Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium (original) (raw)
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Potentiation of ATP-induced Ca2+ mobilisation in human retinal pigment epithelial cells
Interaction of signalling pathways directs the functional output of many cells. This study investigated the consequences of activating adenosine and adrenergic receptors on ATP-induced Ca2+ responses in human retinal pigment epithelial (RPE) cells. Intracellular Ca2+ concentration ([Ca2+]i) of human RPE cells in primary culture was monitored using Fura-2. Cyclic adenosine monophosphate (cAMP) concentration was measured using an enzyme-linked immunosorbent assay. Both ATP and UTP (10 μm) increased [Ca2+]i in human RPE cells. Adenosine (10 nm–10 μm) had no effect on resting [Ca2+]i, but potentiated a sub-threshold response to ATP (100 nm) when ATP was added in the presence of adenosine. The potentiation occurred with other G-protein receptor agonists such as acetylcholine. Potentiation persisted in Ca-free medium, but was blocked by prior application of thapsigargin. The A1 and A2 adenosine receptor antagonists, DPCPX and MRS1706 (100 nm) respectively, inhibited potentiation in 76±7 and 23±12% of cells, respectively, but the A3 antagonist MRS1191 had no effect. Conversely, agents that activate the cAMP pathway, including isoproterenol (10 μm), forskolin (10 μm), and the protein kinase A (PKA) activator Sp-cBIMPS (1 μm), potentiated the ATP-induced response in the RPE cells. Agents that are known to inhibit the production of cAMP in other systems also caused potentiation, including clonidine (10 μm) and the Gi-activator mastoparan (10 μm). Under resting conditions, cAMP concentration in RPE cells was 7·1±0·5 pmol mg−1 protein. Isoproterenol (10 μm) and forskolin (10 μm) increased levels to 104·6±5·2 and 113·7±4·2 pmol mg−1 protein, respectively, while adenosine, clonidine, and mastoparan (all 10 μm) had no significant effect on cAMP levels. These data indicate that whilst activation of A1 and A2 adenosine receptors and α2 and β adrenergic receptors does not influence basal Ca2+ levels, stimulation of these receptors can potentiate Ca2+ signalling by cAMP dependent and independent mechanisms in human RPE cells.
The Journal of Physiology, 1999
The retinal pigment epithelium (RPE) carries out a number of roles that are essential for the maintenance and viability of the neurosensory retina. These roles include phagocytosis of shed rod and cone outer segments, melanin synthesis and recycling and regulation of subretinal volume via ioncoupled fluid absorption (Steinberg & Miller, 1979; Zinn & Benjamin-Henkind, 1979; Clark, 1986). In order to carry out these diverse functions, the RPE must be able to detect and respond to paracrine signals coming from the adjacent choroidal andÏor neural retinal tissue andÏor via systemic sources. A number of metabotropic receptors have been identified on the RPE including those for dopamine, acetylcholine, adrenaline (epinephrine) and adenosine (Friedman et al. 1988; Dearry et al. 1990; Frambach et al. 1990). Activation of these receptors by their respective signalling molecules has been linked to changes in light-evoked responses (Dearry et al. 1990; Gallemore & Steinberg, 1990), phagocytic ability (Gregory et al. 1994) and ion and fluid transport across the RPE (Edelman & Miller, 1991; Joseph & Miller, 1992). Recently, in monolayers of bovine and rat RPE, extracellular adenosine 5'-triphosphate (ATP) and uridine triphosphate (UTP) were demonstrated to induce changes in intracellular Ca¥ and transepithelial ion and fluid movement (Stalmans & Himpens, 1997; Peterson et al. 1997). A role for intracellular ATP has also recently been demonstrated for the activation of a delayed inwardly rectifying K¤ current (IK(IR)) in isolated bovine RPE cells (Hughes & Takahira, 1998). These findings support the presence of metabotropic purinoceptors and suggest that ATP may act as an important paracrine signal in the RPE. Purinoceptors are divided into two main classes, P1 and P2, based on their selectivity for adenosine and ATP, respectively (Burnstock & Kennedy, 1985). Adenosine or P1
PloS one, 2018
Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the e...
The Journal of General Physiology, 1984
In the frog retinal pigment epithelium (RPE), the cellular levels of cyclic AMP (cAMP) were measured in control conditions and after treatment with substances that are known to inhibit phosphodiesterase (PDE) activity (isobutyl-1-methylxanthine, SQ65442) or stimulate adenylate cyclase activity (forskolin). The cAMP levels were elevated by a factor of 5-7 compared with the controls in PDE-treated tissues and by a factor of 18 in forskolin-treated tissues. The exogenous application of cAMP (1 mM), PDE inhibitors (0.5 mM), or forskolin (0.1 mM) all produced similar changes in epithelial electrical parameters, such as transepithelial potential (TEP) and resistance (Rt), as well as changes in active ion transport. Adding 1 mM cAMP to the solution bathing the apical membrane transiently increased the short-circuit current (SCC) and the TEP (apical side positive) and decreased Rt. Microelectrode experiments showed that the elevation in TEP is due mainly to a depolarization of the basal mem...
Biophysical Journal, 1995
Membrane current and light response were recorded from rods of monkey and guinea pig by means of suction electrodes. The correlation between adaptation and the Na+/K+ pump was investigated by measuring light-dependent changes in sensitivity with and without inhibition of Na+/K+ ATPase by strophanthidin. Strophanthidin was found to reduce the dark current, to slow the time course of the photoresponse, and to increase light sensitivity. At concentrations between 20 and 500 nM, the pump inhibitor suppressed in a reversible way the current re-activation occurring during prolonged illumination and modified the light-dependent decrease in sensitivity, which in control conditions approximates to a Weber-Fechner function. The effects of the pump inhibitor on the adaptive properties of rods are associated with an increased time constant of the membrane current attributed to the operation of the Na+:Ca2+,K+ exchanger. The effects of rapid application of the pump inhibitor on the current re-activation are consistent with the idea that significant changes in the internal sodium occur in rods of mammals during background illumination and that they play an important role in the process of light adaptation.
Fluid transport across retinal pigment epithelium is inhibited by cyclic AMP
Proceedings of the National Academy of Sciences, 1982
Fluid transport across the retinal pigment epithelium (bullfrog) has been measured. These experiments were carried out by using a capacitance probe technique and a waterimpermeable chamber that allowed the measurements to be made with an accuracy of 0.5-1.0 nl/min. With identical Ringer's solution on both sides of the epithelium, and in the absence of a hydrostatic driving force, the direction ofnet fluid movement is from the retina to the choroid (absorption). The net transport rate, approximately 10 nl/min (4.8 jl/cm2'hr), is comparable to that ob-
Calcium pump in the disk membranes isolated from bovine retinal rod outer segments
Journal of Photochemistry and Photobiology B: Biology, 1994
The existence of a Ca'+ pump in rod outer segment disks of bovine retina is strongly suggested by the isolation on sodium dodecyl sulfate polyacrylamide gel electrophoresis of a hydrovlamine-sensitive phosphorylated intermediate (E-P) of molecular mass of about 100 kDa as well as by measurements of active calcium transport and adenosine 5'-triphosphate (ATP) hydrolysis.
Mechanisms of Ion Transport Across the Mouse Retinal Pigment Epithelium Measured In Vitro
Investigative Opthalmology & Visual Science, 2020
To examine ion transport across the mouse retinal pigment epithelium (RPE), measured by the short-circuit current (I SC) and transepithelial resistance (TER). METHODS. Sheets of RPE from mice (C57BL6/J) with retina, choroid, and sclera attached were mounted in Ussing chambers (0.031-cm 2 aperture) and Krebs solution. The I SC and TER were recorded with voltage clamps. Receptors implicated in ion transport were blocked or stimulated by ligands applied to both sides. RESULTS. The mean initial I SC was −12.0 ± 3.9 μA/cm 2 (basolateral negative), and mean TER was 67.1 ± 8.0 ohm•cm 2. RPE preparations remained stable for 3 hours, with I SC decreasing by 0.078 ± 0,033 μA/cm 2 /hr. Adenosine triphosphate (100 μM) increased I SC by 2.22 ± 0.41 μA/cm 2 (P = 0.003). Epinephrine (100 μM) increased I SC by 1.14 ± 0.19 μA/cm 2 (P = 0.011). Bumetanide (100 μM) reduced I SC by 1.72 ± 0.73 μA/cm 2 (P = 0.027). Ouabain (1 mM) induced a biphasic response: an I SC increase from −7.9 ± 2.4 to −15.49 ± 2.12 μA/cm 2 and then a decrease to −3.7 ± 2.2 μA/cm 2. Ouabain increased TER by 15.3 ± 4.8 ohm•cm 2. These compounds were added sequentially. Apical [K + ] o at zero mM transiently increased I SC by 3.36 ± 1.06 μA/cm 2. Ba ++ decreased I SC from −10.4 ± 3.1 to −6.6 ± 1.8 μA/cm 2 (P = 0.01). Ba ++ reversed the K +-free response, with I sc decreasing further from −5.65 ± 1.24 to −3.37 ± 0.79 μA/cm 2 (P = 0.029). CONCLUSIONS. The I SC and TER can be recorded from the mouse RPE for 3 hours. Adrenergic and purinergic receptors affect murine RPE ion transport. Sodium-potassium adenosine triphosphatase plays a role in net ion transport across mouse RPE, and Na-K-2Cl cotransporter activity partly accounts for transepithelial ion transport. Mimicking lightinduced changes, low subretinal [K + ] o increases ion transport transiently, dependent on K + channels.
Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium
The Journal of General Physiology, 1992
Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ iono...