Cytochemical localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity in the facial nerve of reserpinized guinea pigs.

Ion-transporting Na,K-ATPase plays an essential role in nerve conduction. To clarify the cytochemical effects of reserpine on transport Na,K-ATPase activity, the localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the facial nerves of normal and reserpinized guinea pigs using a cerium-based method. In the normal facial nerve, the reaction product of K-NPPase activity was observed on the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was localized to the paranodal and nodal axolemma. In the reserpinized nerves, reaction product was detectable on the nodal axolemma but was undetectable on the other parts of the axolemma. Nodal K-NPPase was not affected by reserpine treatment. Therefore, the transport Na,K-ATPase on the nodal axolemma might differ from that on the other parts of the axolemma. Allowing reserpinized animals to survive. Two different ouabain-sensitive K-NPPase reactivities, "reserpine-sensitive" and "reserpine-resistant," might be present in the facial nerve of guinea pigs.

Cytochemical localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase activity in the choroid plexus of normal and reserpinized guinea pigs.

High doses of reserpine induce depletion of biogenic amines. The K-NPPase activity of choroid plexus was determined after one-shot reserpine administration using cerium-based cytochemistry. In normal untreated animals, reaction product was found on the microvilli of the choroidal epithelium but was almost undetectable 3 and 7 days after reserpinization. At 20 days after reserpinization, however, it was detectable. These findings suggested that reserpine decreased the choroidal Na,K-ATPase activity, and that catecholamines might be essential to maintain normal choroidal Na,K-ATPase activity. (J Histochem Cytochem 46: 975-976, 1998)

A fact database for toxicological data at the National Institute of Hygienic Sciences, Japan.

The computerized fact database for the toxicity data of chemicals was constructed at the National Institute of Hygienic Sciences, Tokyo, Japan (biological database, BL-DB). The BL-DB stores data on mutagenicity, teratogenicity, carcinogenicity, and other toxicological tests of chemicals that appeared in the scientific literature. The BL-DB includes information about chemical identification, test system, results of the assays, and a bibliography. The system consists of five modules: data collection, data maintenance, data search, data downloading, and backup. ADABAS is used as a core database management system. Many kinds of test data are stored with the same formats; therefore, users can retrieve data of different toxicological data by the same manner. A user of the BL-DB can use about 50 kinds of commands to interact with the system, and the majority of fields are defined as search fields, thereby facilitating retrieval of target data through many ways. Currently, there are mainly data for the mutagenicity, especially on the Salmonella/microsome assay and the rodent micronucleus assay. These data can be retrieved and used for structure-activity relationship studies.

Cytochemical localization of Na+/K+-ATPase activity in cochlear strial marginal cells after various catecholamine administrations.

Sodium/potassium-activated adenosine triphosphatase (Na+/K+-ATPase) activity in the kidney and brain is high, and is regulated by catecholamines. Na+/K+-ATPase activity is also high in the basolateral infoldings of the strial marginal cells, where it aids in maintaining the characteristic electrolyte composition of the endolymph. To clarify the involvement of humoral control in strial function, particularly the role of catecholamines, the K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of strial marginal cells was investigated in guinea pigs using a cerium-based cytochemical method. The effects of reserpine, serotonin (5-HT), norepinephrine (NE), epinephrine (EP), both alone and in combination, were studied. High doses of reserpine cause depletion of sympathetic substances. Strial K+-NPPase activity was decreased after reserpine or dopamine treatment, and was increased after 5-HT, NE, and EP treatment. After reserpinization, repeated treatment with 5-HT, NE, or EP led to detectable strial enzyme activity. Thus, exogenous 5-HT, NE, and EP were able to restore strial K+-NPPase activity in the reserpine-treated animals. These results suggested that biogenic amines regulate strial K+-NPPase activity. Thus, the function of the stria vascularis may be regulated by the opposing actions of these catecholamines, and 5-HT.

Tremorgenic indole alkaloids. Studies directed toward the assembly of the A, F, and I rings of penitrem D: observation of an unexpected stereochemical outcome.

[formula: see text] In this Letter we demonstrate the viability of a highly stereoselective tandem Mannich cyclization-grammine fragmentation/addition cascade, critical for assembly of the A and F rings of penitrem D. We also explored simultaneous execution of this tactic with concurrent construction of ring I. Reinvestigation of a model system provided an explanation for the unanticipated stereochemical outcome at C(28).

Effects of dopamine hydrochloride (Inovan(R)) on ouabain-sensitive, K+-dependent, p-nitrophenylphosphatase activity of choroid plexus in guinea pigs.

Dopamine hydrochloride (Inovan(R)) is used for the treatment of acute circulatory insufficiency. In the present study, the Na-K ATPase activity of the choroid plexus was determined normal and after repeated injections of dopamine hydrochloride using cerium-based cytochemistry. In the normal guinea pigs, the reaction product was detected on the microvilli of the choroidal epithelium, but was almost undetectable after seven repeated injections of dopamine hydrochloride. These findings suggested that high doses of dopamine hydrochloride decreased the choroidal Na-K ATPase activity.

Reserpine inhibits the NaK ATPase activity of the stria vascularis in the cochlea.

The function of the stria vascularis in the cochlea is believed to be the production of endolymph. The mechanisms that maintain or control the function of the stria vascularis, however, remain unclear. In the present study, the effects of one-shot reserpine administration on the NaK ATPase activity of the stria vascularis in guinea pigs were investigated. NaK ATPase activity was shown to be completely inhibited 3 to 20 days after reserpinization, and was detectable again 60 days after reserpinization. As reserpine is an adrenergic neuron blocker, these results suggest that catecholamines may play an essential role in the maintenance or control of NaK ATPase activity, and that the stria vascularis may be one of the target organs of catecholamines.

Impaired Ranvier node sodium-potassium adenosine triphosphatase may induce facial palsy.

To clarify the part of the neuron essential for myelinated nerve conduction, the cytochemical localization of potassium ion (K+)-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the normal and reserpine-treated facial nerve of guinea pigs. In the normal animals, K-NPPase activity was localized to the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was observed along the paranodal and nodal axolemma. In reserpinized nerves, K-NPPase activity was absent along the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, however, enzyme activity was detectable only in the nodal axolemma. The reserpinized animals demonstrated no evidence of facial palsy. Because K-NPPase is essential for nerve conduction, these results indicate that the location of enzyme activity in reserpinized animals, namely the nodal axolemma, may be of prime importance in saltatory nerve conduction.

Effects of epinephrine on ouabain-sensitive, K(+) -dependent P-nitrophenylphosphatase activity in strial marginal cells of guinea pigs.

In strial marginal cells, Na+/K(+)-ATPase activity is abundant, and contributes to maintain the characteristic electrolyte composition of the cochlear endolymph. In the present study, to clarify the relationship between epinephrine and strial Na+/K(+)-ATPase activity, the ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and guinea pigs treated with reserpine, epinephrine, and reserpine plus epinephrine. In our previous study, K(+)-NPPase activity had almost completely decreased 3 to 20 days after reserpine administration. In the present study, at 10 days after reserpinization and following repeated epinephrine treatment, enzyme activity was detectable. These results suggest that exogenous epinephrine was able to restore strial K(+)-NPPase activity in the reserpine-treated animals, and that epinephrine might increase strial Na+/K(+)-ATPase activity.

Effect of peroral glycerol administration on inner ear fluid electrolytes of guinea pigs.

After the oral administration of 50% glycerol (12 mL/kg), serum, CSF and inner ear fluids from scala tympani perilymph, scala vestibuli perilymph, and scala media endolymph were collected from normal guinea pigs under sodium pentobarbital anesthesia (25-35 mg/kg). The sodium and potassium concentrations were determined by microflame photometry. Increases in sodium concentrations were found in CSF, scala tympani perilymph, scala vestibuli perilymph, and cochlear endolymph. No significant change was observed in the serum. These sodium increases were considered to be due to the dehydration caused by the osmotic action of glycerol. Potassium concentration was increased only in scala tympani perilymph. Oral administration of glycerol was found to be more gradual and effective in dehydration compared to intravenous injection.

Effect of norepinephrine on ouabain-sensitive, K+-dependent p-nitrophenylphosphatase activity in strial marginal cells of the cochlea in normal and reserpinized guinea pigs.

On the basolateral infoldings of the strial marginal cells in the cochlea, Na K ATPase activity is abundant. To clarify the humoral control by norepinephrine, K-NPPase activity of strial marginal cells in the cochlea was investigated in normal, reserpine, norepinephrine (NE), reserpine plus NE-treated guinea pigs using a cerium-based method. K-NPPase activity was almost completely decreased 3-20 days after reserpine administration. At 10 days after reserpinization and following NE repeated treatment, enzyme activity was detectable. These results suggested that norepinephrine might restore and regulate strial K-NPPase activity.

Dopamine inhibits the Na-K ATPase activity of the stria vascularis in the cochlea. In vivo ultracytochemical study.

It is believed that the function of the stria vascularis in the cochlea is to produce endolymph. However, the mechanisms that maintain and control the function of the stria vascularis remain unclear. In the present study, to clarify the role of humoral substances in the stria vascularis, a cerium-based method was used to investigate the ultracytochemical effects of intraperitoneal dopamine administration on ouabain-sensitive, K(+)-dependent, p-nitrophenylphosphatase (K-NPPase) activity, which is the second step in the formation of the Na-K ATPase complex, in the stria vascularis of guinea pigs. Na-K ATPase activity was shown to be completely inhibited after dopamine administration. This observation suggests that dopamine inhibits Na-K ATPase activity in the marginal cells of the stria vascularis, and that the stria vascularis may have dopamine receptors. Catecholamines may play an important role in the maintenance and/or control of stria vascularis function.

Effects of intravenous glycerol injection on inner ear fluid electrolytes.

Under sodium pentrobarbital anesthesia (20-30 mg/kg, i.p.), normal guinea pigs received an intravenous injection of glycerol (1.0 ml/kg). Serum, cerebrospinal fluids (CSF) and inner ear fluids were collected from the scala tympani perilymph, scala vestibuli perilymph and the scala media endolymph. The sodium and potassium concentrations were assessed using microflame photometry. Increases in sodium concentration were found in the CSF and the scala tympani perilymph; no significant changes were observed in the serum, scala vestibuli perilymph or the scala media endolymph. These sodium increases were considered to be a result of the dehydration process caused by the osmotic agent glycerol. Increases in potassium concentration were found only in the scala vestibuli perilymph.

Effects of reserpinization on the electrolytes distribution in inner ear fluids of guinea pig.

Reserpine (2 mg/kg and 10 mg/kg, i.p.), an adrenergic blocker, was administered to normal guinea pigs, and samples of serum, CSF, scala tympani perilymph, scala vestibuli perilymph and cochlear endolymph were collected. The concentrations of Na and K in these fluids were then assessed to observe electrolyte distributions. Both Na and K concentrations were reduced in serum. In CSF, the Na concentration was markedly depressed, while the K concentration remained unchanged. Scala tympani perilymph showed a pattern of electrolyte changes similar to that in CSF. In scala vestibuli perilymph, the Na concentration was unaltered, but the K concentration was lowered. Cochlear endolymph exhibited no change of the Na concentration but there was a marked decrease in the K concentration. These changes are thought to be attributable to the indirect blockage of energy producing systems in membrane transport in the inner ear by the reserpine-induced depletion of catecholamines.

Effect of glycerol on the EP decrease caused by furosemide.

A change in endocochlear potential (EP) following furosemide injection was observed in regard to prior glycerol administration in two groups of guinea pigs. In one group, various doses of furosemide (20, 30, 40, 50 mg/kg) were injected, while in the other, glycerol (50 v/v %, 1 ml/kg) was injected prior to the furosemide (20, 30, 40 mg/kg) injection. In the glycerol-furosemide group, the decrease in EP was 40% greater than in the furosemide group. Therefore, glycerol was thought to potentiate the EP lowering action of furosemide. Such an effect of glycerol was assumed to be resulted by facilitating the access of furosemide to the site of action in the stria vascularis.

Ultracytochemical study of ouabain-sensitive K(+)-dependent p-nitro-phenylphosphatase activity in stria vascularis of reserpinized guinea pigs.

In order to clarify the effect of reserpine on the Na(+)-K+ active transport in the stria vascularis we investigated the localization of ouabain-sensitive K(+)-dependent p-nitro-phenylphosphatase (K-NPPase) activity in the stria vascularis of reserpinized guinea pigs using the cerium-based method. The K-NPPase activity in marginal cells differed from cell to cell, and was almost completely absent in some cells. These results are consistent with the previous results and suggest that Na(+)-K+ active transport in the stria vascularis may be inhibited by reserpine administration.

Cytochemical effects of in vitro dopamine treatment on the Na-KATPase activity in strial marginal cells.

Dopamine is often used clinically for the treatment of patients with shock. In a previous study, the Na-KATPase (K-NPPase) activity of strial marginal cells was inhibited after the repeated in vivo administration of dopamine hydrochloride. In the present study, the K-NPPase activity of strial marginal cells was determined using a cerium-based cytochemistry after an in vitro incubation with dopamine. The enzyme reaction product was found in untreated normal strial marginal cells, but it was almost completely undetectable after an in vitro treatment with 10 mM dopamine. This finding suggested that dopamine directly inhibited the Na-KATPase activity of strial marginal cells, and that these cells might have a dopamine receptor.

The developing electrolytes concentrations of inner ear fluids in guinea pigs.

Cochlear endolymph, scala tympani perilymph, and scala vestibuli perilymph were collected from fetal, neonatal, and mature guinea pigs. The concentrations of Na and K in these fluids were then assessed using a microflame photometer. The electrolyte composition in the endolymph was already characterized by a high concentration of K and a low concentration of Na by the 49th day of gestation.

Effect of glycerol on furosemide-caused EP decrease in guinea pig neonates.

Furosemide, a loop diuretic, reduces the endocochlear potential in guinea pigs. In our previous experiment using adult guinea pigs, glycerol was thought to open the blood-cochlea barrier and to help furosemide to reach its site of action in the stria vascularis. The present study was designed to determine whether or not the same potentiation by glycerol occurs in neonate guinea pigs. In one group, furosemide (10-30 mg/kg) was administered, while in another group, 50 v/v% glycerol (1 ml/kg) was administered 15 minutes prior to the injection of furosemide. Furosemide reduced the EP significantly more in neonates than in adults. This effect was not potentiated in the glycerol-pretreated group. From these findings, it seemed conceivable that the neonate blood-cochlea barrier still remains immature in some functions.

Effects of glycerol on the inner ear fluid electrolytes of guinea pigs--oral and intravenous administration.

Under sodium pentobarbital anesthesia (25-35 mg/kg), serum, CSF and inner ear fluids from scala tympani perilymph, scala vestibuli perilymph, and scala media endolymph were collected from normal guinea pigs after the oral administration of glycerol (50%, 12 ml/kg) or the intravenous injection of glycerol (1.0 ml/kg). The sodium and potassium concentrations were assessed by microflame photometry. The electrolyte dynamics of the inner ear fluids were compared after these two routes of administration. Serum: There was no significant change in Na or K levels after either route of administration. CSF: The Na concentration increased rapidly after both intravenous and oral administrations of glycerol and remained high until the end of the experiment. The K concentration did not change significantly after intravenous injection. Scala tympani perilymph: The Na concentration increased transiently, after intravenous glycerol, while after oral administration it increased slowly and steadily. The K concentration increased only after oral administration. Scala vestibuli perilymph: The Na level increased slowly and steadily only after oral administration of glycerol, and the K level increased slightly after intravenous injection. Scala media endolymph: No changes in the K level occurred after either route of administration. After oral administration the Na level increased. These experiments show that dehydration persists longer after oral than after intravenous administration of glycerol. In the diagnosis of Meniere's disease, oral administration of glycerol is more effective than intravenous injection.