively the presence of popliteal cysts, may assist in the diagnosis of rupture, and furthermore will assist in quantitative, sequential assessment of patients with painful knees and calves.

Grey scale ultrasonography and arthrography in evaluation of popliteal cysts.

Ultrasonography will reliably detect popliteal cysts of clinically significant size and improvements in ultrasound imaging have enhanced the value of ultrasound in relation to arthrography in the assessment of cyst rupture. Forty-eight knees in 25 patients suspected of having a popliteal cyst were examined by ultrasonography followed immediately by arthrography. Popliteal cysts were demonstrated in 40% (19/48) by ultrasound and in 46% (22/48) by arthrogram. Arthrography detected small cysts not seen with ultrasound, but altrasonic scanning showed cysts which did not fill on arthrography. A ruptured cyst or deep venous thrombosis was suspected in 10 patients. Rupture was confirmed in two patients by arthrography, in both of whom soft tissue changes and attenuation of the distal margin of the cysts were shown by ultrasound.