Catestatin is involved in neuropathic pain mediated by purinergic receptor P2X4 in the spinal microglia of rats.

Neuropathic pain is defined as a type of chronic pathological pain that often results from nerve damage or disease. The purinergic receptor P2X4 is mainly expressed on the cell surface of spinal dorsal horn microglia and is known to be involved in neuropathic pain. Catestatin (CST) is an endogenous peptide derived from chromogranin A. Here, we attempted to identify how CST function in neuropathic pain. Rat model of chronic constriction injury (CCI) was used and experimental results indicated that mechanical and thermal pain sensitivities were significantly increased in CCI rats. The group of CCI rats that received intrathecal CST injection (CCI + CST) exhibited higher P2X4 mRNA and protein levels compared with the CCI group. Moreover, the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2) in the CCI + CST group was higher than in the CCI group. This suggested that CST might aggravate neuropathic pain by enhancing P2X4 receptor expression in spinal microglia, and that the ERK1/2 pathway might be key in the development of neuropathic pain.

Establishing the Quantitative Relationship Between Lanreotide Autogel®, Chromogranin A, and Progression-Free Survival in Patients with Nonfunctioning Gastroenteropancreatic Neuroendocrine Tumors.

The objective of this work was to establish the quantitative relationship between Lanreotide Autogel® (LAN) on serum chromogranin A (CgA) and progression-free survival (PFS) in patients with nonfunctioning gastroenteropancreatic neuroendocrine tumors (GEP-NETs) through an integrated pharmacokinetic/pharmacodynamic (PK/PD) model. In CLARINET, a phase III, randomized, double-blind, placebo-controlled study, 204 patients received deep subcutaneous injections of LAN 120 mg (n = 101) or placebo (n = 103) every 4 weeks for 96 weeks. Data for 810 LAN and 1298 CgA serum samples (n = 632 placebo and n = 666 LAN) were used to develop a parametric time-to-event model to relate CgA levels and PFS (76 patients experienced disease progression: n = 49 placebo and n = 27 LAN). LAN serum profiles were described by a one-compartment disposition model. Absorption was characterized by two parallel pathways following first- and zero-order kinetics. As PFS data were considered informative dropouts, CgA and PFS responses were modeled jointly. The LAN-induced decrease in CgA levels was described by an inhibitory E MAX model. Patient age and target lesions at baseline were associated with an increment in baseline CgA. Weibull model distribution showed that decreases in CgA from baseline reduced the hazard of disease progression significantly (P < 0.001). Covariates of tumor location in the pancreas and tumor hepatic tumor load were associated with worse prognosis (P < 0.001). We established a semimechanistic PK/PD model to better understand the effect of LAN on a surrogate endpoint (serum CgA) and ultimately the clinical endpoint (PFS) in treatment-naive patients with nonfunctioning GEP-NETs.

Microneedle delivery of autoantigen for immunotherapy in type 1 diabetes.

Antigen specific immunotherapy mediated via the sustained generation of regulatory T cells arguably represents the ideal therapeutic approach to preventing beta cell destruction in type 1 diabetes. However, there is a need to enhance the efficacy of this approach to achieve disease modification in man. Previous studies suggest that prolonged expression of self-antigen in skin in a non-inflammatory context is beneficial for tolerance induction. We therefore sought to develop a dry-coated microneedle (MN) delivery system and combine it with topical steroid to minimise local inflammation and promote prolonged antigen presentation in the skin. Here we show that a combination of surface-modified MNs coated with appropriate solvent systems can deliver therapeutically relevant quantities of peptide to mouse and human skin even with hydrophobic peptides. Compared to conventional "wet" intradermal (ID) administration, "dry" peptide delivered via MNs was retained for longer in the skin and whilst topical hydration of the skin with vehicle or steroid accelerated loss of ID-delivered peptide from the skin, MN delivery of peptide was unaffected. Furthermore, MN delivery resulted in enhanced presentation of antigen to T cells in skin draining lymph nodes (LNs) both 3 and 10days after administration. Repeated administration of islet antigen peptide via MN was effective at reducing antigen-specific T cell proliferation in the pancreatic LN, although topical steroid therapy did not enhance this. Taken together, these data show auto-antigenic peptide delivery into skin using coated MNs results in prolonged retention and enhanced antigen presentation compared to conventional ID delivery and this approach may have potential in individuals identified as being at a high risk of developing type 1 diabetes and other autoimmune diseases.

Chronic Administration of Catestatin Improves Autonomic Function and Exerts Cardioprotective Effects in Myocardial Infarction Rats.

Catestatin (CST), which is emerging as a novel cardiac modulator, can protect the heart against excessive sympathetic drive in hypertensive cardiomyopathy. The aim of this study is to investigate whether exogenous CST decreases excessive cardiac sympathetic drive and improves autonomic function and exerts cardioprotective effects in myocardial infarction (MI) rats. Rats were divided into a sham group, MI group, and MI plus CST (MI + CST) group. Four weeks later, the autonomic function of the animals was assessed by analyzing heart rate variability (HRV) and measuring plasma catecholamine. Cardiac function was evaluated via echocardiography. Electrophysiological characteristics were assessed in Langendorff-perfused hearts. Compared to the MI group, the chronic administration of CST significantly increased the standard deviation of normal-normal intervals (P < .01) and low-frequency (LF) and high-frequency (HF) HRV and decreased the ratio of LF-HF HRV (P < .01 for all). Additionally, the level of plasma catecholamine was reduced in the MI + CST group compared to the MI group (P < .01). Treatment with CST significantly increased ejection fraction (EF) and fraction shorting (FS) and significantly decreased the left ventricular end-systolic diameter and left ventricular end-diastolic diameter at 28 days postmyocardial infraction (P < .05 for all). After MI, the ventricular repolarization duration, such as QTc intervals and action potential duration (APD) at 90% repolarization, was prolonged, and this prolongation could be decreased by CST (P < .05 for all). The CST also increased the threshold of ADP alternans (P < .01). Moreover, ventricular arrhythmias were induced in 83% of the MI group but only 33% of the MI + CST group (P < .05). These results suggested that the chronic administration of CST plays a role in cardioprotection in MI rats, which may function by decreasing the cardiac sympathetic drive and improving autonomic function.

Obligatory role for endothelial heparan sulphate proteoglycans and caveolae internalization in catestatin-dependent eNOS activation.

The chromogranin-A peptide catestatin modulates a wide range of processes, such as cardiovascular functions, innate immunity, inflammation, and metabolism. We recently found that the cardiac antiadrenergic action of catestatin requires a PI3K-dependent NO release from endothelial cells, although the receptor involved is yet to be identified. In the present work, based on the cationic properties of catestatin, we tested the hypothesis of its interaction with membrane heparan sulphate proteoglycans, resulting in the activation of a caveolae-dependent endocytosis. Experiments were performed on bovine aortic endothelial cells. Endocytotic vesicles trafficking was quantified by confocal microscopy using a water-soluble membrane dye; catestatin colocalization with heparan sulphate proteoglycans and caveolin 1 internalization were studied by fluorimetric measurements in live cells. Modulation of the catestatin-dependent eNOS activation was assessed by immunofluorescence and immunoblot analysis. Our results demonstrate that catestatin (5 nM) colocalizes with heparan sulphate proteoglycans and induces a remarkable increase in the caveolae-dependent endocytosis and caveolin 1 internalization, which were significantly reduced by both heparinase and wortmannin. Moreover, catestatin was unable to induce Ser(1179) eNOS phosphorylation after pretreatments with heparinase and methyl-ß-cyclodextrin. Taken together, these results highlight the obligatory role for proteoglycans and caveolae internalization in the catestatin-dependent eNOS activation in endothelial cells.

Microglial cathepsin B contributes to the initiation of peripheral inflammation-induced chronic pain.

Interleukin (IL)-1ß and IL-18 play critical roles in the induction of chronic pain hypersensitivity. Their inactive forms are activated by caspase-1. However, little is known about the mechanism underlying the activation of pro-caspase-1. There is increasing evidence that cathepsin B (CatB), a typical lysosomal cysteine protease, is involved in the pro-caspase-1 activation and the subsequent maturation of IL-1ß and IL-18. In this context, CatB is considered to be an important molecular target to control chronic pain. However, no information is currently available about the role of CatB in chronic pain hypersensitivity. We herein show that CatB deficiency or the intrathecal administration of CA-074Me, a specific CatB inhibitor, significantly inhibited the induction of complete Freund's adjuvant-induced tactile allodynia in mice without affecting peripheral inflammation. In contrast, CatB deficiency did not affect the nerve injury-induced tactile allodynia. Furthermore, CatB deficiency or CA-074Me treatment significantly inhibited the maturation and secretion of IL-1ß and IL-18 by cultured microglia following treatment with the neuroactive glycoprotein chromogranin A (CGA), but not with ATP. Moreover, the IL-1ß expression in spinal microglia and the induction of tactile allodynia following the intrathecal administration of CGA depended on CatB, whereas those induced by the intrathecal administration of ATP or lysophosphatidic acid were CatB independent. These results strongly suggest that CatB is an essential enzyme for the induction of chronic inflammatory pain through its activation of pro-caspase-1, which subsequently induces the maturation and secretion of IL-1ß and IL-18 by spinal microglia. Therefore, CatB-specific inhibitors may represent a useful new strategy for treating inflammation-associated pain.

Catestatin has an unexpected effect on the intrathecal actions of PACAP dramatically reducing blood pressure.

This study focuses on presympathetic neurons of the rostral ventrolateral medulla (RVLM) that regulate sympathetic vasomotor tone. Many neurotransmitters are colocalized in RVLM neurons and are released under specific conditions to modulate efferent homeostatic responses. Of particular interest here are two peptides colocalized in catecholaminergic RVLM neurons: catestatin and pituitary adenylate cyclase-activating polypeptide (PACAP). Chromogranin A-derived catestatin is a potent endogenous noncompetitive nicotinic and adrenoreceptor antagonist. Catestatin impairs adenylate cyclase and phospholipase C action: mechanisms engaged by PACAP. Although PACAP and catestatin are likely coreleased, the possible effects of this are unknown. We aimed to determine whether catestatin affects the normal sympathoexcitatory but isotensive responses to intrathecal PACAP. Urethane-anesthetized, vagotomized, ventilated Sprague-Dawley rats (n = 22) were given an intrathecal injection of catestatin at different times prior to intrathecal administration of PACAP-38. Arterial pressure, splanchnic sympathetic nerve activity, heart rate, and reflex responses to baroreceptor and chemoreceptor activation were recorded. The key findings of this study are that pretreatment with catestatin time dependently enhances the PACAP-38 effect on mean arterial pressure and enhances sympathetic barosensitivity and chemosensitivity. The time-scale of the effect of catestatin on the response to PACAP-38 strongly suggests that catestatin is either causing changes in gene expression to exert its effects, or modifying intracellular mechanisms normally engaged by PAC(1) receptors. The ability of catestatin pretreatment to enhance barosensitivity and chemosensitivity after PACAP-38 injection supports the hypothesis that catestatin manipulates the intracellular environment within sympathetic neurons in a way that increases responses to PACAP.

The N-terminal fragment of chromogranin A, vasostatin-1 protects mice from acute or chronic colitis upon oral administration.

BACKGROUND: Vasostatin-1 (VS-1), the N-terminal fragment of chromogranin A (CgA), decreases the permeability of endothelial cells in vitro and in vivo. AIMS: Here, we investigated whether a similar effect could be observed also on intestinal epithelial cells (IECs) in vitro and whether VS-1 could have favorable effects on animal models of acute or chronic colitis, which are characterized by increased permeability of the intestinal epithelium. METHODS: In vitro, VS-1 was tested on IEC monolayers showing increased permeability, on mechanically injured IEC monolayers, and on the production of the chemokine IL-8/KC by lipopolysaccharide (LPS)-stimulated IECs. In vivo, VS-1 was tested in animal models of dextran sodium salt (DSS)-induced acute or chronic colitis. RESULTS: In vitro, VS-1 inhibited increased permeability of IECs induced by interferon-γ and tumor necrosis factor-α. Moreover, VS-1 promoted healing of mechanically injured IEC monolayers, most likely through stimulation of cell migration, rather than cell proliferation. Eventually, VS-1 inhibited LPS-induced production of IL-8. In vivo, VS-1 exerted protective effects in animal models of acute or chronic colitis upon oral, but not systemic administration. CONCLUSIONS: VS-1 is therapeutically active in animal models of acute or chronic, DSS-induced colitis. The mechanisms underlying this effect are likely to be multiple, and may include inhibition of enhanced intestinal permeability, repair of injured intestinal mucosae, and inhibition of the production of IL-8/KC and possibly other inflammatory cytokines.

Catestatin, a chromogranin A-derived peptide, is sympathoinhibitory and attenuates sympathetic barosensitivity and the chemoreflex in rat CVLM.

Hypertension is a major cause of morbidity. The neuropeptide catestatin [human chromogranin A-(352-372)] is a peptide product of the vesicular protein chromogranin A. Studies in the periphery and in vitro studies show that catestatin blocks nicotine-stimulated catecholamine release and interacts with ß-adrenoceptors and histamine receptors. Catestatin immunoreactivity is present in the rostral ventrolateral medulla (RVLM), a key site for blood pressure control in the brain stem. Recently, we reported that microinjection of catestatin into the RVLM is sympathoexcitatory and increases barosensitivity. Here, we report the effects of microinjection of catestatin (1 mM, 50 nl) into the caudal ventrolateral medulla (CVLM) in urethane-anesthetized, bilaterally vagotomized, artificially ventilated Sprague-Dawley rats (n = 8). We recorded resting arterial pressure, splanchnic sympathetic nerve activity, phrenic nerve activity, heart rate, and measured cardiovascular homeostatic reflexes. Homeostatic reflexes were evaluated by measuring cardiovascular responses to carotid baroreceptor and peripheral chemoreceptor activation. Catestatin decreased basal levels of arterial pressure (-23 ± 4 mmHg), sympathetic nerve activity (-26.6 ± 5.7%), heart rate (-19 ± 5 bpm), and phrenic nerve amplitude (-16.8 ± 3.3%). Catestatin caused a 15% decrease in phrenic inspiratory period (T(i)) and a 16% increase in phrenic expiratory period (T(e)) but had no net effect on the phrenic interburst interval (T(tot)). Catestatin decreased sympathetic barosensitivity by 63.6% and attenuated the peripheral chemoreflex (sympathetic nerve response to brief hypoxia; range decreased 39.9%; slope decreased 30.1%). The results suggest that catestatin plays an important role in central cardiorespiratory control.

Cutting edge: vasostatin-1-derived peptide ChgA29-42 is an antigenic epitope of diabetogenic BDC2.5 T cells in nonobese diabetic mice.

Mechanistic and therapeutic insights in autoimmune diabetes would benefit from a more complete identification of relevant autoantigens. BDC2.5 TCR transgenic NOD mice express transgenes for TCR Vα1 and Vß4 chains from the highly diabetogenic BDC2.5 CD4(+) T cell clone, which recognizes pancreatic ß cell membrane Ags presented by NOD I-A(g7) MHC class II molecules. The antigenic epitope of BDC2.5 TCR is absent in ß cells that do not express chromogranin A (ChgA) protein. However, characterization of the BDC2.5 epitope in ChgA has given inconclusive results. We have now identified a ChgA29-42 peptide within vasostatin-1, an N-terminal natural derivative of ChgA as the BDC2.5 TCR epitope. Having the necessary motif for binding to I-A(g7), it activates BDC2.5 T cells and induces an IFN-γ response. More importantly, adoptive transfer of naive BDC2.5 splenocytes activated with ChgA29-42 peptide transferred diabetes into NOD/SCID mice.

Global disturbances in autonomic function yield cardiovascular instability and hypertension in the chromogranin a null mouse.

We reported previously that chromogranin A (Chga) knockout (KO) mice are hypertensive and hyperadrenergic. Here we sought to determine the basis of such alterations by probing physiological, biochemical, and pharmacological responses to perturbations of the autonomic nervous system. In the conscious state, KO mice had substantially elevated basal high blood pressure (BP) and heart rate (HR); immobilization stress caused increments in systolic BP and HR in both wild-type (WT) and KO mice, with higher maxima but blunted increments in the KO state. Catestatin (CST; CHGA(352-372)) selectively diminished stress-induced increments in BP and HR in KO mice, implicating CST as an antihypertensive peptide, even in stressful conditions. Heightened plasma catecholamines in KO mice returned to WT level after CST. Stress caused further increments in catecholamines in WT mice but no change in KO mice. KO mice displayed diminished baroreflex sensitivity in response to either phenylephrine or sodium nitroprusside, accounting for exaggerated pressor and depressor responses to these compounds; baroreceptor function was normalized by CST. To probe the relative roles of endogenous/basal sympathetic vs. parasympathetic tone in control of BP and HR, we used the muscarinic-cholinergic antagonist atropine or the beta-adrenergic antagonist propranolol; HR and BP responses to each antagonist were exaggerated in KO animals. We conclude that ablation of Chga expression results in global disturbances in autonomic function, both sympathetic and parasympathetic, that can be abrogated (or rescued), at least in part, by replacement of CST. The results point to mechanisms whereby CHGA and its CST fragment act to control cardiovascular homeostasis.

Catestatin attenuates the effects of intrathecal nicotine and isoproterenol.

Catestatin (Cts; human chromogranin A(352-372)) is a neuropeptide derived from chromogranin A (ChgA). In the periphery it is released from the terminals of preganglionic neurons. In the adrenal medulla it inhibits catecholamine release by non-competitively antagonizing nicotinic cholinergic receptors. ChgA is present in the central nervous system, but the extent to which it is present within bulbospinal sympathoexcitatory neurons is unknown. We investigated the distribution of ChgA in the brainstem and its relationship to sympathoexcitatory neurons by combining immunofluorescence and in situ hybridization. A possible role for Cts in modulating the effect of other neurotransmitter systems in the spinal cord was examined by intrathecal injection of Cts, in conjunction with nicotine (1 microg-100 microg) and isoproterenol (0.12 microg-2.5 microg), in the anesthetized rat. Cts attenuated the hypotensive effect of isoproterenol on mean arterial pressure (maximum dose, 2.5 microg isoproterenol; -27 mmHg pre-Cts to -18 mmHg post-Cts), splanchnic sympathetic nerve activity (at 2.5 microg isoproterenol; 10.5% pre-Cts to 2.4% post-Cts), HR (at 2.5 microg isoproterenol; 1.1% pre-Cts to -1.6% post-Cts), and the dp/dt max of carotid pulse pressure (at 2.5 microg isoproterenol 17.3% pre-Cts to 9.3% post-Cts). Cts attenuated the hypertensive effect of nicotine on mean arterial pressure (at 10 microg nicotine, 19.3 mmHg pre-Cts to 6.8 mmHg post-Cts), splanchnic sympathetic nerve activity (at 10 microg nicotine, 10.7% pre-Cts to 4.5% post-Cts), and HR (at 10 microg nicotine, 4.1% pre-Cts to 2.0% post-Cts). The results indicate that Cts antagonizes both central nicotinic acetylcholine receptors and beta-adrenoceptors that are involved in cardiovascular regulation in vivo.

Human recombinant chromogranin A-derived vasostatin-1 mimics preconditioning via an adenosine/nitric oxide signaling mechanism.

The acidic protein chromogranin A (CgA) is the precursor of several regulatory peptides generated by specific proteolytic processes. Human recombinant CgA NH(2)-terminal fragment STA-CgA(1-78) (hrSTA-CgA(1-78)), containing vasostatin-1 (CgA(1-76)) domain, exerts a negative inotropic effect and counteracts the beta-adrenergic positive inotropic effect on the rat heart. We hypothesized an involvement of nitric oxide (NO)-dependent pathway in both cardiodepression and cardioprotection by hrSTA-CgA(1-78). We also hypothesized an involvement of adenosine A(1) receptor and protein kinase C (PKC) in cardioprotection by hrSTA-CgA(1-78). Therefore, we evaluated whether 1) the cardioinhibition mediated by hrSTA-CgA(1-78) involves the G(i/o) proteins/NO-dependent signal transduction cascade, 2) hrSTA-CgA(1-78) induces ischemic preconditioning-like protective effects on the myocardium, and 3) inhibition of NO synthase (NOS), adenosine A(1) receptor, or PKC affects hrSTA-CgA(1-78) protection. Using the isolated rat heart, we found that the reduction of left ventricular pressure (LVP), rate-pressure product, and maximal values of the first derivative of LVP elicited by hrSTA-CgA(1-78) at 33 nM is abolished by blocking G(i/o) proteins with pertussis toxin, scavenging NO with hemoglobin, and blocking NOS activity with N(G)-monomethyl-l-arginine or N(5)-(iminoethyl)-l-ornithine, soluble guanylate cyclase with 1H-[1,2,4]oxadiazole-[4,4-a]quinoxalin-1-one, and protein kinase (PKG) with KT5823. Data suggest the involvement of the G(i/o) proteins/NO-cGMP-PKG pathway in the hrSTA-CgA(1-78)-dependent cardioinhibition. When given before 30 min of ischemia, hrSTA-CgA(1-78) significantly reduced the size of the infarct from 64 +/- 4 to 32 +/- 3% of the left ventricular mass. This protective effect was abolished by either NOS inhibition or PKC blockade and was attenuated, but not suppressed, by the blockade of A(1) receptors. These results suggest that hrSTA-CgA(1-78) activity triggers two different pathways: one of these pathways is mediated by A(1) receptors, and the other is mediated by NO release. As with repeated brief preconditioning ischemia, hrSTA-CgA(1-78) may be considered a stimulus strong enough to trigger both pathways, which may converge on PKC.