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Robert J Parmer

Publications and source records attributed to Robert J Parmer.

15 recordsLinked to original sources
PubMed2006

Cell-surface actin binds plasminogen and modulates neurotransmitter release from catecholaminergic cells.

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by approximately 90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to beta/gamma-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Actins↗
PubMed2005

Plasminogen receptors: the sine qua non of cell surface plasminogen activation.

Localization of plasminogen and plasminogen activators on cell surfaces promotes plasminogen activation and serves to arm cells with the broad spectrum proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration including macrophage recruitment during the inflammatory response, tissue remodeling, wound healing, tumor cell invasion and metastasis and skeletal myogenesis. Cell associated plasmin on platelets and endothelial cells is optimally localized for promotion of clot lysis. In more recently recognized functions that are likely to be independent of matrix degradation, cell surface-bound plasmin participates in prohormone processing as well as stimulation of intracellular signaling. This issue of Frontiers in Bioscience on Plasminogen Receptors encompasses chapters focusing on the kinetics of cell surface plasminogen activation and the regulation of plasminogen receptor activity as well as the contribution of plasminogen receptors to the physiological and pathophysiological processes of myogenesis, muscle regeneration and cancer. The molecular identity of plasminogen receptors is cell-type specific, with distinct molecular entities providing plasminogen receptor function on different cells. This issue includes chapters on the well studied plasminogen receptor functions.

Animals↗
PubMed2005

Genetic variation at the human alpha2B-adrenergic receptor locus: role in blood pressure variation and yohimbine response.

Exaggerated response to alpha2-adrenergic receptor (alpha2-AR) blockade by yohimbine in normotensive subjects is an intermediate phenotype that predicts increased risk for development of hypertension. Here, we assessed the 3 alpha2-AR loci (alpha2A, alpha2B, alpha2C) as candidate genes for their influence on baseline and yohimbine-mediated increase in mean arterial pressure. Because initial results with 173 individuals implicated a possible association of yohimbine response with genetic variation at a site in the alpha2B-AR gene, but not at sites in the other 2 alpha2-AR, we sequenced the alpha2B-AR gene (4.4 kb, including 1.2 kb upstream and 1.9 kb distal to the coding sequence) in those subjects and an additional 81 individuals to search for other alpha2B-AR variants. We identified 25 polymorphisms, of which 14 are previously unreported, and 2 major haplotypes that differ by the presence/absence of a 9-bp in-frame deletion that encodes Glu301 to Glu303. Frequency differences in haplotypes were observed between blacks and whites but did not predict response to yohimbine. Genotyping of 2 additional white cohorts, including 1269 individuals with extremes in blood pressure selected from >50,000 subjects, also failed to reveal an association of the 2 major alpha2B-AR haplotypes with differences in blood pressure. Thus, despite considerable polymorphism in alpha2-AR genes, such variation is not a major determinant of variability in yohimbine response and by inference, in susceptibility to essential hypertension.

Adrenergic alpha-Antagonists↗
PubMed2004

Human sympathetic activation by alpha2-adrenergic blockade with yohimbine: Bimodal, epistatic influence of cytochrome P450-mediated drug metabolism.

BACKGROUND: alpha2-Adrenergic blockade responses suggest adrenergic dysfunction in hypertension. alpha2-Blockade is also used to treat autonomic dysfunction. However, pharmacokinetic determinants of yohimbine disposition are not understood. METHODS: We evaluated alpha2-blockade with intravenous yohimbine in 172 individuals. Specific cytochrome P450 (CYP) isoform-mediated metabolism was investigated. Results were evaluated by ANOVA and by maximum likelihood analysis for bimodality of response distributions. RESULTS: Yohimbine metabolism to 11-hydroxy-yohimbine displayed greater than 1000-fold variability, with 17 individuals showing no metabolism. Nonmetabolizers differed from others in ethnicity but not in age, sex, body habitus, blood pressure, heart rate, or family history of hypertension. Bimodality of metabolism was suggested by frequency histogram, as well as maximum likelihood and cluster analysis. Among ethnic groups, subjects of European ancestry had the highest frequency of nonmetabolism. In vitro oxidation suggested that the major route of metabolism (lowest Michaelis-Menten constant and greatest intrinsic clearance) was likely via CYP2D6 to 11-hydroxy-yohimbine. In vivo genotypes at both CYP2D6 and CYP3A4 were necessary to predict metabolism (overall F = 3.03, P =.005); an interaction of alleles at these 2 loci (interaction F = 3.05, P =.033) suggested an epistatic effect on drug metabolism in vivo. Nonmetabolizers had greater activation of sympathetic nervous system activity. Yohimbine increased blood pressure, an effect mediated hemodynamically by elevation of cardiac output rather than systemic vascular resistance. Blood pressure and cardiac output responses did not differ by metabolizer group. CONCLUSIONS: We conclude that heterogeneous, bimodally distributed yohimbine metabolism depends on common genetic variation in both CYP2D6 and CYP3A4 and contributes to differences in sympathetic neuronal response to alpha2-blockade. These results have implications for both diagnostic and therapeutic uses of this alpha2-antagonist.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed2003

Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells.

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

Adrenal Medulla↗
PubMed2003

Diminished renal kallikrein responses to mineralocorticoid stimulation in African Americans: determinants of an intermediate phenotype for hypertension.

BACKGROUND: Hypertension is a complex trait with an ill-defined genetic predisposition, in which renal mechanisms seem to be involved even at the early stages. Renal kallikrein excretion is diminished in patients with hypertension, and perhaps even in the early, prehypertensive phases of the syndrome. African Americans, a group at increased risk of developing hypertension, have especially diminished kallikrein expression, coupled with decreased renal excretion of K(+), a known stimulant of kallikrein expression, suggesting an environmental mechanism for their kallikrein deficit. We, therefore, tested whether short-term indirect (K(+)) or direct (fludrocortisone) stimulation of mineralocorticoid activity might be capable of restoring kallikrein excretion in African Americans. METHODS: Nineteen healthy normotensive young men (n = 10 white, n = 9 African Americans) were treated with the following sequence of four oral medications, each for 1 week: placebo, KCl (120 mEq/day), placebo, and the mineralocorticoid fludrocortisone (0.4 mg/day). At each stage, we measured vital signs, excretion of kallikrein, aldosterone and electrolytes, and serum renin. Results were evaluated by two-way, repeated measures ANOVA. RESULTS: African Americans had diminished urinary excretion of not only kallikrein (P =.007), but also K(+) (P <.001) and aldosterone (P =.015). Kallikrein responses to mineralocorticoid stimulation were substantially blunted in African Americans, whether achieved indirectly (by supplemental K(+); P =.019) or directly (by the exogenous mineralocorticoid fludrocortisone; P =.027), despite achievement of substantial increments in K(+) excretion after KCl (P =.002), and multiple other mineralocorticoid effects after fludrocortisone (P =.005). The kallikrein increment after KCl was best predicted by renin activity (P =.001) rather than ethnicity. Potassium chloride did not lower blood pressure (BP) in either group (P >.4). CONCLUSIONS: Restoration of K(+) and aldosterone secretion to levels found in whites does not normalize kallikrein excretion or lower BP in African Americans, at least in the short term. Nor does exogenous mineralocorticoid stimulation fully restore kallikrein expression in African Americans. Therefore, the diminution of kallikrein biosynthesis in African Americans seems to involve mechanisms at or distal to the aldosterone receptor, and perhaps at the level of the kallikrein gene itself.

Adult↗
PubMed2002

Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A.

Chromogranin A (CgA) is the index member of the chromogranin/secretogranin (or 'granin') family of regulated secretory proteins that are ubiquitously distributed in amine- and peptide-containing secretory granules of endocrine, neuroendocrine and neuronal cells. Because of their abundance and such widespread occurrence, granins have often been used as prototype proteins to elucidate mechanisms of protein targeting into dense-core secretory granules. In this study, we used a series of full-length, point mutant or truncated CgA-green fluorescent protein (GFP) chimeras to explore routing of CgA in neuroendocrine PC12 cells. Using sucrose gradient fractionation and 3D deconvolution microscopy to determine the subcellular localization of the GFP chimeras, as well as secretagogue-stimulated release, the present study establishes that a CgA-GFP fusion protein expressed in neuroendocrine PC12 cells is trafficked to the dense core secretory granule and thereby sorted to the regulated pathway for exocytosis. We show that information necessary for such trafficking is contained within the N-terminal but not the C-terminal region of CgA. We find that CgA's conserved N-terminal hydrophobic Cys(17)-Cys(38) loop structure may not be sufficient for sorting of CgA into dense-core secretory granules, nor is its stabilization by a disulfide bond necessary for such sorting. Moreover, our data reveal for the first time that the CgA(77-115) domain of the mature protein may be necessary (though perhaps not sufficient) for trafficking CgA into the regulated pathway of secretion.

Amino Acid Sequence↗
PubMed2002

Localization of regulatory elements mediating constitutive and cytokine-stimulated plasminogen gene expression.

The activity of plasmin, the major enzyme responsible for dissolving fibrin clots, is regulated by plasminogen activators, plasminogen activator inhibitors, alpha(2)-antiplasmin, and inflammatory mediators. Recent studies suggest that plasmin activity can be regulated also at the level of plasminogen gene expression. In this study, we characterized the murine plasminogen promoter and 5'-flanking region. The major transcription start site was identified at -83 bp relative to the ATG translational initiation codon. A series of 5'-flanking sequences up to 2400 bp upstream of the transcription initiation site were fused to the luciferase reporter gene and transfected into hepatocytic cells. A 106-bp 5'-flanking region of the murine plasminogen gene demonstrated sufficient functional promoter activity in plasminogen-expressing cells. IL-6 treatment stimulated luciferase activity driven by the 5'-flanking region and an intact consensus IL-6-responsive element at -791, was required for maximal stimulation by this cytokine. These results indicate the presence of regulatory elements in the 5'-flanking region of the murine plasminogen promoter that may regulate murine plasminogen gene expression and, hence, plasmin activity.

Amino Acid Motifs↗
PubMed2002

Early decline in the catecholamine release-inhibitory peptide catestatin in humans at genetic risk of hypertension.

BACKGROUND: Hypertension is a complex trait with an ill-defined genetic predisposition, in which adrenergic mechanisms seem to be involved even at the early stages. Chromogranin A is a pro-hormone stored and released with catecholamines by exocytosis; its fragment catestatin, formed in vivo, inhibits further catecholamine release as an antagonist at the physiologic trigger for secretion, the neuronal nicotinic cholinergic receptor. METHODS: We measured catestatin by radioimmunoassay in n = 277 subjects stratified by blood pressure (n = 61 hypertensive, n = 216 normotensive), and if normotensive by genetic risk of developing hypertension: family history positive (n = 176) versus negative (n = 40). Maximum likelihood analysis tested for bimodality. Involvement of catestatin in pathophysiology was probed by measurements of catecholamines and leptin, and the hemodynamic responses to environmental (cold) stress. RESULTS: The normotensive offspring of patients with hypertension already had diminished catestatin (P = 0.024), and family history was a better predictor of catestatin than age, ethnicity or gender (P = 0.014). Greater catestatin variance among family history-positive individuals (P = 0.021) suggested heterogeneity in this group, and a bimodal distribution (P < 0.001) identified 4.3% of individuals in a lower mode of catestatin values, all with positive family histories (P = 0.05). Catestatin correlated inversely with body mass index (r = -0.215, r(2) = 0.046, n = 276, P < 0.001) and plasma leptin (r = -0.203, r(2) = 0.041, n = 212, P = 0.003), while body mass index and leptin correlated directly (r = 0.59, r(2) = 0.350, n = 212, P < 0.001). Family history-positive individuals had greater epinephrine excretion (P = 0.037) in addition to diminished catestatin, suggesting an inhibitory effect of catestatin on chromaffin cells in vivo. Low plasma catestatin predicted enhanced pressor response to a sympathoadrenal stressor (cold stress; r = -0.184, r(2) = 0.034, n = 211, P = 0.007), suggesting an adrenergic mechanism whereby diminished catestatin might predispose to later development of hypertension. In white subjects, diminished catestatin also predicted greater systemic vascular resistance responses to cold stress (r = -0.307, r(2) = 0.094, n = 75, P = 0.007), a relationship not found in Blacks (r = 0.122, r(2) = 0.015, n = 94, P = 0.243). CONCLUSIONS: We conclude that catestatin is diminished early in the course of development of hypertension, even in the normotensive offspring of patients with the disease. Low catestatin predicts augmented adrenergic pressor responses, suggesting a mechanism whereby diminished catestatin might increase the risk for later development of hypertension.

Adult↗
PubMed2002

The local chromaffin cell plasminogen/plasmin system and the regulation of catecholamine secretion.

Chromaffin cells express components of the plasminogen/plasmin system, including its major activator, tissue plasminogen activator (t-PA), and high-affinity cellular receptors for plasminogen, which promote local concentration and activation of plasminogen at the cell surface. Our studies suggest that plasmin participates in local neuroendocrine prohormone processing and that perturbation of this system profoundly affects the secretory characteristics of the cells. These results suggest the presence of a local, functionally active, chromaffin cell plasminogen/plasmin system that plays a major role in the regulation of catecholamine release from catecholaminergic cells.

Animals↗
PubMed2002

Catecholaminergic pathways, chromaffin cells, and human disease.

Recent studies demonstrate major effects of adrenal medullary and catecholaminergic pathways on a wide variety of normal physiologic and regulatory events. Alterations in these pathways, involving changes in catecholamines or in proteins and peptides costored and coreleased with catecholamines, may lead to profound changes in autonomic, cardiovascular, neuroendocrine, metabolic, nociceptive, and immune function. These findings have important implications for a variety of human disease states. In addition, molecules associated with catecholaminergic function may provide novel diagnostic and therapeutic strategies for human disease and suggest specific genetic loci as important and fruitful targets for further genetic and pharmacogenetic studies.

Catecholamines↗
PubMed2002

Plasminogen has a broad extrahepatic distribution.

Plasmin is the major enzyme that dissolves fibrin in the vasculature and the predominant source of its zymogen, plasminogen, is liver. However, plasmin has a broad substrate spectrum and, if present in other tissues, may perform additional functions. We tested the hypothesis that plasminogen is expressed broadly extrahepatically. A sensitive and specific isotopic quantitative RT-PCR assay was developed to detect plasminogen mRNA from total RNA isolated from C57BL/6J mice tissues. Plasminogen mRNA was detected in adrenal, kidney, brain, testis, heart, lung, uterus, spleen, thymus and gut. Of these tissues, adrenal had the highest plasminogen mRNA content. In situ hybridization was utilized to localize plasminogen mRNA expressing cell types. Besides hepatocytes, positive cells were identified in both adrenal and kidney medullae and cortexes. Plasminogen mRNA expression was detected in cerebral, hippocampal and cerebellar neurons. Plasminogen mRNA was detected in cells in early stages of spermatogenesis in testis, present in the cortex and medulla of the thymus and in splenic white and red pulps. Our results suggest that the plasminogen gene is expressed broadly in extrahepatic tissues. Thus, tissues separated by local anatomic barriers as well as tissues accessible to circulating plasminogen have the capacity to provide local sources of plasminogen.

Animals↗