ABSTRACT
Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.
ABSTRACT
Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.
ABSTRACT
In the inner ear, there is considerable evidence that extracellular adenosine 5'-triphosphate (ATP) plays an important role in auditory neurotransmission as a neurotransmitter or a neuromodulator, although the potential role of adenosine signalling in the modulation of auditory neurotransmission has also been reported. The activation of ligand-gated ionotropic P2X receptors and G protein-coupled metabotropic P2Y receptors has been reported to induce an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) in inner hair cells (IHCs), outer hair cells (OHCs), spiral ganglion neurons (SGNs), and supporting cells in the cochlea. ATP may participate in auditory neurotransmission by modulating [Ca(2+)](i) in the cochlear cells. Recent studies showed that extracellular ATP induced nitric oxide (NO) production in IHCs, OHCs, and SGNs, which affects the ATP-induced Ca(2+) response via the NO-cGMP-PKG pathway in those cells by a feedback mechanism. A cross-talk between NO and ATP may therefore exist in the auditory signal transduction. In the present article, I review the role of NO on the ATP-induced Ca(2+) signalling in IHCs and OHCs. I also consider the possible role of NO in the ATP-induced Ca(2+) signalling in SGNs and supporting cells.
ABSTRACT
CONCLUSION: This study demonstrates that the co-administration of lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) (LPS/TNF-alpha) can induce the expression of inducible nitric oxide synthase (iNOS), which results in the generation of apoptosis in cultured human nasal microvascular endothelial cells (HNMECs). Since LPS and TNF-alpha have been suggested to play an important role in the pathophysiology of nasal disease, we conclude that microvascular leakage may therefore contribute to the inflammatory process in nasal disease, such as allergic rhinitis and asthma. MATERIALS AND METHODS: The HNMECs were obtained from the inferior turbinate and subsequently cultured. The expression of iNOS induced by both the LPS and TNF-alpha was investigated by fluorescent immunohistochemistry, using confocal laser microscopy. The DNA-binding dye, Hoechist 33342, was also used to analyze the apoptosis in the HNMECs. RESULTS: The fluorescent immunohistochemistory study demonstrated that LPS and TNF-alpha induced the expression of iNOS in HNMECs. LPS/TNF-alpha remarkably augmented the expression of iNOS in HNMECs in comparison to stimulation by either LPS or TNF-alpha alone. LPS/TNF-alpha also induced apoptosis in HNMECs. 1400W, a highly selective inhibitor of iNOS, inhibited both the expression of iNOS and the apoptosis induced by LPS/TNF-alpha.
Subject(s)
Apoptosis/drug effects , Endothelium, Vascular/drug effects , Escherichia coli , Lipopolysaccharides/pharmacology , Nasal Mucosa/blood supply , Nitric Oxide Synthase Type II/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Capillary Permeability/drug effects , Cells, Cultured , Enzyme Induction/drug effects , Humans , In Vitro Techniques , Microcirculation/drug effects , Microscopy, Confocal , Microscopy, Fluorescence , Platelet Endothelial Cell Adhesion Molecule-1/metabolismABSTRACT
Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca2+-free solution. L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca2+ concentrations ([Ca2+]i) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca2+ response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca2+]i increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca2+ response via the NO-cGMP-PKG pathway by a feedback mechanism.
Subject(s)
Cell Size , Hair Cells, Auditory, Outer/metabolism , Nitric Oxide/metabolism , Organ of Corti/metabolism , Signal Transduction , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Calcium/metabolism , Carbazoles/pharmacology , Cell Size/drug effects , Cells, Cultured , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Gadolinium/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , Guinea Pigs , Hair Cells, Auditory, Outer/drug effects , Hypotonic Solutions , Indoles/pharmacology , Kinetics , Membrane Potentials , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Organ of Corti/cytology , Organ of Corti/drug effects , Osmotic Pressure , Phorbol Esters/pharmacology , Potassium/metabolism , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2/metabolism , S-Nitroso-N-Acetylpenicillamine/pharmacology , Signal Transduction/drug effects , Sodium Chloride/metabolism , Suramin/pharmacology , TRPV Cation Channels/agonists , TRPV Cation Channels/metabolismABSTRACT
Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca(2+)-free solution. L-N(G)-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca(2+) response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca(2+)](i) increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca(2+) response via the NO-cGMP-PKG pathway by a feedback mechanism.
Subject(s)
Cochlea/metabolism , Hair Cells, Auditory, Outer/metabolism , Nitric Oxide/metabolism , Signal Transduction , Water-Electrolyte Balance , Animals , Calcium/metabolism , Carbazoles/pharmacology , Cell Size , Cells, Cultured , Cochlea/cytology , Cochlea/drug effects , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Gadolinium/pharmacology , Guanylate Cyclase/metabolism , Guinea Pigs , Hair Cells, Auditory, Outer/drug effects , Hypotonic Solutions/metabolism , Indoles/pharmacology , Mechanoreceptors/drug effects , Mechanoreceptors/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Phorbol Esters/pharmacology , Potassium/metabolism , Protein Kinase Inhibitors/pharmacology , S-Nitroso-N-Acetylpenicillamine/pharmacology , Signal Transduction/drug effects , TRPV Cation Channels/agonists , TRPV Cation Channels/metabolism , Up-Regulation , Water-Electrolyte Balance/drug effectsABSTRACT
Recently, a negative feedback effect of nitric oxide (NO) on the adenosine 5'-triphosphate (ATP)-induced Ca2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca2+ -sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor, and suramin, a P2 receptor antagonist. ATP failed to induce NO production in the Ca2+ -free solution. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca2+ concentrations ([Ca2+]i), while L-NAME inhibited it. SNAP accelerated ATP-induced Mn2+ quenching in fura-2 fluorescence, while L-NAME suppressed it. 8-Bromoguanosine-cGMP, a membrane permeable analog of cGMP, mimicked the effects of SNAP. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase inhibited the ATP-induced [Ca2+]i increase. Selective neuronal NOS inhibitors, namely either 7-nitro-indazole or 1-(2-trifluoromethylphenyl) imidazole, mimicked the effects of L-NAME regarding both ATP-induced Ca2+ response and NO production. Immunofluorescent staining of neuronal nitric oxide synthase (nNOS) in isolated OHCs showed the localization of nNOS in the apical region of OHCs. These results suggest that the ATP-induced Ca2+ influx via a direct action of P2X receptors may be the principal source for nNOS activity in the apical region of OHCs. Thereafter, NO can be produced while conversely enhancing the Ca2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.
Subject(s)
Adenosine Triphosphate/pharmacology , Calcium Signaling/drug effects , Calcium/metabolism , Cochlea/cytology , Hair Cells, Auditory, Outer/metabolism , Nitric Oxide/physiology , Analysis of Variance , Animals , Blotting, Western/methods , Calcium Signaling/physiology , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Diagnostic Imaging/methods , Drug Interactions , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique/methods , Guinea Pigs , Nitric Oxide Donors/pharmacology , Penicillamine/analogs & derivatives , Penicillamine/pharmacology , Time FactorsABSTRACT
Transient receptor potential vanilloid 4, the Ca2+-permeable cation channel has been proposed as an osmosensitive and a mechanosensitive channel. We investigated functional expression of transient receptor potential vanilloid 4 in inner hair cells, outer hair cells, and spiral ganglion neurons of the mouse cochlea. Transient receptor potential vanilloid 4 mRNA and protein were expressed in inner hair cells, outer hair cells, and spiral ganglion neurons on the basis of the findings of reverse transcriptase-polymerase chain reaction, single-cell reverse transcriptase-polymerase chain reaction, and immunohistochemistry, whereas they were negative in transient receptor potential vanilloid 4-/- mice cochleae. Hypotonic stimulation and 4-alpha-phorbol 12,13-didecanoate, a transient receptor potential vanilloid 4 synthetic activator, increased the intracellular Ca2+ concentrations in wild-type outer hair cells, whereas in transient receptor potential vanilloid 4-/- mice, outer hair cells failed to exhibit a Ca2+ response to both stimulations. In conclusion, transient receptor potential vanilloid 4 may function as an osmosensory and a mechanosensory receptor in the cochlea.
Subject(s)
Cochlea/cytology , Cochlea/metabolism , Gene Expression Regulation/physiology , Neurons/metabolism , TRPV Cation Channels/physiology , Animals , Blotting, Northern/methods , Blotting, Western/methods , Calcium/metabolism , Chelating Agents/pharmacology , Drug Interactions , Egtazic Acid/pharmacology , Gene Expression Regulation/genetics , Hypotonic Solutions/pharmacology , Immunohistochemistry/methods , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/drug effects , Phorbols/pharmacology , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Ruthenium Red/metabolism , TRPV Cation Channels/deficiencyABSTRACT
We recently demonstrated that extracellular adenosine 5'-triphosphate (ATP) induced nitric oxide (NO) production in the inner hair cells (IHCs) of the guinea pig cochlea, which inhibited the ATP-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) by a feedback mechanism [Shen, J., Harada, N. & Yamashita, T. (2003) Neurosci. Lett., 337, 135-138]. We herein investigated the role of the NO-cGMP pathway and neuronal NO synthase (nNOS) in the ATP-induced Ca(2+) signalling in IHCs using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye DAF-2. Fura-2 fluorescence-quenching experiments with Mn(2+) showed that ATP triggered a Mn(2+) influx. L-N(G)-nitroarginine methyl ester (L-NAME), a nonspecific NOS inhibitor, accelerated the ATP-induced Mn(2+) influx while S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, suppressed it. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase, and KT5823, an inhibitor of cGMP-dependent protein kinase, enhanced the ATP-induced [Ca(2+)](i) increase. 8-Bromoguanosine-cGMP, a membrane-permeant analogue of cGMP mimicked the effects of SNAP. Moreover, the effects of 7-nitroindazole, a selective nNOS inhibitor, mimicked the effects of L-NAME regarding both the enhancement of the ATP-induced Ca(2+) response and the attenuation of NO production. Immunofluorescent staining of nNOS using a single IHC revealed that nNOS was distributed throughout the IHCs, but enriched in the apical region of the IHCs as shown by intense staining. In conclusion, the ATP-induced Ca(2+) influx may be the principal source for nNOS activity, which may interact with P2X receptors in the apical region of IHCs. Thereafter, NO can be produced and conversely inhibits the Ca(2+) influx via the NO-cGMP-PKG pathway by a feedback mechanism.
Subject(s)
Adenosine Triphosphate/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Cyclic GMP/metabolism , Hair Cells, Auditory, Inner/metabolism , Nerve Tissue Proteins/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , Adenosine Triphosphate/pharmacology , Animals , Calcium Signaling/drug effects , Cyclic GMP/analogs & derivatives , Cyclic GMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Feedback, Physiological/physiology , Fluoresceins , Fura-2 , Guinea Pigs , Hair Cells, Auditory, Inner/drug effects , Immunohistochemistry , Manganese/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase Type I , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
To investigate the interaction between Ca(2+) and nitric oxide (NO) in inner hair cells of the guinea pig cochlea (IHCs), the extracellular adenosine 5'-triphosphate (ATP)-induced NO production and the effects of NO on ATP-induced increase of intracellular Ca(2+) concentrations ([Ca(2+)](i)) were investigated in IHCs using the NO-sensitive dye DAF-2 and the Ca(2+)-sensitive dye Fura-2. Extracellular ATP induced an increase in DAF-2 fluorescence, which thus indicates NO production in IHCs. The ATP-induced NO production was mainly due to Ca(2+) influx through the activation of P2 receptor. L-N(G)-nitroarginine methyl ester, a NO synthesis inhibitor, enhanced the ATP-induced [Ca(2+)](i) increase in IHCs while S-nitroso-N-acetylpenicillamine, a NO donor, inhibited it. We conclude that NO inhibits the ATP-induced [Ca(2+)](i) increase in IHCs by a negative-feedback mechanism.
