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Biomedical subjects

D T O'Connor

Publications and source records attributed to D T O'Connor.

At least 19 recordsLinked to original sources
PubMed2001

Proteolytic cleavage of chromogranin A (CgA) by plasmin. Selective liberation of a specific bioactive CgA fragment that regulates catecholamine release.

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.

Amino Acid Sequence↗
PubMed2001

Neuroendocrine-specific and gastrin-dependent expression of a chromogranin A-luciferase fusion gene in transgenic mice.

BACKGROUND AND AIMS: Chromogranin A (CgA) is a multifunctional acidic protein specifically expressed in neuroendocrine cells. In the stomach, CgA is found predominantly in enterochromaffin-like (ECL) cells, where it is regulated by gastrin. We investigated the ability of a promoter fragment comprising 4.8 kb of 5'-flanking DNA of the mouse CgA (mCgA) gene to direct cell-specific expression as well as gastrin responsiveness in the gastroenteropancreatic neuroendocrine system. METHODS: Two independent lines of mCgA 4.8 kb-luc transgenic mice were created. Transgene expression was assessed by determination of luciferase activity and reverse-transcription polymerase chain reaction analysis of luciferase messenger RNA. Cell specificity of transgene expression was investigated by immunohistochemical analysis. The influence of hypergastrinemia on transgene expression was determined after repeated omeprazole injections. RESULTS: In both transgenic lines, mCgA 4.8 kb-luc expression paralleled the expression pattern of the endogenous CgA gene. ECL cells were identified as the major gastric cell population expressing the transgene. Omeprazole treatment stimulated expression of the transgene and the endogenous CgA gene selectivity in the gastric corpus (3-4-fold). CONCLUSIONS: mCgA 5'-flanking DNA (4.8 kb) contain the major cis-regulatory element(s) required for cell-specific CgA expression in the neuroendocrine system and gastrin-responsiveness in the gastric corpus. Further analysis of the CgA promoter in transgenic studies may elucidate the general molecular mechanisms underlying cell-specific gene expression in the gastroenteropancreatic neuroendocrine system.

Animals↗
PubMed2001

Molecular genetics of essential hypertension: recent results and emerging strategies.

Efforts to identify hypertension-predisposition genetic loci have focused largely on candidate gene strategies, in which specific candidates have been tested for linkage and association with blood pressure or the diagnosis of hypertension. A variety of candidate genes have been investigated, including loci involving the renin-angiotensin-aldosterone system, sodium epithelial channel, catecholaminergic/adrenergic function, renal kallikrein system, alpha-adducin, and others involving lipoprotein metabolism, hormone receptors, and growth factors. These studies, and more recently, several genome-wide scans, have yielded highly promising results suggesting a number of potential candidate genes and genomic regions that may contribute to blood pressure variation. The results also point to the need for more robust phenotypes that are intermediate in the pathogenetic development of high blood pressure. Additional methods and strategies for improving genetic studies of human hypertension include comparative genomics, in which results from animal studies are used to target potential blood pressure loci, the use of newly developed quantitative tests of linkage and association, comprehensive single-nucleotide polymorphism discovery in candidate loci, and the use of single-nucleotide polymorphisms in cladistic/haplotype analyses and genome-wide searches.

Animals↗
PubMed2001

Early alteration in glomerular reserve in humans at genetic risk of essential hypertension: mechanisms and consequences.

Essential hypertension has a familial predisposition, but the phenotype of elevated blood pressure has delayed penetrance. Because the kidney is a crucial determinant of blood pressure homeostasis, we studied early glomerular alterations in still-normotensive young subjects at genetic risk of hypertension. Thirty-nine normotensive adults (mean age 29 to 31 years), stratified by genetic risk (parental family history [FH]) of hypertension (26 with positive FH [FH+], 13 with negative FH [FH-]), underwent intravenous infusion of mixed amino acids. Before and during amino acid administration, we measured glomerular filtration rate (GFR), putative second messengers of amino acids (nitric oxide [NO.] metabolites and cGMP), serum insulin and amino acid concentrations, and the FE(Li)+ as an index of renal proximal tubular reabsorption. The FH+ group had a blunted GFR rise in response to amino acids (2.43+/-8.16% versus 31.0+/-13.4% rise, P:=0.0126). The amino acid-induced change in GFR correlated (r=0.786, P:<0.01) with the change in urinary NO. metabolite excretion; a diminished rise in urinary NO. metabolite excretion in the FH+ group (P:=0.0105) suggested a biochemical mechanism for the different GFR responses between FH groups: a relative inability to convert arginine to NO. The FH+ group had a far lower initial cGMP excretion at baseline (261+/-21.1 versus 579+/-84.9 nmol. h(-1)/1.73 m(2), P:=0.001), although cGMP did not change during the amino acid infusion (P:=0.703). FH status, baseline GFR, and baseline serum insulin jointly predicted GFR response to amino acids (P:=0.0013), accounting for approximately 45% of the variance in GFR response. Decline in FE(Li)+, an inverse index of proximal tubular reabsorption, paralleled increase in GFR (r=-0.506, P:=0.01), suggesting differences in proximal tubular reabsorption during amino acids between the FH groups. GFR response to amino acid infusion was blunted in the FH+ group despite significantly higher serum concentrations of 6 amino acids (arginine, isoleucine, leucine, methionine, phenylalanine, and valine) in the FH+ group, suggesting a novel form of insulin resistance (to the amino acid-translocating action of insulin) in FH+ subjects. We conclude that blunted glomerular filtration reserve in response to amino acids is an early-penetrance phenotype seen even in still-normotensive subjects at genetic risk of hypertension and is linked to impaired formation of NO. in the kidney. Corresponding changes in GFR and fractional excretion of Li(+) suggest that altered proximal tubular reabsorption after amino acids is an early pathophysiologic mechanism. Resistance to the amino acid-translocating actions of insulin may play a role in the biological response to amino acids in this setting. This glomerular reserve phenotype may be useful in genetic studies of renal traits preceding or predisposing to hypertension.

Adult↗
PubMed2000

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.

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.

Amino Acid Sequence↗
PubMed2000

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.

Adrenal Medulla↗
PubMed2000

Heredity and the autonomic nervous system in human hypertension.

Because the complex phenotype of human hypertension is at least in part genetically determined, how individual genes ultimately contribute to the disease is not well understood. By contrast, intermediate phenotypes are traits associated with complex disease, but which may display simpler genetic properties such as greater heritability, more consistent and earlier penetrance and bimodality, and may suggest particular candidate susceptibility genes. Because autonomic nervous system activity is altered in hypertension, we examined biochemical, physiologic, and pharmacologic autonomic traits that fulfill at least some of these properties. Such biochemical, physiologic, or pharmacologic autonomic traits may be especially valuable as phenotypic anchor points in linkage or association studies probing the genetic basis of human hypertension.

Autonomic Nervous System↗
PubMed2000

Beta2-adrenergic receptor polymorphisms at codon 16, cardiovascular phenotypes and essential hypertension in whites and African Americans.

Beta2-adrenergic receptors (beta2-AR) contribute to cardiovascular regulation by influencing several functions and previous studies suggest that a decreased function of the beta2-AR may be involved in essential hypertension. Beta2-AR are polymorphic and certain polymorphisms of these receptors are of functional importance. We focus here on the Arg16-->Gly16 beta2-AR polymorphism, which shows enhanced agonist-promoted downregulation of the receptor and which, in two recent studies, yielded opposite results in terms of association with essential hypertension: an increased frequency of the Gly16 variant in African-Caribbean hypertensives and of the Arg16 variant in offspring of Norwegian white hypertensive parents. In the current study, we genotyped 243 subjects, including both African-American and white individuals, for codon 16 polymorphism and assessed blood pressure and cardiovascular function using impedance cardiography and pressor sensitivity to phenylephrine. We found similar patterns of cardiovascular function and expression of hypertension with the two genotypes of codon 16. There was no statistically significant difference in the overall allelic distribution of the two genotypes: among African-Americans, 51% of the hypertensives and 50% of the normotensives carried the Arg16 allele, whereas among the white subjects 40% of the hypertensives and 47% of the normotensives were carriers of the Arg16 allele. Although we observed a statistically significant increase in the Arg16/Gly16 heterozygotes in the African-American population, the Gly16 allele was not significantly increased in the African-Americans compared to whites. These findings indicate that the codon 16 polymorphisms are not associated with hypertension in a mixed American study population nor do they appear to substantially impact on a variety of hemodynamic variables.

Adult↗
PubMed2000

Renal kallikrein excretion: role of ethnicity, gender, environment, and genetic risk of hypertension.

BACKGROUND AND OBJECTIVE: Alterations in renal kallikrein excretion are well-described in hypertension, and kallikrein excretion may predict risk of developing hypertension, but kallikrein excretion has not been directly compared across several ethnic strata, nor have the effects of ethnicity, gender, environment, and genetic risk of hypertension been simultaneously considered as determinants of kallikrein. METHODS: We investigated determinants of kallikrein excretion in a cross-section of n = 204 normotensive subjects stratified by ethnicity (119 Caucasian, 33 African-American, 52 Asian), gender (109 men, 95 women), environment (spontaneous electrolyte intake/excretion), and heredity (genetic risk (family history) of hypertension). Results were interpreted by analysis of variance (with Bonferroni post hoc comparison corrections), analysis of covariance, multiple linear regression, and maximum likelihood. RESULTS: Urinary kallikrein activity varied substantially (F = 5.30, P = 0.006) across the three ethnic groups, with African-American values approximately 50% lower than Caucasian (P = 0.005) or Asian (P = 0.02). Ethnicity and gender (T = 3.24, P = 0.001) had independent effects on kallikrein, with women excreting approximately 50% more kallikrein than men, regardless of ethnicity. Subjects at genetic risk of hypertension were over-represented (P = 0.048) in the lower stratum of a bimodal distribution of kallikrein excretion (chi-square = 29.6, P < 0.001). Potassium excretion was diminished in African-Americans (P < 0.001 to P = 0.002), and in a multivariate analysis, potassium excretion was the strongest correlate of kallikrein excretion (T = 4.10, P = 0.0001). In a subset of Caucasian and African-American individuals, African-Americans exhibited diminished excretion of not only kallikrein and potassium, but also aldosterone (P = 0.003), suggesting a mechanistic link between potassium and kallikrein excretion in their ethnic variations. CONCLUSIONS: Kallikrein excretion is influenced by several independent determinants, both hereditary (gender, ethnicity, and genetic risk of hypertension) and environmental (potassium intake and excretion). Ethnicity and environment may interact uniquely to influence kallikrein, as demonstrated by the case of African-Americans with diminutions of both kallikrein and potassium excretion. These results suggest a mechanism whereby kallikrein excretion is diminished in African-Americans, as well as therapeutic strategies to correct this deficiency. Finally, the identified determinants of kallikrein excretion will require analytic adjustment during genetic studies of this 'intermediate phenotype' in hypertension. Journal of Human Hypertension (2000) 14, 461-468

Adult↗
PubMed2000

Circulating amylin in human essential hypertension: heritability and early increase in individuals at genetic risk.

BACKGROUND: Human essential hypertension is a complex trait with poorly understood genetic determination. Insulin resistance is frequently associated with this trait. OBJECTIVE: To determine whether a potentially pathogenic feature of the insulin-resistant state, circulating amylin (islet amyloid polypeptide, co-released with insulin from pancreatic islet beta-cells), is already increased in prehypertensive individuals (normotensive persons at genetic risk of hypertension because of family history), whether such individuals already differ in their amylin response to beta-cell stimulation, and whether plasma amylin concentration is heritable. Such features could establish increased circulating amylin as a hereditary 'intermediate phenotype' useful in genetic analyses of hypertension. METHODS: Plasma amylin and insulin were measured in 283 medication-free individuals stratified by blood pressure status (82 hypertensive and 201 normotensive), and genetic risk (family history) of hypertension. Differences in means were tested by ANOVA, variances by F test, and frequency distributions by maximum likelihood analysis. Co-release of amylin and insulin was provoked by intravenous infusion of mixed amino acids. The effect of antihypertensive treatment was evaluated after monotherapy with either angiotensin converting enzyme inhibition or calcium-channel blockade in hypertension. RESULTS: Plasma amylin was increased in hypertension (P= 0.027), and body mass index was a strong predictor of increased circulating amylin (P = 0.0001). Plasma amylin and plasma renin activity were not correlated (P = 0.395), and effective antihypertensive monotherapy with either angiotensin converting enzyme inhibition or calcium-channel blockade did not affect either amylin (P = 0.87-0.97) or insulin (P= 0.55-0.59). Among normotensive individuals, those at genetic risk of hypertension (with positive family history) already had increased concentrations of amylin (P< 0.001), despite exhibiting no difference in blood pressure or body mass index compared with the family-history-negative group; however, among normotensive individuals, both family history (P = 0.043) and body mass index (P= 0.0059) were significant predictors of increased concentrations of amylin. By maximum likelihood analysis, plasma amylin was distributed heterogeneously in the normotensive individuals, with two modes best explaining the distribution (chi2 = 77.4, P< 0.001), and family-history-positive individuals completely accounting for the upper mode (chi2 = 4.63, P = 0.031). Family-history-positive normotensive individuals showed greater plasma amylin concentrations both before and during beta-cell stimulation by amino acid infusion (P = 0.014). Black (n = 111) and white (n = 172) individuals did not differ in mean (P = 0.946) or variance (P = 0.172) of plasma amylin concentrations. CONCLUSIONS: These results suggest that plasma amylin concentration is in part determined by heredity. Both basal and stimulated plasma amylin excess may identify a subgroup of individuals bearing an inherited predisposition to hypertension. Measurement of amylin might identify a useful 'intermediate phenotype' in the genetic analysis of essential hypertension and its relationship to insulin resistance.

Adult↗
PubMed2000

Plasma hydrogen peroxide production in human essential hypertension: role of heredity, gender, and ethnicity.

Oxygen free radicals, including hydrogen peroxide, may mediate oxidative stress in target organ tissues and contribute to cardiovascular complications in hypertension. To examine heritability of hydrogen peroxide production, we investigated this trait in a family-based cohort consisting of family members (n=236) ascertained through probands (n=57) with essential hypertension. Significant effects on hydrogen peroxide production were found for gender and ethnicity, with men having greater values than women (P<0.001) and white subjects having greater values than black subjects (P=0.025). Hydrogen peroxide production correlated directly with plasma renin activity (P=0.015), suggesting an important interaction between circulating oxygen radicals and the renin-angiotensin system and a potential mechanism for lower hydrogen peroxide values observed in blacks. Heritability estimates from familial correlations revealed that approximately 20% to 35% of the observed variance in hydrogen peroxide production could be attributed to genetic factors, suggesting a substantial heritable component to the overall determination of this trait. Hydrogen peroxide production negatively correlated with cardiac contractility (r=-0.214, P=0.001) and renal function (r=-0.194, P=0.003). In conclusion, these results indicate that hydrogen peroxide production is heritable and is related to target organ function in essential hypertension. Genetic loci influencing hydrogen peroxide production may represent logical candidates to investigate as susceptibility genes for cardiovascular target organ injury.

Adult↗
PubMed2000

Malignant pheochromocytoma. Chromaffin granule transmitters and response to treatment.

Chromaffin granule transmitters such as chromogranin A and catecholamines have been used in the diagnosis of pheochromocytoma, but the diagnostic and prognostic value of chromogranin A have not been explored in malignant pheochromocytoma. We evaluated these transmitters in patients with pheochromocytoma (n=27), both benign (n=13) and malignant (n=14). Patients with benign pheochromocytoma were studied before and after surgical excision (n=6), whereas patients with malignant pheochromocytoma were evaluated before and after combination chemotherapy with regular cycles of cyclophosphamide/dacarbazine/vincristine (nonrandomized trial in n=9). During treatment, patient responses to chemotherapy were divided according to anatomic and clinical criteria: responders (n=5) versus nonresponders (n=4). Plasma chromogranin A rose progressively (P<0.0001) from control subjects (48.0+/-3.0 ng/mL) to benign pheochromocytoma (188+/-40.5 ng/mL) to malignant pheochromocytoma (2932+/-960 ng/mL). Parallel changes were seen for plasma norepinephrine (P<0.0001), though plasma epinephrine was actually lower in malignant than benign pheochromocytoma (P=0.0182). In bivariate analyses, chromogranin A, norepinephrine, and epinephrine discriminated between pheochromocytoma and control subjects (all P<0.0001), whereas in a multivariate analyses, norepinephrine was the best discriminator (P:=0.011). Chromogranin A was significantly different in benign versus malignant pheochromocytoma on both bivariate (P=0.0003) and multivariate (P:=0.011) analyses. After excision of benign pheochromocytoma, chromogranin A (P=0.028), norepinephrine (P=0.047), and epinephrine (P=0.037) all fell to values near normal. During chemotherapy of malignant pheochromocytoma (n=9), plasma chromogranin A (P=0.047) and norepinephrine (P=0.02) fell but not epinephrine. In 5 responders to chemotherapy, there were significant declines in chromogranin A (P=0.03) and norepinephrine (P=0.03) but not epinephrine; in 4 nonresponders, none of the transmitters changed. Plasma chromogranin A varied longitudinally with tumor response and relapse. We conclude that plasma chromogranin A is an effective tool in the diagnosis of pheochromocytoma, and markedly elevated chromogranin A may point to malignant pheochromocytoma. During chemotherapy of malignant pheochromocytoma, chromogranin A can be used to gauge tumor response and relapse.

Adolescent↗
PubMed2000

Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion.

Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.

Adrenal Glands↗
PubMed2000

Neuroendocrine cell type-specific and inducible expression of the chromogranin B gene: crucial role of the proximal promoter.

Chromogranin B, a soluble acidic secretory protein, is widely distributed in neuroendocrine and neuronal cells, although not in other cell types. To identify the elements governing such widespread, yet selective, expression of the gene, we characterized the isolated mouse chromogranin B promoter. 5'-Promoter deletions localized neuroendocrine cell type-specific expression to the proximal chromogranin B promoter (from -216 to -91 bp); this region contains an E box (at [-206 bp]CACCTG[-201 bp]), four G/C-rich regions (at [-196 bp]CCCCGC[-191 bp], [-134 bp]CCGCCCGC[-127 bp], [-125 bp]GGCGCCGCC[-117 bp], and [-115 bp]CGGGGC[-110 bp]), and a cAMP response element (CRE; at [-102 bp]TGACGTCA[-95 bp]). A 60-bp core promoter region, defined by an internal deletion from - 134 to -74 bp upstream of the cap site and spanning the CRE and three G/C-rich regions, directed tissue-specific expression of the gene. The CRE motif directed cell type-specific expression of the chromogranin B gene in neurons, whereas three of the G/C-rich regions played a crucial role in neuroendocrine cells. Both the endogenous chromogranin B gene and the transfected chromogranin B promoter were induced by preganglionic secretory stimuli (pituitary adenylyl cyclase-activating polypeptide, vasoactive intestinal peptide, or a nicotinic cholinergic agonist), establishing stimulus-transcription coupling for this promoter. The adenylyl cyclase activator forskolin, nerve growth factor, and retinoic acid also activated the chromogranin B gene. Secretagogue-inducible expression of chromogranin B also mapped onto the proximal promoter; inducible expression was entirely lost upon internal deletion of the 60-bp core (from 134 to -74 bp). We conclude that CRE and G/C-rich domains are crucial determinants of both cell type-specific and secretagogue-inducible expression of the chromogranin B gene.

Animals↗