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Correspondence to: Naoyuki Kanoh, Dept. of Otolaryngology, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya, Hyogo 663, Japan.
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Summary |
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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. (J Histochem Cytochem 45:1129-1135, 1997)
Key Words: Na,K-ATPase, reserpine-sensitive, reserpine-resistant, K-NPPase cytochemistry, saltatory conduction, Ranvier node, facial nerve, guinea pig
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Introduction |
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Membrane-bound, ouabain-sensitive, K+-dependent adenosine triphosphatase (Na,K-ATPase) is inserted into the plasma membrane, where it generates and maintains high potassium and low sodium concentrations in the cytoplasm (1 (McK1) and
2 (McB2) subunits of the Na+,K+-ATPase. The abundance of the
2 subunit isoform was reduced by 41% in vas deferens homogenates obtained from animals treated with reserpine compared with untreated controls. Using a cerium-based method (
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Materials and Methods |
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The intratemporal portion of bilateral facial nerves was obtained from 13 Hartley guinea pigs weighing 400-500 g with a normal Prayer's reflex. The experimental animals were divided into the following three groups. The first group was sacrificed at 3 days after one-shot reserpine (10 mg/kg IP) administration (n = 5). The second group was sacrificed at 7 days after one-shot reserpine (10 mg/kg IP) administration (n = 3). The third group consisted of normal untreated animals (n = 5) as a positive control for enzyme reactivity.
Preparation of Tissues
Under deep ketamine hydrochloride (IM injection) anesthesia, the animals were perfused through the heart with a fixative containing 2% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. After decapitation, the temporal bones were excised under a stereo microscope in the same fixative. The bilateral facial nerves were carefully dissected from the temporal bones and immersed in the same fixative for 1 hr at 4C. The procedure for determining enzyme activity was performed on treated and untreated facial nerves simultaneously.
Cytochemical Procedure
The tissues were cut into sections 40 µm thick with a Microslicer (Dosaka EM; Kyoto, Japan). The tissue samples were rinsed with 50 mM Tricine buffer, pH 7.5, for 15 min and incubated in the medium according to
Procedure for Electron Microscopy
After cytochemical incubation, the tissues were postfixed with 2% OsO4 in 0.2 M cacodylate buffer, pH 7.4, for 1 hr at room temperature. The specimens were then dehydrated through a graded series of alcohol solutions and propylene oxide, and then embedded in Spurr's epoxy resin (
The animal use protocol was approved by the Institutional Animal Care and Use Committee of the Hyogo College of Medicine, and all experiments were performed in accordance with the guidelines of the Declaration of Helsinki.
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Results |
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In the axon, cytochemically fine granular reaction product was clearly deposited on the cytoplasmic side of the entire axolemma but was hardly visible on the microtubules in the axoplasm (Figure 1a and Figure 1b). In the nodes of Ranvier, the reaction product was localized to the nodal, paranodal, and internodal portions of the axolemma. Enzyme activity was also localized to the cytoplasmic side of the plasma membrane in the terminal paranodal loop of Schwann cells (Figure 1c). In animals sacrificed at 3 days after one-shot reserpine administration, the enzyme reaction was undetectable on the internodal, paranodal axolemma, and Schmidt-Lanterman incisure (Figure 2a), whereas it was detectable on the Ranvier node axolemma (Figure 2b).
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In the control samples, formation of reaction product was almost completely inhibited when 10 mM ouabain was included in the medium (Figure 3a and Figure 3b) and reaction product was undetectable in a substrate-free medium (Figure 4a and Figure 4b) and in a medium in which K+ had been replaced with Na+ (Figure 5a and Figure 5b).
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Table 1 and Table 2 summarize the results obtained from the five normal and eight reserpinized guinea pigs, respectively. In untreated normal animals, K-NPPase activity was detectable in 617 internodal axolemmae of a total of 707 (87.2%) nerve fibers and was also noted in 95 nodal axolemmae of 104 (91.3%) nodes of Ranvier. However, in the 3 days after reserpinization, enzyme activity was only detectable in 152 internodal axolemmae of 635 (23.9%) but was clearly detectable in 90 nodal axolemmae of 102 (88.2%) investigated cases. At 7 days after reserpinization, enzyme activity was detectable only in 35 internodal axolemmae of 510 (6.9%) but was clearly detected in 48 nodal axolemmae of the 58 (82.8%) cases investigated. Reaction product was detectable on the nodal axolemma in the facial nerve but was almost undetectable on other parts of the axolemma after reserpine treatment.
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Discussion |
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Ion-transporting ATPase is a membrane-bound enzyme that couples ATP hydrolysis to the active transport of sodium and potassium ions and plays a crucial role in nerve conduction (
In previous cytochemical studies of the nervous system, Na,K-ATPase activity was reported to be localized on the synaptic plasma membrane (
Na,K-ATPase contains two polypeptide subunits: the or catalytic subunit and the ß or glycoprotein subunit. In 1979, Sweadner described two forms of the catalytic unit of Na,K-ATPase, the
type, which has a low affinity for ouabain, and the
+ type, which has a high affinity for it. The enzyme detected in the current experimental study was almost completely inhibited by ouabain, and therefore appeared to be the
+ form of Na,K-ATPase. Recently, three
-isoforms,
1,
2, and
3, have been sequenced in the rat brain (
-polypeptides have also been detected immunohistochemically in the brain (
1 is the original
-isoform, and that
2 and
3 are contained in the
+ isoform (
Reserpine, an adrenergic neuron blocker, belongs to the family rauwolfia alkaloids and is employed clinically for treatment of hypertension. Pharmacologically, this compound releases biological amines such as norepinephrine, epinephrine, dopamine, and serotonin from storage or binding sites in the central and peripheral nervous system. Therefore, high doses of reserpine induce depletion of these amines, inhibiting reabsorption in the storage site and preventing recombination at the binding sites. The purpose of the present study was to evaluate the cytochemical effects of catecholamine depletion on transport Na,K-ATPase activity in the guinea pig facial nerve. The dose of reserpine used in the present study (10 mg/kg) was believed to be high enough to completely abolish the activity of catecholamines (
In the cochlea, K-NPPase activity in the stria vascularis was shown to be almost completely decreased after reserpine administration (
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Acknowledgments |
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Supported by a Grant-in-Aid (08671998) for Science Research from the Ministry of Education, Science and Culture of Japan.
The author thanks Prof Harumichi Seguchi and Associate Prof Teruhiko Okada, Department of Anatomy and Cell Biology, Kochi Medical School, for their valuable comments.
Received for publication June 20, 1996; accepted February 21, 1997.
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