Urocortin 2 lowers blood pressure and reduces plasma catecholamine levels in mice with hyperadrenergic activity.

Exaggerated adrenergic activity is associated with human hypertension. The peptide urocortin 2 (Ucn 2) inhibits catecholamine synthesis and secretion from adrenal chromaffin cells in vitro and administration to mammals lowers blood pressure (BP). The chromogranin A-null mouse (Chga-/-) manifests systemic hypertension because of excessive catecholamine secretion from the adrenal and decreased catecholamine storage. In the present study, we investigated whether systemic administration of Ucn 2 could reduce BP and adrenal and plasma levels of catecholamines in vivo. Ucn 2 peptide was administered to freely moving, conscious Chga-/- and wild-type control mice. Telemetry and HPLC measured changes in BP and catecholamine levels, respectively. In both groups of mice, Ucn 2 dose-dependently decreased BP, and this effect was mediated by corticotropin factor-receptor type 2. However, in Chga-/- mice, the maximal percentage decrease of systolic BP from basal systolic BP was 37% compared with only a 23% reduction in wild-type mice (P=0.04). In Chga-/- mice only, Ucn 2 decreased adrenal and plasma levels of catecholamines as well as adrenal levels of tyrosine hydroxylase protein and phosphorylation. In vitro mechanistic studies demonstrated that Ucn 2 reduces both catecholamine secretion and tyrosine hydroxylase promoter activity, suggesting that the exaggerated action of Ucn 2 to reduce BP in the Chga-/- mouse is mediated through inhibition of both catecholamine synthesis and secretion. The data suggest that Ucn 2 may be therapeutically useful in regulating the exaggerated sympathoadrenal function of hyperadrenergic hypertension.

Conserved regulatory motifs at phenylethanolamine N-methyltransferase (PNMT) are disrupted by common functional genetic variation: an integrated computational/experimental approach.

The adrenomedullary hormone epinephrine transduces environmental stressors into cardiovascular events (tachycardia and hypertension). Although the epinephrine biosynthetic enzyme PNMT genetic locus displays both linkage and association to such traits, genetic variation underlying these quantitative phenotypes is not established. Using an integrated suite of computational and experimental approaches, we elucidate a functional mechanism for common (minor allele frequencies > 30%) genetic variants at PNMT. Transcription factor binding motif prediction on mammalian PNMT promoter alignments identified two variant regulatory motifs, SP1 and EGR1, disrupted by G-367A (rs3764351), and SOX17 motif created by G-161A (rs876493). Electrophoretic mobility shifts of approximately 30-bp oligonucleotides containing ancestral versus variant alleles validated the computational hypothesis. Queried against chromaffin cell nuclear protein extracts, only the G-367 and -161A alleles shifted. Specific antibodies applied in electrophoretic gel shift experiments confirmed binding of SP1 and EGR1 to G-367 and SOX17 to -161A. The in vitro allele-specific binding was verified in cella through promoter reporter assays: lower activity for -367A haplotypes cotransfected by SP1 (p = 0.002) and EGR1 (p = 0.034); and enhanced inhibition of -161A haplotypes (p = 0.0003) cotransfected with SP1 + SOX17. Finally, we probed cis/trans regulation with endogenous factors by chromatin immunoprecipitation using SP1/EGR1/SOX17 antibodies. We describe the systematic application of complementary computational and experimental techniques to detect and document functional genetic variation in a trait-associated regulatory region. The results provide insight into cis and trans transcriptional mechanisms whereby common variation at PNMT can give rise to quantitative changes in human physiological and disease traits. Thus, PNMT variants in cis may interact with nuclear factors in trans to govern adrenergic activity.

A dynamic pool of calcium in catecholamine storage vesicles. Exploration in living cells by a novel vesicle-targeted chromogranin A-aequorin chimeric photoprotein.

Chromaffin vesicles contain very high concentration of Ca2+ (approximately 20-40 mM total), compared with approximately 100 nM in the cytosol. Aequorin, a jellyfish photoprotein with Ca(2+)-dependent luminescence, measures [Ca2+] in specific subcellular compartments wherein proteins with organelle-specific trafficking domains are fused in-frame to aequorin. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgA-Aeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. hCgA-Aeq and cytoplasmic aequorin (Cyto-Aeq) luminescence displayed linear functions of [Ca2+] in vitro, over >5 log10 orders of magnitude (r > 0.99), and down to at least 10(-7) M sensitivity. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca2+]ves at granule interior pH (approximately 5.5). In the cytoplasm, Cyto-Aeq accurately determined [Ca2+]cyto under both basal ([Ca2+]cyto = 130 +/- 35 nM) and exocytosis-stimulated conditions, confirmed by an independent reference technique (Indo-1 fluorescence). The hCgA-Aeq chimera determined vesicular free [Ca2+]ves = 1.4 +/- 0.3 microM under basal conditions indicating that >99% of granule total Ca2+ is in a "bound" state. The basal free [Ca2+]ves/[Ca2+]cyto ratio was thus approximately 10.8-fold, indicating active, dynamic Ca2+ uptake from cytosol into the granules. Stimulation of exocytotic secretion revealed prompt, dynamic increases in both [Ca2+](ves) and [Ca2+]cyto, and an exponential relation between the two (y = 0.99 x e(1.53x), r = 0.99), reflecting a persistent [Ca2+]ves/[Ca2+]cyto gradient, even during sharp increments of both values. Studies with inhibitors of Ca2+ translocation (Ca(2+)-ATPase), Na+/Ca(+)-exchange, Na+/H(+)-exchange, and vesicle acidification (H(+)-translocating ATPase), documented a role for these four ion transporter classes in accumulation of Ca2+ inside the vesicles.

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.

Chromaffin cell catecholamine secretion: bisindolylmaleimide compounds exhibit novel and potent antagonist effects at the nicotinic cholinergic receptor in pheochromocytoma cells.

Activation of protein kinase C (PKC) stimulates nicotine-induced catecholamine secretion. PKC down-regulation by prolonged pretreatment with phorbol 12-myristate 13-acetate diminished nicotine-induced catecholamine secretion only slightly (approximately 16%), suggesting substantial PKC independence of nicotinic receptor activation. However, we found that bisindolylmaleimide compounds (which are also putative PKC chemical inhibitors) dramatically inhibited nicotine-induced catecholamine secretion (IC(50) values of approximately 24-37 nM). This inhibition was specific for the nicotinic cholinergic receptor. Catecholamine secretion induced by other nicotinic agonists (such as epibatidine, anatoxin, or cytisine) was also powerfully antagonized by bisindolylmaleimide II (IC(50) values of approximately 60-90 nM). Even high-dose nicotinic agonists failed to overcome the inhibition by bisindolylmaleimide II, suggesting noncompetitive nicotinic antagonism by this class of compounds. Nicotinic inhibition by bisindolylmaleimide seemed not to be readily reversible. Structure-activity studies of bisindolylmaleimide compounds revealed that bisindolylmaleimides I through III are the most potent nicotinic antagonists at the nicotinic cholinergic receptor in PC-12 cells (IC(50) < or =37 nM), whereas bisindolylmaleimide IV and V have far less nicotinic antagonist activity (IC(50) >1 microM); the active compounds I through III have cationic tails at an indole nitrogen, whereas the least potent compounds IV and V do not. By contrast, a free NH within the maleimide ring is crucial for PKC inhibition by this class of compounds. We conclude that bisindolylmaleimides I through III are some of the most potent noncompetitive neuronal nicotinic antagonists, indeed the most potent such antagonists we have observed in PC-12 cells. Nicotinic antagonism of these compounds seems to be independent of PKC inhibition.