ARTICLE

Cytochemical Localization of Ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase Activity in the Facial Nerve of Reserpinized Guinea Pigs

Correspondence to: Naoyuki Kanoh, Dept. of Otolaryngology, Hyogo College of Medicine, 1-1 Mukogawacho, Nishinomiya, Hyogo 663, Japan.

	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

	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 (Skou 1965 ; Whittam and Wheeler 1970 ). The transport of Na⁺ and K⁺ across the cell membrane of neurons appears to be a primary prerequisite for conduction of nerve impulses. Gatherer et al. 1979 reported that the Na⁺,K⁺-ATPase activity of the guinea pig vas deferens was reduced after 5 days of treatment with reserpine. Kanoh and Makimoto 1984 reported that the K concentration in the cochlear endolymph decreased and that the sum of the Na and K concentrations of each compartment of the cochlea was almost the same after reserpine administration. They speculated that these phenomena might be due to the inhibition of active K⁺ transport in the stria vascularis. Hershman et al. 1993 addressed this issue with monoclonal antibodies directed against the 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 (Kobayashi et al. 1987 ), Kanoh et al. 1993a and Kanoh 1994 investigated the localization of K-NPPase activity in the stria vascularis of reserpinized guinea pigs and found that K-NPPase activity was decreased from Day 3 to Day 20 after reserpinization. K-NPPase is the second component of the Na,K-ATPase complex, which represents the dephosphorylation step in the sodium pump cycle (Judah et al. 1962 ). In the present study, to clarify the cytochemical effects of reserpine, the localization of K-NPPase activity in the temporal portion of the facial nerve of guinea pigs was examined using a cerium-based method.

	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 Kobayashi et al. 1987 . The incubation medium contained 50 mM Tricine buffer, pH 7.5, 2 mM cerium chloride as the capture agent, 10 mM magnesium chloride, 50 mM potassium chloride, and 2 mM p-NPP (Mg salt) as the substrate, 5% sucrose, 2.5 mM levamisole (an inhibitor of alkaline phosphatase), and 0.00015% Triton X-100 to facilitate penetration of the incubation medium (Robinson 1985 ). The medium was filtered through 0.22-µm pore size filters after the pH was adjusted to 7.5. Incubation was performed for 45 min at 37C in a shaking incubator. Tissue samples were washed for 10 min with 0.1 mM Tris-maleate buffer at pH 6.0, then processed for electron microscopy. In control experiments to determine negative enzyme reactivity, tissue samples were incubated in a substrate-free medium, in a medium containing 10 mM ouabain, and in a medium in which K⁺ had been replaced with Na⁺.

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 (Spurr 1969 ). Thin sections were cut on an Ultracut OmU₄ ultramicrotome (C. Reichert; Vienna, Austria) and picked up with copper grids. Uncontrasted ultrathin sections were then observed under a JEM-1200 electron microscope (JEOL; Tokyo, Japan).

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.

	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).

View larger version (132K): [in this window] [in a new window]   Figure 1. Normal facial nerve. (a) Ouabain-sensitive K-NPPase activity in a longitudinal section of an axon in the facial nerve of guinea pig. Reaction product was localized to the internodal axolemma (arrowheads). Bar = 1 µm. (b) Higher magnification of the internodal axolemma seen in a. Re-action product was localized to the internodal axolemma (arrowheads). Bar = 1 µm. (c) Ranvier node. Reaction product was noted on the nodal axolemma (arrows). Bar = 0.1 µm.

View larger version (116K): [in this window] [in a new window]   Figure 2. Reserpinized facial nerve. (a) K-NPPase activity in a longitudinal section of an axon at 3 days after reserpinization. Reaction product was almost undetectable in the internodal axolemma (asterisks). (b) Ranvier node. Reaction product was localized to the nodal axolemma (arrows). Bars = 1 µm.

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).

View larger version (103K): [in this window] [in a new window]   Figure 3. Control study (ouabain treatment). (a) Axon cylinder (longitudinal section). Reaction product was undetectable on the internodal axolemma (asterisks) when incubated with 10 mM ouabain. (b) Ranvier node. The positive reaction was almost completely inhibited by 10 mM ouabain, including the nodal axolemma (arrow). Bars = 1 µm.

View larger version (98K): [in this window] [in a new window]   Figure 4. Control study (substrate-free). (a) Axon (longitudinal section). Reaction product was undetectable on the internodal axolemma (asterisks) incubated in a substrate-free medium. Bar = 0.1 µm. (b) Ranvier node. Positive reaction was undetectable on the nodal axolemma (arrow). Bar = 1 µm.

View larger version (95K): [in this window] [in a new window]   Figure 5. Control study (K+ replacement with Na+). (a) Axon (longitudinal section). Reaction product was undetectable on the internodal axolemma (asterisks). (b) Ranvier node. Positive reaction was undetectable on the nodal axolemma (arrow). Bars = 1 µm.

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.

	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 (Albers et al. 1989 ). In the present study, the cerium-based method of Kobayashi et al. 1987 was employed to detect K-NPPase activity. The major advantages of this method are its ability to detect ouabain-sensitive K-NPPase activity at physiological pH and the absence of nonspecific deposits and reaction product diffusion.

In previous cytochemical studies of the nervous system, Na,K-ATPase activity was reported to be localized on the synaptic plasma membrane (Vorbrodt et al. 1982 ; Inomata et al. 1983 ; Nasu 1983 ), the axolemma of axons and dendrites (Stahl and Broderson 1976 ; Broderson et al. 1978 ; Vorbrodt et al. 1982 ; Inomata et al. 1983 ; Nasu 1983 ), and the astrocytic foot processes (Mrsulja et al. 1985 ) in the brain, and on the nodal axolemmae, and the paranodal regions of Schwann cells in peripheral nerves, including the sciatic nerve (Vorbrodt et al. 1982 ; Mrsulja et al. 1985 ; Ariyasu and Ellisman 1989 ). Therefore, there appears to be no significant difference in the localization of Na,K-ATPase activity between the facial nerve (Kanoh et al. 1993b , Kanoh et al. 1994 ; Kanoh and Kumoi 1994 ) and other peripheral nerves, such as the sciatic nerve.

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 (Shull et al. 1986 ; Herrera et al. 1987 ; Orlowski and Lingrel 1988 ), and three -polypeptides have also been detected immunohistochemically in the brain (Arystarkhova et al. 1989 ; Shyjan and Levenson 1989 ; Urayama et al. 1989 ). It is now known that 1 is the original -isoform, and that 2 and 3 are contained in the + isoform (Schneider et al. 1988 ).

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 (Wakada 1980 ). This dose is 500-1000 times higher than that in clinical use. The biological half-lives of reserpine in serum and blood were also reported to be 271 hr and 386 hr, respectively (Maass et al. 1969 ). Therefore, for evaluating the effects of reserpine on K-NPPase activity, Day 3 and Day 7 after reserpine administration represent the reasonable checkpoints.

In the cochlea, K-NPPase activity in the stria vascularis was shown to be almost completely decreased after reserpine administration (Kanoh et al. 1993a , Kanoh et al. 1994 ). In a paper to be published in the near future, K-NPPase activity in the kidney was reported to be similarly suppressed after reserpine administration. In the present study, reaction product was detectable on the nodal axolemma but was almost undetectable on other parts of the axolemma and Schmidt-Lanterman incisures after reserpine treatment, i.e., transport Na,K-ATPase localized to the Ranvier nodes was not affected by reserpine administration, whereas transport Na,K-ATPase detected on other parts of the axolemma was decreased by reserpine treatment. Therefore, two different ouabain-sensitive K-NPPase reactivities, "reserpine-sensitive" and "reserpine-resistant," might be present in the facial nerve. Even when transport Na,K-ATPase activity on the internodal axolemma was almost completely eliminated, reserpinized animals displayed no facial palsy (Kanoh and Sakagami 1996 ), and could survive. In 1991, Mata et al. reported two different immunoreactivities in the nervous system. They evaluated immunocytochemical localization in the sciatic and optic nerves of the rat using a polyclonal antiserum raised against the denatured catalytic subunit of brain Na⁺,K⁺-ATPase. Immunoreactivity was detected along the internodal axolemma of myelinated fibers in both nerves and was undetectable on the nodal axolemma. Although the mechanism of suppression of K-NPPase activity in the facial nerves requires further consideration, the current results suggest that the nodal and internodal axolemma may contain different enzymes and that, because reserpine induces the depletion of sympathetic substances, transport Na,K-ATPase may be partially maintained by catecholamines. Other explanations at the molecular level will be forthcoming in the near future.

	Acknowledgments

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.

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

Albers RW, Siegel GJ, Stahl WC (1989) Membrane transport. In Siegel GJ, Agranoff BW, Albers RW, Molinoff PB, eds. Basic Neurochemistry. 4th Ed. New York, Raven Press, 49-70

Ariyasu RG, Ellisman MH (1989) The distribution of (Na⁺-Ka⁺) ATPase is continuous along the axolemma of unensheathed axons from spinal roots of 'dystrophic' mice. J Neurocytol 16:239-248

Arystarkhova EA, Lakhtina OE, Modyanov NN (1989) Immunodetection of Na,K-ATPase alpha 3-isoform in renal and nerve tissues. FEBS Lett 250:545-548 [Medline]

Broderson SH, Patton DL, Stahl WL (1978) Fine structural localization of potassium-stimulated p-nitrophenylphosphatase activity in dendrites of the cerebral cortex. J Cell Biol 77:13-17

Gatherer WT, Fedan JS, Westfall DP, Goto K, Fleming WW (1979) Involvement of the sodium-potassium pump in the mechanism of postjunctional supersensitivity of the vas deferens of the guinea pig. J Pharmacol Exp Ther 210:27-36 [Free Full Text]

Herrera VL, Emanuel JR, Ruiz Opazo N, Levenson R, Nadal Ginard B (1987) Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications. J Cell Biol 105:1855-1865 [Abstract/Free Full Text]

Hershman KM, Taylor DA, Fleming WW (1993) Adaptive supersensitivity in the guinea pig vas deferens is associated with a reduction in the abundance of the alpha 2 subunit isoform of Na⁺/K⁺-ATPase. Mol Pharmacol 43:833-837 [Abstract]

Inomata K, Mayahara H, Fujimoto K, Ogawa K (1983) Ultrastructural localization of ouabain-sensitive potassium-dependent p-nitrophenylphosphatase (Na⁺-K⁺-ATPase) activity in the central nervous system of the rat. Acta Histochem Cytochem 16:277-285

Judah JD, Ahmed K, McLean AEM (1962) Ion transport and phosphoproteins of human red cells. Biochim Biophys Acta 65:472-484

Kanoh N (1994) Reserpine inhibits the Na,K-ATPase activity of the stria vascularis in the cochlea. Laryngoscope 104:197-200 [Medline]

Kanoh N, Kobayashi T, Okada T, Seguchi H (1994) Cytochemical study of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (Na-K ATPase) activity in the cat facial nerve. Eur Arch Otorhinolaryngol 251:238-240 [Medline]

Kanoh N, Kumoi T (1994) Cytochemical study of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase activity in guinea pig facial nerve. ORL J Otorhinolaryngol Relat Spec 56:143-145 [Medline]

Kanoh N, Kumoi T, Kobayashi T, Okada T, Seguchi H (1993a) Ultracytochemical study of ouabain-sensitive K⁺-dependent p-nitrophenylphosphatase activity in the stria vascularis of reserpinized guinea pigs. Acta Otolaryngol (Stockh) 113:142-145 [Medline]

Kanoh N, Kumoi T, Okada T, Seguchi H (1993b) Ultracytochemical localization of the K⁺-dependent p-nitrophenylphosphatase activity in the normal cat facial nerve and after experimentally induced paralysis. Acta Histochem Cytochem 26:435-439

Kanoh N, Makimoto K (1984) Effects of reserpinization on the electrolytes distribution in inner ear fluids of guinea pig. Acta Otolaryngol (Stockh) 98:98-104 [Medline]

Kanoh N, Sakagami M (1996) Impaired Ranvier node sodium-potassium adenosine triphosphatase may induce facial palsy. Laryngoscope 106:1180-1183 [Medline]

Kobayashi T, Okada T, Seguchi H (1987) Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent, p-nitrophenylphosphatase activity at physiological pH. J Histochem Cytochem 35:601-611 [Abstract]

Maass AR, Jenkins B, Shen Y, Tannenbaum P (1969) Studies on absorption, excretion, and metabolism of ³H-reserpine in man. Clin Pharmacol Ther 10:366-371 [Medline]

Mata M, Fink DJ, Ernst SA, Siegel GJ (1991) Immunocytochemical demonstration of Na⁺-,K⁺-ATPase in internodal axolemma of myelinated fibers of rat sciatic and optic nerves. J Neurochem 57:184-192 [Medline]

Mrsulja BJ, Zalewski AA, Coping G (1985) Ultracytochemical localization of ouabain-sensitive K⁺-dependent, p-nitrophenyl-phosphatase in myelin. Brain Res 343:154-158 [Medline]

Nasu F (1983) Ultracytochemical demonstration of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (Na⁺-K⁺-ATPase) activity in the rat hippocampal formation. Acta Histochem Cytochem 16:368-373

Orlowski J, Lingrel JB (1988) Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs. J Biol Chem 263:10436-10442 [Abstract/Free Full Text]

Robinson JM (1985) Improved localization of intracellular sites of phosphatase using cerium and cell permeabilization. J Histochem Cytochem 33:749-754 [Abstract]

Schneider JW, Mercer RW, Gilmore-Hebert M, Utset MF, Lai C, Greene A, Benz EJ (1988) Tissue specificity, localization in brain, and cell-free translation of mRNA encoding the A3 isoform of Na⁺, K⁺-ATPase. Proc Natl Acad Sci USA 85:284-288 [Abstract/Free Full Text]

Shull GE, Greeb J, Lingrel JB (1986) Molecular cloning of three distinct forms of Na⁺, K⁺-ATPase alpha-subunit from rat brain. Biochemistry 25:8125-8132 [Medline]

Shyjan AW, Levenson R (1989) Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits in rat tissue membranes. Biochemistry 28:4531-4535 [Medline]

Skou JC (1965) Enzymatic basis for active transport of Na⁺ and K⁺ across cell membrane. Physiol Rev 45:596-617 [Free Full Text]

Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31-43 [Medline]

Stahl WL, Broderson SH (1976) Histochemical localization of potassium-stimulated p-nitrophenylphosphatase activity in the somatosensory cortex of the rat. J Histochem Cytochem 24:731-739 [Abstract]

Sweadner KJ (1979) Two molecular forms of (Na⁺+K⁺)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin. J Biol Chem 254:6060-6067 [Abstract/Free Full Text]

Urayama O, Shutt H, Sweadner KJ (1989) Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. J Biol Chem 264:8271-8280 [Abstract/Free Full Text]

Vorbrodt AW, Lossinsky AS, Wisniewski HM (1982) Cytochemical localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (transport ATPase) in the mouse central and peripheral nervous system. Brain Res 243:225-234 [Medline]

Wakada AR (1980) A comparison of rates of depletion and recovery of noradrenaline stores of peripheral and central noradrenergic neurons after reserpine administration. Importance of neuronal activity. Br J Pharmacol 68:93-98 [Medline]

Whittam R, Wheeler KP (1970) Transport across cell membranes. Annu Rev Physiol 32:21-60 [Medline]

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