Car8 dorsal root ganglion expression and genetic regulation of analgesic responses are associated with a cis-eQTL in mice

Abstract

Carbonic anhydrase-8 (Car8 mouse gene symbol) is devoid of enzymatic activity, but instead functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) to regulate this intracellular calcium release channel important in synaptic functions and neuronal excitability. Causative mutations in ITPR1 and carbonic anhydrase-8 in mice and humans are associated with certain subtypes of spinal cerebellar ataxia (SCA). SCA mice are genetically deficient in dorsal root ganglia (DRG) Car8 expression and display mechanical and thermal hypersensitivity and susceptibility to subacute and chronic inflammatory pain behaviors in mice. In this report we show that DRG Car8 expression is variable across 25 naive-inbred strains of mice, and this cis-regulated eQTL (association between rs27660559, rs27706398, and rs27688767 and DRG Car8 expression; P<1×10−11) is correlated with nociceptive responses in mice. Next, we hypothesized that increasing DRG Car8 gene expression would inhibit intracellular calcium release required for morphine antinociception and might correlate with antinociceptive sensitivity of morphine and perhaps other analgesic agents. We show that mean DRG Car8 gene expression is directly related to the dose of morphine or clonidine needed to provide a half-maximal analgesic response (r=0.93, P<0.00002; r=0.83, P<0.0008, respectively), suggesting that greater DRG Car8 expression increases analgesic requirements. Finally, we show that morphine induces intracellular free calcium release using Fura 2 calcium imaging in a dose-dependent manner; and V5-Car8WT overexpression in NBL cells inhibits morphine-induced calcium increase. These findings highlight the ‘morphine paradox’ whereby morphine provides antinociception by increasing intracellular free calcium, while Car8 and other antinociceptive agents work by decreasing intracellular free calcium. This is the first study demonstrating that biologic variability associated with this cis-eQTL may contribute to differing analgesic responses through altered regulation of ITPRI-dependent calcium release in mice.

Impact of human CA8 on thermal antinociception in relation to morphine equivalence in mice

“…In contrast, we demonstrated that murine AAV8-V5-Car8WT showed inhibition of ITPR1-mediated cytosolic calcium release in conjunction with decreased thermal hypersensitivity 6 . We also found that greater DRG Car8 expression antagonizes morphine analgesia concomitant with regulation of morphine-induced ITPR-mediated calcium release 8 . The disadvantage of μ opioids, such as of morphine, is the development of analgesic tolerance seen after morphine administration 14 , which may involve pathways that increase intracellular calcium release such as IP3 binding to IP3 receptors 17 .…”

“…We constructed vectors containing the WT CA8 cDNA with a V5 tag ( V5-CA8WT ) and CA8 MT cDNA sequence (S100P), which served as a negative control ( V5-CA8MT ) 8 , 9 . We assessed the effects of V5-CA8 overexpression on thermal nociceptive thresholds for 19 days after SN injection and DRG transduction before and after carrageenan injection and inflammation in the hindpaw of naive C57BL/6J mice (Fig.…”

“…The Car8 protein is known to inhibit inositol 1,4,5-triphosphate receptor-1 (ITPR1) to decrease calcium release 6 , whereas morphine depends on increased ITPR1-dependent calcium release to produce analgesia 7 . In another study, we addressed the morphine ‘paradox’ in which we found that murine DRG Car8 gene expression is highly variable across inbred strains of mice and genetically regulates the analgesic effects of morphine in an antagonistic manner 8 . We surmised that the antagonism between Car8 expression and morphine may be related to their opposing effects on ITPR1-mediated calcium release 8 .…”

“…In another study, we addressed the morphine ‘paradox’ in which we found that murine DRG Car8 gene expression is highly variable across inbred strains of mice and genetically regulates the analgesic effects of morphine in an antagonistic manner 8 . We surmised that the antagonism between Car8 expression and morphine may be related to their opposing effects on ITPR1-mediated calcium release 8 .…”

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice

“…Twenty-four hours before assay, 100K to 250K cells/mL in 1 or 0.5 mL 12 or 24-well plates respectively and then split onto 15 mm glass coverslips (Propper, Long Island, NY) coated with poly-lysine overnight and laminin for 2 h at RT. On the day of the assay, cells were rinsed with perfusate buffer (Buffer 1), 85 then incubated in Buffer 1 containing 0.1 μM Fura-2/AM (Molecular Probes) and 0.012% pluronic F-127 (Sigma) for 45 min at RT (21-22OC) and washed twice with Buffer 1. After another 10 min incubation in Buffer 1 at RT, coverslips were transferred to a RC-42 LP open bath chamber in the QE-1 platform, loaded onto a Leica DMI 6000 B inverted microscope and perfused with Ca2+ free Buffer 2 (Buffer 2, all concentrations in mM: 130 NaCl; 4.7 KCl; 2.3 MgSO4; 5 Glucose; 20 HEPES; 10 EGTA; 1.2 KH2PO4).…”