Excitation and adaptation of Limulus ventral photoreceptors by inositol 1,4,5 triphosphate result from a rise in intracellular calcium

Single pressure injections of 1-10 pl of inositol 1,4,5 triphosphate (IP3) or inositol 4,5 bisphosphate [I(4,5)P2] excite Limulus ventral photoreceptors by inducing rapid bursts of inward current. After excitation by IP3, responses to subsequent injections of IP3 or light flashes are often reversibly diminished (adapted). Single injections of IP3 and I(4,5)P2 are effective at concentrations in the injecting pipette of 20 microM to 1 mM. Single injections of inositol 1,4 bisphosphate are ineffective at concentrations of 100-500 microM. Excitation by IP3 or I(4,5)P2 is accompanied by a rise in intracellular free calcium, as indicated by aequorin luminescence. Prior injection of calcium buffer solutions containing 100 mM EGTA greatly diminishes the total charge transferred across the plasma membrane during excitation by IP3 or I(4,5)P2, which suggests that a rise in Cai is necessary for excitation by the inositol polyphosphates. Adaptation of the response to light by IP3 is also abolished by prior injection of EGTA. In the same cells, the response to brief light flashes is slowed and diminished in amplitude by the injection of calcium buffer, but the charge transferred during the response is not significantly diminished. This suggests that light has access to a pathway of excitation in the presence of EGTA that is not accessible to intracellularly injected IP3.     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

Light-induced oxygen consumption in Limulus ventral photoreceptors does not result from a rise in the intracellular sodium concentration

Illumination of Limulus ventral photoreceptors leads to an increase in the intracellular concentration of sodium, [Na+]i, and to an increase in the consumption of O2 (delta QO2). After a 1-s light flash, it takes approximately 480 s for [Na+]i to return to within 10% of its preillumination level, whereas delta QO2 takes approximately 90 s. Thus, the delta QO2 is complete long before [Na+]i has returned to its resting level. Pressure injection of Na+ into the cell in order to elevate [Na+]i to the same levels as attained by illumination causes a rise in [Na+]i that returns to baseline with the same time course as the light-induced rise in [Na+]i. However, the injection of Na+ does not lead to an increase of the consumption of O2. We conclude that activation of the Na pump by a rise in [Na+]i is not a factor involved in the light-induced activation of O2 consumption in these cells.     

A direct demonstration that inositol-trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors

Inositol-trisphosphate was pressure-injected into Limulus ventral photoreceptors; these injections induced electrical responses that mimic several aspects of the electrical responses induced by light. Single cells were also injected with aequorin. Injections of inositol-trisphosphate into such cells induced an increase in luminescence from the intracellular aequorin, even in the absence of extracellular calcium ions. These aequorin responses show directly that inositol-trisphosphate induces an increase in ionized calcium concentration within intact and functioning cells that arises from release of calcium ions from intracellular stores.     

Pharmacological inhibition of intracellular calcium release blocks acid-induced bone resorption

In vivo chronic metabolic acidosis induces net Ca2+ efflux from bone, and incubation of neonatal mouse calvariae in medium simulating physiological metabolic acidosis induces bone resorption. It appears that activation of the proton (H+) receptor OGR1 in the osteoblast leads to an increase in intracellular Ca2+, which is associated with an increase in cyclooxygenase 2 (COX2) and PGE2-induced receptor activator of NF-κB ligand (RANKL) and H+-induced osteoclastic bone resorption. To support this hypothesis, we tested whether intracellular Ca2+ signaling was integral to H+-induced bone resorption by determining whether 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and 2-aminoethoxydiphenyl borate (2-APB), inhibitors of inositol trisphosphate-mediated Ca2+ signaling, would block H+-induced bone resorption in cultured neonatal calvariae and, if so, would do so by inhibiting H+-induced stimulation of COX2 and RANKL in osteoblastic cells. We found that H+-induced bone resorption is significantly inhibited by TMB-8 and 2-APB. Both compounds also inhibit H+-induced stimulation of COX2 protein in calvariae and COX2 mRNA and protein levels in primary osteoblasts. H+-induced stimulation of RANKL in calvarial cultures, as well as primary cells, is also completely inhibited by TMB-8 and 2-APB. These results support the hypothesis that H+ stimulation of net Ca2+ efflux from bone, mediated by COX2- and subsequent PGE2-induced RANKL production, is initiated in the osteoblast via activation of Ca2+ signaling.     

Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors

This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose- dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 microM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation.     

Lipoxygenase inhibitors suppress intracellular calcium rise induced by ionomycin in rat thymocytes

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 15S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration [( Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 microM whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.     

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.     

Changes in the intracellular calcium concentration upon activation of rat superior cervical ganglion neurons

Changes in the intracellular calcium concentration induced by activation of neurons of the isolated intact rat superior cervical ganglion were recorded. It is concluded that stimulation within the physiological range of frequencies can effectively increase the intracellular calcium concentration in these neurons. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 400–402, July–October, 2007.     

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+-extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.     

Calcitonin inhibits the rise of intracellular calcium induced by thyrotropin-releasing hormone in GH3 cells

Both human and salmon calcitonins markedly inhibit the TRH-stimulated rise in intracellular [Ca2+] in GH3 cells. Calcitonin also inhibits prolactin release from these cells. Both [Ala] salmon calcitonin and salmon calcitonin (1-23) peptide amide also inhibit this rise in [Ca2+] and also inhibit TRH-stimulated prolactin release from GH3 cells as well as from primary pituitary cell cultures. It is likely that calcitonin inhibits prolactin release in the pituitary by decreasing the extent of the rise of intracellular calcium concentration. Neither an intact disulfide bond at the amino terminus nor residues 24-32 of the carboxyl terminus of salmon calcitonin are required for this inhibition.     

Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neurons

The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 +/- 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 +/- 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge.     

Sustained intracellular calcium rise mediates neuronal mitophagy in models of autosomal dominant optic atrophy

Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca²⁺-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca²⁺ levels, and decreased axonal mitochondrial density. Chelation of Ca²⁺ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca²⁺ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca²⁺-calcineurin-AMPK signaling cascade.Subject terms: Cell biology, Neurological disorders     

A "melanopic" spectral efficiency function predicts the sensitivity of melanopsin photoreceptors to polychromatic lights

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photosensitive retinal ganglion cells expressing the photopigment melanopsin. These mRGCs are especially important contributors to circadian entrainment, the pupil light reflex, and other so-called nonimage-forming (NIF) responses. The spectral sensitivity of melanopsin phototransduction has been addressed in several species by comparing responses to a range of monochromatic stimuli. The resultant action spectra match the predicted profile of an opsin:vitamin A-based photopigment (nomogram) with a peak sensitivity (λ(max)) around 480 nm. It would be most useful to be able to use this spectral sensitivity function to predict melanopsin's sensitivity to broad-spectrum, including "white," lights. However, evidence that melanopsin is a bistable pigment with an intrinsic light-dependent bleach recovery mechanism raises the possibility of a more complex relationship between spectral quality and photoreceptor response. Here, we set out to empirically determine whether simply weighting optical power at each wavelength according to the 480-nm nomogram and integrating across the spectrum could predict melanopsin sensitivity to a variety of polychromatic stimuli. We show that pupillomotor and circadian responses of mice relying solely on melanopsin for their photosensitivity (rd/rd cl) can indeed be accurately predicted using this methodology. Our data therefore suggest that the 480-nm nomogram may be employed as the basis for a new photometric measure of light intensity (which we term "melanopic") relevant for melanopsin photoreception. They further show that measuring light in these terms predicts the melanopsin response to light of divergent spectral composition much more reliably than other methods for quantifying irradiance or illuminance currently in widespread use.     

Parathyroid hormone causes a transient rise in intracellular ionized calcium in vascular smooth muscle cells

The effect of parathyroid hormone on intracellular calcium concentration in vascular smooth muscle cells in culture was studied. Human PTH 1-34 (hPTH (1-34)) caused a transient rise in intracellular calcium in a dose-dependent manner at physiological concentrations. The effect of PTH was mimicked by dibutyryl cyclic AMP and inhibited by a PTH receptor antagonist. The effect of PTH was increased in parallel with extracellular calcium concentration and a sustained response was observed when extracellular calcium was 2 mM or higher. The PTH action was blocked by nisoldipin, a calcium antagonist, but not by ouabain, a Na, K-ATPase inhibitor. These data indicate that PTH increases intracellular calcium through its receptor via opening calcium channels. A possible role of this effect in the regulation of vascular tone is also discussed.     

Leptin inhibits lysophosphatidic acid-induced intracellular calcium rise by a protein kinase C-dependent mechanism

Leptin communicates the status of body energy stores to the central nervous system, regulating appetite, metabolic rate, and neuroendocrine functions. These effects are mediated by leptin binding and activation of the cognate cell surface receptor, a member of type I cytokine receptor family, which lead to the activation of receptor-associated kinases of the Janus family. In this work, we demonstrate that leptin inhibits the l-alpha-lysophosphatidic acid (LPA)-induced intracellular calcium mobilization in a dose-dependent manner in HEK-293 cells stably expressing full-length leptin receptor (OB-Rb). This action appears to be selective, as it was not observed when other signaling families, such as VIP or EGF, were studied. Pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin, reversed the effect of leptin, pointing to PI3K as an intermediate molecule involved in this process. An unspecific protein kinase C (PKC) inhibitor, staurosporine, disrupted the inhibitory action of leptin. Furthermore, intracellular levels of phosphorylated PKCepsilon and PKCdelta rose to a maximum 5 min after leptin administration, suggesting that these atypical PKC isoforms are involved in the observed cross-desensitization. To define the regions of the OB-Rb intracellular domain required for the cross-desensitization, a series of C-terminal deletion mutants were transfected into HEK-293 cells. C-terminal truncation that removed the consensus Box 3 motif of OB-Rb prevented leptin action, indicating that heterologous desensitization over LPA was exerted at the level of this intracellular motif. Our date demonstrate that leptin plays a key role in the regulation of the earliest signaling pathways activated by growth factors, such as LPA, through a signaling pathway involving PKCdelta and PKCepsilon coupled to Box 3 motif of the OB-Rb through PI3K.