Plasma membrane Ca2+-ATPase isoforms in frog crista ampullaris: identification of PMCA1 and PMCA2 specific splice variants.

Ca2+ ions play a pivotal role in inner ear hair cells as they are involved from the mechano-electrical transduction to the transmitter release. Most of the Ca2+ that enters into hair cells via mechano-transduction and voltage-gated channels is extruded by the plasma membrane Ca2+-ATPases (PMCAs) that operate in both apical and basal cellular compartments. Here, we determined the identity and distribution of PMCA isoforms in frog crista ampullaris: we showed that PMCA1, PMCA2 and PMCA3 are expressed, while PMCA4 appears to be negligible. We also identify PMCA1bx, PMCA2av and PMCA2bv as the major splice variants produced from PMCA1 and PMCA2 genes. PMCA2av appears to be the major Ca2+-pump operating at the apical pole of the cell, even if PMCA1b is also expressed in the stereocilia. PMCA1bx is, instead, the principal PMCA of hair cell basolateral compartment, where it is expressed together with PMCA2 (probably PMCA2bv) and PMCA3. Frog crista ampullaris hair cells lack a Na/Ca exchanger, therefore PMCAs are the only mechanism of Ca2+ extrusion. The coexpression of specific isozymes in the different cellular compartments responds to the need of a fine regulation of both basal and dynamic Ca2+ levels at the apical and basal pole of the cell.

Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish.

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.

Prostaglandin E synthases in zebrafish.

OBJECTIVE: Prostaglandin E synthases (PGESs) are being explored as antiinflammatory drug targets as alternatives to cyclooxygenase (COX)-2. Located downstream of the cyclooxygenases, PGESs catalyze PGE(2) formation, and deletion of microsomal (m)-PGES-1 abrogates inflammation. We sought to characterize the developmental expression of COX and PGES in zebrafish. METHODS AND RESULTS: We cloned zebrafish cytosolic (c) and m-PGES orthologs and mapped them to syntenic regions of chromosomes 23 and 5. cPGES was widely expressed during development and was coordinately regulated with zCOX-1 in the inner ear, the pronephros, and intestine. COX-2 and mPGES-1 exhibited restricted expression, dominantly in the vasculature of the aortic arch. However, the enzymes were anatomically segregated within the vessel wall. Experiments with antisense morpholinos and with nonsteroidal antiinflammatory drugs suggest that these genes may not be critical for development. CONCLUSIONS: mPGES-1 is developmentally coregulated with COX-2 in vasculature. Given the high fecundidity and translucency of the zebrafish, this model may afford a high throughput system for characterization of novel PGES inhibitors. Microsomal prostaglandin E synthase (mPGES)-1, located downstream of COX-2, may represent a novel antiinflammatory drug target. Zebrafish cytosolic (c) PGES-1 and COX-1 were coordinately expressed; mPGES-1 and COX-2 were expressed particularly in the vasculature. Zebrafish may afford a high throughput system for detection of novel PGES inhibitors.

Ecto-ATPase activity sites in vestibular tissues: an ultracytochemical study in frog semicircular canals.

The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction. Electron-dense precipitates, indicating ecto-adenosine-triphosphatase (ecto-ATPase) activity, were found at the outer surface of plasma membranes of crista hair cells and supporting cells of the sensory epithelium, transitional cells and undifferentiated cells of the ampullar wall and dark cells constituting the secretory epithelium. Non-sensory cells of the ampulla usually exhibited reaction deposits at the level of both apical and basolateral membranes coming into contact with the endolymph and the perilymph respectively, while cells constituting the sensory epithelium showed evident differences in relation to their position. Hair cells and supporting cells of the peripheral regions exhibited clear reaction products both at the level of apical and basolateral membranes, while those of the isthmus region showed abundant reactivity only at the level of their apical membranes. Of particular interest was the observation that hair cell stereocilia exhibited an abundant ecto-ATPase activity, thus suggesting a possible colocalization of enzymatic sites with purinergic receptors and mechanotransduction channels. This strategic expression of ecto-ATPase sites could provide a rapid mechanism of ATP removal able to rapidly restore the sensitivity of transduction channels. In conclusion, the widespread distribution of ecto-ATPase sites at the level of sensory and non-sensory cells of the frog semicircular canals suggests that ATP may have a key role in controlling vestibular function.

Purine and pyrimidine nucleotide-sensitive phospholipase A(2) in ampulla from frog semicircular canal.

This study was attempted to characterize pharmacologically the P2Y receptors triggering phospholipase A(2) (PLA(2)) activation in ampulla from frog semicircular canal. A microassay was developed to screen the abilities of UTP analogs to stimulate [(3)H]arachidonic acid release by labeled ampullas. At 26 degrees C UTP induced a dose-dependent and saturable increase of PLA(2) activity (apparent activation constant 1.3 +/- 0.4 microM, Hill coefficient 0.9 +/- 0.2, maximal stimulating factor 2.0 +/- 0.1). The rank order of potency of agonists for PLA(2) activation was UTP > or = UDP > adenosine 5'-O-(2-thiodiphosphate) = adenosine 5'-O-(3-thiotriphosphate) > or = ATP = 2-methylthio-ATP > or = ADP = diadenosine tetraphosphate > or = alpha,beta-methylene-ATP = CTP > 2' and 3'-O-(4-benzoylbenzoyl)-ATP > or = AMP = UMP >> uridine and adenosine. UTP- and 2-methylthio-ATP-induced PLA(2) activations were inhibited by U-73122, GF-109203X, and methyl arachidonyl fluorophosphate. Basal activity was stimulated by phorbol ester and epinephrine and reduced by vasotocin, isoproterenol, prostaglandin E(2), cAMP, and forskolin. H-89 restored the cAMP- and forskolin-inhibited PLA(2) activities. Results indicate that P2Y receptor-mediated PLA(2) stimulation requires phopholipase C and protein kinase C activations and basal activity is inhibited by agonist-stimulated cAMP-dependent mechanisms.

UTP binding and phosphoinositidase C activation in ampulla from frog semicircular canal.

Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [(3)H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[(3)H]inositol-loaded ampullas were determined. Specific [(3)H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 microM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/microgram protein; dissociation constant (K(D1)) = 0.4 microM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/microgram protein; K(D2) = 10 microM). The stereospecificity pattern for UTP analog recognition was UMP > UDP >/= ADP = UTP = dTTP > adenosine 5'-O-(3-thiotriphosphate) = ATP = CTP = 2'-and 3'-O-4-(benzoylbenzoyl)-ATP (Bz-ATP) >/= AMP >/= 2-methylthio-ATP = alpha,beta-methylene-ATP > uridine = diadenosine tetraphosphate (Ap(4)A); cAMP and adenosine were inactive. Antagonist recognition pattern was DIDS = pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) = reactive blue 2 > suramin. The rank order of potencies for agonist-induced PLC activation was UDP >/= UTP >/= Ap(4)A >/= UMP = Bz-ATP; uridine was inactive. UTP-stimulated PLC activity was inhibited by DIDS = reactive blue 2 = PPADS > suramin. These results suggest that the population of [(3)H]UTP-labeled binding sites is heterogeneous, with a low number of high-affinity UTP receptors whose function(s) need to be determined and a large number of moderate-affinity receptors triggering PLC activation.

Localization of Ca-ATPase in frog crista ampullaris.

The distribution of Ca-ATPase in frog crista ampullaris was mapped ultracytochemically by using a one-step lead citrate reaction. Electron-dense precipitates, as an expression of Ca-ATPase activity, were observed on the surface of stereocilia and on the apical membrane surrounding the cuticular plate of hair cells. Sensory cells of the isthmus region showed more reactivity than those of the peripheral regions of the crista. No reaction products were detectable on the basolateral membranes and in cytoplasmatic organelles. Supporting cells of the crista showed a quite variable Ca-ATPase reaction on microvilli and on basolateral membranes. The presence of an evident reactivity on the stereocilia is consistent with the existence of an apical calcium microdomain involved in the mechano-transduction process and supports the current view that calcium ions enter the stereocilia during natural stimulation. On the other hand, the lack of an observable reactivity on the basolateral membrane of hair cells suggests that in semicircular canals other mechanisms of active transport of calcium ions across the plasma membrane, such as Na-Ca exchange, may be involved in homeostasis of the ion.

Purine and pyrimidine nucleotide-sensitive phosphoinositidase C in ampulla from frog semicircular canal.

A microassay was developed to screen the abilities of ATP analogues to stimulate phosphoinositidase C in single ventral regions (including dark cells and sensory cells) of ampullas microdissected from posterior vertical semicircular canals of Rana ridibundo and labeled with myo-[3H]inositol. ATP induced a dose-dependent and saturable increase of total [3H]linositol phosphate production accompanied by an equivalent decrease in the [3H]phosphoinositide pool. The rank order of analogues revealing agonistic potencies for phosphoinositidase C activation was as follows: uridine 5'-triphosphate > or = adenosine 5'-O-[3-thiophosphate] tetralithium > adenosine 5'-O-[2-thiodiphosphate] trilithium > or = ATP > or = ADP = inosine 5'-triphosphate > or = guanosine 5'-triphosphate > or = 2-methylthio-adenosine 5'-triphosphate tetrasodium > or = 2'-desoxy-thymidine 5'-triphosphate > or = cytidine 5'-triphosphate = (alpha, beta)-methyl ATP > AMP, whereas adenosine 3',5'-cyclic monophosphate and adenosine were almost devoid of activity. For antagonists, 1,4'-diisothiocyanostilbene-2, 2'-disulfonic acid was far more active than suramin for competitive inhibition of ATP-induced enzyme stimulation, whereas reactive blue 2 acted as a noncompetitive inhibitor. Results indicate that the putative P2 receptors triggering phosphoinositidase C activation in ventral ampullary epithelium from frog semicircular canal exhibit mainly the functional properties of P2Y and P2U receptors.

Histochemical demonstration of 11 beta-hydroxysteroid dehydrogenase in ampullae of semicircular canals.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) plays a major role in the protection of the mineralocorticoid (type I) receptor. The cellular mechanism of aldosterone selectivity relies on the coexpression of mineralocorticoid receptors and 11 beta HSD in the same cells. An 11 beta HSD activity has been localized in the ampullae of the semicircular canal by histochemical technique which links steroid metabolism with the deposition of formazan deposition. Oxidation of 11 beta-hydroxyandrostenedione was observed in the dark cell region. Oxidation of 11 beta-hydroxyandrostenedione was nicotine-adenine dinucleotide (NAD) dependent. These results demonstrate the presence of a dehydrogenase activity separate from the nicotineamide-adenine dinucleotide phosphate (NADP) dependent 11 beta HSD.

Acid phosphatase activity in the chick's inner ear.

The cellular localization of distribution of acid phosphatase (AP) in the plastic sections of newly hatched chick's inner ear were investigated utilizing an azo-coupling method. AP activity as evidenced by azo dye deposits were well defined and localized in various cells of membranous labyrinth. Intense AP activity was detectable in the supranuclear area of hair cells in the basilar papilla and vestibular sensory hair cells. As in the case of the other sites of AP activity, marked AP activity was seen in the supranuclear area of the transitional epithelia of crista ampullaris and in the supranuclear area, or diffusely in the cytoplasm of the dark cells at the base of crista ampullaris. The columnar cells and the cells of tegmentum vasculosum showed moderate to strong AP activity. The statoacoustic and vestibular ganglion cells showed various degrees of AP activity. On the AP activity of statoacoustic or vestibular ganglion, in comparison between the sections from JB-4 Plus embedded specimens and those from LR White embedded specimen, the latter could more intensely demonstrate AP activity than the former. Moreover, sections fixed in 2.5% paraformaldehyde demonstrated more intense AP activity than those fixed in 2.5% glutaraldehyde.

Adenylate cyclase in the semicircular canal. Hormonal stimulation and ultrastructural localization.

The modulation of the cyclic AMP (cAMP) production and the cytochemical localization of adenylate cyclase were studied in isolated semicircular canal epithelium of the frog. The basal cAMP content, as measured by radioimmunoassay, was 344 +/- 37.8 fmoles/structure/5 min (mean +/- SEM, n = 41). This content was increased 6- to 8-fold by forskolin (10(-7) M to 10(-5) M). Among the tested drugs, only prostaglandin E2, isoproterenol, and vasotocin increased the cAMP production: 1.7-fold by prostaglandin E2 (1.5 X 10(-7) M) and isoproterenol (10(-6) M), and 1.3- and 3.3-fold by vasotocin at 10(-8) M and 10(-7) M, respectively. The addition of alpha 2-adrenergic agonists blunted the stimulatory effect of vasotocin. The adenylate cyclase was evidenced in both the basolateral and apical membranes of the dark cells. Vasotocin stimulated only the apical adenylate cyclase of dark cells. These results indicated that the adenylate cyclase located in the apical dark cells of the semicircular canal was stimulated by the antidiuretic hormone which may be involved in the regulation of the endolymph secretion.

Antidiuretic hormone stimulation of adenylate cyclase in semicircular canal epithelium.

Basal adenosine 3',5'-cyclic monophosphate (cAMP) content and the modulation of its production were studied in the frog's semicircular canal epithelium. This epithelium secretes endolymph, a K(+)-rich, positively polarized fluid. The basal cAMP content measured by microradioimmunoassay was 244 +/- 14.2 fmol/structure per 5 min (n = 30). This content was increased about 8 times by 10(-5) M forskolin. Vasotocin, the frog antidiuretic hormone, increased the cAMP production by factors of 1.3 and 3.3 at concentrations of 10(-8) M and 10(-7) M, respectively. This stimulatory effect of vasotocin was blunted by the addition of alpha 2-adrenergic agonists, such as 10(-8) M-10(-5) M norepinephrine, in the presence of 10(-5) M propranolol, or 10(-5) M clonidine. Prostaglandin E2 at a concentration of 10(-8) M, which did not affect the cAMP production, did not modify the response to vasotocin. Glucagon (10(-6) M), calcitonin (10(-6) M), and parathyroid hormone (10 units/ml) did not affect the cAMP content. Prostaglandin E2 (10(-7) M) and the beta-adrenergic agonist isoproterenol (10(-6) M) stimulated the cAMP production by a factor of 1.6. These results indicate that the frog semicircular canal is a target of both vasotocin and catecholamines and that catecholamines through alpha 2-receptors modulate vasotocin-induced cAMP generation. Further, this interaction might be of physiological relevance in the modulation of ion transport in this structure.

Adenylate cyclase and carbonic anhydrase in the semicircular canal epithelium of the frog Rana esculenta. An ultrastructural cytochemical localization.

Because the secretion of endolymph has been localized in the ampullar part of the frog semicircular canal, we attempted to determine by cytochemical methods the ultrastructural localization of two enzymes that are assumed to play a role in endolymph secretion: carbonic anhydrase and adenylate cyclase. Functionally, the epithelium of the frog semicircular canal can be schematically divided into three areas: sensory (crista ampullaris), secretory (dark cells), and non-sensory and nonsecretory (transitional and undifferentiated cells) areas. Carbonic anhydrase activity was widely distributed in dark cells. Dark cell labeling disappeared in the presence of acetazolamide. The other cells of the canal did not show any carbonic anhydrase labeling except for the supporting cells of the sensory cells. Adenylate cyclase activity was found on the basolateral and apical membranes of dark cells, and on the apical membrane of sensory cells; weak labeling was also observed in the other epithelial cells. In the apical membrane of the dark cells, adenylate cyclase labeling was dependent on the presence of vasotocin, the frog antidiuretic hormone. The dark cells of the frog semicircular canal thus possess the enzyme equipment needed for the secretion of endolymph and its possible hormonal regulation.

Possible cholinergic neurotransmission in the cristae ampullares of the chick inner ear.

The presence of choline acetyltransferase (ChAT) was investigated in the chick inner ear in order to assess the possible role of acetylcholine (ACh) in neurotransmission within the vestibular labyrinth. ChAT activity found in homogenates of isolated chick vestibular cristae ampullares is of the same order of magnitude and has similar properties as its homologous enzyme in nervous tissue from various regions. These findings are in accord with a probable neurotransmitter role of ACh in the chick vestibular labyrinth.