Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues.

Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. Studies have shown that enkephalins play an important role in behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance. Our global hypothesis is that enkephalins are released from non-neuronal tissues in response to brief ischemia or exercise, and that this release contributes to cardioprotection. To identify tissues that could serve as potential sources of enkephalins, we used real-time PCR, Western blot analysis, ELISA, immunofluorescence microscopy, and ex vivo models of enkephalin release. We found widespread expression of preproenkephalin (pPENK) mRNA and production of the enkephalin precursor protein proenkephalin (PENK) in rat and mouse tissues, as well as in tissues and cells from humans and pigs. Immunofluorescence microscopy with anti-enkephalin antisera demonstrated immunoreactivity in rat tissues, including heart and skeletal muscle myocytes, intestinal and kidney epithelium, and intestinal smooth muscle cells. Finally, isolated tissue studies showed that heart, skeletal muscle, and intestine released enkephalins ex vivo. Together our studies indicate that multiple non-neuronal tissues produce PENK and release enkephalins. These data support the hypothesis that non-neuronal tissues could play a role in both local and systemic enkephalin-mediated effects.

Cytokine activation of nuclear factor kappa B in vascular smooth muscle cells requires signaling endosomes containing Nox1 and ClC-3.

Reactive oxygen species (ROS) are mediators of intracellular signals for a myriad of normal and pathologic cellular events, including differentiation, hypertrophy, proliferation, and apoptosis. NADPH oxidases are important sources of ROS that are present in diverse tissues throughout the body and activate many redox-sensitive signal transduction and gene expression pathways. To avoid toxicity and provide specificity of signaling, ROS production and metabolism necessitate tight regulation that likely includes subcellular compartmentalization. However, the constituent elements of NADPH oxidase-dependent cell signaling are not known. To address this issue, we examined cytokine generation of ROS and subsequent activation of the transcription factor nuclear factor kappaB in vascular smooth muscle cells (SMCs). Tumor necrosis factor-alpha and interleukin (IL)-1beta stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22(phox), integral membrane subunits of NADPH oxidase, coimmunoprecipitated with early endosomal markers in SMCs. ClC-3, an anion transporter that is primarily found in intracellular vesicles, also colocalized with Nox1 in early endosomes and was necessary for tumor necrosis factor-alpha and interleukin-1beta generation of ROS. Cytokine activation of nuclear factor kappaB in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-alpha and interleukin-1beta, NADPH oxidase generates ROS within early endosomes and that Nox1 cannot produce sufficient ROS for cell signaling in the absence of ClC-3. These data best support a model whereby ClC-3 is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signaling endosomes.

Altered GABAergic function accompanies hippocampal degeneration in mice lacking ClC-3 voltage-gated chloride channels.

Mice lacking ClC-3 chloride channels, encoded by the Clcn3 gene, undergo neurodegeneration of the hippocampal formation and retina [Neuron, 29 (2001) 185-196; Genes Cells, 7 (2002) 597-605]. We independently created a mouse lacking the Clcn3 gene which demonstrated similar central nervous system abnormalities, including early postnatal degeneration of retinal photoreceptors. However, we observed a characteristic spatial-temporal sequence of hippocampal neurodegeneration that differs from the pattern previously reported. Anterior-to-posterior degeneration and astrogliosis of the dentate gyrus and hippocampus progressed over months. Sequential loss of hippocampal neuronal subpopulations began in the dentate gyrus and progressed to CA3, followed by CA1 neurons. Projection neurons of the entorhinal cortex degenerated, secondary to the loss of their synaptic targets within the hippocampal formation. Other characteristics of the Clcn3(-/-) mice included an abnormal gait, kyphosis, and absence of hindlimb escape extension upon tail elevation. Spontaneous seizures were observed in four adult Clcn3(-/-) mice, and one mouse died during the event. We hypothesized that neuronal injury may be related to recurrent seizures. Clcn3(-/-) mice had normal serum electrolytes and pH, and exhibited neither hyperglycemia nor rebound hypoglycemia following a glucose load. They displayed a greatly reduced susceptibility to pentylenetetrazole-induced seizures and an abnormally prolonged sedation to benzodiazepines. There was no change in vulnerability to kainic acid-induced seizures. Immunostaining revealed a progressive loss of GABA synthesizing cells in the dentate gyrus. The death of these cells was preceded by increased GABA(A) receptor immunoreactivity. These data suggest that GABA(A) inhibitory neurotransmission is altered in Clcn3(-/-) mice. The increase in GABA(A) receptor density may represent a compensatory response either to chronic excessive excitatory stimuli or reduced inhibitory input from local GABAergic interneurons within the dentate gyrus.