Spermine and spermidine as gating molecules for inward rectifier K+ channels.

Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.

DFMO reduces cortical infarct volume after middle cerebral artery occlusion in the rat.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is induced in ischemic tissue and may mediate vasogenic edema and delayed neuronal death. We determined the effects of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC, on infarct size and ODC activity in a rat model of transient focal ischemia. DFMO blocked the ischemia-induced increase in ODC and significantly reduced infarct volumes by 57-45%, depending upon the treatment regimen. These studies suggest that polyamine metabolism plays a role in the development of cerebral infarction after focal ischemia and that DFMO may be useful in limiting injury after a stroke.

Impairment in cochlear function produced by chloramphenicol and noise.

Ototoxic interaction between chloramphenicol and noise was studied in two separate investigations. In the first study, permanent ototoxicity was demonstrated in a group of rats which were subjected to short-duration, high-intensity noise and were then given chloramphenicol orally. The anatomical damage in this group was consistent with observed changes in cochlear round window recordings of cochlear microphonics at 4 kHz and of the N1 component of the eighth nerve action potential. In the second study, a temporary depression in the function of the cochlea was observed in rats subjected to the noise-chloramphenicol regimen used in the first study. Depressions in recordings of the round window similar to those in the first study were seen only during the first five days of recordings. After the fifth day, the recordings of the round window were normal, indicating recovery from a temporary shift in threshold produced by chloramphenicol and noise. Incidence of purulent otitis media was found in 57 and 0% of the animals in the first and second studies, respectively. The combination of chloramphenicol and noise appears to be responsible for the production of temporary cochlear deficits. The addition of the third variable, otitis media, appears to result in permanent impairment of the cochlea.

Inhibition of renal ornithine decarboxylase by aminoglycoside antibiotics in vitro.

The inhibition of renal ornithine decarboxylase (ODC) by aminoglycoside antibiotics was characterized in the postmitochondrial fraction of a kidney homogenate from adult pigmented guinea pigs. Enzymatic activity was defined as the rate of decarboxylation of [14C]ornithine sensitive to a specific ODC inhibitor, alpha-difluoromethylornithine (DFMO). The Km for ornithine was 61 +/- 32 microM. There were two forms of the enzyme with respect to their affinity for pyridoxal phosphate (PLP): (I) Km = 2.1 +/- 1.8 microM; (II) Km = 36.2 +/- 12.7 microM. Putrescine, a known ODC inhibitor, acted competitively on the renal enzyme with Ki = 1.7 +/- 1.4 mM. Aminoglycoside antibiotics inhibited ODC by an uncompetitive mechanism with inhibitor constants of comparable magnitude: neomycin, Ki = 1.3 +/- 0.1 mM; gentamicin, Ki = 1.6 +/- 0.1 mM; kanamycin, Ki = 1.9 +/- 0.2 mM; and netilmicin, Ki = 1.7 +/- 0.2 mM. Neomycin inhibited both forms of the enzyme (low and high affinity for PLP) uncompetitively with similar inhibitor constants (1.5 +/- 0.3 and 1.8 +/- 0.4 mM respectively), suggesting a single mechanism of action. Inhibition of ODC suggests that aminoglycoside-polyamine interactions may be an important component of the sequence of biochemical events associated with aminoglycoside toxicity.

Activation of ornithine decarboxylase and accumulation of putrescine after traumatic brain injury.

Activation of ornithine decarboxylase (ODC), the initial enzyme in polyamine synthesis, and accumulation of putrescine are thought to mediate pathological processes in the ischemic and traumatized brain. Past studies have separately investigated either ODC or polyamines after head injury. The purpose of the present study was to quantify both ODC activity and polyamines in the rat parietal cortex before and after controlled cortical impact injury. Adult, male rats underwent a right craniectomy and were subjected to a 5 m/sec, 2-mm deformation impact injury. Rats were sacrificed 1, 4, 8, and 24 h postimpact and tissues from the injured (right) and contralateral (left) hemisphere were analyzed for ODC and polyamines. ODC activity was determined by measuring the decarboxylation of [14C]ornithine to putrescine. Putrescine, spermidine, and spermine were determined by high performance liquid chromatography. Cortical impact induced a 10- to 20-fold increase in ODC activity and a 4- to 5-fold increase in putrescine in the ipsilateral cortex. Spermidine and spermine did not significantly increase in the ipsilateral (right) cortex compared to controls (right cortex). In contrast, there was a slight increase in spermidine content in the contralateral (left) cortex after injury. The delayed increase in ODC activity and accumulation of putrescine may mediate pathophysiological changes observed after head injury.

Effects of anesthesia on polyamine metabolism and water content in the rat brain.

Because variances have been noted in brain putrescine levels of anesthetized rats (control, SHAM-operated), we investigated the effects of several anesthetics on polyamine metabolism and water content in the adult rat brain. Short duration (5 min) anesthesia was studied in three groups: ketamine:xylazine [KX; 40 and 8 mg/kg, respectively, intraperitoneal injection (IP)], urethane (UR; 1.5 g/kg, IP), and isoflurane (IF, initially 3.5% in 100% O2, followed by a maintenance dose of 2.5% IF in 100% O2). Effects of IF at longer duration (30 min) were also studied because this paradigm is often used in our laboratory. Control rats received no anesthesia (NA). Following decapitation, tissue samples were obtained from 3 bilateral brain regions: parietal cortex, motor area (CPm); parietal cortex, somatosensory area (CPs); and the pyriform cortex (CPF). The polyamines, spermidine and spermine, and their precursor, putrescine, were quantified by HPLC-fluorometric detection and brain water content was determined by wet-to-dry weight measures. KX decreased putrescine (54%) and spermidine (20%) in the CPs, increased spermine (24%) in the CPF, and increased water content in all brain regions. UR decreased putrescine (51%) and slightly increased water content (0.7%) in the CPF. Short duration IF decreased putrescine and spermidine in all brain regions; decreased spermine in the CPm, and increased water content in the CPF (0.8%). In contrast, longer duration IF increased putrescine (181%) and spermidine (23%) in the CPm, with no change in water content. Anesthetics produce region-specific changes in putrescine, polyamines, and water content in the rat brain which could contribute to the experimental variability.

Inhibition of inner ear ornithine decarboxylase by neomycin in-vitro.

We quantitated the activity of ornithine decarboxylase (ODC) in homogenates and subcellular fractions of inner ear tissues from the rat and guinea pig and demonstrate inhibition of cochlear ODC by the aminoglycoside neomycin. Subcellular fractionation showed the enzyme associated with the post-mitochondrial supernatant fraction in each of the tissues: Specific activities of ODC, defined as alpha-difluoromethylornithine (DFMO)-sensitive decarboxylation of ornithine, in the supernatant fractions of combined inner ear tissues were: guinea pig = 44 +/- 4 pmoles CO2 produced/hour/mg protein, and rat = 133 +/- 30. In the guinea pig, supernatant fractions of the lateral wall tissues (stria vascularis and spiral ligament) had specific activities of 62 +/- 25, those of the organ of Corti (plus VIIIth nerve) 64 +/- 41. The ototoxic aminoglycoside neomycin produced a dose-dependent inhibition of ODC with half-maximal inhibition observed at 50 microM drug and almost complete inhibition at 100 microM. This is the first report of the presence of ODC in the inner ear and its inhibition by neomycin. Since both the ODC-inhibitors, DFMO and neomycin, can cause hearing loss in patients and experimental animals it is suggested that inhibition of ODC may be an important factor in the ototoxicity of these drugs.

Auditory aspects of seizure in the genetically epilepsy prone rat.

The organ of Corti of Genetically Epilepsy Prone Rats was examined anatomically and electrophysiologically using scanning electron microscopy (SEM) and electrophysiological recording of alternating current cochlear potentials (ACCP) and N1, a volume conductor recording of the primary auditory afferent action potentials. ACCPs for GEPRs with low intensity seizures (Acoustic Response Score (ARS) = 2 or 3) and high intensity seizures (ARS = 9) showed similar impairment in cochlear function. Approximately a 25-35 dB shift in input-output functions was present in GEPRs as compared to controls. SEM revealed several types of possible genetic abnormalities which explain the deficits in cochlear function and could serve as the basis for seizure predisposition in these animals.

Postnatal developmental changes in inner ear ornithine decarboxylase (ODC).

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cellular growth, differentiation and development. Although ODC has been quantitated in cochlear tissues of the adult rat, it has not been assessed quantitatively in developing inner-ear tissues. The purpose of the present study was to quantitate ODC in cochlear tissues of the rat during the period of development of hearing. Cochlear ODC was significantly elevated throughout the period of cochlear maturation in that it increased rapidly during the first 10 days, peaked on day 10 and then declined thereafter. ODC in the lateral wall/organ of Corti tissues was significantly higher than in the cochlear nerve in developing, but not in adult rats. Further examination of separate cochlear tissues from 10-day old rats revealed that ODC activity was higher in the organ of Corti than in the lateral wall or cochlear nerve. Postnatal changes in ODC paralleled functional maturation of hearing and the hypersensitive period for aminoglycoside ototoxicity in the rat. Since aminoglycosides have been shown to inhibit ODC in vitro, aminoglycoside inhibition of polyamine synthesis may mediate the hypersensitivity of developing animals to the effects of these drugs.

Postnatal changes in cochlear polyamine metabolism in the rat.

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, is increased in the developing rat cochlea, suggesting that polyamine biosynthesis is important in cochlear development. Although cochlear polyamines have been detected in adult rats, they have not been identified in developing rats. We quantified polyamines in the developing and mature rat cochlea and further characterized ODC in the early postnatal period. Putrescine and spermidine in combined tissues of the organ of Corti and lateral wall of the cochlea were highest during the first 10 postnatal days, then declined to adult levels shortly thereafter. Spermine demonstrated a similar developmental trend. A high spermidine to spermine ratio was noted during this period as was rapidly increasing ODC activity. A high spermidine/spermine ratio was also noted in the cochlear nerve of developing and mature rats, suggesting that spermidine may be necessary for function and maintenance of the nerve. This is the first report of polyamines in the developing rat cochlea. The period of increased polyamine synthesis coincides with the critical period for ototoxicity induced by alpha-difluoromethylornithine, a specific ODC inhibitor, and the period of rapid cochlear development.

Distortion product otoacoustic emissions and outer hair cell defects in the hyt/hyt mutant mouse.

Thyroid hormone plays an important role in hearing development. Hereditary hypothyroidism is frequently associated with sensorineural hearing loss as identified in both animal models and human patients. Building upon our original demonstration of congenital deafness and hair cell abnormality in a hyt/hyt mouse model which carries an autosomal recessive mutation causing hereditary hypothyroidism, we investigated the functional capacity of the outer hair cell (OHC) system in these animals using distortion product otoacoustic emissions (DPOAEs). In particular, the amplitude and detection features of DPOAEs were correlated with measures of the auditory brainstem response (ABR) as well as the cellular structure and ultrastructure of the organ of Corti. Input-output (I/O) functions for the 2f(2)-f(1) DPOAEs were obtained for frequencies from 2 to 18 kHz. The thresholds were significantly higher and amplitudes were significantly lower in the homozygous mice (hyt/hyt) than in both heterozygous mice (hyt/+) and wild-type controls at DPOAE frequencies recorded above 6 kHz. Hearing thresholds were significantly elevated in the mutant compared to control mice. In addition, morphological studies revealed consistent inner ear defects in hyt/hyt animals including distortion of the tectorial membrane, dysplasia of the tunnel of Corti and distinct OHC abnormalities. The most striking histopathological finding was a contiguous membrane along the apices of all of the OHC stereocilia. Such ultrastructural changes in the stereocilia of the OHC may limit the deflection of the stereocilia and therefore affect an active cochlear function that produces otoacoustic emissions as well as cause a failure to evoke the normal action potentials in the auditory nerve. From both functional and morphologic evaluations, it was concluded that the OHC system is the most susceptible to the developmental effects of congenital hypothyroidism in the hyt/hyt mouse. The normal OHCs with well-developed ciliary bundles are crucial to maintain the activity of biological mechanisms within the cochlea.

Increased susceptibility of male rats to kanamycin-induced cochleotoxicity.

Although clinical observations suggest that males are more susceptible than females to ototoxic drugs, controlled experimental studies investigating gender susceptibility have not been performed. Aminoglycosides initially attack the cochlea's outer hair cells (OHCs). We investigated the effects of the aminoglycoside, kanamycin, on electrophysiological function of OHCs in male and female rats. Animals were grouped by gender and treated with kanamycin (400 mg/kg/day kanamycin base, intramuscular injection) or equivolume normal saline. Administration was continued until distortion product otoacoustic emissions (DPOAEs) suggested a loss in OHC function in kanamycin-treated rats. Males treated with kanamycin showed changes in DPOAE thresholds and amplitudes as early as treatment day 10 which spread to all test frequencies by treatment day 13. In contrast, females treated with kanamycin did not show significant changes in thresholds or amplitudes until treatment day 22. The mechanism of increased male susceptibility to kanamycin cochleotoxicity has not been determined.

Localization of ornithine decarboxylase (ODC) in the cochlea of the immature rat.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents.

Pharmacokinetics of kanamycin in the developing rat.

The developing rat is hypersensitive to aminoglycoside ototoxicity during the period of anatomical and functional development of the cochlea. Toxicity is expressed only after a few days of treatment when kanamycin is given during the most sensitive period for production of ototoxicity (postnatal days 11-20). In contrast, when the drug is administered after the 20th postnatal day, the same dose and duration of treatment do not produce an ototoxic effect. Only after prolonged treatment (e.g., > or = 20 days) is there an observed effect. We characterized the pharmacokinetics of kanamycin in the serum of 12- and 25-day-old rats and observed a greater than 2.5-fold increase in elimination half-life in the 12- versus 25-day-old rat. The longer duration half-life of kanamycin in younger rats may explain the hypersensitivity of immature mammals to aminoglycoside ototoxicity.

Sensitive developmental periods for kanamycin ototoxic effects on distortion-product otoacoustic emissions.

The developing rat is hypersensitive to aminoglycoside toxicity, which is expressed early on as a destruction of outer hair cells (OHC). In the current study, distortion-product otoacoustic emissions (DPOAE), which specifically measure the micromechanical activity of OHCs, were used to assess functional effects of administering a regimen of kanamycin to three groups of neonatal rats representing discrete postnatal developmental periods. In this manner, pigmented rats were treated at postnatal days 1-10, 11-20, and 21-30. A series of input-output (I/O) functions obtained for the 2f1-f2 DPOAE during the post-treatment period indicated that detection thresholds were significantly elevated for the animals treated on postnatal days 1-10 and 11-20, with the greatest elevations observed at the higher test frequencies.

Postnatal development of 2f1-f2 otoacoustic emissions in pigmented rat.

Distortion-product emissions at 2f1-f2 were measured in developing, pigmented rats to determine the characteristics of the functional onset and maturation of these emitted responses. Distortion-product testing was conducted on postnatal days 12, 14, 18, 21, 24, 28, and 51. The geometric-mean frequencies of the primaries were tested at one-half octave intervals, between 4 and 11.3 kHz. No emissions were detected on postnatal day 12; however, by day 14, emissions were measured at geometric-mean frequencies between 5.7 and 11.3 kHz, but not at 4 kHz. By the 18th postnatal day, all animals had measurable emissions at 4, 5, 7, 8, and 11.3 kHz. Both the functional onset and maturation of high- to midfrequency emissions developed before those elicited by lower-frequency primaries. Response/growth or input/output functions for the higher frequencies exhibited 'adult-like' properties that included an increase in maximum amplitude and 'saturation' at high levels of stimulation by the third to fourth postnatal week. Maturation of the lower-frequency responses progressed at a slower rate. These results differ somewhat from those reported previously for the neonatal albino rat. However, the present findings were based upon a wider frequency range of primary tones, lower levels of acoustic stimulation, and a measuring system with a significantly lower noise floor. These results are consistent with documented periods of the anatomical maturation of the rat outer hair-cell system and establish a functional 'baseline' for future studies utilizing agents that damage the developing cochlea.

Effects of cisplatin and thiosulfate upon auditory brainstem responses of guinea pigs.

Two side effects which limit the use of cisplatin in cancer chemotherapy are severe nephrotoxicity and ototoxicity. The concurrent administration of sodium thiosulfate with cisplatin reportedly protects from cisplatin nephrotoxicity, however, protection from ototoxicity has not been documented. The purpose of this study was to examine the efficacy of using thiosulfate to ameliorate the ototoxic effects of cisplatin. Toward this end, the effects of cisplatin alone, cisplatin administered concurrently with sodium thiosulfate (CIS/THIO), and sodium thiosulfate alone on the auditory brainstem response (ABR) of guinea pigs were compared. ABR waveforms, comparing latencies, amplitudes and response thresholds, were monitored before, immediately after, and 30 days post treatment. Sodium thiosulfate administered with cisplatin (CIS/THIO) consistently protected animals from hearing loss and surprisingly yielded significant increases in amplitude when compared to baseline and saline controls. However, ABRs of CIS/THIO animals returned toward baseline values after 30 days.

Cochlear polyamines: markers of otitis media-induced cochlear damage.

High-performance liquid chromatography (HPLC) set to the femtomole [corrected] sensitivity level was used to identify and quantify the polyamines spermidine and spermine as well as the diamine putrescine in the different tissues of the inner ears of guinea pigs with experimentally induced otitis media. The tissues examined were the lateral wall (stria vascularis and the spiral ligament), the organ of Corti, and the cochlear nerve. The difference in polyamine profile in the different tissues of the control noninfected guinea pigs suggests a relation to the particular function of each of these tissues [see erratum notice re: preceding sentence]. The difference in polyamine profile in infected different inner ear tissues compared to controls encourages the assumption that the polyamines may be involved in a repair process of the inner ear after injury and that they may be considered as biochemical markers for inner ear damage secondary to acute otitis media.

Kanamycin depletes cochlear polyamines in the developing rat.

Developing mammals are more sensitive to aminoglycoside antibiotics and other ototoxic agents than adults, with maximum sensitivity occurring during the period of anatomic and functional maturation of the cochlea. For the aminoglycoside antibiotics, the hypersensitive period in rats occurs during the second and third postnatal weeks. Toxicity is initially expressed as outer hair cell (OHC) damage in the high-frequency, basal region of the cochlea. Distortion-product otoacoustic emissions (DPOAEs), physiologic measures of OHC function, are particularly sensitive to aminoglycoside exposure during the period of rapid cochlear physiologic development. Toxicity is characterized by increased DPOAE thresholds and decreased amplitudes. The mechanism of developmental sensitivity to aminoglycosides is unknown. A potential biochemical target of aminoglycosides is the ornithine decarboxylase (ODC)-polyamine pathway. ODC activity is elevated in the developing rat cochlea, aminoglycosides inhibit cochlear ODC in developing rats, and alpha-difluoromethylornithine (a specific ODC inhibitor) impairs development of cochlear function. In the present study we demonstrate an incomplete polyamine response to aminoglycoside damage, characterized by inhibition of the polyamines spermidine and spermine and accumulation of putrescine in the organ of Corti. Aminoglycoside inhibition of polyamine synthesis may mediate developmental ototoxic hypersensitivity by interfering with developmental and repair processes.

Kanamycin inhibits cochlear-renal ODC in neonatal rats.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is important in development and regeneration. We hypothesize that aminoglycoside inhibition of ODC mediates developmental hypersensitivity to aminoglycoside ototoxicity. Kanamycin effects on ODC activity (decarboxylation of ornithine) in vitro were determined in the postmitochondrial fraction of cochlear and renal homogenates from 11-day-old rats. Kanamycin inhibited cochlear and renal ODC by an uncompetitive mechanism. For the cochlear enzyme, the inhibitor constant (Ki) for kanamycin was 99 +/- 25 mumol/L; for the renal enzyme, the Ki = 1.5 +/- 0.1 mmol/L. In vivo effects of kanamycin on cochlear, renal, brain ODC activity were determined in rats treated with kanamycin (400 mg/kg/day, intramuscularly) or saline during postnatal days 11 through 20, the hypersensitive period for ototoxicity. Rats were killed on postnatal days 12, 14, 16, and 20 and ODC was assayed. Kanamycin significantly inhibited ODC in the lateral wall-organ of Corti and kidney (ANOVA alpha = 0.05), but had no effect on cochlear nerve and no consistent inhibitory effect in the brain. These results suggest that ODC is a potential target of kanamycin in susceptible tissues and may be a contributing factor in developmental sensitivity to the drug by inhibiting repair and developmental processes mediated by ODC.