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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. [ABSTRACT FROM AUTHOR]

Previous studies in our laboratory have indicated that adenylate cyclase of the stria vascularis is strongly inhibited in vitro by ethacrynic acid and furosemide. In order to test whether the in vitro effects upon the enzyme are also present under in vivo conditions, ethacrynic acid was perfused perilymphatically for 15 min and 20 min at a concentration of 10(-3) M. Cyclic AMP of the stria vascularis was reduced by 27% and 34%, respectively, but ATP also declined significantly, suggesting unspecific effects. When ethacrynic acid was applied intravenously at a dosage of 50 mg/kg, and the endolymphatic potential allowed to decline to -10mV, no significant changes in cyclic AMP and ATP were seen. The absence of effects upon cyclic AMP in the early stage of systemic intoxication with ethacrynic acid is strong evidence against a mediating role of adenylate cyclase in the ototoxic action of ethacrynic acid. When a bolus of 3 x 10(-2) M furosemide was applied intra-arterially the endolymphatic potential declined at the exceedingly rapid rate of about 10 mV/sec, strongly suggesting that the action of the drug takes place in the vicinity of the capillaries of the stria vascularis. In view of the proposition that adenylate cyclase appears to be located primarily at eh luminal aspect of the stria vascularis, this constitutes further evidence against a role of the enzyme in the mediation of the specific ototoxic effects of loop diuretics. Other recent evidence against a mediatory role of the adenylate cyclase--cyclic AMP system is discussed. [ABSTRACT FROM AUTHOR]

The levels of 19 amino acids in utricular endolymph, vestibular and cochlear perilymph, and cerebrospinal fluid of guinea pigs were determined using gradient elution reverse phase high performance liquid chromatography of the o-phthaldialdehyde-ethanethiol adducts with fluorescence detection. Aspartate and glutamate were significantly higher in endolymph than in perilymph, in agreement with earlier results on cochlear fluids based on enzymatic fluorometric techniques. All other amino acids tested were significantly lower in the endolymph, in most cases by an order of magnitude. Vestibular perilymph and perilymph of scala vestibuli are virtually identical. Amino acid levels were all higher in perilymph of scala vestibuli than in cerebrospinal fluid; two by an order of magnitude. All differences were statistically significant, with the exception of aspartate. Amino acid levels in perilymph of scala tympani were highly variable dependent upon sampling technique, and no definite values are therefore presented. Comparisons with results from other laboratories, technical pitfalls, and possible implications and interpretations of the results are presented. [ABSTRACT FROM AUTHOR]

The purpose of the reported experiments was to measure the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in the organ of Corti in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions the concentrations of ATP, ADP, and AMP amounted to 15.8, 3.9, and 0.53 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since ischemia of 65 seconds leads to an increase of 155%. Under normal conditions the adenylate energy charge, the energy status, and the phosphorylation state amounted to 0.83, 4.1, and 2.5 gram wet weight/mumole, respectively. Within 10 minutes of ischemia the energy charge had declined by 26%, the energy status by 50%, and the phosphorylation state by 76%. The apparent equilibrium constant of adenylate kinase of the organ of Corti was found to be 0.55. The potential significance of these data and their relationship to the situation in the stria vascularis are discussed. [ABSTRACT FROM AUTHOR]

Levels of 19 free amino acids in cochlear endolymph and perilymph of scala vestibuli of the guinea pig were determined using reverse phase high performance liquid chromatography (HPLC) of the o-phthaldialdehyde-ethanethiol derivatives with fluorescence detection. Aspartate and glutamate were found to be significantly higher in endolymph than in perilymph, confirming results from another study using a different analytical method. The remaining 17 amino acids were significantly lower in the endolymph, in many cases by an order of magnitude. The data are compared with results on utricular endolymph and vestibular perilymph obtained by HPLC in another study from our laboratory. Possible implications and interpretations of the results are discussed. [ABSTRACT FROM AUTHOR]

By means of vascular perfusion via the anterior inferior cerebellar artery with a blood substitute containing the perfluorochemical FC 47 as oxygen carrier, it is possible to maintain normal or near normal levels of the cochlear microphonics and the endolymphatic potential of the guinea pig for periods of 90 min. or longer. Following 60 min. of perfusion with artificial blood, the levels of ATP and 5′AMP in the stria vascularis and the organ of Corti are comparable to those of nonperfused control animals maintained at optimal metabolic conditions. Following the same period of perfusion, the appearance of the organ of Corti is normal, but small vacuoles, presumably deposits of FC 47, are visible in the marginal cells of the stria vascularis. Preliminary experiments concerning the survival time and the revival time of the cochlear potentials, as well as the response to furosemide, ouabain, and mersalyl are presented to illustrate the value of this method in elucidating various biochemical and pharmacological problems of the cochlea. [ABSTRACT FROM AUTHOR]

The purpose of the reported experiments was to measure the strial concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions, the concentrations of ATP, ADP, and AMP were found to be 11.4, 3.7, and 0.6 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since its concentration doubles within 6 sec. of ischemia and reaches a peak level of about 1500% of the control following 65 sec. of ischemia. Under normal conditions, the 'adenylate energy charge,' the 'energy status,' and the 'phosphorylation state' amount to 0.84, 3.0, and 1.52 gram wet weight/μmole, respectively. In ischemia of 10 min. duration, the adenylate energy charge decreases 3 fold, the energy status 7 fold and the phosphorylation state 14 fold. The size of the adenylate pool shows a slight increase in the earliest stage of ischemia, but declines progressively thereafter. The apparent equilibrium constant of strial adenylate kinase was found to be 0.48. The advantages and limitations of the different adenylate ratios, as indicators of metabolic health and as regulatory parameters, are discussed. [ABSTRACT FROM AUTHOR]

The influence of various toxic substances and of drugs with ototoxic side effects upon energy generation, energy utilization, and membrane processes of the cochlea were studied. None of the drugs tested interfered with energy generation to as great an extent as did anoxia or cyanide and 2,4-dinitrophenol. Ouabain produced a pronounced interference with energy utilization of the stria vascularis. The 'loop' diuretics ethacrynic acid and furosemide produced a reduction of energy utilization of a lesser degree than did ouabain. The 'loop' diuretics do not seem to exert their toxic action upon strial Na+K+-ATPase, but may act by interfering with strial adenylate cyclase. Aminoglycoside antibiotics and diuretic and nondiuretic mercurials seem to exert their primary noxious action upon cochlear function by interfering with membrane processes of the structures bounding the cochlear duct. [ABSTRACT FROM AUTHOR]

Ischemic changes in the levels of glucose, glycogen, ATP and P-creatine are determined under 'closed system' conditions in the organ of Corti, stria vascularis, ganglion spirale, cochlear nerve and vestibular sensory epithelia. From the resting levels of these compounds the total energy reserve in terms of equivalents of high energy phosphate, both preformed and potentially available from anaerobic glycolysis, is computed. The energy reserves are highest in the organ of Corti, intermediate in stria vascularis, spiral ganglion and vestibular structures, and lowest in the cochlear nerve. The rate of depletion of these energy reserves in ischemia is used as an indicator of the energy requirements of the respective tissues. The metabolic rate is by far the highest in the stria vascularis, intermediate in ganglion spirale and cochlear nerve and lowest in the organ of Corti and vestibular structures. There is no correlation between the total energy reserve and the initial energy use rates. The obtained data are compared with the dynamic patterns of the corresponding biopotentials and with pertinent results of enzymatic and respirometric studies; in addition, ischemic changes in glucose and lactate levels of perilymph are described. [ABSTRACT FROM AUTHOR]

A method is described for the dissection of single outer hair cells in the freeze-dried state. The average dry weight of outer hair cells from the third and fourth cochlear turn in the guinea pig is of the order of one nanogram, with variations depending upon the precise location. The measured dry weight is in line with the wet weight estimated from the volume of living outer hair cells maintained under iso-osmotic conditions. The lipid fraction of outer hair cells amounts to about 21 percent of the dry weight. Average ATP levels in outer hair cells of the upper two turns range between 12-15 mmoles/kg dry weight, with no significant difference between rows or turns. [ABSTRACT FROM AUTHOR]

Further biochemical and biophysical characterization of two low-molecular-weight, strongly acidic proteins that are present at extremely high levels in the organ of Corti, tentatively named OCP-I and OCP-II, is presented. The two proteins are also present, although at much lower levels, in the vestibular end-organs and a variety of other inner ear tissues; they have not been observed in other systems. OCP-I and II are highly soluble and do not contain appreciable amounts of carbohydrate. The two proteins, originally described in the guinea pig, are compared electrophoretically with the corresponding proteins in several other mammalian species. Preliminary data on the amino acid composition of the two proteins are presented. Moreover, the amino-terminal sequence of a 22-residue segment of OCP-II is shown and compared to the sequences of known proteins.