Protein kinases regulate glycine receptor binding in brain stem auditory nuclei after unilateral cochlear ablation

Abstract

Glycinergic synaptic inhibition is part of acoustic information processing in brain stem auditory pathways and contributes to the regulation of neuronal excitation. We found previously that unilateral cochlear ablation (UCA) in young adult guinea pigs decreased [ 3 H]strychnine binding activity in several brain stem auditory nuclei. This study determined if the UCA-induced deficit could be regulated by protein kinase C (PKC), protein kinase A (PKA) or Ca 2+ /calmodulin-dependent protein kinase II (CaMKII). The specific binding of [ 3 H]strychnine was measured in slices of the dorsal (DCN), posteroventral (PVCN) and anteroventral (AVCN) cochlear nucleus (CN), the lateral (LSO) and medial (MSO) superior olive, and the inferior colliculus (IC) 145 days after UCA. Tissues from age-matched unlesioned animals served as controls. UCA induced deficits in specific binding in the AVCN, PVCN and LSO on the ablated side and in the MSO bilaterally. These deficits were reversed by 3 μM phorbol 1,2-dibutyrate, a PKC activator, or 0.2 mM dibutyryl-cAMP, a PKA activator. However, 50 nM Ro31-8220, a PKC inhibitor, and 2 μM H-89, a PKA inhibitor, had no effect in unlesioned controls and after UCA. In contrast, 4 μM KN-93, a CaMKII inhibitor, relieved or reversed the UCA-induced binding deficits and elevated binding in the IC. These findings suggest a UCA-induced down regulation of glycine receptor synthesis may have occurred via reduced phosphorylation of proteins that control receptor synthesis; this effect was reversed by diminishing CaMKII activity or increasing PKC and PKA activity.

Mechanisms of Glycine Release in Mouse Brain Stem Slices

“…Efflux rate constants (910 -3 min -1 ) ± SEM k 1 k 2 k 2 (50 mM K + ) Basal (control) 3.64 ± 0.14 [41] 3.15 ± 0.12 [16] 6.84 ± 0.33 [16] t-ACPD 3.70 ± 0.24 [8] 3.30 ± 0.20 [4] 6.08 ± 0.67 [4] DHPG 4.72 ± 0.37** [8] 3.89 ± 0.08** [4] 7.35 ± 0.73 [8] + AIDA 3.91 ± 0.07 [4] 3.13 ± 0.14* [4] 7.52 ± 0.42 [6] 2R,4R-APDC 3.81 ± 0.29 [8] 2.95 ± 0.16 [4] 8.10 ± 0.62 [4] L-AP4 3.96 ± 0.33 [7] 3.10 ± 0.40 [4] 6.94 ± 0.38 [4] L-SOP 4.77 ± 0.29** [8] 3.74 ± 0.39** [4] 10.52 ± 1.12** [4] + CPPG 3.36 ± 0.32*** [4] 3.00 ± 0.18 [4] 6.03 ± 0.48 [12] AIDA 3.80 ± 0.26 [8] 3.42 ± 0.29 [4] 6.41 ± 0.37 [4] CPPG 3.88 ± 0.20 [8] 3.29 ± 0.31 [4] 6.07 ± 0.61 [4] The slices were preloaded for 30 min with 10 lM stimulatory effect of glycine further confirms the involvement of transporters in the release. In Na + -free media the transporters were not functional, and accordingly, addition of extracellular glycine caused no effect.…”

“…Table 1 Effects of ion channel inhibitors and compounds involved in the second messenger systems on glycine release from mouse brain stem slices Concentration (mM) Efflux rate constants (910 -3 min -1 ) ± SEM k 1 k 2 k 2 (50 mM K + ) Basal (control) 3.64 ± 0.14 [41] 3.15 ± 0.12 [16] 6.84 ± 0.33 [16] DIDS 0.5 3.62 ± 0.13 [22] 3.11 ± 0.14 [11] 6.73 ± 0.57 [11] SITS 2.0 3.02 ± 0.25* [16] 2.44 ± 0.29* [8] 6.69 ± 0.32 [8] 9-AC 0. 2 3.23 ± 0.20 [8] 2.67 ± 0.14 [4] 5.37 ± 0.40 [4] Riluzole 0.1 3.35 ± 0.22 [11] 2.75 ± 0.29 [4] 4.86 ± 0.70** [7] Genistein 0.001 3.41 ± 0.25 [16] 3.00 ± 0.17 [8] 4.11 ± 0.40** [7] Quinacrine The drugs were added at the beginning of the superfusion and 50 mM K + at 30 min. The results show the efflux rate constants ± SEM (910 -3 min -1 ) for the time intervals of 20-30 min (k 1 ) and for 32-40 min (k 2 ) with the number of independent experiments in parenthesis.…”

“…However, the properties of glycinergic pathways, release mechanisms and their regulation remain poorly characterized in this region. Glycinergic pathways have been implicated in the functions of many nuclei, for instance in the brain stem auditory nuclei [4][5][6][7], substantia nigra [8], inferior colliculus [9], locus coeruleus [10,11] and periaqueductal grey [12], thus participating in all vital functions in the brain stem.…”

Glycine and glycine receptor signalling in non-neuronal cells

“…Downstream events of such factor(s) could at least imply protein kinase activity. In that respect, it has been shown that, in the retina and in auditory nuclei, protein kinases modulate strychnine binding and, hence, GlyR expression (Salceda and Aguirre-Ramirez, 2005 ; Yan et al, 2007 ). Figure 1 summarizes the current findings on glycine and GlyR signalling in macroglial cells.…”

Modulators of the Inhibitory Glycine Receptor

“…Conformational changes induced by phosphorylation were delineated, , and phosphorylation was shown to affect the kinetics of GlyR channel activation and desensitization ,, as well as current amplitudes and single-channel conductance of recombinant GlyRs expressed in Xenopus oocytes. Phosphorylation-dependent changes on receptor surface expression and internalization ,,, and motility in the membrane were described. Phosphorylation modulates ethanol sensitivity of GlyRs − and GlyR-mediated nociceptive signaling. , Aspects of GlyR phosphorylation have been discussed and reviewed in detail. ,,,,, …”