The discovery of the new mechanism: Celastrol improves spinal cord injury by increasing cAMP through VIP-ADCYAP1R1-GNAS pathway.

Spinal cord injury (SCI) is a debilitating condition that results in significant impairment of motor function and sensation. Despite the ongoing efforts to develop effective treatments, there are currently very limited options available for patients with SCI. Celastrol, a natural anti-inflammatory compound extracted from Tripterygium wilfordii, has been shown to exhibit anti-inflammatory and anti-apoptotic properties. In this study, we aimed to explore the therapeutic potential of celastrol for SCI and elucidate the underlying molecular mechanisms involved. We found that local tissue often experiences a significant decrease in cAMP content and occurrs apoptosis after SCI. However, the treatment of celastrol could promote the production of cAMP by up-regulating the VIP-ADCYAP1R1-GNAS pathway. This could effectively inhibit the phosphorylation of JNK and prevent apoptosis, ultimately improving the exercise ability after SCI. Together, our results reveal celastrol may be a promising therapeutic agent for the treatment of SCI.

Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides.

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand-binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C-terminus of the Gα subunit (G-peptides) on signaling from two Gi-coupled receptors, adenosine A1 receptor (A1 R) and cannabinoid receptor 1 (CB1 ). We expressed A1 R and CB1 fusions with G-peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G-peptides. We used agonists N6 -Cyclopentyladenosine (CPA) and 5'-N-Ethylcarboxamidoadenosine (NECA) for A1 R and 2-Arachidonoylglycerol (2-AG) and WIN 55,212-2 mesylate (WN) for CB1 . G-peptides derived from Gαi and Gαq enhance agonist-dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi-coupled signaling. In contrast, both G-peptides suppress agonist-dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq-coupled signaling. Taken together with our previous studies on Gs-coupled receptors, this study provides an extended model for the allosteric effects of G-peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.

Secretogranin III as a novel target for the therapy of choroidal neovascularization.

Wet age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. The advent of anti-vascular endothelial growth factor (VEGF) drugs represents a major breakthrough in wet AMD therapy but with limited efficacy to improve visual acuity. Secretogranin III (Scg3, SgIII) was recently discovered as a novel angiogenic factor with VEGF-independent mechanisms. Scg3-neutralizing monoclonal antibody (mAb) was reported to alleviate pathological retinal neovascularization in oxygen-induced retinopathy mice and retinal vascular leakage in diabetic mice with high efficacy and disease selectivity. Herein we investigated whether Scg3 is a novel angiogenic target for CNV therapy in mouse models. We found that anti-Scg3 ML49.3 mAb inhibited Scg3-induced proliferation and Src phosphorylation in human retinal microvascular endothelial cells. Intravitreal injection of Scg3-neutralizing polyclonal antibodies (pAb) or mAb significantly attenuated laser-induced CNV leakage, CNV 3D volume, lesion area and vessel density. Furthermore, subcutaneous administration of Scg3-neutralizing pAb or mAb significantly prevented Matrigel-induced CNV. The efficacy of anti-Scg3 pAb or mAb was comparable to VEGF inhibitor aflibercept. These findings suggest that Scg3 plays an important role in CNV pathogenesis and that anti-Scg3 mAb efficiently ameliorates laser- or Matrigel-induced CNV.

Cholesterol biosynthesis pathway intermediates and inhibitors regulate glucose-stimulated insulin secretion and secretory granule formation in pancreatic beta-cells.

Cholesterol is reportedly abundant in the endocrine secretory granule (SG) membrane. In this study, we examined the involvement of cholesterol biosynthesis intermediates and inhibitors in insulin secretion and SG formation mechanisms. There are two routes for the supply of cholesterol to the cells: one via de novo biosynthesis and the other via low-density lipoprotein receptor-mediated endocytosis. We found that insulin secretion and content are diminished by ß-hydroxy-ß-methylglutaryl-coenzyme A inhibitor lovastatin but not by lipoprotein depletion from the culture medium in MIN6 ß-cells. Cholesterol biosynthesis intermediates mevalonate, squalene, and geranylgeranyl pyrophosphate enhanced glucose-stimulated insulin secretion, and the former two increased insulin content. The glucose-stimulated insulin secretion-enhancing effect of geranylgeranyl pyrophosphate was also confirmed in perifusion with rat islets. Morphologically, mevalonate and squalene increased the population of SGs without affecting their size. In contrast, lovastatin increased the SG size with reduction of insulin-accumulating dense cores, leading to a decrease in insulin content. Furthermore, insulin was secreted in a constitutive manner, indicating disruption of regulated insulin secretion. Because secretogranin III, a cholesterol-binding SG-residential granin-family protein, coincides with SG localization based on the cholesterol composition, secretogranin III may be associated with insulin-accumulating mechanisms. Although the SG membrane exhibits a high cholesterol composition, we could not find detergent-resistant membrane regions using a lipid raft-residential protein flotillin and a fluorescent cholesterol-Si-pyrene probe as markers on a sucrose-density gradient fractionation. We suggest that the high cholesterol composition of SG membrane with 40-50 mol% is crucial for insulin secretion and SG formation functions.

The antimicrobial peptides derived from chromogranin/secretogranin family, new actors of innate immunity.

Chromogranins/secretogranins are members of the granin family present in secretory vesicles of nervous, endocrine and immune cells. In chromaffin cells, activation of nicotinic cholinergic receptors induces the release, with catecholamines, of bioactive peptides resulting from a natural processing. During the past decade, our laboratory has characterized new antimicrobial chromogranin-derived peptides in the secretions of stimulated bovine chromaffin cells. They act at the micromolar range against bacteria, fungi, yeasts, and are non-toxic for the mammalian cells. They are recovered in several biological fluids involved in defence mechanisms (human serum, neutrophil secretions and saliva). These new antimicrobial peptides demonstrate the major role of the adrenal medulla in innate immunity. In this review we focus on the antimicrobial peptides derived from human and bovine chromogranin A (CGA), chromogranin B (CGB) and secretogranin II (SGII) emphasizing their direct action against pathogens and their effects on immune cells.

Inhibition of insulin secretion by betagranin, an N-terminal chromogranin A fragment.

Betagranin, an N-terminal fragment of chromogranin A, results from a proteolytic processing, and is co-secreted with insulin. While other chromogranin A-derived peptides negatively modulate hormone secretion, the role of betagranin in pancreatic beta-cells is so far unknown. We have recently shown that pancreatic islet betagranin levels are down-regulated in obese, leptin-deficient mice. In the present study, we have investigated the distribution of betagranin in primary mouse islets and cells of the MIN6 line and have evaluated its effects on insulin secretion. We showed that betagranin co-localizes with insulin within secretory granules and strongly inhibited insulin secretion in response to both glucose and potassium, by blocking the influx of calcium. The data demonstrated a hitherto unknown inhibitory effect of betagranin on insulin secretion.

The chromogranin A fragment catestatin: specificity, potency and mechanism to inhibit exocytotic secretion of multiple catecholamine storage vesicle co-transmitters.

BACKGROUND: Secretory granules of chromaffin cells and neurons co-store and release, by exocytosis, the acidic soluble protein chromogranin A (human, CHGA; rodent, Chga) along with catecholamines, neuropeptides and adenosine triphosphate (ATP). CHGA serves as a pro-protein and upon proteolytic cleavage it generates active peptides, including catestatin (human CHGA352-372), first discovered in adrenal medullary chromaffin granules. Studies in our laboratory demonstrated that catestatin acts at the nicotinic acetylcholine receptor to inhibit catecholamine secretion. However, the specificity of catestatin to exert nicotinic-cholinergic antagonism among its co-transmitters is not clearly known, nor is the potential effect of catestatin on multiple vesicle co-transmitters understood. AIM: Here we probed the specificity of catestatin's actions among its co-transmitters: catecholamines, ATP, and neuropeptide Y (NPY). METHODS: We studied the effects of each transmitter on exocytotic secretion of its co-transmitters from PC12 chromaffin cells, stimulating secretion by triggering physiological pathways at multiple sites. RESULTS: We observed that, among chromaffin granule co-transmitters, only catestatin and NPY inhibited catecholamine release induced by nicotinic-cholinergic stimulation; catestatin was more than tenfold more potent than NPY in this setting. We also stimulated norepinephrine secretion by other chromaffin cell agonists: catestatin blocked norepinephrine release induced by nicotine, but not by other agents (such as membrane depolarization) acting at later stages in the secretory pathway, nor by agents acting on other receptor classes. By contrast, NPY acted less specifically, blocking norepinephrine release triggered by either nicotine or membrane depolarization. Catestatin inhibited nicotinic-cholinergic co-release of all classes of chromaffin granule co-transmitters: catecholamines, chromogranins, neuropeptides, and ATP. Naturally occurring variants of human catestatin (Gly364Ser and Pro370Leu) exhibited parallel changes in potency to inhibit secretion of catecholamines and ATP. CONCLUSION: We conclude that, among the chromaffin granule co-transmitters, catestatin acts as the most specific and potent inhibitor of physiological pathway (nicotinic-cholinergic) stimulated secretion. Furthermore, catestatin generally inhibits nicotinically triggered exocytotic release of multiple co-transmitters from chromaffin granules. The results have physiological and pharmacological implications for co-transmission in the sympathochromaffin system.

Interactions of chromogranin A-derived vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine.

Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.

Recombinant N-terminal fragments of chromogranin-A modulate cardiac function of the Langendorff-perfused rat heart.

In this study we tested the hypothesis that vasostatins could act as myocardial modulators in the mammalian heart. Using the Langendorff-perfused rat heart, the cardiac effects of the two recombinant human CGA N-terminal fragments STA-CGA1-78 and STA-CGA1-115, containing the vasostatin-1 (CGA 1-76) and vasostatin-2 (CGA 1-113) sequences, respectively, were evaluated at concentrations of 11 / 165 nM. Cardiac performance was evaluated by analyzing left ventricular pressure (LVP) and the rate pressure product (RPP: HR x LVP), used as indexes of contractile activity and cardiac work, respectively. Under basal conditions, STA-CGA1-78 at all concentrations tested elicited a dose-dependent negative inotropism (LVP variations ranging from -9.6% +/- 2 to -23% +/- 2.9) without affecting coronary pressure (CP). In contrast, STA-CGA1-115 increased CP at 110 and 165 nM without affecting inotropism. Both STA-CGA1-78 and STA-CGA1-115 counteracted the cardio-stimulatory effects of isoproterenol (ISO). The ISO-dependent positive chronotropism was unaffected by STA-CGA1-78, while being reduced by STA-CGA1-115. Both peptides abolished the ISO-induced positive inotropism without modifying either the beta-adrenergic-dependent coronary dilation or the ouabain-induced positive inotropism. The analysis of the percentage of variations of RPP in terms of EC50 values of ISO alone (-8.5 +/- 0.3; r2 = 0.88) and in presence of STA-CGA1-78 (11, or 33, or 65 nM: -7.7 +/- 0.15, r2 = 0.97; -7.7 +/- 0.15, r2 = 0.97; -7.8 +/- 0.78, r2 = 0.55, respectively) revealed a non-competitive type of antagonism of STA-CGA1-78. Taken together, these data suggest vasostatins as novel cardioregulatory peptides in mammals.

Chromogranin A: a surprising link between granule biogenesis and hypertension.

Chromogranin A (CHGA) and its derived peptides, which are stored and released from dense-core secretory granules of neuroendocrine cells, have been implicated as playing multiple roles in the endocrine, cardiovascular, and nervous systems. In this issue of the JCI, Mahapatra et al. present in vivo evidence for 2 important functions of CHGA: the regulation of catecholamine-containing dense-core chromaffin granule biogenesis in the adrenal gland and the control of blood pressure. Obliteration of CHGA expression in a KO mouse model led to decreased size and number of chromaffin granules as well as hypertension in these animals. Transgenic expression of human Chga and exogenous injection of human catestatin, a CHGA-derived nicotinic cholinergic antagonist, restored normal blood pressure in these mice. These results suggest a coupled relationship between CHGA-mediated chromaffin granule biogenesis, necessary for catecholamine storage, and catestatin-induced inhibition of cholinergic-stimulated catecholamine release, which regulates autonomic control of blood pressure.

Inhibitory influence of chromogranin A N-terminal fragment (vasostatin-1) on the spontaneous contractions of rat proximal colon.

Very little is known about the role played by CGA and its fragments in the gastrointestinal physiology. We have studied the role of CGA N-terminal fragments in the regulation of intestinal smooth muscle contractility by measuring the influence of recombinant CGA 1-78 (VS-1) and synthetic CGA 7-57 peptides on the spontaneous mechanical activity of rat proximal colon in vitro. The mechanical activity was recorded as changes in the intraluminal pressure. VS-1 (0.1-30 nM) and CGA 7-57 (10-300 nM) produced concentration-dependent inhibitory effects, characterized by a progressive decrease in the mean amplitude of circular muscle spontaneous contractions, without affecting the resting tone. The response to VS-1 was antagonised by anti-CGA monoclonal antibodies (mAb5A8, B4E11, 7D1 or 4D5) but not by an irrelevant antibody, indicating that the effect was specific. The inhibitory responses to VS-1 and to CGA 7-57 were significantly reduced by pre-treatment of the preparations with N(omega)-nitro-l-arginine methyl ester (l-NAME) (300 microM), 1H-(1,2,4) oxadiazolo-(4,3-a) quinoxalin-1-one (ODQ) (10 microM), apamin (0.1 microM) or tetrodotoxin (TTX) (1 microM). The results suggest that VS-1 plays an inhibitory modulatory role on spontaneous contractions rat colon circular muscle, through mechanisms involving in part neural release of nitric oxide.

New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A.

Catestatin (bCGA(344-364)), an endogenous peptide of bovine chromogranin A, was initially characterized for its effect on the inhibition of catecholamine release from chromaffin cells. Catestatin and its active domain (bCGA(344-358)) were identified in chromaffin cells and in secretion medium. The present study identified a potent antimicrobial activity of bCGA(344-358) in the lowmicromolar range against bacteria, fungi and yeasts, without showing any haemolytic activity. Confocal laser microscopy demonstrated penetration of the rhodaminated peptide into the cell membranes of fungi and yeasts and its intracellular accumulation. Time-lapse videomicroscopy showed arrest of fungal growth upon penetration of the labelled peptide into a fungal filament. We identified several catestatin-containing fragments in the stimulated secretion medium of human polymorphonuclear neutrophils, suggesting the N-terminal sequence of catestatin (bCGA(344-358)) (named cateslytin) as a novel component of innate immunity.

3-Formylchromones IV. The rearrangement of 3-formylchromone enamines as a simple, facile route to novel pyrazolo[3,4-b]pyridines and the synthetic utility of the latter.

One-pot and facile preparations of 6-(2-hydroxy-5-R-benzoyl)-4-methyl-2-R1- pyrazolo[3,4-b]pyridines 4a-o are described, using the reaction of 3-formyl chromones 1 with 5-amino-1-R1-pyrazoles 2. An enamine-intermediate 2-ethyloxy-6-R-3-(3-methyl-1- phenylpyrazol-5-ylaminomethylene)chroman-4-one 3 was isolated at lower temperatures. Acyloxy-derivatives 5 of compounds 4 were obtained by acylation with acid chlorides or acid anhydrides. Coumarins 6 substituted at the 3- and 4-positions were prepared from the pyrazolo[3,4-b]pyridines 4 by condensation reactions and hydrazones 7 were formed from their reaction with 2,4-dinitrophenyl hydrazine. Reactions under microwave irradiation proceeded significantly faster and with high yields.

Synergistic amplification of beta-amyloid- and interferon-gamma-induced microglial neurotoxic response by the senile plaque component chromogranin A.

Activation of the microglial neurotoxic response by components of the senile plaque plays a critical role in the pathophysiology of Alzheimer's disease (AD). Microglia induce neurodegeneration primarily by secreting nitric oxide (NO), tumor necrosis factor-alpha (TNFalpha), and hydrogen peroxide. Central to the activation of microglia is the membrane receptor CD40, which is the target of costimulators such as interferon-gamma (IFNgamma). Chromogranin A (CGA) is a recently identified endogenous component of the neurodegenerative plaques of AD and Parkinson's disease. CGA stimulates microglial secretion of NO and TNFalpha, resulting in both neuronal and microglial apoptosis. Using electrochemical recording from primary rat microglial cells in culture, we have shown in the present study that CGA alone induces a fast-initiating oxidative burst in microglia. We compared the potency of CGA with that of beta-amyloid (betaA) under identical conditions and found that CGA induces 5-7 times greater NO and TNFalpha secretion. Coapplication of CGA with betaA or with IFNgamma resulted in a synergistic effect on NO and TNFalpha secretion. CD40 expression was induced by CGA and was further increased when betaA or IFNgamma was added in combination. Tyrphostin A1 (TyrA1), which inhibits the CD40 cascade, exerted a dose-dependent inhibition of the CGA effect alone and in combination with IFNgamma and betaA. Furthermore, CGA-induced mitochondrial depolarization, which precedes microglial apoptosis, was fully blocked in the presence of TyrA1. Our results demonstrate the involvement of CGA with other components of the senile plaque and raise the possibility that a narrowly acting agent such as TyrA1 attenuates plaque formation.

Effect of acetic acid or trypsin application on rat colonic motility in vitro and modulation by two synthetic fragments of chromogranin A.

The hypothesis that Chromogranin A (CgA)-derived peptides are involved in mechanisms modulating altered colonic motility was tested. Rat distal colonic strips were studied using an organ bath technique. Acetic acid (AA)-induced effects were characterized on spontaneous mechanical activities (SMA) in the presence of CgA4-16 or CgA47-66. In preparations with mucosa, AA induced a transient hyperactivity followed by a decrease in tone. The first phase is sensitive to tetrodotoxin (TTX) and capsaicin. The second phase was sensitive to BAYK8644 but insensitive to L-nitro-arginine-methyl-ester (L-Name)/apamin together. CgA4-16 or CgA47-66 alone produced no change on SMA. The administration of CgA4-16 prior to AA increased the duration of the excitatory component and reduced tone inhibition. CgA47-66 prior to AA only decreased duration of the excitatory phase. In preparations without mucosa, AA decreased tone. This effect was sensitive to BAYK8644 and CgA4-16. Trypsin decreased basal tone. This effect was suppressed by TTX, BAYK8644 or L-Name/apamin and were reduced by CgA4-16. AA-induced effects on rat colonic motility in vitro may be mediated through activation of primary afferents and an action at L-Type calcium channels. CgA-derived peptides are shown to decrease AA-induced effects on motility.

Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery.

Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA(1-128)), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA(1-76)), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA(7-40) and bCgA(47-70) with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA(7-57), bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60-150 mm Hg, the rat peptide (rCgA(7-57)) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin). Neither Rh-bCgA(7-40) nor Rh-bCgA(47-70) stained the endothelial layer, while Rh-bCgA(47-70) but not Rh-bCgA(7-40) stained the smooth muscle compartment. Analogously, bCgA(47-66) but not bCgA(7-40) reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA(7-57) and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA(47-66)), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA.

Mucosal neuroendocrine cell abnormalities in patients with chronic constipation.

OBJECTIVES: The aim of this study was to examine the distribution of neuroendocrine cells (NEC) in patients with chronic constipation (CC) as a means of establishing a relationship between pathology, symptomatology and treatment. METHODS: Rectal biopsy specimens from 43 patients with CC (aged 17-82 years) and 20 age-matched normal controls were examined histopathologically using haematoxylin and eosin, and immunohistochemically using antibodies against chromogranin-A (Ch-A) and serotonin (5-HT) to detect NEC. The number of positive NEC per 70 crypts (CR) was counted and expressed as the ratio of NEC/CR. CC patients were divided into groups based on management, then compared using NEC/CR. RESULTS: CC was managed conservatively in 29 patients (group A) and invasively in 14. Of these 14, 10 had normal histology (group B) and four had typical histopathological signs of intestinal neuronal dysplasia (IND; group C). All control specimens were unremarkable. In controls, NEC/CR was 0.94+/-0.33 for Ch-A and 0.32+/-0.08 for 5-HT. In group A, NEC/CR was 2.23+/-0.13 for Ch-A and 1.02+/-0.06 for 5-HT. In group B, it was 2.79+/-0.18 for Ch-A and 1.72+/-0.33 for 5-HT. In group C, it was 3.12+/-0.22 for Ch-A and 2.32+/-0.14 for 5-HT. The increase in Ch-A and 5-HT immunoreactive cells in groups B and C compared with controls was greater (p<0.01, p<0.05) than the increase seen in group A compared with controls (p<0.01, p<0.05). CONCLUSIONS: These results demonstrate that increased numbers of NEC may play a role in the abnormal bowel function seen in CC, and may have some relationship with the development of giant submucosal ganglia in IND.

Influence of vasostatins, the chromogranin A-derived peptides, on the working heart of the eel (Anguilla anguilla): negative inotropy and mechanism of action.

We have studied the effects of exogenous human recombinant Vasostatin-1 (VS-1), Vasostatin-2 (VS-2) and the human Chromogranin A (CGA) 7-57 synthetic peptides on the mechanical performance of the isolated and perfused working eel (Anguilla anguilla) heart. Under basal conditions, the three peptides decreased stroke volume (SV) and stroke work (SW), thus exerting negative inotropism. The VS-1-mediated negative inotropism was abolished by exposure to inhibitors of either Gi/o protein (pertussis toxin; PTx) or M1 muscarinic receptors (Pirenzepine) or calcium (Lantanum and Diltiazem) and potassium (Ba2+, 4-aminopyridine, tetraethylammonium, glibenclamide) channels, while it required an intact endocardial endothelium (EE). Using NG-monomethyl-L-arginine (L-NMMA) as an inhibitor of nitric oxide (NO) synthase (NOS), and hemoglobin as a NO scavenger, we demonstrated the obligatory role of NO signaling in mediating the vasostatin response. Pretreatment with either a specific inhibitor of soluble guanylate cyclase (GC) 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ), or the inhibitor of the cGMP-activated protein kinase (PKG) KT5823, abolished the VS-1-mediated inotropism, indicating the cGMP-PKG component as a crucial target of NO signaling. Of note, VS-1 was effective in counteracting the adrenergic (Isoproterenol and Phenylephrine)-mediated positive inotropism. These findings provide the first evidence that vasostatins exert cardiotropic action in fish, thus suggesting their long evolutionary history as well as their species-specific mechanisms of action.

Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen.

Chromogranin B (CgB) is a major matrix protein in secretory granules/large dense-cored vesicles and a precursor for smaller peptides. In an earlier study, we have identified a secretolytin-like peptide (KR-11, pCgB(637-647)) from porcine chromaffin granules. Further evidence is presented here to show the processing of chromogranin B to this peptide during axonal transport in the splenic nerve and its release in the spleen upon various conditions of stimulation. Immunohistochemical staining showed that in the porcine spleen chromogranin B and NPY completely colocalize in nerve fibres and varicosities in blood vessel walls and trabeculae, and along the loose network of smooth muscle cells in the red pulp, as identified by their alpha-smooth muscle cell actin content. No antibacterial activity was found for the porcine secretolytin-like peptide, KR-11. The change of one amino acid residue (Thr-->Asn) in the porcine homologous fragment of secretolytin appears to be responsible for its loss of antibacterial activity.

The catecholamine release-inhibitory "catestatin" fragment of chromogranin a: naturally occurring human variants with different potencies for multiple chromaffin cell nicotinic cholinergic responses.

The catestatin fragment of chromogranin A is an endogenous inhibitor of nicotinic cholinergic transmission, functioning in negative feedback control of catecholamine secretion. We explored naturally occurring polymorphisms in the amino acid sequence of catestatin. Three human variants were identified: Gly364Ser, Pro370Leu, and Arg374Gln. Variants were tested for ability to inhibit four nicotinic processes. The rank order of potency for inhibition of catecholamine secretion was Pro370Leu > wild type > Gly364Ser > Arg374Gln. Decrease in potency was paralleled by decline in Hill slope, suggesting that negative cooperativity at ascending dose might underlie loss of potency. Several lines of evidence indicated that each variant acted as a nicotinic antagonist: potency to inhibit secretion paralleled inhibition of agonist-triggered (22)Na(+) uptake (r = 0.986); variants inhibited secretion with similar potency when triggered by several nicotinic agonists, though not by agents using other secretory pathways or bypassing the nicotinic receptor; and blockade of secretion was noncompetitive with agonist. Variants also inhibited desensitization of secretion after prior agonist exposure and stimulation of secretory protein biosynthesis by agonist. Rank order of variant inhibitory potency for all four nicotinic processes was identical (Pro370Leu > wild type > Gly364Ser > Arg374Gln), suggesting mediation by similar combinations of receptor alpha/beta subunits and that crucial catestatin residues are likely to be identical across the four processes. When catestatin variants were mixed in likely heterozygotic (1:1 M ratio) combinations, the inhibitory curve was left-shifted onto that of the more potent variant in the combination, suggesting phenotypic dominance. The results have quantitative implications for interindividual variations in human nicotinic signaling.