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T Yorio

Publications and source records attributed to T Yorio.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of a transformed rat retinal ganglion cell line.

The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801). In conclusion, a transformed rat retinal cell line, RGC-5, has certain characteristics of retinal ganglion cells based on Thy-1 and Brn-3C expression and its sensitivity to glutamate excitotoxicity and neurotrophin withdrawal. These cells may be valuable in understanding of retinal ganglion cell biology and physiology including in vitro manipulations in experimental models of glaucoma.

Animals↗
PubMed2001

Inhibition of Na(+)/K(+)-atpase by endothelin-1 in human nonpigmented ciliary epithelial cells.

Endothelin-1 (ET-1), a potent vasoconstrictor, lowers intraocular pressure in mammals, either by enhancing the outflow of aqueous humor (AH) via the trabecular meshwork and Schlemm's canal or by reducing AH formation at the ciliary epithelium. Aqueous humor production occurs by passive diffusion of water coupled with active transport of ions, mainly involving Na(+):K(+):2Cl(-) cotransporter and Na(+)/K(+)-ATPase pump from serosal to aqueous side. Presently, we have evaluated the effects of ET-1 on Na(+):K(+):2Cl(-) cotransport and Na(+)/K(+)-ATPase activity in HNPE cells using (86)Rb(+) uptake. ET-1 (100 pM-100 nM) decreased mean (86)Rb(+) uptake by 15% during a 15-min uptake period. ET-1's effect was not prevented by BQ610, an ET(A) receptor antagonist, but was blocked by BQ788, an ET(B) receptor antagonist. ET-1's effect was mimicked by sarafotoxin, an ET(B) agonist. ET-1-induced reduction in (86)Rb(+) uptake was additive with bumetanide, a selective inhibitor of Na(+):K(+):2Cl(-) cotransporter but not with ouabain, a selective inhibitor of the Na(+)/K(+)-ATPase. ET-1 did not affect iberiotoxin-sensitive maxi K(+) channels. This suggests that ET-1-induced reduction in (86)Rb(+) uptake is mediated through the inhibition of the Na(+)/K(+)-ATPase via an ET(B)-like receptor. These findings are consistent with an ET-1 effect on active ion transport activity in HNPE cells that could explain the reduction in aqueous humor production and the lowering of intraocular pressure.

Antihypertensive Agents↗
PubMed2000

Dependence of insulin secretion from permeabilized pancreatic beta-cells on the activation of Ca(2+)/calmodulin-dependent protein kinase II. A re-evaluation of inhibitor studies.

Previous studies utilizing inhibitors of the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) to address the role of this enzyme in insulin secretion have produced contradictory results. In the current study, these inconsistencies have been addressed by evaluating the effect of various CaM kinase II inhibitors to decrease Ca(2+)-induced insulin secretion from permeabilized beta-cells. KN-93 (2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlo rocinnamyl)-N-methylbenzylamine) markedly inhibited both CaM kinase II activation and insulin secretion in parallel in alpha-toxin-permeabilized beta-cells. These effects were specific since they were not mimicked by the inactive analog, KN-92 (2-[N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methy lbenzylamine). In contrast, KN-62 (1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine) , while reported to be similar to KN-93 with respect to mechanism of action, did not inhibit Ca(2+)-induced activation of CaM kinase II or insulin secretion in these cell preparations. All three agents suppressed Ca(2+) influx in intact beta-cells induced by depolarization in the presence of elevated extracellular potassium although to different extents. The synthetic peptide inhibitors of CaM kinase II, [Ala(286)]CaMK 281-302 and AIP (autocamtide-2-related inhibitory peptide), strongly inhibited Ca(2+)-induced insulin secretion from electropermeabilized islets, an effect that also correlated with an equivalent inhibition of CaM kinase II activation. This re-evaluation (i) explains a lack of effect of KN-62 on insulin secretion from permeabilized cells based on its inability to inhibit CaM kinase II activation in these preparations; (ii) has revealed that CaM inhibitors, either chemical or peptide in nature, that are capable of preventing enzyme activation uniformly suppress Ca(2+)-sensitive insulin secretion; and (iii) cautions the use of KN-62/93/92 as selective inhibitors of CaM kinase II in intact cell studies. These observations reinforce the suggestion that CaM kinase II plays an important role in insulin exocytosis in the beta-cell.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Microfilament network is needed for the endocytosis of water channels and not for apical membrane insertion upon vasopressin action.

In the current study, a novel role for the microfilaments in vasopressin-induced water transport in toad urinary bladders, a popular model for the mammalian collecting duct, was established. Vasopressin-induced water transport was not affected by cytochalasin D (CD, 20 microM) or latrunculin B (Lat B, 0.5-2 microM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels and insertion of membranes into the apical membrane are microfilament-independent. After the removal of vasopressin, bladders treated with CD or Lat B continued to transport water at least 2-3-fold greater than those that received the vehicle. Furthermore, the enhanced water transport was inhibited by HgCl2 (1 mM), a potent inhibitor of water channel-mediated water flow, suggesting that the enhanced water flow was through water channels. In addition, Lat B and CD inhibited vasopressin-induced endocytosis of horseradish peroxidase (HRP), a fluid endocytotic marker. These results suggested that although microfilaments are not needed for the initial trafficking of water channels to the apical side, the microfilament network is essential for the retrieval of water channels following their insertion into apical membranes.

Actin Cytoskeleton↗
PubMed2000

Cholinergic and adrenergic modulation of the Ca2+ response to endothelin-1 in human ciliary muscle cells.

PURPOSE: To determine the cholinergic (carbachol, CCH) and adrenergic (norepinephrine, NE) modulation of Ca2+ response to endothelin-1 in human ciliary smooth muscle (HCSM) cells. METHODS: Intracellular calcium levels were measured using the Fura-2 calcium imaging system in HCSM cells treated either singly with endothelin-1 (ET-1; 2-200 nM), CCH (1-100 microM), NE (0.1-10 microM) or isoproterenol (ISO; 1 microM) or in combinations of CCH, NE, or ISO with ET-1. Intracellular cAMP levels after NE and ISO treatments were also measured using a radioimmunoassay. RESULTS: Endothelin-1 dose-dependently increased [Ca2+]i and was characteristically biphasic (peak [Ca2+]i for ET-1: 2 nM, 517 +/- 73 nM; 20 nM, 785 +/- 65 nM; and 200 nM, 2564 +/- 359 nM). Carbachol also dose-dependently increased [Ca2+]i; however, subsequent additions of ET-1 (200 nM) resulted in lower [Ca2+]i (100 microM CCH + ET-1; 300 +/- 21 nM) compared with that observed with 200 nM ET-1 alone (2564 +/- 359 nM). Norepinephrine pretreatment also decreased ET-1-induced [Ca2+]i (10 microM NE + ET-1; 619 +/- 64 nM) compared with ET-1 alone, and NE's effect could be reversed by propranolol (beta-adrenergic antagonist) treatment. Neither CCH nor NE was able to completely abolish ET-1's ability to mobilize calcium in HCSM cells. Isoproterenol (a beta-agonist) mimicked NE's effect on ET-1-induced [Ca2+]i (1 microM ISO + ET-1; 254 +/- 56 nM). Both ISO and NE elevated [cAMP] in HCSM cells. CONCLUSIONS: In HCSM cells, CCH and ET-1 can activate common as well as specific [Ca2+]i pools. The reduction in ET-1-induced [Ca2+]i after NE/ISO treatment appears to be due to elevated cAMP levels via beta-receptor activation, suggesting the existence of receptor cross talk. The ability of CCH and NE to modulate ET-1's actions on HCSM may be relevant to the regulation of ciliary muscle contraction and aqueous humor outflow.

Adrenergic Agonists↗
PubMed1999

Identification of endothelin converting enzyme-1 in human non-pigmented ciliary epithelial cells.

Endothelins (ETs), potent vasoactive peptides, are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin-converting enzyme (ECE). Although the role of ocular ET's are uncertain, they are elevated in the aqueous humor of normal as well as glaucomatous eyes and have been shown to lower the intraocular pressure for prolonged periods of time. In the current study, an endothelin-converting enzyme-1 (ECE-1) has been identified and its activity has been studied in SV-40 transformed human non-pigmented ciliary epithelial (HNPE) cells. Western blotting using polyclonal antibodies against ECE-1, detected a 124 kDa protein in the plasma membrane but not in the cytosol. Further characterization of the enzymatic activity of ECE-1 (conversion of Big ET-1 to ET-1) using the plasma membrane fraction of HNPE cells was performed by a novel assay involving(125)I-Big ET-1 (substrate; 80 fmoles mg(-1)protein) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as fmoles(125)I-ET-1 produced mg protein(-1)time(-1)) increased linearly with time and was similar to that observed in rat lung tissue. ECE-1 activity was enhanced with increasing concentrations of substrate ((125)I-Big ET-1; 30-200 fmoles mg protein(-1)180 min(-1)) as well as with increasing concentrations of protein (5-20 microgram proteins at the substrate concentration of 80 fmoles mg protein(-1)180 min(-1)). Treatments with CGS-26303 (100 micrometer), an inhibitor of ECE-1 and thiorphan (2 mM; a metalloprotease inhibitor), significantly decreased ECE-1 activity. Furthermore, both, acidification of the reaction buffer (from pH 7.4 to 6.4) and the addition of a metal chelator, EGTA (2 mM) decreased ECE-1 activity by nearly 60%. These results suggest that the ECE-1 localized in HNPE cells, is a neutral pH-sensitive metalloprotease which is similar in its activity to that observed in lung tissue and is essential for the production of ET-1 in HNPE cells. The physiological importance of the unusual proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure.

Aspartic Acid Endopeptidases↗
PubMed1999

Phenylarsine oxide inhibits phosphate uptake in human ciliary non-pigmented epithelial cells.

Phenylarsine oxide (PAO), a sulfhydryl modifying reagent and a widely used inhibitor for tyrosine phosphatases and endocytosis, was tested on the level of phosphorylation in human nonpigmented ciliary epithelial ocular (HNPE) cells. Pretreatment with (PAO, 10 microM) for 30 min followed by incubation with 32Pi to stimulate endogenous phosphorylation surprisingly resulted in a total reduction in 32Pi labeled proteins. PAO (10-50 microM) dose-dependently inhibited both sodium-dependent and -independent phosphate uptake in cells. p-Hydroxymercuribenzoate (pHMB, 10 microM), another sulfhydryl modifying reagent failed to mimic PAO effects. However, metabolic inhibitors (iodoacetamide (0.1 mM) and 2,4-dinitrophenol (DNP, 0.5 mM) also mimicked PAO effects, suggesting that the inhibition of ATP production may be responsible for attenuation of both phosphate uptake mechanisms. However, sodium-dependent phosphate uptake in isolated plasma membrane vesicles pretreated with PAO was also significantly lower than control vesicles treated with dimethlysulfoxide (DMSO), suggesting that PAO may be directly targeting a component of the sodium-dependent cotransporter. It is suggested that PAO is a novel inhibitor of phosphate uptake in HNPE cells that acts indirectly by inhibiting ATP production and directly by inhibiting the Na-dependent cotransporter.

Arsenicals↗
PubMed1998

Regulation of endothelin-1 in human non-pigmented ciliary epithelial cells by tumor necrosis factor-alpha.

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) by the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle thus enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regulation of endothelin-1 (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effect of tumor necrosis factor-alpha(TNF-alpha; 10 nm), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and release in SV-40 transformed human ciliary non-pigmented epithelial cells (HNPE). ET-1 and Big endothelin-1 (Big ET-1) immunoreactivity was time-dependently increased following TNF-alphatreatment. Phorbol esters (PMA), activators of PKC, also raised the immunoreactive levels of ET-1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nm), decreased ET-1 levels in TNF-alpha-stimulated cells. Pre-treatment with phosphoramidon (1 micron) an ECE-inhibitor, followed by TNF-alpha stimulation, decreased ir-ET-1 levels. Cycloheximide (9 micron), a protein synthesis inhibitor, decreased TNF-alpha-stimulated levels for ir-ET-1 and ir-Big ET-1, suggesting that TNF-alpha may be directly regulating ET-1 expression at the ET-1 gene. Our data indicates that TNF-alpha regulates ET-1 levels in HNPE cells possibly by activating PKC either to stimulate protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an important role in aqueous humor dynamics following cytokine activation.

Cells, Cultured↗
PubMed1998

Endothelin receptor A is expressed and mediates the [Ca2+]i mobilization of cells in human ciliary smooth muscle, ciliary nonpigmented epithelium, and trabecular meshwork.

PURPOSE: To identify which endothelin receptor subtype is expressed and is functional in the human ciliary body and trabecular meshwork, tissues that regulate aqueous humor dynamics. METHODS: Immunocytochemistry was used to characterize the primary culture cells of normal human ocular cells. Endothelin receptor gene expression was probed with reverse transcription of polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) mobilization was measured with video image microscopy using Fura-2AM as a fluorescent probe. RESULTS: Identities of primary cultures, human ciliary smooth muscle (HCSM), ciliary nonpigmented epithelial (HCE), and trabecular meshwork (HTM) cells were confirmed by immunocytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ETA) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ETB receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca2+]i of HCSM cells was increased from 57 +/- 7 nM to 328 +/- 108 nM (n = 23; mean +/- SE; P < 0.05) by 1 nM endothelin-1 (ET-1). In HCE cells, [Ca2+]i increased from 40 +/- 3 nM to 90 +/- 10 nM (n = 55) (P < 0.001) with the same concentration of ET-1. Similarly, ET-1 (1 nM) increased the [Ca2+]i from 51 +/- 6 nM to 185 +/- 47 nM (n = 19) (P < 0.001) in the HTM cells. The agonist for ETB, S6c, had no effect on [Ca2+]i transients in all three cell types. No ETB receptor expression was detected in these cell types under the experimental and culture conditions. CONCLUSION: ETA receptor is expressed and is possibly responsible for mediating the signal for [Ca2+]i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.

Calcium↗
PubMed1998

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium.

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.

Calcium↗
PubMed1998

Effect of temperature on apical membrane remodeling in ADH-stimulated toad urinary bladders.

Pretreatment and removal of vasopressin (ADH) in toad urinary bladder renal model tissues induces endocytosis at 25 degrees C. The objective of the current study is to determine if apical membrane remodeling, as well as transepithelial water flow, can be affected by lowering the temperature to 15 degrees C. Control toad urinary bladders in the presence of an osmotic gradient at either 25 degrees C or 15 degrees C when visualized by scanning electron microscopy (SEM) show a typical apical membrane surface with no apparent surface differences. ADH-treated tissues following 15-min stimulation at 25 degrees C or 15 degrees C revealed a propagation of apical microvilli on their surface membranes. After 15 min following removal of ADH, bladder tissues at 25 degrees C or 15 degrees C showed surface invaginations involving over 44% and 80% of granular cells, respectively. The rate of water flow in tissues at 15 degrees C remained elevated compared to tissues held at 25 degrees C. This was consistent with the observation that ADH-stimulated tissues following washout at 15 degrees C still had marked apical membrane surface involvement. However, at 30 min and 60 min postwashout, ADH-stimulated tissues at 15 degrees C recovered considerably, with a reduction in the number of shallow apical membrane invaginations involving fewer than 33% and 20% of granular cells respectively. This may indicate that the membrane undergoes continuous remodeling even at cold temperature conditions but with a different half-time. Control bladder tissues subjected to transmission electron microscopy (TEM) reveal a dense cytoplasmic profile with a scattered distribution of secretory granules, rough ER cisternae, mitochondria, and little or no vacuolation. In contrast, ADH-stimulated bladder tissues displayed a vacuolated cytoplasm, expanded rough ER cisternae, and ruffled basolateral membranes. These observations suggest that the apical membrane undergoes considerable reorganization following cessation of hormone action and that lowering the temperature reduces the rate of membrane remodeling and thus may provide a means to monitor the processes of endocytosis and the mechanisms responsible for water channel retrieval.

Animals↗
PubMed1998

Aquaporins (water channels): role in vasopressin-activated water transport.

The discovery of water channels (aquaporins) was a breakthrough in research on water transport. Aquaporins are a family of intrinsic membrane proteins that function as water-selective channels (except aquaporin-3 and aquaporin-7, which are permeable to urea and glycerol as well) in the plasma membranes of many cells. Aquaporin-0 (MIP26) functions to maintain fluid balance in the lens. Aquaporin-1 is involved in water reabsorption in the kidney's proximal tubules and the thin descending Henle's loop, aqueous humor formation in eye, cerebrospinal fluid formation in brain, and airway hydration in lung. Aquaporin-2 is the only water channel that is activated by vasopressin to enhance water reabsorption in the kidney collecting duct. Aquaporin-3 also contributes to water reabsorption in the kidney collecting duct but is unresponsive to vasopressin. It also appears that aquaporin-3 may contribute to cornea transparency. Aquaporin-4 is involved in cerebrospinal fluid transport in brain, water transport in the kidney collecting duct, aqueous humor transport in the eye, and airway hydration in the lung. Aquaporin-5 apparently is coupled to fluid secretion in exocrine tissues. Although the exact function of aquaporin-6 is not known due to its uncertain localization, its restricted presence in the kidney may suggest a potential role in water transport. Aquaporin-7 appears to play a role in the cryopreservation of the sperm whereas aquaporin-8 is responsible for the secretion of pancreatic juice. The major focus of this review is a discussion of aquaporins in renal epithelia, and particularly the mechanisms associated with vasopressin-mediated water transport involving aquaporin-2 and the signal transduction pathways linked to vasopressin action.

Animals↗
PubMed1997

The ATP-depleting reagent iodoacetamide induces the degradation of protein kinase C alpha (PKC alpha) in LLC-PK1 pig kidney cells.

The alkylating reagent iodoacetamide, a potent inhibitor of sulfhydryl proteases, was found to stimulate the selective degradation of protein kinase C alpha (PKC alpha) isoform (80 KDa). Treatment of LLC-PK1 cells with iodoacetamide (0.5-15 mM) for 30-90 minutes at room temperature allowed by western blotting on total cell homogenate, revealed the appearance of an 50 KDa band that was still recognized with the antibody. However, iodoacetamide (15 mM) resulted in the total disappearance of the 80 KDa protein. Serine protease inhibitors, metalloprotease inhibitors and leupeptin failed to prevent the degradation of PKC alpha. The degradation persisted at 4 degrees C and in the absence of Ca2+. Iodoacetamide had no direct effect on purified PKC alpha. PKC activities in iodoacetamide-treated cells were also inhibited. In conclusion, the degradation of PKC alpha is a novel phenomenon. The degradation process could not be prevented by known protease inhibitors or in the absence of Ca2+ or by incubation at 4 degrees C and appears to involve interactions with unknown cellular intermediates.

Adenosine Triphosphate↗
PubMed1997

The effects of elevated glucose on Na+/K(+)-ATPase of cultured bovine retinal pigment epithelial cells measured by a new nonradioactive rubidium uptake assay.

The effects of stimulated hyperglycemia on the Na+/K(+)-ATPase activity of cultured bovine retinal pigment epithelial (RPE) cells were investigated. Total Rb+ uptake, measured by a chromatographic method, was decreased 20-30% by 55.5 mM glucose relative to 5.55 mM glucose for culture periods of 2 to 28 days. An acute hyperglycemic stress (< 1 week) had no effect on ouabain-inhibition of Rb+ uptake or ouabain binding to RPE cells (IC50 = 55 nM for both processes) and did not alter the IC50 value (near 10 nM) for binding of strophanthidin, another selective Na+/K(+)-ATPase inhibitor. A small increase in the apparent K(m) of Rb+ for Na+/K(+)-ATPase accompanied the decrease in maximal Rb+ uptake at 55.5 mM glucose. The continuous presence of AL-1576, an aldose reductase inhibitor (ARI), normalized the effect of severe hyperglycemia on Rb+ uptake in the chronic (28 days) but not the acute exposure protocols. Thus, decreased efficiency of Na+/K(+)-ATPase caused by chronic accumulation of intracellular sorbitol can account for previously reported functional and structural alterations in the RPE cell layer of diabetic rodents. The results of the present study suggest that hyperglycemia-induced loss of Na+/K(+)-ATPase function in RPE cells, which responds to aldose reductase inhibitor treatment, contributes to the pathogenesis of diabetic retinopathy.

Aldehyde Reductase↗
PubMed1997

Mechanism of vasopressin-induced increase in intracellular Ca2+ in LLC-PK1 porcine kidney cells.

Analysis of the signal transduction cascade of vasopressin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in LLC-PK1 cells was performed. First, a comparison of the effect of vasopressin on [Ca2+]i in LLC-PK1 cells with that produced in rat hepatocytes was performed [an intracellular mobilizing mechanism involving a V1 receptor coupled to the production of inositol 1,4,5-trisphosphate (IP3)]. Second, the effect of known inhibitors of intracellular Ca2+ mobilization on vasopressin Ca2+ response in LLC-PK1 cells was studied. Vasopressin induced a transient increase in [Ca2+]i in both LLC-PK1 cells and hepatocytes. In contrast to the single [Ca2+]i spike seen in LLC-PK1 cells, vasopressin induced an average of two to three [Ca2+]i spikes in hepatocytes. The V1 antagonist (Pmp1-O-Me-Tyr2-[Arg8]vasopressin, 1 microM) abolished vasopressin Ca2+ response in both cell types. Inhibitors of intracellular Ca2+ mobilization, thapsigargin (5 microM) and U-73122 (3 microM), abolished the Ca2+ response by vasopressin in LLC-PK1 cells. The results suggest that vasopressin-induced increase in [Ca2+]i in LLC-PK1 cells is mediated via a V1-like receptor and involves the mobilization of intracellular Ca2+ through an IP3- or thapsigargin-sensitive Ca2+ pool.

Animals↗
PubMed1997

Bicarbonate and CO2 transport across the skin of the leopard frog, Rana pipiens: effect of PGF2alpha.

The amphibian skin represents an important organ for osmoregulation and, like the mammalian kidney, maintains acid-base balance by secreting protons or base. However, the lack of a reliable and accurate method to measure the contribution of unidirectional fluxes of HCO3- ions to this mechanism has been an obstacle for the determination of the role of bicarbonate in epithelial acid-base homeostasis. Recently, one of us developed a method that allows for the reliable determination of transepithelial fluxes of bicarbonate, and this method was applied to determine unidirectional fluxes of (14)CO2 and H(14)CO3 under a variety of conditions. We report that the combined CO2 and HCO3- mucosal-to-serosal flux under 5% CO2 was 40% larger than the opposing flux, giving a net flux in the mucosal-to-serosal direction. This net flux was inhibited by acetazolamide. In CO2-free conditions, there was no detectable net flux; however, acetazolamide and PGF(2alpha) attenuated the mucosal-to-serosal flux and established an apparent secretion of HCO3-. A model is presented that depicts twelve vectors or components to the CO2 plus HCO3- fluxes in the frog skin. This model can accurately reproduce the experimental values measured from unidirectional fluxes of CO2 and HCO3- under a variety of conditions and can explain the effects of PGF(2alpha) on unidirectional 14C-labeled fluxes as a consequence of inhibition of H+ secretion to the apical bath, similar to what was previously suggested by our laboratory using a different methodological approach. The present method, utilizing radiolabeled HCO3-, may be useful as a means to evaluate the mechanism of action of hormones and drugs that may regulate acid-base homeostasis by altering proton and bicarbonate transport processes.

Animals↗