Xiurong Sun

Connections Between Connexins, Calcium, and Cataracts in the Lens

There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was ∼0.5 S/cm2 of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was ∼1.0 S/cm2 and calcium varied from ∼500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to ∼2 μM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of Cx46 causes loss of coupling of mature fiber cells; the efflux path for calcium is therefore blocked; calcium accumulates in the central cells; at concentrations above ∼1 μM (from the center to about half way out of a 3-wk-old lens) Lp82 is activated; Lp82 cleaves cytoplasmic proteins (crystallins) in central cells; and the cleaved proteins aggregate and scatter light.

Isoform-specific function and distribution of Na/K pumps in the frog lens epithelium.

Abstract. Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (IP), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-IP blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, IP in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole.

Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na+ that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens’ circulation of Na+ was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.

α‐Adrenergic effects on Na+‐K+ pump current in guinea‐pig ventricular myocytes

1 The whole‐cell patch clamp was employed to study Na+‐K+ pump current (Ip) in acutely isolated myocytes. α‐Adrenergic receptors were activated with noradrenaline (NA) after blocking β‐adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). 2 Activation of α‐receptors by NA increased Ip in a concentration‐dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nm[Ca2+]i the K0.5 was 219 nm NA whereas at 1.4 μm [Ca2+]i it was 3 nm. 3 The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14‐15 %. 4 Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the α‐receptor‐mediated stimulation of Ip at either high or low[Ca2+]i. 5 The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str. 6 Phenylephrine, methoxamine and metaraminol, three selective α1‐adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an α1‐antagonist, but was unaffected by yohimbine, an α2‐antagonist. 7 We conclude noradrenaline activates ventricular α1‐receptors, which are specifically coupled via PKC to increase Na+‐K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.

Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart

Abstract We have previously shown activation of α1-adrenergic receptors increases Na+-K+ pump current (Ip) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on Ip. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased IP at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K0.5) was 6 µM at 15 nM cytosolic [Ca2+] ([Ca2+]i) and13 nM at 314 nM [Ca2+]i. The maximal increase at either [Ca2+]i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC8), increased Ip similarly. The effect of PMA on IP was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α1-adrenergic agonist effects both were sensitive to [Ca2+]i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased Ip uniformly at all voltages. However, they differed in that α1-activation caused a maximum increase of 15% vs 30% via PMA, and α1-effects were less sensitive to [Ca2+]i than PMA effects. These results demonstrate that activation of PKC causes an increase in Ip in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α1-adrenergic activation to Ip is entirely through PKC, however α1-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.

The effects of age on lens transport.

PURPOSE Age-related nuclear cataracts involve denaturation and aggregation of intracellular proteins. We have documented age-dependent changes in membrane transport in the mouse lens to see what might initiate changes in the intracellular milieu. METHODS Microelectrode-based intracellular impedance studies of intact lenses were used to determine gap junction coupling conductance, fiber and surface cell membrane conductances, effective extracellular resistivity, and intracellular voltage. Fiber cell connexin expression was detected by Western blotting. Intracellular hydrostatic pressure was measured with a microelectrode/manometer system. Concentrations of intracellular sodium and calcium were measured by intracellular injection of sodium-binding benzofuran isophthalate and Fura2, respectively. RESULTS In adult lenses, as age increased: fiber cell gap junction coupling conductance declined significantly, correlating with decreases in Cx46 and Cx50 labeling in Western blots; fiber and surface cell membrane conductances did not change systematically; effective extracellular resistivity increased monotonically; center to surface gradients for intracellular pressure, sodium, calcium, and voltage all increased, but in an interdependent manner that moderated changes. In newborn pup lenses, there were changes that did not simply fit with the above paradigm. CONCLUSIONS In newborn pup lenses, the observed changes may relate to growth factors that are not related to age-dependent changes seen in adult lenses. The major change in adult lenses was an age-dependent decrease in gap junction coupling, probably due to oxidative damage leading to degradation of connexin proteins. These changes clearly lead to compromise of intracellular homeostasis and may be a causal factor in age-related nuclear cataracts.

Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

Autocrine A2 in the T-System of Ventricular Myocytes Creates Transmural Gradients in Ion Transport: A Mechanism to Match Contraction with Load?

Transmural heterogeneities in Na/K pump current (IP), transient outward K(+)-current (Ito), and Ca(2+)-current (ICaL) play an important role in regulating electrical and contractile activities in the ventricular myocardium. Prior studies indicated angiotensin II (A2) may determine the transmural gradient in Ito, but the effects of A2 on IP and ICaL were unknown. In this study, myocytes were isolated from five muscle layers between epicardium and endocardium. We found a monotonic gradient in both Ip and Ito, with the lowest currents in ENDO. When AT1Rs were inhibited, EPI currents were unaffected, but ENDO currents increased, suggesting endogenous extracellular A2 inhibits both currents in ENDO. IP- and Ito-inhibition by A2 yielded essentially the same K0.5 values, so they may both be regulated by the same mechanism. A2/AT1R-mediated inhibition of IP or Ito or stimulation of ICaL persisted for hours in isolated myocytes, suggesting continuous autocrine secretion of A2 into a restricted diffusion compartment, like the T-system. Detubulation brought EPI IP to its low ENDO value and eliminated A2 sensitivity, so the T-system lumen may indeed be the restricted diffusion compartment. These studies showed that 33-50% of IP, 57-65% of Ito, and a significant fraction of ICaL reside in T-tubule membranes where they are transmurally regulated by autocrine secretion of A2 into the T-system lumen and activation of AT1Rs. Increased AT1R activation regulates each of these currents in a direction expected to increase contractility. Endogenous A2 activation of AT1Rs increases monotonically from EPI to ENDO in a manner similar to reported increases in passive tension when the ventricular chamber fills with blood. We therefore hypothesize load is the signal that regulates A2-activation of AT1Rs, which create a contractile gradient that matches the gradient in load.

Aquaporin 0 Modulates Lens Gap Junctions in the Presence of Lens-Specific Beaded Filament Proteins

Purpose The objective of this study was to understand the molecular and physiologic mechanisms behind the lens cataract differences in Aquaporin 0-knockout-Heterozygous (AQP0-Htz) mice developed in C57 and FVB (lacks beaded filaments [BFs]) strains. Methods Lens transparency was studied using dark field light microscopy. Water permeability (Pf) was measured in fiber cell membrane vesicles. Western blotting/immunostaining was performed to verify expression of BF proteins and connexins. Microelectrode-based intact lens intracellular impedance was measured to determine gap junction (GJ) coupling resistance. Lens intracellular hydrostatic pressure (HP) was determined using a microelectrode/manometer system. Results Lens opacity and spherical aberration were more distinct in AQP0-Htz lenses from FVB than C57 strains. In either background, compared to wild type (WT), AQP0-Htz lenses showed decreased Pf (approximately 50%), which was restored by transgenic expression of AQP1 (TgAQP1/AQP0-Htz), but the opacities and differences between FVB and C57 persisted. Western blotting revealed no change in connexin expression levels. However, in C57 AQP0-Htz and TgAQP1/AQP0-Htz lenses, GJ coupling resistance decreased approximately 2.8-fold and the HP gradient decreased approximately 1.9-fold. Increased Pf in TgAQP1/AQP0-Htz did not alter GJ coupling resistance or HP. Conclusions In C57 AQP0-Htz lenses, GJ coupling resistance decreased. HP reduction was smaller than the coupling resistance reduction, a reflection of an increase in fluid circulation, which is one reason for the less severe cataract in C57 than FVB. Overall, our results suggest that AQP0 modulates GJs in the presence of BF proteins to maintain lens transparency and homeostasis.