Jun Ichikawa

Comparison of the expression of two immediate early gene proteins, FosB and Fos in the rat preoptic area, hypothalamus and brainstem during pregnancy, parturition and lactation

Medial preoptic area (MPA), supraoptic nucleus (SON), magnocellular (MaPVN) and parvocellular (PaPVN) paraventricular hypothalamic nuclei, and mesencephalic lateral tegmentum (MLT) are involved in maternal behavior, parturition and lactation. This study investigated the FosB and Fos immunoreactivity in these regions of virgin, pregnant, parturient, lactating, and lactating-arrested rats. The patterns of FosB and Fos expression were compared between the sections taken from the same animals. Quantitative immunohistochemistry revealed a significant increase in the numbers of FosB-positive neurons in the MPA, SON, MaPVN, and MLT of parturient and lactating females as compared with pregnant or virgin animals. In lactating rats, the numbers of FosB-positive neurons in the MPA, PaPVN, and MLT were increased, but the numbers in the SON and MaPVN were decreased as compared with parturient females. Many Fos-positive neurons were also seen in parturient and lactating rats, and the patterns of Fos expression in each region were quite similar to those of FosB. Moreover, double-labeling immunohistochemistry revealed that: (1) many FosB-positive nuclei were observed in oxytocin and vasopressin neurons of the SON and PVN in parturient rats; (2) within FosB-positive neurons, 89.5% in the MPA, 86.8% in the MLT of parturient rats, and 92% in the MPA and 90.8% in the MLT of lactating animals were also Fos-positive. Only a small number of FosB and Fos-positive neurons were seen in females that were killed in the early stage of parturition. Removal of the litters immediately after parturition completely eliminated FosB and Fos expression in each region in the dams. Taken together, the present results suggest that FosB expression is co-involved with Fos in the neural activation during parturition and lactation in rats.

Critical Role of TRPC1-Mediated Ca2+ Entry in Decidualization of Human Endometrial Stromal Cells

Decidualization is an ovarian steroid-induced remodeling/differentiation process of uterus essential for embryo implantation and placentation. Here, we investigated the possible involvement of enhanced Ca²⁺ dynamics in the decidualization process in human endometrial stromal cells (hESC) in its connection with a recently emerging nonvoltage-gated Ca²⁺ entry channel superfamily, the transient receptor potential (TRP) protein. Combined application of 17β-estradiol (E₂) (10 nM) and progesterone (P₄) (1 μM) for 7-14 d resulted in morphological changes of hESC characteristic of decidualization (i.e. cell size increase), whereas sole application of E₂ exerted little effects. A 7- to 14-d E₂/P₄ treatment greatly increased the expression level of decidualization markers IGF binding protein-1 (IGFBP-1) and prolactin and also up-regulated the expression of TRPC1, a canonical TRP subfamily member that has been implicated in store-operated Ca²⁺ influx (SOC) in other cell types. In parallel with this up-regulation, SOC activity in hESC, the nuclear translocation of phosphorylated cAMP responsive element binding protein (p-CREB) and the expression of Forkhead box protein 01 were enhanced significantly. Small interfering RNA knockdown of TRPC1 counteracted the E₂/P₄-induced up-regulation of IGFBP-1 and prolactin and enhancement of SOC activity together with the inhibition of hESC size increase, p-CREB nuclear translocation, and FOXO1 up-regulation. Coadministration of SOC inhibitors SK&F96365 or Gd³⁺ with E₂/P₄ also suppressed the up-regulation of IGFBP-1 and hESC size increase. Similar inhibitory effects were observed with extracellularly applied TRPC1 extracellular loop 3-directed antibody, which is known to bind a near-pore domain of TRPC1 channel and block its Ca²⁺ transporting activity. These results strongly suggest that up-regulation of TRPC1 protein and consequent enhancement of SOC-mediated Ca²⁺ influx may serve as a crucial step for the decidualization process of hESC probably via p-CREB-dependent transcriptional activity associated with FOXO1 activation.

Cell density-dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem cells. We investigated intracellular Ca2+ signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca2+ levels ([Ca2+]i). The order of potency followed ATP = UTP > ADP = UDP. ATP‐induced rise in [Ca2+]i was suppressed by U73122 and suramin, but not by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), suggesting the functional expression of G protein‐coupled P2Y2 receptors. RT‐PCR and immunohistochemical studies also showed the expression of P2Y2 receptors. [Ca2+]i response to UTP changed with cell density. The UTP‐induced rise in [Ca2+]i was greatest at high density. Vmax (maximum Ca2+ response) and EC50 (agonist concentration that evokes 50% of Vmax) suggest that the amount and property of P2Y2 receptors were changed by cell density. Note that UTP induced Ca2+ oscillation at only medium cell density. Pharmacological studies indicated that UTP‐induced Ca2+ oscillation required Ca2+ influx by store‐operated Ca2+ entry. Carbenoxolone, a gap junction blocker, enhanced Ca2+ oscillation. Immunohistochemical and quantitative real‐time PCR studies revealed that proliferating cell nuclear antigen (PCNA)‐positive cells declined but the mRNA expression level of the P2Y2 receptor increased as cell density increased. Co‐application of fetal calf serum with UTP induced Ca2+ oscillation at high cell density. These results suggest that the different patterns observed for [Ca2+]i mobilization with respect to cell density may be associated with cell cycle progression. J. Cell. Physiol. 219: 372–381, 2009.

EGF enhances Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

Effects of epidermal growth factor (EGF) on the intracellular Ca2+ ([Ca2+]i) responses to nucleotides, Ca2+ release from thapsigargin‐sensitive stores and capacitative Ca2+ entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti‐PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S‐phase of cell cycle. EGF treatment apparently increased the number of PCNA‐stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca2+]i response. EGF treatment for 1–2 days in culture, however, enhanced [Ca2+]i responses including [Ca2+]i increase by ATP, UTP and other nucelotides, Ca2+ release from thapsigargin‐sensitive stores, as well as capacitative Ca2+ entry. Genistein, a tyrosine kinase inhibitor, prevented EGF‐induced cell proliferation and the [Ca2+ ]i responses in a dose‐dependent manner. These results indicate that EGF treatment enhances Ca2+ mobilization and capacitative Ca2+ entry, well correlated with cellular proliferation in mammary epithelial cells. Copyright

TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells

Ca2+ influx is important for cell cycle progression, but the mechanisms involved seem to vary. We investigated the potential roles of transient receptor potential (TRP) channels and store‐operated Ca2+ entry (SOCE)‐related molecules STIM (stromal interaction molecule)/Orai in the cell cycle progression of rat bone marrow stromal cells (BMSCs), a reliable therapeutic resource for regenerative medicine.

In vitro changes in capacitative Ca2+ entry in neuroblastoma X glioma NG108-15 cells

Changes in capacitative Ca2+ entry were studied in neuroblastoma x glioma NG108-15 cells with fura-2 fluorescence measurements in the following three culture conditions. The application of thapsigargin (250 nM) with a Ca2+-free solution depleted intracellular Ca2+ stores and the capacitative Ca2+ entry was induced by the addition of extracellular Ca2+ in the cells cultured in the medium for proliferation. The capacitative Ca2+ entry decreased in the cells cultured in the medium for neuronal differentiation. When these cells resumed proliferation after changing the culture media to the initial medium for proliferation, the capacitative Ca2+ entry increased again and exceeded the level in the initial proliferation state. These results suggested that the capacitative Ca2+ entry occurred more intensely at the proliferation state than at the neuronally differentiated state.

Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II

•  Ca2+/calmodulin (CaM)‐dependent kinase II (CaMKII) plays pivotal roles in diverse Ca2+‐mediated cellular functions including the physiology/pathophysiology of the cardiovascular system, through modulation of a variety of Ca2+‐permeable channels such as a non‐voltage‐gated Ca2+ channel TRPC6. •  In this study, we investigated the molecular mechanism underlying its positive regulation by CaMKII with chimera, deletion and site‐directed mutagenesis approaches. •  The results indicate that two spatially separated sites of TRPC6 channel, i.e. a distal part of the C‐terminal inositol‐1,4,5‐trisphosphate receptor/CaM binding domain and Thr487 located on the putative first intracellular loop, are crucial for the CaMKII‐mediated regulation of TRPC6 channels. •  This mechanism may serve as an effective positive feedback regulation of Ca2+ influx through TRPC6 channels, in concert with intracellular and transmembrane Ca2+ mobilization upon phospholipase C‐coupled receptor stimulation by neurohormonal factors, thereby fine‐tuning the cardiovascular functions. •  Disruption of these could lead to pathological states such as cardiac hypertrophy and arrhythmia, hypertension and atherosclerosis.

Cortical field potentials preceding self-paced forelimb movements and influences of cerebellectomy upon them in rats.

Seven rats were well trained to move lever to the left by right forelimb at self-pace (self-paced forelimb movements). Cortical field potentials associated with self-paced forelimb movements were recorded by electrodes implanted chronically on the surface and at a 2.0 mm depth in the forelimb motor cortex on the left side. A surface-negative, depth-positive potential starting about 1.0 s prior to the movement was recorded in the rostral part of the forelimb motor cortex. Further we found that the premovement potential was eliminated by the cerebellar hemispherectomy on the right side. This suggests the participation of the cerebellar hemisphere in preparing the activity of the motor cortex before self-paced forelimb movements in rats, by cerebello-thalamo-cortical projections.

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches

Aims Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4. Methods and results Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent Kd = ∼500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+- and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (≥ six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced. Conclusions These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

Serum-free medium with osteogenic supplements induces adipogenesis in rat bone marrow stromal cells

Adult BMSCs (bone marrow stromal cells) contain MSCs (mesenchymal stem cells). MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and myoblasts and are thus considered useful in tissue engineering for therapeutic and clinical purposes. FCS (fetal calf serum) is usually included in the differentiation medium, but the clinical application of FCS may pose a problem for some patients. To improve the efficiency and safety of BMSC cultivation, the effect of SF (serum‐free) conditions on the osteogenic differentiation of rat BMSCs was examined. In the presence of 10% FCS and osteogenic supplements, the cells formed mineralized von Kossa‐positive deposits. Under SF conditions with osteogenic supplementation, however, the cells possessed cytosolic lipid vacuoles that could be stained with Oil Red O. The mRNA expression of PPARγ (peroxisome proliferator‐activated receptor γ), an adipogenic marker, increased under the SF condition with osteogenic supplements. These data indicate that rat BMSCs differentiate into adipocytes under SF conditions even with osteogenic supplementation and that FCS is needed to induce proper osteogenic differentiation in rat BMSCs.