Sushil K. Mahata

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.

Chromogranin A is an autoantigen in type 1 diabetes

Autoreactive CD4+ T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4+ T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-Ag7 in an atypical manner, occupying only the carboxy-terminal half of the I-Ag7 peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice

The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress-induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Galphaq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.

Brain PPAR-γ promotes obesity and is required for the insulin–sensitizing effect of thiazolidinediones

In adipose tissue, muscle, liver and macrophages, signaling by the nuclear receptor peroxisome proliferator–activated receptor-γ (PPAR-γ) is a determinant of insulin sensitivity and this receptor mediates the insulin–sensitizing effects of thiazolidinediones (TZDs). As PPAR-γ is also expressed in neurons, we generated mice with neuron-specific Pparg knockout (Pparg brain knockout (BKO)) to determine whether neuronal PPAR-γ signaling contributes to either weight gain or insulin sensitivity. During high-fat diet (HFD) feeding, food intake was reduced and energy expenditure increased in Pparg-BKO mice compared to Ppargf/f mice, resulting in reduced weight gain. Pparg-BKO mice also responded better to leptin administration than Ppargf/f mice. When treated with the TZD rosiglitazone, Pparg-BKO mice were resistant to rosiglitazone-induced hyperphagia and weight gain and, relative to rosiglitazone-treated Ppargf/f mice, experienced only a marginal improvement in glucose metabolism. Hyperinsulinemic euglycemic clamp studies showed that the increase in hepatic insulin sensitivity induced by rosiglitazone treatment during HFD feeding was completely abolished in Pparg-BKO mice, an effect associated with the failure of rosiglitazone to improve liver insulin receptor signal transduction. We conclude that excess weight gain induced by HFD feeding depends in part on the effect of neuronal PPAR-γ signaling to limit thermogenesis and increase food intake. Neuronal PPAR-γ signaling is also required for the hepatic insulin sensitizing effects of TZDs.

The Extended Granin Family: Structure, Function, and Biomedical Implications

The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers.

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 μM 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.

Catecholamine release-inhibitory peptide catestatin (chromogranin A352-372): Naturally occurring amino acid variant Gly364Ser causes profound changes in human autonomic activity and alters risk for hypertension

Background— Chromogranin A, coreleased with catecholamines by exocytosis, is cleaved to the catecholamine release–inhibitory fragment catestatin. We identified a natural nonsynonymous variant of catestatin, Gly364Ser, that alters human autonomic function and blood pressure. Methods and Results— Gly364Ser heterozygotes and controls underwent physiological and biochemical phenotyping, including catecholamine production, chromogranin A precursor, and its catestatin product. Case-control studies replicated effects of the gene on blood pressure in the population. Gly364Ser displayed diminished inhibition of catecholamine secretion from cultured neurons. Gly/Ser heterozygotes displayed increased baroreceptor slope during upward deflections (by ≈47%) and downward deflections (by ≈44%), increased cardiac parasympathetic index (by ≈2.4-fold), and decreased cardiac sympathetic index (by ≈26%). Renal norepinephrine excretion was diminished by ≈26% and epinephrine excretion by ≈34% in Gly/Ser heterozygotes. The coalescent dated emergence of the variant to ≈70 000 years ago. Gly364Ser was in linkage disequilibrium with 1 major Chromogranin A promoter haplotype, although promoter haplotypes did not predict autonomic phenotypes. The 364Ser variant was associated with lower diastolic blood pressure in 2 independent/confirmatory groups of patients with hypertension; genotype groups differed by ≈5 to 6 mm Hg, and the polymorphism accounted for ≈1.8% of population diastolic blood pressure variance, although a significant gene-by-sex interaction existed, with an enhanced effect in men. Conclusions— The catestatin Gly364Ser variant causes profound changes in human autonomic activity, both parasympathetic and sympathetic, and seems to reduce risk of developing hypertension, especially in men. A model for catestatin action in the baroreceptor center of the nucleus of the tractus solitarius accounts for these actions.

Mechanism of cardiovascular actions of the chromogranin A fragment catestatin in vivo.

Catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL), reduces catecholamine secretion from chromaffin cells in vitro. We investigated the effects of this peptide on catecholamine release and blood pressure in vivo. Intravenous catestatin reduced pressor responses to activation of sympathetic outflow by electrical stimulation in rats, and the catestatin effect persisted even after adrenergic (alpha plus beta) blockade. Catestatin did not alter plasma norepinephrine levels, but increased plasma epinephrine 11-fold. Catestatin also blunted pressor responses to exogenous neuropeptide Y agonists. A control peptide (chromogranin A(141-160)) did not alter pressor or catecholamine responses to electrical stimulation. Pretreatment with a histamine H1 receptor antagonist blocked both the vasodepressor response to catestatin and the elevation in plasma epinephrine. Catestatin elevated endogenous circulating histamine 21-fold, and exogenous histamine mimicked both the epinephrine elevation and the vasodepressor actions of catestatin. We conclude that catestatin is a potent vasodilator in vivo whose actions appear to be mediated, at least in part, by histamine release and action at H1 receptors.

Distribution of secretoneurin, a peptide derived from secretogranin II, in rat brain: An immunocytochemical and radioimmunological study

The distribution of secretoneurin, a peptide derived from its precursor secretogranin II by proteolytic processing, was studied in the central nervous system of the rat by immunocytochemistry and radioimmunoassay and compared to the distribution of secretogranin II messenger RNA by using in situ hybridization. With a specific antiserum a distinct staining of fibers and to a lesser extent also of perikarya was observed throughout the central nervous system. A high density of immunoreactive fibers and terminals was found in several brain areas, i.e. the lateral septum, the medial parts of the amygdala, some medial thalamic nuclei, the hypothalamus, habenula, nucleus interpeduncularis, locus coeruleus, nucleus tractus solitarii, the substantiae gelatinosae of the caudal trigeminal nucleus and of the spinal cord. The quantitative distribution as measured by a radioimmunoassay agreed well with the varying densities of immunoreactivity found by immunocytochemistry. The highest concentrations of this peptide were present in the hypothalamus, in particular, in the median eminence and are comparable to those of the most highly concentrated neuropeptides. The distribution of immunopositive perikarya corresponded well with that of secretogranin II messenger RNA obtained by in situ hybridization. The pattern of secretoneurin expression in rat brain was widespread and unique, partially overlapping with established chemical transmitters and neuropeptides. The functional significance of this new brain peptide remains to be established.

The Antihypertensive Chromogranin A Peptide Catestatin Acts as a Novel Endocrine/Paracrine Modulator of Cardiac Inotropism and Lusitropism

Circulating levels of catestatin (Cts; human chromogranin A352-372) decrease in the plasma of patients with essential hypertension. Genetic ablation of the chromogranin A (Chga) gene in mice increases blood pressure and pretreatment of Chga-null mice with Cts prevents blood pressure elevation, indicating a direct role of Cts in preventing hypertension. This notable vasoreactivity prompted us to test the direct cardiovascular effects and mechanisms of action of wild-type (WT) Cts and naturally occurring human variants (G364S-Cts and P370L-Cts) on myocardial and coronary functions. The direct cardiovascular actions of WT-Cts and human variants were determined using the Langendorff-perfused rat heart. WT-Cts dose-dependently increased heart rate and coronary pressure and decreased left ventricular pressure, rate pressure product and both positive and negative LVdP/dt. WT-Cts not only inhibited phospholamban phosphorylation, but also the inotropic and lusitropic effects of WT-Cts were abolished by chemical inhibition of beta2-adrenergic receptors, Gi/o protein, nitric oxide or cGMP, indicating involvement of beta2-adrenergic receptors-Gi/o protein-nitric oxide-cGMP signaling mechanisms. In contrast, G364S-Cts did not affect basal cardiac performance but abolished isoproterenol-induced positive inotropism and lusitropism. P370L-Cts decreased rate pressure product and inhibited only isoproterenol-induced positive inotropism and lusitropism by 70%. Cts also inhibited endothelin-1-induced positive inotropism and coronary constriction. Taken together, the cardioinhibitory influence exerted on basal mechanical performance and the counterregulatory action against beta-adrenergic and endothelin-1 stimulations point to Cts as a novel cardiac modulator, able to protect the heart against excessive sympathochromaffin overactivation, e.g. hypertensive cardiomyopathy.