Feedback inhibition by calcium limits the release of calcium by inositol trisphosphate in Limulus ventral photoreceptors

Injection of inositol 1,4,5 trisphosphate (InsP3) into Limulus ventral photoreceptors elevates the concentration of intracellular calcium ions and as a consequence depolarizes the photoreceptor. This InsP3-induced elevation can be inhibited by a prior injection of calcium or InsP3 delivered 1 s earlier. Recovery from this inhibition has a half-time of between 1.5 and 5 s at 20 degrees C. Calcium released by InsP3 therefore inhibits further release of calcium from InsP3-sensitive calcium stores. This feedback inhibition may protect the calcium stores from depletion during prolonged bright illumination. Feedback inhibition, rather than periodic depletion of calcium stores, may also underlie the oscillatory bursts of InsP3-induced calcium release that have been observed in many cell types.     

Inositol 1,4,5-trisphosphate-induced calcium release is necessary for generating the entire light response of limulus ventral photoreceptors

The experiments reported here were designed to answer the question of whether inositol 1,4,5-trisphosphate (IP3)-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. For this purpose the membrane-permeable IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2APB) (Maruyama, T., T. Kanaji, S. Nakade, T. Kanno, and K. Mikoshiba. 1997. J. Biochem. (Tokyo). 122:498-505) was used. Previously, 2APB was found to inhibit the light activated current of Limulus ventral photoreceptors and reversibly inhibit both light and IP3 induced calcium release as well as the current activated by pressure injection of calcium into the light sensitive lobe of the photoreceptor (Wang, Y., M. Deshpande, and R. Payne. 2002. Cell Calcium. 32:209). In this study 2APB was found to inhibit the response to a flash of light at all light intensities and to inhibit the entire light response to a step of light, that is, both the initial transient and the steady-state components of the response to a step of light were inhibited. The light response in cells injected with the calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was reversibly inhibited by 2APB, indicating that these light responses result from IP3-mediated calcium release giving rise to an increase in Cai. The light response obtained from cells after treatment with 100 microM cyclopiazonic acid (CPA), which acts to empty intracellular calcium stores, was reversibly inhibited by 2APB, indicating that the light response after CPA treatment results from IP3-mediated calcium release and a consequent rise in Cai. Together these findings imply that IP3-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors.     

Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2-aminoethoxydiphenylborate

2-Aminoethoxydiphenylborate (2-APB) inhibits the extent of inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) release from cerebellar microsomes with a potency that is dependent upon the InsP(3) concentration used. At high InsP(3) concentrations (10 microM), the concentration of 2-APB required to cause half-maximal InsP(3)-induced Ca(2+) release (IC(50)) was greater than 1 mM, while at 0.25 microM InsP(3) this reduced to 220 microM. The fact that the inhibition of the extent of InsP(3)-induced Ca(2+) release (IICR) by 2-APB was not restored to control levels by high concentrations of InsP(3), in addition to the fact 2-APB did not substantially inhibit [3H]InsP(3) binding to its receptor, indicates that the inhibition is not competitive in nature. Since the cooperativity of IICR as a function of InsP(3) was reduced in the presence of 2-APB (Hill coefficient changing from 1.9 in the absence of 2-APB to 1.4 in the presence of 1 mM 2-APB), this suggests that it is acting as an allosteric inhibitor. 2-APB also reduces the rate constants for IICR. In cerebellar microsomes this release process is biphasic in nature, with a fast and slow phase. 2-APB appears particularly to affect the fast-phase component. Although 2-APB does not inhibit the ryanodine receptor, it does inhibit the Ca(2+) ATPase activity as well store-operated Ca(2+) entry channels, which may limit its use as a specific membrane permeant InsP(3) receptor inhibitor.     

Injection of inositol trisphosphorothioate into Limulus ventral photoreceptors causes oscillations of free cytosolic calcium

Limulus ventral photoreceptors contain calcium stores sensitive to release by D-myo-inositol 1,4,5 trisphosphate (InsP3) and a calcium-activated conductance that depolarizes the cell. Mechanisms that terminate the response to InsP3 were investigated using nonmetabolizable DL-myo-inositol 1,4,5 trisphosphorothioate (InsPS3). An injection of 1 mM InsPS3 into a photoreceptor's light-sensitive lobe caused an initial elevation of cytosolic free calcium ion concentration (Cai) and a depolarization lasting only 1-2 s. A period of densensitization followed, during which injections of InsPS3 were ineffective. As sensitivity recovered, oscillations of membrane potential began, continuing for many minutes with a frequency of 0.07-0.3 Hz. The activity of InsPS3 probably results from the D-stereoisomer, since L-InsP3 was much less effective than InsP3. Injections of 1 mM InsP3 caused an initial depolarization and a period of densensitization similar to that caused by 1 mM InsPS3, but no sustained oscillations of membrane potential. The initial response to InsPS3 or InsP3 may therefore be terminated by densensitization, rather than by metabolism. Metabolism of InsP3 may prevent oscillations of membrane potential after sensitivity has recovered. The InsPS3-induced oscillations of membrane potential accompanied oscillations of Cai and were abolished by injection of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. Removal of extracellular calcium reduced the frequency of oscillation but not its amplitude. Under voltage clamp, oscillations of inward current were observed. These results indicate that periodic bursts of calcium release underly the oscillations of membrane potential. After each burst, the sensitivity of the cell to injected InsP3 was greatly reduced, recovering during the interburst interval. The oscillations may, therefore, result in part from a periodic variation in sensitivity to a constant concentration of InsPS3. Prior injection of calcium inhibited depolarization by InsPS3, suggesting that feedback inhibition of InsPS3-induced calcium release by elevated Cai may mediate desensitization between bursts and after injections of InsPS3.     

Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.     

Variants of TRP ion channel mRNA present in horseshoe crab ventral eye and brain

Transient receptor potential (TRP) channels mediate light-induced Ca(2+) entry and the electrical response in Drosophila photoreceptors. The role of TRP channels in other invertebrate photoreceptors is unknown, particularly those, exemplified by Limulus ventral eye photoreceptors, in which calcium release from intracellular stores is prominent. We have amplified cDNA encoding three variants of a Limulus TRP channel. LptrpA and LptrpBencode proteins of 896 and 923 amino acids, differing by a 27 amino acid insert within the C-terminus. LptrpC encodes an alternative 63 amino acid sequence in the pore domain compared with LptrpB. LptrpB and LptrpC are present in ventral eye mRNA, while LptrpA is only present in brain mRNA. In situ hybridization indicates the presence of Lptrp in photoreceptors of the Limulus ventral eye. Some canonical TRP channels can be activated by diacylglycerol analogs. Injection of a diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), into Limulus photoreceptors can activate an inward current with electrical characteristics similar to the light-activated current. However, simultaneous elevation of cytosolic calcium concentration appears to be necessary. Illumination attenuates the response to OAG injections and vice versa. These results provide molecular and pharmacological evidence for a TRP channel in Limulus ventral eye that may contribute to the light-sensitive conductance.     

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release,inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels

The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.     

Facilitation of the responses to injections of inositol 1,4,5-trisphosphate analogs in Limulus ventral photoreceptors.

Injection of inositol 1,4,5-trisphosphate and its metabolically resistant analogs InsP3S3 and L-chiro-2,3,5-InsP3 into the ventral photoreceptors of Limulus results in the release of calcium from internal stores and in a current flow into the cells. We show here that the dependence of the current response on the amount of analog injected is supralinear. The injections also facilitate the responses to subsequent injections. We analyze the kinetics of the responses either by very slow application of the analogs directly into the lobe that is sensitive to InsP3 and light or by delivering a pulse into the nonsensitive lobe of the cell, in both cases creating a ramp of rising concentration in the sensitive region. Typically, a long latent period was followed by a strong brief inward current. The ratio between the latency and the duration of the response, defined as twice the time from half-amplitude to the peak of the response, reaches values greater than 10. Our analysis shows that this value cannot be attained within realistic models whose only nonlinearity is the cooperative binding of the ligand to its receptor. The observed ratio, however, can be achieved with a positive feedback model. Treatments that lead to partial depletion of calcium stores reversibly increase the latency of the response. We conclude that the mechanisms of the response of Limulus ventral eye to the metabolically resistant analogs of InsP3 probably involves a positive feedback mechanism and that the carrier of the feedback is likely to be Ca2+.     

Inositol 1,4,5 trisphosphate releases calcium from specialized sites within Limulus photoreceptors

We have investigated the subcellular distribution and identity of inositol trisphosphate (InsP3)-sensitive calcium stores in living Limulus ventral photoreceptor cells, where light and InsP3 are known to raise intracellular calcium. We injected ventral photoreceptor cells with the photoprotein aequorin and viewed its luminescence with an image intensifier. InsP3 only elicited detectable aequorin luminescence when injected into the light-sensitive rhabdomeral (R)-lobe where aequorin luminescence induced by light was also confined. Calcium stores released by light and InsP3 are therefore localized to the R-lobe. Within the R-lobe, InsP3-induced aequorin luminescence was further confined around the injection site, due to rapid dilution and/or degradation of injected InsP3. Prominent cisternae of smooth endoplasmic reticulum are uniquely localized within the cell beneath the microvillar surface of the R-lobe (Calman, B., and S. Chamberlain, 1982, J. Gen. Physiol., 80:839-862). These cisternae are the probable site of InsP3 action.     

2-Aminoethoxydiphenyl borate inhibits inositol 1,4,5-trisphosphate receptor function,ubiquitination and downregulation,but acts with variable characteristics in different cell types

2-Aminoethoxydiphenyl borate (2-APB) is a putative, membrane-permeable inhibitor of inositol 1,4,5-trisphosphate (InsP(3)) receptors, but it is the case that little is known about its action at the InsP(3) receptor level. Thus, we examined the effects of 2-APB on InsP(3) receptor-mediated effects in a range of cell types expressing different complements of InsP(3) receptor types. In experiments with permeabilized cells we found that 2-APB could inhibit InsP(3)-induced release of stored Ca(2+), but also that it released Ca(2+), and that the prevalence of these two effects varied between different cell types and did not correlate with the expression of a particular receptor type. These effects of 2-APB reflected an interaction distal to the ligand binding site of InsP(3) receptors, since InsP(3) binding was unaffected by 2-APB. In intact cells, we found only inhibitory effects of 2-APB on Ca(2+) mobilization, and that variation between cell types in the characteristics of this inhibition appeared to be due to differential entry of 2-APB. 2-APB also inhibited InsP(3) receptor ubiquitination and proteasomal degradation, which again was cell type dependent. In total, these data reveal a remarkable degree of variation between cell types in the effects of 2-APB, showing that its usefulness as a specific and universal inhibitor of InsP(3) receptors is limited. However, the ability of 2-APB to inhibit InsP(3) receptor ubiquitination and degradation indicates that 2-APB may block InsP(3)-induced conformational changes in the receptor, resulting in perturbation of multiple regulatory events.     

Intracellular injection of heparin and polyamines. Effects on phototransduction in limulus ventral photoreceptors

Heparin is thought to inhibit InsP3 binding to receptors involved in the intracellular release of Ca2+. Injection of heparin into Limulus ventral photoreceptors to high intracellular concentrations reduces the amplitude and slows the rate of rise of voltage-clamp currents induced by brief flashes, tends to make the responses to long flashes more "square," and tends to block the light-induced rise in [Ca2+]i detected by arsenazo III. In these ways, intracellular heparin mimics the effects of high concentrations of intracellular BAPTA or EGTA. In addition, the effects of heparin are attenuated by prior injection of BAPTA to high intracellular concentrations. Neomycin and spermine are thought to inhibit phospholipase C activity. Injections of spermine or neomycin to low intracellular concentrations largely mimic the effects of intracellular heparin. These findings suggest that the predominant effect of polyamines is to inhibit light-induced production of InsP3 by phospholipase C activity and thereby reduce the light-induced increase in [Ca2+]i. Our findings suggest that excitation can proceed in the absence of InsP3-induced increases in [Ca2+]i, but (a) the gain and speed of transduction are reduced and (b) adaptation is largely blocked.     

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.     

Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes.

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca(2+) signals and replenishment of Ca(2+) stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP(3)R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca(2+) entry between 1-10 microm. 2-APB activated authentic SOCs because the entry was totally selective for Ca(2+) (no detectable entry of Ba(2+) or Sr(2+) ions), and highly sensitive to La(3+) ions (IC(50) 30-100 nm). To assess the role of InsP(3)Rs in this response, we used the DT40 triple InsP(3)R-knockout (ko) cell line, DT40InsP(3)R-ko, in which the absence of full-length InsP(3)Rs or InsP(3)R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP(3)R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP(3)R-ko, cells indicating InsP(3)Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca(2+) entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca(2+) release from stores in intact DT40wt cells but not in DT40InsP(3)R-ko cells, indicating an InsP(3)R-mediated effect. In contrast, 2-APB blocked InsP(3)Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP(3)Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP(3)R activation but did not alter InsP(3)R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP(3)Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca(2+) entry.     

A lingering elevation of Cai accompanies inhibition of inositol 1,4,5 trisphosphate-induced Ca release in Limulus ventral photoreceptors

Injection of inositol 1,4,5 trisphosphate (InsP3) into Limulus ventral photoreceptors causes an elevation of intracellular free Ca concentration (Cai) and depolarizes the photoreceptors. When measured with the photoprotein aequorin, the InsP3-induced Cai increase follows the time course of depolarization and declines within 1-2 s. However, sensitivity to further injections of InsP3 remains suppressed for several tens of seconds. The possibility that the suppression of Ca release (feedback inhibition) is due to a small lingering elevation of Cai, below the existing detection limit of aequorin, was investigated by measuring Cai with Ca-sensitive electrodes. Double-barreled, Ca- selective microelectrodes were used to pressure inject InsP3 and measure Cai at the same point. Light or InsP3 injections into the light- sensitive compartment depolarized the photoreceptors and induced an elevation of Cai that persisted for tens of seconds. Injections of InsP3 during the decay of Cai showed that sensitivity to InsP3 recovered as resting Cai approached the prestimulus level. The relationship between elevated Cai and feedback inhibition was very steep. An elevation of Cai of 1 microM or more was associated with inhibitions of 79 +/- 12.4% (SEM; n = 7) for the InsP3-induced Cai increase and of 76 +/- 8% for depolarizations. With a residual Cai elevation of 0.01 microM or less, the mean inhibition was 10 +/- 7.4% for InsP3-induced Cai increase and 6.6 +/- 4% for InsP3-induced depolarization. Injections of InsP3 into a light-insensitive compartment within the cell induced elevations of Cai with no associated depolarizations or feedback inhibition. To verify that a sustained elevation of Cai is necessary for inhibition of InsP3-induced Cai increase and depolarization, we injected ethyleneglycol-bis-(beta- aminoethylether)-N,N'-tetraacetic acid (EGTA) between two injections of InsP3. Injection of 1 mM EGTA or the related Ca chelator BAPTA, delivered 750 ms after the first injection of InsP3, restored the peak depolarization caused by the second injection of InsP3 to > 80 +/- 3% of control, compared with 13 +/- 8% without an intervening injection of EGTA. Measurement of Cai with aequorin showed that an intervening injection of EGTA partially restored the InsP3-induced Cai increase. The results suggest that feedback inhibition of InsP3-induced Cai increase and depolarization is mediated by a lingering elevation of Cai and not by depletion of intracellular Ca stores.     

The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback

Microvillar photoreceptors of invertebrates exhibit a light-induced rise in the intracellular concentration of free calcium (Cai) that results in part from release of calcium from an intracellular compartment. This light-induced release of calcium appears to result from a cascade of reactions that involve rhodopsin, a GTP-binding protein and a phospholipase-C which releases inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) from the plasma membrane; the Ins(1,4,5)P3 acts to release calcium from smooth endoplasmic reticulum. In the ventral photoreceptor of the horseshoe crab Limulus polyphemus not all of the endoplasmic reticulum is subject to calcium release by Ins(1,4,5)P3. Only endoplasmic reticulum in the light-sensitive region of the cell is competent to release calcium in response to Ins(1,4,5)P3. The release of calcium by Ins(1,4,5)P3 in ventral photoreceptors appears to be subject to feedback inhibition through elevated Cai. We suggest that this feedback inhibition contributes to sensory adaptation in the photoreceptor and may account for oscillatory membrane responses sometimes observed with large injections of Ins(1,4,5)P3.     

The role of the inositol phosphate cascade in visual excitation of invertebrate microvillar photoreceptors

The identity of the transmitter(s) involved in visual transduction in invertebrate microvillar photoreceptors remains unresolved. In this study, the role of inositol 1,4,5-trisphosphate (IP3) was examined in Limulus ventral photoreceptors by studying the effects on the light response of heparin and neomycin, agents that inhibit the production or action of IP3. Both heparin and neomycin reduce responses to brief flashes of light and the transient component of responses to steps of light, and also inhibit IP3-induced calcium release, indicating that IP3 plays a direct role in invertebrate visual excitation. The effects of BAPTA, a calcium buffer, were also examined and shown to be consistent with a role for IP3-mediated calcium release in visual excitation. However, all three agents fail to block the plateau component of the response to a step of light, indicating that a single pathway involving IP3 and calcium cannot solely be responsible for visual excitation in invertebrates. We suggest that the inositol phosphate cascade and a second parallel process that is not dependent on IP3 are involved in the production of the light response.     

Temperature-dependent calcium-induced calcium release via InsP3 receptors in mouse olfactory ensheathing glial cells

Cooling can induce Ca(2+) signaling via activation of temperature-sensitive ion channels such as TRPM8, TRPA1 and ryanodine receptor channels. Here we have studied the mechanism of cooling-evoked Ca(2+) signaling in mouse olfactory ensheathing cells (OECs), a specialized type of glial cells in the olfactory nerve layer of the olfactory bulb. Reducing the temperature from above 30°C to 28°C and below triggered Ca(2+) transients that persisted in the absence of external Ca(2+), but were suppressed after Ca(2+) store depletion by cyclopiazonic acid. Cooling-evoked Ca(2+) transients were present in mice deficient of TRPM8 and TRPA1, and were not inhibited by ryanodine receptor antagonists. Inhibition of InsP(3) receptors with 2-APB and caffeine entirely blocked cooling-evoked Ca(2+) transients. Moderate Ca(2+) increases, as evoked by flash photolysis of NP-EGTA (caged Ca(2+)) and cyclopiazonic acid, triggered InsP(3) receptor-mediated Ca(2+) release at 22°C, but not at 31°C. The results suggest that InsP(3) receptors mediate Ca(2+)-induced Ca(2+) release in OECs, and that this Ca(2+) release is temperature-sensitive and can be suppressed at temperatures above 28°C.     

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.     

InsP(3)-induced Ca(2+) release in permeabilized invertebrate photoreceptors: a link between phototransduction and Ca(2+) stores

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.