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The synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], the immediate precursor of intracellular signals generated by calcium-mobilizing hormones and growth factors, is initiated by the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate [Ptdlns(4)P] by phosphatidylinositol 4-kinase (PtdIns 4-kinase). Although cells contain several Ptdlns 4-kinases, the enzyme responsible for regulating the synthesis of hormonesensitive PtdIns(4,5)P2 pools has not been identified. In this report we describe the inhibitory effect of micromolar concentrations ofwortmannin (WT) on the synthesis of hormonesensitive PtdIns(4)P and Ptdlns(4,5)P2 pools in intact adrenal glomerulosa cells, and the presence of a WT-sensitive Ptdlns 4-kinase in adrenocortical extracts. In addition to its sensitivity to the PtdIns 3-kinase inhibitor WT, this enzyme is distinguished from the recognized membrane-bound Ptdlns 4-kinases by its molecular size and weak membrane association. Inhibition of this These results indicate that a specific WT-sensitive Ptdlns 4-kinase is critical for the maintenance of the agonist-sensitive polyphosphoinositide pool in several cell types.Phosphatidylinositol 4,5-phosphate [PtdIns(4,5)P2] is a critical plasma-membrane precursor of several of the intracellular messengers that mediate the actions of many hormones, transmitters, and growth factors (2-4). PtdIns(4,5)P2 has also been implicated in other cellular functions, including regulation of the activity of certain actin-binding proteins (5) and of clathrin assembly and, hence, receptor endocytosis (6). Hormone-sensitive and -insensitive pools of inositolphospholipids have been described (7-9), as well as a nuclear inositolphospholipid system that appears to be regulated independently of receptor-activated inositolphospholipid turnover (10). The production of polyphosphoinositides from phosphatidylinositol (PtdIns) is therefore of vital importance for multiple aspects of cell regulation.The first step in the production of Ptdlns(4,5)P2 is catalyzed by Ptdlns 4-kinases, several forms of which have been isolated from mammalian sources as well as from yeast (11-13). Two membrane-bound mammalian Ptdlns 4-kinases have been characterized, a 56-kDa type II enzyme that is inhibited by adenosine and an -200-kDa type III enzyme that is relatively insensitive to adenosine. The mammalian type I enzyme, which phosphorylates PtdIns at the 3 position, is largely cytoplasmic in resting cells and contains 110-kDa catalytic and 85-kDaThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. regulatory subunits (14, 15). The catalytic domain has significant homology with the Saccharomyces cerevisiae PtdIns 3-kinase Vps34p (16) and with a recently cloned yeast cytoplasmic PtdIns 4-kinase (13). The latter enzyme, which has a molecular mass of 125 kDa, was isolated from the cytosolic fraction and appea...

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 4-kinases is the first step in the synthesis of polyphosphoinositides, the lipid precursors of intracellular signaling molecules. We have recently identified a cytosolic PtdIns 4-kinase (cPI4K) in the bovine adrenal cortex that is distinguished from previously known PtdIns 4-kinases by its sensitivity to the PtdIns 3-kinase inhibitor wortmannin (WT). The present study has further characterized this soluble enzyme and compared its properties to those of the membrane-bound, type II PtdIns 4-kinase activity of the adrenal cortex and the type III enzyme of bovine brain. The enzymatic activity of adrenal cPI4K was inhibited not only by WT (IC50 approximately 50 nM) but also by LY-294002 (IC50 approximately 100 microM), another inhibitor of PtdIns 3-kinase, and neither compound affected type II PtdIns 4-kinase at concentrations that inhibited cPI4K. In contrast to the type II enzyme, cPI4K had a significantly higher Km for ATP, was relatively insensitive to inhibition by adenosine (Ki approximately 800 microM vs approximately 40 microM), had lower affinity for PtdIns, and was not inhibited by Ca2+ ions. These properties identify the WT-sensitive adrenal cPI4K as a type III PtdIns 4-kinase that is distinct from the tightly membrane-bound, Ca2+- and adenosine-sensitive, type II PtdIns 4-kinase. The type III PtdIns 4-kinase prepared from bovine brain exhibited similar kinetic parameters as the adrenal cPI4K, and was also inhibited by WT with an IC50 of 30-50 nM. Since WT inhibits the synthesis of agonist-regulated phosphoinositide pools in intact cells at micromolar concentrations, these findings indicated that type III rather than type II PtdIns 4-kinases are responsible for the maintenance of the precursor phospholipids required for intracellular signaling through the inositol phosphate/Ca2+ pathway.

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Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins.

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KYI 1784. RP-1776 selectively inhibited the binding of PDGFBB to the extracellular domain of the PDGF /^receptor with an IC50 value of 11±6/im. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGFj3-receptor induced by PDGFBB. These results suggested that RP-1776 antagonizes the signaling of PDGFBB probably through the inhibition of PDGFBB binding to the PDGF^-receptor. Platelet-derived growth factor (PDGF) is a potent mitogen and chemotactic molecule for various cells, released from activated platelets. h2) PDGFis also expressed in embryonal tissues, and has neurotrophic activity for neuronal cells in rat brain, suggesting involvement of

Profound vasodilation of the kidneys and other nonreproductive organs transpires during early pregnancy. Because nitric oxide (NO) was found to mediate renal vasodilation and hyperfiltration in conscious pregnant rats, and endogenous endothelin (ET) was suggested to be vasodilatory in the renal circulation of nonpregnant rats, we tested whether endothelin mediates the NO-dependent changes in the renal circulation during pregnancy. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious pregnant and virgin rats before and during infusion of 30 μg/min RES-701-1 (a selective ETB receptor subtype antagonist). Baseline GFR and ERPF were significantly increased by 35% in gravid rats relative to virgin controls. During infusion of RES-701-1, the pregnant rats responded more robustly, showing a greater decline in both GFR and ERPF such that renal function converged in the two groups of rats. ERPF also converged in pregnant and virgin rats during infusion of SB-209760, a nonselective ETA/B receptor subtype antagonist. Combined infusion of N ω-nitro-l-arginine methyl ester [l-NAME, an NO synthase (NOS) inhibitor] and RES-701-1 reduced GFR and ERPF to levels comparable to those reached with either agent given alone, suggesting inhibition of a common vasodilatory pathway. RES-701-1 and SB-209670 significantly lowered the cGMP content of small renal arteries from gravid and virgin rats in vitro, strengthening the link between the renal endothelial ETBreceptor subtype and NO. Importantly, we showed that RES-701-1 is not a direct inhibitor of NOS. We conclude that endothelin mediates the NO-dependent changes in the renal circulation of conscious rats during pregnancy.