Abstract
Calmodulin (CAL) is an ubiquitous calcium binding protein which regulares calcium-mediated cellular reactions. Using immunohistochemical techniques Slepecky et al [8] demostrated a selective accumulation of CAL in the inner and outer hair cells of the organ of Corti (OC). In this study we tried to quantitate the CAL levels in OC and its substructures for the purpose of estimating a possible role of CAL as a regulatory protein using quantitative polyacrylamide gel electrophoresis.
Tissues of the inner ear and other organs were obtained from pigmented guinea pigs and chinchillas. The temporal bones were quickly removed from the skull after decapitation under general anesthesia, followed by freeze-drying under vacuum for 96 hours at-40°C. Specimens of various inner ear structures including OC substructures were then dissected stereomicroscopically at 20°C and a relative humidity of 40% or less, and weighed on quartz fiber balances. Freeze-dried specimens ranging from approxiately 1μg to 7μEg dry weight and CAL standard series were subjected to two dimensional polyacrylamide gel electrophoresis (2-D PAGE) with isoelectric focusing in the first dimension and SDS gel electrophoresis in the second essentially by the method of O' Farrel [9]. These gels were followed by staining with silver by a modification of the method of Oakley [11]. The densities of identitied CAL spots were then measured with Laser densitometer and the CAL levels were calculated from the standard curve in each experiment.
The results of this study were summarized as follow. The CAL level in OC was estimated 2400 mg/kg wet weight, the highest among other inner ear structures or tissues analysed in guinea pig. This level reached as much as three times higher than that in the cerebral cortex or the cerebellum. The CAL level was very low in the stria vascularis and the Reisner's membrane. Among the substructures in OC of chinchilla it was found that the CAL level is by far the highest in the outer heir cells, approximately 6% of total protein. For comparison, the highest known accumulation of CAL (in the head of spermatosona) amounts to 12% [13]. CAL was cosiderably lower in the inner hair cells (about 1%), and was not detectable with usual protein loads in other cell types of OC. These results suggest that the calcium dependent intracellular response is performed actively in the outer hair cells of OC. The relative distribution of CAL in OC is very similar to that of glycogen, which reaches the exceedingly high level (600mmol/kg dry weight) in the outer hair cells [14]. This circumstantial evidence suggests that the activation of the glycogen phosphorylase kinase is an important role of CAL in the outer hair cells, whereby ultimate energy for contractile motility is mobilized from glycogen.
The mechanisms responsible for a slow sustained shortening in the outer hair cells are still not known, but the idea is supported in many laboratories that the actin-myosin interaction could induce the shortening in a manner similar to that which occurs in the smooth muscle and non-muscle cells. Consequently, CAL-induced activation of myosin light-chain kinase is likely to take place as a part of the contractile process.
