Cyclic AMP modulation of ion transport across frog retinal pigment epithelium. Measurements in the short-circuit state (original) (raw)
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The Journal of general …, 1984
A modified version of a capacitance probe technique has been used to measure fluid transport across the isolated retinal pigment epithelium (RPE)-choroid of the bullfrog. The accuracy of this measurement is 0 .5-1 .0 nl/ min. Experiments carried out in the absence of external osmotic or hydrostatic gradients show that the RPE-choroid transports fluid from the retinal to the choroid side of the tissue at a rate of-10 nl/min (4-6 ul/cm' .h). Net fluid absorption (.Jv) was abolished within 10 min by the mitochondrial uncoupler 2,4-dinitrophenol. It was also inhibited (70%) by the removal of bicarbonate from the bulk solutions bathing the tissue. Ouabain caused a slow decrease in ,Jv (no effect at 10 min, 70% at 3 h), which indicates that RPE fluid transport is not directly coupled to the activity of the Na-K pump located at the apical membrane of this epithelium. In contrast to ouabain, cyclic AMP (cAMP) produced a quick decrease in Jv (84% within 5 min). Radioisotope experiments in the open circuit show that cAMP stimulated secretory fluxes of Na and Cl, which accounted for the observed cAMP-induced decrease in Jv. The direction of net fluid absorption, the magnitudes of the net ionic fluxes in the open circuit, and the dependence ofJ v on external bicarbonate concentration strongly suggest that fluid absorption is generated primarily by the active absorption of bicarbonate .
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-
The electrogenic sodium pump of the frog retinal pigment epithelium
The Journal of Membrane Biology, 1978
It was previously shown that ouabain decreases the potential difference across an in vitro preparation of bullfrog retinal pigment epithelium (RPE) when applied to the apical, but not the basal, membrane and that the net basal-to-apical Na + transport is also inhibited by apical ouabain. This suggested the presence of a Na+-K § pump on the apical membrane of the RPE. In the present experiments, intracellular recordings from RPE cells show that this pump is electrogenic and contributes approximately-10 mV to the apical membrane potential (VAp). Apical ouabain depolarized Vae in two phases. The initial, fast phase was due to the removal of the direct, electrogenic component. In the first one minute of the response to ouabain, VAe depolarized at an average rate of 4.4 +0.42 mV/min (n= 10, mean • and VAe depolarized an average of 9.6_+0.5 mV during the entire fast phase. A slow phase of membrane depolarization, due to ionic gradients running down across both membranes, continued for hours at a much slower rate, 0.4 mV/min. Using a simple diffusion model and K+-specific microelectrodes, it was possible to infer that the onset of the ouabain-induced depolarization coincided with the arrival of ouabain molecules at the apical membrane. This result must occur if ouabain affects an electrogenic pump. Other metabolic inhibitors, such as DNP and cold, also produced a fast depolarization of the apical membrane. For a decrease in temperature of-~10~ the average depolarization of the apical membrane was 7.1 _+3.4mV (n =5) and the average decrease in transepithelial potential was 3.9 +_0.3 mV (n= 10). These changes in potential were much larger than could be explained by the effect of temperature on an RT/F electrodiffusion factor. Cooling the tissue inhibited the same mechanism as ouabain, since prior exposure to ouabain greatly reduced the magnitude of the cold effect. Bathing the tissue in 0 mM [K +] solution for 2 hr inhibited the electrogenic pump, and subsequent re-introduction of 2mM [K +] solution produced a rapid membrane hyperpolarization. We conclude that the electrogenic nature of this pump is important to retinal function, since its contribution to the apical membrane potential is likely to affect the transport of ions, metabolites, and fluid across the RPE.
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.
Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium
The Journal of Membrane Biology, 1978
We have studied the hyperpolarizing, electrogenic pump located on the apical membrane of the retinal pigment epithelium (RPE) in an in vitro preparation of bullfrog RPE-choroid. Changes in RPE [K+]i alter the current produced by this pump. Increasing [K+]o in the solution perfusing the basal membrane increases RPE [K+]~ (measured with a K+-specific microelectrode), and also depolarizes the apical membrane. This depolarization is due to a decrease in electrogenic pump current flowing across the apical membrane resistance, since it is abolished when the pump is inhibited by apical ouabain, by cooling the tissue, or by 0 mM [K+]o outside the apical membrane. Removal of C1-from the solution perfusing the basal membrane abolishes the K § apical depolarization by preventing the entry of K § (as KC1) into the cell. We conclude that the increase in [K+]~ causes the decrease in pump current. This result is consistent with the finding that [K+]~ is a competitive inhibitor of the Na §-K + pump in red blood cells. It is possible that the light-evoked changes in [K+]o in the distal retina could alter RPE [K+]i, and thus could affect the pump from both sides of the apical membrane. Any change in pump current is likely to influence retinal function, since this pump helps to determine the composition of the photoreceptor extracellular space.
Investigative Ophthalmology & Visual Science, 2006
PURPOSE. To investigate the effects of 8-bromo-cAMP (cAMP) on porcine ciliary transepithelial short-circuit current (Isc) and transport of chloride (Cl Ϫ) and sodium (Na ϩ). METHODS. With Ussing-type chambers, cAMP-induced changes in Isc, electrical resistance (ER), and transepithelial 36 Cl Ϫ and 22 Na ϩ fluxes were measured. Drugs were applied to the nonpigmented epithelium (NPE) and/or pigmented epithelium (PE) side(s). The effect of IBMX (1, 5, or 10 M; 3-isobutyl-1methylxanthine) on Isc-increase induced by 8-bromo-cAMP on the PE side was also tested. RESULTS. On the NPE side, a single concentration (10 M, 100 M, or 1 mM) of 8-bromo-cAMP induced a biphasic (transient peak followed by sustained plateau) Isc increase. On the PE side, 8-bromo-cAMP induced a similar but delayed biphasic Isc increase at 1 mM, a slight plateau-Isc increase at 100 M, and no Isc increase at 10 M. In the concentration-response curve, the cAMP-induced peak-Isc increase became significant at a concentration 10,000 times lower on the NPE than on the PE side. At 10 M, the cumulative cAMP-induced Isc-increase reached its maximum on the NPE side, but was virtually nonexistent on the PE side. IBMX (a phosphodiesterase inhibitor) but not 8-CPT-6-Phe-cAMP (higher permeability than 8-bromo-cAMP) significantly increased the peak-Isc concentration-response curve induced by 8-bromo-cAMP (10 nM-1 mM) on the PE side. On the NPE but not the PE side, 10 M 8-bromo-cAMP induced a significant but transient increase in net PE-to-NPE 36 Cl Ϫ flux (1.03 Ϯ 0.18 Eq/min per square centimeter; P Ͻ 0.001). Neither ER nor transepithelial 22 Na ϩ flux was changed after cAMP exposure. CONCLUSIONS. In porcine ciliary processes, apparently on the NPE side, cAMP triggers a biphasic (transient peak followed by a sustained plateau) Isc increase. Only the peak-Isc increase involves an increase in net PE-to-NPE Cl Ϫ transport. (Invest
Cyclic AMP-dependent Stimulation of Basolateral K+Conductance in the Rabbit Conjunctival Epithelium
Experimental Eye Research, 2000
The regulation of Na + transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl − secretion and Na + absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl − free media. In this situation, the short-circuit current (I sc ) measures an amiloride-resistant Na + absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 µ) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na + -dependent I sc by $ 3n5-4n5 µA cm −# (minimal 40 % increase) and reduced transepithelial resistance (R t ) by at least 7 % (P 0n05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 µ), which in itself inhibited the I sc by 0n5 µA cm −# , attenuated the I sc responses of the cAMP-elevating agents (P 0n05, unpaired data). In reverse, H-89 promptly decreased the I sc by 1n5-2n5 µA cm −# and increased R t by 5 % (P 0n05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the I sc by 12 and 22 % respectively over baseline and reduced R t by 6 % (P 0n05). Similarly, in the presence of a transepithelial K + gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1n8 µA cm −# and decreased the R t by 4 % (P 0n05), changes that were reversed by subsequent H-89 addition. Under Cl − rich conditions, pretreatment with 5 m Ba# + reduced the basal I sc by 59 % and blocked the cAMP-induced I sc stimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba# + -inhibitable (voltage insensitive) basolateral K + conductance in rabbit conjunctival epithelial cells. The action of Cl − secretogogues acting via cAMP on basolateral K + channel activity indicates that endogenous levels of cAMP may play a role in the regulation of Cl − secretion and Na + absorption in the conjunctiva.
Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium
American Journal of Physiology-Cell Physiology, 1995
Intracellular microelectrode techniques were employed to characterize the blocker sensitivity of the K+ conductance (gK) at the apical membrane of the toad retinal pigment epithelium (RPE). Increasing the K+ concentration in the apical bath ([K+]o) from 2 to 5 mM produced a rapid depolarization of the apical membrane potential (VA). The addition of 0.5 mM Ba2+ or 5 mM Cs+ to the apical bath rapidly depolarized VA and increased the transepithelial resistance and ratio of apical-to-basolateral membrane resistance. In the presence of apical Ba2+ or Cs+, the response of VA to delta [K+]o was markedly reduced, indicating that these ions are effective blockers of apical gK. The Ba(2+)- and Cs(+)-induced decreases in the apparent apical-to-basolateral membrane conductance ratio were concentration dependent, with apparent dissociation constants of 17 microM and 0.5 mM, respectively. The apparent blocker sensitivity of apical gK is similar to that previously demonstrated for the inwardly rec...
Passive ionic properties of frog retinal pigment epithelium
The Journal of Membrane Biology, 1977
The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was-88inV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8-20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductance T~ of each membrane. For the apical membrane: TK~0.52, Tnco3_~0.39 and TNa-~0.05 , and its specific resistance was estimated to be 6000-7000f~ cm 2. For the basal membrane: TK~0.90 and its specific resistance was estimated to be 400-1200~ cm 2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at ll0mM. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered "tight". The shunt pathway did not distinguish between anions (HCOi, C1-, methylsulfate, isethionate) but did distinguish between Na + and K-. The pigment epithelial cells and the photoreceptors are closely associated in the vertebrate retina. Apical processes from the pigment epithelial cells lie alongside and, in some cases, surround the photoreceptor outer segments. The epithelium also separates the sensory retina from its choroidal blood supply and transports salts and metabolites to and from the retina (