Calcium Sets the Physiological Value of the Dominant Time Constant of Saturated Mouse Rod Photoresponse Recovery

Background: The rate-limiting step that determines the dominant time constant (tD) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca 2+-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that tD is modulated by background light in mouse rods. Methodology/Principal Findings: We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca 2+ shortens the dominant time constant (tD) of saturated photoresponse recovery, i.e., when extracellular free Ca 2+ is decreased from 1mMto,25 nM, the tD is reversibly increased,1.5–2-fold. Conclusions: We conclude that the increase in tD during low Ca 2+ treatment is not due to increased [cGMP], increased [Na +] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that aCa 2+-dependent mechanism controls the life time of active PDE

Retinal Temperature Determination Based on Photopic Porcine Electroretinogram

Objective: Subthreshold retinal laser therapy (SLT) is a treatment modality where the temperature of the retinal pigment epithelium (RPE) is briefly elevated to trigger the therapeutic benefits of sublethal heat shock. However, the temperature elevation induced by a laser exposure varies between patients due to individual differences in RPE pigmentation and choroidal perfusion. This study describes an electroretinography (ERG)-based method for controlling the temperature elevation during SLT. Methods: The temperature dependence of the photopic ERG response kinetics were investigated both ex vivo with isolated pig retinas and in vivo with anesthetized pigs by altering the temperature of the subject and recording ERG in different temperatures. A model was created for ERG-based temperature estimation and the feasibility of the model for controlling SLT was assessed through computational simulations. Results: The kinetics of the photopic in vivo flash ERG signaling accelerated between 3.6 and 4.7%/degrees C, depending on the strength of the stimulus. The temperature dependence was 5.0%/degrees C in the entire investigated range of 33 to 44 degrees C in ex vivo ERG. The simulations showed that the method is suitable for determining the steady-state temperature elevation in SLT treatments with a sufficiently long laser exposure and large spot size, e.g., during > 30 s laser exposures with > 3 mm stimulus spot diameter. Conclusions: The described ERG-based temperature estimation model could be used to control SLT treatments such as transpupillary thermotherapy. Significance: The introduced ERG-based method for controlling SLT could improve the repeatability, safety, and efficacy of the treatment of various retinal disorders.Peer reviewe

Thermal Activation and Photoactivation of Visual Pigments

A visual pigment molecule in a retinal photoreceptor cell can be activated not only by absorption of a photon but also ‘‘spontaneously’’ by thermal energy. Current estimates of the activation energies for these two processes in vertebrate rod and cone pigments are on the order of 40–50 kcal/mol for activation by light and 20–25 kcal/mol for activation by heat, which has forced the conclusion that the two follow quite different molecular routes. It is shown here that the latter estimates, derived from the temperature dependence of the rate of pigment-initiated ‘‘dark events’’ in rods, depend on the unrealistic assumption that thermal activation of a complex molecule like rhodopsin (or even its 11-cis retinaldehyde chromophore) happens through a simple process, somewhat like the collision of gas molecules. When the internal energy present in the many vibrational modes of the molecule is taken into account, the thermal energy distribution of the molecules cannot be described by Boltzmann statistics, and conventional Arrhenius analysis gives incorrect estimates for the energy barrier. When the Boltzmann distribution is replaced by one derived by Hinshelwood for complex molecules with many vibrational modes, the same experimental data become consistent with thermal activation energies that are close to or even equal to the photoactivation energies. Thus activation by light and by heat may in fact follow the same molecular route, starting with 11-cis to all-trans isomerization of the chromophore in the native (resting) configuration of the opsin. Most importantly, the same model correctly predicts the empirical correlation between the wavelength of maximum absorbance and the rate of thermal activation in the whole set of visual pigments studied

Regulation of intracellular pH in salamander retinal rods

1. We measured intracellular pH (pHi) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'-bis(carboxyethyl)- 5(and -6)-carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pH,. There was no sharp change between the value obtained from the first exposure of dark-adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min-' either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3--free solution at pH 7 5, the rods had a steady-state pHi of 7'09 + 0-02 (n = 46) and a buffering power (,Bi) of 24 + 1 mm (pH unit)f' (n = 48). The buffering power was virtually constant in the pH range 6-6-8-0. In the same range, pHi depended linearly on perfusion pH (pHO) with regression coefficients of 0 4-0 5. 4. There were no significant differences between the inner and outer segment of intact rods as regards steady-state pH, or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate-free perfusate was completely blocked by decreasing the extracellular Nae concentration to 7 mm, and slowed by 86% by applying 1 mm amiloride. 6. Introduction of 2% C02-13 mm HC03- caused an alkalinization that was often preceded by a transient acidification. Steady-state pHi was on average 0.1 pH units higher than in nominally bicarbonate-free solution. The mean acid extrusion rate, calculated on the assumption that C02-HC03- behaves as an open system, was 19% higher (31 + 2 mm h-1) than in a solution buffered only by Hepes (26 + 2 mm h-). 7. In the presence of CO2-HC03-, 100 ,m 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Clconcentration to 7 mm raised pH,, but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pHi by both Na+-H+ exchange and mechanism(s) involving HC03--Cl- exchange. In the present conditions, the Na+-H+ exchanger appears as the dominant mechanism for acid extrusion

Visual Neuroscience Flash Responses of Mouse Rod Photoreceptors in the Isolated Retina and Corneal Electroretinogram: Comparison of Gain and Kinetics

PURPOSE. To examine the amplification and kinetics of murine rod photoresponses by recording ERG flash responses in vivo and ex vivo from the same retina. We also aimed to evaluate the two available methods for isolating the rod signal from the ERG flash response, that is, pharmacology and paired flash method on the isolated retina. METHODS. Dark-adapted ERG responses to full-field flashes of green light were recorded from anesthetized (ketamine/ xylazine) C57BL/6N mice. ERG flash responses to homogenous light stimuli arriving from the photoreceptor side were then recorded transretinally from the same retinas, isolated and perfused with Ringer's or Ames' solution at 378C. The responses were analyzed to determine the a-wave kinetics as well as the estimated flash sensitivity and kinetics of the full rod responses derived with the paired flash protocol. The analysis was complemented with pharmacologic blockade of glutamatergic transmission in the isolated retina. RESULTS. The a-waves were of comparable size, sensitivity and kinetics in vivo and in the isolated retina, but the onset of the b-wave was delayed in the isolated retina. The Lamb-Pugh activation constants determined for the a-waves were similar in both preparations. The kinetics of the derived photoreceptor responses were similar in both conditions, although the responses were consistently slightly slower ex vivo. This was not explicable as a direct effect of ketamine or xylazine on the photoreceptors or as their indirect effect through hyperglycemia, as tested on the isolated retina. CONCLUSIONS. Through comparison to the corneal ERG, the transretinal ERG is a valuable tool for assaying the physiologic state of isolated retinal tissue. The rod photoreceptor responses of the intact isolated retina correspond well to those recorded in vivo. The origin of their faster kinetics compared to single cell recordings remains to be determined. (Invest Ophthalmol Vis Sci. 2012;53:5653-5664

Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity

BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC(50) values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (I(Na) ) and hERG K(V) 11.1 currents (I(hERG) ) in a concentration-dependent manner; IC(50) values were 23.2 and 10.0 μM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 μM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.Peer reviewe

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1 alpha in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1 alpha dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1 alpha dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.Peer reviewe