Louisville, Louisville, Kentucky 40292; and Department of Surgery and Departments of Pharmacology and. Toxicology (G.H.G.), University of Texas Medical ...
0013.7227/93/1332-0461$03.00/0 Endocrinology Copyright 0 1993 by The Endocrine
Vol. 133, No. 2 Printed in U.S.A.
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Parastatin (Porcine Chromogranin Chromogranin A-Derived Peptide, Parathyroid Cell Secretion* BRIGITTE H. FASCIOTTO, AND DAVID V. COHN
CHRISTIANE
A. TRAUSS,
GEORGE
A347-419), a Novel Inhibits H. GREELEY,
Departments of Biological and Biophysical Sciences and Biochemistry, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292; and Department of Surgery and Departments of Pharmacology and Toxicology (G.H.G.), University of Texas Medical Branch, Galveston, Texas 77550 ABSTRACT Chromogranin A (CgA), previously referred to as secretory proteinI, is a 50-kilodalton protein present in secretory granules of many endocrine and neuroendocrine cells. In the parathyroid it is present and cosecreted with PTH in response to hypocalcemia. CgA appears to be a precursor of bioactive peptides including pancreastatin, P-granin, vasostatin, and chromostatin. The presence of several highly conserved pairs of basic amino acids, putative cleavage sites, in the CgA molecule suggests that other yet unidentified bioactive peptides might exist within the molecule. We tested this speculation by subjecting porcine parathyroid CgA to digestion by endoproteinase Lys-C. Resulting CgAderived peptides were isolated by reverse-phase Cl8 HPLC and tested for their ability to affect low-Ca*+ stimulated secretion by porcine
parathyroid cells. We characterized one peptide, which we named parastatin, that inhibited secretion of both PTH and CgA in a dosedependent fashion over the range of 0.2-0.6 pM. Parastatin migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mol wt of 11,000. Edman degradation yielded the sequence L-S-F-R-A-P-A-Y-G-F-R-G-P-G-L corresponding to residues 347-361 of porcine CgA. Amino acid analysis of endoproteinase Lys-C and endoproteinase Asp-N-generated fragments indicated that parastatin corresponds to residues 347-419 of CgA. A synthetic NH,terminal fragment of rat parastatin corresponding to residues I-19 was as inhibitory as intact porcine parastatin on parathyroid gland secretion. These results extend the concept that CgA is a precursor of biologically active peptides. (Endocrinology 133: 461-466, 1993)
C
acid secretion by rabbit parietal cells (27). Eiden (28) and Huttner and Benedum (29) noted that the sequenceof pancreastatin bore a close similarity to a central region of bovine CgA (bCgA). Subsequently, lacangelo et al. (17) deduced the amino acid sequence of porcine CgA (pCgA) from its complementary DNA and noted that the sequencecomprisedby residues 240-288 corresponded exactly to that of pancreastatin. Additionally, it was reported that intact CgA inhibited glucose-stimulated insulin release when perfused through the rat pancreas(23), and parathyroid (30) and catecholamine (31) secretion when added to dispersed cells in culture, but each of these actions required proteolytic processing of the molecule to smaller peptides. In addition to pancreastatin, other CgA-derived peptides have been reported to affect secretion of specific cell types including chromostatin (bCgA124-143) (32), bCgAld10(33, 34), vasostatin (CgA1& (35), and P-granin (CgAIml14)(36). In the parathyroid, as well as other cell types, we thought it was possible that additional, as yet unidentified, regions of CgA are bioactive. We tested this possibility in the present study by cleaving intact porcine CgA with endoproteinase Lys-C and assaying generated fragments. We describebelow a hitherto unreported fragment which we have named parastatin (pCgA347-419) that strongly inhibited low Ca’+-stimulated parathyroid secretion. A rat CgA sequencecorresponding to parastatinl_lg is as active as intact parastatin.
HROMOGRANIN A (CgA), alternatively referred to as secretory protein-I (1) is a 50-kilodalton (kDa) acidic protein present in most endocrine and neuroendocrine cells. It is cosecreted via a regulated pathway with the resident hormones of the cell (2-5). In the parathyroid CgA coexists with PTH in individual secretory granules (6, 7) and is cosecretedwith PTH in responseto hypocalcemic stimulation (I, 8). The physiological function of CgA in the parathyroid and other tissues has been imprecisely defined. Increasing evidence supports its role as a precursor of biologically active hormones or neuropeptides (9-11). The earliest clue that suggestedthat CgA was a precursor peptide was the presence in the molecule of 8-10 (depending upon animal species) conserved basic amino acid pairs (12-18). Such pairs of basic amino acids serve as favored cleavage sitesto yield bioactive peptides (17) in the casesof several prohormones including proopiomelanocortin (19) and proPTH (20). That CgA is a precursor peptide was supported by the isolation from pig pancreas of pancreastatin, a carboxyamidated, 49-amino acid peptide that strongly inhibited glucose- and glucagon-stimulated insulin secretion by the pancreas(21-24), hypocalcemic-stimulated PTH and CgA secretion by porcine parathyroid cells (25), cholecystokinin-induced amylase release by the exocrine pancreas (26), and
Materials
Received February 26, 1993. Address all correspondence and requests for reprints to: Dr. David V. Cohn, University of Louisville, Department of Biological and Biophysical Sciences, School of Dentistry, Louisville, Kentucky 40292. * This work was supported by NIH Grant DK-38296.
Preparation
of
and Methods
dispersed parathyroid
cells
Porcine parathyroid cells were obtained by collagenase fresh glands as described previously (25). The cells (106/ml) 461
digestion of were prein-
PARASTATIN
462
INHIBITS
PARATHYROID
cubated at 37 C for 1 h in phosphate-saline-supplemented medium (120 rnM NaCl, 5.6 rnM KCl, 1 rnM Mach, 1 rnM NaH2P04, 5 rnM glucose, 0.5 rnM ascorbic acid, 15 mM HEPES, pH 7.45, 5 mM glutamic acid, 5.5 mM fumaric acid, and 5 rnM lactic acid) with 0.5% heat-inactivated dialyzed porcine serum and containing 1.0 mM CaCIZ.
Assay for inhibition of secretion of CgA and PTH CgA-derived peptides
PTH was analyzed by RIA Institute, San Juan Capistrano,
Digestion of CgA by endoproteinase of parastatin
using CA).
the
Lys-C and isolation
SDS-PAGE
sequencing
and amino acid composition
NHz-Terminal sequencing was performed by automated Edman radation on an Applied Biosystems (Foster City, CA) gas-phase
degse-
of
rat parastatin
1-19
We had available a panel of rat CgA-related peptides that were synthesized by Prof. N. Yanaihara (University of Shizuoka, Shizuoka, Japan) employing the method of Merrifield (39). One of these, rCgA359-389, contains a sequence (rCgA371-389) that corresponds in all but three residues to porcine parastatirr-ig (substitution of Arg for Pro at position 6, Asp for Gly at position 12, and Pro for Leu at position 17). The purity of this peptide was confirmed by HPLC and amino acid analysis and was at least 99% pure. This peptide was digested with endoproteinase Lys-C, and the resulting fragments were separated by HPLC as described above. The peptide whose amino acid composition corresponded to parastatinlmig was collected and tested for biological activity as described above for CgA-derived fragments.
Sources of other peptides
and reagents
Synthetic bovine chromostatin was obtained ratories (Belmont, CA). All other reagents were suppliers and were the highest grade available.
Statistical
Five to 10 rg of calibrating polypeptides or samples were separated using a discontinuous Tricine-SDS-PAGE as described by Schagger and von Jagow (37). The gel was made of a separating gel (16.5%T, 3%C), a spacer-gel (lO%T, 3%C), and a stacking gel (4%T, 3%C) (%T = percent of acrvlamide and bis-acrvlamide and %C = percent of bis-acrvlamide). The anode buffer was 0.2 M Tris HCl, pH 6.9 and the cathode buffer was 0.1 M Tris HCl, pH 8.25, 0.1 M Tricine, 0.1% SDS. The samples were boiled 5 min at 95 C in SDS-sample buffer and applied to the gel. After electrophoresis, the gels were fixed and stained with brilliant blue G-colloidal concentrate (Sigma Chemical Co., St. Louis, MO; 38). Low molecular weight markers from Bio-Rad (Richmond, CA) and the MWSDS-l 75 molecular weight marker kit from Sigma (Sigma Chemical Co.) and glucagon (Sigma Chemical Co., 3.5 kDa) were used to calibrate the gels.
NH2-Terminal
quencer. Amino acid compositions were obtained with the Pica-Tag methodology (Waters Chromatography Division, Millipore Corp.) using a standard amino acid mixture (Pierce, Rockford, IL) as reference. The calculated molar amount of one amino acid in the sample was selected as a base to determine the molar ratio of the other amino acids in the sample.
Generation
This enzyme cleaves protein chains on the COOH-terminal side of Lvs residues. In order to obtain conditions for complete digestion of &A, 100.pg samples of highly purified porcine parathyroid CgA, prepared in our laboratory (30), were incubated for various times at 37 C with endoproteinase Lys-C (Boehringer Mannheim, Indianapolis, IN) in 100 rnM Tris HCl, pH 8.5, .at a protein:enzyme ratio of I;OOO:l. The reaction mixtures were examined bv SDS-PAGE as described below. Under these conditions all of the Cgk was digested by 2 h. For preparatory studies, 2-mg samples were digested for 2 h under the same condition, and the reaction mixture was applied to a 0.78 X 30 cm Ci8 FBondapak column (Waters Chromatography Division, Millipore Corp., Milford, MA) using a Waters Model 845 HPLC controller. The column was eluted at 2.5 ml/min with consecutive linear gradients as follows: acetonitrile in 0.1% trifluoroacetic acid: O-49% from 5-100 min; 4970% from 100-I 10 min. Absorbance was monitored at 212 nm. Fractions were collected, lyophilized, and maintained at -80 C until assayed. This procedure was repeated as required to generate and isolate parastatin (fraction P).
Polypeptide
Endo. 1993 Vol 133. No 2
SECRETION
by
Radioactive assay for CgA. The preincubated cells were centrifuged and resuspended in 0.5 ml fresh phosphate-saline-supplemented medium containing either 0.5 or 2.5 mM CaC12 and 50 PCi [3H]lysine (2.77 teraBecquerels/mmol; Amersham Corp., Arlington Heights, IL). CgA-derived peptides were added to the cells, and incubation was initiated. After 3 h incubation, the cells and medium were separated by centrifugation and stored at -80 C until analyzed. Newly synthesized (i.e. radioactive) secreted CgA was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography as previously described (30). Density of the bands was integrated with a Bio-Image Visage 60 system (Millipore, Ann Arbor, MI). Relative secretion is presented as integrated optical density (IOD) per pg cell protein, At all times, integration of the radioactive bands was maintained within the linear range of calibration standards. We previously showed that secretion of radioactive CgA parallels that of the immunoactive species (25). RIA. When indicated, secreted Allegro intact PTH kit (Nichols
CELL
from Peninsula obtained from
Labovarious
analysis
The data were expressed as mean + SD of three to five samples analyzed statistically by nonpaired Student’s t test.
and
Results The HPLC profile digestion of 2 mg CgA with endoproteinase Lys-C for 2 h is presented in Fig. 1A. There were three absorption peaks at the injection front due primarily to buffer components and other small residues generated by cleavage of dibasic lysine pairs. We tested each major fraction eluting beyond the injection front region (fraction D through T) for inhibitory activity on CgA secretion by parathyroid cells incubated at 0.5 mM Ca2+ (Fig. 1B). Only fraction I’, referred to subsequently as parastatin was active. In the experiment shown it inhibited CgA secretion by 77% at a dose of 4.25 pg/ml (equivalent to 0.5 PM based on the sequence of parastatin determined subsequently). Inhibitory activity was dose-dependent over the range 0.2-0.6 PM with 50% inhibition (IDso) of about 0.4 PM (Fig. 2). Parastatin was tested for inhibitory action on iPTH secretion (Table 1). Secretion of iPTH at 0.5 mM Ca*’ (regulated plus constitutive secretion) was about twice that at 2.5 mM Ca*+ (constitutive secretion). Parastatin at either 10 or 100 nM did not affect secretion of iPTH but at 1 PM inhibited secretion to 42% of maximum. Structural
characterization
of
parastatin
Figure 3 shows a typical SDS-PAGE pattern for parastatin. The sample migrated as a broad single band with an apparent mol wt of 11,000. Edman degradation yielded the partial NH*-terminal amino acid sequence L-S-F-R-A-P-A-Y-G-FR-G-P-G-L. This sequence exactly matches that of residues 347-361 of pCgA. The amino acid composition determined
PARASTATIN
-0.24
INHIBITS
PARATHYROID
CELL
SECRETION
463
c 0
20
40
60
60
100
120
MINUTES
Parastatin
(PM)
FIG. 2. Dose-response curve for the inhibition of CgA secretion by parastatin (fraction P). Parathyroid cells were incubated for 3 h with [3H]lysine at 0.5 mM Ca2+ with the indicated concentrations of parastatin. Secreted radioactive CgA was determined as described in Muterials and Methods. The data are presented as mean ? SD of three or four samples. *, P < 0.02; **, P < 0.001. TABLE 1. Effect of parastatin (fraction P), parastatiri-is, and chromostatin on secretion of iPTH by parathyroid cells in culture Secreted iPTH (pg/fig cell protein)
Condition
-DEfGHIJKLMNOPQRST
FRACTION
1. Separation and activity of peptides derived by endoproteinase Lys-C digestion of porcine CgA. A, HPLC elution profile of peptides generated from a 2-mg sample of CgA after a 2-h digestion at 37 C. Peptides were separated on a reverse-phase C,, FBondapak column using an acetonitrile:trifluoroacetic acid gradient as described in Muterials and Methods. B, Fractions D through T were tested for inhibition of CgA secretion of CgA by parathyroid cells as described in Materials and Methods. The cells were incubated at 0.5 mM Ca*+ with [3H]lysine and indicated fraction for 3 h. Relative secretion represents mean IOD + SD per pg cell protein of three samples. The dashed horizontal line with error bar represents the average relative secretion + SD in the presence of fraction D-T. Only Fraction P was significantly different from the mean secretion of all fractions. *, P < 0.02. FIG.
for parastatin is listed in Table 2 together with the theoretical compositions for CgA fragments consisting of residues 347419, 347-391, and 347-430. The COOH-terminal residue for the first two of these fragments is Lys which represents putative cleavage sites for Endoproteinase Lys-C. The last fragment would be formed if the proteinase did not cleave at a Lys residue beyond position 346. The composition of parastatin exactly matched fragment CgA347-419,which would be formed if the endoproteinase did not cleave at Lye.391and
Control (0.5 mM Ca*+) Parastatin (10 nM) Parastatin (100 nM) Parastatin (1 FM) Parastatinl-lg (10 nM) Parastatin,.ig (100 nM) Parastatini.ig (I PM) Parastatinlmlg (5 pM) Chromostatin (1 PM) Ca*+ (2.5 mM)
10.9 + 1.2 12.3 + 2.3 10.0 f 1.3 8.8 + 0.2" 12.5 + 2.8 11.6 + 0.6 7.7 + Lob 6.0 + 0.9* 11.2 2 1.4 5.9 + 1.3*
The cells were incubated for with the indicated peptides as Maximum inhibition is defined are presented as mean + SD for a Different from 0.5 mM Ca’+, b Different from 0.5 mM Ca2+,
+ % -+ 8.2 * 6.2 3.5 z
\
0 -28 18 42" -32 -14 64'
98* -6
100
4
:,‘: AL,:,,.> .‘
‘I
Inhibition (%)
3 h at 0.5 mM Ca’+, alone (control) or described in Materials and Methods. as that achieved at 2.5 mM Ca*+. Data 3-10 samples per condition. P < 0.02. P < 0.001.
123
21.5 17.0
Maximum
.’ :. \
._\\ : \~.~,,
: ,. _,
:~
._// \ ” ‘: ._\ .,\,,‘ ‘. \ \ :
,,;:
\
:I +Parastotin
Lys392.
In order to ascertain that cleavage did not occur at Lyss9* or Ly~392, we digested parastatin with endoproteinase AspN, an enzyme that cleaves on the -NH, side of Asp residues. The resulting digest was separated by reverse phase HPLC and the amino acid compositions of separated peaks were determined. Table 3 lists the composition of peak C, one of the generated fragments. The amino acid composition of this peak corresponds to CgA374-419(or parastatin,,& confirming the lack of cleavage at positions 391 and 392. The reason for
FIG. 3. Peptide SDS-polyacrylamide gel electrophoresis of parastatin. Lane 1, low molecular size markers (Bio-Rad, 14.4-92.5 kDa). Lane 2, molecular size-SDS-17 kit (Sigma, 2.51-17.0 kDa). Lane 3, glucagon (Sigma, 3.5 kDa). Lane 4, 10 pg parastatin.
this may be due to the strong acidic environment at those sites created by the five acidic Glu residues that immediately flank the Lys residues.
PARASTATIN TABLE
2. Amino
acid composition
INHIBITS
PARATHYROID
+ 0.4 c 1.1 f 0.7 k 0.3 * 0.1 + 0.6 + 0.2 + 0.5 + 0.3 + 0.7 t 0.1 + 0.2 + 0.3 f 0.1 + 0.4 2.0* 2.9 f 0.2 0
3 16 7 6 0 9 0 6 5 2 2 0 0 1 9 2.0 3 1
1 6 4 5 0 7 0 3 4 2 2 0 0 0 6 2.0 1 1
3 18 8 7 0 11 0 7 6 2 3 0 0 1 11 2.0 3 1
’ *SD of four separate preparations. *Two Phe residues were taken as the base for the calculation composition.
TABLE parastatin
3. Amino acid composition by endoproteinase Asp-N Amino acid Asp/Asn Glu/Gln Ser GUY His Arg Thr Ala Pro ‘br Val Met CYS Ile Leu Phe LYS Unknown
rParastatm,.,B
10
Residues found” 3.7 15.5 7.3 6.0 0.1 8.6 0.3 5.7 5.1 2.2 2.3 0.1 0.2 1.3 8.8
Asp/Am Glu/Gln Ser GUY His Arz Thr Ala Pro Tyr Val Met CYS Ile Leu Phe LYS Unknown
Endo. 1993 Vol 133. No 2
SECRETION
of parastatin 1
Amino acid
CELL
of peak digestion
Residues found 1.9 10.8 4.8 2.5 0.0 3.1 0.0 3.8 2.3 -0 2.0* 0.0 0.0 1.0 6.0 0.0 2.6 0.0
C, derived
porcine
of
(control, 0.58 f 0.19) and 59% at 5 PM (0.24 + 0.08). As such, it was as potent as intact parastatin. Chromostatin (CgA124-143), a strong inhibitor of stimulated catecholamine secretion by adrenal chromaffin cells (32), was tested and did not affect secretion of iPTH at a concentration of 1 PM (Table 1).
from
Discussion
Theory, C~&wm 2.0 14.0 4.0 2.0 0.0 3.0 0.0 4.0 2.0 1.0 2.0 0.0 0.0 1.0 6.0 0.0 3.0 1.0
a Due to a contaminant in the parastatin preparation, Tyr were unreliable. *Two Val residues were taken as the base for the composition.
Parastatin
FIG. 4. Amino acid sequence of parastatin (CgA347-419) and the biologically active fragment corresponding to rat parastatinlelg. The amino acids in rat parastatin that differ from the homologous sequence of porcine parastatin are shaded.
the values calculation
for of
The sequence of parastatin is based on the deduced sequence of pCgA (17) and is presented in Fig. 4. The deduced sequenceof CgA has an unidentified amino acid at position 386 (corresponding to residue 40 of parastatin) that from our compositional results and the determined nucleotide triplet AXX in the pCgA cDNA (17) could be Ser or Asn. Rat parastatinl-ls, prepared as described in Materials and Methods was tested for its effect on parathyroid secretion. It reduced relative secretion of iPTH by 62% at 1 PM and by 98% at 5 PM (Table 1) and, in an experiment not listed in the table, that of newly synthesized CgA by 53% at 1 PM (0.27 + 0.12 IOD/pg cell protein), compared to 0.5 mM Ca*+
The present report contributes to the growing body of direct evidence that CgA is a precursor of a panel of biologically active peptides. We previously showed that both pancreastatin and CgA inhibited low Ca’+-stimulated parathyroid cell secretion (25, 40). CgA gained potency during incubation, and antibodies against pancreastatin or CgA potentiated secretion above the maximum level achieved at low Ca*+. These results suggested that CgA was processed in the medium and that CgA-related peptides had a physiological inhibitory function. We have now characterized parastatin, a hitherto undescribed peptide derived from porcine CgA, that inhibits low Ca’+-stimulated parathyroid cell secretion. Parastatin contains 73 amino acids corresponding to pCgA residues347419. It is acidic with a calculated p1 of 4.79 (ChargPro analysis, PC/Gene, IntelliGenetic Inc., Mountain View, CA). On SDS-PAGE it migrates as a peptide of mol wt 11,000, substantially greater than its theoretical mol wt 8,326. In this regard parastatin exhibits similar anomalous migratory behavior on gels as does its parent CgA that migrates as a protein of 70 kDa compared to its true molecular size of 50 kDa (13, 41). The regions of CgA in the human, bovine, rat, and mouse speciesthat correspond to porcine parastatin are highly conserved (85-90% similarity). Three pairs of basic amino acid residuesexist in parastatin that could serve as cleavage sites, raising the possibility that the molecule upon generation from the parent CgA is itself cleaved into smaller bioactive peptide( Indeed, we show here that parastatinl-ls, the sequencethat extends to the first pair of basic amino acids, is as active as the entire peptide. Leduc et al. (42) have shown that fragmentation of bCgA with plasmakallikrein, an enzyme able to cleave prohormone
PARASTATIN
INHIBITS
PARATHYROID
in vivo, yielded a fragment corresponding to CgA352m35s. This fragment corresponds to parastatins-il. Since we find that parastatinl-lg exhibits inhibitory activity equivalent to the full parastatin molecule, it is possible that parastatin5-i1 may itself be active. Parastatin now joins several other CgA-related peptides that exhibit biological activity on a variety of cell types. These peptides include pancreastatin (pCgA240-288), which inhibits PTH and insulin release from parathyroid and pancreatic islet cells, respectively (21, 22, 25); chromostatin (CgA124-143), which inhibits release of catecholamines from adrenal chromaffin cells (32); /3-granin (CgA1m114), which inhibits PTH secretion (36); CgAI-40, which stimulates calcitonin generelated peptide release and inhibits calcitonin and PTHrelated peptide release (33, 34); and vasostatin (CgAlmT6), which has been recently reported to reduce vascular tension (35). Although parastatin acted to inhibit parathyroid secretion at a relatively high concentration compared to pancreastatin (10e6 vs. 10p9, respectively) (25), we cannot judge what a physiologically correct concentration might be. This would particularly be the case should parastatin act as an autocrine or paracrine agent, since it could exist at a relatively high concentration in the extracellular milieu immediately surrounding the secreting cell and exert its action, and yet upon dilution in the extracellular fluid and plasma be inactive on peripheral tissue cells. The existence of several active fragments of CgA, each from a unique and non-overlapping region of CgA, and several of which act in the same manner on secretion, might seem surprising. For example, parastatin, pancreastatin, and /3-granin each block low-Ca2+-stimulated PTH secretion. If, however, the proteolytic processing of the molecule were cell specific, then only one of many possible inhibitory peptides would be generated in situ. This suggestion has precedence in the posttranslational processing of POMC that differs in the several cell types in which it is formed (19). Moreover, evidence exists that processing of CgA is, indeed, cell or tissue specific. For example, secretion of immunoactive pancreastatin from a human pancreatic carcinoid cell line (43) and a human pancreatic islet cell tumor (44) has been reported, and pancreastatin has been isolated from various porcine tissues (45), including the pancreas (46). Yet Drees and Hamilton (47) were unable to detect pancreastatin either in parathyroid secretory vesicles or in incubation medium. On the other hand, fi-granin was isolated from pancreatic P-cells (48, 49) and was found to be secreted by parathyroid cells (36). In addition, several reports show that the fragmentation patterns of CgA-derived peptides vary in different cell types (50, 51). Existence of unique processing patterns would agree with the patterns of activity already discerned for some of the CgA-related fragments. Specifically, chromostatin, but not pancreastatin, blocks adrenal cell secretion, whereas we report herein that pancreastatin, but not chromostatin, inhibits parathyroid secretion. The task remains to establish whether or not various bioactive peptides derived from CgA are physiological moie-
CELL
SECRETION
ties and not merely pharmacological agents. This will require that experimental data meet certain criteria. In the specific case of parastatin, it will be helpful to show: 1) that it is produced within the parathyroid or from secreted CgA upon the latter interacting with a target tissue; and 2) that it is subject to physiological regulation, for example by Ca2+ concentrations over the physiological range. Another type of evidence would be a demonstration that a specific parastatin receptor exists, such as has been reported for chromostatin in the adrenal (52). Acknowledgments We thank A. Nishijima for expert technical assistance, Drs. J. W. Hamilton and J, Rouse (Veterans Administration Medical Center, Kansas City, MO) for performing N-terminal sequencing experiments, and Prof. N. Yanaihara (University of Shizuoka, Shizuoka, Japan) for providing CgA-derived peptides in collaboration with Dr. Greeley. We thank Fischer Packing Company and Montfort Inc. (Louisville, KY) for allowing us to collect porcine parathyroid glands.
References 1. Cohn DV, Zangerle R, Fischer-Colbrie R, Chu LLH, Elting JJ, Hamilton JW, Winkler H 1982 Similarity of secretory protein-I from parathyroid gland to chromogranin A from adrenal medulla. Proc Nat1 Acad Sci USA 79:6056-6059 2. Hogue-Angeletti R 1986 Chromogranins and neuroendocrine secretion Lab Invest 55:387-390 3. O’Connor DT 1983 Chromogranin: widespread immunoreactivity in polypeptide hormone producing tissues and in serum. Regul Pept 6:263-280 4. Cohn DV, Elting JJ, Frick M, Elde R 1984 Selective localization of the secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat. Endocrinology 114:1963-1974 5. Winkler H, Apps DK, Fischer-Colbrie R 1986 The molecular function of adrenal chromaffin granules: established facts and unresolved topics. Neuroscience 18:261-290 6. Arps H, Dietel M, Lauritzen B, Elting JJ, Niendorf A, Cohn DV 1987 Co-localization of parathyroid hormone and secretory proteinI in bovine parathyroid glands: a double immunocytochemical study at the electron microscopical level. Bone Miner 2:175-183 7. Ravazzola M, Orci L, Habener JF, Potts Jr JT 1978 Parathyroid secretory protein: immunocytochemical localization within cells that contain parathyroid hormone. Lancet 2:371-372 JJ, Cohn DV 1978 The effect of calcium and magnesium 8. Morrissey on the secretion of parathormone and parathyroid secretory protein by isolated porcine parathyroid cells. Endocrinology 103:2081-2090 R, Dean WL, Cohn DV 1987 New sug9. Gorr S-U, Kumarasamy gestions for the physiological role of secretory protein-I. Bone Miner 2:251-255 10. Cohn DV, Fasciotto BH, Gorr S-U, Parkins F, Shioi J, Levine MA, Greeley Jr GH 1990 The putative role of secretory protein-I/ chromogranin A as a precursor for regulatory hormones. Prog Clin Biol Res 332:51-66 H, Fischer-Colbrie R 1992 The chromogranins A and B: 11. Winkler the first 25 years and future perspectives. Neuroscience 49:497-528 12. Iacangelo A, Affolter HU, Eiden LE, Herbert E, Grimes M 1986 Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine tissues. Nature 323:82-86 13. Ahn TG, Cohn DV, Gorr S-U, Ornstein DL, Kashdan MA, Levine MA 1987 Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence. Proc Nat1 Acad Sci USA 84:5043-5047 14. Iacangelo A, Okayama H, Eiden LE 1988 Primary structure of rat chromogranin A and distribution of its mRNA. FEBS Lett 227:115121
466
PARASTATIN
INHIBITS
PARATHYROID
15. Konecki DS, Benedum UM, Gerdes H-H, Huttner WB 1987 The primary structure of human chromogranin A and pancreastatin. J Biol Chem 262:17026-17030 LJ, Ahn TG, Levine MA, Allison A, Cohen PS, Cooper 16. Helman MJ, Cohn DV, Israel MA 1988 Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA. J Biol Chem 263:11559-11563 17. Iacangelo AL, Fischer-Colbrie R, Keller KJ, Brownstein MJ, Eiden LE 1988 The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin. Endocrinology 122: 2339-2341 18. Grimes M, Iacangelo A, Eiden LE, Godfrey B, Herbert E 1987 Chromogranin A: the primary structure deduced from cDNA clones reveals the presence of pairs of basic amino acids. Ann N Y Acad Sci 493:351-378 19. Eipper BA, Mains RE 1980 Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides. Endocr Rev l:l-17 20. Cohn DV, MacGregor RR, Chu LLH, Kimmel JR, Hamilton JW 1972 Calcemic fraction-A: biosynthetic peptide precursor of parathyroid hormone. Proc Nat1 Acad Sci USA 69:1521-1526 21. Tatemoto K, Efendic S, Mutt V, Makk G, Feistner GJ, Barchas JD 1986 Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 324:476-478 22. Efendic S, Tatemoto K, Mutt V, Quan C, Chang D, &tenson CG 1987 Pancreastatin and islet hormone release. Proc Nat1 Acad Sci USA 84:7257-7260 23. Greeley Jr GH, Thompson JC, Ishizuka J, Cooper CW, Levine MA, Gorr S-U, Cohn DV 1989 Inhibition of glucose-stimulated insulin release in the perfused rat pancreas by parathyroid secretory motein(chromozranin-A). Endocrinologv 124:1235-1238 24. Peiro E, Miralles”P, Silvestre RA, Villaiueva ML, Marco J 1989 Pancreastatin inhibits insulin secretion as induced by glucagon, vasoactive intestinal peptide, gastric inhibitory peptide, and 8-cholecystokinin in the perfused rat pancreas. Metabolism 38:679-682 25. Fasciotto BH, Gorr S-U, DeFranco DJ, Levine MA, Cohn DV 1989 Pancreastatin, a presumed product of chromogranin-A (secretory protein-I) processing, inhibits secretion from porcine parathvroid I ’ cells in culture. End&inology 125:1617-1622 26. Funakoshi A, Mivasaka K. Nakamura R. Kitani K. Funakoshi S. Tamamura H, Fujii N, Yajima H 1988 Bioactivity of synthetic human pancreastatin on exocrine pancreas, Biochem Biophys Res Commun 156:1237-1242 27. Lewis JJ, Zdon MJ, Adrian TE, Modlin IM 1988 Pancreastatin: a novel peptide inhibitor of parietal cell secretion, Surgery 104:10311036 28. Eiden LE 1987 Is chromogranin a prohormone? Nature 325:301 29. Huttner WB, Benedum UM 1987 Chromogranin A and pancreastatin. Nature 325:305 30. Fasciotto BH, Gorr S-U, Cohn DV 1992 Autocrine inhibition of parathyroid cell secretion requires proteolytic processing of chromogranin A. Bone Miner 17:323-333 31. Simon J-P, Bader M-F, Aunis D 1988 Secretion from chromaffin cells is controlled by chromogranin A-derived peptides. Proc Nat1 Acad Sci USA 85:1712-1716 32. Galindo E, Rill A, Bader M-F, Aunis D 1991 Chromostatin, a 20. amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion. Proc Nat1 Acad Sci USA 88:1426-1430 33. Deftos LJ, Hogue-Angeletti R, Chalberg C, Tu S 1989 PTHrP secretion is stimulated by CT and inhibited by CgA peptides. Endocrinology 1251563-565 34. Deftos LJ, Hogue-Angeletti R, Chalberg C, Tu S 1990 A chromogranin A-derived peptide differentially regulates the secretion of calcitonin gene products. J Bone Miner Res 5:989-991
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1993 No 2
Aardal S, Helle KB 1992 The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels. Regul Pept 41:9-18 Drees BM, Rouse J, Johnson J, Hamilton JW 1991 Bovine parathyroid glands secrete a 26.kDa N-terminal fragment of chromograninA which inhibits parathyroid cell secretion. Endocrinology 129:3381-3387 Schagger H, von Jagow G 1987 Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368-379 Neuhoff V, Arold N, Taube D, Ehrhardt W 1988 Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255-262 Merrifield RB 1964 Solid phase peptide synthesis. I. Synthesis of a tetrapeptide. J Am Chem Sot 85:2149-2154 Fasciotto BH, Gorr S-U, Bourdeau AM, Cohn DV 1990 Autocrine regulation of parathyroid secretion: inhibition of secretion by chromogranin-A (secretory protein-I) and potentiation of secretion by chromogranin-A and pancreastatin antibodies. Endocrinology 127:1329-1335 Cohn DV, Morrissey JJ, Hamilton JW, Shofstall RE, Smardo FL, Chu LLH 1981 Isolation and partial characterization of secretory protein-I from bovine parathyroid glands. Biochemistry 20:41354140 Leduc R, Hendy GN, Seidah NG, Chretien M, Lazure C 1990 Fragmentation of bovine chromogranin A by plasma kallikrein. Life Sci 46:1427-1433 Jeng YJ, Townsend Jr CM, Nagasawa S, Chuo S, Kern K, Yanaihara N, Ferrar RS, Hill FL, Thompson JC, Greeley Jr GH 1991 Regulation of pancreastatin release from a human pancreatic carcinoid cell line in vitro. Endocrinology 128:220-225 Funakoshi A, Tateishi K, Tsuru M, Jimi A, Wakasugi H, Ikeda Y, Kono A 1990 Pancreastatin producing cell line from human pancreatic islet cell tumor. Biochem Biophys Res Commun 168:741746 Borglum Jensen TD, Holst JJ, Fahrenkrug J 1991 Characterization of immunoactive pancreastatin in porcine tissues. Stand J Clin Lab Invest 51:681-691 astenson CG, Efendic S, Holst JJ 1989 Pancreastatin-like immunoreactivity and insulin are released in parallel from the perfused porcine pancreas. Endocrinology 124:2986-2990 Drees BM, Hamilton JW 1992 Pancreastatin and bovine parathyroid cell secretion. Bone Miner 17:335-346 Hutton JC, Davidson HW, Grimaldi KA, Peshavaria M 1987 Biosynthesis of betagranin in pancreatic beta-cells, Identification of a chromogranin A-like precursor and its parallel processing with proinsulin. Biochem J 244:449-456 Hutton JC, Davidson HW, Peshavaria M 1987 Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20V kDa N-terminal fragment (betagranin)-by the concerted action of a Ca*+-dependent endooeotidase and carboxvueutidase H (EC il I 3.4.17.10). Bi&hem J 24434’57-464 Deftos LJ, Gazdar AF, Hogue-Angeletti R, Mullen PS, Burton DW 1990 Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells. Bone Miner 9:169-178 Simon J-P, Bader M-F, Aunis D 1989 Proteolytic processing of chromogranin A in cultured chromaffin cells. Biochim Biophys Acta 1051:123-130 Galindo E, Mendez M, Calvo S, Gonzalez-Garcia C, Cena V, Hubert P, Bader M-F, Aunis D 1992 Chromostatin receptors control calcium channel activity in adrenal chromaffin cells. J Biol Chem 267:407-412
