Mechanisms of Ion Transport Across the Mouse Retinal Pigment Epithelium Measured In Vitro (original) (raw)
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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...
Phorbol ester modulation of active ion transport across the rabbit conjunctival epithelium
Experimental eye …, 1999
Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial transporters. The present work examined the influence of phorbol esters, which are known activators of PKC isoenzymes, on the short-circuit current (I sc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the I sc measures a Na +-dependent, bumetanide-inhibitable Cl − transport in the basolateral-to-apical direction plus an amiloride-resistant Na + absorptive process in the opposite direction. Additions of phorbol 12-myristate-13-acetate (PMA) to the basolateral bathing media did not affect the transepithelial electrical parameters ; but its introduction to the apical bath at 1 and 10 µ elicited a transient ($ 2 min duration) I sc spike followed by a sustained reduction relative to the control level. Such PMA-elicited I sc reductions were from 14n0p2n0 to 3n1p0n8 µA cm −# (p...'s, n l 3) at 1 µ and from 16n5p1n9 to 4n6p0n7 µA cm −# (n l 22) at 10 µ. The former concentration failed to produce extensive I sc reductions in 3 other experiments. Similar results were obtained with phorbol 12,13-dibutyrate (PDBu). Its apical administration at 0n1 µ reduced the I sc from 18n5p4n1 to 7n8p2n0 (n l 3), and from 16n5p2n9 to 6n9p1n2 (n l 7) when introduced at 1 µ. The phorbol-evoked I sc reductions occurred without a simultaneous change in transepithelial resistance (R t). However, after about 15-20 min, R t gradually declined by about 25 %. In contrast to these results, treatment with a phorbol ester known not to activate PKC (4-α-PMA) did not affect the electrical parameters when added at 10 µ. PMA-and PDBu-evoked I sc reductions could be obtained with conjunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl −-free media, and (3) pretreated with amphotericin B, changes consistent with a likely inhibition of the basolateral Na + \K + pump. Such I sc inhibitions were attenuated with conjunctiva pre-exposed to 1 µ staurosporine, a nonselective kinase inhibitor known to suppress PKC activity. Staurosporine, in itself, produced a rapid 26 % I sc inhibition (n l 15) along with a 17% R t increase upon its apical introduction. These electrical responses were less extensive in Cl −-free media and absent in amphotericin B-treated conjunctiva, suggesting the presence of a kinase-mediated regulation of apical channels for both Na + and Cl −. Overall, these results imply that in addition to previously demonstrated epinephrine-elicited, up-regulation of Cl − secretion, mechanisms may also exist, via PKC activation, to suppress Na + \K + pumping and consequently reduce transepithelial transport rates.
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...
J Gen Physiol, 1990
Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (-30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K +] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K +] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, CI reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),CI cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and C1, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and CI in the ratio of 1:1:2.
The Journal of General Physiology, 1990
Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (-30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K +] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K +] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, CI reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),CI cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and C1, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and CI in the ratio of 1:1:2.
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
Ion transport across the epithelium of the rabbit caecum
Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1992
The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36C1 and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/CI-symport in the apical barrier.
Retinal pigment epithelial transport mechanisms and their contributions to the electroretinogram
Progress in Retinal and Eye Research, 1997
The translocation of ions, fluid and macromolecules across epithelia is made possible by the asymmetric distribution of transport proteins, enzymes and receptors in two physically distinct plasma membrane domains that form the apical and basolateral sides of the cell. Each side faces a different extracellular environment. In the back of the vertebrate eye, the retinal pigment epithelium (RPE) apical membrane receives a continuous stream of paracrine signals that are generated by a variety of retinal neurons in the light and dark. These signals help regulate RPE function, and conversely, alterations in RPE function can modify the activity of retinal neurons. At the basolateral surface, there is a continual exchange of nutrienl:s and waste products, along with a flow of hormonal signals from the choroidal blood supply, all of which serve to maintain the health and integrity of the distal retina and in particular, the photoreceptors. This review provides an integrated summary of the apical and basolateral membrane and intracellular signaling mechanisms that mediate the vectorial traffic of ions and fluid across the RPE. These same mechanisms help regulate the chemical milieu within the cell and in the extracellular spaces that surround the cell. They also generate specific components of the electrical signals that are recorded clinically across the intact human eye, the electroretinogram (ERG) and the electrooculogram (EOG). The last part of this review is focused on the light-induced photoreceptor-dependent decrease in subretinal potassium concentration ([K]o) that occurs in the intact eye and serves as a paracrine signal for the RPE. This signal plays a central role in regulating RPE physiology and in mediating retina/RPE interactions, following transitions between light and dark; it is mirrdcked in vitro by a small (3 mM) change in [K]o on the apical side of the epithelium. The clinical implications are discussed in terms of the transport mechanisms that regulate hydration of the subretinal space and that potentially mediate fluid absorption out of the retina.