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Please see also Cash J. D. DDAVP and factor VIII: a tale from Edinburgh. This issue, pp. 619±621. Mannucci P. M. Desmopressin (DDAVP) and factor VIII: the tale as viewed from Milan (and Malmo È ). This issue, pp. 622±624. Kaufmann J. E. et al. Desmopressin (DDAVP) induces NO production in human endothelial cells via V2 receptor-and cAMP-mediated signaling. This issue, pp. 821±828.Summary. The synthetic analog of vasopressin desmopressin (DDAVP) is widely used for the treatment of patients with von Willebrand disease (VWD), hemophilia A, several platelet disorders, and uremic bleeding. DDAVP induces an increase in plasma levels of von Willebrand factor (VWF), coagulation factor VIII (FVIII), and tissue plasminogen activator (t-PA). It also has a vasodilatory action. In spite of its extensive clinical use, its cellular mechanism of action remains incompletely understood. Its effect on VWF and t-PA as well as its vasodilatory effect are likely explained by a direct action on the endothelium, via activation of endothelial vasopressin V2R receptor and cAMP-mediated signaling. This leads to exocytosis from Weibel Palade bodies where both VWF and t-PA are stored, as well as to nitric oxide (NO) production via activation of endothelial NO synthase. The mechanism of action of DDAVP on FVIII plasma levels remains to be elucidated. The hemostatic effect of DDAVP likely involves additional cellular effects that remain to be discovered.

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Please see also Cash J. D. DDAVP and factor VIII: a tale from Edinburgh. This issue, pp. 619±621. Mannucci P. M. Desmopressin (DDAVP) and factor VIII: the tale as viewed from Milan (and Malmo È ). This issue, pp. 622±624. Kaufmann J. E., Vischer U.M. Cellular mechanisms of the hemostatic effects of desmopressin (DDAVP). This issue, pp. 682±689.Summary. The hemostatic agent desmopressin (DDAVP) also has strong vasodilatory effects. DDAVP is a selective agonist for the vasopressin V2 receptor (V2R), which is coupled to cAMPdependent signaling. DDAVP-induced vasodilation may be due to endothelial NO synthase (eNOS) activation. This hypothesis implies cAMP-mediated eNOS activation. It also implies wide extrarenal, endothelial V2R expression. We show that in human umbilical vein endothelial cells (HUVECs) the cAMP-raising agents forskolin and epinephrine increase NO production, as measured by a L-NMMA-inhibitable rise in cellular cGMP content. They also increase eNOS enzymatic activity, in a partly calcium-independent manner. cAMP-mediated eNOS activation is associated with phosphorylation of residue Ser1177, in a phosphatidyl inositol 3-kinase (PI3K)-independent manner. HUVECs do not express V2R. However, after heterologous V2R expression, DDAVP induces cAMP-dependent eNOS activation via Ser1177 phosphorylation. We have previously found V2R expression in cultured lung endothelial cells. By real time quantitative RT-PCR, we now ®nd a wide V2R distribution notably in heart, lung and skeletal muscle. These results indicate that DDAVP and other cAMP-raising agents can activate eNOS via PI3K-independent Ser1177 phosphorylation in human endothelial cells. This mechanism most likely accounts for DDAVP-induced vasodilation.

Background information. NPY (neuropeptide Y) may have an effect on the properties of vascular endothelial cells such as pro-angiogenic effects and potentiation of noradrenaline-induced vasoconstriction. In HUVEC (human umbilical-vein endothelial cells), immunoreactive neuropeptide Y has been detected, but NPY synthesis, storage and secretion have not been studied. The aim of the present study was to establish NPY expression, storage and cellular transducing effects in HUVEC.Results. HUVEC contain 0.19 fmol of NPY/µg of protein and 0.46 fmol of pro-NPY/µg of protein, as measured by ELISA. RT (reverse transcriptase)-PCR confirmed the expression of NPY in HUVEC. Immunofluorescence revealed the presence of NPY in small punctate structures, with a fluorescence pattern different from that observed for von Willebrand factor, indicating distinct storage compartments. Double labelling for NPY and Rab3A demonstrated similar granular patterns, with at least partial co-localization. Electron microscopy showed NPY immunoreactivity in vesicle-like cytoplasmic structures, of a fine fibrillar texture, as well as in mitochondria and in the nucleus. A similar general distribution pattern was also obtained for Rab3A. Y1 and Y2 receptors were expressed in HUVEC as assessed by RT-PCR, and they were functional since NPY induced a 42 nM intracellular calcium increase within 100 s, representing 22% of the histamine-induced response. In contrast with histamine, NPY did not induce acute von Willebrand factor secretion.Conclusions. HUVEC produce, store and respond to NPY, suggesting an autocrine regulatory role for NPY in the endothelium.Introduction NPY (neuropeptide Y) is a 36-amino-acid peptide involved in the regulation of the cardiovascular system.

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and pro-insulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide-A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.

Proinsulin is converted into insulin by the action of two endoproteases. Type I (PC1/PC3) is thought to cleave between the B-chain and the connecting peptide (C-peptide) and type II (PC2) between the C-peptide and the A-chain. An acidic region immediately C-terminal to the point of cleavage at the B-chain/C-peptide junction is well conserved throughout evolution and has been suggested to be important for proinsulin conversion [Gross, Villa-Komaroff, Kahn, Weir and Halban (1989) J. Biol. Chem. 264, 21486-21490]. We have here compared the precise role of this region as a whole and just the first acidic residue C-terminal to the point of cleavage in processing of proinsulin by PC3. To this end, several mutations were introduced in this region of human proinsulin (native sequence, B-chain RREAEDL C-peptide): RRPAEDL (C1Pro mutant); RRLAEDL (C1Leu mutant); RRL (C1-C4del mutant); RRE (del-C1Glu mutant). Mutant and native cDNAs were stably transfected into AtT20 (pituitary corticotroph) cells, in which PC3 is known to be the major conversion endoprotease, and kinetics of proinsulin conversion were studied (pulse-chase/HPLC analysis of proinsulin-related peptides). The results show that the acidic region following the B-chain/C-peptide junction is indeed important for PC3 cleavage at this site, and that the reduced cleavage observed for the C1-C4del mutant proinsulin can be partially overcome by replacing the acidic region with a single acidic residue (del-C1Glu mutant). Replacing only the first residue of the acidic region with leucine (C1Leu mutant) has no impact on conversion, whereas its replacement with proline (C1Pro mutant) almost completely abolishes cleavage at the B-chain/C-peptide junction without affecting that at the C-peptide/A-chain junction.

Proinsulin conversion to insulin occurs in secretory granules of pancreatic beta-cells. This processing has been suggested to require both the endoproteases PC2 and PC3 with each cleaving at only one of the two sites linking the insulin A- and B-chains with C-peptide. To evaluate this in an appropriate cellular setting, conversion of human proinsulin was followed in GH3 (rat pituitary) cells normally unable to convert this prohormone but equipped with the regulated secretory pathway. For this purpose, human proinsulin was expressed in GH3 cells, alone or in combination with PC2 and/or PC3, using recombinant adenoviruses. Cells were infected with the given adenoviruses and 24 h later were pulse-chased. Kinetics of proinsulin conversion were monitored by reverse-phase high-performance liquid chromatography. It was observed that while the two endoproteases do display a preference for a single site of cleavage (PC2 at the A-chain/C-peptide junction; PC3 at the B-chain/C-peptide junction) and act in a synergistic manner to promote proinsulin conversion, either PC2 or PC3 alone can cleave at both sites to fully convert proinsulin to insulin. These results also show that a cell can be successfully infected by three different recombinant adenoviruses.