PACAP in adult sensory neurons - implications in injury and pain

The data presented in this thesis deal with the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) in adult sensory neurons. We have used different injury and pain models to determine the involvement of PACAP during such conditions. The role of neurotrophins for PACAP expression was also investigated. PACAP is one of the excitatory neuropeptides expressed in small sized DRG neurons. The level of PACAP expression in such neurons is increased following peripheral inflammation but is decreased following sciatic nerve injury and instead induced in medium to large sized neurons. Data showed that under normal conditions and upon peripheral inflammation PACAP mRNA was expressed in trkA positive DRG L5 neurons. Intrathecally applied NGF dramatically increased both the number and the level of PACAP mRNA expression in small to medium sized neurons. The increased expression of PACAP after peripheral inflammation was inhibited by anti-NGF treatment. Hence, NGF seems to be a strong positive regulator of PACAP expression and appears to be responsible for the increased levels of PACAP expression during inflammation. In the formalin test, mice lacking the PACAP preferring receptor, PAC1 had a profoundly decreased nociceptive response in the late, inflammatory phase suggesting that the PAC1 receptor is important in the mediation of inflammatory pain. Intrathecally applied NT-3 reduced PACAP expression in intact neurons. Thus, while NGF appears to promote PACAP expression NT-3 appears to antagonise it. The dramatic increase of PACAP expression following nerve injury occurs in trkC positive, medium to large sized neurons and can be effectively mitigated by NT-3 and to a lesser degree by NGF. PAC1-/- mice had greater injury-induced changes in the expression of other neuropeptides, especially galanin, implying neuroregulatory functions of the PAC1 receptor after injury

Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation

Expression of pituitary adenylate cyclase-activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT-3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4-L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT-3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT-3 infusion, suggesting a role for NT-3 in returning the 'injured phenotype' back towards an 'Intact phenotype'. NGF dramatically up-regulated PACAP expression in trkA-positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT-3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT-3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small-medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons

Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-[beta H1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients

Comparing the impact of conventional pesticide and use of a transgenic pest-resistant crop on the beneficial carabid beetle Pterostichus melanarius

The potential impact of a chemical pesticide control method has been compared with that of transgenic plants expressing a protease inhibitor conferring insect resistance by utilising a tritrophic system comprising the crop plant Brassica napus (L.) (Oilseed rape), the pest mollusc Deroceras reticulatum (Müller) and the predatory carabid beetle Pterostichus melanarius (Illiger). Cypermethrin, as the most widely used pesticide in UK oilseed rape (OSR) cultivation, was selected as the conventional treatment. OSR expressing a cysteine protease inhibitor, oryzacystatin-1 (OC-1), was the transgenic comparator. In feeding trials, D. reticulatum showed no significant long-term effects on measured life history parameters (survival, weight gain, food consumption) as a result of exposure to either the cypermethrin or OC-1 treatment. However, D. reticulatum was able to respond to the presence of the dietary inhibitor by producing two novel proteases following exposure to OC-1-expressing OSR. Similarly, P. melanarius showed no detectable alterations in mortality, weight gain or food consumption when feeding on D. reticulatum previously fed either pesticide-contaminated or GM plant material. Furthermore, as with the slug, a novel form of protease, approximately M(r) 27 kDa, was induced in the carabid in response to feeding on slugs fed OC-1-expressing OSR

Clinical and biochemical characteristics of study participants and correlations between characteristics and the secretagogin (SCGN) level.

<p>Clinical and biochemical characteristics of study participants and correlations between characteristics and the secretagogin (SCGN) level.</p

Secretagogin was released from human beta cells after induction of cellular stress.

<p>(a) Secretagogin release, (b) insulin secretion and (c) intracellular secretagogin level of EndoC-βH1 cells treated for 24h with stress inducers, either tunicamycin (10 μg/ml), thapsigargin (1 μM) or cytokine cocktail (IFN-γ (40 ng/ml), IL1-β (20 ng/ml), TNF-α (40 ng/ml)). All inducers were dissolved in DMSO (1:1000) and control cells were incubated in DMSO (1:1000). Data are presented as mean±SD, statistical differences were calculated using one-way ANOVA analysis, n = 4. **** <i>p</i><0.0001.</p

Plasma level of human secretagogin was increased as a result of human islet transplant failure in mice.

<p>(a) Plasma glucose levels in human islet-transplanted nu/nu mice. Mice (n = 7) were injected with alloxan to ablate the mouse beta cells, at day -3, followed by transplantation with human islets (1000 IEQ) under the kidney capsule at day 0. Plasma glucose level was monitored over 40 days. (b) Human secretagogin level (c) and human C-peptide level were measured in mouse plasma of both normoglycemic and hyperglycemic mice 40 days after human islet transplantation. Data are presented as mean±SD and statistical differences were calculated using Student's unpaired t-test.</p