Performance of a Novel Research-Use-Only Secretoneurin ELISA in Patients with Suspected Acute Coronary Syndrome: Comparison with an Established Secretoneurin Radioimmunoassay.

BACKGROUND: Circulating secretoneurin (SN) concentrations, as measured by established radioimmunoassay (RIA), risk stratify patients with cardiovascular disease. We now report data for a recently developed research-use-only SN enzyme-linked immunosorbent assay (ELISA) in patients with suspected acute coronary syndrome (ACS). METHODS: SN ELISA was developed according to industry standards and tested in 401 unselected chest pain patients. Blood samples were drawn <24 h from admission, and we adjudicated all hospitalizations as ACS or non-ACS. The mean follow-up was 6.2 years. RESULTS: SN ELISA with 2 monoclonal sheep anti-SN antibodies has a measuring range of 10-250 pmol/L and demonstrates excellent analytical precision and accuracy across the range of SN concentrations. SN measured by ELISA and RIA correlated in the chest pain patients: rho = 0.39, p < 0.001. SN concentrations were higher in ACS patients (n = 161 [40%]) than in non-ACS patients (n = 240) for both assays, with an area under the curve (AUC) of 0.66 (95% CI: 0.61-0.71) for ELISA and 0.59 (0.54-0.65) for RIA. SN concentrations were also higher in nonsurvivors (n = 65 [16%]) than survivors, with an AUC of 0.72 (0.65-0.79) for ELISA versus 0.64 (0.56-0.72) for RIA, p = 0.007, for difference between assays. Adjusting for age, sex, blood pressure, previous myocardial infarction, atrial fibrillation, and heart failure in multivariable analysis, SN concentrations as measured by ELISA, but not RIA, remained associated with mortality, with a hazard ratio of 1.71 (1.03-2.84), p = 0.038. CONCLUSIONS: The novel SN ELISA has excellent performance, higher AUC for diagnosis, and superior prognostic accuracy compared to the established RIA in chest pain patients.

Is secretoneurin a new hormone?

Numerous small potentially bioactive peptides are derived from the selective processing of the ~600 amino acid secretogranin II (SgII) precursor, but only the 31-42 amino acid segment termed secretoneurin (SN) is well-conserved from sharks to mammals. Both SNa and SNb paralogs have been identified in some teleosts, likely arising as a result of the specific genome duplication event in this lineage. Only one copy of the putative lamprey SgII (188 amino acids) could be identified which gives rise to a divergent agnathan SN that contains the signature YTPQ-X-LA-X(7)-EL sequence typical of the central core of all known SN peptides. In rodent models, SN has regulatory effects on neuroinflammation and neurotransmitter release, and possesses therapeutic potential for the induction of angiogenesis. The wide distribution of SN in neuroendocrine neurons and pituitary cells suggests important endocrine roles. The clearest example of the endocrine action of SN is the stimulatory effects on pituitary luteinizing hormone release from goldfish pituitary and mouse LßT2 gonadotroph cells, indicative of an important role in reproduction. Several lines of evidence suggest that the SN receptor is most likely a G-protein coupled protein. Microarray analysis of SN effects on dispersed goldfish pituitary cells in vitro reveals novel SN actions that include effects on genes involved in notch signaling and the guanylate cyclase pathway. Intracerebroventricular injection of SN increases feeding and locomotory behaviors in goldfish. Given that SgII appeared early in vertebrate evolution, SN is an old peptide with emerging implications as a new multifunctional hormone.

Chromogranin peptides in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder which primarily affects motor neurons. Eight cases of ALS and seven control cases were studied with semiquantitative immunocytochemistry for chromogranin A, chromogranin B and secretogranin II that are soluble constituents of large dense core vesicles, synaptophysin as a membrane protein of small synaptic vesicles and superoxide dismutase 1. Among the chromogranin peptides, the number and staining intensity of motor neurons was highest for chromogranin A. In ALS, the staining intensity for chromogranin peptides and synaptophysin was significantly lower in the ventral horn of ALS patients due to a loss in immunoreactive motor neurons, varicose fibers and varicosities. For all chromogranins, the remaining motor neurons displayed a characteristic staining pattern consisting of an intracellular accumulation of immunoreactivity with a high staining intensity. Confocal microscopy of motor neurons revealed that superoxide dismutase 1-immunopositive intracellular aggregates also contained chromogranin A, chromogranin B and secretogranin II. These findings indicate that there is a loss of small and large dense core vesicles in presynaptic terminals. The intracellular co-occurrence of superoxide dismutase 1 and chromogranins may suggest a functional interaction between these proteins. This study should prompt further experiments to elucidate the role of chromogranins in ALS patients.

Immunohistochemical staining of human islet cells with region-specific antibodies against secretogranins II and III.

Chromogranins and secretogranins belong to the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In earlier publications we have described the development of region-specific antibodies against CgA and CgB. In this study we describe antibodies to SgII and SgIII and their usefulness for immunohistochemical staining. Peptides homologous to defined parts of secretogranins II and III were selected and synthesized. Antibodies were raised and immunostainings were performed on normal human pancreas. The SgII 154-165 (N-terminal secretoneurin), SgII 172-186 (C-terminal secretoneurin) and SgIII antibodies immunostained all insulin-immunoreactive cells, most of the glucagon cells and some of the pancreatic polypeptide cells. The SgII 225-242 antibody immunostained only the insulin-containing cells. None of the antibodies immunostained the somatostatin cells. This study is the first observation of the expression of SgIII in human tissues, where we show expression of SgIII in three of the four major islet cell types in human pancreas.

Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases.

By means of highly sensitive radioimmunoassays, the levels of substance P (SP) and secretoneurin (SN) were detected in vitreous aspirates of patients with macular holes which served as controls, in patients with nonproliferative diabetic retinopathy (DR), active proliferative diabetic retinopathy (active PDR), inactive PDR, rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR). Furthermore, SN-like immunoreactivities were characterized by reversed phase-HPLC. The concentration of SN was more than 20-fold higher in macular holes when compared with SP and reversed phase HPLC revealed evidence that the vitreous levels of SN represent authentic SN. SN was significantly decreased in patients with nonproliferative DR, active PDR and inactive PDR by more than 70% which seems to result from a reduced expression and/or secretion from the cilary epithelium and a reduced release from the retina both due to diabetes mellitus. By contrast SP was increased in rhegmatogenous retinal detachment most obviously due to an enhanced outflow of the peptide through retinal breaks. Despite their proangiogenic activities, SP and SN are unlikely to be involved in the pathogenesis of neovascularizations in DR because of their unchanged and reduced levels, respectively, but the low levels of both peptides may facilitate the regression of vasoproliferations following laser photocoagulation.

Wavy multistratified amacrine cells in the monkey retina contain immunoreactive secretoneurin.

The goals of this study were to describe the morphology, neurotransmitter content and synaptic connections of neurons in primate retinas that contain the neuropeptide secretoneurin. Amacrine cells were labeled with antibodies to secretoneurin in macaque and baboon retinas. Their processes formed three distinct plexuses in the inner plexiform layer: one in the outermost stratum, one in the center and one in the innermost stratum. In light microscopic double immunolabeling experiments, GABA was colocalized with secretoneurin in these cells, but glycine transporter 1 and Substance P were not. ON bipolar cell axon terminals labeled with antibody to the cholecystokinin precursor, G6-gly, have ON responses to stimulation of short wavelength sensitive (S) cones. Axons of these bipolar cells made contacts with amacrine cell dendrites containing secretoneurin. Secretoneurin-IR amacrine cells also made contacts with retinal ganglion cell dendrites labeled with antibody to the photopigment melanopsin, which have OFF responses to stimulation of S cones. Using electron microscopic immunolabeling, 436 synapses from macaque retina were analyzed. Axons from bipolar cells were identified by their characteristic synaptic ribbons; their synaptic densities were asymmetric like those of excitatory synapses in the brain. Amacrine cells made and received conventional synapses with symmetric synaptic densities, like those of inhibitory synapses in the brain. Ganglion cell dendrites were identified by their absence of presynaptic specializations; they received inputs from both amacrine cells and bipolar cells. The majority of inputs to the secretoneurin-IR amacrine cells were from other amacrine cells, but they also received 21% of their input from bipolar cells. They directed most of their output, 54%, to amacrine cells, but there were many synapses onto bipolar cell axons and ganglion cell dendrites, as well. The synaptic connections were very similar in the three plexuses with one notable exception; output synapses to bipolar cells were significantly less common in the innermost one, where the S-ON bipolar cells terminate. Taken together, these findings suggest that the secretoneurin-IR amacrine cells in primates receive excitatory input from S-ON bipolar cells and, in turn, inhibit intrinsically photosensitive retinal ganglion cells.

Fecal chromogranins and secretogranins are increased in patients with ulcerative colitis but are not associated with disease activity.

BACKGROUND: Little is known of the importance of chromogranins (Cg) and secretogranins (Sg) in ulcerative colitis (UC). We therefore investigated fecal levels of CgA, CgB, SgII and SgIII, and their association with inflammatory activity, disease duration and medical therapy in UC. METHODS: Analyses of CgA, CgB, SgII, SgIII and calprotectin in stool samples from 41 UC patients and 29 healthy controls were performed. Two stool samples, during relapse and remission, respectively, were obtained from each UC patient. RESULTS: The levels of fecal CgA and SgII were higher in UC patients with active disease as compared to healthy controls. CgB and SgII were positively correlated with disease duration, but none of the granins were positively correlated with calprotectin, Mayo score, CRP or serum concentrations of TNF in UC patients with active disease. Also UC patients in remission had higher levels of CgA, CgB, SgII, and SgIII as compared to healthy controls. However, levels of fecal CgA, CgB, SgII and SgIII were lower during active disease relative to remission. Moreover, fecal levels of CgA and SgII were higher in UC patients in remission treated with thiopurines than in thiopurine-naïve patients in remission. CONCLUSION: Fecal chromogranins and secretogranins are increased in UC but are not associated with disease activity, but seem to increase with duration of the disease. Thus, fecal granins might reflect structural changes associated with chronicity of disease, or medical therapy.

Increased fecal levels of chromogranin A, chromogranin B, and secretoneurin in collagenous colitis.

Interactions between the enteric nervous system and the immune system are suggested to play an important role in the pathophysiology of inflammatory bowel disease (IBD). This study aims to determine if chromogranin A (CgA), chromogranin B (CgB), and secretoneurin (SN) are detectable in feces (F) from patients with collagenous colitis (CC) and to compare the levels found in patients with ulcerative colitis (UC) and Crohn's disease (CD) before and during treatment. Patients with CC (n = 12) were studied before and after 3, 7, 28, and 56 days of treatment. Patients with IBD (UC, n = 21; CD, n = 11) were studied before and after 28 and 56 days of treatment. Clinical data were recorded, and fecal samples were collected at each occasion. F-CgA, F-CgB, and F-SN were measured by RIA. Eleven patients with CC, 21 with UC, and 10 with CD achieved remission. On inclusion, CC patients had higher levels of F-CgA, F-CgB, and F-SN than patients with IBD and controls. Patients with IBD expressed markedly lower levels of F-SN than controls. During treatment, F-SN in CC patients decreased to control levels but remained low in IBD patients. No change was found in F-CgA or F-CgB in any of the groups. In conclusion, CgA, CgB, and SN are detectable in feces, and CC patients express higher values than patients with IBD and controls. During treatment, F-SN decreased to control levels in CC. These findings suggest that the enteric nervous system is clearly involved in the pathophysiology of CC.

Secretoneurin, substance P and neuropeptide Y in the oxygen-induced retinopathy in C57Bl/6N mice.

In this study, we investigated whether the proangiogenic neuropeptides secretoneurin (SN), substance P (SP), and neuropeptide Y (NPY) contribute to the development of abnormal neovascularization in the oxygen-induced retinopathy (OIR) model in mice. By exposing litters of C57Bl/6N mice to 75% oxygen from postnatal day 7 (P7) until postnatal day 11 (P11) and then returning them to normoxic conditions, retinal ischemia and subsequent neovascularization on the retinal surface were induced. Retinae were dissected on P9, P11, P12-P14, P16 and P20, and the concentrations of SN, SP, NPY and VEGF determined by radioimmunoassay or ELISA. The levels of SN and SP increased in controls from P9 until P16 and from P9 until P14, respectively, whereas the levels of NPY were high at P9 and decreased thereafter until P20, suggesting that NPY may participate in the development of the retina. However, dipeptidyl peptidase IV (DPPIV) and the NPY-Y2 receptor were not detectable in the immature retina indicating that NPY is not involved in the physiological vascularization in the retina. Compared to controls, OIR had no effect on the levels of SN, whereas levels of both SP and NPY slightly decreased during hyperoxia. Normalization of the levels of SP, and to a more pronounced extent of NPY, was significantly delayed during relative hypoxia. This clearly indicates that these three neuropeptides are not involved in the pathogenesis of neovascularization in OIR. Moreover, since there were no differences in the expression of two vessel markers in the retina of NPY knockout mice versus controls at P14, NPY is also not involved in the delayed development of the intermediate and deep vascular plexus in the retina in this animal model.

Chromogranins A and B and secretogranin II: evolutionary and functional aspects.

Chromogranins/secretogranins or granins are a class of acidic, secretory proteins that occur in endocrine, neuroendocrine, and neuronal cells. Granins are the precursors of several bioactive peptides and may be involved in secretory granule formation and neurotransmitter/hormone release. Characterization and analysis of chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) in distant vertebrate species confirmed that CgA and CgB belong to related monophyletic groups, probably evolving from a common ancestral precursor, while SgII sequences constitute a distinct monophyletic group. In particular, selective sequences within these proteins, bounded by potential processing sites, have been remarkably conserved during evolution. Peptides named vasostatin, secretolytin and secretoneurin, which occur in these regions, have been shown to exert various biological activities. These conserved domains may also be involved in the formation of secretory granules in different vertebrates. Other peptides such as catestatin and pancreastatin may have appeared late during evolution. The function of granins as propeptide precursors and granulogenic factors is discussed in the light of recent data obtained in various model species and using knockout mice strains.

Effects of a depot formulation of the GnRH agonist leuprorelin on the ultrastructure of male rat pituitary gonadotropes.

To clarify the acute and chronic effects of GnRH agonists on pituitary gonadotropes, changes in the ultrastructure of male rat gonadotropes were examined immunocytochemically and morphometrically after the administration of a one-month depot formulation of the GnRH agonist, leuprorelin. Immediately after the depot administration, the relative amounts of secretory granules drastically decreased in gonadotropes concomitantly with a marked increase in the plasma LH level. After the acute hyperstimulated phase, secretory granules in gonadotropes were gradually restored although the newly synthesized granules were less densely immunolabeled for LHbeta; their relative amounts and sizes were still significantly smaller than the controls after depot treatment for 28 days. Eighty-four days after the leuprorelin depot administration, however, the ultrastructural characteristics of pituitary gonadotropes appeared to recover as observed in controls: there were no significant differences in the relative amounts, sizes, and labeling densities for LHbeta of secretory granules, and the amounts of chromogranin A (CgA) and secretogranin II (SgII) were restored in secretory granules to control levels. When the rats were repeatedly treated with the leuprorelin depot at intervals of 4 weeks, the expression and intracellular storage levels of gonadotropins remained highly suppressed, judging from the labeling density for LHbeta. These findings suggest that the depot formulation of the GnRH agonist could suppress both the biosynthesis and release of gonadotropins for a month by synergistically depleting the intracellular storage of secretory granules at the onset of the treatment and by inducing the subsequent desensitization of the GnRH receptor signaling.