John W. Bigbee

The mammalian amiloride-insensitive non-specific salt taste receptor is a vanilloid receptor-1 variant

The amiloride‐insensitive salt taste receptor is the predominant transducer of salt taste in some mammalian species, including humans. The physiological, pharmacological and biochemical properties of the amiloride‐insensitive salt taste receptor were investigated by RT‐PCR, by the measurement of unilateral apical Na+ fluxes in polarized rat fungiform taste receptor cells and by chorda tympani taste nerve recordings. The chorda tympani responses to NaCl, KCl, NH4Cl and CaCl2 were recorded in Sprague‐Dawley rats, and in wild‐type and vanilloid receptor‐1 (VR‐1) knockout mice. The chorda tympani responses to mineral salts were monitored in the presence of vanilloids (resiniferatoxin and capsaicin), VR‐1 antagonists (capsazepine and SB‐366791), and at elevated temperatures. The results indicate that the amiloride‐insensitive salt taste receptor is a constitutively active non‐selective cation channel derived from the VR‐1 gene. It accounts for all of the amiloride‐insensitive chorda tympani taste nerve response to Na+ salts and part of the response to K+, NH4+ and Ca2+ salts. It is activated by vanilloids and temperature (> 38°C), and is inhibited by VR‐1 antagonists. In the presence of vanilloids, external pH and ATP lower the temperature threshold of the channel. This allows for increased salt taste sensitivity without an increase in temperature. VR‐1 knockout mice demonstrate no functional amiloride‐insensitive salt taste receptor and no salt taste sensitivity to vanilloids and temperature. We conclude that the mammalian non‐specific salt taste receptor is a VR‐1 variant.

Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

Two pole air gap electrospinning: Fabrication of highly aligned, three-dimensional scaffolds for nerve reconstruction

We describe the structural and functional properties of three-dimensional (3D) nerve guides fabricated from poly-ε-caprolactone (PCL) using the air gap electrospinning process. This process makes it possible to deposit nano-to-micron diameter fibers into linear bundles that are aligned in parallel with the long axis of a cylindrical construct. By varying starting electrospinning conditions it is possible to modulate scaffold material properties and void space volume. The architecture of these constructs provides thousands of potential channels to direct axon growth. In cell culture functional assays, scaffolds composed of individual PCL fibers ranging from 400 to 1500 nm supported the penetration and growth of axons from rat dorsal root ganglion. To test the efficacy of our guide design we reconstructed 10mm lesions in the rodent sciatic nerve with scaffolds that had fibers 1 μm in average diameter and void volumes >90%. Seven weeks post implantation, microscopic examination of the regenerating tissue revealed dense, parallel arrays of myelinated and non-myelinated axons. Functional blood vessels were scattered throughout the implant. We speculate that end organ targeting might be improved in nerve injuries if axons can be directed to regenerate along specific tissue planes by a guide composed of 3D fiber arrays.

Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth

Acetylcholinesterase (AChE) can promote neurite outgrowth through a mechanism that is independent of its role in hydrolyzing the neurotransmitter acetylcholine. It has been proposed that this neuritogenic capacity of AChE may result from its intrinsic capacity to function in adhesion. In this study we directly tested this hypothesis using neuroblastoma cell lines that have been engineered for altered cell‐surface expression of AChE. Using a microtiter‐plate adhesion assay and the electrical cell‐substrate impedance‐sensing (ECIS) method, we demonstrate that the level of cell‐substratum adhesion of these cells directly correlates with their level of AChE expression. Furthermore, this adhesion is blocked by either an anti‐AChE antibody or a highly specific AChE inhibitor (BW284c51), both of which have also been shown to block neurite outgrowth. In addition, cells that overexpress AChE showed enhanced neurite initiation. By employing cell lines with different levels of AChE expression in two types of cell‐substratum adhesion assays, our current studies provide evidence for an adhesive function for AChE. These results, together with the fact that AChE shares sequence homology and structural similarities with several known cell adhesion molecules, support the hypothesis that AChE promotes neurite outgrowth, at least in part, through an adhesive function. J. Neurosci. Res. 63:165–175, 2001.

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.

Opioid Addiction and Pregnancy: Perinatal Exposure to Buprenorphine Affects Myelination in the Developing Brain

Buprenorphine is a μ‐opioid receptor partial agonist and κ‐opioid receptor antagonist currently on trials for the management of pregnant opioid‐dependent addicts. However, little is known about the effects of buprenorphine on brain development. Oligodendrocytes express opioid receptors in a developmentally regulated manner and thus, it is logical to hypothesize that perinatal exposure to buprenorphine could affect myelination. To investigate this possibility, pregnant rats were implanted with minipumps to deliver buprenorphine at 0.3 or 1 mg/kg/day. Analysis of their pups at different postnatal ages indicated that exposure to 0.3 mg/kg/day buprenorphine caused an accelerated and significant increase in the brain expression of all myelin basic protein (MBP) splicing isoforms. In contrast, treatment with the higher dose caused a developmental delay in MBP expression. Examination of corpus callosum at 26‐days of age indicated that both buprenorphine doses cause a significant increase in the caliber of the myelinated axons. Surprisingly, these axons have a disproportionately thinner myelin sheath, suggesting alterations at the level of axon‐glial interactions. Analysis of myelin associated glycoprotein (MAG) expression and glycosylation indicated that this molecule may play a crucial role in mediating these effects. Co‐immunoprecipitation studies also suggested a mechanism involving a MAG‐dependent activation of the Src‐family tyrosine kinase Fyn. These results support the idea that opioid signaling plays an important role in regulating myelination in vivo and stress the need for further studies investigating potential effects of perinatal buprenorphine exposure on brain development.

Morphological and proliferative responses of cultured Schwann cells following rapid phagocytosis of a myelin-enriched fraction

SummaryCultured Schwann cells were found to phagocytose exogenously applied myelin membranes within 1 h. However, the resulting proliferative response required an additional 9 h of incubation. Treatment with ammonium chloride, a lysosomal inhibitor, delayed the appearance of the proliferative response to the myelin membranes by 12 h. Processing of myelin within the Schwann cells was followed by the appearance of immunocytochemically detectable myelin basic protein which was first visible at 4 h. Similar to the proliferative response, the appearance of immunoreactive material was delayed by the addition of ammonium chloride. Schwann cells were observed initially to ingest myelin fragments at their distal-most tips after which time the myelin phagosomes collected in the perinuclear region and fused with lysosomes. Phagocytic Schwann cells had a notable increase in Golgi membranes and microfilaments and contained widely dilated, rough endoplasmic reticulum cisternae. In purified cell cultures, Schwann cells phagocytosed myelin slower than macrophages, but displayed phagocytic abilities much greater than fibroblasts. The ability of cultured Schwann cells to phagocytose myelin rapidly suggests that these cells may aid in the breakdown and removal of myelin during Wallerian degeneration. These data further confirm the mitogenic effect of myelin and its possible role during nerve regeneration.

Metabolic Gene Remodeling and Mitochondrial Dysfunction in Failing Right Ventricular Hypertrophy Secondary to Pulmonary Arterial Hypertension

Background— Right ventricular (RV) dysfunction (RVD) is the most frequent cause of death in patients with pulmonary arterial hypertension. Although abnormal energy substrate use has been implicated in the development of chronic left heart failure, data describing such metabolic remodeling in RVD remain incomplete. Thus, we sought to characterize metabolic gene expression changes and mitochondrial dysfunction in functional and dysfunctional RV hypertrophy. Methods and Results— Two different rat models of RV hypertrophy were studied. The model of RVD (SU5416/hypoxia) exhibited a significantly decreased gene expression of peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr;, peroxisome proliferator-activated receptor-&agr; and estrogen-related receptor-&agr;. The expression of multiple peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; target genes required for fatty acid oxidation was similarly decreased. Decreased peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr; expression was also associated with a net loss of mitochondrial protein and oxidative capacity. Reduced mitochondrial number was associated with a downregulation of transcription factor A, mitochondrial, and other genes required for mitochondrial biogenesis. Electron microscopy demonstrated that, in RVD tissue, mitochondria had abnormal shape and size. Lastly, respirometric analysis demonstrated that mitochondria isolated from RVD tissue had a significantly reduced ADP-stimulated (state 3) rate for complex I. Conversely, functional RV hypertrophy in the pulmonary artery banding model showed normal expression of peroxisome proliferator-activated receptor-&ggr; coactivator-1&agr;, whereas the expression of fatty acid oxidation genes was either preserved or unregulated. Moreover, pulmonary artery banding-RV tissue exhibited preserved transcription factor A mitochondrial expression and mitochondrial respiration despite elevated RV pressure-overload. Conclusions— Right ventricular dysfunction, but not functional RV hypertrophy in rats, demonstrates a gene expression profile compatible with a multilevel impairment of fatty acid metabolism and significant mitochondrial dysfunction, partially independent of chronic pressure-overload.

Evaluating neuronal and glial growth on electrospun polarized matrices: bridging the gap in percussive spinal cord injuries

One of the many obstacles to spinal cord repair following trauma is the formation of a cyst that impedes axonal regeneration. Accordingly, we examined the potential use of electrospinning to engineer an implantable polarized matrix for axonal guidance. Polydioxanone, a resorbable material, was electrospun to fabricate matrices possessing either aligned or randomly oriented fibers. To assess the extent to which fiber alignment influences directional neuritic outgrowth, rat dorsal root ganglia (DRGs) were cultured on these matrices for 10 days. Using confocal microscopy, neurites displayed a directional growth that mimicked the fiber alignment of the underlying matrix. Because these matrices are generated from a material that degrades with time, we next determined whether a glial substrate might provide a more stable interface between the resorbable matrix and the outgrowing axons. Astrocytes seeded onto either aligned or random matrices displayed a directional growth pattern similar to that of the underlying matrix. Moreover, these glia-seeded matrices, once co-cultured with DRGs, conferred the matrix alignment to and enhanced outgrowth exuberance of the extending neurites. These experiments demonstrate the potential for electrospinning to generate an aligned matrix that influences both the directionality and growth dynamics of DRG neurites.

Acetylcholinesterase antibody treatment results in neurite detachment and reduced outgrowth from cultured neurons: further evidence for a cell adhesive role for neuronal acetylcholinesterase.

Data from our laboratory and others demonstrate that acetylcholinesterase (AChE) is expressed transiently by neurons during periods of neurite outgrowth preceding synaptogenesis, suggesting an extrasynaptic function for this molecule. These findings, along with reports that AChE shares amino acid sequence homology and structural similarities with known cell adhesion molecules, have led to the theory that, during development, AChE may exert a morphogenic effect through cell adhesion. To further test this hypothesis, we have examined the effects of an AChE monoclonal antibody (MAB304) on neurite outgrowth in primary cultures of rat dorsal root ganglion (DRG) neurons. Short‐term, high‐concentration antibody treatment produced a rapid detachment of established DRG neurites, which was followed by regrowth upon removal of the antibody from the culture medium. This effect appeared to be site‐specific, because other AChE antibodies that were able to detect AChE immunocytochemically failed to produce this disadhesion. Long‐term, low‐concentration antibody exposure produced a 50% reduction in total area of outgrowth, in which neurites were more densely packed and interlaced compared with the neurites in control cultures. These results extend our previous observations on the outgrowth perturbing effects of AChE inhibitor treatment and provide further evidence that AChE may support neurite outgrowth through a cell adhesive role. J. Neurosci. Res. 53:454–464, 1998.