Insulin induces cobalt uptake in a subpopulation of rat cultured primary sensory neurons

Abstract

Previous findings show that both the vanilloid receptor 1 and the insulin receptor are expressed on small primary sensory neurons. As insulin evokes activity in second messengers which could induce opening of the vanilloid receptor 1, we examined, by using the cobalt-uptake technique, whether or not insulin can activate cultured rat primary sensory neurons through activating the vanilloid receptor 1. Capsaicin (50, 100 and 500 nm) induced concentration-dependent labelling in primary sensory neurons. Preincubation of cells in insulin (10 micromoles) for 10 min followed by a 2-min wash did not produce significant change in the capsaicin-induced labelling. Coapplication of insulin (10 micromoles) with capsaicin, however, potentiated the 50 and 100 nm capsaicin-evoked staining. Insulin itself also produced cobalt labelling in a concentration-dependent manner. The size-frequency distributions of neurons showing capsaicin- or insulin-induced cobalt accumulation were similar. The insulin-induced cobalt labelling was significantly reduced by the tyrosine kinase inhibitor, tyrphostin AG1024, the vanilloid receptor 1 antagonists, ruthenium red and capsazepine, the protein kinase inhibitor, staurosporine and the phospholipase C inhibitor neomycin. Double immunostaining of cultured primary sensory neurons and sections from dorsal root ganglia revealed that about one-third of the cells coexpress the insulin receptor and vanilloid receptor 1. These findings suggest that insulin activates a subpopulation of primary sensory neurons, probably through phosphorylation- and/or phosphatidylinositol(4,5)biphosphate hydrolysis-evoked activation of the vanilloid receptor 1. Although the insulin-induced activation of vanilloid receptor 1 seems to be a short-lived effect in vitro, in vivo it might play a role in the development of burning pain sensation in hyperinsulinism.

Localization of the Endocannabinoid-Degrading Enzyme Fatty Acid Amide Hydrolase in Rat Dorsal Root Ganglion Cells and Its Regulation after Peripheral Nerve Injury

“…TRPV1-mediated activation of cultured DRG neurons was assessed by cobalt uptake, as described previously (Sathianathan et al, 2003). Coverslips were washed for 2 min in buffer solution (in mM: 57.5 NaCl, 5 KCl, 2 MgCl 2 , 10 HEPES, 12 glucose, and 139 sucrose, pH 7.4) and then preincubated for 5 min in buffer containing one of the following: the FAAH enzyme inhibitor URB597 (cyclohexylcarbamic acid 3-carbamoyl biphenyl-3-yl ester) (Mor et al, 2004;Fegley et al, 2005); buffer solution only (control); SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide] (CB 1 antagonist); URB597 ϩ SR141716; or URB597 and the TRPV1 antagonist capsazepine (Table 1).…”

“…After background subtraction, images of labeled and nonlabeled cells were captured in grayscale, and their average optical intensity was measured within defined cell areas using QWIN image analysis software. Frequency distributions of optical intensity data (representing Co-ppt levels in Ͼ100 cells after each active or vehicle treatment) were fitted to Gaussian curves to determine the intensity threshold for Co-ppt-positive cells, as described previously (Sathianathan et al, 2003). Negative and positive control experiments were performed for each culture, in which control buffer or 10 M capsaicin was added to the cobalt-containing buffer, respectively.…”

Anandamide produced by Ca2+-insensitive enzymes induces excitation in primary sensory neurons

“…Cobalt uptake was assessed as described [24,34,38,39]. Briefly, cells attached to the coverslips were washed in a buffer containing (in mM): NaCl 57.5, ethanol and the coverslips were mounted on glass slides with glycerol.…”

“…Briefly, cells attached to the coverslips were washed in a buffer containing (in mM): NaCl 57.5, ethanol and the coverslips were mounted on glass slides with glycerol. The mean gray value of more than 100 cells which were chosen randomly but systemically was established by a Leica light microscope attached to a PC running the QWin software package (Leica), and analysed with the ImageJ software package (NIH, USA) as described [24,34,38,39].…”

TRPV1 Gates Tissue Access and Sustains Pathogenicity in Autoimmune Encephalitis

“…To use abundant glucose, the PANEM complex must sustain high volume one-way traffic of insulin that makes the PANEM complex an insulin-rich milieu comparable to some extent to pancreatic islets. Insulin receptor ligation by TRPV1 + terminals raises TRPV1 currents, resetting activation thresholds to room temperature (50,51). Thus, there is potential for TRPV1 activation outside of its normal temperature constraints where specific TRPV1 alleles may confer a risk scenario.…”