We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.Chromogranin A, the major soluble protein in catecholamine storage vesicles, not only stabilizes the core of such vesicles (1) but also serves as a prohormone cleaved into several biologically active peptides, including the insulin release-inhibitory fragments pancreastatin (2) and ␤-granin (3-5), the vasodilator vasostatin (6, 7), and the parathyroid hormone release-inhibitory parastatin (8). Recently we described an additional chromogranin A fragment, catestatin (bovine chromogranin A 344 -364), which acts specifically as a potent (IC 50 ϳ200 -400 nM) nicotinic cholinergic antagonist to inhibit catecholamine release (9, 10), suggesting a novel autocrine feedback mechanism controlling sympathochromaffin exocytosis. Initial studies of catestatin relied on synthetic peptides (10); hence, the existence of the peptide in vivo remains unexplored, as do its precise cleavage sites from chromogranin A.In this study, we explored processing of the catestatin region of chromogranin A in secretory granules of chromaffin cells and sympathetic nerves, as well as in human pheochromocytomas. We documented catestatin cleavage from chromogranin A and determined the precise endogenous cleavage sites bounding catestatin in bovine and human chromaffin granules by chromatographic separations coupled with amino-terminal amino acid sequencing, immunoprecipitation, and matrix-assisted laser desorption ionization (MALDI) 1 mass spectrometry. We confirmed catestatin's regulated secretion by chromaffin cells. A major form of catestatin was processed at dibasic sites (bovine chromogranin A 332-364 or human chromogranin A 340 -372 ), while a smaller form was also identified (bovine chromogranin A 343-362 ). Both larger and smaller size forms were synthesized; each displayed specific antagonism of nicotinic cholinergicstimulated catecholamine release, while the smaller form had greater potency of inhibition. MATERIALS AND METHODS Preparation of TissueFractions-All preparative steps on tissues or protein fractions were conducted at 0 -4°C. Bovine adrenal medullary chromaffin granules were prepared by centrifugation on 0.3 M/1.6 M sucrose density step gradients, as described previously (10). After granule hypotonic lysis and centrifugal removal of granule membranes, the soluble proteins and peptides in the supernatant were size-fractionated on a 2.6 ϫ 80-cm Sephacryl S-300 column (Amersham Pharmacia Biotech), eluting with the volatile buffer 0.3 M ammonium acetate, pH 6.5, as described previously (11). The buffer was removed by lyophilization before further studies. In some experiments, bovine chromaffin granule proteins/peptides (200 l c...

Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.

Successful invasion of the maternal vascular system by trophoblast cells is a prerequisite for the establishment of a normal hemochorial placenta. Transforming growth factor-beta (TGFbeta) has been implicated in the regulation of trophoblast invasiveness into the uterus. Endoglin is a component of the TGFbeta receptor complex that binds beta1 and beta3 isoforms and is expressed at high levels on syncytiotrophoblast throughout pregnancy and is also transiently up-regulated on extravillous trophoblasts differentiating along the invasive pathway. We investigated the role of endoglin in a serum-free human villous explant culture system that allows the study of trophoblast outgrowth, migration, and invasion and mimics events occurring in anchoring villi during the first trimester of gestation. Addition to explant cultures from 5-8 weeks gestation of a monoclonal antibody to endoglin or of antisense endoglin oligonucleotides significantly stimulated trophoblast outgrowth and migration. These responses were specific, as incubation of explants with nonimmune IgG or sense and scrambled oligonucleotides had no effect. Antisense endoglin-induced trophoblast outgrowth and migration were accompanied by cell division of villous-associated trophoblasts within the proximal region of the forming column and by the characteristic switch in integrins observed in anchoring villi in situ. Treatment of villous explants with antibody and antisense oligonucleotides to endoglin also resulted in an increased fibronectin release into the culture medium. The stimulatory effect of antisense endoglin on fibronectin production was overcome by the addition of exogenous TGFbeta2, but not TGFbeta1 and -beta3. These findings suggest that endoglin expression in the transition from polarized to nonpolarized trophoblasts in anchoring villi is necessary for mediation of the inhibitory effect of TGFbeta1 and/or TGFbeta3 on trophoblast differentiation along the invasive pathway.

Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of labor contractions. The expression of connexin-43 (Cx-43), the major myometrial gap junction protein, is increased during delivery (associated with an increase in the plasma estradiol/progesterone ratio) and after estradiol treatment of ovariectomized nonpregnant rats. However, Cx-43 is only 1 member of at least 16 proteins encoded by this family of gap junction genes. Using a RT-PCR method, we identified the presence of another member of this family, Cx-26, in laboring rat myometrium. The temporal expression pattern of Cx-26 was assessed using Northern and Western analyses. In contrast to Cx-43, whose expression is low throughout the pregnancy but increases immediately before the onset of labor (day 23), the expression of Cx-26 increased on day 17, reached maximal levels between days 19-21, and fell to low levels before the onset of labor. Treatment of pregnant rats with progesterone beginning on day 20 (which blocks both the increase in Cx-43 expression and the onset of labor) maintained the elevated expression of Cx-26. Induction of preterm labor in rats after ovariectomy on day 17 inhibited the normal preterm increase in Cx-26 transcripts. Progesterone treatment of these animals reversed the effects of ovariectomy. Immunofluorescence data identified Cx-26 antigen in the cell membranes of myometrial cells and in the luminal and glandular epithelium of the endometrium in the late pregnant (day 21) uterus. These data suggest that the role of gap junction formation in the myometrium in relation to the maintenance of pregnancy and the onset of labor is much more complex than previously recognized. Myometrial cell-cell communication is afforded by at least two different gap junction proteins, Cx-43 and Cx-26, that not only exhibit temporally distinct patterns of expression but are also subject to differential regulation.