Phylogenetic conservatism in the presence of a neurotensin-related hexapeptide in neurons of globus pallidus

The vast majority of the pallidal neurons of the hamster, pigeon, caiman and turtle basal telencephalon were positively labeled by an antiserum against LANT-6, a neurotensin-like hexapeptide. In sharks also, LANT-6-positive neurons were observed in the apparent equivalent of the globus pallidus. These results, which imply the coexistence of a LANT-6-like peptide with gamma-aminobutyric acid (GABA) in pallidal neurons, suggest that a LANT-6-like peptide may be an important and evolutionarily conserved neurotransmitter/neuromodulator in pallidal neurons.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25594/1/0000138.pd

Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein

A LANT6-like substance that is distinct from neuromedin N is present in pallidal and striatal neurons in monkeys

The basal ganglia of rhesus and squirrel monkeys were examined using immunohistochemical techniques with antibodies against the neurotensin-related hexapeptides Lys8-Asn9-Neurotensin(8-13) (LANT6) and Neuromedin N. A high percentage of neurons in both segments of globus pallidus and many large neurons of the striatum were found to label for LANT6, but not Neuromedin N. Previous studies have shown that LANT6 or a LANT6-like substance is present in many pallidal neurons in a wide range of vertebrate species. The current results indicate that a LANT6-like substance that is distinct from Neuromedin N is also present in many pallidal neurons in primates. This raises the possibility that this substance may be involved in neurotransmission between the pallidum and its projection targets.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26572/1/0000111.pd

NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9

<p>Abstract</p> <p>Background</p> <p>Sustained neuroinflammation strongly contributes to the pathogenesis of pain. The clinical challenge of chronic pain relief led to the identification of molecules such as cytokines, chemokines and more recently matrix metalloproteinases (MMPs) as putative therapeutic targets. Evidence points to a founder member of the matricial CCN family, NOV/CCN3, as a modulator of these inflammatory mediators. We thus investigated the possible involvement of NOV in a preclinical model of persistent inflammatory pain.</p> <p>Methods</p> <p>We used the complete Freund's adjuvant (CFA)-induced model of persistent inflammatory pain and cultured primary sensory neurons for <it>in vitro </it>experiments. The mRNA expression of NOV and pro-inflammatory factors were measured with real-time quantitative PCR, CCL2 protein expression was assessed using ELISA, MMP-2 and -9 activities using zymography. The effect of drugs on tactile allodynia was evaluated by the von Frey test.</p> <p>Results</p> <p>NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. <it>In vitro</it>, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1β- and TNF-α-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-α-induced MMP-9 expression through β₁integrin engagement. <it>In vivo</it>, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia.</p> <p>Conclusions</p> <p>This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.</p

Detergent-free extraction of a functional low-expressing GPCR from a human cell line

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene–maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state