L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Cav1.2, Cav1.3, and ...Cav1.4) expressed in the retina. While Cav1.2 is expressed in all retinal cells including the Müller glia and neurons, Cav1.3 and Cav1.4 are expressed in the retinal neurons with Cav1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Cav1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Cav1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Cav1.3 are not associated with severe vision impairment in humans or in Cav1.3-null (Cav1.3-/-) mice. However, a failure to regulate Cav1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Cav1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Cav1.3 in retinal physiology and function that has been undervalued thus far. Through ex vivo and in vivo electroretinogram (ERG) recordings and immunohistochemical staining, we found that Cav1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Cav1.3 decreased ex vivo ERG a- and b-wave amplitudes. In Cav1.3-/- mice, their scotopic ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Cav1.3-/- mice retinas. Hence, Cav1.3 plays a more prominent role in retinal physiology and function than previously reported.
The affinity of cGMP-gated ion channels (CNGCs) for cGMP in chick retinal cone photoreceptors is under circadian control. Here we report that dopamine (DA) and D2 receptor agonists evoke ...phase-dependent shifts in the affinity of CNGCs for activating ligand. Inside-out patch recordings from cultured chick cones were performed at circadian time (CT) 4-7 and CT 16-19 on the second day of constant darkness. Exposing intact cells to DA or the D2 agonist quinpirole for 2 hr before patch excision caused a significant increase in the K(D) for cGMP during the night (CT 16-19) but had no effect during the day (CT 4-7). DA or quinpirole treatment had no effect on the Hill slope or the average number of channels per patch. The effect of DA was blocked by the D2 antagonist eticlopride and was not mimicked by D1 agonists or blocked by D1 antagonists. By contrast, a brief (15 min) exposure to DA or quinpirole caused a decrease in K(D) during the subjective day and had no effect during the subjective night. Thus, the effect of D2 agonists depends on both the duration of agonist exposure and the time of day. Application of DA or quinpirole evoked a transient activation of the MAP kinase Erk (extracellular signal-related kinase) during the day but caused a sustained inhibition during the night. Conversely, D2 agonists caused activation of Ca2+/calmodulin-dependent protein kinase II during the night and inhibited this enzyme during the day. A circadian oscillator in cones appears to regulate the nature of the transduction cascade used by D2 receptors.
Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological ...angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (K.sub.Ca 3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking K.sub.Ca 3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional K.sub.Ca 3.1 in ECs contributes to EC activation and EC-dependent angiogenesis.
Extracellular signal-regulated kinase (ERK) participates in numerous cellular functions including circadian-related activities. In the retina, the activity of ERK is under circadian control. However, ...it is not clear whether acute illumination changes or the circadian clocks in the retina have a larger impact on ERK activity, and the cellular distribution of activated ERK (pERK) as a function of circadian time in cone photoreceptors is not known. Chick embryos were exposed to the light or dark for various lengths of time after 12:12
h light–dark (LD) cycles, or on the second day of constant darkness after LD entrainment. Retinas were excised after various exposure times and relative ERK activity was determined by western immunoblotting. We also performed immunohistochemical and immunocytochemical stainings on circadian entrained retina sections and dissociated retina cells. There is about a fourfold difference in ERK activity between retinas harvested at circadian time (CT) 4 and CT 16, and the internal circadian control of ERK activity in the retina overcomes external light exposure. Also, during the subjective night, pERK was more apparent in the outer segment of cones, while pERK distribution was more uniform throughout the photoreceptors during the subjective day. Our results imply that the activity of retinal ERK is influenced more by circadian oscillators than acute illumination changes. Hence, the circadian oscillators in retina photoreceptors play a major role in the regulation of photoreceptor physiology, which leads to the circadian control of light sensitivity in photoreceptors.
A subpopulation of avian amacrine cells expresses somatostatin-14 (SS14) and somatostatin-28 (SS28), which provide a potential efferent limb for light-dependent regulation of photoreceptors. Here, we ...demonstrate that SS14 and SS28 modulate cone photoreceptor cGMP-gated channels (CNGCs) through multiple mechanisms. In chicken cones cultured in constant darkness for 2 d after previous entrainment to light-dark (LD) cycles or in cells maintained in LD, application of 100 nm SS14 or 100 nm SS28 for either 15 min or 2 h caused a decrease in the sensitivity of CNGCs to cGMP during the night, at circadian time 16 (CT16)-CT20 or zeitgeber time 16 (ZT16)-ZT20. SS14 had no effect during the day (CT4-CT8 or ZT4-ZT8). These effects persist in cells pretreated with pertussis toxin (PTX) and, like dopamine, may work to reinforce long-term circadian fluctuations in CNGCs driven by oscillators within the photoreceptors themselves. In contrast, a 15 min exposure to SS28 caused a seemingly paradoxical increase in the sensitivity of CNGCs to cGMP during the early day (ZT4-ZT6), but only in cones maintained in LD. This effect of SS28 desensitizes rapidly, is blocked by pretreatment with PTX, and is selectively mimicked by the cyclohexapeptide agonist MK-678. This transient response also requires activation of phospholipase C and protein kinase C. The transient response to SS28 may play a role in photoreceptor adaptation to rapid changes in ambient illumination. These data also show that photoreceptor responses to at least some peptide neurotransmitters depend on the previous history of light exposure.
To investigate the role of tyrosine phosphorylation in circadian regulation of cGMP-gated cation channels (CNGCs) of chicken cone photoreceptors.
Chick retinas were studied on the second day of ...constant darkness (DD) after several days of entrainment to 12:12 hr light-dark (LD) cycles in vitro. Inside-out patch recordings were made during the subjective day and subjective night to quantify circadian changes in the sensitivity of CNGCs to activation by cGMP after treatment with various tyrosine kinase and tyrosine phosphatase inhibitors. Immunoprecipitation and immunoblot analysis were also used to examine tyrosine phosphorylation of CNGCs and closely associated proteins after separation by conventional and two-dimensional SDS-PAGE.
Treatment with tyrosine kinase inhibitors caused a significant decrease in K(1/2) for cGMP activation of CNGCs in patches excised from cones during the subjective day, but had no effect on K(1/2) during the subjective night. Conversely, treatment with a tyrosine phosphatase inhibitor caused a significant increase in the K(1/2) of CNGCs in patches excised during the subjective night but had no effect on channel K(1/2) during the subjective day. Broad spectrum serine-threonine phosphatase inhibitors had no effect. An 85-kDa tyrosine polypeptide that coimmunoprecipitated with CNGC alpha-subunits was detectable at higher levels during the subjective day than during the subjective night. CNGC alpha-subunits were not tyrosine phosphorylated as a function of the time of day.
Circadian control of cone CNGCs appears to entail elevated daytime tyrosine phosphorylation of an approximately 85-kDa auxiliary protein or another subunit of the CNGCs.
Circadian oscillators of chicken retinal cone photoreceptors modulate the gating properties of cGMP-gated channels (CNGCs) such that they have a higher apparent affinity for cGMP during the ...subjective night. This effect is driven in part by cAMP, which acts through Erk MAP kinase to initiate a cascade leading to modulation of CGNCs. Here, we show that cAMP effects on the gating properties CNGCs persist when protein synthesis is blocked. The effects is cAMP also persist when calcium influx through
L-type channels is blocked by nitrendipine. The mechanisms whereby cAMP modulates CNGCs therefore differ from those previously reported to underline regulation of melatonin synthesis and secretion.
Peptide Lv is a small endogenous secretory peptide that is proangiogenic through hyperpolarizing vascular endothelial cells (ECs) by enhancing the current densities of K
3.1 channels. However, it is ...unclear how peptide Lv enhances these currents. One way to enhance the current densities of ion channels is to promote its trafficking and insertion into the plasma membrane. We hypothesized that peptide Lv-elicited K
3.1 augmentation occurs through activating the mitogen-activated protein kinase kinase 1 (MEK1)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathways, which are known to mediate ion channel trafficking and membrane insertion in neurons. To test this hypothesis, we employed patch-clamp electrophysiological recordings and cell-surface biotinylation assays on ECs treated with peptide Lv and pharmaceutical inhibitors of ERK and Akt. Blocking ERK or Akt activation diminished peptide Lv-elicited EC hyperpolarization and increase in K
3.1 current densities. Blocking PI3K or Akt activation decreased the level of plasma membrane-bound, but not the total amount of K
3.1 protein in ECs. Therefore, the peptide Lv-elicited EC hyperpolarization and K
3.1 augmentation occurred in part through channel trafficking and insertion mediated by MEK1-ERK and PI3K-Akt activation. These results demonstrate the molecular mechanisms of how peptide Lv promotes EC-mediated angiogenesis.
Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological ...angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (KCa3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking KCa3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional KCa3.1 in ECs contributes to EC activation and EC-dependent angiogenesis.
L-type voltage-gated calcium channels (LTCCs) regulate tonic neurotransmitter release from sensory neurons including retinal photoreceptors. There are three types of LTCCs (Ca
1.2, Ca
1.3, and Ca
...1.4) expressed in the retina. While Ca
1.2 is expressed in all retinal cells including the Müller glia and neurons, Ca
1.3 and Ca
1.4 are expressed in the retinal neurons with Ca
1.4 exclusively expressed in the photoreceptor synaptic terminals. Mutations in the gene encoding Ca
1.4 cause incomplete X-linked congenital stationary night blindness in humans. Even though Ca
1.3 is present in the photoreceptor inner segments and the synaptic terminals in various vertebrate species, its role in vision is unclear, since genetic alterations in Ca
1.3 are not associated with severe vision impairment in humans or in Ca
1.3-null (Ca
1.3
) mice. However, a failure to regulate Ca
1.3 was found in a mouse model of Usher syndrome, the most common cause of combined deafness and blindness in humans, indicating that Ca
1.3 may contribute to retinal function. In this report, we combined physiological and morphological data to demonstrate the role of Ca
1.3 in retinal physiology and function that has been undervalued thus far. Through
and
electroretinogram (ERG) recordings and immunohistochemical staining, we found that Ca
1.3 plays a role in retinal light responses and synaptic plasticity. Pharmacological inhibition of Ca
1.3 decreased
ERG a- and b-wave amplitudes. In Ca
1.3
mice, their dark-adapted ERG a-, b-wave, and oscillatory potential amplitudes were significantly dampened, and implicit times were delayed compared to the wild type (WT). Furthermore, the density of ribbon synapses was reduced in the outer plexiform layer of Ca
1.3
mice retinas. Hence, Ca
1.3 plays a more prominent role in retinal physiology and function than previously reported.
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