Evidence for water-permeable channels in auditory hair cells in the leopard frog.

Auditory hair cells in the amphibian papilla (APHCs) of the leopard frog, Rana pipiens pipiens, have a significantly higher permeability to water than that observed in mammalian hair cells. The insensitivity of water permeability in frog hair cells to extracellular mercury suggests that an amphibian homologue of the water channel aquaporin-4 (AQP4) may mediate water transport in these cells. Using immunocytochemistry, we show that an AQP4-like protein is found in APHCs. Rabbit anti-AQP4 antibody was used in multiple-immunohistochemical staining experiments along with AP hair cell and hair bundle markers in leopard frog and mouse tissue. AQP4 immunoreactivity was found in the basal and apical poles of the APHCs and shows uniform immunoreactivity. This study provides the first identification and localization of an AQP4-like protein in the amphibian inner ear. We also report a more direct measure of hyperosmotically-induced volume changes in APHCs that confirms previous findings. The presence of water channels in anuran APHCs constitutes a novel physiological difference between amphibian and mammalian hair cell structure and function.

Osmotic properties of auditory hair cells in the leopard frog: evidence for water-permeable channels.

When amphibian papillar hair cells (APHCs) of the leopard frog, Rana pipiens pipiens, are osmotically challenged, they exhibit a characteristically asymmetric (rectifying) response: small decreases (5%, or less) in the extracellular solution's osmolarity do not significantly affect the cells' volume; larger decreases produce a relatively slow volume increase in APHCs, while exposure to a hyperosmotic medium leads to rapid shrinking of these cells. Furthermore, the rate of volume change appears to be a function of the rate of extracellular osmotic change. These characteristics make the application of methods devised for the estimation of the osmotic permeability coefficient (P(f)) for semipermeable membranes - i.e., those with significant permeability only to water - to APHC membrane rather futile. We have, therefore, devised a method that takes both the permeability to solutes as well as the kinetics of the osmolarity change into consideration, in order to obtain estimates of P(f) that are to a large degree independent of these factors. We have compared the new and earlier methods. Using the new method, we have estimated the P(f) of APHCs' plasma membrane to be in the 10(-2)-cm/s range, and thus significantly larger than those reported for lipid bilayers. APHC's membrane P(f) appears to be cell-size independent and insensitive to extracellular mercury. These results suggest that APHCs express water-permeable channels in their plasma membrane. Furthermore, we suggest that asymmetric and rate dependent shape changes produced by osmolarity changes in APHCs imply the presence of significant permeability to solutes. The significance of transmembrane solute transport and water channel expression in amphibian auditory hair cells is discussed.

Slow motility in hair cells of the frog amphibian papilla: myosin light chain-mediated shape change.

Using video, fluorescence and confocal microscopy, quantitative analysis and modeling, we investigated intracellular processes mediating the calcium/calmodulin (Ca(2+)/CaM)-dependent slow motility in hair cells dissociated from the rostral region of amphibian papilla, one of the two auditory organs in frogs. The time course of shape changes in these hair cells during the period of pretreatment with several specific inhibitors, as well as their response to the calcium ionophore, ionomycin, were recorded and compared. These cells respond to ionomycin with a tri-phasic shape change: an initial phase of iso-volumetric length decrease; a period of concurrent shortening and swelling; and the final phase of increase in both length and volume. We found that both the myosin light chain kinase inhibitor, ML-7, and antagonists of the multifunctional Ca(2+)/CaM-dependent kinases, KN-62 and KN-93, inhibit the iso-volumetric shortening phase of the response to ionomycin. The type 1 protein phosphatase inhibitors, calyculin A and okadaic acid induce minor shortening on their own, but do not significantly alter phase 1 response. However, they appear to counter effects of the inhibitors of Ca(2+)/CaM-dependent kinases. We hypothesize that an active actomyosin-based process mediates the iso-volumetric shortening in the frog rostral amphibian papillar hair cells.

Slow motility in hair cells of the frog amphibian papilla: Ca2+-dependent shape changes.

We investigated the process of slow motility in non-mammalian auditory hair cells by recording the time course of shape change in hair cells of the frog amphibian papilla. The tall hair cells in the rostral segment of this organ, reported to be the sole recipients of efferent innervation, were found to shorten in response to an increase in the concentration of the intracellular free calcium. These shortenings are composed of two partially-overlapping phases: an initial rapid iso-volumetric contraction, followed by a slower length decrease accompanied with swelling. It is possible to unmask the iso-volumetric contraction by delaying the cell swelling with the help of K+ or Cl- channel inhibitors, quinine or furosemide. Furthermore, it appears that the longitudinal contraction in these cells is Ca2+-calmodulin-dependent: in the presence of W-7, a calmodulin inhibitor, only a slow, swelling phase could be observed. These findings suggest that amphibian rostral AP hair cells resemble their mammalian counterparts in expressing both a Ca2+-calmodulin-dependent contractile structure and an "osmotic" mechanism capable of mediating length change in response to extracellular stimuli. Such a mechanism might be utilized by the efferent neurotransmitters for adaptive modulation of mechano-electrical transduction, sensitivity enhancement, frequency selectivity, and protection against over-stimulation.

Purinergic signaling in the rabbit ciliary body epithelium.

In the past forty years, a wealth of information has accumulated that points to the presence of adenosine and adenine nucleotides in the anterior segment of the eye and a number of hypotheses have been introduced to describe the possible role of these agents in the regulation of aqueous humor flow. However, in the absence of a generally accepted model for the cellular and molecular mechanisms of aqueous humor formation by the ciliary body epithelium, efforts to identify the signal transduction pathway(s) responsible for regulation of the ion and water transport have not been successful. This article briefly reviews the evidence for (i). the presence in aqueous humor of adenine nucleotides, cyclic adenosine monophosphate and adenosine, their metabolic product, (ii). the possible role of these agents in the regulation of aqueous humor dynamics, and (iii). the expression of ecto-nucleotidases, receptors, and second messengers that may mediate such regulation. Finally, a model for the regulation of aqueous humor formation by adenosine and ATP is proposed.

Ectonucleotidases of the rabbit ciliary body nonpigmented epithelium.

PURPOSE: To investigate the expression of both the message and function of ENPP1 (a member of the ectonucleotide pyrophosphatase/phosphodiesterase family, also known as PC-1), NTPD1 (a member of the ectonucleoside 5'-triphosphate diphosphohydrolase family, CD39), and ecto-5'-nucleotidase (CD73) in rabbit ciliary body nonpigmented epithelial (NPE) cells. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to reveal the presence of mRNAs of ectonucleotidases in NPE cells. Real-time fluorescence ratio imaging of the intact fura-2-loaded NPE cells was used to record changes in the intracellular calcium concentration. RESULTS: RT-PCR analysis revealed the expression of mRNAs for ENPP1, NTPD1, and ecto-5'-nucleotidase, but not NTPD2 (ecto-ATPase, or CD39L1), in the rabbit NPE cells. The ENPP1 inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), and to a lesser degree the nonspecific ectonucleotidase antagonist 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine 5-triphosphate (ARL 67156), reduced the [Ca(2+)](i) increase elicited by the combination of acetylcholine (ACh) and cAMP. However, both inhibitors significantly enhanced the [Ca(2+)](i) increase generated by uridine triphosphate (UTP). The ecto-5'-nucleotidase inhibitor alphabeta-meADP significantly diminished the [Ca(2+)](i) increase evoked by ACh+cAMP, but not that generated by UTP. The A(1)-specific adenosinergic receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) significantly blocked the response to ACh+cAMP. CONCLUSIONS: These observations suggest that rabbit NPE cells possess at least three distinct ectonucleotidases capable of catalyzing the stepwise hydrolysis of adenine and pyrimidine nucleotides, as well as cAMP, thus shaping the purinergic-receptor-coupled signaling in these cells.

P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium.

PURPOSE: To identify and characterize P2 purinergic receptors and their signaling pathways in the epithelial cells of the rabbit ciliary body. METHODS: Real-time fluorescence ratio imaging of the intact fura-2-loaded nonpigmented ciliary body epithelial (NPE) cells of rabbit were used to record changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to a number of purinergic agonists and antagonists. The effects of some of these drugs on the inositol phosphate (IP) levels in ciliary processes were also examined. RESULTS: Adenosine diphosphate (ADP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) dose dependently increased the [Ca(2+)](i) and IP levels. The [Ca(2+)](i) increases induced by ADP and UTP were distinguishable, both kinetically and pharmacologically. The effect of ADP on [Ca(2+)](i) was mimicked by a number of P2Y(1)-selective agonists, and was blocked by three P2Y(1)-receptor-specific antagonists. The [Ca(2+)](i) increases elicited by ADP (or its analogs) and UTP were additive. CONCLUSIONS: Rabbit ciliary body epithelium possesses both P2Y(1) and P2Y(2) metabotropic purinergic receptor subtypes, which differentially use the IP(3)/Ca(2+) second-messenger pathway.