Estradiol effects on the dopamine transporter – protein levels, subcellular location, and function

Background

The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause.

Results

The activity of the DA transporter (DAT) was measured by the specific uptake of ³H-DA. We found that low concentrations (10^-14 to 10^-8 M) of 17β-estradiol (E₂) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E₂ exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-α (ERα) were elevated in cell extracts by a 20 ng/ml 2 day NGFβ treatment, while ERβ was not. DAT, ERα and ERβ were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E₂. At cell densities above 15,000 cells per well, the E₂-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E₂ treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20–30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5–60 min E₂ treatment, though immunoblot analyses demonstrated no total cellular loss of protein.

Conclusion

Our results suggest that physiological levels of E₂ may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.     

Intracellular Ca2+ regulating proteins in vascular smooth muscle cells are altered with type 1 diabetes due to the direct effects of hyperglycemia

Background

Diminished calcium (Ca²⁺) transients in response to physiological agonists have been reported in vascular smooth muscle cells (VSMCs) from diabetic animals. However, the mechanism responsible was unclear.

Methodology/Principal Findings

VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP₃R) and the SR Ca²⁺ pumps (SERCA 2 and 3). Generally, a decrease in IP₃R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP₃R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca²⁺ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca²⁺ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca²⁺ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca²⁺ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca²⁺ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.

Conclusions/Significance

This work demonstrates that the previously-reported reduced Ca²⁺ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP₃R Ca²⁺ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.     

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Background

Previous studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44^mapk) and ERK2 (p42^mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca²⁺ and Ca²⁺-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F_2α.

Results

Pretreatment of the cells with the Ca²⁺ chelators BAPTA-AM or EGTA, as well as the Ca²⁺ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca²⁺/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF_2α.

Conclusion

The present data indicate that Ca²⁺ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.     

Atypical properties of a conventional calcium channel β subunit from the platyhelminth Schistosoma mansoni

Background

Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP₃/Ca²⁺ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca²⁺ signaling and InsP₃-induced Ca²⁺ release from the endoplasmic reticulum (ER).

Results

Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca²⁺]_i changes into a sustained increase. Intraluminal Ca²⁺ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP₃-induced Ca²⁺ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP₃ receptor Ca²⁺ channel for InsP₃. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP₃-induced Ca²⁺ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP₃/Ca²⁺ signaling pathway for 5-HT, because IBMX potentiated Ca²⁺-dependent Cl^- transport activated by a threshold 5-HT concentration.

Conclusion

This report shows, for the first time for an insect system, that cAMP can potentiate InsP₃-induced Ca²⁺ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP₃/Ca²⁺ signaling pathways enhances transepithelial electrolyte transport.     

On the role of extracellular Ca2+ for prolactin release and adenosine 3′:5′-monophosphate formation induced by thyroliberin in cultured rat pituitary cells

In cultured rat pituitary tumour cells (GH₃ cells) the absence of extracellular Ca⁺⁺ or addition of NaEGTA reduced spontaneous prolactin (PRL) release and abolished the stimulatory effect of thyroliberin (TRH). Readdition of CaCl₂, but not of equimolar concentrations of MgCl₂ increased spontaneous hormone release, and restored the effect of TRH. The calcium ionophore, A-23187, induced PRL release during normal calcium conditions, but not when an excess NaEGTA was present. TRH increased cyclic AMP accumulation in the presence and the absence of extracellular calcium. The effect of TRH on PRL release and cyclic AMP formation occured concomitantly with an increased efflux of ⁴⁵Ca²⁺. Intracellular electrophysiological recordings from the same single cells before and after TRH activation showed increased frequency and duration of the Ca²⁺ dependent action potentials. We conclude that TRH elevates the Ca²⁺ influx which depends on the depolarizing action current, and this effect is probably linked to formation of cyclic AMP and PRL release.     

Puerarin Suppresses Proliferation of Endometriotic Stromal Cells Partly via the MAPK Signaling Pathway Induced by 17?-estradiol-BSA

Background

Puerarin is a major isoflavonoid compound extracted from Radix puerariae. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E₂-BSA).

Methodology

ESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway.

Principal Findings

Our data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E₂). E₂-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E₂-BSA.

Conclusions/Significance

Puerarin suppresses proliferation of ESCs induced by E₂-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.     

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

Background

Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca²⁺-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca²⁺ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca²⁺ influx mediated by mechanosensitive Ca²⁺ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.

Results

We here identified and investigated OsMCA1, the sole rice homolog of putative Ca²⁺-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca²⁺ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca²⁺ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca²⁺-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.

Conclusions

We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca²⁺-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca²⁺ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.     

Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons

Background

Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP₃-gated thapsigargin sensitive intracellular calcium (Ca²⁺) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn²⁺) that has been reported to play a role in cell signaling.

Results

In primary cultured cortical cells (neurons) labeled with intracellular fluorescent Zn²⁺ indicators, we showed that intracellular regions of Zn²⁺ staining co-localized with the endoplasmic reticulum (ER). The latter was identified with ER-tracker Red, a marker for ER. The colocalization was abolished upon exposure to the Zn²⁺ chelator TPEN, indicating that the local Zn²⁺ fluorescence represented free Zn²⁺ localized to the ER in the basal condition. Blockade of the ER Ca²⁺ pump by thapsigargin produced a steady increase of intracellular Zn²⁺. Furthermore, we determined that the thapsigargin-induced Zn²⁺ increase was not dependent on extracellular Ca²⁺ or extracellular Zn²⁺, suggesting that it was of intracellular origin. The applications of caged IP₃ or IP₃-3Kinase inhibitor (to increase available IP₃) produced a significant increase in intracellular Zn²⁺.

Conclusions

Taken together, these results suggest that Zn²⁺ is sequestered into thapsigargin/IP₃-sensitive stores and is released upon agonist stimulation.     

Tectoridin,a poor ligand of estrogen receptor α, exerts its estrogenic effects via an ERK-dependent pathway

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17β-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER α as compared to 17β-estradiol and genistein. Despite poor binding to ER α, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER α at Ser¹¹⁸. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

Dephosphorylation of Ca²⁺ channels by the Ca²⁺-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca²⁺-dependent inactivation of Ca²⁺ current in rat pituitary tumor (GH₃) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca²⁺ to the channel protein, the alternative ``calcineurin hypothesis' has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH₃ cells. To determine if calcineurin plays a part in the voltage- and/or Ca²⁺-dependent components of dihydropyridine-sensitive Ca²⁺ current decay, we rapidly altered the intracellular Ca²⁺ buffering capacity of GH₃ cells by flash photolysis of DM-nitrophen, a high affinity Ca²⁺ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca²⁺ current inactivation in a two-pulse voltage protocol with Ca²⁺ as the charge carrier, but had no effect when Ba²⁺ was substituted for Ca²⁺. Despite confirmation of the abundance of calcineurin in the GH₃ cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca²⁺-dependent inactivation of Ca²⁺ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca²⁺-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca²⁺-dependent inactivation after photolysis. The results provide strong evidence against Ca²⁺-dependent dephosphorylation as the mechanism of Ca²⁺ current inactivation in GH₃ cells, but support the alternative idea that Ca²⁺-dependent inactivation reflects a direct effect of intracellular Ca²⁺ on channel gating. Received: 12 August 1996/Revised: 21 October 1996     

The pharmacodynamic action of the cyclic AMP phosphodiesterase inhibitor rolipram on prolactin producing rat pituitary adenoma (GH4C1) cells

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low K_m cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH₄C₁) cells in culture (ED₅₀=5·10^–8 M). This effect is due to a selective inhibition of the low K_m cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca²⁺/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the _i family (G _i2 orG _k). Rolipram did not affect phosphoinositide metabolism (i.e. IP₃ accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20–1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity.Long term incubation of GH₄C₁ cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of -receptor number, AC activation and cAMP accumulation, while Ro 20–1724 brought about a marked down-regulation and desensitization of the AC complex.In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in -adrenoceptor AC downregulation. These features are not shared by the other drugs tested.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca²⁺ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH₃) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca²⁺ concentrations and inhibited by higher (μM range) concentrations of the cation. A 2–3-fold stimulation of the activity in response to Ca²⁺ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca²⁺ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca²⁺ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca²⁺ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca²⁺. Mg²⁺ concentrations in the incubation also influenced the Ca²⁺ concentration dependence of adenylate cyclase; at high Mg²⁺ more Ca²⁺ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH₃ cells only in the presence of Ca²⁺; as Ca²⁺ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca²⁺ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH₃ cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca²⁺. Increasing free Ca²⁺ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

HSV-mediated expression of interleukin-4 in dorsal root ganglion neurons reduces neuropathic pain

Background

Triptans, 5-HT_1B/ID agonists, act on peripheral and/or central terminals of trigeminal ganglion neurons (TGNs) and inhibit the release of neurotransmitters to second-order neurons, which is considered as one of key mechanisms for pain relief by triptans as antimigraine drugs. Although high-voltage activated (HVA) Ca²⁺ channels contribute to the release of neurotransmitters from TGNs, electrical actions of triptans on the HVA Ca²⁺ channels are not yet documented.

Results

In the present study, actions of zolmitriptan, one of triptans, were examined on the HVA Ca²⁺ channels in acutely dissociated rat TGNs, by using whole-cell patch recording of Ba²⁺ currents (I_Ba) passing through Ca²⁺ channels. Zolmitriptan (0.1–100 μM) reduced the size of I_Ba in a concentration-dependent manner. This zolmitriptan-induced inhibitory action was blocked by GR127935, a 5-HT_1B/1D antagonist, and by overnight pretreatment with pertussis toxin (PTX). P/Q-type Ca²⁺ channel blockers inhibited the inhibitory action of zolmitriptan on I_Ba, compared to N- and L-type blockers, and R-type blocker did, compared to L-type blocker, respectively (p < 0.05). All of the present results indicated that zolmitriptan inhibited HVA P/Q- and possibly R-type channels by activating the 5-HT_1B/1D receptor linked to G_i/o pathway.

Conclusion

It is concluded that this zolmitriptan inhibition of HVA Ca²⁺ channels may explain the reduction in the release of neurotransmitters including CGRP, possibly leading to antimigraine effects of zolmitriptan.     

Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle

Background

The multisubunit (α_1S,α₂-δ, β_1a and γ₁) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca²⁺ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca²⁺ channel. To more fully investigate the function of the γ₁ subunit in these two processes, we produced mice lacking this subunit by gene targeting.

Results

Mice lacking the DHPR γ₁ subunit (γ₁ null) survive to adulthood, are fertile and have no obvious gross phenotypic abnormalities. The γ₁ subunit is expressed at approximately half the normal level in heterozygous mice (γ₁ het). The density of the L-type Ca²⁺ current in γ₁ null and γ₁ het myotubes was higher than in controls. Inactivation of the Ca²⁺ current produced by a long depolarization was slower and incomplete in γ₁ null and γ₁ het myotubes, and was shifted to a more positive potential than in controls. However, the half-activation potential of intramembrane charge movements was not shifted, and the maximum density of the total charge was unchanged. Also, no shift was observed in the voltage-dependence of Ca²⁺ transients. γ₁ null and γ₁ het myotubes had the same peak Ca²⁺ amplitude vs. voltage relationship as control myotubes.

Conclusions

The L-type Ca²⁺ channel function, but not the SR Ca²⁺ release triggering function of the skeletal muscle dihydropyridine receptor, is modulated by the γ₁ subunit.     

The role of sphingosine kinase 1/sphingosine-1-phosphate pathway in the myogenic tone of posterior cerebral arteries

Aims

The goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, we assessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signaling pathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions.

Methods and Results

We measured pressure-induced myogenic response, Ca²⁺ concentration, and 20 kDa myosin light chain phosphorylation (MLC₂₀) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevated transmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca²⁺ and in the presence of mechanosensitive ion channel and voltage-sensitive Ca²⁺ channel blockers. Pressure-induced myogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented by treatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P further augmented pressure-induced myogenic responses. Pressure induced an increase in Ca²⁺ concentration leading to the development of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-induced increased Ca²⁺ concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenic tone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenous S1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), and CAY10444 (S1P3). MLC₂₀ phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg and exogenous S1P further increased MLC₂₀ phosphorylation. The pressure-induced increase of MLC₂₀ phosphorylation was inhibited by pre-treatment of arteries with SKI.

Conclusions

Our results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenic responses in rabbit PCAs under normal physiological conditions.     

Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes

Background

It has been found that gap junction-associated intracellular Ca²⁺ [Ca²⁺]_i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca²⁺ signaling, in particular the basal [Ca²⁺]_i activities, is unclear.

Methods and Results

Global and local Ca²⁺ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca²⁺ transients and local Ca²⁺ sparks in monolayer NRVMs and Ca²⁺ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP₃ butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca²⁺ signal and LY uptake by gap uncouplers, whereas blockade of IP₃ receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca²⁺ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca²⁺ signaling regulation in cardiomyocytes.

Conclusions

These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca²⁺ signaling in normal ventricular myocytes, in which IP₃/IP₃ receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca²⁺ activities in cardiomyocytes.