Activation of the G protein Gq/11 through tyrosine phosphorylation of the alpha subunit.

Various receptors coupled to the heterotrimeric guanine nucleotide-binding protein Gq/11 stimulate formation of inositol-1,4,5-trisphosphate (IP3). Activation of these receptors also induces protein tyrosine phosphorylation. Formation of IP3 in response to stimulated receptors that couple to Gq/11 was blocked by protein tyrosine kinase inhibitors. These inhibitors appeared to act before activation of Gq/11. Moreover, stimulation of receptors coupled to Gq/11 induced phosphorylation on a tyrosine residue (Tyr356) of the Galphaq/11 subunit, and this tyrosine phosphorylation event was essential for Gq/11 activation. Tyrosine phosphorylation of Galphaq/11 induced changes in its interaction with receptors. Therefore, tyrosine phosphorylation of Galphaq/11 appears to regulate the activation of Gq/11 protein.

Rapid induction of neutrophil apoptosis by sulfasalazine: implications of reactive oxygen species in the apoptotic process.

Accumulating evidence indicates that neutrophils are crucially involved in the pathogenesis of inflammatory bowel diseases and rheumatoid arthritis. We therefore investigated the effect of sulfasalazine (SSZ), which is widely used in the treatment of these diseases, on neutrophil apoptosis in vitro. The apoptosis of neutrophils was determined by morphology, a DNA histogram of propidium iodide-stained nuclei, and DNA fragmentation. SSZ rapidly accelerated the rate of spontaneous neutrophil apoptosis within clinically relevant concentrations. This effect is unique to neutrophils because other types of leukocytes and a number of leukocyte cell lines are resistant to SSZ. Neutrophil apoptosis caused by SSZ was abrogated by a tyrosine kinase inhibitor, a protein kinase A inhibitor, and antioxidants. The subsequent results provided pharmacological evidence that the phosphorylation of tyrosine kinase and protein kinase A and generation of reactive oxygen species are involved in SSZ-mediated neutrophil apoptosis. These data suggest that SSZ-induced neutrophil apoptosis may account, in part, for the clinical benefits of SSZ on inflammatory bowel diseases and rheumatoid arthritis.

A new highly selective metabotropic excitatory amino acid agonist: 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid.

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was, however, shown to be a specific and rather potent agonist at mGlu6, approximately 4 times weaker than the nonselective excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu6 as well as mGlu2. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu6. It is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology and physiological role of mGlu6. We describe a new versatile synthesis of this homologue of AMPA and the synthesis of compound 10.

(S)-homo-AMPA, a specific agonist at the mGlu6 subtype of metabotropic glutamic acid receptors.

Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has already become a standard agonist for the pharmacological characterization of mGlu6 (Trends Pharmacol. Sci. Suppl. 1997, 37-39), and we here report the resolution, configurational assignment, and pharmacology of (S)- (6) and (R)- (7) Homo-AMPA. Using the "Ugi four-component condensation", 3-(3-ethoxy-5-methylisoxazol-4-yl)propanal (10) was converted into the separable diastereomeric derivatives of 6 and 7, compounds 12 and 11, respectively. Deprotection of 12, in one or two steps, gave extensively racemized 6, which was converted in low yield into 6 (99.0% ee) through several crystallizations. 6 (99.7% ee) and 7 (99.9% ee) were finally obtained by preparative chiral HPLC. The configurational assignments of 6 and 7 were based on 1H NMR spectroscopic studies on 12 and 11, respectively, and circular dichroism studies on 6 and 7. Values of optical rotations using different solvents and the chiral HPLC elution order of 6 and 7 supported the results of the spectroscopic configurational assignments. The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 = 58 +/- 11 microM) comparable in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 = 20 +/- 3 microM). Although Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist (IC50 = 131 +/- 18 microM).

Structure-activity relationships of new agonists and antagonists of different metabotropic glutamate receptor subtypes.

1. We investigated the agonist and antagonist activities of 22 new phenylglycine and phenylalanine derivatives for metabotropic glutamate receptors (mGluRs) by examining their effects on the signal transduction of mGluR1, mGluR2 and mGluR6 subtypes expressed in Chinese hamster ovary cells. This analysis revealed several structural characteristics that govern receptor subtype specificity of the agonist and antagonist activities of phenylglycine derivatives. 2. Hydroxyphenylglycine derivatives possessed either an agonist activity on mGluR1/mGluR6 or an antagonist activity on mGluR1. 3. Carboxyphenylglycine derivatives showed an agonist activity on mGluR2 but an antagonist activity on mGluR1. 4. alpha-Methylation or alpha-ethylation of the carboxyphenylglycine derivatives converts the agonist property for mGluR2 to an antagonist property, thus producing antagonists at both mGluR1 and mGluR2. 5. Structurally-corresponding phenylalanine derivatives showed little or no agonist or antagonist activity on any subtypes of the receptors. 6. This investigation demonstrates that the nature and positions of side chains and ring substituents incorporated into the phenylglycine structure are critical in determining the agonist and antagonist activities of members of this group of compounds on different subtypes of the mGluR family. 7. We also tested two alpha-methyl derivatives of mGluR agonists. (2S, 1'S, 2'S)-2-(2-Carboxycyclopropyl)glycine (L-CCG-I) is a potent agonist for mGluR2 but alpha-methylation of this compound changes its activity to that of an mGluR2-selective antagonist. In contrast, alpha-methylation of L-2-amino-4-phosphonobutyrate (L-AP4) results in retention of an agonist activity on mGluR6. Thus, alpha-methylation produces different effects, depending on the chemical structures of lead compounds and/or on the subtype of mGluR tested.

Prostaglandin E2 facilitates excitatory synaptic transmission in the nucleus tractus solitarii of rats.

Prostaglandin E2 (PGE2) binding sites are rich in the nucleus tractus solitarii (NTS). We studied the effects of PGE2 on evoked excitatory postsynaptic currents (eEPSCs) and miniature EPSCs (mEPSCs) in voltage-clamped neurons in rat NTS slices. eEPSCs and mEPSCs, mediated by non-NMDA glutamate receptors, fluctuated in size from event to event. In 37.5% of neurons, PGE2 increased the mean size of eEPSCs and changed the size distribution non-proportionally. In 42.9% of neurons, PGE2 increased the frequency of mEPSCs keeping the skewed size distribution unchanged. However, PGE2 did not modulate postsynaptic non-NMDA receptor sensitivity. We propose that size distributions of eEPSCs before and after PGE2 application are predictable from those of mEPSCs by quantal analysis with multinomial distribution. Our results suggest that PGE2 facilitates evoked and spontaneous release of glutamate vesicles.

Cutaneous vasculitis in a patient with dermatomyositis without muscle involvement.

A 74-year-old female patient with cutaneous ulcerations and typical dermatomyositis (DM) skin rash had no muscle disease for a 1-year and 5 months period. Histological examination of the skin ulceration indicated vascular occlusion without cellular infiltration. Cutaneous ulceration is a very rare manifestation of adult-onset DM patients without inflammatory myopathy.

[Liver disease in systemic lupus erythematosus].

Liver disease in 193 patients (17 male and 176 female) with systemic lupus erythematosus (SLE) at Kawasaki Municipal Hospital were analyzed. Abnormal transaminase levels were found in 78 case (40.4%). Among them, there were 35 patients whose liver disease were identified. There were 12 patients whom no cause could be found other than SLE. Other liver disease were as follows: fatty liver in 9 cases, virus infection in 5 cases, gall stone and/or cholecystitis in 3 cases, drug allergy in 2 cases, autoimmune hepatitis 2 cases, primary biliary cirrhoses in 1 case. Liver disease with systemic lupus erythematosus was frequent, but there was no severe case.

Studies on exogenous and endogenous interaction of gastrin and secretin in a case of achalasia.

Studies were carried out in a case of achalasia. Administration of secretin caused relaxation of the spastic condition of LES, and high levels of serum gastrin and lower levels of plasma secretin are suggested to be related with the abnormally spastic condition of LES in the patient.

Prevention of neutrophil apoptosis by monosodium urate crystals.

The effects of monosodium urate (MSU) crystals on apoptosis of cultured peripheral blood neutrophils were investigated. MSU crystals at low concentrations decreased the rate of spontaneous apoptosis of neutrophils in a dose- and time-dependent manner. The culture supernatant of MSU crystal stimulated neutrophils also promoted a delay in neutrophil apoptosis. MSU crystals at higher concentrations rapidly caused cell lysis. These findings indicated that MSU crystals are capable of amplifying the inflammatory responses of gouty arthritis by decreasing the rates of neutrophil apoptosis at lower concentrations and inducing cell lysis at higher concentrations.

In-vivo induction of monocyte chemotactic and activating factor in patients with chronic renal failure.

BACKGROUND: Monocyte chemotactic and activating factor (MCAF) is a novel inflammatory cytokine belonging to the chemokine superfamily and stimulates chemotaxis and activation of monocytes. Increased production of inflammatory cytokines has been shown in patients with end-stage renal disease (ESRD). This study was thus conducted to determine plasma MCAF in patients with ESRD. METHODS: Plasma levels of MCAF were determined by ELISA. Gene expression of MCAF in PBMC was assessed by RT-PCR followed by southern blot hybridization. RESULTS: Plasma MCAF in 72 patients with long-term haemodialysis (HD) (162.4 +/- 58.2 pg/ml) and eight uraemic patients not yet dialysed (167.6 +/- 57.7 pg/ml) was found to exceed significantly the level in 24 normal subjects (86.0 +/- 19.4 pg/ml). MCAF before HD session in long-term HD patients was the same whether HD was carried out with either cellulosic (CUP) or synthetic (PMMA) membrane dialysers. Intradialytic increase in plasma MCAF during a single HD session was observed in both patient groups dialysed with CUP or PMMA membranes. The results of RT-PCR analysis indicated that haemodialysis stimulates the gene expression of MCAF in PBMC in vivo. CONCLUSIONS: The present results indicate that increased levels of plasma MCAF may promote the activation of monocytes in patients with ESRD.

Serum gastrin and gastrin-like immunoreactivity of gastrointestinal mucosa in fetal, neonatal and adult rats.

Serum gastrin concentration became became detectable in rats at one week after birth (56 pg/ml), and a rapid rise to 198 pg/ml was seen at two weeks after birth. Gradual decreases were seen again at three weeks and four weeks, and thereafter it attained the adult level of 132 +/- 17.6 pg/ml. In regards to the gastrin-like immunoreactivity (GLI) in the gastrointestinal mucosa during the fetal period, the values were extremely low even in the antral mucosa. However, the GLI showed an increase during the neonatal suckling period, and was accompanied by a remarkable increase at the commencement of feeding. This GLI coincided with so-called little gastrin, and while an increase accompanied by the development of growth was seen, no qualitative changes were evident. GLI values were high in the duodenal mucosa followed by the antrum, and positive evidence was also seen in the corpus of the stomach and jejunum. High values of GLI in the mucosae were gradually seen at two weeks after birth coinciding with the rise in serum gastrin levels, and the significance of the increases was discussed from the anlge of the trophic effect of gastrin.

Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1.

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate >> ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.

[Two cases of rheumatoid arthritis presenting autoimmune hepatic diseases].

We report on two cases of rheumatoid arthritis (RA) presenting autoimmune hepatic diseases. The first patient, who had been diagnosed as RA at the age of 63, was hospitalized in order to undergo surgery for total left knee replacement at the age of 69. She acquired acute serum hepatitis as a result of blood transfusion she received during the operation. Five years later, she visited our clinic suffering from polyarthritis. She was found to have hyper-alkaline phosphatase (ALP) and hyper rGTP, but no AMA. The second patient, a 60-year-old female whose onset of RA was at the age of 45, complained of general fatigue, and was admitted to the hospital because of persistent liver dysfunction. When corticosteroid was administered to these patients, ALP and rGTP levels in the first case, and AST and ALT levels in the second case were reduced to values in the normal range. ANA in the first case continued to register negative, but ANA in the second case became positive after the patient developed acute hepatitis. Both patients were found to have anti-p25 triplet liver/kidney microsome antibody. We discuss the clinical significance of this antibody.