Fluorescent probes for intracellular Mg2+ measurement

Recently, fluorescent probes are widely used as tools for dynamical measurement of ion distributions and concentrations in cells. They are highly sensitive, and offer imaging by the use of fluorescent microscopy in easily and less cell damaging way. This paper discusses the selectivity and optical character of the three novel Mg(2+) fluorescent probes. KMG-20AM offers ratiometric quantative measurement of Mg(2+), KMG-104 provides high-sensitive qualitative analysis and 3-D measurement. With those improved Mg(2+) fluorescent probes, the physiological and pathological role of Mg(2+) are going to be more and more clear.     

Modulation of intracellular Ca2+ by estrogen

Regulatory pathways of intracellular Ca(2+) by estrogen can be classified into two, both of which function in the plasma membrane. One is the rapid rise of intracellular Ca(2+) caused by the physiologic dose of estrogen via estrogen receptor from both extracellular and intracellular sources. The other is the inhibition of the agonist-induced increase in intracellular Ca(2+) by the supraphysiologic dose of estrogen, and this action is not likely to be mediated by estrogen receptor.     

Plasma and intracellular Mg2+ concentrations in pre-eclampsia

Plasma and intra-erythrocytic magnesium concentrations were determined in 27 patients with pre-eclampsia and in 22 healthy pregnant women. In the pre-eclamptic women, the Mg2+ concentrations were measured before and after treatment with Mg2+ salts and after delivery. The plasma Mg2+ concentration was not significantly different in the pre-eclamptic and the healthy pregnant women. The intra-erythrocytic Mg2+ concentration before treatment with Mg2+ was significantly lower in the pre-eclamptic patients than in the healthy pregnant women [1.33 +/- 0.29 versus 1.01 +/- 0.16 mmol/l (means +/- s.d.); P less than 0.05] and increased after treatment with Mg2+ to 1.19 +/- 0.24 mmol/l. Lowered cellular Mg2+ concentrations in pre-eclampsia may contribute to the development of hypertension in this disorder.     

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

Background  

Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function.     

Interrelationship between growth factor-induced pH changes and intracellular Ca2+.

Many mitogens cause rapid changes in intracellular pH and Ca2+. We studied the patterns of pH and Ca2+ changes after exposure of murine fibroblasts to platelet-derived growth factor (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca2+ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. PDGF and bombesin caused a rapid (maximum change, less than 2 min) cytoplasmic acidification of 0.03 pH unit followed by a slower (5-10 min) alkalinization of approximately 0.11 pH unit above the resting pH of 6.88. PMA caused alkalinization without causing the early acidification. Bradykinin caused rapid acidification without the slower net alkalinization. Ionomycin also caused acidification without subsequent alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca2+ response were also determined for each agent. PDGF and bombesin caused a transient increase in cytoplasmic Ca2+ from a resting level of 85 +/- 12 nM to 190 +/- 12 nM within 2 min and return to baseline within 5 min. PMA caused no change in intracellular Ca2+. Bradykinin caused the most rapid (maximum response, less than 20 sec) increase in intracellular Ca2+. For each agonist, the Ca2+ transient could be blocked by buffering intracellular Ca2+ with quin-2. In Ca2+-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. We propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca2+. However, growth factor-induced alkalinization via the Na+/H+ exchanger is independent of changes in Ca2+.     

Antagonist-induced intracellular sequestration of rabbit bradykinin B(2) receptor

In a contractility assay based on the rabbit jugular vein, the structurally related drugs NPC 17731 or icatibant (1 to 3 nmol/L) were insurmountable antagonists of bradykinin (BK) B(2) receptors (B(2)Rs). After ample washing (3 hours), the antagonism exerted by these peptides was not reversible. By contrast, the antagonist LF 16. 0687 (30 to 100 nmol/L) was competitive and reversible. A rabbit B(2)R-green fluorescent protein (B(2)R-GFP) conjugate was expressed in mammalian cells. In COS-1 cells, it exhibited an affinity for [3H]BK (K(D)=1.61 nmol/L) similar to that of the wild-type rabbit B(2)R. The stably expressed construction in HEK-293 cells was functionally active (phospholipase A(2) assay), and the antagonists mentioned above retained their respective surmountable or insurmountable behavior. Competition of [(3)H]BK binding to B(2)R-GFP by the antagonists or BK was largely reversible after a 3-hour washout period at 0 degrees C; at 37 degrees C, icatibant or NPC 17731 effects were not reversible. B(2)R-GFP was visualized in the plasma membranes of HEK-293 cells and rapidly internalized in response to BK. NPC 17731 or icatibant slowly translocated B(2)R-GFP into cells over 24 hours, whereas LF 16.0687 had no effect on the subcellular distribution of B(2)R-GFP. Cell extract immunoblotting with anti-GFP antibodies revealed a 101- to 105-kDa protein that was not significantly degraded on 24 hours of cell treatment with any of the ligands but was translocated in part to the 15 000-g pellet of the extract on treatment with BK or the noncompetitive antagonists. NPC 17731 and icatibant are noncompetitive, nonequilibrium antagonists that promote the cellular sequestration of rabbit B(2)R expressed in an heterologous system.     

von Willebrand factor storage and multimerization: 2 independent intracellular processes

The von Willebrand factor propeptide, vW AgII, has been shown to be required for the formation of vWF multimers and sorting of vWF to storage granules; whether these 2 processes are independent events has been unclear. Chimeric constructs of human and canine vWF were developed to further define these processes and to determine whether they are independent intracellular events. Cells expressing only mature vWF (Deltapro) produced vWF dimers that were not stored in AtT-20 cells; whereas the expression of vW AgII alone resulted in vW AgII granular storage. Expression of vW AgII in trans with Deltapro resulted in the multimerization of vWF and colocalized storage of vW AgII and vWF. Expression of canine vW AgII in trans or cis with human Deltapro resulted in the multimerization of human vWF, with no storage of human vWF but with normal storage of canine vW AgII. This dissociation of functions indicates that the signals for multimerization of vWF are different from the signals for trafficking of vWF to storage and demonstrates that vWF storage and multimerization are 2 independent intracellular processes. vW AgII contains the signal(s) required for trafficking to storage, and only through interaction with vW AgII is vWF chaperoned into granules. (Blood. 2000;96:1808-1815)     

Prostaglandin E2-induced luteinizing hormone-releasing hormone release involves mobilization of intracellular Ca+2

The present in vitro experiments were performed to examine the involvement of Ca+2 in the mechanism by which prostaglandin E2 (PGE2) induces LHRH release from the median eminence (ME) of the hypothalamus. Stepwise decreases in the Ca+2 concentration of the incubation medium reduced the LHRH response to PGE2. Nevertheless, neither complete omission of Ca+2 (residual Ca+2 concentration, 3.5 microM) nor chelation of residual Ca+2 with EGTA prevented the stimulatory effect of the PG, suggesting that a significant portion of the PGE2 effect on LHRH release is independent of extracellular Ca+2. Blockade of Ca+2 influx with verapamil, a Ca+2 entry blocker, demonstrated that this component of the PGE2 effect is completely independent of inward Ca+2 movement. Depletion of intraterminal Ca+2 stores by exposure of the MEs to the Ca+2 ionophore A23187 in medium without Ca+2 containing EGTA almost completely obliterated the subsequent LHRH response to PGE2, indicating that normal intraterminal Ca+2 levels are important for the PGE2 effect to occur. Preloading the ME terminals with 45Ca+2 and subsequent stimulation with PGE2 demonstrated that even in the absence of extracellular Ca+2, PGE2 stimulates Ca+2 efflux from the terminals, and this Ca+2 movement occurs temporarily associated with LHRH release. Depolarization of ME terminals with 56 mM K+ in the presence of normal Ca+2 concentration resulted in massive efflux of 45Ca+2 and a greater LHRH response than that produced by PGE2, suggesting that the effect of PGE2 is not the consequence of a nonspecific general depolarization of ME nerve terminals. Thus, although a full LHRH response to (exogenous) PGE2 necessitates normal extraterminal Ca+2 concentrations, the results indicate that translocation of Ca+2 from intracellular stores is an event involved in the mechanism by which PGE2 releases LHRH.     

Effects of prostaglandin F2 alpha on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes

OBJECTIVE: We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2 alpha (PGF2 alpha) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. METHODS: Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. RESULTS: PGF2 alpha (3 nmol l-1-3 mumol l-1 increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2 alpha, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2 alpha (3 mumol l-1 caused a reversible rise in intracellular pH of 0.08 +/- 0.01 pH units (n = 5, p < 0.05). The Na(+)-H+ exchanger inhibitor, HOE 694 (10 mumol l-1, abolished the PGF2 alpha-induced rise in pH and the increase in cell shortening. PGF2 alpha-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 mumol l-1. CONCLUSION: The positive inotropic action of PGF2 alpha appears to be mediated via activation of the Na(+)-H+ exchanger with the possible involvement of PKC. This suggests that PGF2 alpha-produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium.     

Clioquinol and pyrithione activate TRPA1 by increasing intracellular Zn2+

The antifungal and amoebicidal drug clioquinol (CQ) was withdrawn from the market when it was linked to an epidemic of subacute myelo-optico-neuropathy (SMON). Clioquinol exerts its anti-parasitic actions by acting as a Cu/Zn chelator and ionophore. Here we show that local injections of CQ produce mechanical hyperalgesia and cold hypersensitivity through a mechanism involving TRPA1 in mice. We also show that CQ activates TRPA1 in a Zn²⁺-dependent manner. Using a different Zn²⁺-ionophore, zinc pyrithione (ZnPy), we demonstrate that low, nanomolar concentrations of intracellular Zn²⁺ ([Zn²⁺]_i) stimulate TRPA1. Direct application of Zn²⁺ to the intracellular face of excised, inside-out patches activates TRPA1 with an EC₅₀ value of 7.5 ± 1 nM. TRPA1 is expressed in a subpopulation of nociceptive dorsal root ganglion (DRG) neurons, where it acts as a sensory receptor for environmental irritants and oxidants. Using cultured DRG neurons from wild-type and TRPA1-deficient mice, we demonstrate that TRPA1 is the principal excitatory receptor for increased [Zn²⁺]_i in DRG neurons. In conclusion, we have discovered that TRPA1 acts a sensor of intracellular Zn²⁺, and that Zn²⁺ ionophores, such as CQ and ZnPy, activate TRPA1 by increasing [Zn²⁺]_i. We also demonstrate that CQ-evoked mechanical hyperalgesia and cold allodynia require TRPA1 in vivo.     

AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na⁺/H⁺-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.     

异丙肾上腺素对心肌细胞内钙释放和肌浆网内钙容量的影响

目的 :心肌细胞膜上的 β-肾上腺素受体的激活对兴奋 -收缩耦联 （ECC）过程有重要的调节作用。本课题利用异丙肾上腺素 （ISO,1μmol/ L）激活 β-肾上腺素受体从而研究其对源自心肌细胞肌浆网的胞内钙释放 （ECC的重要环节 ）和肌浆网内钙容量的影响 ,进而分析钙释放与钙容量之间的关系。方法 :局部场刺激作用于成年大鼠心肌细胞 ,促使后者产生动作电位 ,进而诱发胞内钙瞬变 （ACT） ,由 ACT可估测胞内钙释放。肌浆网内钙容量则由咖啡因 （2 0 m mol/ L）诱发的钙瞬变 （CCT）估测。实验结果均由 Zeiss L SM- 5 10激光共聚焦显微镜系统记录。结果 :ISO作用下的 ACT峰值为 10 .2 9± 0 .35 （n=13）比正常情况下的 5 .74± 0 .2 7（n=18）高 （P<0 .0 1）。 ISO作用下的CCT峰值为 11.2 3± 0 .2 9（n=13）比正常情况下的 7.6 2± 0 .2 4 （n=18）高 （P<0 .0 1）。结论 :ISO可明显地提高心肌细胞内钙释放量和肌浆网内的钙容量。不管有无 ISO存在 ,胞内钙释放量总是只占肌浆网内钙容量的一部分。在正常情况下 ,心肌的钙释放量有较大的储备能力 ,且此储备可因β-肾上腺素受体的激活而动员。     

Down-regulated insulin receptors in HepG2 cells have an altered intracellular itinerary

The delivery of insulin and the insulin receptor into an intracellular compartment may be important for eliciting some of the biologic responses of the cell to the hormone. Internalization of insulin-receptor complexes in cells from hyperinsulinemic type II diabetic patients is diminished, suggesting a possible role for this cellular process in insulin resistance. To examine whether hyperinsulinemia contributes to defective insulin-receptor processing in vitro, cultured hepatoma cells (HepG2) were incubated with high concentrations of (500 ng/ml) insulin from 1-3 days. Insulin induced a decrease in the number of total and surface insulin receptors within 24 hours; however, the hormone did not mediate a change in the number of intracellular receptors. The cellular itinerary of control and down-regulated receptors were then compared. Insulin mediated internalization of down-regulated receptors was impaired compared to control receptors; however, the down-regulated receptors that were internalized recycled back to the plasma membrane more efficiently. By covalently labeling the insulin receptor with the photoactive insulin derivative, 125I-NAPA-DP-insulin, it was demonstrated that the rates of receptor degradation of down-regulated and control receptors were similar. These results suggest that incubating HepG2 cells with high concentrations of insulin alters the cellular itinerary of the insulin receptor.     

Monoclonal antibodies increase intracellular Ca2+ in sea urchin spermatozoa.

Changes in intracellular free Ca2+ ([Ca2+]i) of sea urchin (Strongylocentrotus purpuratus) spermatozoa were measured using the fluorescent Ca2+ indicators fura-2 and indo-1. The intracellular pH (pHi) of sperm was also determined. The fucose sulfate-rich glycoconjugate component of egg jelly induced increases in [Ca2+]i and pHi in sperm and induced the acrosome reaction. Monoclonal antibodies (mAbs) to external domains of a 210-kDa glycoprotein of the sperm plasma membrane induced a 23-fold increase in [Ca2+]i (vs. 9-fold for fucose sulfate-rich glycoconjugate), but the mAbs did not cause the pHi to increase and did not induce the acrosome reaction. When the mAb treatment which induced an increase in [Ca2+]i was combined with an NH4Cl treatment, which increased the pHi, the acrosome reaction was induced. mAb-induced increases in [Ca2+]i were dependent on millimolar concentrations of extracellular Ca2+ and were reversed by placing sperm in Ca2+-free seawater or by chelating Ca2+ with EGTA. The mAb-induced [Ca2+]i increase was sensitive to the pH of the seawater, although mAb binding was not. The data show that increased [Ca2+]i and pHi are necessary for induction of the acrosome reaction and suggest that the 210-kDa protein may play a role in regulating Ca2+ entry into the spermatozoan. These mAbs make it possible to separate the increase in [Ca2+]i from the increase in pHi and may be useful in the elucidation of the regulatory role of Ca2+ in sperm physiology.     

The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants

The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.     

The intracellular Ca2+-homeostasis influences the frequency-dependent force-generation in man

The present study invesitgates the effect of stimulation frequency and external Ca²⁺-concentration on intracellular systolic and diastolic Ca²⁺ as well as on the force-frequency relationship (FFR, 0.5 to 3.0 Hz, 1.0 mmol/l extracellular Ca²⁺) in human myocardium using fura-2 AM loaded electrically stimulated right atrial muscle strips (coronary bypass surgery, n = 15, age: 60.0 ± 1.9 years). The FFR was positive (3.0 vs. 0.5 Hz: 184 ± 43 % of basal value) and linked to an increase in peak systolic (R340/380 _sys, 119 ± 7 %) as well as diastolic Ca²⁺ (R340/380_ED, Δ fura-2 ratio +0.20 ± 0.02). After elevating the extracellular Ca²⁺ concentration from 1.0 to 2.4 mmol/l, force of contraction (FOC) increased from 0.5 up to 1.0 Hz (128 ± 8 %) and declined after further augmentation of stimulation frequency (3.0 Hz: 87 ± 15 %). However, this decrease in FOC was accompanied by an increase in diastolic Ca²⁺ (Δ fura-2 ratio +0.45 ± 0.08), while systolic Ca²⁺ declined at high stimulation frequencies. In conclusion, the frequency-dependent force generation is accompanied by an increase in both systolic and diastolic Ca²⁺ levels. Thus, especially at high stimulation frequencies the Ca²⁺-lowering mechanisms may become crucial and may be responsible for the blunted force-frequency relationship in failing human myocardium. Received: 2 November 1998, Returned for revision: 30 November 1998, Revision received: 3 February 1999, Accepted: 10 February 1999     

Insulin decreases intracellular oxidative stress in patients with type 2 diabetes mellitus

Patients affected by diabetes mellitus have oxidative stress with an impaired glutathione (GSH) redox state. The objective of this study was to determine the influence of insulin on oxidative stress, defined as a reduced intracellular GSH/GSH disulfide (GSSG) ratio and lipid peroxidation by plasma thiobarbituric acid reactive substances (TBARSs) in patients with type 2 diabetes. Two experimental interventions were used: (1) measurement of GSH/GSSG ratio after insulin incubation in erythrocytes from 10 type 2 diabetic patients, and (2) measurement of intraerythrocytic GSH/GSSG ratio and plasma TBARS in 14 type 2 diabetic patients during an in vivo hyperinsulinemic condition obtained from a euglycemic hyperinsulinemic clamp study. We confirmed that our patients underwent oxidative stress as shown by the significant difference in intracellular GSH/GSSG ratio in diabetic patients as compared to controls (13.56+/-3.84 vs 27.89+/-8.37, P<.0001). We found a significant elevation in the GSH/GSSG ratio after 2 hours of incubation with insulin in erythrocytes from diabetic patients (11.56+/-1.98 to 15.61+/-2.62, P<.001). During the clamp studies, GSH/GSSG ratio had already increased after 60 minutes and even more after 120 minutes (baseline, 15.04+/-4.19; at 60 minutes, 19.74+/-6.33; at 120 minutes, 25.33+/-11.15; P<.0001). On the contrary, no significant changes were observed in plasma TBARS (3.59+/-0.77 to 3.56+/-0.83, NS). We conclude that insulin in patients with type 2 diabetes mellitus can reduce intracellular oxidative stress through increased GSH/GSSG ratio.