Search PubMed⌕ Search

Biomedical subjects

R J Parmer

Publications and source records attributed to R J Parmer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Neurotrophin activation of catecholamine storage vesicle protein gene expression: signaling to chromogranin a biosynthesis.

Nerve growth factor differentiates precursor cells into sympathetic neurons. Does acquisition of a "neuronal" phenotype after nerve growth factor involve biosynthesis of chromogranin A, the major soluble protein in chromaffin granule cores? Nerve growth factor activated chromogranin A gene expression 7.6-fold in PC12 pheochromocytoma cells, and similarly activated PC12-transfected mouse, rat or human chromogranin A promoter/reporter constructs. Chromogranin A promoter 5'-deletions narrowed the nerve growth factor response element to a region from - 77 to - 61 bp upstream of the cap site, a region containing the chromogranin A cyclic AMP response element (TGACGTAA). Three different site-directed mutations of the cyclic AMP response element each reduced the nerve growth factor effect by >90%. Transfer of the cyclic AMP response element to a heterologous (thymidine kinase) promoter activated that promoter approximately 5-fold after nerve growth factor, while transfer of a cyclic AMP response element point-gap mutant (TGA-GTAA) to a heterologous promoter abolished the nerve growth factor effect. These findings indicate that the cyclic AMP response element in cis is, at least in part, both necessary and sufficient to activate the chromogranin A gene. Chemical blockade of the nerve growth factor receptor TrkA or the mitogen-activated protein kinase pathway component MEK substantially diminished nerve growth factor-induced expression of chromogranin A. By contrast, the response of chromogranin A to nerve growth factor was not impaired after blockade of phospholipase C-gamma or phosphoinositide-3 kinase. Chemical blockade of TrkA, Ras, MEK or mitogen-activated protein kinase similarly inhibited nerve growth factor activation of chromogranin A. Expression of constitutively activated Ras, Raf or MEK mutants increased chromogranin A promoter activity. Expression of dominant negative (inhibitory) mutants of Sos, Ha-Ras, Rafl, mitogen-activated protein kinase, ribosomal protein S6 serine kinase II (CREB kinase) or CREB (KCREB) each inhibited the nerve growth factor-induced increase in chromogranin A promoter activity. Thus, each component of the mitogen-activated protein kinase pathway is crucially involved in relaying the nerve growth factor signal in trans to the chromogranin A gene, in the following proposed sequence: nerve growth factor --> TrkA --> Shc/Grb2/Sos --> Ras --> Raf --> MEK --> mitogen-activated protein kinase --> ribosomal protein S6 serine kinase II --> CREB cyclic AMP response element.

Alkaloids↗
PubMed1999

Catecholamine storage vesicle protein expression in genetic hypertension.

Chromogranin A expression is heritable in humans, and both plasma chromogranin A concentration and its releasable adrenal and sympathetic neuronal pools are augmented in established essential (hereditary) hypertension. To evaluate chromogranin A further as a simpler or "intermediate phenotype" in the complex trait of hypertension, we studied chromogranin A expression in the spontaneously hypertensive rat (SHR), a rodent model of essential hypertension. Both plasma (p < 0.0001) and adrenal medullary (p = 0.003 to p < 0.0001) chromogranin A were elevated in the SHR, even at the earliest stages (3-4 weeks of age). In the adult adrenal gland, both chromogranin A (p=0.005) and norepinephrine (p=0.011) were increased in the SHR, while dopamine beta-hydroxylase activity was diminished (p < 0.0001). Chromogranin A mRNA expression was also elevated in the SHR adrenal medulla (p = 0.017). Differences in chromogranin A processing were not noted between SHR and Wistar Kyoto control (WKY) rats. In an SHR x WKY genetic intercross, control of the adrenal chromogranin A phenotype by a single major locus was suggested by comparison of phenotypic variance of the F2 vs F1 generations, and by bimodal frequency histogram (3:1 ratio), confirmed by maximum likelihood analysis (chi2 = 74.6, p < 0.000001) in the F2 generation. However, microsatellite alleles at a surrogate locus (Ighe) 12.7 cM from chromogranin A (Chga), on rat chromosome 6, failed to co-segregate with blood pressure in an F2 generation (F = 0.06, p = 0.94). In another rodent model of hereditary hypertension, the genetically hypertensive mouse (BPH/2), adrenal chromogranin A (p=0.018) and norepinephrine (p = 0.004) were actually diminished. We conclude that over-expression of chromogranin A is a variable feature of mammalian genetic hypertension. In one rodent model (the SHR), over-expression of chromogranin A is largely controlled by a single genetic locus, but the chromogranin A locus itself is not directly linked to determination of the blood pressure elevation of the SHR.

Adrenal Medulla↗
PubMed1999

Variability in phenylephrine response and essential hypertension: a search for human alpha(1B)-adrenergic receptor polymorphisms.

Genetic polymorphisms in drug receptors, in particular adrenergic receptors, may contribute to intersubject differences in pharmacologic response. We tested patients and first-degree normotensive and hypertensive relatives of patients with essential hypertension and found substantial intersubject variability in blood pressure response to infusion of the alpha(1)-adrenergic agonist phenylephrine. Because response to phenylephrine depends upon interaction with alpha(1B)-adrenergic receptors, we tested whether polymorphisms in this receptor contribute to the variable responses. Accordingly, we developed a polymerase chain reaction-based method, generating four exon-spanning fragments, to identify polymorphisms in the coding sequence of the two exons of the human alpha(1B)-adrenergic receptor. We sequenced the entire coding sequence of exon 1 from 51 subjects and exon 2 from 16 of these 51 subjects. Compared with the published sequence for the alpha(1B)-adrenergic receptor, we found one amino acid addition in exon 2 at position 368 (Arg) and one substitution (Arg371Gly) in all subjects. We thus suggest we have defined the correct coding sequence of the human alpha(1B) receptor. We found two "silent" polymorphisms in exon 1, one of which occurred in 3 of 51 subjects. These polymorphisms were unrelated to blood pressure status or response to phenylephrine. The 95% confidence intervals for expression of polymorphisms in exons 1 and 2 were 0 to 11%. Our data reveal that although phenylephrine response varies in humans, frequent polymorphisms in the coding sequence of the human alpha(1B)-adrenergic receptor appear not to account for this variation or for the increased blood pressure in patients with essential hypertension.

Adult↗
PubMed1998

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a beta-strand/loop/beta-strand structure secured by hydrophobic interactions and predictive of activity.

A novel fragment of chromogranin A, known as 'catestatin' (bovine chromogranin A344-364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29-35.5% identity, general alignment score 44-57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, IPKM, and 2IG2. Each of these sequences exists in nature as a beta-strand/loop/beta-strand structure, stabilized by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic beta-strand. Circular dichroism in the far ultraviolet showed substantial (63%) beta-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.

Animals↗
PubMed1998

Targeting of tissue plasminogen activator to the regulated pathway of secretion.

Tissue plasminogen activator is a serine protease that plays the dominant role in removal of fibrin from the vascular tree by activating plasminogen to the primary fibrinolytic enzyme, plasmin. Tissue plasminogen activator has a widespread neuroendocrine distribution in addition to its expression by endothelial cells. Within neuroendocrine cells, secretory proteins are sorted into one of two pathways: regulated or constitutive. Proteins entering the regulated pathway are concentrated and stored in vesicles, and subsequently released upon stimulation by a secretagogue. In contrast, in the constitutive pathway, newly synthesized protein is not stored but is transported directly to the cell surface and secreted even in the absence of an extracellular signal. The focus of this article is to review recent studies demonstrating that tissue plasminogen activator is targeted to the regulated secretory pathway in neuroendocrine cells and to discuss the physiological implications of the trafficking of tissue plasminogen activator to regulated secretory vesicles.

Journal Article↗
PubMed1998

Racial differences in renal kallikrein excretion: effect of the ovulatory cycle.

BACKGROUND: Renal kallikrein excretion is diminished in essential hypertension, especially in African-Americans, and evidence exists for a major gene effect on the kallikrein phenotype. In addition, urinary kallikrein excretion differs by gender, with ovulating females having greater kallikrein excretion than males or postmenopausal females. Recent studies have shown that renal kallikrein excretion varies in females during the ovulatory cycle, with levels rising during the luteal phase and returning during the follicular phase to levels that are similar to those of males. In family studies, gender differences in urinary kallikrein excretion were present in white subjects, but not black subjects. We therefore hypothesized dysregulation of kallikrein biosynthetic responses in African-Americans. METHODS: We determined urinary kallikrein activity [chromogenic substrate S2266 (D-val-leu-arg-paranitroanilide) assay; in microU/mg creatinine] in white (N = 15) and black (N = 11) ovulating females during the ovulatory cycle. Serum progesterone, estrogen, plasma renin activity as well as urinary aldosterone, and urinary electrolytes were determined to investigate changes between mid-follicular and mid-luteal phases in the two groups. RESULTS: White and black groups were matched for age, body mass index, blood pressure, heart rate and renal function. Ovulatory cycle phases were confirmed by serum progesterone determinations, which increased significantly in whites and blacks to a comparable degree [0.84 +/- 0.14 nmol/liter (mid-follicular) to 29.77 +/- 4.70 nmol/liter (mid-luteal) in whites, 0.67 +/- 0.08 nmol/liter (mid-follicular) to 28.62 +/- 5.83 nmol/liter (mid-luteal) in blacks; P < 0.001 for cycle effect, P = NS for race effect and race X cycle interaction]. Urinary kallikrein activity increased from 623 +/- 86 microU/mg creatinine (mid-follicular) to 948 +/- 142 microU/mg creatinine (mid-luteal) in whites, but did not change in blacks during the ovulatory cycle [239 +/- 73 microU/mg creatinine (mid-follicular] to 244 +/- 41 microU/mg creatinine (mid-luteal)]. Two-way ANOVA revealed significant effects on urinary kallikrein for race (P < 0.001), cycle (P < 0.05), and race X cycle interaction (P < 0.05). Thus, white females had higher urinary kallikrein than black females, and demonstrated a significant increase in urinary kallikrein excretion during the ovulatory cycle, whereas no significant change in urinary kallikrein activity was seen in the black group. Enzyme kinetic studies and mixing studies demonstrated that these racial differences in renal kallikrein excretion were quantitative, rather than due to qualitative differences in the renal kallikrein enzyme or due to the presence of a kallikrein inhibitor. CONCLUSIONS: These results suggest pronounced blunting of menstrual cycle changes in urinary kallikrein excretion in black females. Blunted urinary kallikrein responses during the ovulatory cycle are consistent with dysregulation of renal kallikrein biosynthetic responses in African-Americans, a group at increased risk for hypertension.

Adult↗
PubMed1998

Expression of altered alpha2-adrenergic phenotypic traits in normotensive humans at genetic risk of hereditary (essential) hypertension.

BACKGROUND: Essential (hereditary) hypertension is a common, though complex, trait with substantial heritability, but a still-obscure mode of inheritance. In this disorder with relatively late onset, knowledge of phenotypes with earlier penetrance would aid genetic analyses, as well as assessment of risk. OBJECTIVE: Because alpha2-adrenergic receptor alterations are among the most heritable in experimental genetic hypertension, we hypothesized enhanced expression of alpha2-adrenergic phenotypic traits in still-normotensive humans at genetic risk of hypertension. METHODS: We evaluated hemodynamic (blood pressure, cardiac output, systemic vascular resistance, stroke volume, and cardiac contractility) and biochemical (plasma drug, catecholamine, renin, and chromogranin A levels) responses to alpha2-adrenergic blockade with intravenous yohimbine in 84 normotensive subjects stratified by genetic risk of essential hypertension (67 with positive family histories and 17 with negative family histories of hypertension), as well as 18 subjects with established essential hypertension. Results were evaluated by analysis of variance, normal likelihood ratio test, and by maximum likelihood analysis for bimodality (i.e. mixtures) of response distributions. RESULTS: Blood pressure rose (P<0.001) during alpha2-adrenergic blockade, with greater response (P<0.001) in members of the hypertensive than in members of the normotensive group. Hemodynamically, the rise in blood pressure resulted from an increase in cardiac output (P<0.001), with associated increases in stroke volume (P=0.002) and cardiac contractility (P=0.006), without an overall change in systemic vascular resistance. Biochemically, plasma norepinephrine (P<0.001), epinephrine (P=0.001), and chromogranin A (P=0.02) rose, suggesting augmentation of efferent exocytotic sympathoadrenal activity. Cardiac output and stroke volume responses were correlated to increments in plasma catecholamines (especially epinephrine) for the positive group, but not for the negative group. Baseline plasma catecholamines predicted increments of stroke volume after administration of yohimbine (P=0.003-0.007) for the positive but not for the negative group. Simultaneous comparison of means and variances of cardiac output and stroke volume alpha2-adrenergic responses, by using a normal likelihood ratio test, revealed highly significant (P=0.025 to P<0.0001) differences between the groups of subjects with and without family histories of hypertension. Frequency histogram suggested that there was a bimodal distribution of responses of stroke volume to alpha2-adrenergic blockade for the normotensive group with positive family histories of hypertension; maximum likelihood analysis strongly rejected the hypothesis of a unimodal distribution, whereas the hypothesis of bimodality could not be rejected (chi2=18.4, P=0.0004). The second (exaggerated) mode of response of stroke volume to alpha2-adrenergic blockade, defined by maximum likelihood analysis, was found for 9.5% of subjects in the normotensive group with positive family histories of hypertension, and was characterized by significantly different responses of cardiac output (P=0.001), stroke volume (P<0.001), contractility (P<0.001), heart rate (P=0.03), systemic vascular resistance (P<0.001), and epinephrine (P<0.001). Even prior to alpha2-adrenergic blockade, baseline stroke volume (P=0.01), heart rate (P=0.04), systemic vascular resistance (P=0.005), and catecholamine (P=0.001-0.005) values for this subgroup were different than control values. CONCLUSIONS: We conclude that heterogeneous, bimodally distributed hemodynamic responses to alpha2-adrenergic blockade in subjects with positive family histories of hypertension suggest a discrete subgroup with early expression of perhaps Mendelian traits associated with risk of later development of hypertension. Such phenotypic traits ('intermediate phenotypes'), with earlier penetrance than hypertension itself, can be

Adolescent↗
PubMed1998

Proadrenomedullin N-terminal 20 peptide: minimal active region to regulate nicotinic receptors.

Proadrenomedullin N-terminal 20 peptide (PAMP-[1-20]; ARLDVASEFRKKWNKWALSR-amide) is a potent hypotensive and catecholamine release-inhibitory peptide released from chromaffin cells. We studied the mechanism of PAMP action and how its function is linked to structure. We tested human PAMP-[1-20] on catecholamine secretion in PC12 pheochromocytoma cells and found it to be a potent, dose-dependent (IC50 approximately 350 nmol/L) secretory inhibitor. Inhibition was specific for nicotinic cholinergic stimulation since PAMP-[1-20] failed to inhibit release by agents that bypass the nicotinic receptor. Nicotinic cationic (22Na+,45Ca2+) signal transduction was disrupted by this peptide, and potencies for inhibition of 22Na+ uptake and catecholamine secretion were comparable. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. N- and C-terminal PAMP truncation peptides indicated a role for the C-terminal amide and refined the minimal active region to the C-terminal 8 amino acids (WNKWALSR-amide), a region likely to be alpha-helical. PAMP also blocked (EC50 approximately 270 nmol/L) nicotinic cholinergic agonist desensitization of catecholamine release, as well as desensitization of nicotinic signal transduction (22Na+ uptake). Thus, PAMP may exert both inhibitory and facilitatory effects on nicotinic signaling, depending on the prior state of nicotinic stimulation. PAMP may therefore contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release.

Adrenomedullin↗
PubMed1998

Binding of human single chain urokinase to Chinese Hamster Ovary cells and cloning of hamster u-PAR.

The plasminogen activator, urokinase (u-PA), interacts with the u-PA receptor (u-PAR) which results in enhanced plasminogen activation on cell surfaces. The u-PAR is comprised of three homologous domains of approximately 90 amino acids, defined by the pattern of disulfide bonds. Domain 1 (amino acids 1-87) binds the ligand. Within this domain, Y57, and a site between residues 47 and 53, have been suggested as ligand contact points. Intradomain interactions also contribute to the interaction of u-PA and u-PAR. The interaction of u-PA with its receptor exhibits some species specificity. Previous studies have shown that human u-PA does not bind to the murine u-PAR and murine u-PA does not recognize human u-PAR. However, human u-PA does interact with bovine cells with high affinity. To further examine the interaction of the human ligand with the u-PAR of a different species, we characterized the binding of human 125I single chain u-PA (scu-PA) to hamster cells. Chinese Hamster Ovary (CHO) cells bound human scu-PA with high affinity and capacity (Kd = 1.13 +/- 0.8 nM; Bmax = 5.45 +/- 0.98 x 10(4) sites/cell). In ligand blotting with human 125I-scu-PA, major bands migrating with apparent Mr's of 74, 49 and 38 kDa were observed. The cDNA of hamster u-PAR was cloned and a single 1.4 kb mRNA species identified in Northern blots of CHO cell RNA. For comparison, we also cloned u-PAR cDNA from human THP-1 cells. Our human sequence was identical to those published for U937 and endothelial cells. These sequences were aligned with the published sequences for the murine, bovine and rat u-PAR's to obtain a consensus sequence for five species. The cysteine residues could be aligned for all species. Y57, which has been suggested as a ligand contact point was also conserved across species. In addition, 5 of the 7 amino acids between amino acids 47 and 53 were conserved in all species. Gly283, the most likely glycosyl-phosphatidyl inositol attachment site, was also conserved in all species. The conservation of these amino acid residues across all five species, attests to their importance in u-PAR function. In addition, the results of our studies suggest that the hamster may be a useful small animal model for studies of human urokinase function.

Amino Acid Sequence↗
PubMed1997

Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist.

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.

Amino Acid Sequence↗
PubMed1997

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.

How do chromaffin cell secretory stimuli program resynthesis of secreted peptides and amines? We previously showed that the physiologic nicotinic cholinergic signal for secretion also activates the biosynthesis of chromogranin A, the major protein released with catecholamines. Here, we examine signal transduction pathways whereby secretory stimuli influence exocytotic secretion versus chromogranin A transcription. Both secretion and transcription depended on initial nicotinic-triggered sodium entry into the cytosol, followed by calcium entry through -type voltage-gated channels. When calcium entered through -type channels, activation of secretion paralleled activation of transcription (r = 0.897, P = 0.002). Calcium entry from intracellular stores or through calcium ionophore channels activated secretion, though not transcription. Nicotinic-stimulated transcription depended upon protein kinase C activation; nicotine caused translocation of protein kinase C to the cell membrane fraction, and inhibition of protein kinase C blocked activation of transcription, while activation of protein kinase C mimicked nicotine effects. Transcriptional responses to both nicotine and protein kinase C mapped principally onto the chromogranin A promoter's cAMP response element (TGACGTAA; CRE box). KCREB, a dominant negative mutant of the CRE-binding protein CREB, blunted activation of chromogranin A transcription by nicotine, phorbol ester, or membrane depolarization. We conclude that activation of chromogranin A transcription by secretory stimulation in chromaffin cells is highly dependent upon precise route of calcium entry into the cytosol; transcription occurred after entry of calcium through -type channels on the cell surface, and was mediated by protein kinase C activation. The trans-acting factor CREB ultimately relays the secretory signal to the chromogranin A promoter's CRE box in cis.

Animals↗
PubMed1997

Regulation of plasminogen gene expression by interleukin-6.

Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.

Animals↗
PubMed1997

Tissue plasminogen activator (t-PA) is targeted to the regulated secretory pathway. Catecholamine storage vesicles as a reservoir for the rapid release of t-PA.

Tissue-type plasminogen activator (t-PA) is a serine protease that plays a central role in the regulation of intravascular thrombolysis. The acute release of t-PA in vivo is induced by a variety of stimuli including exercise, trauma, and neural stimulation. These types of stimuli also result in sympathoadrenal activation and exocytotic release of amines and proteins from catecholamine storage vesicles of the adrenal medulla and sympathetic neurons. Therefore, we tested the hypothesis that t-PA is packaged in and released directly from catecholamine storage vesicles, using several chromaffin cell sources including the rat pheochromocytoma PC-12 chromaffin cell line, primary cultures of bovine adrenal chromaffin cells, and human pheochromocytoma. t-PA was expressed in chromaffin cells as detected by Northern blotting, immunoprecipitation of [35S]Met-labeled t-PA, and specific t-PA enzyme-linked immunosorbent assay of cell homogenates. In addition, chromaffin cell t-PA was enzymatically active by fibrin zymography. To explore the subcellular localization of the expressed t-PA, PC-12 cells were labeled with [3H]norepinephrine, homogenized, and subjected to sucrose density fractionation. [3H]Norepinephrine and t-PA antigen were co-localized to the same subcellular fraction with a major peak at 1.4 M sucrose, consistent with the buoyant density of catecholamine storage vesicles. In addition, catecholamine storage vesicle lysates isolated from human pheochromocytoma tumors were enriched approximately 30-fold in t-PA antigen, compared with tumor homogenate. Furthermore, exposure of PC-12 cells or primary bovine adrenal chromaffin cells to chromaffin cell secretagogues (60 microM nicotine, 55 mM KCl, or 2 mM BaCl2) resulted in co-release of t-PA in parallel with catecholamines. These data demonstrate that t-PA is expressed in chromaffin cells, is sorted into the regulated pathway of secretion, and is co-released with catecholamines by chromaffin cell stimulation. Catecholamine storage vesicles may be an important reservoir and sympathoadrenal activation an important physiologic mechanism for the rapid release of t-PA. In addition, expression of t-PA by chromaffin cells suggests a role for this protease in the proteolytic processing of chromaffin cell proteins.

Animals↗
PubMed1996

Stimulus-transcription coupling in pheochromocytoma cells. Promoter region-specific activation of chromogranin a biosynthesis.

To explore stimulus-transcription coupling in pheochromocytoma cells, we studied the biosynthetic response of chromogranin A, the major soluble protein co-stored and co-released with catecholamines, to chromaffin cells' physiologic nicotinic cholinergic secretory stimulation. Chromogranin A mRNA showed a time-dependent 3.87-fold response to nicotinic stimulation, and a nuclear run-off experiment indicated that the response occurred at a transcriptional level. Transfected chromogranin A promoter/luciferase reporter constructs were activated by nicotinic stimulation, in time- and dose-dependent fashions, in both rat PC12 pheochromocytoma cells and bovine chromaffin cells. Cholinergic subtype agents indicated that nicotinic stimulation was required. Promoter deletions established both positive and negative nicotinic response domains. Transfer of candidate promoter domains to a heterologous (thymidine kinase) promoter conferred region-specific nicotinic responses onto that promoter. A proximal promoter domain (from -93 to -62 base pairs) was activated in copy number- and distance-dependent fashion, and thus displayed features of a promoter element. Its activation was sufficient to account for the overall positive response to nicotine. Within this proximal region, a cAMP response element (CRE) was implicated as a major nicotinic response element, since a CRE point-gap mutation decreased nicotinic induction, transfer of CRE to a thymidine kinase promoter augmented the promoter's response to nicotine, and nicotine activated the CRE-binding protein CREB through phosphorylation at serine 133. We conclude that secretory stimulation of pheochromocytoma cells also activates the biosynthesis of the major secreted protein (chromogranin A), that the activation is transcriptional, and that a small proximal domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine.

Animals↗
PubMed1996

Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.

Chromogranins A and B and secretogranin II are a family of acidic proteins found in neuroendocrine secretory vesicles; these proteins contain multiple potential cleavage sites for proteolytic processing by the mammalian subtilisin-like serine endoproteases PC1 and PC2 (prohormone convertases 1 and 2), and furin. We explored the role of these endoproteases in chromogranin processing in AtT-20 mouse pituitary corticotropes. Expression of inducible antisense PC1 mRNA virtually abolished PC1 immunoreactivity on immunoblots. Chromogranin A immunoblots revealed chromogranin A processing, from both the NH2 and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells. After antisense PC1 induction, an approximately 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an approximately 50 kD NH2-terminal and an approximately 30 kD COOH-terminal fragment declined in abundance. Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [35S]Methionine/cysteine pulse-chase metabolic labeling in AtT-20 antisense PC1 and antisense furin cells revealed reciprocal changes in secreted chromogranin A COOH-terminal fragments (increased approximately 82 kD and decreased approximately 74 kD forms, as compared with wild-type AtT-20 cells) indicating decreased cleavage, while AtT-20 cells overexpressing PC2 showed increased processing to and secretion of approximately 71 and approximately 27 kD NH2-terminal chromogranin A fragments. Antisense PC1 specifically abolished regulated secretion of both chromogranin A and beta-endorphin in response to the usual secretagogue, corticotropin-releasing hormone. Moreover, immunocytochemistry demonstrated a relative decrease of chromogranin A in processes (where regulated secretory vesicles accumulate) of AtT-20 cells overexpressing either PC1 or PC2. These results demonstrate that chromogranin A is a substrate for the endogenous endoproteases PC1 and furin in vivo, and that such processing influences its trafficking into the regulated secretory pathway; furthermore, lack of change in chromogranin B and secretogranin II cleavage after diminution of PCl suggests that the action of PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.

Animals↗
PubMed1996

Hereditary intermediate phenotypes in African American hypertension.

OBJECTIVE: Essential hypertension is a heterogeneous and multifactorial disorder and is at least twice as frequent among African Americans as in the general population. Inheritance of high blood pressure is complex, with the gene(s) responsible for hypertension still remaining elusive. A useful strategy for investigating the heritability of hypertension is to evaluate 'intermediate phenotypes'--simple Mendelian or monogenic traits that are associated with hypertension. These intermediate steps may identify potential pathophysiological factors that antedate the development of high blood pressure and suggest candidate genes. We are attempting to identify and characterize several such intermediate phenotypes, in particular as these might apply to hypertension in African Americans. METHODS: We studied several physiological and biochemical candidate intermediate phenotypes in untreated black and white patients with essential hypertension and in their normotensive counterparts stratified by genetic risk of hypertension. RESULTS AND CONCLUSIONS: Promising intermediate phenotypes, which may be useful for studies in African American families, include baroreceptor sensitivity to low and high pressure stimuli, cold pressor test responses, and biochemical markers such as plasma chromogranin A, dopamine-beta-hydroxylase and urinary kallikrein excretion. Identification of genes involved in complex traits such as hypertension may be facilitated by the intermediate phenotype approach, combined with recent advances in quantitative genetics and linkage mapping. Further studies are needed to pinpoint the nature of genes in African American hypertension.

Arousal↗