Akihiro Nezu

Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations

Inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger that elicits a wide range of spatial and temporal Ca2+ signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP3 biosensors that exhibit strong pH stability and varying affinities for IP3, as well as a method for the quantitative measurement of cytosolic concentrations of IP3 ([IP3]i) in single living cells. We applied this method to elucidate IP3 dynamics during agonist-induced Ca2+ oscillations, and we demonstrated cell type-dependent differences in IP3 dynamics, a nonfluctuating rise in [IP3]i and repetitive IP3 spikes during Ca2+ oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP3 spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP3]i spike peak was preceded by a Ca2+ spike peak. These results suggest that repetitive IP3 spikes in HSY-EA1 cells are passive reflections of Ca2+ oscillations, and are unlikely to be essential for driving Ca2+ oscillations. In addition, the interspike periods of Ca2+ oscillations that occurred during the slow rise in [IP3]i were not shortened by the rise in [IP3]i, indicating that IP3-dependent and -independent mechanisms may regulate the frequency of Ca2+ oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP3 and Ca2+.

Interplay between Calcium, Diacylglycerol, and Phosphorylation in the Spatial and Temporal Regulation of PKCα-GFP

The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCα fused to green fluorescent protein (PKCα-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCα-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCα-GFP to the cytosol and caused a stepwise accumulation of PKCα-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCα-GFP. Ionomycin induced a transient translocation of PKCα-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCα-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCα-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCα-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCα.

Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca2+ release and entry in DT40 B lymphocytes

We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca(2+) release and entry using a mutant DT40 cell line (IP(3)R-KO) in which all three IP(3)R genes had been disrupted. GFP-IP(3)R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP(3)R3 apparently co-localized with the plasma membrane. The application of IP(3) to permeabilized WT (wild-type) DT40 cells induced Ca(2+) release from internal stores. Although this did not occur in IP(3)R-KO cells it was restored by expression of GFP-IP(3)R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca(2+) response in IP(3)R-KO cells, whereas these treatments induced oscillatory or transient Ca(2+) responses in GFP-IP(3)R3-expressing IP(3)R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca(2+) entry in WT and GFP-IP(3)R3-expressing IP(3)R-KO cells but not in IP(3)R-KO cells. This BCR-mediated Ca(2+) entry was observed in the presence of La(3+), which blocks capacitative Ca(2+) entry. Thapsigargin depleted Ca(2+) stores and led to Ca(2+) entry in IP(3)R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca(2+) entry was completely blocked by La(3+), suggesting that the BCR-mediated Ca(2+) entry pathway is distinct from the capacitative Ca(2+) entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP(3)R in both IP(3)-induced Ca(2+) release and BCR-mediated Ca(2+) entry.

A Conserved Hydrophobic Tetrad near the C Terminus of the Secretory Na+-K+-2Cl- Cotransporter (NKCC1) Is Required for Its Correct Intracellular Processing

Little is known about the intracellular folding and trafficking of integral membrane proteins. Here we identify a hydrophobic amino acid tetrad (ILLV) close to the C terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) that is important for the proper intracellular processing of this protein. This tetrad appears in a C-terminal sequence pattern that is conserved across species in a number of members of the NKCC1 gene family (slc12) of electroneutral salt transporters. We studied the effects of various mutations of these amino acids on NKCC1 transiently transfected into HEK-293 cells. Our results show that mutation of two of these residues to alanine leads to a >50% reduction in expression and complex glycosylation levels and that multiple mutations to alanine have cumulative effects. By contrast, scrambling of these amino acids, or mutation of other nearby conserved C-terminal residues, has little effect on these parameters. Mutation of ILLV to AAAA reduces complex glycosylation of NKCC1 by ∼90% and results in a protein that does not form stable dimers and is retained in the endoplasmic reticulum in a highly aggregated state. Our results are consistent with the hypothesis that mutation of the hydrophobic tetrad ILLV to AAAA leads to the ab initio misfolding and concomitant aggregation of this NKCC1 mutant, resulting in its retention in the endoplasmic reticulum.

Activation of β-adrenoceptors does not cause any change in cytosolic Ca2+ distribution in rat parotid acinar cells

The effects of the beta-adrenoceptor agonist isoproterenol on the distribution of cytosolic Ca2+ concentrations were studied with digital imaging microscopy in fura-2-loaded rat parotid acinar cells. At concentrations < 10 microM, isoproterenol did not cause any measurable change in cytosolic Ca2+ concentration ([Ca2+]i). Monitoring of [Ca2+]i in selected areas of the acinar cells failed to show that stimulation with isoproterenol causes a localized rise in [Ca2+]i at the apical region close to the lumen. As the maximum response of amylase exocytosis is observed at 0.1 or 1 microM isoproterenol [Tanimura, A., Matsumoto, Y., Tojyo, Y., 1990. Evidence that isoproterenol-induced Ca2+-mobilization in rat parotid acinar cells is not mediated by activation of beta-adrenoceptors. Biochim. Biophys. Acta, 1055, pp. 273-277], the data obtained here indicate that the isoproterenol-induced amylase exocytosis is not accompanied by Ca2+ mobilization. The high concentration (100 microM) of isoproterenol caused a small but significant increase in [Ca2+]i, particularly in the apical region. This response was completely attenuated by the alpha-adrenoceptor antagonist phentolamine, but not by the beta-adrenoceptor antagonist propranolol, indicating that the isoproterenol-induced increase in [Ca2+]i resulted from an activation of alpha-adrenoceptors. Further, the effect of cyclic AMP on Ca2+ release from intracellular Ca2+ stores was studied in saponin-permeabilized acinar cells using the lipophilic Ca2+ indicator Calcium Green C18. Cyclic AMP had no effect on the Ca2+ release, while the same acinar cells responded strongly to inositol 1,4,5-trisphosphate. This result does not support the hypothesis that cyclic AMP directly stimulates Ca2+ mobilization in rat parotid acinar cells.

Thrombin-induced Ca2+ mobilization in human gingival fibroblasts is mediated by protease-activated receptor-1 (PAR-1).

By reverse transcription (RT)-PCR analyses, human gingival fibroblasts (HGFs) were demonstrated to express mRNAs for protease-activated receptor-1 (PAR-1) and PAR-3, although the expression of PAR-3 was much weaker than that of PAR-1. The mRNAs for PAR-2 and PAR-4 were not found by RT-PCR. Furthermore, PAR activation was studied by monitoring cytosolic Ca(2+) concentration ([Ca(2+)]i) in cultured HGFs loaded with fura-2. At concentrations > 0.1 nM, alpha-thrombin caused a transient increase in [Ca(2+)]i in a concentration-dependent manner, and the maximum response was obtained with 10 nM alpha-thrombin. After the [Ca(2+)]i response, the HGFs were completely desensitized to the second stimulation with alpha-thrombin. The PAR-1 agonist peptide SFLLRN produced approximately the same transient [Ca(2+)]i response as alpha-thrombin. After stimulation with SFLLRN, the HGFs did not respond to alpha-thrombin, indicating that treatment with SFLLRN results in complete desensitization to alpha-thrombin. The PAR-2 and PAR-4 agonist peptides had no effect on [Ca(2+)]i in HGFs. These results suggest that alpha-thrombin-induced Ca(2+) mobilization in HGFs is solely mediated by PAR-1.

Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the beta-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca(2+) signalling as intracellular Ca(2+) stores, changes in cytosolic free Ca(2+) ion concentration ([Ca(2+)](i)) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca(2+)](i) induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca(2+) wave towards the basolateral region. The magnitude of the [Ca(2+)](i) response and the speed of the Ca(2+) wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP(3)R) was performed on zymogen granule membranes and microsomes using anti-IP(3)R antibodies. The immunoreactivity of all three IP(3)Rs was clearly observed in the microsomal preparations. Although a weak band of IP(3)R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca(2+) signalling in rat parotid acinar cells.

Visualization of Ins(1,4,5)P3 dynamics in living cells: two distinct pathways for Ins(1,4,5)P3 generation following mechanical stimulation of HSY-EA1 cells

In the present study, the contribution of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] generation on the mechanical-stimulation-induced Ca2+ response was investigated in HSY-EA1 cells. Mechanical stimulation induced a local increase in the cytosolic concentration of Ins(1,4,5)P3 ([IP3]i), as indicated by the Ins(1,4,5)P3 biosensor LIBRAvIII. The area of this increase expanded like an intracellular Ins(1,4,5)P3 wave as [IP3]i increased in the stimulated region. A small transient [IP3]i increase was subsequently seen in neighboring cells. The phospholipase C inhibitor U-73122 abolished these Ins(1,4,5)P3 responses and resultant Ca2+ releases. The purinergic receptor blocker suramin completely blocked increases in [IP3]1 and the Ca2+ release in neighboring cells, but failed to attenuate the responses in mechanically stimulated cells. These results indicate that generation of Ins(1,4,5)P3 in response to mechanical stimulation is primarily independent of extracellular ATP. The speed of the mechanical-stimulation-induced [IP3]i increase was much more rapid than that induced by a supramaximal concentration of ATP (1 mM). The contribution of the Ins(1,4,5)P3-induced Ca2+ release was larger than that of Ca2+ entry in the Ca2+ response to mechanical stimulation in HSY-EA1 cells.

Possible mechanisms regulating ATP- and thimerosal-induced Ca2+ oscillations in the HSY salivary duct cell line

The ATP-induced oscillatory changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)) were analysed in HSY cells, a salivary ductal cell line from human parotid, using a fluorescence ratio imaging system. At concentrations higher than 1 microM, ATP caused sinusoidal [Ca(2+)](i) oscillations due to the periodic release and reuptake of Ca(2+) by intracellular Ca(2+) stores. The phorbol ester 4beta-phorbol 12,13-dibutyrate (PDBu) changed the [Ca(2+)](i) oscillations to a single spike. The inhibitory effect of PDBu on the [Ca(2+)](i) signals was reversed by protein kinase C (PKC) inhibitors such as staurosporine and chelerythrine chloride. However, preincubation of the cells with the PKC inhibitors did not affect the pattern of the ATP-induced [Ca(2+)](i) oscillations. The desensitization of the [Ca(2+)](i) response observed during prolonged stimulation with ATP was also not prevented by the PKC inhibitors. Incubation of HSY cells with the sulphydryl reagent thimerosal, which enhances the sensitivity of inositol 1,4,5-trisphosphate (IP(3)) receptors, caused repetitive Ca(2+) release from intracellular Ca(2+) stores resulting in baseline spikes of [Ca(2+)](i). The thimerosal-induced [Ca(2+)](i) oscillations did not change in the presence of PDBu and the phospholipase C inhibitor U73122. Thus, we could not provide evidence that negative feedback by PKC plays a central role in the regulation of ATP-induced [Ca(2+)](i) oscillations. These results suggest that the [Ca(2+)](i) oscillations, at least the baseline spikes, in HSY cells can be generated without stimulating the formation of IP(3).

Expression of functional Stim1-mKO1 in rat submandibular acinar cells by retrograde ductal injection of an adenoviral vector

OBJECTIVE Adenoviruses are used for gene transfer to salivary glands cells in vivo. We constructed an adenovirus vector that expressed a fusion protein of human Stim1 and the fluorescent protein mKO1 (Ad-Stim1-mKO1), and used it to investigate the molecular dynamics and functions of exogenously expressed proteins in living salivary acinar cells. DESIGN Ad-Stim1-mKO1 was transferred to rat submandibular glands by retrograde ductal injection. Expression and distribution of Stim1-mKO1 were examined by confocal microscopy. In addition, the effects of Stim1-mKO1 on store-operated Ca(2+) entries were examined in fura-2-loaded cells. RESULTS The expression of Stim1-mKO1 was detected in approximately 40% of rat submandibular acini, whereas the expression in HSY-EA1 cells was ∼80%. Confocal microscopy revealed Stim1-mKO1 fluorescence along the endoplasmic reticulum-like network in the cytoplasm of both HSY-EA1 and dispersed acinar cells. The depletion of intracellular Ca(2+) stores with thapsigargin (ThG), a sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitor, led to the translocation of Stim1-mKO1 to the peripheral region in these cells. In addition, expression of Stim1-mKO1 enhanced store-operated Ca(2+) entry in these cells. CONCLUSIONS We succeeded in expressing Stim1-mKO1 fluorescent protein in the salivary glands of live animals by retrograde ductal injection of an adenoviral vector. This method allowed us to investigate the functions and molecular dynamics of these expressed molecules in living salivary acinar cells.