Masafumi Fujimoto

A novel non‐peptide endothelin antagonist isolated from bayberry, Myrica cerifera

A potent non‐peptide ET receptor antagonist, myrireron caffeoyl ester (50–235), was isolated from the bayberry, Myrica cerifera. This compound selectively antagonized specific binding of [125I]ET‐1, but not of [125I]ET‐3, to rat cardiac membranes, ET‐1‐induced increase in the intracellular free calcium concentration in Swiss 3T3 fibroblasts, and ET‐1‐induced contraction of rat aortic strips. Thus, 50–235 is the first non‐peptide ETA receptor antagonist. This compound can be useful for studying the physiological role of endothelin and exploring its role in various diseases.

Neuropeptide Y stimulates DNA synthesis in vascular smooth muscle cells

Neuropeptide Y (NPY) stimulated DNA synthesis in porcine aortic smooth muscle cells in a concentration-dependent manner. [Leu31, Pro34]NPY, a Y1-specific agonist, was several hundred times more potent than NPY(13-36), which preferentially bound to Y2 receptors, for stimulating DNA synthesis. On the other hand, human pancreatic polypeptide had no effect. The potency of NPY and related peptides for stimulating DNA synthesis paralleled their potency for increasing the cytosolic free Ca2+ concentration in the cells. Pertussis toxin treatment completely blocked both effects of the peptides. Thus, NPY may induce Ca2+ mobilization and stimulation of DNA synthesis in vascular smooth muscle cells via Y1 receptors whose signal transduction system involves pertussis toxin-sensitive GTP-binding protein(s).

Effects of endothelin B receptor agonists on amyloid β protein (25-35)-induced neuronal cell death

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ET(B) receptor) is associated with neuronal survival in the brain. In the Alzheimers disease (AD) brain, accumulation of amyloid beta protein (Abeta) is thought to cause neuronal cell death via apoptosis. In the present study, we investigated effects of ET(B) receptor agonists on Abeta-induced neuronal cell death. In primary cultures of rat cortical neurons, Abeta(25-35) caused neuronal cell death in a concentration- and time-dependent manner. Abeta(25-35)-induced neuronal cell death was accompanied by chromatin condensation and DNA fragmentation, exhibiting apoptotic features. ET-3 and IRL-1620, ET(B) receptor agonists, significantly prevented neurons from undergoing Abeta(25-35)-induced cell death. Prior to cell death, Abeta increased concentration of intracellular Ca(2+) ([Ca(2+)](i)). Nimodipine, an L-type voltage-sensitive Ca(2+) channel (L-VSCC) blocker, suppressed the Abeta-induced Ca(2) influx, and attenuated Abeta-induced neuronal apoptosis. On the other hand, omega-conotoxin GIVA, an N-type VSCC blocker and omega-conotoxin MVIIC and omega-agatoxin IVA, P/Q-type VSCC blockers, had no effect. ET-3 and IRL-1620 significantly blocked Abeta(25-35)-induced Ca(2) influx. Furthermore, BQ788, an ET(B) receptor antagonist, inhibited both an anti-apoptotic effect and an L-VSCC-inactivating effect of ET(B) receptor agonists. In conclusion, ET(B) receptor agonists exhibit a protective effect against neurotoxicity of Abeta. Furthermore, these agonists appear to act as anti-apoptotic factors by blocking of L-VSCCs.

Effects of S-2474, a novel nonsteroidal anti-inflammatory drug, on amyloid β protein-induced neuronal cell death

The accumulation of amyloid β protein (Aβ) in the brain is a characteristic feature of Alzheimers disease (AD). Clinical trials of AD patients with nonsteroidal anti‐inflammatory drugs (NSAIDs) indicate a clinical benefit. NSAIDs are presumed to act by suppressing inhibiting chronic inflammation in the brain of AD patients. In the present study, we investigated effects of S‐2474 on Aβ‐induced cell death in primary cultures of rat cortical neurons. S‐2474 is a novel NSAID, which inhibits cyclo‐oxygenase‐2 (COX‐2) and contains the di‐tert‐butylphenol antioxidant moiety. S‐2474 significantly prevented neurons from Aβ(25 – 35)‐ and Aβ(1 – 40)‐induced cell death. S‐2474 ameliorated Aβ‐induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely. Prior to cell death, Aβ(25 – 35) generated prostaglandin D2 (PGD2) and free radicals from neurons. PGD2 is a product of cyclo‐oxygenase (COX), and caused neuronal cell death. S‐2474 significantly inhibited the Aβ(25 – 35)‐induced generation of PGD2 and free radicals. The present cortical cultures contained little non‐neuronal cells, indicating that S‐2474 affected neuronal survival directly, but not indirectly via non‐neuronal cells. Both an inhibitory effect of COX‐2 and an antioxidant effect might contribute to the neuroprotective effects of S‐2474. In conclusion, S‐2474 exhibits protective effects against neurotoxicity of Aβ. Furthermore, the present study suggests that S‐2474 may possess therapeutic potential for AD via ameliorating degeneration in neurons as well as suppressing chronic inflammation in non‐neuronal cells.

Neurotrophic Activity of Neudesin, a Novel Extracellular Heme-binding Protein, Is Dependent on the Binding of Heme to Its Cytochrome b5-like Heme/Steroid-binding Domain

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinΔHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.

Novel binding sites of 15-deoxy-Δ12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.

Neuropeptide Y Receptor in Vascular Smooth Muscle

Abstract: 125I‐Bolton‐Hunter (125I‐BH) neuropeptide Y (NPY) was used to identify specific high‐affinity NPY binding sites in porcine aortic smooth muscle membrane fractions and to characterize the binding sites in comparison with those in porcine hippocampal membrane fractions. Ca2+, but not Mg2+ or Mn2+, enhanced specific 125I‐BH‐NPY binding in aortic smooth muscle, while all of the cations did in hippocampus. The fast, saturable, and selective binding was to a single population of sites, with a KD of 0.99 ± 0.11 nM and a Bmax of 0.35 ± 0.06 fmol/mg protein. GTP and its non‐hydrolyzable analogues reduced 125I‐BH‐NPY binding dose dependently. Neither calcium channel blockers nor nor‐adrenaline had any effect on the binding. Among structurally related peptides, peptide YY displaced 125I‐BH‐NPY binding as potently as NPY, while human, avian, and rat pancreatic polypeptides displaced 125I‐BH‐NPY binding 100 times less potently than NPY. The C‐terminal fragment NPY(13–36) for inhibiting 125I‐BH‐NPY binding in aortic smooth muscle was ∼40 times less potent than in the hippocampus. When we examined the effect of these peptides on cytosolic free Ca2+ ([Ca2+]i) in cultured porcine aortic smooth muscle cells, only the peptides showing high affinity for 125I‐BH‐NPY binding sites increased [Ca2+]i. These results indicate that the NPY binding sites labeled by 125I‐BH‐NPY in aortic smooth muscle are functional receptors and have different properties from those in the hippocampus with respect to dependency on divalent cations, sensitivity to GTP analogues, and affinity for NPY(13–36) and pancreatic polypeptides.

Molecular cloning and characterization of the angiotensin receptor subtype in porcine aortic smooth muscle

A cDNA encoding porcine aortic smooth muscle angiotensin II (AII) receptor has been isolated using the homology screening approach and sequenced. Specific binding of [125I]AII was found in COS-7 cells transfected with the cDNA (Kd = 0.37 nM, Bmax = 1 approximately 3 x 10(4)/cell) and was displaced with unlabeled AII-related peptides and DUP753 in the order of [Sar1,Ile8]AII = AII > des-Asp1-[Ile8]AII = DUP753 > angiotensin I = angiotensin III. EXP655 had no effect on [125I]AII binding. COS-7 cells transfected with the cDNA responded to AII by only a small increase in the concentration of intracellular free Ca2+. However, electrophysiological study of the receptor expressed in Xenopus laevis oocytes provided strong evidence that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of Ca2+. Northern blot analysis in cultured porcine aortic smooth muscle cells demonstrated that the expression of this gene varies with the culture media. These results indicate that the cDNA encodes the functional and regulated AT1 subtype of AII receptors.

Proposed mechanisms of stimulation and inhibition of guanylate cyclase with reference to the actions of chlorpromazine, phospholipases and triton X-100

Abstract Particulate guanylate cyclase activity in the homogenate of guinea pig tracheal muscle was activated prominently after treatment with phospholipase-A or C. Even after the stimulation by phospholipases or Triton X-100, most of the activity was still associated with the particulate fraction. Phenothiazine tranquilizers and imipramine strongly inhibited the activity, whereas haloperidol did not. These results suggest that (i) particulate guanylate cyclase is associated fimly with the membrane and perturbation of the membrane architecture rather than solubilization of the enzyme is accountable for the stimulation, and (ii) stabilization of membranes in such a way that it affects the function of mucarinic cholinergic receptors inhibits the activity.

Functions of MAPR (Membrane-Associated Progesterone Receptor) Family Members As Heme/Steroid-Binding Proteins

Progesterone receptor membrane component 1 (PGRMC1), PGRMC2, neudesin, and neuferricin all contain a cytochrome b5-like heme/steroid-binding domain and belong to the membrane-associated progesterone receptor (MAPR) family. Their amino acid sequences are well conserved among vertebrates, from humans to zebrafish. MAPR family genes are abundantly expressed in the central nervous system and exhibit neurotrophic effects in neural cells. During lipid metabolism, PGRMC1 regulates cholesterol synthesis, and neudesin plays a role in adipogenesis. Their bioactivities are dependent on the binding of heme to their cytochrome b5-like heme/steroid-binding domains. Conversely, it has been reported that the binding of steroids to MAPR family proteins induces biological responses that are unrelated to the nuclear steroid receptors. The interaction between PGRMC1 and progesterone promotes cell survival and damage resistance by progesterone. Moreover, MAPR family proteins exhibit a unique expression pattern in breast cancer, indicating the possibility of using MAPR family members as drug target in breast cancer. In this review, we summarize the identification, structure, and bioactivity of members of the MAPR family, and present an essential overview of the current understanding of their physiological roles.