Endolymphatic Perfusion Research Articles

Endocytosis of MPO in Marginal Cells Is Regulated by PKC, Protein Phosphatase, ERK and PI3-K Signaling Cascades, but Not by PKA and MEK Signaling Cascades

Endocytosis of marginal cells plays a key role in maintaining the homeostasis of endocytosis and function of the organ of Corti. How the signaling cascade is involved in the regulation of endocytosis is an important issue at present. To investigate the regulation of endocytosis in marginal cells of the stria vascularis by the signaling network, we perfused MPO, an endocytosis tracer, with PMA, OA, staurosporin, PAO, PD98059, SB20580 or wortmannin into the cochlear duct. After 30 min endolymphatic perfusion, the tissues were fixed and the distribution of MPO was examined by electron microscopy. We explored the functions of PKC, RTK, PI3-K, PTP, and PP1/2A in MPO endocytosis and defined the MPO endocytic route. MPO endocytosis was strongly dependent on PKC, ERK, PTP, PP1/2A and PI3-K signaling networks, but not on PKA and MEK signaling networks. The MPO endocytic pathways are clathrin-, GPI-AP-, and caveolae-independent.

Ca2+ regulation of endocochlear potential in marginal cells

We examined the effect of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca(2+) chelator or inhibitors of Ca(2+)-permeable channels or Ca(2+)-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca(2+) channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca(2+)-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the gamma subunit of epithelial Na(+) channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 muM amiloride or 10 muM phenamil. Taken together, these findings suggest that [Ca(2+)](c) regulated by endoplasmic Ca(2+)-pump and Ca(2+)-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na(+) across the basolateral membrane in marginal cells.

Presence and regulation of epithelial sodium channels in the marginal cells of stria vascularis

Conclusion. This study indicates that epithelial Na+-selective channels (ENaC) recycle Na+ via clathrin-mediated endocytosis in the marginal cells of the stria vascularis and that clathrin-independent endocytosis appeared to be modulated by the amount of Na+ transported. These results suggest the presence of ENaC in the luminal membrane of marginal cells and that ENaC are an efficient pathway for the uptake of Na+ from the endolymph. Objective. The ENaC found in many transporting epithelia play a key role in the regulation of salts and water homeostasis, cellular pH, cell volume, and cell function. Both biochemical and physiological approaches have been used to identify, characterize, and quantify this important channel, but its location in the marginal cells of the stria vascularis has not been fully clarified. The aim of this study was to determine the localization and regulation of ENaC. Materials and methods. Forty healthy female guinea pigs were used: 20 for the control experiment, 10 for the amiloride experiment, and 10 for the aldosterone experiment. We perfused cationized ferritin (CF) and microperoxidase (MPO) as tracers for clathrin-mediated and clathrin-independent endocytosis, respectively, into the cochlear duct. After 30 min of endolymphatic perfusion, the tissues were fixed and CF- and MPO-loaded endosomes within the marginal cell were observed by transmission electron microscopy. The numbers of CF- and MPO-loaded endosomes were compared between the three groups. Results. In the amiloride group, the numbers of CF- and MPO-loaded endosomes decreased in comparison with the control. In the aldosterone group, the numbers of CF- and MPO-loaded endosomes decreased and increased, respectively. Recently, it has been reported that ENaC are endocytosed via clathrin-mediated endosomes and aldosterone decreases the rate of endocytosis of ENaC. In this study, the results of the aldosterone experiment were consistent with those of recent studies.

A method for introducing non-silencing siRNA into the guinea pig cochlea in vivo

The aim of this preliminary study was to establish the methodology by which siRNA can be introduced into the adult guinea pig cochlea in vivo whilst preserving auditory function with a view to using targeted siRNAs to knockdown genes essential for auditory transduction. Initially a fluorescently tagged non-silencing siRNA complexed with a lipid-based transfection reagent was introduced into the perilymphatic compartment of the cochlea. Although auditory function was fully preserved, siRNA uptake was only observed in cells lining the perilymphatic space that are not critically involved in auditory transduction and therefore of little interest. Another approach was therefore adopted, in which siRNA was introduced directly into the scala media (endolymphatic compartment) of the apical (fourth) cochlear turn by slow pressure injection. During endolymphatic perfusion, the endocochlear potential (EP) and compound action potential (CAP) thresholds for basal turn frequencies from 6 to 20 kHz could be preserved, while CAP thresholds for 1–4 kHz were often elevated by 10–20 dB. CAP thresholds and EP were preserved 24 and 48 h after perfusion in some animals but reduced in others. siRNA uptake was observed predominantly in marginal and intermediate cells of the stria vascularis in all cochlear turns but not in cells of the organ of Corti.

Endocytosis of microperoxidase in the marginal cells of stria vascularis

Objective Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. Methods We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5–60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. Results Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. Conclusion The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

Coxsackie adenovirus receptor and ανβ3/ανβ5 integrins in adenovirus gene transfer of rat cochlea

This study was designed to determine whether Coxsackie adenovirus receptor (CAR) and alpha nu beta3/alpha nu beta5 integrin co-receptors are involved in adenovirus gene transfer in the rat cochlea. We find that CAR and integrin co-receptors are expressed in every cell subtype transduced by the adenoviral vector Ad5 DeltaE1-E3/cytomegalovirus/green fluorescent protein (GFP) on cochlear slices in vitro. The spiral ganglion neurons, which do not express CAR, were not transduced by the virus. Blocking these receptors by monoclonal antibodies decreased transgene expression, whereas disrupting tight junctions with ethylenediaminetetraacetic acid led to an increased transgene expression. However, sensory hair cells and strial cells also expressing CAR and alpha nu integrins were not transduced by the vector. GFP expression was also studied in vivo. Perilymphatic perfusion of adenovirus in vivo did not affect hearing and only cells lining the perilymphatic spaces were transduced. Endolymphatic perfusion resulted in low-frequency hearing loss and although some cells of the organ of Corti were efficiently transduced, the sensory and the strial cells were not. Transduced sensory and strial cells were occasionally observed in cochleas after single shot of adenovirus. Pretreatment with anti-CAR and anti-alpha nu antibodies decreases GFP expression in vivo, suggesting that the CAR/alpha nu integrin pathway is involved in adenovirus transduction in the cochlea.

Endolymphatic Perfusion with EGTA-Acetoxymethyl Ester Inhibits Asphyxia- and Furosemide-Induced Decrease in Endocochlear Potential in Guinea Pigs

We examined the effect of the Ca(2+) concentration in the endolymph ([Ca](e)) or in the endolymphatic surface cells ([Ca](i)) on the endocochlear potential (EP) by using an endolymphatic or perilymphatic perfusion technique, respectively. (i) A large increase in [Ca](e) up to approximately 10(-3) M with a fall in the EP was induced by transient asphyxia ( approximately 2 min) or by the intravenous administration of furosemide (60 mg/kg), and a significant correlation was obtained between the EP and p[Ca](e) (= -log [Ca](e), r = 0.998). (ii) Perfusion of the endolymph with 10 mM EGTA for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP (DeltaEP(asp)), suggesting that neither [Ca](e) nor the Ca(2+) concentration gradient across the stria vascularis contributed directly to the generation of the EP in the condition of low [Ca](e). In contrast, endolymphatic perfusion with high Ca(2+) (more than 10 mM) produced a decrease in EP and a significant correlation was obtained between the EP and the Ca(2+) concentration of perfusion solution (r = 0.982), suggesting that Ca(2+) permeability may exist across the stria vascularis. (iii) The administration of a Ca(2+) chelator, EGTA-acetoxymethyl ester (AM, 0.3 mM), to the endolymph, which produced a gradual increase in EP, suppressed significantly, by 60-80%, DeltaEP(asp) or furosemide-induced changes in EP. In contrast, perilymphatic administration of 0.5 mM EGTA-AM caused no significant suppression of the DeltaEP(asp). These findings suggest that [Ca](i) plays an important role in generating/maintaining a large positive EP.

Endocytosis of Cationized Ferritin in the Marginal Cells of Stria Vascularis

To investigate the properties of endocytosis in the marginal cells of stria vascularis (SV), we perfused cationized ferritin (CF), an endocytosis tracer, into the cochlear duct. After five minutes to two hours of endolymphatic perfusion, the tissues were fixed and the distribution of CF was observed by transmission electron microscopy. CF was endocytosed by the marginal cells. CF-loaded vesicles were represented mainly by coated vesicles. The time-course of endocytosis was as follows. After five minutes perfusion, CF was distributed over the surface of the apical membrane and CF-loaded vesicles were seen very close to the apical membrane. After 30 minutes perfusion, a number of CF-loaded small endosomes and CF-loaded large vacuolar endosomes were observed at the supranuclear region. After one hour and two hours perfusion, CF was transferred to the multivesicular body. After two hours perfusion, acid phosphatase (ACPase) activity, a marker of lysosomes, was investigated. CF was not transferred to lysosomes until after two hours, because ACPase activity was not seen in CF-loaded vesicles. In this paper, we discuss the basic endocytosis properties of marginal cells.

Location and function of the epithelial Na channel in the cochlea.

In the cochlea, endolymph is a K-rich and Na-poor fluid. The purpose of the present study was to check the presence and to assess the role of epithelial Na channel (ENaC) in this organ. alpha-, beta-, and gamma-ENaC subunit mRNA, and proteins were detected in rat cochlea by RT-PCR and Western blot. alpha-ENaC subunit mRNA was localized by in situ hybridization in both epithelial (stria vascularis, spiral prominence, spiral limbus) and nonepithelial structures (spiral ligament, spiral ganglion). The alpha-ENaC-positive tissues were also positive for beta-subunit mRNA (except spiral ganglion) or for gamma-subunit mRNA (spiral limbus, spiral ligament, and spiral ganglion), but the signals of beta- and gamma-subunits were weaker than those observed for alpha-subunit. In vivo, the endocochlear potential was recorded in guinea pigs under normoxic and hypoxic conditions after endolymphatic perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear potential when Na-rich but not when K-rich solutions were perfused. In conclusion, ENaC subunits are expressed in epithelial and nonepithelial cochlear structures. One of its functions is probably to maintain the low concentration of Na in endolymph.

Response of cochlear potentials to presumed alterations of ionic conductance: endolymphatic perfusion of barium, valinomycin and nystatin.

Two models ('single-pump' and 'two-pump') of transepithelial potassium movement by the marginal cells of the stria vascularis have been proposed in the literature. Their validity was considered by exposing the endolymphatic (luminal) surface to agents (barium, valinomycin and nystatin) which are known to alter specific cellular membrane conductances in other tissues. This was accomplished by the use either of injections or of a relatively satisfactory technique for perfusion of scala media, which is described. Injection of barium caused the endocochlear potential (EP) to increase in normal animals and had no effect on the EP of deaf, Waltzing guinea pigs. Perfusion of the ionophores caused a decline in the EP in both normal and Waltzing guinea pigs. Only the 'two-pump' model (Na/K-ATPase-mediated cation pump on the basolateral membrane and rheogenic K transporter at the luminal membrane) is consistent with the results. The cellular heterogeneity of the cochlear duct, however, introduces a measure of uncertainty into this interpretation.

Effect of cholera toxin upon cyclic AMP (cAMP) levels in tissues lining cochlear duct

Feldman and Brusilow [Proc. Natl. Acad. Sci. USA 73, 1761 (1976)] reported that endolymphatic injection of cholera toxin (adenylate cyclase stimulator) results in endolymphatic hydrops, the pathological substrate of Meniere's disease. The authors postulated that the situation is similar to the intestine, where the cholera toxin mediated elevation of cAMP levels causes profuse fluid secretion. We found that perilymphatic application of cholera toxin caused an approximately 2× increase of cAMP in Reissner's membrane (RM), but no significant changes in stria vascularis (SV) and organ of Corti (OC). Endolymphatic perfusion produced no changes. However, when cholera toxin, in combination with theophylline (phosphodiesterase inhibitor) was perfused perilymphatically, a dramatic increase in cAMP levels occurred in RM (80×), a substantial increase in OC (8×), but only a small increase in SV (2×). Endolymphatic perfusion caused a 20×, 4×, and 3.5× increase, respectively. These results indicate that RM may be more important in cochlear fluid homeostasis than generally assumed. Note, however, that the relatively low level of strial cAMP may be due to clearance via the vasculature. [Supported by NIH.]

Ototoxicity of kanamycin sulfate and the barriers in the inner ear.

The effect of kanamycin sulfate administered by three routes on the function of the stria vascularis was monitored electrophysiologically in guinea pigs. The three routes were intramuscular injection, perilymphatic perfusion, or endolymphatic perfusion. Neither systemic administration of 500 mg/kg of body weight per day for 7 to 12 days nor perilymphatic perfusion of 10(-3) M kanamycin affected the endocochlear dc potential (EP). However, with perfusion of kanamycin 10(-3) M in the endolymphatic space, the EP declined severely. Moreover, the decline in the EP was greater with higher concentrations of kanamycin in the endolymphatic perfusate. Furosemide given by each of the three routes produced an approximately equal decrease in the EP. The effects of kanamycin on the cells of the stria vascularis and the evidence for the perilymphatic-endolymphatic and blood-cochlear barriers are discussed.