An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells.

Adenylyl cyclase (AC) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across AC isoforms, and reverse transcription-polymerase chain reaction (RT-PCR), coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca(2+), the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca(2+). Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate AC activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. Although a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC5/6, will down-modulate levels of cAMP, thus fine-tuning and gradating the hair-cell response to dopamine D1A. As predicted by the trout saccular hair cell model, evidence has been obtained for the first time that hair cells of mammalian otolithic vestibular end organs (rat/mouse saccule/utricle) express dopamine D1A and D2L receptors, and each receptor co-localizes with AC5/6, with a marked presence of all three proteins in subcuticular regions of type I vestibular hair cells. A putative efferent, presynaptic source of dopamine was identified in tyrosine hydroxylase-positive nerve fibers which passed from underlying connective tissue to the sensory epithelia, ending on type I and type II vestibular hair cells and on afferent calyces.

Eukaryotic initiation factor 2alpha kinase and phosphatase activity during postischemic brain reperfusion.

When ischemic brain is reperfused, there is in vulnerable neurons immediate inhibition of protein synthesis associated with a large increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 [eIF2alpha, phosphorylated form eIF2alpha(P)]. We examined eIF2alpha kinase and eIF2alpha(P) phosphatase activity in brain homogenate postmitochondrial supernatants obtained from rats after 3 to 30 min of global brain ischemia (cardiac arrest), after 5 min of ischemia and 5 min of reperfusion (5R), and after 10 min of ischemia and 90 min reperfusion (90R). Because it has been suggested that PKR might be specifically responsible for producing eIF2alpha(P) during reperfusion, we also examined in brain homogenates from wild-type and PKR0/0 C57BL/6J x 129/SV mice the effect of 5 min of ischemia and 5 min of reperfusion on eIF2alpha(P). Cytosolic brain eIF2alpha(P) in the 5R and 90R rats was 18- and 23-fold that of nonischemic controls without any change in the rate of eIF2alpha(P) dephosphorylation. There was no change in eIF2alpha kinase activity between 3 and 30 min of ischemia but an 85% decrease in the 5R group; the 90R group was similar to controls. In wild-type and PKR0/0 mice total eIF2alpha was identical, and there was an identical 16-fold increase in eIF2alpha(P) at 5 min of reperfusion. Our observations contradict hypotheses that PKR activation, loss of eIF2alpha(P) phosphatase activity, or any general increase in eIF2alpha kinase activity are responsible for reperfusion-induced phosphorylation of eIF2alpha, and we suggest that the mechanism may involve regulation of the availability of eIF2alpha to a kinase.