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P S Reinach

Publications and source records attributed to P S Reinach.

At least 19 recordsLinked to original sources
PubMed2001

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells.

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 microm RG-13022) and cPLA2 activity (10 microm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 microg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 microm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 microm H-89); 2) Erk1/2 (15 microm PD98059) or 3) p38 (15 microm SB203580) activity. Stimulation protein kinase C activity (0.1 microm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 microm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.

Animals↗
PubMed2001

Characterization of human taurine transporter expressed in insect cells using a recombinant baculovirus.

A recombinant baculovirus system was used to express the human taurine transporter in Sf9 cells and characterize its mediated uptake activity. This uptake process exhibited: (i) Na(+) dependence, (ii) larger inhibition of taurine transport by competing beta-amino acids than by alpha- and gamma-amino acids, (iii) apparent Michaelis constant, K(t), for taurine transport of 1.6 +/- 0.2 microM, and (iv) a maximal velocity, V(max), of 262 +/- 18 pmol/mg protein per 15 min. Coexpression of a molecular chaperone, human calnexin, enhanced taurine transporter activity by 43%. During development of taurine transporter expression, exposure to tunicamycin (10 microg/ml) decreased taurine transport activity by 76%. The taurine transporter linked to glutathione S-transferase (GST) was expressed to determine whether this conjugate also elicits taurine transport activity. Even though transport activity was markedly decreased, its Na(+) dependence was still evident. Coexpression of calnexin enhanced expression of this conjugated transporter activity by 54%. Immunoblot analysis revealed that calnexin did not change the amount of GST-taurine transporter conjugate or its molecular mass (i.e., 58.4-68.0 kDa). However, tunicamycin decreased its molecular mass. Taken together, taurine transport activity in a baculovirus expression system has characteristics similar to its wild-type counterpart. Stimulation of transport activity by coexpression with calnexin suggests the importance of transporter folding for optimal transport activity. Glycosylation of the transporter also increases its transport activity. Finally, GST-taurine transporter conjugate usage may aid transporter purification even though its transport activity decreases.

Animals↗
PubMed2001

Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport.

Although Na(+)-K(+)-2Cl(-) cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [(3)H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s(-1). Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na(+)-K(+)-2Cl(-) cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.

Amino Acid Sequence↗
PubMed2001

Differential expression of Na:K:2Cl cotransporter, glucose transporter 1, and aquaporin 1 in freshly isolated and cultured bovine corneal tissues.

Little is known about whether culturing corneal limiting layers causes changes in the expression of their membrane transporter proteins from those present in fresh tissues. Accordingly, we compared mRNA abundance of three well-described types of transporters: water channel aquaporin 1 (AQP1), glucose transporter (GLUT1), and Na:K:2Cl cotransporter (NKCC), as well as NKCC protein levels in fresh bovine corneal epithelium and endothelium with those in their cultured counterparts. Abundance of mRNA encoding AQP1, GLUT1, and NKCC was quantified by a lysate nuclease protection assay. NKCC transcription was further characterized by Northern blotting. All data were normalized to cell DNA and protein contents. In the fresh epithelium, in all three cases mRNA levels were two to four times higher than in the endothelium. Expression of AQP1 and GLUT1 was 10 to 12 times higher than that of NKCC. After the third passage, the endothelial cell mRNA abundance in each case decreased 2- to 3-fold. Passage-dependent decreases were also observed in NKCC protein expression in the epithelial cells. In both corneal layers, there was a qualitative correlation between NKCC mRNA and protein levels. Both in fresh and cultured epithelial and endothelial cells, a shark NKCC1 DNA probe hybridized with mRNAs of two different lengths (about 5.0-5.5 and 7.0-7.5 kb). An anti-NKCC T4 monoclonal antibody recognized two major proteins with apparent molecular masses of 190 to 200 and 150 to 160 kDa. In summary, membrane transporter function in culture may not be always indicative of their role in fresh tissue since in cultured cells AQP1, GLUT1, and NKCC mRNA levels declined. Furthermore, in both epithelial and endothelial cells, there is expression of two different proteins and mRNAs that possibly encode for secretory (NKCC1) and absorptive (NKCC2) isoforms.

Animals↗
PubMed2001

Corneal epithelial wound healing.

One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. Furthermore, the smooth optical properties of the corneal epithelial surface are sustained through this renewal process. The rate of renewal is dependent on a highly integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. One experimental approach to characterize these three aspects of the renewal process has been to study the kinetics and dynamics of corneal re-epithelialization in a wound-healing model. This effort has employed in vivo and in vitro studies. From such studies it is evident that the appropriate integration and coordination of corneal epithelial proliferation, adhesion, migration, and cell demise is dependent on the actions of a myriad of cytokines. Our goal here is to provide an overview into how these mediators and environmental factors elicit control of cellular proliferation, adhesion, migration, and apoptosis. To this end we review the pertinent literature dealing with the receptor and the cell signaling events that are responsible for mediating cytokine control of corneal epithelial renewal. It is our hope that a better appreciation can be obtained about the complexity of the control processes that are responsible for assuring continuous corneal epithelial renewal in health and disease.

Apoptosis↗
PubMed2001

Activation of a CFTR-mediated chloride current in a rabbit corneal epithelial cell line.

PURPOSE: To determine whether there is gene expression and functional activity of cystic fibrosis transmembrane conductance regulator protein (CFTR) in an SV40-immortalized rabbit corneal epithelial cell line, tRCE. METHODS: Both whole-cell and cell-attached patch-clamp techniques were used to examine the biophysical characteristics of the cAMP-dependent chloride current. The molecular identity of this conductance was evaluated using RT-PCR analysis. RESULTS: In whole-cell patch-clamp studies, a cAMP-dependent chloride conductance was further facilitated by the known CFTR activator genistein (20 microM). Kinetic analysis of cell-attached patches containing few channels ascertained that genistein increased the chloride channel activity by increasing channel open probability (via an increased channel open time and a decreased channel closed time). In addition, in the presence of a reduced forskolin concentration (i.e., 100 nM), the chloride conductance generated could be augmented by the nonspecific phosphodiesterase enzyme inhibitor, IBMX (100 microM), implicating the importance of intracellular cAMP in the regulation of this conductance. Furthermore, this conductance exhibited voltage-dependent inhibition in the presence of the CFTR chloride channel blocker glibenclamide (250 microM), but was DIDS insensitive (500 microM). Consistent with the presence of a CFTR-mediated chloride conductance, the expression of CFTR-mRNA was detected using RT-PCR. Sequence analysis of the product revealed 99.4% homology to that described for rabbit CFTR. CONCLUSIONS: In tRCE cells, there is gene expression and functional CFTR activity. Its presence may have important therapeutic implications in corneal epithelial diseases resulting from declines in transepithelial secretory and fluid transport activity.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Adaptation by corneal epithelial cells to chronic hypertonic stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity.

We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 microm bumetanide, proliferation measured as (3)H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive (86)Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC.

Adaptation, Physiological↗
PubMed2000

Okadaic acid suppresses TPA-induced differentiation by stimulating G1/S transition in human myeloblastic leukaemia ML-1 cells.

The association between the phosphorylation status of the retinoblastoma protein, pRb and changes in cell cycle control caused by either protein kinase C (PKC) or protein kinase A (PKA) stimulation was evaluated in human myeloblastic leukaemia ML-1 cells. TPA-induced PKC activation resulted in dephosphorylation of pRb and subsequently induced ML-1 differentiation based on morphological changes and CD14 expression. In the present study, we showed that inhibition of protein phosphatases (PP-1 and PP-2a) prevented the TPA-induced differentiation in ML-1 cells. Preinhibition of PP-1 and PP-2a activities with 1-100 nM okadaic acid dose-dependently blunted the decrease in the phosphorylation status of pRb obtained with TPA and overrode cell cycle arrest. PKA stimulation with 8-chlorophenylthio-cAMP (100 microM) decreased cell proliferation by 65% and the distribution of cells in the G1 phase significantly increased from 38% to 83% concomitant with a 34% decline in the number of cells present in the S phase. In addition, PKA stimulation significantly decreased the pRb phosphorylation status but did not elicit CD14 expression, indicating that cAMP-induced dephosphorylation of pRb cannot by itself trigger differentiation in ML-1 cells.

Cell Differentiation↗
PubMed2000

Fluid transport by cultured corneal epithelial cell layers.

BACKGROUND/AIMS: Fluid transport across the in vitro corneal epithelium is short lived, hence difficult to detect and characterise. Since stable rates of fluid transport across several cultured epithelial cell layers have been demonstrated, the behaviour of confluent SV40 transformed rabbit corneal epithelial cells (tRCEC) grown on permeable supports was examined. METHODS: Fluid transport was determined with a nanoinjector volume clamp; the specific electrical resistance of the layers was 184 (SEM 9) Omega cm(2). tRCEC layers transported fluid (from basal to apical) against a pressure head of 3 cm H(2)O for 2-3 hours. RESULTS: In the first hour, the rate of fluid transport was 5.2 (0.5) microl/h/cm(-2) (n=23), which is comparable with that found in other epithelia. Fluid transport was completely inhibited in 15-30 minutes by either 100 microM ouabain (n=6), 50 microM bumetanide (n=6), or 1 microM endothelin-1 (ET-1; n=6). Preincubation with 10 microM BQ123 (an ET(A) receptor antagonist) obviated inhibition by ET-1 (n=6). ET-1 also caused a 22% decrease in specific resistance. CONCLUSIONS: Fluid transport appears to depend on transepithelial Cl(- )transport since (1) their directions are the same (stroma-->tear), and (2) both bumetanide and ouabain inhibit it with similar time course. tRCEC appear useful to investigate aspects of the physiology and pharmacology of fluid transport across this layer, including receptor mediated control of this process.

Animals↗
PubMed2000

Inhibitory effect of PGE2 on EGF-induced MAP kinase activity and rabbit corneal epithelial proliferation.

PURPOSE: To determine in rabbit corneal epithelial cells in culture whether epidermal growth factor (EGF)-induced increases in prostaglandin (PG) E2 production inhibit both the extracellular signal-regulated kinase 2 (Erk-2), a mitogen-activated protein kinase (MAPK), cascade activation, and the mitogenic response to this growth factor. METHODS: Serum starvation for 24 to 36 hours was used to synchronize cultures of SV40-transformed rabbit corneal epithelial (RCE) cells. The effects of exogenous PGE2, inhibition of PGE2 synthesis, and modulation of protein kinase A (PKA) activity on EGF-induced Erk-2 activation were assessed by immunoprecipitation, kinase assays, and Western blot analysis. PGE2 synthesis was measured by using enzyme-linked immunosorbent assay. [3H]-Thymidine incorporation was used to measure RCE cell proliferation rates. RESULTS: EGF (5 ng/ml) significantly increased PGE2 production in a time-dependent manner up to 94%+/-8% after 3 hours. EGF-induced PGE2 production was suppressed by AACOCF3, a phospholipase A2 (cPLA2) inhibitor. EGF-induced Erk-2 activation reached a maximal level at 15 minutes, followed by a decline toward the control level after 3 hours. In the presence of either PGE2 (50 microg/ml) or 8-CPT-cAMP (100 microM), the EGF-induced Erk-2 activation was lessened. PKA was activated by applications of EGF or PGE2 and suppressed by AACOCF3. On the other hand, either inhibition of PGE2 production with AACOCF3 or H-89, a PKA inhibitor, enhanced EGF-induced Erk-2 activity. Raf-1 activity was stimulated by EGF to maximal activity at 5 minutes and returned toward its control level after 60 minutes. As with the dependence of Erk-2 activity on PKA activity, in the presence of H-89, the EGF-induced Raf-1 activation was significantly enhanced. DNA synthesis was increased 59%+/-5% (n = 4) after EGF stimulation, indicating a mitogenic effect of EGF in RCE cells. Inhibition of cPLA2 activity with AACOCF3 increased DNA synthesis in RCE cells by another 64% relative to the effect of EGF alone. In contrast, with either PGE2 or 8-CPT-cAMP present the mitogenic response to EGF was totally suppressed. CONCLUSIONS: EGF-induced increases in PGE2 production dampened the mitogenic response to this growth factor. This suppression appears to be a consequence of PGE2-elicited increases in PKA activity, which leads to inhibition of EGF-induced activation of MAPK cascades at the level of Raf-1 and further affects downstream events including Erk-2. These results indicate that the mitogenic response to EGF in vivo in the proliferating basal cell layer may be dependent on the level of its PKA activity.

Animals↗
PubMed1999

Fluoxetine inhibits K(+) transport pathways (K(+) efflux, Na(+)-K(+)-2Cl(-) cotransport, and Na(+) pump) underlying volume regulation in corneal endothelial cells.

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K(+) efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 microM by 26%, and 50 microM totally. Fluoxetine had a triphasic effect on K(+) efflux; from 20 to 100 microM it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 microM by 25%, and 50 microM completely. Bumetanide (1 microM) inhibited RVI by 43%. In a Cl(-)-free medium, fluoxetine (50-500 microM) progressively inhibited bumetanide-insensitive K(+) influx. The inhibitions of RVI and K(+) influx induced by fluoxetine 20 to 50 microM were similar to those induced by 1 microM bumetanide and by Cl(-)-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K(+) channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K(+) efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 microM inhibits NKCC, and at 100-1000 microM inhibits the Na(+) pump. One possible explanation for these findings is that fluoxetine could interact with K(+)-selective sites in K(+) channels, the NKC cotransporter and the Na(+) pump.

Animals↗
PubMed1998

ETA receptor mediated inhibition of intracellular pH regulation in cultured bovine corneal epithelial cells.

The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na:H exchange (NHE) and vacuolar H+-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pHi). Furthermore, we characterized the effects on pHi regulation of exposure to 1 microM ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2',7' Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pHi was 7.1 in NaCl (nominally HCO3-free) Ringers. Inhibition of NHE with 100 microM dimethylamiloride (DMA) rapidly decreased pHi by 0.37 units. Similarly, selective inhibition of V-type H+-ATPase with 10 microM bafilomycin A1 decreased pHi by 0.22 units. Following acid loading in NaCl Ringers with a 20 mm NH4Cl prepulse, pHi recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 microM DMA or 20 microM bafilomycin A1. Based on decreases in H+ efflux resulting from selective inhibition of NHE and V-type H+ pump activity, NHE activity accounts for 76% of the pHi recovery following acid loading. Under control conditions, ET-1 (1 microM) had no effect on pHi whereas ET-1 completely suppressed pHi recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ETA because in the presence of BQ-123 (10 microM), a selective ETA receptor antagonist, pHi recovery was completely restored. Suppression of pHi recovery also occurred following stimulation of protein kinase C (PKC) with 10(-7) m phorbol myristate (PMA) whereas 10(-7) m 4 alpha phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pHi recovery after inhibition of PKC with 0.5 microM calphostin C suggesting that the inhibition of pHi recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca2+-dependent calmodulin stimulated CaM II kinase with KN-62 (10 microM) reversed the suppression of pHi recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 microM phenylarsine oxide, 10 nm okadaic acid, 10 microM cyclosporin A1 or 20 microM BAPTA, also obviated the suppression of pHi recovery by ET-1. Therefore ETA receptor mediated inhibition of pHi regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H+ pump. This alteration may prevent them from becoming adequately stimulated to elicit pHi recovery in response to acid loading.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release.

The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tauvr) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+]i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 microM cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 microM ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 microM ryanodine; 2.10 microM CPA and 100 microM ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.

Animals↗
PubMed1997

Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells.

PURPOSE: Bovine corneal epithelial cells (BCEC) were cultured to determine whether endothelin (ET) receptor subtype stimulation affects ET isoform expression (ET-1, ET-2, and ET-3) through capacitative Ca2+ influx. To probe in the isolated bovine corneal epithelium (BCE) for ET isoform and ET (i.e., ETA and ETB) receptor gene expression. METHODS: [Ca2+]i transients were characterized with microfluorometry. Endothelin isoform and ET receptor gene expression were probed with RNase protection analysis. Enzyme-linked immunosorbent assay was used to measure levels of ET-1-like immunoreactivity (ET-1-LI) in conditioned medium. RESULTS: ET-1 (10(-6) M) increased [Ca2+]i more than twofold. After treatment with 10(-7) M alltrans retinoic acid (an inducer of differentiation), 10(-6) M sarafotoxin (S-6-c) (a selective ETB agonist), had a similar effect. Preincubation with either 5 microM U73122 (an inhibitor of IP8 formation) or 10 microM cyclopiazonic acid, which depletes intracellular Ca2+ store content, eliminated ET agonist-mediated [Ca2+]i increases. With a nominally Ca(2+)-free solution containing 10 microM cyclopiazonic acid, simultaneous 10(-6) M ET-1 and extracellular Ca2+ additions transiently increased [Ca2+]i twofold, whereas 10(-6) M S-6-c increased it by only 20%. This augmentation was eliminated by preexposure to either BQ123 (10 microM), selective ETA receptor antagonist, U73122 (5 microM), or SKF 96365 (3 x 10(-5) M), an inhibitor of stores-operated channels. ET-1, ET-2 isoforms, and ET receptor mRNAs were identified. S-6-c (10(-6) M) increased the level of ET-1-LI after 12 hours by approximately ninefold. CONCLUSIONS: In BCEC, capacitative calcium influx is involved in mediating a positive feedback relationship between ETB receptor stimulation and ET protein expression. Identification of ET-1 and ET-2 gene expression in BCE strengthens the notion that this regulation could be autocrine mediated.

Amino Acid Sequence↗
PubMed1996

Characterization of the muscarinic receptor subtypes in the bovine corneal epithelial cells.

Muscarinic receptor subtypes in the bovine corneal epithelial cells (BCE) were characterized on the basis of their: 1) ligand binding properties, 2) linkage to Ca2+ and cAMP cell signaling pathways, and 3) gene transcripts. Receptor subtypes, m1 and m2, are indicated by competition experiments using subtype-selective muscarinic receptor ligands. [3H]N-methylscopolamine ([3H]-MS) binding was displaced with IC50s of: 1) 1 microM for the m1 antagonist, pirenzipine; 2) 51 microM for the competitive m2 antagonist, AFDX-116; 3) 100 microM for the competitive m3 antagonist, 4-DAMP. In fural2 loaded BCE, carbachol (0.001 - 100 microM) increased intracellular Ca2+ concentration ([Ca2+]i), and these responses were significantly suppressed if they were preincubated with either atropine (1 microM) or 1 microM pirenzipine. In the absence of extracellular Ca2+, these carbachol-induced increases in [Ca2+]i were depressed. A considerable fall occurred with the presence of extracellular Ca2+ and 1 microM verapamil, an L-type Ca2+ channel blocker. These responses suggest that carbachol increases Ca2+ influx through an L-type Ca2+ channel in the plasma membrane, in addition to mobilizing Ca2+ from an intracellular store. BCE also possessed muscarinic receptors which were negatively linked to cAMP production insofar as: 1) preincubation with 10 microM carbachol significantly suppressed the increases in cAMP accumulation induced by isoproterenol (1 - 25 microM); 2) this blunting effect of carbachol on cAMP production was eliminated when the BCE were preincubated with either 1 microM AFDX-116, or 100 ng/ml pertussis toxin. The results of probing for muscarinic receptor gene expression are partially consistent with the ligand binding and functional assays. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of m2 but not m1, m3 or m4 gene transcripts. In summary, we obtained pharmacological and functional evidence for m1 and m2 receptors in BCE. However, only the m2 gene transcript could be detected.

Animals↗
PubMed1996

Endothelin protein expression in tear glands of the rabbit.

PURPOSE: Endothelin-1 (ET-1) has been found to accelerate rabbit corneal epithelial wound healing in vivo and proliferation of rabbit and bovine epithelial cells in vitro. We have now determined that endothelin-like immunoreactivity (ET-LI) is present in the tear fluid of New Zealand white rabbits, and we have addressed the question of whether it is produced by the tear glands (lacrimal gland and Harderian gland). METHODS: Tears were obtained with capillary pipettes and lacrimal gland fluid was collected by cannulation. ET-LI was determined with a sandwich-enzyme immunoassay. The reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine if there are gene transcripts for prepro ET-1 in tear glands. ET-LI in the tear glands was evaluated using immunohistochemistry. RESULTS: The tears contained 13.85 +/- 3.94 pg/ml (mean +/- SEM, n = 6) ET-LI and the lacrimal gland fluid had 24.90 +/- 6.00 pg/ml (n = 6). The plasma contained 0.89 +/- 0.20 pg/ml (n = 6). Gene transcripts were identified for prepro ET-1 in the lacrimal and Harderian glands. Specific staining for ET-LI was found in the epithelial cells of the ducts of the lacrimal gland and the acinar cells of the Harderian gland. CONCLUSIONS: The higher concentrations of ET-LI in the tears and the lacrimal gland fluid than in plasma along with prepro ET-1 gene transcripts in the tear glands suggest that they secrete ET-1.

Animals↗