Physiol. Genomics Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 25: 470-479, 2006. First published March 22, 2006; doi:10.1152/physiolgenomics.00295.2005
1094-8341/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
25/3/470    most recent
00295.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Greenwood, T. A.
Right arrow Articles by O’Connor, D. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Greenwood, T. A.
Right arrow Articles by O’Connor, D. T.
Received 30 November 2005; accepted in final form 22 February 2006.
Physiological Genomics 25:470-479 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Pleiotropic effects of novel trans-acting loci influencing human sympathochromaffin secretion

Tiffany A. Greenwood 2,4, Fangwen Rao 1, Mats Stridsberg 7, Nitish R. Mahapatra 1, Manjula Mahata 1, Elizabeth O. Lillie 1, Sushil K. Mahata 1,6, Laurent Taupenot 1, Nicholas J. Schork 2,4,5 and Daniel T. O’Connor 1,3,5,6

1 Departments of Medicine
2 Psychiatry
3 Pharmacology
4 Polymorphism Research Laboratory
5 Center for Human Genetics and Genomics, University of California-San Diego, La Jolla
6 Veterans Affairs San Diego Healthcare System, San Diego, California
7 Department of Medical Sciences, Clinical Chemistry, University Hospital, Uppsala, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Family studies have suggested a genetic contribution to variation in blood pressure, but the genes responsible have thus far eluded identification. The use of intermediate phenotypes associated with hypertension, such as chromogranin plasma concentrations, may assist the discovery of hypertension-predisposing loci. We measured the concentrations of four chromogranin A (CHGA) and B (CHGB) peptides in 742 individuals from 235 nuclear families. The CHGA- and CHGB-derived peptides displayed significant heritability and revealed significant genetic correlations, most strikingly observed between CHGA361–372 (catestatin) and CHGB439–451. A 5-cM microsatellite genome scan revealed significant and suggestive evidence for linkage on several chromosomes for three of the peptides. Subsequent bivariate linkage analysis for peptides CHGA361–372 and CHGB439–451, which showed evidence for convergent linkage peaks on chromosomes 2, 7, and 13, resulted in increased evidence for linkage to these regions, suggesting pleiotropic effects of these three loci on multiple chromogranin traits. Because CHGA itself is on chromosome 14q32, and CHGB itself is on chromosome 20pter-p12, the pleiotropic regions on chromosomes 2, 7, and 13 must represent trans-acting quantitative trait loci coordinately affecting CHGA/CHGB biosynthesis and/or exocytotic secretion, likely by regulating efferent sympathetic outflow, a conclusion consistent with the in vitro studies presented here of the dual control of both exocytosis and transcription of these peptides by secretory stimuli in chromaffin cells. The results suggest a new approach to heritable autonomic control of circulation and the genetic basis of cardiovascular diseases such as systemic hypertension.

exocytosis; chromaffin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HYPERTENSION IS A polygenic, heterogeneous disorder with a substantial environmental component. Although investigations into the genetic determinants of variation in blood pressure have found heritabilities ranging from ~30% to ~50% (72), the vast majority of genetic loci responsible for this variation have thus far remained elusive. The use of biochemical or physiological traits that represent intermediate pathogenic steps in the development of hypertension, so-called "intermediate phenotypes," may aid in the discovery of hypertension-predisposing loci (27, 42, 43, 62). Because such traits are more likely to be influenced by the action of a single gene, they should be less prone to environmental influences that confound the genetic dissection of a complex trait such as hypertension.

Genes in a wide variety of biochemical pathways have been implicated in blood pressure control that are likely to influence a susceptibility to hypertension (42). Of these pathways, those influencing the release of catecholamines may be some of the most important, because the release of catecholamines into the circulation is elevated not only in patients with essential hypertension but also in their normotensive first-degree relatives (16). Catecholamine release is controlled, in part, by members of the chromogranin family of proteins (32, 34, 69), which makes this family of proteins logical candidates for the exploration of sympathoadrenal activity in essential hypertension. The chromogranins comprise a family of acidic, soluble proteins that are stored in dense-core secretory granules throughout the endocrine and nervous systems and are coreleased by exocytosis with hormones, neurotransmitters, and neuropeptides (69). Chromogranin A (CHGA) was first isolated from chromaffin cells of the adrenal medulla (6, 24), whereas chromogranin B (CHGB) was initially characterized in a rat pheochromocytoma cell line (30). Human CHGA and CHGB loci have been mapped to chromosomes 14q32 and 20pter-p12 (33).

Several members of the chromogranin family undergo aggregation induced by low-pH and high-calcium levels. CHGA and CHGB form homodimeric complexes at pH 7.5, with progression to tetrameric stoichiometry at a secretory granule interior pH of 5.5 (80). CHGB also binds calcium at high capacity (93 mol calcium/mol CHGB at pH 5.5) within secretory granules (80). Chromogranins have been found to interact with other components of the matrix of the secretory granule, such as catecholamines, serotonin, and histamine, suggesting that they contribute to the formation of secretory granules (8, 21, 25, 73, 78). CHGA, in particular, appears to be crucial for the formation of secretory granules and sequestration of hormones in neuroendocrine cells (28, 32). Within chromaffin cells and sympathetic axons, CHGB may play a role in the sorting and trafficking of peptide hormone and neuropeptide precursors to secretory granules as well as an important role in granulogenesis (39). In addition, an amino-terminal domain of CHGB binds to the secretory vesicle membrane (79).

Chromogranins also have extracellular roles in the neuroendocrine system that are dependent on their functions as prohormones. Extensive posttranslational proteolytic processing at dibasic cleavage sites of CHGA and CHGB form biologically active peptides that have autocrine, paracrine, and endocrine activities. In humans, these granins are cleaved to produce a series of peptides: CHGA to vasostatins I and II [CHGA1–76 and CHGA1–115 (1)], pancreastatin [CHGA250–301 (44, 65)], catestatin [CHGA352–372 (34, 54)], and parastatin [CHGA357–428(14)]; and CHGB to GAWK [CHGB420–493 (9)], CCB [CHGB597–656 (9)], and PE-11 [CHGB536–545 (29)]. Granins also produce the endogenous antimicrobial/bacteriolytic peptides chromacin (CHGA176–197), chrombacin [CHGB597–657 (38)], and secretolytin [CHGB647–657 (60)], which may play a role in the neuroendocrine/sympathoadrenal stress response to systemic infection, perhaps providing innate immunity (69).

A role for CHGA in hypertension has been proposed, based on the tight coupling of the action of catecholamine on blood pressure and the CHGA-regulated formation of dense-core granules (71, 75). CHGA is overexpressed in catecholaminergic cells in rodent models of genetic [spontaneously hypertensive rat (41, 48)] and acquired [renovascular (60, 62)] hypertension, suggesting augmented sympathoadrenal activity in the pathogenesis of these syndromes. Genetic ablation of the CHGA locus in the mouse results in dysregulated, elevated sympathochromaffin secretion and severe hypertension (32). Thus, to identify candidate regions of the genome likely harboring loci contributing to hypertension susceptibility, we first assessed the genetic contribution to quantitative CHGA and CHGB expression by measuring the heritability of several circulating fragments. We then performed linkage analysis with genomic DNA markers at an ~5-cM density in 235 families. Finally, we coupled our heritability and linkage results with our in vitro studies of catecholamine and chromogranin release.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sample.
A sample of 742 individuals, constituting 235 nuclear families with 405 possible sibling pairs and 968 parent-offspring pairs, was recruited from southern California. The protocol was approved by the University of California-San Diego Institutional Review Board, and each subject gave written informed consent. The average family consisted of four individuals in two generations; 149 of these families were collected as part of a twin sample collection (81), 91 of which contained a self-reported monozygotic twin pair and 58 of which contained a self-reported dizygotic twin pair. When available, parents and additional siblings were also collected to form nuclear families. For these analyses, one twin from each monozygotic twin pair was removed before the genotyping, whereas all dizygotic twin pairs remained in the sample. After the genotyping, the zygosity of all self-reported dizygotic twin pairs was confirmed before sib-pair linkage analyses. The remaining 86 families in the sample were collected as nuclear families. The number of possible sibling pairs that could be formed per nuclear family ranged from 1 to 36 [given by (n) x (n – 1)/2]. Ethnicity was defined by self-identification of the ethnicity of the subject’s four grandparents. Of these families, 160 were Caucasian (of European ancestry), 22 were Hispanic (Mexican-American), 21 were African-American, and 32 were of other ethnicities or of mixed ancestry. The ages of the offspring in the sample ranged from 14 to 78 yr with a mean of 37.5 yr. One hundred twenty-five families (~53%) had a positive family history for hypertension (in a first-degree relative before the age of 60 yr), 82 families had a negative family history, and 28 families had an indeterminate/unknown family history (43, 44); 545 individuals were normotensive and 88 individuals were hypertensive with 109 individuals, primarily parents, having missing data. None of the subjects had a history of renal failure.

Genotyping.
Microsatellite genotyping of all individuals was performed by the National Heart, Lung, and Blood Institute Mammalian Genotyping Service at the Center for Medical Genetics, Marshfield Clinic, Marshfield, WI. Microsatellite markers were from screening sets 13 and 52. Genetic map positions for the 730 autosomal markers, which had an average intermarker distance of ~5 cM, were also provided by the Marshfield Clinic (http://research.marshfieldclinic.org/genetics). The genotype data were curated and Mendelian errors were identified using the Pedstats module of Merlin (3). The program Graphical Representation of Relationships (GRR) was used to confirm family relationships and twin zygosity (2). Among the 742 individuals genotyped, there was a 9.8% missing genotype data rate and 1.9% apparent genotyping error rate as identified by Mendelian inconsistencies in the genotypes.

Biochemical phenotyping.
EDTA-anticoagulated plasma was obtained from each subject and stored at –70°C before being assayed. Chromogranin region-specific radioimmunoassays based on synthetic peptides were performed as previously described (55–59). 125I radiolabeling of each peptide was enabled by either an endogenous or adventitious (terminal) tyrosine residue. Polyclonal rabbit antisera were developed to the synthetic chromogranin regions as previously described (45, 58, 59).

Statistical analyses.
Because variance-components approaches are not robust to departures from multivariate normality, we attempted to correct this to the extent possible for our phenotypes (5). The values for CHGB312–331 and CHGB439–451 were multiplied by 10 to increase the SD above 0.5, as suggested in the documentation for sequential oligogenic linkage analysis routines (SOLAR) (4). The trait values for CHGA116–439 were all log transformed and multiplied by 10 to reduce the kurtosis below 0.8 and increase the standard deviation above 0.5. Only values for the transformed phenotypes are reported. The values for CHGA361–372 were relatively normally distributed and thus remained unchanged.

Heritability (hr2) estimates were obtained via the variance component methodology implemented in the SOLAR version 2.1.2 linkage analysis package (4). This maximum likelihood method assumes a multivariate normal distribution of phenotypes in a pedigree and can accommodate a defined set of covariates (49, 50). The null hypothesis of no heritability (Formula) is tested by comparing a "full" model, which assumes that some fraction of the phenotypic variation is explained by genetic factors, with a "reduced" model, which assumes that no variation is explained by genes, using likelihood ratio tests. Covariates (age and gender) that were significant in the heritability analysis were retained and considered in the linkage analyses.

Two-point and multipoint quantitative trait linkage analyses of the microsatellite marker data were conducted using the SOLAR package, which employs a variance-component-based algorithm. Missing genotypes are imputed and assessed probabilistically by conditioning on all other linked marker data and pedigree structure, and the proportion of marker alleles shared identical by descent among all relative pairs is estimated independently for all autosomal markers. Linkage is assessed by fitting a polygenic model that does not incorporate genotype information provided by marker loci and comparing it with models that incorporate genotype data either with a specific marker (two-point analysis) or with multiple markers (multipoint). The log (base 10) of the ratio of the likelihoods of the marker-specific and polygenic models is the log of the odds (LOD) score, a traditional measure of genetic linkage. Because there are many different methodologies that can be used to assess linkage for quantitative traits, we also took advantage of the regression-based method implemented in the software Merlin-regress of the Merlin linkage analysis package (3, 53). We felt it necessary to incorporate both SOLAR and Merlin because there are advantages and disadvantages to each method. For example, Merlin-regress has been shown to be more robust to issues involving incomplete marker informativity (10, 17, 51, 52).

Bivariate genetic ({rho}G) and environmental ({rho}E) correlations were computed for pairs of phenotypes using SOLAR. {rho}G represents the genetic correlation [the cross-product of the h2 values for any two phenotypes or the coefficient of shared genetic determination, a measure of pleiotropy], whereas {rho}E is the environmental correlation (13). We tested two hypotheses for genetic correlations: whether there was evidence for pleiotropy ({rho}G significantly different from 0) and whether or not the pleiotropy was complete ({rho}G significantly different from 1). When convergent linkages were observed for single traits, bivariate linkage analyses were also run for those traits in combination, within SOLAR.

CHGA and CHGB secretion assays.
PC12 cells grown in 10-cm plates were incubated (in duplicates) with mock buffer versus the secretagogues nicotine (Sigma, 60 µM), pituitary adenylate cyclase-activating polypeptide (PACAP; Calbiochem, 0.2 µM), or BaCl2 (2 mM) in 3 ml release buffer [150 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM HEPES (pH 7); in the case of stimulation by BaCl2, the release buffer did not contain CaCl2] for 30 min.

For studies of CHGA and CHGB secretion, the release medium was collected and centrifuged at 1,000 rpm for 5 min to remove any suspended cells, the supernatant was transferred into a polypropylene tube, and an equal volume (3 ml) of 1% trifluroacetic acid (TFA; Sigma) was added. The mixture was loaded onto an equilibrated (by washing with 1 ml of 100% acetonitrile once, followed by 3 ml of 1% TFA three times) C-18 Sep column (Peninsula Laboratories; San Carlos, CA). After the column was washed with 3 ml of 1% TFA three times, proteins were eluted with 3 ml of 60% acetonitrile-1% TFA into a polypropylene tube. The eluant was frozen in dry ice-ethanol bath and evaporated to dryness by a lyophilizer. The lyophilized sample was dissolved in 100 µl of SDS-PAGE sample buffer (Invitrogen), 15 µl of each sample were run on a 10% polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane for immunoblot detection of CHGA and CHGB. The membrane was blocked in Tris-buffered saline (TBS) containing 5% nonfat milk and 0.1% Tween 20 for 1 h at room temperature, followed by incubation with a goat polyclonal antibody (Santa Cruz Biotechnology, 1:300 dilution) raised against the COOH-terminal 20 amino acids of human CHGA overnight at 4°C. After being washed, the membrane was incubated with a donkey anti-goat IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, 1:5,000 dilution) for 1 h, followed by chemiluminescent detection of CHGA bands by Supersignal West Pico reagent (Pierce). The membrane was stripped, and CHGB bands were detected using a goat polyclonal antibody (Santa Cruz Biotechnology, 1:300 dilution) raised against the COOH-terminal 19 amino acids of human CHGB, following the same method as CHGA. The relative intensities of the bands were measured using NIH ImageJ (version 1.31) software, and the stimulated secretion values were expressed with respect to mock secretion of CHGA and CHGB.

As a control for this chromogranin secretion experiment, the same batch of PC12 cells was subjected to [3H]norepinephrine secretion assays as described previously (38, 69, 70) using the same stimuli for the same period of time (30 min).

CHGA and CHGB transcription assays.
To probe the regulation of CHGA and CHGB biosynthesis, we used a transfected chromogranin promoter/luciferase reporter system, as previously described (30, 63, 64, 66, 77). We expressed promoter/reporter plasmids with a 1,133-bp mouse CHGA promoter (77) or a 1,303-bp mouse CHGB promoter (33) in PC12 cells in the presence or absence of secretory stimulation by three chromaffin cell secretogogues: the nicotinic cholinergic agonist nicotine (63, 66) and the PAC1 G protein-coupled receptor (GPCR) agonist PACAP (22, 63, 64, 68). After 48 h, cell lysates were harvested for assays of luciferase reporter activity and cellular protein content, as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Heritability.
Table 1 reports the descriptive statistics and heritability estimates for each phenotype. All biochemical phenotypes were relatively normally distributed after transformation. Heritability estimates for all CHGA and CHGB fragments were highly significant (P < 0.0001) and ranged from 0.45 to 0.91. Age was a significant covariate for CHGA116–439 and CHGB439–451, whereas gender was a significant covariate for CHGA361–372 and CHGB439–451.


View this table:
[in this window]
[in a new window]
  		Table 1. Descriptive statistics and heritability estimates of the phenotypes employed in the analyses

 
Genetic and environmental correlations.
Analysis of {rho}G and {rho}E between these fragments revealed significant {rho}G values between CHGA116–439 and CHGB439–451, CHGA361–372 and CHGB312–331, and CHGA361–372 and CHGB439–451,as shown in Table 2. Significant {rho}E values were observed between CHGA116–439 and CHGA361–372, CHGA116–439 and CHGB439–451, and CHGB439–451 and SCG2154–165.


View this table:
[in this window]
[in a new window]
  		Table 2. {rho}E and {rho}G and genetic values observed between all peptides

 
Univariate linkage analyses.
The linkage results from the genome-wide scan are summarized in Table 3. Only those phenotypes for which a multipoint LOD > 1.5 was observed using SOLAR and confirmed using Merlin are reported. Linkage peaks were observed for CHGA361–372 on chromosomes 1, 2, 4, 5, 7, 13, and 17; for CHGB312–331 on chromosome 7; and for CHGB439–451 on chromosomes 2, 7, and 13. No consistent linkage peaks were observed for CHGA116–439 or SCG2154–165 alone.


View this table:
[in this window]
[in a new window]
  		Table 3. Summary of multipoint LOD scores >1.5 obtained using SOLAR and confirmed using Merlin and the highest 2-point LOD score observed in the region

 
Of the LOD peaks observed for CHGA361–372 and CHGB439–452, four peaks were significant at the genome-wide level using SOLAR. Because these two chromogranin fragments had very high heritabilities, and sib-pair linkage power varies directly with trait heritability (Ref. 48; http://statgen.iop.kcl.ac.uk/gpc/), the higher resolution of the CHGA361–372 and CHGB439–452 LOD peaks in these linkage analyses is perhaps not unexpected. However, only one of these linkage peaks retained statistical significance at the genome-wide level using Merlin-regress.

Bivariate (pleiotropic) linkage analyses.
CgA361–372 and CgB439–451 mapped to approximately the same regions on chromosomes 2 and 13. Because these two phenotypes were also found to be genetically correlated (Table 2), we conducted bivariate linkage analysis for these phenotypes using SOLAR in an effort to improve evidence for linkage and increase the resolution. The results of the univariate and bivariate analyses are shown in Fig. 1 and Table 4. Bivariate linkage analysis of CgA361–372 and CgB439–451 on chromosome 2 resulted in a maximum LOD score of 3.97 at 18 cM. The maximum LOD scores for CgA361–372 and for CgB439–451 alone were 1.68 at 16 cM and 3.10 at 20 cM, respectively. Bivariate linkage analysis of CgA361–372 and CgB439–451 on chromosome 13 resulted in a maximum LOD score of 4.61 at 74 cM. The maximum LOD scores for CgA361–372 and CgB439–451 alone were 2.51 at 74 cM and 4.13 at 72 cM, respectively.


Figure 1
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Sequential oligogenic linkage analysis routines (SOLAR) multipoint linkage results for chromosomes 2 (A) and 13 (B). Results for chromogranin A (CHGA361–372) are indicated by a thin solid line, and results for chromogranin B (CHGB439–451) are indicated by a dotted line. The bivariate results for CHGA361–372 and CHGB439–451 are represented by a thick solid line. LOD, log of the odds score.

 

View this table:
[in this window]
[in a new window]
  		Table 4. Summary of bivariate (two-trait) linkage results and comparison with individual trait multipoint results

 
CgA361–372, CgB312–331, and CgB439–451 all mapped to nearby regions on chromosome 7. The maximum LOD scores for CgA361–372, CgB312–331, and CgB439–451 alone were 2.13 at 76 cM, 2.74 at 83 cM, and 1.81 at 103 cM, respectively. Because significant genetic correlations were observed between CgA361–372 and CgB312–331 and between CgA361–372 and CgB439–451, bivariate linkage analyses were performed for these phenotypes. The results of the univariate and bivariate analyses are shown in Fig. 2. Bivariate linkage analysis of CgA361–372 and CgB312–331 resulted in a maximum LOD score of 3.49 at 77 cM. Analysis of CgA361–372 and CgB439–451 resulted in a maximum LOD score of 3.08 at 74 cM. Although the {rho}G value between CgB312–331 and CgB439–451 did not reach statistical significance in this sample using SOLAR, bivariate linkage of these two phenotypes resulted in a maximum LOD score of 3.39 at 74 cM.


Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. SOLAR multipoint linkage results for chromosome 7. A: results for CHGA361–372 (thin solid line), CHGB312–331 (dashed line), and bivariate CHGA361–372 and CHGB312–331 (thick solid line). B: results for CHGA361–372 (thin solid line), CHGB439–451 (dotted line), and bivariate CHGA361–372 and CHGB439–451 (thick solid line). C: results for CHGB312–331 (dashed line), CHGB439–451 (dotted line), and bivariate CHGB312–331 and CHGB439–451 (thick solid line).

 
Coordinate control of secretion and transcription of CHGA and CHGB.
To test whether chromaffin cell stimuli such as nicotine, PACAP, and BaCl2 activate secretion of CHGA and CHGB simultaneously, we treated PC12 chromaffin cells with these secretagogues (vs. mock stimulation) and analyzed the release media by immunoblot analysis. As shown in Fig. 3,Go nicotine (acting at the physiological control point for secretion, the nicotinic cholinergic receptor) stimulated secretion of CHGA and CHGB by 3.37 ± 0.17- and 17.46 ± 0.59-fold, respectively; PACAP (acting on its PAC1 GPCR) activated secretion of CHGA and CHGB by 1.57 ± 0.08- and 4.89 ± 0.16-fold, respectively; and Ba2+ (blocking cell surface K+ channels and thereby depolarizing the cell membrane) triggered secretion of CHGA and CHGB by 4.55 ± 0.26- and 17.59 ± 0.51-fold, respectively. Thus the rank order of efficacy of secretagogues to activate exocytotic corelease of both CHGA and CHGB was Ba2+ > nicotine > PACAP. This observation is consistent with the rank order of their effects on norepinephrine secretion. Thus several chromaffin cell secretagogues functioning through very different upstream targets and signaling pathways (see Fig. 5) coordinately stimulate cosecretion of catecholamines as well as the peptides CHGA and CHGB.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3. Simultaneous activation of CHGA (CgA), CHGB (CgB), and catecholamine release from PC12 cells by chromaffin cell secretory stimuli. A: immunoblot detection of CHGA and CHGB in the release buffer from PC12 cells treated with mock versus nicotine (60 µM), pituitary adenylate cyclase-activating polypeptide (PACAP; 0.2 µM), or BaCl2 (2 mM). B: comparison of endogenous CHGA and CHGB secretion with [3H]norepinephrine (NE) release from PC12 cells after stimulation by chromaffin cell secretagogues. Results are shown as means ± SE for triplicate or quadruplicate determinations.

 

Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4. Transcription of transfected CHGA and CHGB promoter/luciferase reporter plasmids in PC12 cells. Results are shown for a 1,133-bp mouse CHGA promoter and 1,303-bp mouse CHGB promoter. Plasmids were transfected, whereupon the cells were treated with the indicated secretogogues (vs. mock stimulation) and harvested at 48 h for measurement of the luciferase reporter in cell lysates. Results were normalized to micrograms of cell protein. RLU, relative light units. Results are shown as means ± SE for triplicate or quadruplicate determinations.

 

Figure 5
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5. Pleiotropy: potential pathways, mechanisms, and candidates. Coordinate control of CHGA and CHGB biosynthesis and exocytotic secretion in chromaffin cells is illustrated in response to the physiological secretory stimuli acetylcholine (ACh) and PACAP. Points of shared physiological control suggest gene products whose encoding loci can be examined for polymorphism to explain the observed pleiotropy. nAChR, nicotinic ACh receptor; ER, endoplasmic reticulum; PAC1R, PACAP PAC1 receptor.

 
Using transfected promoter/reporter plasmids, we tested whether the biosynthesis of CHGA and CHGB was similarly controlled by secretory stimulation. As shown in Fig. 4, both PACAP and nicotine stimulated transcription of both CHGA and CHGB, and, in each case, the rank order of potency was PACAP > nicotine. This rank order contrasts with the effects of the same stimuli on secretion, in which nicotine > PACAP (Fig. 3). It is apparent that both PACAP and nicotine stimulate transcription of both CHGA and CHGB, but PACAP is a more powerful trigger to transcription (of either CHGA or CHGB) than is nicotine.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Overview.
These data provide evidence that circulating peptides derived from members of the chromogranin family display high levels of heritability (up to ~91%, CHGB439–451; Table 1), and their concentrations reveal significant {rho}G values as well as genetic linkage to markers on several chromosomes. Evidence for pleiotropy was first suggested by analysis of the {rho}G for chromogranin peptides in our sample (Table 2). In particular, CHGA361–372 and CHGB439–451 showed especially significant evidence for {rho}G. Because these peptides also gave evidence for linkage to the same regions on chromosomes 2, 7, and 13, we performed bivariate linkage analysis on composite traits formed from these two peptides, which yielded higher LOD scores than linkage for either of the individual traits considered alone (Table 4 and Figs. 1 and 2). Because the genes encoding CHGA and CHGB reside on chromosomes 14q32 and 20pter-p12, respectively (34), these pleiotropic regions must represent trans-quantitative trait loci (QTLs) affecting CHGA/CHGB biosynthesis or secretion, likely by regulating efferent sympathetic outflow (Fig. 5).

Genetic complexity.
Contrary to expectation, we did not observe cis-QTLs for CHGA/CHGB peptide expression at the CHGA/CHGB loci themselves. We (74) have already studied polymorphism at the human CHGA locus and documented the effect of promoter variants on CHGA release into the bloodstream. However, the relative power of linkage (effect of a locus on a trait during meiotic cosegregation) versus association (effect of a particular allele on a trait in a population) may be quite different, depending on the degree of trait-determining locus heterogeneity (across the genome) versus allelic heterogeneity (at a locus); indeed, association may be considerably more powerful than linkage in many circumstances (47). However, linkage (the approach in this study) does offer the advantage of genome-wide searching with a relatively small number of markers (typically ~400–800 microsatellites).

While significant, the {rho}G values between CHGA and CHGB fragments in plasma (e.g., CHGA116–439 with CHGB439–451, {rho}G = 0.531, P < 0.01) were less than complete (i.e., substantially <1). This is, perhaps, not unexpected considering that after initial synthesis, CHGA and CHGB are subject to further posttranslational modification in the form of proteolytic cleavage (7, 12, 21) to generate the fragments recognized by immunoassays, and such cleavage varies among the chromogranins from tissue site to site (7, 21).

While {rho}G values for fragment concentrations between CHGA/CHGB peptide pairs were typically significant (e.g., CHGA116–439 with CHGB439–451, {rho}G = 0.531, P < 0.01), {rho}G values for peptide pairs within CHGA (CHGA116–439 with CHGA361–372, {rho}G = 0.097) or within CHGB (CHGB313–331 with CHGB439–451, {rho}G = 0.172) were typically not significant (Table 2). Apparently the relatively high interchromogranin {rho}G values reflect coordinate/shared genetic control of biosynthesis/release, whereas the relatively low intrachromogranin {rho}G values reflect the influence of other factors on each trait, such as shared environment (e.g., CHGA116–439 with CHGA361–372, {rho}E = –0.649, P < 0.01). Indeed, the negative sign on this {rho}E value is consistent with the typical inverse relationship seen between the plasma concentrations of CHGA116–439 and CHGA361–372 (43, 57), likely representing interindividual differences in the degree of proteolytic processing of the CHGA precursor into the catestatin product (12, 70).

Potential cellular mechanisms of pleiotropy.
What might be the mechanism of such shared genetic determination (or {rho}G)? We found that CHGA, CHGB, and catecholamines were coreleased by exocytotic secretory stimuli of chromaffin cells with similar rank orders of secretagogue efficacy (Ba2+ > nicotine > PACAP; Fig. 4). CHGA and CHGB also displayed shared transcriptional control in chromaffin cells, once again with similar rank orders of stimulus efficacy (this time: PACAP > nicotine; Fig. 4). Transcriptional activation by secretory stimuli illustrates the concept of "stimulus-transcription coupling" (or "stimulus-secretion-synthesis coupling"), a process whereby the secretory cell replenishes the just-released transmitters that we have described for the individual members of the chromogranin family (31, 35, 63, 64). Figure 5 synthesizes these results and illustrates potential control points for coordinate control of CHGA and CHGB biosynthesis and secretion, which, in turn, might suggest candidate processes or genetic loci for pleiotropy. For example, a QTL influencing efferent preganglionic sympathetic tone to sympathochromaffin cells would be expected to perturb both synthesis and secretion of CHGA and CHGB. Alterations in the elements of the intracellular signal transduction pathways whereby chromaffin cell secretogogues (acetylcholine and PACAP) activate either transmitter exocytosis or resynthesis would also represent logical possible sites for a pleiotropic QTL. Our pleiotropic human linkage results (Figs. 1 and 2 and Table 4) do not allow us to distinguish among the multiple possible mechanisms underlying such pleiotropy, nor to distinguish whether the mechanism(s) governing trans-QTL-mediated cosecretion of CHGA and CHGB is predominantly at the level of exocytosis or transcription (or both). Of note, elevated sympathetic tone is frequently described in human hypertension as well as the first-degree relatives of patients with the syndrome (42, 62). In rodent genetic models of hypertension, sympathetic tone and chromogranin expression are also deranged (18, 19, 41), and an early increase in sympathetic activity may cosegregate with later increased blood pressure in rat genetic hypertension (27).

CHGA361–372 is representative of the catestatin fragment of CHGA, which inhibits the release of catecholamines from sympathoadrenal chromaffin cells by blocking the neuronal nicotinic receptor, the physiological trigger for secretion (34, 54, 68). Catestatin also prevents the desensitization of catecholamine release from chromaffin cells that is induced by repeated nicotinic agonist stimulation (35). Catestatin may thus contribute to an autocrine negative-feedback mechanism that regulates catecholamine release within the sympathoadrenal system. Because excess sympathetic activity has been implicated in the development of hypertension (42), a disturbance of the catestatin mechanism may be a contributing factor. Indeed, recent studies have shown a decrease in plasma catestatin levels in patients with hypertension as well as in normotensive individuals with genetic risk for hypertension (43), and catestatin replacement "rescues" the elevated blood pressure in the hypertensive mouse CHGA targeted ablation model (32). Although catestatin levels were reduced in these individuals, plasma CHGA levels and catecholamine excretion were augmented, suggesting a possible processing defect of CHGA (40, 61). Thus a novel QTL influencing catestatin production could have implications for autonomic control of the circulation and hence for the development of hypertension.

Positional candidate genetic loci.
An examination of the regions beneath the CHGA361–372 and CHGB439–451 bivariate linkage peaks on chromosomes 2, 7, and 13 provides evidence for positional candidate genes that might influence exocytosis, based on neural expression or involvement in secretion or receptor signal transduction. The 1-LOD interval within the linkage region on chromosome 2 (2p25.2–25.1, 14–24 cM, ~6.7–10.6 Mb) contained 17 genes, one of which (YWHAQ, 14-3-3 protein {tau}) is an activator of the rate-limiting step in catecholamine biosynthesis (tyrosine hydroxylase). The 1-LOD interval beneath the peak on chromosome 7 (7p13–11.22, 66–85 cM, ~44–70 Mb) contained 74 genes, 6 of which are potential candidate genes: soluble NSF attachment receptor protein Ykt6 (a potential mediator of exocytosis), calcium/calmodulin-dependent protein kinase type II ß-chain, DOPA decarboxylase (in the catecholamine biosynthetic pathway), calcitonin gene-related peptide receptor component protein, potassium channel tetramerization domain containing 7, and calneuron 1 (a calcium-binding protein). There were 20 genes located beneath the 1-LOD interval under the linkage peak on chromosome 13 (13q31.2–32.3, 68–80 cM, ~88–99 Mb), 2 of which suggested candidate gene status: probable GPCR 80 and Ras-related protein Rap-2a. Feitosa et al. (15) also found linkage of chromosomes 7 and 13 to body mass index.

This is the initial report on genome-wide linkage for multiple chromogranin fragments. Our previous CHGB312–331 linkage scan in the UGRP/CEPH extended pedigrees (23) found a LOD peak on chromosome 11, which was not replicated in this sample. However, neither of these two clinical samples was randomly sampled from the population, and the two samples differed in ascertainment criteria. The UGRP/CEPH pedigrees were collected based on the availability of very large sibships (typically 6–12 siblings), coupled initially with the availability of both parents and all four grandparents (11, 22, 23, 37, 76). Such criteria are likely to select for healthy families with considerable longevity and against chronic disease with associated premature mortality. In contrast, our family sample was ascertained in the setting of a blood pressure study (81) with ~16% of the subjects already diagnosed with hypertension and another ~36% at genetic risk of developing hypertension by virtue of positive family history. Thus the two different samples (Utah and San Diego) likely differ substantially in genetic predisposition toward cardiovascular disease and hence autonomic function (42).

Perspectives.
In summary, these studies provide evidence for coordinate genetic control of exocytosis of multiple sympathochromaffin cell cotransmitters. Figure 5 summarizes our findings and proposes a model for neuronal and endocrine cellular events, both secretory and transcriptional, likely to give rise to our observations. These results have implications for heritable autonomic control of the circulation and the genetic basis of cardiovascular diseases such as systemic hypertension.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the Department of Veterans Affairs and National Institutes of Health.


   ACKNOWLEDGMENTS
 
We appreciate the technical assistance of Payam Mahboubi and Guangfa Zhang. Phenotyping was conducted in the University of California-San Diego National Institutes of Health (NIH)-sponsored General Clinical Research Center (NIH Grant RR-00827). Microsatellite genotyping was performed by the NIH Mammalian Genotyping Service at the Center for Medical Genetics, Marshfield Clinic, Marshfield, WI (Dr. James L. Weber).


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. T. O’Connor or N. J. Schork, Dept. of Medicine and Center for Human Genetics and Genomics, Univ. of California-San Diego, and Veterans Affairs San Diego Healthcare System (0838), 9500 Gilman Dr., La Jolla, CA 92093-0838 (e-mail: doconnor{at}ucsd.edu or nschork{at}ucsd.edu).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aardal S, Helle KB, Elsayed S, Reed RK, and Serck-Hanssen G. Vasostatins, comprising the N-terminal domain of chromogranin A, suppress tension in isolated human blood vessel segments. J Neuroendocrinol 5: 405–412, 1993.[CrossRef][Web of Science][Medline]
  2. Abecasis GR, Cherny SS, Cookson WO, and Cardon LR. GRR: graphical representation of relationship errors. Bioinformatics 17: 742–743, 2001.[Abstract/Free Full Text]
  3. Abecasis GR, Cherny SS, Cookson WO, and Cardon LR. Merlin–rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30: 97–101, 2002.[CrossRef][Web of Science][Medline]
  4. Almasy L and Blangero J. Multipoint quantitative-trait linkage analysis in general pedigrees. Am J Hum Genet 62: 1198–1211, 1998.[CrossRef][Web of Science][Medline]
  5. Amos CI. Robust variance-components approach for assessing genetic linkage in pedigrees. Am J Hum Genet 54: 535–543, 1994.[Web of Science][Medline]
  6. Banks P and Helle K. The release of protein from the stimulated adrenal medulla. Biochem J 97: 40C–41C, 1965.[Medline]
  7. Barbosa JA, Gill BM, Takiyyuddin MA, and O’Connor DT. Chromogranin A: posttranslational modifications in secretory granules. Endocrinology 128: 174–190, 1991.[Abstract/Free Full Text]
  8. Bargsten G and Grube D. Serotonin storage and chromogranins: an experimental study in rat gastric endocrine cells. J Histochem Cytochem 40: 1147–1155, 1992.[Abstract]
  9. Benjannet S, Leduc R, Adrouche N, Falgueyret JP, Marcinkiewicz M, Seidah NG, Mbikay M, Lazure C, and Chretien M. Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues. FEBS Lett 224: 142–148, 1987.[CrossRef][Web of Science][Medline]
  10. Cordell HJ. Bias toward the null hypothesis in model-free linkage analysis is highly dependent on the test statistic used. Am J Hum Genet 74: 1294–1302, 2004.[CrossRef][Web of Science][Medline]
  11. Drayna D, Coon H, Kim UK, Elsner T, Cromer K, Otterud B, Baird L, Peiffer AP, and Leppert M. Genetic analysis of a complex trait in the Utah Genetic Reference Project: a major locus for PTC taste ability on chromosome 7q and a secondary locus on chromosome 16p. Hum Genet 112: 567–572, 2003.[Web of Science][Medline]
  12. Eskeland NL, Zhou A, Dinh TQ, Wu H, Parmer RJ, Mains RE, and O’Connor DT. Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway. J Clin Invest 98: 148–156, 1996.[Web of Science][Medline]
  13. Falconer DS and Mackay TFC. Introduction to Quantitative Genetics. Essex, UK: Longman, 1996.
  14. Fasciotto BH, Trauss CA, Greeley GH, and Cohn DV. Parastatin (porcine chromogranin A347–419), a novel chromogranin A-derived peptide, inhibits parathyroid cell secretion. Endocrinology 133: 461–466, 1993.[Abstract/Free Full Text]
  15. Feitosa MF, Borecki IB, Rich SS, Arnett DK, Sholinsky P, Myers RH, Leppert M, and Province MA. Quantitative-trait loci influencing body-mass index reside on chromosomes 7 and 13: the National Heart, Lung, and Blood Institute Family Heart Study. Am J Hum Genet 70: 72–82, 2002.[CrossRef][Web of Science][Medline]
  16. Ferrier C, Cox H, and Esler M. Elevated total body noradrenaline spillover in normotensive members of hypertensive families. Clin Sci (Lond) 84: 225–230, 1993.[Medline]
  17. Franke D and Ziegler A. Weighting affected sib pairs by marker informativity. Am J Hum Genet 77: 230–241, 2005.[Medline]
  18. Fries RS, Mahboubi P, Mahapatra NR, Mahata SK, Schork NJ, Schmid-Schoenbein GW, and O’Connor DT. Neuroendocrine transcriptome in genetic hypertension: multiple changes in diverse adrenal physiological systems. Hypertension 43: 1301–1311, 2004.[Abstract/Free Full Text]
  19. Friese RS, Mahboubi P, Mahapatra NR, Mahata SK, Schork NJ, Schmid-Schonbein GW, and O’Connor DT. Common genetic mechanisms of blood pressure elevation in two independent rodent models of human essential hypertension. Am J Hypertens 18: 633–652, 2005.[CrossRef][Web of Science][Medline]
  20. Gill BM, Barbosa JA, Dinh TQ, Garrod S, and O’Connor DT. Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing. Regul Pept 33: 223–235, 1991.[CrossRef][Web of Science][Medline]
  21. Gorr SU, Shioi J, and Cohn DV. Interaction of calcium with porcine adrenal chromogranin A (secretory protein-I) and chromogranin B (secretogranin I). Am J Physiol Endocrinol Metab 257: E247–E254, 1989.[Abstract/Free Full Text]
  22. Greenwood TA, Cadman PE, Stridsberg M, Nguyen S, Taupenot L, Schork NJ, and O’Connor DT. Genome-wide linkage analysis of chromogranin B expression in the CEPH pedigrees: implications for exocytotic sympathochromaffin secretion in humans. Physiol Genomics 18: 119–127, 2004.[Abstract/Free Full Text]
  23. Hall MA, Norman PJ, Thiel B, Tiwari H, Peiffer A, Vaughan RW, Prescott S, Leppert M, Schork NJ, and Lanchbury JS. Quantitative trait loci on chromosomes 1, 2, 3, 4, 8, 9, 11, 12, and 18 control variation in levels of T and B lymphocyte subpopulations. Am J Hum Genet 70: 1172–1182, 2002.[CrossRef][Web of Science][Medline]
  24. Helle KB. Some chemical and physical properties of the soluble protein fraction of bovine adrenal chromaffin granules. Mol Pharmacol 2: 298–310, 1966.[Abstract/Free Full Text]
  25. Iacangelo AL and Eiden LE. Chromogranin A: current status as a precursor for bioactive peptides and a granulogenic/sorting factor in the regulated secretory pathway. Regul Pept 58: 65–88, 1995.[CrossRef][Web of Science][Medline]
  26. Judy WV, Watanabe AM, Murphy WR, Aprison BS, and Yu PL. Sympathetic nerve activity and blood pressure in normotensive backcross rats genetically related to the spontaneously hypertensive rat. Hypertension 1: 598–604, 1979.[Abstract/Free Full Text]
  27. Kailasam MT, O’Connor DT, and Parmer RJ. Hereditary intermediate phenotypes in African American hypertension. Ethn Health 1: 117–128, 1996.[Medline]
  28. Kim T, Tao-Cheng JH, Eiden LE, and Loh YP. Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis. Cell 106: 499–509, 2001.[CrossRef][Web of Science][Medline]
  29. Kroesen S, Marksteiner J, Leitner B, Hogue-Angeletti R, Fischer-Colbrie R, and Winkler H. Rat brain: distribution of immunoreactivity of PE-11, a peptide derived from chromogranin B. Eur J Neurosci 8: 2679–2689, 1996.[CrossRef][Web of Science][Medline]
  30. Lee RW and Huttner WB. Tyrosine-O-sulfated proteins of PC12 pheochromocytoma cells and their sulfation by a tyrosylprotein sulfotransferase. J Biol Chem 258: 11326–11334, 1983.[Abstract/Free Full Text]
  31. Mahapatra NR, Mahata M, Datta AK, Gerdes HH, Huttner WB, O’Connor DT, and Mahata SK. Neuroendocrine cell type-specific and inducible expression of the chromogranin B gene: crucial role of the proximal promoter. Endocrinology 141: 3668–3678, 2000.[Abstract/Free Full Text]
  32. Mahapatra NR, O’Connor DT, Vaingankar SM, Hikim AP, Mahata M, Ray S, Staite E, Wu H, Gu Y, Dalton N, Kennedy BP, Ziegler MG, Ross J, and Mahata SK. Hypertension from targeted ablation of chromogranin A can be rescued by the human ortholog. J Clin Invest 115: 1942–1952, 2005.[CrossRef][Web of Science][Medline]
  33. Mahata SK, Kozak CA, Szpirer J, Szpirer C, Modi WS, Gerdes HH, Huttner WB, and O’Connor DT. Dispersion of chromogranin/secretogranin secretory protein family loci in mammalian genomes. Genomics 33: 135–139, 1996.[CrossRef][Web of Science][Medline]
  34. Mahata SK, O’Connor DT, Mahata M, Yoo SH, Taupenot L, Wu H, Gill BM, and Parmer RJ. Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist. J Clin Invest 100: 1623–1633, 1997.[Web of Science][Medline]
  35. Mahata SK, Mahata M, Parmer RJ, and O’Connor DT. Desensitization of catecholamine release. The novel catecholamine release-inhibitory peptide catestatin (chromogranin a344–364) acts at the receptor to prevent nicotinic cholinergic tolerance. J Biol Chem 274: 2920–2928, 1999.[Abstract/Free Full Text]
  36. Mahata SK, Mahata M, Wen G, Wong WB, Mahapatra NR, Hamilton BA, and O’Connor DT. The catecholamine release-inhibitory "catestatin" fragment of chromogranin A: naturally occurring human variants with different potencies for multiple chromaffin cell nicotinic cholinergic responses. Mol Pharmacol 66: 1180–1191, 2004.[Abstract/Free Full Text]
  37. Malhotra A, Cromer K, Leppert MF, and Hasstedt SJ. The power to detect genetic linkage for quantitative traits in the Utah CEPH pedigrees. J Hum Genet 50: 69–75, 2005.[CrossRef][Web of Science][Medline]
  38. Metz-Boutigue MH, Goumon Y, Lugardon K, Strub JM, and Aunis D. Antibacterial peptides are present in chromaffin cell secretory granules. Cell Mol Neurobiol 18: 249–266, 1998.[CrossRef][Web of Science][Medline]
  39. Natori S and Huttner WB. Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor. Proc Natl Acad Sci USA 93: 4431–4436, 1996.[Abstract/Free Full Text]
  40. O’Connor DT. Plasma chromogranin A. Initial studies in human hypertension. Hypertension 7: I76–I79, 1985.[Web of Science][Medline]
  41. O’Connor DT, Takiyyuddin MA, Printz MP, Dinh TQ, Barbosa JA, Rozansky DJ, Mahata SK, Wu H, Kennedy BP, Ziegler MG, Wright FA, Schlager G, and Parmer RJ. Catecholamine storage vesicle protein expression in genetic hypertension. Blood Press 8: 285–295, 1999.[CrossRef][Web of Science][Medline]
  42. O’Connor DT, Insel PA, Ziegler MG, Hook VY, Smith DW, Hamilton BA, Taylor PW, and Parmer RJ. Heredity and the autonomic nervous system in human hypertension. Curr Hypertens Rep 2: 16–22, 2000.[Medline]
  43. O’Connor DT, Kailasam MT, Kennedy BP, Ziegler MG, Yanaihara N, and Parmer RJ. Early decline in the catecholamine release-inhibitory peptide catestatin in humans at genetic risk of hypertension. J Hypertens 20: 1335–1345, 2002.[CrossRef][Web of Science][Medline]
  44. O’Connor DT, Cadman PE, Smiley C, Salem RM, Rao F, Smith J, Funk SD, Mahata SK, Mahata M, Wen G, Taupenot L, Gonzalez-Yanes C, Harper KL, Henry RR, and Sanchez-Margalet V. Pancreastatin: multiple actions on human intermediary metabolism in vivo, variation in disease, and naturally occurring functional genetic polymorphism. J Clin Endocrinol Metab 90: 5414–5425, 2005.[Abstract/Free Full Text]
  45. Portela-Gomes GM and Stridsberg M. Region-specific antibodies against chromogranin B display different immunoreactivity in the human pancreatic islet cell types. Ann NY Acad Sci 971: 341–344, 2002.[Web of Science][Medline]
  46. Purcell S, Cherny SS, and Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 19: 149–150, 2003.[Abstract/Free Full Text]
  47. Risch N and Merikangas K. The future of genetic studies of complex human diseases. Science 273: 1516–1517, 1996.[Abstract/Free Full Text]
  48. Schober M, Howe PR, Sperk G, Fischer-Colbrie R, and Winkler H. An increased pool of secretory hormones and peptides in adrenal medulla of stroke-prone spontaneously hypertensive rats. Hypertension 13: 469–474, 1989.[Abstract/Free Full Text]
  49. Schork NJ. Extended multipoint identity-by-descent analysis of human quantitative traits: efficiency, power, and modeling considerations. Am J Hum Genet 53: 1306–1319, 1993.[Web of Science][Medline]
  50. Schork N. The design and use of variance component models in the analysis of human quantitative pedigree data. Biometrical J 35: 387–405, 1993.[CrossRef]
  51. Schork NJ and Greenwood TA. Inherent bias toward the null hypothesis in conventional multipoint nonparametric linkage analysis. Am J Hum Genet 74: 306–316, 2004.[CrossRef][Medline]
  52. Schork NJ and Greenwood TA. Got bias? The authors reply. Am J Hum Genet 75: 723–727, 2004.[CrossRef]
  53. Sham PC, Purcell S, Cherny SS, and Abecasis GR. Powerful regression-based quantitative-trait linkage analysis of general pedigrees. Am J Hum Genet 71: 238–253, 2002.[CrossRef][Web of Science][Medline]
  54. Simon JP, Bader MF, and Aunis D. Secretion from chromaffin cells is controlled by chromogranin A-derived peptides. Proc Natl Acad Sci USA 85: 1712–1716, 1988.[Abstract/Free Full Text]
  55. Stridsberg M, Oberg K, Li Q, Engstrom U, and Lundqvist G. Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours. J Endocrinol 144: 49–59, 1995.[Abstract/Free Full Text]
  56. Stridsberg M, Angeletti RH, and Helle KB. Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods. J Endocrinol 165: 703–714, 2000.[Abstract]
  57. Stridsberg M. Measurements of chromogranins and chromogranin-related peptides by immunological methods. Adv Exp Med Biol 482: 319–327, 2000.[Web of Science][Medline]
  58. Stridsberg M, Eriksson B, Oberg K, and Janson ET. A panel of 11 region-specific radioimmunoassays for measurements of human chromogranin A. Regul Pept 117: 219–227, 2004.[CrossRef][Web of Science][Medline]
  59. Stridsberg M, Eriksson B, Oberg K, and Janson ET. A panel of 13 region-specific radioimmunoassays for measurements of human chromogranin B. Regul Pept 125: 193–199, 2005.[Medline]
  60. Strub JM, Garcia-Sablone P, Lonning K, Taupenot L, Hubert P, Van Dorsselaer A, Aunis D, and Metz-Boutigue MH. Processing of chromogranin B in bovine adrenal medulla Identification of secretolytin, the endogenous C-terminal fragment of residues 614–626 with antibacterial activity. Eur J Biochem 229: 356–368, 1995.[Web of Science][Medline]
  61. Takiyyuddin MA, Cervenka JH, Hsiao RJ, Barbosa JA, Parmer RJ, and O’Connor Chromogranin A DT. Storage and release in hypertension. Hypertension 15: 237–246, 1990.[Abstract/Free Full Text]
  62. Takiyyuddin MA, Parmer RJ, Kailasam MT, Cervenka JH, Kennedy B, Ziegler MG, Lin MC, Li J, Grim CE, Wright FA, and O’Connor DT. Chromogranin A in human hypertension. Influence of heredity. Hypertension 26: 213–220, 1995.[Abstract/Free Full Text]
  63. Tang K, Wu H, Mahata SK, Taupenot L, Rozansky DJ, Parmer RJ, and O’Connor DT. Stimulus-transcription coupling in pheochromocytoma cells. Promoter region-specific activation of chromogranin A biosynthesis. J Biol Chem 271: 28382–28390, 1996.[Abstract/Free Full Text]
  64. Tang K, Wu H, Mahata SK, Mahata M, Gill BM, Parmer RJ, and O’Connor DT. Stimulus coupling to transcription versus secretion in pheochromocytoma cells. Convergent and divergent signal transduction pathways and the crucial roles for route of cytosolic calcium entry and protein kinase C. J Clin Invest 100: 1180–1192, 1997.[Web of Science][Medline]
  65. Tatemoto K, Efendic S, Mutt V, Makk G, Feistner GJ, and Barchas JD. Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion. Nature 324: 476–478, 1986.[CrossRef][Medline]
  66. Taupenot L, Mahata SK, Wu H, and O’Connor DT. Peptidergic activation of transcription and secretion in chromaffin cells. Cis and trans signaling determinants of pituitary adenylyl cyclase-activating polypeptide (PACAP). J Clin Invest 101: 863–876, 1998.[Web of Science][Medline]
  67. Taupenot L, Mahata M, Mahata SK, and O’Connor DT. Time-dependent effects of the neuropeptide PACAP on catecholamine secretion: stimulation and desensitization. Hypertension 34: 1152–1162, 1999.[Abstract/Free Full Text]
  68. Taupenot L, Mahata SK, Mahata M, Parmer RJ, and O’Connor DT. Interaction of the catecholamine release-inhibitory peptide catestatin [human chromogranin A(352–372)] with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism. Regul Pept 95: 9–17, 2000.[CrossRef][Web of Science][Medline]
  69. Taupenot L, Harper KL, and O’Connor DT. The chromogranin-secretogranin family. N Engl J Med 348: 1134–1149, 2003.[Free Full Text]
  70. Taylor CV, Taupenot L, Mahata SK, Mahata M, Wu H, Yasothornsrikul S, Toneff T, Caporale C, Jiang Q, Parmer RJ, Hook VY, O’Connor DT. Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A: determination of cleavage sites in hormone storage granules. J Biol Chem 275: 22905–22915, 2000.[Abstract/Free Full Text]
  71. Videen JS, Mezger MS, Chang YM, and O’Connor DT. Calcium and catecholamine interactions with adrenal chromogranins. Comparison of driving forces in binding and aggregation. J Biol Chem 267: 3066–3073, 1992.[Abstract/Free Full Text]
  72. Ward R. Familial aggregation and genetic epidemiology of blood pressure. In: Hypertension: Pathophysiology, Diagnosis, and Management, edited by Laragh JH and Brenner BM. New York: Raven, 1990, p. 81–100.
  73. Watkinson A and Dockray GJ. Functional control of chromogranin A and B concentrations in the body of the rat stomach. Regul Pept 40: 51–61, 1992.[CrossRef][Web of Science][Medline]
  74. Wen G, Mahata SK, Cadman P, Mahata M, Ghosh S, Mahapatra NR, Rao F, Stridsberg M, Smith DW, Mahboubi P, Schork NJ, O’Connor DT, and Hamilton BA. Both rare and common polymorphisms contribute functional variation at CHGA, a regulator of catecholamine physiology. Am J Hum Genet 74: 197–207, 2004.[CrossRef][Web of Science][Medline]
  75. Westermann R, Stogbauer F, Unsicker K, and Lietzke R. Calcium-dependence of chromogranin A-catecholamine interaction. FEBS Lett 239: 203–206, 1988.[CrossRef][Medline]
  76. White R, Leppert M, Bishop DT, Barker D, Berkowitz J, Brown C, Callahan P, Holm T, and Jerominski L. Construction of linkage maps with DNA markers for human chromosomes. Nature 313: 101–105, 1985.[CrossRef][Medline]
  77. Wu H, Rozansky DJ, Webster NJ, and O’Connor DT. Cell type-specific gene expression in the neuroendocrine system. A neuroendocrine-specific regulatory element in the promoter of chromogranin A, a ubiquitous secretory granule core protein. J Clin Invest 94: 118–129, 1994.[Web of Science][Medline]
  78. Yoo SH and Albanesi JP. High capacity, low affinity Ca2+ binding of chromogranin A. Relationship between the pH-induced conformational change and Ca2+ binding property. J Biol Chem 266: 7740–7745, 1991.[Abstract/Free Full Text]
  79. Yoo SH and Kang YK. Identification of the secretory vesicle membrane binding region of chromogranin B. FEBS Lett 406: 259–262, 1997.[CrossRef][Web of Science][Medline]
  80. Yoo SH, So SH, Huh YH, and Park HY. Inositol 1,4,5-trisphosphate receptor/Ca2+ channel modulatory role of chromogranins A and B. Ann NY Acad Sci 971: 300–310, 2002.[Web of Science][Medline]
  81. Zhang L, Rao F, Wessel J, Kennedy BP, Rana BK, Taupenot L, Lillie EO, Cockburn M, Schork NJ, Ziegler MG, and O’Connor DT. Functional allelic heterogeneity and pleiotropy of a repeat polymorphism in tyrosine hydroxylase: prediction of catecholamines and response to stress in twins. Physiol Genomics 19: 277–291, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J Am Coll CardiolHome page
Y. Chen, F. Rao, J. L. Rodriguez-Flores, M. Mahata, M. M. Fung, M. Stridsberg, S. M. Vaingankar, G. Wen, R. M. Salem, M. Das, et al.
Naturally Occurring Human Genetic Variation in the 3'-Untranslated Region of the Secretory Protein Chromogranin A Is Associated With Autonomic Blood Pressure Regulation and Hypertension in a Sex-Dependent Fashion
J. Am. Coll. Cardiol., October 28, 2008; 52(18): 1468 - 1481.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
P.-a. B. Shih and D. T. O'Connor
Hereditary Determinants of Human Hypertension: Strategies in the Setting of Genetic Complexity
Hypertension, June 1, 2008; 51(6): 1456 - 1464.
[Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
R. M. Salem, P. E. Cadman, Y. Chen, F. Rao, G. Wen, B. A. Hamilton, B. K. Rana, D. W. Smith, M. Stridsberg, H. J. Ward, et al.
Chromogranin A Polymorphisms Are Associated With Hypertensive Renal Disease
J. Am. Soc. Nephrol., March 1, 2008; 19(3): 600 - 614.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
F. Rao, J. Wessel, G. Wen, L. Zhang, B. K. Rana, B. P. Kennedy, T. A. Greenwood, R. M. Salem, Y. Chen, S. Khandrika, et al.
Renal Albumin Excretion: Twin Studies Identify Influences of Heredity, Environment, and Adrenergic Pathway Polymorphism
Hypertension, May 1, 2007; 49(5): 1015 - 1031.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
25/3/470    most recent
00295.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Greenwood, T. A.
Right arrow Articles by O’Connor, D. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Greenwood, T. A.
Right arrow Articles by O’Connor, D. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.