Biomedical subjects

J Schacht

Publications and source records attributed to J Schacht.

At least 19 recordsLinked to original sources

PubMed1995

P2y purinergic receptors coupled to phosphoinositide hydrolysis in tissues of the cochlear lateral wall.

The tissues of the lateral wall of the cochlea, spiral ligament and stria vascularis, maintain homeostasis of the inner ear through the production of endolymph and regulation of cochlear blood flow. We examined the effects of purinergic agonists on the release of inositol phosphates (InsPs) in intact isolated lateral wall tissues of the Fischer-344 rat. After preincubation with myo[3H]inositol, stimulation with the P2 purinergic agonist ATP-gamma-S resulted in a concentration-dependent increase in the formation of [3H]InsPs. The P2 purinergic agonist alpha,beta-methylene ATP (AMP-CPP) was a weaker stimulant, and the P1 purinergic agonist adenosine was ineffective. These results are consistent with the presence of P2y receptors and a purinergically controlled InsP3 second messenger system in the cochlear lateral wall.

Adenosine Triphosphate↗

PubMed1995

Second messengers in the cochlea.

Second messengers are vital to the regulation of nearly every aspect of cellular physiology. Evidence is reviewed for the role of second messengers in cochlear fluid homeostasis, hair cell adaptation, motility and synaptic activity. We suggest that the elucidation of the role of second messengers in the regulation of cochlear physiology will be important for the therapeutic management of otopathologies and the side effects of ototoxic agents.

Adenosine Monophosphate↗

PubMed1994

Detection and characterization of nitric oxide synthase in the mammalian cochlea.

The messenger molecule nitric oxide (NO) is involved in blood flow regulation, cytotoxicity, and neural signalling, processes that are important in the physiology and pathophysiology of the mammalian cochlea. However, neither the presence of NO nor its synthetic enzyme, NO synthase, has been established in the peripheral auditory system. NO synthase activity, measured as the enzymatic conversion of radioactive arginine to citrulline, was predominantly soluble in the auditory nerve, lateral wall, vestibule and cochlear neuroepithelium. N-methyl-L-arginine and trifluoperazine inhibited NO synthase activity in the lateral wall and auditory nerve. Histochemical staining by NADPH-diaphorase localized NOS activity to the lateral wall and the neuronal elements of the organ of Corti. Based on these results, the predominant NO synthase isoform in the cochlea is the neuronal type-I isoform.

Amino Acid Oxidoreductases↗

PubMed1994

Sulfhydryl compounds and antioxidants inhibit cytotoxicity to outer hair cells of a gentamicin metabolite in vitro.

Aminoglycoside antibiotics such as gentamicin have long been known to destroy cochlear and vestibular hair cells in vivo. In the cochlea outer hair cells are preferentially affected. In contrast, gentamicin will not damage outer hair cells in vitro unless it has been enzymatically converted to a cytotoxic metabolite. Several potential inhibitors of this enzymatic reaction were tested in an in vitro assay against outer hair cells isolated from the guinea pig cochlea. Viability of hair cells (viable cells as per cent of total number of cells observed) averaged about 70% under control conditions. Addition of metabolized gentamicin significantly reduced viability to less than 50% in one hour. Sulfhydryl compounds (glutathione, dithioerythritol) and antioxidants (vitamin C, phenylene diamine, trolox) prevented the cytotoxic actions of the gentamicin metabolite. Inhibitors of amine oxidases and compounds reportedly protective against renal and acute lethal toxicity of aminoglycosides (poly-L-aspartate and pyridoxal phosphate, respectively) were ineffective as protectants. The results reinforce the hypothesis that gentamicin is enzymatically converted to a cytotoxin and imply the participation of sulfhydryl-sensitive groups or free radicals in this reaction. Alternatively or additionally, sulfhydryl compounds or antioxidants may participate in detoxification reactions.

Animals↗

PubMed1994

Attenuation of gentamicin ototoxicity by glutathione in the guinea pig in vivo.

The effect of glutathione co-therapy on the expression of gentamicin ototoxicity was tested in pigmented guinea pigs. The first group of animals was injected with gentamicin (100 mg/kg body weight/day) for two weeks followed by 10 weeks of rest. A second group received glutathione by gastric gavage immediately prior to each gentamicin injection. Two groups of controls were treated either with saline injections or glutathione gavage alone. Auditory brainstem responses, taken at 2-week intervals, revealed a progressive gentamicin-induced hearing loss reaching a 30 to 40 dB threshold shift at 2 kHz, approximately 60 dB at 8 kHz and 80 dB at 18 kHz. Glutathione co-therapy slowed the progression of hearing loss and significantly attenuated the final threshold shifts by 20 to 40 dB. Morphological evaluation confirmed hair cell loss after gentamicin treatment and protection by glutathione. Drug serum levels were assayed at 2 and 7 days of treatment. There were no differences between the gentamicin (mean = 183 micrograms/ml; range, 90 to 300) and the gentamicin/glutathione group (mean = 164 micrograms/ml; range, 80 to 320). Antimicrobial activity of gentamicin was tested against Staphylococcus aureus and Pseudomonas aeruginosa. A 30-fold molar excess of glutathione did not influence the efficacy of gentamicin. These studies suggest that glutathione protects against ototoxicity by interfering with the cytotoxic mechanism.

Animals↗

PubMed1994

Living isolated cells from inner ear vessels: a new approach for studying the regulation of cochlear microcirculation and vascular permeability.

The spiral modiolar artery with its proximal branches and the microvessels in the spiral ligament and the stria vascularis were microdissected from the guinea pig cochlea. After incubation with proteolytic and collagenolytic enzymes the mixed cell suspension was fractionated by gradient centrifugation. The cells migrated according to their buoyant densities into the fractions of 1.04 g/ml (endothelial cells), 1.06 g/ml (vascular smooth muscle cells obtained from the spiral modiolar artery; strial pericytes) and 1.08 g/ml (pericytes obtained from the spiral ligament). To test for viability cells were loaded with a fluorescent vital stain (BCECF-AM); for identification, cell-specific stains were used. Identity of endothelial cells (ECs) was confirmed using acetylated low density lipoprotein fluorescently labeled with dioctadecyl-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Pericytes were identified immunofluorescently using the method according to Nayak et al. (1988). Vascular smooth muscle cells were stained for F-actin with rhodamin-phalloidin. This in vitro technique may open new approaches to study local factors involved in microcirculation and vessel permeability of various cochlear vascular beds.

Actins↗

PubMed1994

G-proteins coupled to phosphoinositide hydrolysis in the cochlear and vestibular sensory epithelia of the rat are insensitive to cholera and pertussis toxins.

In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoinositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P2y agonists. Such receptor-mediated activation of PLC is expected to be coupled through guanine nucleotide-binding proteins (G-proteins). Although several classes of G-proteins have been identified in the inner ear, nothing is known about the type of G-proteins associated with the phosphoinositide second messenger system in CSE and VSE. Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs). Ten mM NaF plus 10 microM AlCl3 increased basal InsPs accumulation 2-fold in both CSE and VSE of the rat. Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in saponin-permeabilized tissues. Furthermore, release of InsPs stimulated by both carbamylcholine (CCh) and adenosine 5'-O-[3-thiotriphosphate] (ATP-gamma-S) was significantly inhibited by 100 microM guanosine 5'-O-[2-thiodiphosphate] (GDP-beta-S). These results strongly suggest the involvement of G-proteins in the receptor-PLC coupling in CSE and VSE. ADP-ribosylation in membrane fractions of CSE and VSE in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicated the existence of Gs- and G(i)-type G-proteins. However, neither CTX nor PTX affected basal or agonist-stimulated release of InsPs. These observations suggest that muscarinic and P2y purinergic receptors are coupled to PLC via CTX- and PTX-insensitive G-proteins in CSE and VSE.

Adenosine Diphosphate Ribose↗

PubMed1994

Calcium and calmodulin inhibit phosphorylation of a novel auditory nerve protein.

The growing use of cochlear prosthetic devices and demonstrations of direct ototoxic insult to spiral ganglion neurons make it imperative to gain an understanding of intracellular biochemical regulation in primary sensory neurons. Calcium and calmodulin regulate many aspects of neuronal cellular physiology through stimulation of protein kinase activity. We have previously demonstrated the presence of calmodulin-dependent protein kinase substrates in the guinea pig modiolus and, additionally, the presence of two proteins (12 kDa and 81 kDa, designated as p12 and p81) whose phosphorylation is blocked by calcium and calmodulin (Coling and Schacht, 1991). Here, we investigate three models for this unusual regulatory mechanism. The effects of calcium, calmodulin and trifluoperazine on dephosphorylation of both proteins suggests that calmodulin inhibits protein kinase activity. P81 was identified by immunoprecipitation as the myristoylated alanine-rich C kinase substrate (MARCKS), a ubiquitous actin-binding protein. Two observations indicate that MARCKS may be regulated differently in acoustic nerve than in cerebral cortex. 32P incorporation was significantly higher in acoustic nerve than in brain. The calmodulin-dependent block of MARCKS phosphorylation was observed only in acoustic nerve. p12 shares several characteristics with myelin basic protein (MBP). We used a double label assay with 32P autoradiography and immunoblotting to show that p12 is in fact distinct from MBP. We suggest that either p12 or p12 kinase may be either specific to the peripheral auditory system or novel marker proteins for that tissue.

Animals↗

PubMed1994

Effect of aging on myo-inositol and phosphoinositide metabolism in the cochlear and vestibular sensory epithelia of the rat.

Neurotransmission and transmembrane signaling are among the cellular mechanisms affected in the aging nervous system. In the inner ear, the phosphoinositide second messenger cascade is of particular interest as a target of the aging process. In both the cochlear (CSE) and vestibular sensory epithelia (VSE), the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to the second messenger inositol 1,4,5-trisphosphate (InsP3) is coupled to muscarinic cholinergic and P2y purinergic receptors and may be linked to calcium homeostasis. The present study compared the turnover of phosphoinositides (PtdInsPs), receptor-mediated release of inositol phosphates (InsPs), and concentrations of endogenous myo-inositol in the CSE and VSE of young (3 months) and aged (24 months) Fischer-344 rats. In the aged rat, there was a significant increase in [3H]inositol incorporation (per mass of protein) into PtdInsPs plus InsPs in both sensory epithelia while the protein content remained unchanged. In contrast, no age-dependent differences were found when pre-labeled [3H]PtdInsPs were 'chased' with non-radiolabeled myo-inositol indicating that the turnover of these lipids was unaffected. The cholinergic receptor agonist carbamylcholine and the P2 purinergic receptor agonist adenosine 5'-O-(3-thiotriphosphate) stimulated the release of [3H]InsPs two- to six-fold in both organs. This agonist-stimulated release of [3H]InsPs (per mass of protein) was significantly higher in aged animals. However, when the same stimulation was expressed as per cent of control values, there was no age-dependent difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1993

Glutathione S-transferases in the organ of Corti of the rat: enzymatic activity, subunit composition and immunohistochemical localization.

Glutathione S-transferases (GSTs), a family of ubiquitous cytosolic isozymes, catalyze the detoxification of electrophilic substrates with reduced glutathione and participate in intracellular binding and transport of lipophilic substances. This study measured GST activity biochemically in the inner ear of the rat; determined the isozyme profile by Western blotting; and identified, immunohistochemically, the distribution of the mu and pi class GSTs in the organ of Corti. GST enzymatic activity in inner ear tissues ranged from 117 to 348 nmoles glutathione converted/min/mg protein, values somewhat higher than those found in brain (130) and much lower than in liver (1011). Of the GST isoforms, the pi class (identified by antibodies against the Yp subunit) was most prominent, the mu class (Yb1 subunit) clearly evident while the alpha class (Y(a) subunit) was barely detectable on Western blots. Immunocytochemical analysis showed differential distribution of the Yb1 and Yp subunits. The Yb1 subunit was present in the sensory cells, while supporting cells were not specifically stained. At the subcellular level, the isozyme was localized in the apical zones of inner (IHCs) and outer hair cells (OHCs) close to the cuticular plate. The extent of staining, however, varied between OHCs and IHCs. In the OHCs, staining appeared in discrete spots in the apical areas only, whereas in IHCs staining extended further towards the center of the cells. The Yp subunit was mainly localized to Deiters cell processes and pillar cells. Both Yb1 and Yp colocalized with tubulin-specific antibody. The functional significance of GST in the cochlear receptor cells is speculative. However, a role analogous to that in other tissues (detoxification, prostaglandin synthesis) can be assumed. In addition, an association of GST with the microtubule system is possible based on immunohistochemical colocalization with tubulin.

Animals↗

PubMed1993

Receptor-mediated release of inositol phosphates in the cochlear and vestibular sensory epithelia of the rat.

Various neurotransmitters, hormones and other modulators involved in intercellular communication exert their biological action at receptors coupled to phospholipase C (PLC). This enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (DG) which act as second messengers. In the organ of Corti of the guinea pig, the InsP3 second messenger system is linked to muscarinic cholinergic and P2y purinergic receptors. However, nothing is known about the InsP3 second messenger system in the vestibule. In this study, the receptor-mediated release of inositol phosphates (InsPs) in the vestibular sensory epithelia was compared to that in the cochlear sensory epithelia of Fischer-344 rats. After preincubation of the isolated intact tissues with myo-[3H]inositol, stimulation with the cholinergic agonist carbamylcholine or the P2 purinergic agonist ATP-gamma-S resulted in a concentration-dependent increase in the formation of [3H]InsPs in both epithelia. Similarly, the muscarinic cholinergic agonist muscarine enhanced InsPs release in both organs, while the nicotinic cholinergic agonist dimethylphenylpiperadinium (DMPP) was ineffective. The muscarinic cholinergic antagonist atropine completely suppressed the InsPs release induced by carbamylcholine, while the nicotinic cholinergic antagonist mecamylamine was ineffective. Potassium depolarization did not alter unstimulated or carbamylcholine-stimulated release of InsPs in either organ. In both tissues, the P2 purinergic agonist alpha,beta-methylene ATP also increased InsPs release, but the P1 purinergic agonist adenosine did not. These results extend our previous observations in the organ of Corti of the guinea pig to the rat and suggest a similar control of the InsP3 second messenger system in the vestibular sensory epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1993

Chronic electrical stimulation reverses deafness-related depression of electrically evoked 2-deoxyglucose activity in the guinea pig inferior colliculus.

The [14C]-2-deoxyglucose (2-DG) autoradiographic technique was used to study how auditory-related metabolic activity changes with deafness, and how chronic electrical stimulation of the deafened system may modify these changes. Guinea pigs were deafened by administration of kanamycin and ethacrynic acid. After nine weeks of deafness, the basal unstimulated uptake of 2-DG in the inferior colliculus (IC) was lower than in normal hearing control animals. 100 microA of acute cochlear electrical stimulation significantly increased 2-DG uptake in normal hearing animals but did not evoke a significant increase in four or nine week deafened animals. Electrically elicited 2-DG uptake in the IC is therefore depressed by prolonged deafness. In a second series of experiments, after four weeks of deafness, animals were chronically electrically stimulated via a cochlear implant 2.5-3.5 h a day, five days a week for five weeks at 100 microA. Acute cochlear electrical stimulation following this chronic stimulation significantly increased 2-DG uptake in the contralateral IC over unstimulated levels. This suggests that some depressive effects of profound deafness on the auditory brain stem may be reduced or reversed with chronic electrical stimulation by a cochlear implant.

Animals↗

PubMed1993

Membrane stains as an objective means to distinguish isolated inner and outer hair cells.

The use of isolated cochlear outer and inner hair cells has become widespread. While the morphological features of these two cell types in general are sufficiently different to allow discrimination, there are situations where confusion can arise. Small outer hair cells, particularly when they are swollen or distorted, can take on an appearance suggestive of inner hair cells. We describe here two fluorescent membrane stains, 3,3'-dihexyloxacarbocyanine iodide and rhodamine B hexyl ester, as an objective means to distinguish between cochlear hair cell types. Both stains mark the subsurface cisternae of outer hair cells thereby delineating the cell outline, and the interior of the cell shows discrete structure. On the other hand, in inner hair cells, the outline of the cell is not resolved while the interior is diffusely fluorescent. Since the two probes have different excitation and emission wavelengths (fluorescein- and rhodamine-like, respectively), this staining procedure can even be used in the presence of another fluorescent marker (for example, a calcium-indicating dye) by appropriate choice of the membrane stain.

Animals↗

PubMed1993

Biochemical basis of aminoglycoside ototoxicity.

The basis for the development of a rational explanation of aminoglycoside toxicity now appears to exist. The acute effects of these drugs are primarily based on calcium antagonism and block of ion channels. The chronic toxicity requires metabolism, and the expression of tissue-specific toxicity is a balance between synthesis of the toxin and its detoxification. Further investigations into the nature of the toxic metabolite should allow us to combine this information with previously established intracellular actions of aminoglycosides to create a unified hypothesis of action. The ability of glutathione to block toxin formation or to increase detoxification (or both) may have clinical implications for the prevention of aminoglycoside-induced ototoxicity. The clinical use of aminoglycosides has somewhat decreased over the last decade because of the introduction of the less toxic cephalosporins of the third generation and the quinolones, which are effective against Pseudomonas infections. Development of bacterial resistance against aminoglycosides is another factor, although resistance to the cephalosporins is also rapidly becoming a serious problem that eventually will limit their usefulness. Only through a detailed knowledge of the molecular basis of toxicity can we rationally pursue the development of new aminoglycosides with less ototoxic and nephrotoxic potential and devise treatments that will prevent the adverse side effects of these antibiotics.

Aminoglycosides↗

PubMed1992

Formation of a toxic metabolite from gentamicin by a hepatic cytosolic fraction.

We have demonstrated recently that incubation of the aminoglycoside gentamicin with an hepatic post-mitochondrial fraction produces a compound toxic to sensory cells from the inner ear in short-term culture; in contrast, the parent aminoglycoside was non-toxic in vitro (Huang MY and Schacht J, Biochem Pharmacol 40: R11-R14, 1990). In the present study, we investigated the subcellular distribution of the enzymatic activity and the nature of the metabolite. Isolated outer hair cells from the guinea pig cochlea were used to assay for cytotoxicity. The enzyme(s) responsible for this novel reaction of aminoglycosides was exclusively localized to the cytosolic fraction of guinea pig liver. No activity was detected in nuclear, lysosomal/mitochondrial or microsomal preparations. Furthermore, the toxin-forming enzymatic activity was associated with the high molecular weight fraction of the cytosol and did not require low molecular weight components. Filtration of the toxin through molecular weight cut-off membranes showed a molecular size of approximately 500. This evidence is consistent with the toxin being a gentamicin derivative.

Animals↗

PubMed1992

Cholinergic regulation of the phosphoinositide second messenger system in the guinea pig organ of Corti.

The effect of cholinergic agents on the phosphoinositide second messenger system was investigated in the cochlea of the adult guinea pig in vivo and in vitro. In vivo, phospholipids were labeled with [32P]-orthophosphate by perilymphatic perfusion and their hydrolysis assayed in 'chase' experiments with non-radioactive orthophosphate. Carbachol (1 mM) reduced the content of 32P-labeled phosphatidylinositol 4,5-bisphosphate in the organ of Corti from 31% to 21% of total 32P-lipids, indicating stimulated hydrolysis. The pharmacology of this effect was studied in detail in vitro via the release of inositol phosphates from phosphoinositides pre-labeled with 3H-inositol. Release was increased 2-fold by 1 mM carbachol, 1.6-fold by 1 mM muscarine, but was unaffected by dimethylphenylpiperazinium; the stimulation was blocked by 1 microM atropine but not mecamylamine. These responses indicate the coupling of phosphoinositides to a muscarinic receptor. Furthermore, stimulated inositol phosphate release was higher in the base of the organ of Corti than in the apex which correlates with the increased cholinergic efferent innervation of outer hair cells in the basal region. These results suggest that muscarinic-stimulated inositol phosphate release occurs at the level of the outer hair cell and thus may have an important modulatory role in auditory transduction.

Animals↗

PubMed1992

P2 purinoceptors stimulate inositol phosphate release in the organ of Corti.

We examined the effects of purinoceptor agonists on inositol phosphate (IP) release in the guinea-pig organ of Corti. The P2y receptor agonist ATP-gamma-S (200 microM) increased IPs 4-fold; identical concentrations of alpha, beta-methylene ATP, a P2x agonist, and adenosine, a P1 agonist, did not significantly affect IP release. In calcium-free incubations, simulated IP release decreased by 35% indicating partial dependence of ATP-mediated phosphoinositide hydrolysis on calcium influx. ATP-stimulated IP release was not enhanced by the cholinergic agonist carbachol known to increase IPs via muscarinic receptors in the organ of Corti. This is consistent with the notion that ATP and carbachol have a common target, most likely outer hair cells. P2 purinoceptors coupled to the phosphoinositide cascade suggest ATP as an afferent neuromodulator or efferent neurotransmitter in the cochlea.

Animals↗

PubMed1992

Motility of cochlear outer hair cells.

A recent exciting discovery in the physiology of hearing has been that fine tuning and regulatory properties of the mammalian auditory system reside in the micromechanics of the basilar membrane. While the inner hair cells are considered primary afferent transducers, the outer hair cells are postulated to modulate transduction based on their motile properties. These new insights have impacted on our view of auditory processing and are also leading to improved diagnostic procedures (such as those based on otoacoustic emissions) and a better understanding of sensorineural pathology. Outer hair cell motility may be separated into two categories: fast and slow. Fast motility is voltage-driven and frequency-following and provides positive feedback to the motion of the basilar membrane. Slow motility (shape changes measured in milliseconds to seconds) can be triggered through depolarization by potassium, osmotic effects, mechanical stimulation, efferent neurotransmitters, and the elevation of intracellular second messengers and calcium ions. These slow changes may be superimposed upon the fine-tuning provided by the fast motility. This paper discusses the mechanism underlying the shape changes induced by different stimuli, and assesses the role of fast and slow motility in the physiology and pathology of auditory transduction.

Acoustic Stimulation↗