Identification and Functional Distribution of Intracellular Ca2+ Channels in Mouse Lacrimal Gland Acinar Cells

We have determined the presence and cellular distribution of intracellular calcium channels, inositol 1, 4, 5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in adult and postnatal (P10) lacrimal gland acinar cells. Western blot analysis of both P10 cultures and adult tissue identified the presence of each IP3R and RyR isotypes. The immunocytochemistry analysis showed a differential cellular distribution of these calcium channels where the nuclear envelope, endoplasmic reticulum (ER) and Golgi apparatus membranes represent areas with highest levels of channel expression. This IP3R and RyR isotype distribution is confirmed by the immuno-EM results. The findings described in this study are in agreement with published pharmacological data that shows the participation of these channels in the secretion process of the lacrimal gland acinar cells. Furthermore, the differential subcellular distribution between the isoforms could indicate a potential role of these intracellular Ca2+ channels on the regulation of specific cellular functions

Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca2+ channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.CREST of the Japan Science and Technology Corporation; NIH [P30 DK090744]; Human Frontier Science Program [ST00246/2003C]; Deutsche Forschungsgemeinschaft [PE 853/2]; Japan Society for the Promotion of Science; American Heart Association [R10682]info:eu-repo/semantics/publishedVersio

Retinal repair by transplantation of photoreceptor precursors

Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system repair; photoreceptor degeneration initially leaves the inner retinal circuitry intact and new photoreceptors need only make single, short synaptic connections to contribute to the retinotopic map. So far, brain- and retina-derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptors(1-4). Furthermore, there has been no demonstration that transplanted cells form functional synaptic connections with other neurons in the recipient retina or restore visual function. This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation. We hypothesized that committed progenitor or precursor cells at later ontogenetic stages might have a higher probability of success upon transplantation. Here we show that donor cells can integrate into the adult or degenerating retina if they are taken from the developing retina at a time coincident with the peak of rod genesis(5). These transplanted cells integrate, differentiate into rod photoreceptors, form synaptic connections and improve visual function. Furthermore, we use genetically tagged postmitotic rod precursors expressing the transcription factor Nrl (ref. 6) ( neural retina leucine zipper) to show that successfully integrated rod photoreceptors are derived only from immature post-mitotic rod precursors and not from proliferating progenitor or stem cells. These findings define the ontogenetic stage of donor cells for successful rod photoreceptor transplantation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62596/1/nature05161.pd

Effect of calcium-sensing receptor activation in models of autosomal recessive or dominant polycystic kidney disease

Background. Antagonists of relevant Gs protein-coupled and agonists of relevant Gi protein-coupled receptors lower renal cAMP and inhibit growth of renal cysts in animal models of human ARPKD (PCK rat) and/or ADPKD (Pkd2−/WS25 mouse). A calcium-sensing receptor (CaR) is expressed in various tubular segments and couples to Gq, thereby activating phospholipase Cγ, InsP3 generation and calcium mobilization from intracellular stores, and Gi proteins. By both mechanisms, CaR activation could lower intracellular cAMP and inhibit renal cyst growth

Dynamic assembly of ribbon synapses and circuit maintenance in a vertebrate sensory system

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity

Pharmacological Analysis of the Activation and Receptor Properties of the Tonic GABACR Current in Retinal Bipolar Cell Terminals

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABAA receptors (GABAARs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABACRs in their axon terminal, in addition to synaptic GABAAR and GABACR currents, which strongly regulate BC output. The tonic GABACR current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABACRs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABACR current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABACRs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABACR ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABACRs are thought to be ρ1-ρ2 heteromers. To investigate whether GABACRs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABAARs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABACR currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina

Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

The localization of polycystin (PC)1) to the plasma membrane requires coexpression with PC2 and cleavage at the PC1 G protein-coupled receptor proteolytic site. Neither the PC1 binding capacity of PC2 nor its channel function is required for this effect

Localization of metabotropic glutamate receptors in the outer plexiform layer of the goldfish retina

We studied the localization of metabotropic glutamate receptors (mGluRs) in the goldfish outer plexiform layer by light-and electron-microscopical immunohistochemistry. The mGluR1α antibody labeled putative ON-type bipolar cell dendrites and horizontal cell processes in both rod spherules and cone triads. Immunolabeling for mGluR2/3 was absent in the rod synaptic complex but was found at horizontal cell dendrites directly opposing the cone synaptic ribbon. The mGluR5 antibody labeled Müller cell processes wrapping rod terminals and horizontal cell somata. The mGluR7 antibody labeled mainly horizontal cell dendrites invaginating rods and cones and some putative bipolar cell dendrites in the cone synaptic complex. The finding of abundant expression of various mGluRs in bipolar and horizontal cell dendrites suggests multiple sites of glutamatergic modulation in the outer retina

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2

<p>Abstract</p> <p>Background</p> <p>Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes <it>PKD1 </it>and <it>PKD2 </it>account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of <it>PKD2</it>. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.</p> <p>Methods</p> <p>Here we utilized different kidney cell-lines expressing wild-type and mutant <it>PKD2 </it>as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.</p> <p>Results</p> <p>Surprisingly, over-expression of wild-type <it>PKD2 </it>in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated <it>PKD2 </it>augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57^KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.</p> <p>Conclusion</p> <p>Our results indicate the probable involvement of p57^KIP2on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.</p

Drosophila Sperm Swim Backwards in the Female Reproductive Tract and Are Activated via TRPP2 Ion Channels

Sperm have but one purpose, to fertilize an egg. In various species including Drosophila melanogaster female sperm storage is a necessary step in the reproductive process. Amo is a homolog of the human transient receptor potential channel TRPP2 (also known as PKD2), which is mutated in autosomal dominant polycystic kidney disease. In flies Amo is required for sperm storage. Drosophila males with Amo mutations produce motile sperm that are transferred to the uterus but they do not reach the female storage organs. Therefore Amo appears to be a mediator of directed sperm motility in the female reproductive tract but the underlying mechanism is unknown.Amo exhibits a unique expression pattern during spermatogenesis. In spermatocytes, Amo is restricted to the endoplasmic reticulum (ER) whereas in mature sperm, Amo clusters at the distal tip of the sperm tail. Here we show that flagellar localization of Amo is required for sperm storage. This raised the question of how Amo at the rear end of sperm regulates forward movement into the storage organs. In order to address this question, we used in vivo imaging of dual labelled sperm to demonstrate that Drosophila sperm navigate backwards in the female reproductive tract. In addition, we show that sperm exhibit hyperactivation upon transfer to the uterus. Amo mutant sperm remain capable of reverse motility but fail to display hyperactivation and directed movement, suggesting that these functions are required for sperm storage in flies.Amo is part of a signalling complex at the leading edge of the sperm tail that modulates flagellar beating and that guides a backwards path into the storage organs. Our data support an evolutionarily conserved role for TRPP2 channels in cilia