Cyclo-oxygenase enzyme in the perilymph of human inner ear.

CONCLUSIONS: Cyclo-oxygenase-2 (COX-2) enzyme would not appear to be constitutively expressed in human perilymph while it is always induced in the perilymph of patients affected by sensorineural hearing loss (SNHL). The COX-2 isoform may be involved in hearing loss and, therefore, pathological states of the inner ear should possibly be further analyzed to clarify the clinical relevance of prostaglandin and selective COX-2 antagonist therapy. OBJECTIVES: Perilymph samples from a group of patients with bilateral SNHL and another with conductive hearing loss were collected to evaluate the presence of the COX-2 enzyme. The possible correlation between different causes of deafness and the expression of COX-2 in the human ear was studied. METHODS: A prospective clinical study of 14 patients with severe or profound hearing loss who underwent cochlear implant surgery and 4 patients with conductive hearing loss who underwent stapes surgery was carried out. Western blot analysis of perilymph samples was performed with monoclonal anti-human COX-2 antibody. RESULTS: COX-2 enzyme was detected in all patients affected by SNHL and was absent in all those with conductive hearing loss due to otosclerosis.

First clinical experience with beta-trace protein (prostaglandin D synthase) as a marker for perilymphatic fistula.

A diagnosis of perilymphatic fistula is still controversial. Recently, a case report indicated that beta-trace protein (prostaglandin D synthase) might be a potential marker for perilymphatic fluid. In this multicentre clinical case series study beta-trace protein was used as a marker for perilymphatic fluid fistula. Fifteen fluid samples were collected during diagnostic tympanoscopy. In addition, five samples were collected from patients with tympanic membrane perforation for use as as negative controls. Samples were obtained using precision glass capillaries and were analysed for beta-trace protein using laser nephelometry. The diagnosis of perilymphatic fistula was defined by the patient's history, the audiological and vestibular investigation and the findings at tympanoscopy. The cut-off level of beta-trace protein for perilymph-positive samples was chosen at 1.11 mg/l. The sensitivity and specificity were calculated using a 2 x 2 contingency table. There was no false positive result, but in two cases a false negative result was found. The specificity was 1 and the sensitivity was 0.81. The material of this first clinical study is small owing to the rarity of patients undergoing diagnostic tympanoscopy for perilymphatic fluid fistula. However, according to these preliminary results beta-trace protein might be a promising marker in the diagnosis of perilymphatic fluid fistulas.

The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea.

This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.

Ectonucleotidase activity in the perilymphatic compartment of the guinea pig cochlea.

It has been clearly demonstrated that extracellular adenosine 5'-triphosphate (ATP) exerts a potent modulatory activity in the cochlea through its interaction with P2 purinoceptors. However, little is known regarding the metabolism of extracellular ATP in cochlear tissues via ectonucleotidases. This study provides evidence for the presence of ectonucleotidases in the perilymphatic compartment of the guinea pig cochlea. Using microperfusion, ATP (500 microM) was introduced into the cochlear perilymph through the basal turn scala tympani, and effluent was collected from the basal turn scala vestibuli. Samples were subsequently analysed for the presence of adenine metabolites using high performance liquid chromatography (HPLC). Cell viability was evaluated by the activity of the intracellular enzyme lactate dehydrogenase (LDH) in the perfusate. ATP was degraded to 122.8 +/- 9.9 microM (25.0 +/- 5.8%) during the passage through the cochlear perilymphatic compartment. Breakdown of ATP resulted in the formation of adenosine 5'-diphosphate (41.5 +/- 9.0 microM), adenosine 5'-monophosphate (201.3 +/- 15.5 microM), adenosine (108.6 +/- 8.3) and inosine (15.0 +/- 1.5 microM). The degradation of ATP was significantly (P < 0.001, Student's t-test) inhibited in the absence of divalent cations, Ca2+ and Mg2+ in the perfusate. In control experiments, no spontaneous degradation of ATP was observed in vitro. LDH activity was similar during ATP perfusions (2.9 +/- 0.9%) to control perfusions with artificial perilymph (4.2 +/- 1.0%) indicating well preserved cell integrity in the cochlear perilymphatic compartment. The degradation of extracellular ATP in the presence of intact tissues and its inhibition in the absence of divalent cations, a cofactor for ectonucleotidases, provides evidence for ectonucleotidase activity in the perilymphatic fluid space of the cochlea.

[Localization of Na/K ATP-ase in the inner ear]. / De lokalisatie van Na/K ATP-ase in het binnenoor.

The perilymphatic compartment of the inner ear resembles plasma ultrafiltrate, whereas the endolymphatic compartment is characterized by a high potassium and low sodium concentration. Perilymph is considered to be the precursor of endolymph. The chemical composition of endolymph and the generation of the transepithelial potential is regulated by Na-K-activated adenosine triphosphatase. The cytochemical localization of Na/K ATP-ase is demonstrated in the guinea pig inner ear. The mechanism for cation regulation in the endolymphatic compartment is discussed.

[Determination of lactate dehydrogenase isozymes in the cochleae of guinea pigs].

The isozymes of lactate dehydrogenase (LDH) in perilymph and homogenates from vascular stria, Corti's organ, and vestibular membrane of healthy guinea pigs were determined with the micro-homogenate electrophoresis technique. The results were compared with those in serum and cerebrospinal fluid. It was found that electrophoretogram of individual homogenate or perilymph, which showed the isozymes of LDH, had a special pattern different from that in the serum. The factors which might affect LDH in perilymph had been discussed.

Cathepsin D activity in otosclerotic bone and perilymph.

Otosclerotic bone and perilymph samples obtained during stapedectomy operations from patients suffering from pure conductive hearing loss of at least 40 dBs air-bone gap at 500 Hz. Control perilymph samples derived from patients suffering from round window membrane rupture or Meniere's disease. Enzyme activities were determined using a synthetic fluorogenic substrate by fluorescence spectrophotometry. No significant differences have been found between the protein content of otosclerotic and normal or non-otosclerotic perilymph samples. Cathepsin D activity was significantly higher (p less than 0.001) in the otosclerotic perilymph samples. Otosclerotic stapes footplate contained more activity than normal meatal cortical bone as well, though the difference was not significant. Biological significance of the elevated Cathepsin D activity must be considered.

Early effects of acetazolamide on anionic activities of the guinea pig endolymph: evidence for active function of carbonic anhydrase in the cochlea.

The effect of acetazolamide (ACZ) on HCO3- and Cl- activities in inner ear fluid was investigated by ion-selective microelectrode methods. The endocochlear potential, at 81.6 +/- 1.5 mV under normal conditions, was reduced by 4.9 +/- 0.9 mV in 30 min following the ACZ injection. The HCO3- concentrations by 11.5 +/- 1.7 mM in 30 min, while the change of Cl- level was not consistent but showed a tendency toward slight increase in average. The calculated PCO2 in endolymph was 39.6 mmHg. ACZ induced a constant elevation of the endolymph PCO2, with an increase in the venous PCO2 during the initial 20 min and thereafter the endolymph PCO2 attained a plateau despite the continuous increase of the venous PCO2. We postulate that the HCO3- secretion into the endolymph is mediated by carbonic anhydrase (CAH) and is decreased by ACZ due to the inhibition of CAH.

Inhibition of myosin light-chain kinase activity in the organ of Corti by 0.3-5 kilodalton substances of the otosclerotic perilymph.

The guinea pig organ of Corti contains myosin light-chain kinase (MLCK) activity. The upper and lower most parts of the cochlea do not show significantly different activities of the enzyme, which is Ca2+ and calmodulin-dependent. Short-term noise exposure does not cause a significant change. 0.3-5 Kilodalton substances of the otosclerotic perilymph, separated by SG-25 column chromatography, inhibit the MLCK activity in in vitro organ of Corti preparations. This inhibitory action of the perilymph substances can also be observed with the purified MLCK of turkey gizzard. The activity of the enzyme can be specifically inhibited by cyclic AMP-dependent protein kinase.

[Total lactate dehydrogenase activity of perilymph, plasma and cerebrospinal fluid in unstressed and noise stressed guinea pigs]. / Zur Gesamtaktivität der Laktatdehydrogenase von Perilymphe, Plasma und Liquor cerebrospinalis unbelasteter und schallbelasteter Meerschweinchen.

The total activity of lactate dehydrogenase (LDH) of perilymph (PL), plasma, and cerebrospinal fluid (CSF) of unexposed and sound-exposed guinea pigs was examined with due consideration of the principal sources of error. To test the purity of PL samples, protein, potassium, and sodium were determined simultaneously (Table 1). The LDH was analysed fluorometrically. It was found that there are considerable differences in the LDH activities of PL, CSF, and plasma (Table 2). The mean activity of PL was three to four times higher than that of CSF and only about half that of plasma. No significant difference was found between the PL in scala tympani and scala vestibuli. The most frequent LDH values of the individual fluids (Fig. 2) were somewhat lower than the mean values calculated. Immediately (less than or equal to 60 min) after exposing the animals to wide-band noise at an intensity of 140 dB SPL for 10 min, the mean PL values of the scala tympani and scala vestibuli were found to be somewhat higher than in unexposed animals (Table 3). Eighteen hours after the exposure, slightly higher activity was only detectable in PL of the scala vestibuli. The differences were not found to be significant. The LDH values of CSF and plasma remained unchanged both less than or equal to 60 min and 18 h after noise exposure.

The enzymatic mechanism of the otospongiotic disease and NaF action on the enzymatic balance.

Extensive research on enzymes led to the discovery of the innermost mechanism of the otospongiotic disease. The authors present the results of multiple correlations made from February 1976 to September 1980 on 648 samples of perilymph taken during stapedectomies performed on otosclerotic patients. We studied microdosages of three selected enzymes--trypsin, alpha 1 antitrypsin, and alpha 2 macroglobulin--in each of the samples and their relationship with cochlear deterioration expressed in dBs of bone conduction decrease in pure-tone audiometry testing. This study allowed us a better knowledge of the enzymatic mechanism of the otospongiotic disease, based on the previously reported trypsin-alpha 1 antitrypsin balance, but in which alpha 2 macroglobulin appears to play a role as essential as that of alpha 1 antitrypsin. This enzymatic mechanism explains NaF efficiency, which is due in fact to a double action: not only direct trypsin inhibition, but also an overall reduction in enzymatic values in the perilymph of otospongiotic/otosclerotic patients. Current studies could lead to the possibility of future NaF replacement by proteinase inhibitors either of microbial origin, under study by Japanese researchers, or even of synthetic origin, which should be investigated. In fact, the role of NaF therapy could already be taken over by diphosphonates currently under study.

Neurotransmitters in the cochlea and the cochlear nucleus.

The inhibitory efferent transmitter in the cochlear is most likely acetylcholine. The afferent transmitter (between hair cells and primary afferent fibres) is not known. There is some evidence for glutamate (or aspartate) but the high concentrations necessary to activate the afferents when these amino-acids are applied intracochlearly may indicate that their effects is unspecific. A number of other transmitter candidates can be safely ruled out at these synapses. In the cochlear nucleus of transmitter between primary afferents and secondary cells is probably glutamate (or aspartate).

[Enzymatic mechanism of otosclerosis. Action of NaF]. / Méchanisme enzymatique de l'otospongiose. Action du NaF.

The authors present the conclusions they reached after more than four years of multiple correlations made from february 1976 to september 1980. 648 perilymph samples were selected from a total number of 811 samples taken during stapedectomies on otosclerotic patients. These multiple correlations were based on micro-dosages of three selected enzymes (trypsin-alpha 1 antitrypsin and alpha 2 macroglobulin, the fourth cathepsin B having not been found, even in perilymph pools) in each of the selected samples, and on their relationship with the cochlear deterioration expressed in dBs of B.C. decrease and in audiometric stages corrected with reference to the patients' age. This study leads to an enzymatic mechanism, based on the previously reported trypsin-alpha 1 antitrypsin balance, but in which alpha 2 macroglobulin appears to play as essential a role as that of alpha 1 A. This enzymatic mechanism explains NaF efficiency, due in fact to a double action, not only to direct trypsin inhibition, but also to an overall reduction in enzymatic levels in the perilymph of otosclerotic patients. The authors conclude by suggesting the possibility of future NaF replacement by proteinase inhibitors, either of microbial origin currently under study by Japanese researchers, or even of synthetic origin.

Alteration of oxidative enzymes (LDH and MDH) in perilymph after noise exposure.

Chinchillas were exposed to noise (123 dB, 1/2 h, 700--2800 Hz). The activity of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) was studied immediately after exposure or 1, 7, 14, 21, or 28 days later. LDH activity in perilymph doubled immediately after exposure, gradually decreasing to control values in about a month. The activity of MDH not only doubled immediately after the exposure but continued to rise for at least another 24 h, reaching a peak value of 3.5 times normal at 24 h. The activity was still 1.6 times normal even after a month of recovery. The enzyme activity of serum and CSF remained within the control range throughout the experimental period except for an initial increase of serum enzyme activity due to anesthetic. The results demonstrate that the morphological changes induced by a noise exposure that is known to produce permanent threshold shift (PTS) are reflected in a significant elevation of oxidative enzymes in perilymph. Possible mechanisms of the elevation of enzymes in perilymph are discussed.

Enzymatic activity of inner and middle ear fluids in fetal guinea pigs under ambient and high-intensity sound presentation.

The enzymes in perilymph of the inner ear scala and within fluid filling the middle ear cavity were investigated in the fetal guinea pig near term. Perilymph lactic dehydrogenase (LDH) exhibited differential activity between fetal scalae vestibuli and tympani, and LDH, aldolase, phosphohexose isomerase and creatine phosphokinase in perilymph of particular fetal scalae were elevated in activity in activity in comparison to corresponding maternal values. Moreover, LDH displayed different isozyme patterns for antenatal and gravid perilymph. In the middle ear fluid, all of the above enzymes were encountered but varied somewhat from the quantities determined for either prenatal perilymph or maternal serum. After gravid guinea pigs were exposed to a tone of 4 kHz, 100 dB SPL for 2 hours, LDH of the fetal middle ear fluid was reduced in activity 24 hours later. The metabolic significance of the fetal distinctions in the ear fluids and the clinical implications of the current work are discussed.