The loop diuretics ethacrynic acid (EA) and furosemide (FU) were applied systemically to guinea pigs at dosages from 10–100 mg/kg. At high dosages the endolymphatic potential (EP) invariably turned negative. When the EP had reached maximum negative values due to EA, the ATP levels of the stria vascularis were moderately reduced, but P‐creatine levels were normal. In the case of FU both high energy phosphates remained at normal levels. When EA and FU intoxicated ears were subjected to ischemia, the rate of decline of ATP and P‐creatine was markedly less than the ischemic decline in nonintoxicated ears. These results suggest a strong interference with energy utilization, and in the case of EA a moderate impairment of energy generation. In severe intoxication by perilymphatically applied ouabain (10−3 M) strial ATP remained normal but P‐creatine was significantly increased. The reduction of the ischemic decline rate in ouabain intoxicated ears was even more marked than in the case of EA or FU, indicating a very strong interference with energy utilization, presumably due to complete inhibition of Na+K+‐ATPase. The I50 of the endolymphatic potential with regard to perilymphatically applied EA and FU was found to be 10−5 M and 2 x 10−4 M respectively. By K contrast, strial Na+K+‐ATPase was 50% inhibited with 5 x 10−3 M EA and not inhibited at all by FU. It is therefore unlikely that the effect of loop diuretics upon the endolymphatic potential is due to interference with strial Na+K+‐ATPase.
We have shown that collagen accounts for approximately 40% of the total protein of the tectorial membrane (TM) of the guinea pig and have estimated several essential parameters of TM composition including dry weight, wet weight, and water content. The major collagenous protein was definitively identified as type II collagen by SDS‐PAGE, CNBr peptide mapping, and immunoblot assays. Quickfreeze, deep‐etch electron microscopy of the guinea pig TM demonstrated a dense meshwork of fibers embedded in a complex microfibrillar matrix which may consist of proteoglycans; the larger fibers were similar in size and appearance to type II collagen fibers of elastic cartilage. Finally, the comparative free'amino acid profiles of TM strongly suggest that the TM is chemically transparent with respect to endolymph. Thus, the TM appears to consist of a highly hydrated matrix mechanically stabilized by type II collagen fibers.
The possible physicochemical and functional significance of these findings for sound transduction is discussed.
Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) beta 2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins--apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with SP40/40, or cytolysis inhibitor--were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use beta 2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.
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