Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion.

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.

Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord.

Peripheral axotomy-induced sprouting of thick myelinated afferents (A-fibers) from laminae III-IV into laminae I-II of the spinal cord is a well-established hypothesis for the structural basis of neuropathic pain. However, we show here that the cholera toxin B subunit (CTB), a neuronal tracer used to demonstrate the sprouting of A-fibers in several earlier studies, also labels unmyelinated afferents (C-fibers) in lamina II and thin myelinated afferents in lamina I, when applied after peripheral nerve transection. The lamina II afferents also contained vasoactive intestinal polypeptide and galanin, two neuropeptides mainly expressed in small dorsal root ganglion (DRG) neurons and C-fibers. In an attempt to label large DRG neurons and A-fibers selectively, CTB was applied four days before axotomy (pre-injury-labelling), and sprouting was monitored after axotomy. We found that only a small number of A-fibers sprouted into inner lamina II, a region normally innervated by C-fibers, but not into outer lamina II or lamina I. Such sprouts made synaptic contact with dendrites in inner lamina II. Neuropeptide Y (NPY) was found in these sprouts in inner lamina II, an area very rich in Y1 receptor-positive processes. These results suggest that axotomy-induced sprouting from deeper to superficial layers is much less pronounced than previously assumed, in fact it is only marginal. This limited reorganization involves large NPY immunoreactive DRG neurons sprouting into the Y1 receptor-rich inner lamina II. Even if quantitatively small, it cannot be excluded that this represents a functional circuitry involved in neuropathic pain.

Expression of Reverse Cholesterol Transport Pathway Associated Protein Genes in Skeletal Muscle.

Viral and nonviral vectors containing apoAI, apoE or LCAT genes were constructed and transfected into myogenic cells in vitro or injected directly into mouse skeletal muscle. The expression efficiencies of these vectors were assaied to investigate the possibility of ectopic expression of these genes in skeletal muscle and to develop a safe and convenient gene therapy method for atherosclerosis. The results showed that the primary cultured mouse myoblasts, C2C12 cells transfected with pCMVapoE3 expressed human apoE3 successfully and the expressed product was secreted into the medium. Mouse skeletal muscle efficiently expressed apoE3 in vivo after direct plasmid injection. The expression level of Ad-RSV-apoAI in primary cultured mouse myoblasts was correlated with virus titer. Human apoAI was synthesized in mouse skeletal muscle by direct injection of recombinant virus and was secreted into blood continuously up to 30 days functional LCAT was expressed by C2C12 and 293 cells transfected with conventional vector or recombinant AAV plasmid DNA. The expression efficiency of recombinant AAV plasmid DNA was 2-5 times higher than that of conventional plasmid vector. The above results provided experimental data for further studying and developing a gene therapy method for atherosclerosis by enhancement of reverse cholesterol transport using skeletal muscle as target.