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S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4(−/−) knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4(−/−) mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4(−/−) lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4(−/−) knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1−/− and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1−/− lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1−/− cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (−/− cells. HP recovery was shown to be absent in TRPV1−/− lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1−/− mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (–/–) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2–/– peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5–/– and EphA2–/– inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

Glutathione peroxidase 1 (GPX1) and catalase are expressed in the lens epithelial cells and cortical fiber cells, where they detoxify H2O2 to reduce oxidative stress, which is a major cause for cataractogenesis. We sought to find out, between these two enzymes, which is critical for transparency and homeostasis in the aging lens by investigating alterations in the lens's refractive property, transparency, and gap junction coupling (GJC) resistance.Wild-type (C57BL/6J), GPX1 knockout (GPX1In contrast to wild-type (WT) and CATChanges in the refractive and physiological properties of the lens occurred before cataract formation in GPX1

Purpose Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation. Methods Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca2+]i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images. Results In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca2+]i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts. Conclusions Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

Purpose Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient. Methods We measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure. Results Ciliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K. Conclusions These results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

Gap junction-mediated intercellular communication facilitates the circulation of ions, small molecules, and metabolites in the avascular eye lens. Mutants of the lens fiber cell gap junction proteins, connexin46 (Cx46) and connexin50 (Cx50), cause cataracts in people and in mice. Studies in mouse models have begun to elucidate the mechanisms by which these mutants lead to cataracts. The expression of the dominant mutants causes severe decreases in connexin levels, reducing the gap junctional communication between lens fiber cells and compromising the lens circulation. The impairment of the lens circulation results in several changes, including the accumulation of Ca2+ in central lens regions, leading to the formation of precipitates that stain with Alizarin red. The cataract morphology and the distribution of Alizarin red-stained material are similar, suggesting that the cataracts result from biomineralization within the organ. In this review, we suggest that this may be a general process for the formation of cataracts of different etiologies.

Purpose The lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels. One purpose of the current study was to identify the connexin(s) in the hemichannels. Negative pressures are detected by TRPV1, which initiates a cascade that increases surface osmolarity. The increase in osmolarity was initially reported to be through inhibition of Na/K ATPase activity, but a recent study reported it was through stimulation of Na/K/2Cl (NKCC) cotransport. A second purpose of this study was to reconcile these two reports. Methods Intracellular hydrostatic pressures were measured using a microelectrode/manometer system. Lenses from TRPV1 or Cx50 null mice were studied. Specific inhibitors of Cx50 gap junction channels, NKCC, and Akt were used. Results Either knockout of Cx50 or blockade of Cx50 channels completely eliminated the response to positive surface pressures. Knockout of Cx50 also caused a positive drift in surface pressure. The short-term (∼20-minute) response to negative surface pressures was eliminated by blockade of NKCC, but a long-term (∼4-hour) response restored pressure to zero. Both short- and long-term responses were eliminated by knockout of TRPV1 or inhibition of Akt. Conclusions Hemichannels made from Cx50 are required for the response to positive surface pressures. Negative surface pressures first activate NKCC, but a backup system is inhibition of Na/K ATPase activity. Both responses are initiated by TRPV1 and go through PI3K/Akt before branching.

The lens is an avascular organ whose function and survival depend on an internal circulation system. Cx46fs380 mice model a human autosomal dominant cataract caused by a mutant lens connexin. In these mice, fiber cell connexin levels and gap junction coupling are severely decreased. The present studies were conducted to examine components of the lens circulation system that might be altered and contribute to the pathogenesis of cataracts. Lenses from wild-type mice and Cx46fs380 heterozygotes and homozygotes were studied at 2 months of age. Cx46fs380-expressing lens fiber cells were depolarized. Cx46fs380 lenses had increased intracellular hydrostatic pressure and concentrations of Na+ and Ca2+. The activity of epithelial Na+-K+-ATPase was decreased in Cx46fs380 lenses. All of these changes were more severe in homozygous than in heterozygous Cx46fs380 lenses. Cx46fs380 cataracts were stained by Alizarin red, a dye used to detect insoluble Ca2+. These data suggest that the lens internal circulation was disrupted by expression of Cx46fs380, leading to several consequences including accumulation of Ca2+ to levels so high that precipitates formed. Similar Ca2+-containing precipitates may contribute to cataract formation due to other genetic or acquired etiologies.

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.

Purpose We reported previously that aquaporin 0 (AQP0) modulates lens fiber cell gap junction (GJ) channel function. The present study was conducted to find out whether the C-terminal end of AQP0 is involved in this regulation. Methods A mouse model, AQP0ΔC/ΔC, was genetically engineered to express AQP0 with 1-246 amino acids, without the normal intact AQP0 (1-263 amino acids) in the lens. Transparency and focusing of the lens were assessed. Intracellular impedance was measured to determine GJ coupling resistance. Intracellular hydrostatic pressure (HP) was also determined. Western blotting was performed to determine connexin (Cx46 and Cx50) expression levels. Results At postnatal day 10, AQP0ΔC/ΔC mouse lenses relative to age-matched wild-type lenses showed loss of transparency and abnormal optical distortion; GJ coupling resistance increased in the differentiating (1.6-fold) and mature (8-fold) fiber cells; lens HP increased approximately 1.5-fold at the junction between the differentiating and mature fiber cells and approximately 2.0-fold in the center; there was no significant change (P > 0.05) in expression levels of Cx46 or Cx50. Conclusions The increase in GJ coupling resistance was not associated with reduced connexin expression, suggesting either a reduction in the open probability or some physical change in plaque location. The increase in resistance was significantly greater than the increase in HP, suggesting less pressure-driven water flow through each open GJ channel. These changes may lead to a loss of transparency and abnormal optical distortion. Overall, our data demonstrate the C-terminal end of AQP0 is involved in modulating GJ coupling to maintain lens transparency and homeostasis.

Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.

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.

Background— Phosphoinositide 3-kinase (PI3K) p110α plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca 2+ current (I Ca,L ) through the voltage-dependent L-type Ca 2+ channel (LTCC). The aim of this study was to evaluate whether p110α also controls cardiac contractility by regulating the LTCC. Methods and Results— Genetic ablation of p110 α (also known as Pik3ca ), but not p110 β (also known as Pik3cb ), in cardiac myocytes of adult mice reduced I Ca,L and blocked insulin signaling in the heart. p110α-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110α decreased I Ca,L and contractility in canine myocytes. Inhibition of p110β did not reduce I Ca,L . Conclusions— PI3K p110α but not p110β regulates the LTCC in cardiac myocytes. Decreased signaling to p110α reduces the number of LTCCs on the cell surface and thus attenuates I Ca,L and contractility.

The aquaporin (AQP) transmembrane proteins facilitate the movement of water across the plasma membrane. In the lens, AQP0 is expressed in fiber cells and AQP1 in the epithelium. Recently, two individuals were identified with congenital polymorphic autosomal dominant cataract, due to a single nucleotide base deletion mutation in the lens AQP0. The deletion modified the reading frame resulting in the addition of a premature stop codon. In the present study, we examined the water permeability properties, trafficking and dominant negative effects as well as cytotoxicity due to the mutant AQP0 (Delta213-AQP0) protein. The membrane water permeability (P(w)) of Delta213-AQP0 expressing oocytes (14+/-1 microm/s) was significantly lower than those expressing WT-AQP0 (25+/-3 microm/s). P(w) of water injected control oocytes was 13+/-2 microm/s. Co-expression of WT-AQP0 with Delta213-AQP0 significantly lowered the P(w) (18+/-3 microm/s) compared to WT-AQP0. With or without the EGFP tag, WT-AQP0 protein localized in the plasma membranes of oocytes and cultured cells whereas Delta213-AQP0 was retained in the ER. Forster Resonance Energy Transfer (FRET) showed that WT-AQP0 partly localized with the co-expressed Delta213-AQP0. Co-localization studies suggest that the mutant AQP0 gained its dominant function by trapping the WT-AQP0 in the ER through hetero-oligomerization. Incubating the cells with chemical chaperones, namely, TMAO and DMSO, did not correct the folding/trafficking defects. Cell death in the Delta213-AQP0 expressing cells was due to necrosis caused by the accumulation of Delta213-AQP0 protein in the ER in cytotoxic proportions. The data show that replacement of the distal end of the 6th TM domain and the C-terminal domain of AQP0 due to the deletion mutation resulted in the impairment of cell membrane P(w), localization of the mutant protein in the ER without trafficking to the plasma membrane, and cytotoxicity due to the accumulation of the mutant protein. Cataracts in patients with this mutation might have resulted from the above mentioned consequences.

Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent exvivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca(2+) elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca(2+) elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.

We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs) induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2), which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

Mefloquine (MFQ) selectively blocks exogenously expressed gap junction channels composed of Cx50 but not Cx46. The purpose of the current study was to evaluate MFQ effects on wild-type (WT) mouse lenses that express both Cx50 and Cx46 in their outer shell of differentiating fibers (DFs). Lenses in which Cx46 was knocked into both Cx50 alleles (KI) were used as controls; MFQ had no effect on coupling in these lenses. When WT lenses were exposed to MFQ, the DF coupling conductance decreased significantly, suggesting that Cx50 contributes about 57% of the coupling conductance in DF and Cx46 contributes 43%. Remarkably, in the presence of MFQ, the 43% of the channels that remained open did not gate closed in response to a reduction in pH, whereas in the absence of MFQ, the same pH change caused all the DF channels to gate closed. Since MFQ is a selective blocker of Cx50 channels, it appears that Cx46 channels lack pH-mediated gating in the absence of functional Cx50 channels but are pH-sensitive in the presence of Cx50 channels. These results suggest the two types of channels interact and gate cooperatively.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase β (pol β) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol β was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mβ16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mβ16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol β. These two pol β knockdown cell lines (NRK-kcdc and Mβ16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mβ16tsA-wt cells or N2A cells. The levels of pol β mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mβ16tsA-kcdc cells with Mβ16tsA-wt, N2A or NRK-wt cells had no effect on pol β levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol β levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol β in the wt cells. The inability of Mβ16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.

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 approximately 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 approximately 1.0 S/cm2 and calcium varied from approximately 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 approximately 2 microM 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 approximately 1 microM (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.

Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long-term preservation of lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (QO2) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors on (QO2) and partial pressure of oxygen (PO2). The distribution of mitochondria was mapped in living lenses by 2-photon microscopy. We found that a steep gradient of PO2 was maintained within the tissue, leading to PO2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens; however, PO2 gradients extended beyond the boundaries of the mitochondria-containing cell layer, indicating the presence of non-mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion-consumption model. Typical values were 3 x 10(-5) cm(2) s(-1) for the effective diffusion coefficient and a 5 min time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibres (DF) that contained mitochondria and mature fibres (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease in (QO2) and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen-rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of postsurgical nuclear cataract.

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.

Purpose Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ∼2 months in homozygotes and at ≥4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts. Methods Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses. Results Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses. Conclusions Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

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.

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P(Na)/P(Cl) ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P(Na)/P(GSH) ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

Transmural heterogeneities in Na/K pump current (IP), transient outward K+-current (Ito), and Ca2+-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.

β1-Integrin is a heterodimeric transmembrane protein that has roles in both cell–extra-cellular matrix and cell–cell interactions. Conditional deletion of β1-integrin from all lens cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens fibers. Thus, a conditional approach was used to delete β1-integrin solely from the lens fiber cells. This deletion resulted in two distinct phenotypes with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of “clear” conditional knockout lenses revealed that they had profound defects in fiber cell morphology associated with the loss of the F-actin network. Physiological measurements found that the lens fiber cells had a twofold increase in gap junctional coupling, perhaps due to differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In summary, β1-integrin plays a role in maintaining the cellular morphology and homeostasis of the lens fiber cells.

The mammalian lens generates an internal microcirculation that maintains transparency in the avascular lens. Significant progress has been made in characterizing the membrane transport proteins associated with this circulation. By combining physiological and molecular evidence, a more comprehensive understanding of normal lens function and cataractogenesis is emerging.

Lens fiber cell gap junctions contain alpha(3) (Cx46) and alpha(8) (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either alpha(3) or alpha(8) connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from alpha(3) connexin knockout lenses. Here, we present original data from alpha(8) connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell-cell contact of approximately 1 S/cm(2), which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is approximately 0.4 S/cm(2), and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/-) was approximately 70% of normal and 30-40% of the normal for both -/- lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of alpha(3) +/- lenses. In contrast, the coupling of MF in the alpha(8) +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of alpha(3) -/- lenses. Together, these results suggest that alpha(3) connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and alpha(3) connexin -/- lenses. However, in alpha(8) -/- lenses, the pure alpha(3) connexin junctions did not gate closed in the response to acidification. Since alpha(3) connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears alpha(8) connexin regulates the gating of alpha(3) connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that alpha(3) connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears alpha(8) connexin is required for gating in DF.

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 (I P ), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I P 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, I P 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.

Guinea pig ventricular myocytes coexpress two isoforms of the Na+-K+ pump. These two isoforms respond differently to the physical environment and are coupled to autonomic input through different signal transduction cascades. The expression of different isoforms provides each cell type with a mechanism of programming specific responses to environmental changes.

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 micron/sec whereas normal fiber cell membrane pH2O was 17 micron/sec frog, 32 micron/sec rabbit and 43 micron/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 micron/sec for heterozygous and 30 micron/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 micron/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

We have previously shown activation of alpha1-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 microM at 15 nM cytosolic [Ca2+] ([Ca2+]i) and 13 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 alpha1-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 alpha1-activation caused a maximum increase of 15% vs 30% via PMA, and alpha1-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 alpha1-adrenergic activation to Ip is entirely through PKC, however alpha1-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist.

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing α 3 (Cx46) and α 8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous α 3 knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that α 8 remained at similar levels in the three classes of lenses, whereas α 3 was approximately 50% of the normal level in the +/− lenses, and it was absent from the −/− lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In −/− lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (≈−65 mV). The DF remained coupled, but the conductance was reduced to 30–35% of normal. However, the gap junctions in the DF of α 3 −/− lenses remained sensitive to pH. We conclude that α 3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

1. The whole-cell patch clamp was employed to study Na+-K+ pump current (Ip) in acutely isolated myocytes. alpha-Adrenergic receptors were activated with noradrenaline (NA) after blocking beta-adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). 2. Activation of alpha-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 microM [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 alpha-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 alpha1-adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an alpha1-antagonist, but was unaffected by yohimbine, an alpha2-antagonist. 7. We conclude noradrenaline activates ventricular alpha1-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.

The beta-agonist isoproterenol (ISO) reduces the Na/K pump current (Ip) via beta-adrenergic receptors when the intracellular calcium concentration ([Ca2+]i) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 microM), or the adenylyl cyclase activator forskolin (50 microM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 microM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on Ip is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H7 (100 microM) nor the protein phosphatase inhibitor calyculin A (0.5 microM) had any effect on Ip in the absence of ISO. However, H7 could increase Ip and calyculin A could reduce it in the presence of ISO (1 microM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level.

Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p Gly ) was 2.3 ± 0.23 × 10−6 cm sec−1 with MIP vs. 0.92 ± 0.086 × 10−6 cm sec−1 in control oocytes. The p Gly of MIP was independent of concentration from 5 × 10−5 to 5 × 10−2 m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg++, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min−1 cell−1) without changing the binding of glycerol to the kinase (K M ∼ 10 μm).

1. The whole cell patch clamp technique was used to study effects of the beta agonist isoprenaline (Iso) on the current-voltage (I-V) relationship of the Na(+)-K+ pump current (Ip) in acutely isolated guinea-pig ventricular myocytes. 2. The effect of Iso on Ip at high [Ca2+]i (1.4 microM) was voltage dependent. The I-V relationship of Ip in Iso shifted by approximately 30 mV in the negative direction on the voltage axis, increasing Ip at negative voltages but leaving Ip unchanged at positive voltages. 3. Intracellular application of the calmodulin antagonist, calmodulin-dependent protein kinase II fragment 290-309, did not eliminate or reduce the Iso-induced voltage shift, suggesting calmodulin-dependent protein kinase II was not involved. 4. The Iso inhibition of Ip at low [Ca2+]i (15 nM) was not voltage dependent. Ip was reduced by 20 to 30% in the presence of Iso at each holding potential. 5. When the voltage dependence of Ip was largely reduced by substitution of N-methyl-D-glucamine+ for external Na+, the magnitude of the low [Ca2+]i, Iso-induced inhibition of Ip was progressively eliminated by increasing the [Ca2+]i. At a [Ca2+]i of 1.4 microM, this inhibition disappeared. 6. At intermediate values of [Ca2+]i, the I-V curves in Na(+)-containing solution in the presence and the absence of Iso crossed over. The higher the [Ca2+]i, the more positive the voltage at which the two I-V curves intersected. 7. During beta-adrenergic activation our results suggest intracellular Ca2+ has two effects: (a) It prevents protein kinase A (PKA) phosphorylation-induced inhibition of Ip. (b) It causes a PKA phosphorylation-induced shift of the pump I-V relationship in the negative direction on the voltage axis. These effects may have important physiological significance in the regulation of heart rate and cardiac contractility.

The ocular lens generates an internal circulation in order to transport nutrients into the lens. The internal circulation enters at the poles and exits at the equator of the lens. This internal circulation is the result of the localization of membrane proteins and the cellular architecture of the lens. This work presents the development of a finite element model that solves the advection-diffusion equations for ion fluxes, the isotonic transport equations for water flux and the equations for transmembrane ion and water fluxes.