Estrogens attenuate renal injury induced by ischemia/reperfusion (I/R), an effect that is related to nitric oxide production in the post-ischemic kidney. The compound 17beta-estradiol (E(2)-beta) acting via estrogen receptors (ERs) is known to activate endothelial nitric oxide synthase (eNOS) through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. We determined if this pathway contributes to the renoprotective effect of E(2)-beta in the uninephrectomized ischemia reperfusion rat model of acute renal injury. Treatment with E(2)-beta suppressed the I/R-induced increases in blood urea nitrogen, plasma creatinine, urine flow, and fractional excretion of sodium while augmenting creatinine clearance, renal blood flow, and urine osmolality, indicating attenuation of renal injury. Phosphorylation of Akt and eNOS protein was significantly increased 30-60 min after reperfusion in estradiol-treated compared to vehicle-treated rats. The protective effects of E(2)-beta and protein phosphorylation were reversed by the PI3K inhibitor wortmannin or the ER antagonist tamoxifen. Furthermore, the E(2)-beta-induced renoprotective effects were not seen in eNOS knockout mice with renal injury. We conclude that the E(2)-beta-induced renoprotective effect is due to activation of the PI3K/Akt pathway followed by increased eNOS phosphorylation in the post-ischemic kidney.

We found that prostaglandin (PG) D 2 and PGE 2 , which are major PGs in the brain of mammals, powerfully induced the secretion of nerve growth factor (NGF) from cultured mouse astrocytes; PGE 2 or PGD 2 induced an approximately 12-or 19-fold increase in NGF secretion after a 24-h incubation, respectively. Moreover, it was found that the sequential metabolites of PGD 2 , PGJ 2 , v v 12 -PGJ 2 , and 15-deoxy-v v 12;14 -PGJ 2 , induced the NGF secretion to the culture medium strikingly (60^98-fold of the control after a 24-h incubation). NGF secretion induced by the J 2 series of PGs was accompanied by the increased expression of NGF mRNA. These PGs also stimulated the secretion/synthesis of brain-derived neurotrophic factor (BDNF). Our ¢ndings suggest that PGs play a neuroprotective role by inducing NGF and BDNF production in the central nervous system.

The phospholipase A 2 (PLA 2 ) inhibitor PLI␤, purified from the blood plasma of Chinese mamushi snake (Agkistrodon blomhoffii siniticus), is a 160-kDa trimer with three 50-kDa subunits; and it inhibits specifically the enzymatic activity of the basic PLA 2 from its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., and Hayashi, K. (1997) Biochem. J. 325, 527-531). In the present study, the 50-kDa subunit was found to be glycosylated with N-linked carbohydrate, and enzymatic deglycosylation decreased the molecular mass of the 50-kDa subunit to 39-kDa. One 160-kDa trimer of PLI␤ was found to form a stable complex with three basic PLA 2 molecules, indicating that one basic PLA 2 molecule would bind stoichiometrically to one subunit of PLI␤. A cDNA encoding PLI␤ was isolated from a Chinese mamushi liver cDNA library by use of a probe prepared by a polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a 308-residue protein, which contained the sequences of all the peptides derived by lysyl endopeptidase digestion of the subunit. The molecular mass of the mature protein was calculated to be 34,594 Da, and the deduced amino acid sequence contained four potential N-glycosylation sites. The sequence of PLI␤ showed no significant homology with that of the known PLA 2 inhibitors. But, interestingly, it exhibited 33% identity with that of human leucine-rich ␣ 2 -glycoprotein, a serum protein of unknown function. The most striking feature of the sequence is that it contained nine leucine-rich repeats (LRRs), each of 24 amino acid residues and thus encompassing over two-thirds of the molecule. LRRs in PLI␤ might be responsible for the specific binding to basic PLA 2 , since LRRs are considered as the motifs involved in protein-protein interactions.

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␣-Type phospholipase A 2 inhibitory protein (PLI␣) from the serum of the venomous snake Gloydius brevicaudus, GbPLI␣, is one of the protective endogenous proteins that neutralizes its own venom phospholipase A 2 (PLA 2 ), and it is a homotrimer of subunits having a C-type lectin-like domain. The nonvenomous snake Elaphe quadrivirgata has a homologous serum protein, EqPLI␣-LP, that does not show any inhibitory activity against various snake venom PLA 2 s (Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K. (2003) IUBMB Life 55, 539 -545). By constructing GbPLI␣-Eq-PLI␣-LP chimeric proteins, we have mapped the residues important in conferring GbPLI␣ inhibitory activity on region 13-36 in the primary structure of GbPLI␣. Noninhibitory EqPLI␣-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLI␣. Further, mutational analysis of the candidate residues revealed that the individual GbPLI␣ to EqPLI␣-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLI␣ protein with reduced inhibitory activity, with the single N26K substitution having the most significant effect. Residues 13-36 were suspected to be located in the helical neck region of the GbPLI␣ trimer. Therefore, the region of GbPLI␣ responsible for PLA 2 inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.Phospholipases A 2 (PLA 2 s, EC 3.1.1.4) 2 catalyze the hydrolysis of the acyl-ester bond at the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Secretory PLA 2 s are a growing family of low molecular weight, highly disulfide-linked, Ca 2ϩ -requiring secretory enzymes with a His-Asp catalytic dyad and are classified into six main groups (I, II, III, V, X, and XII) according to their primary structures (1). Snake venom is one of the most abundant sources of secretory PLA 2 s, which exhibit a wide variety of pharmacological effects including neurotoxicity and myotoxicity (2). Elapidae venom contains group I PLA 2 s, and Viperidae venom contains group II ones (3). Venomous snakes have three distinct types of PLA 2 inhibitory proteins (PLI␣, PLI␤, and PLI␥) in their blood to protect themselves from the leakage of their own venom PLA 2 s into the circulatory system (4 -6). PLI␣ has only been identified in the blood of Viperidae snakes, such as Protobothrops flavoviridis (renamed from Trimeresurus flavoviridis according to the present taxonomy) (7), Gloydius brevicaudus (renamed from Agkistrodon blomhoffii siniticus) (8), Bothrops asper (9), and Cerrophidion godmani (10). It is a 75-kDa trimeric glycoprotein of 20-kDa subunits having a C-type lectin-like domain (CTLD), which is homologous to that of collectins, such as serum mannose-binding protein (MBP) and lung surfactant-apoproteins (11). G. brevicaudus, B. asper, and C. godmani PLI␣s are composed of three identical subunits, whereas P. flavoviridis PLI␣ is a trimer of two homologous subunits. P. flavoviridis and G. brevicaudus PLI␣s inhibit specifically the group II acidic PLA 2...