Inhibitory effect of nitric oxide on voltage-dependent calcium currents in rat dorsal root ganglion cells

The effect of nitric oxide (NO) on calcium current (I(Ca)) and intracellular calcium concentration ([Ca(2+)](i)) in primarily cultured dorsal root ganglion (DRG) neurons was investigated from neonatal rats. I(Ca) and [Ca(2+)](i) were simultaneously recorded using perforated-patch technique in combination with fluorescence measurement from single DRG neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), inhibited I(Ca) in small-diameter neurons without significant change in voltage-dependence of activation and activation time constants. SNP and SNAP also reduced the transient [Ca(2+)](i) peak accompanied by I(Ca). Inhibition by NO was reproducible, but gradually desensitized. In some DRG neurons, SNP and SNAP increased basal [Ca(2+)](i) in concentration of 10 microM with little effect on NO-induced inhibition of I(Ca). 8-Br-cGMP, a permeable cGMP analog, mimicked the effects of SNP and SNAP. These results suggest that, in DRG neurons, NO has inhibitory effect on I(Ca), which is independent of NO-induced increase of basal [Ca(2+)](i), through cGMP-dependent pathway.     

Novel gating and sensitizing mechanism of capsaicin receptor (TRPV1): tonic inhibitory regulation of extracellular sodium through the external protonation sites on TRPV1

Transient receptor potential V1 (TRPV1) is a nonselective cation channel expressed in nociceptors and activated by capsaicin. TRPV1 detects diverse stimuli, including acid, heat, and endogenous vanilloids, and functions as a molecular integrator of pain perception. Herein we demonstrate a novel regulatory role of extracellular Na(+) ([Na(+)](o)) on TRPV1 function. In human embryonic kidney 293 cells expressing porcine TRPV1, low [Na(+)](o) evoked increases of [Ca(2+)](i) that were suppressed by TRPV1 antagonists and facilitated responses to capsaicin, protons, heat, and an endovanilloid. [Na(+)](o) removal simultaneously elicited a [Ca(2+)](i) increase and outward-rectified current with a reversal potential similar to those of capsaicin. Neutralization of the two acidic residues which confer the proton sensitivity to TRPV1 resulted in a reduction of low [Na(+)](o)-induced responses. In primary culture of porcine sensory neurons, the removal of [Na(+)](o) produced a [Ca(2+)](i) increase and current responses only in the cells responding to capsaicin. Low [Na(+)](o) evoked a [Ca(2+)](i) increase in sensory neurons of wild type mice, but not TRPV1-null mice, and in human embryonic kidney 293 cells expressing human TRPV1. The present results suggest that [Na(+)](o) negatively regulates the gating and polymodal sensitization of the TRPV1 channel. [Na(+)](o) surrounding several proton-sensitive sites on the extracellular side of the pore-forming loop of the TRPV1 channel may play an important role as a brake to suppress the excessive activity of this channel under physiological conditions.     

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.     

Choroid plexus ependymal cells enhance neurite outgrowth from dorsal root ganglion neurons in vitro

The epithelial cells of the choroid plexus are a continuation of the ventricular ependymal cells and are regarded as modified ependymal cells. The present study was carried out to determine the influence of choroid plexus ependymal cells (CPECs) on axonal growth in vitro. Choroid plexuses were dissected from the fourth ventricle of postnatal day-1–10 mice, mechanically dissociated, and plated in fibronectin-coated culture dishes. CPECs had spread into monolayers with few endothelial cells in 3-week cultures. Some macrophages were scattered on the monolayer of CPECs. Dorsal root ganglia (DRG) were excised from mouse fetuses of 14-day gestation, dissociated with trypsin and cocultured on the CPEC monolayers. For comparison, dissociated DRG neurons were cocultured on astrocyte monolayers or cultured on laminin-coated plates. After 4.5 h culturing, the cultures were fixed and immunohistochemically double-stained for neurites and CPECs using antibodies against β-tubulin III and S-100 β, respectively. It was demonstrated that neurons extended many long neurites with elaborate branching on the surface of S-100-stained CPECs. In contrast, DRG neurons cultured on the astrocytes and on the laminin-coated plates had much shorter primary neurites with fewer branches than those cultured on the CPECs. The total length of neurites including primary neurites and their branches, of a single DRG neuron was 285 ± 14, 395 ± 15 and 565 ± 12 μm on the laminin-coated plates, on astrocytes and on CPECs, respectively. Scanning electron microscopy revealed extension of neurites with well-developed growth cones on the ependymal cells. These results suggest that CPECs have a great capacity to promote neurite outgrowth from DRG neurons in vitro.     

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

Interactions between hypoxia and hypercapnic acidosis on calcium signaling in carotid body type I cells

The effects of hypercapnic acidosis and hypoxia on intracellular Ca(2+) concentration ([Ca(2+)](i)) were determined with Indo 1 in enzymatically isolated single type I cells from neonatal rat carotid bodies. Type I cells responded to graded hypoxic stimuli with graded [Ca(2+)](i) rises. The percentage of cells responding was also dependent on the severity of the hypoxic stimulus. Raising CO(2) from 5 to 10 or 20% elicited a significant increase in [Ca(2+)](i) in the same cells as those that responded to hypoxia. Thus both stimuli can be sensed by each individual cell. When combinations of hypoxic and acidic stimuli were given simultaneously, the responses were invariably greater than the response to either stimulus given alone. Indeed, in most cases, the response to hypercapnia was slightly potentiated by hypoxia. These data provide the first evidence that the classic synergy between hypoxic and hypercapnic stimuli observed in the intact carotid body may, in part, be an inherent property of the type I cell.     

In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.     

Altered intracellular calcium homeostasis in cerebellar granule cells of prion protein-deficient mice

Previous studies have indicated that recombinant cellular prion protein (PrP(C)), as well as a synthetic peptide of PrP(C), affects intracellular calcium homeostasis. To analyze whether calcium homeostasis in neurons is also affected by a loss of PrP(C), we performed microfluorometric calcium measurements on cultured cerebellar granule cells derived from prion protein-deficient (Prnp(0/0)) mice. The resting concentration of intracellular free calcium [Ca(2+)](i) was found to be slightly, but significantly, reduced in Prnp(0/0) mouse granule cell neurites. Moreover, we observed a highly significant reduction in the [Ca(2+)](i) increase after high potassium depolarization. Pharmacological studies further revealed that the L-type specific blocker nifedipine, which reduces the depolarization-induced [Ca(2+)](i) increase by 66% in wild-type granule cell somas, has no effect on [Ca(2+)](i) in Prnp(0/0) mouse granule cells. Patch-clamp measurements, however, did not reveal a reduced calcium influx through voltage-gated calcium channels in Prnp(0/0) mice. These data clearly indicate that loss of PrP(C) alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage-gated calcium channels.     

Albumin-bound lipids induce free cytoplasmic calcium oscillations in human osteoblast-like cells

[Ca(2+)](i) oscillations were found in human osteoblast-like cells (hOB cells) exposed to high-lipid bovine serum albumin (BSA), but not when exposed to low-lipid BSA. These [Ca(2+)](i) oscillations were inhibited by heptanol and suramin, which implies that gap junctions and purinergic signalling may be important for these [Ca(2+)](i) oscillations. The high-lipid BSA preparation that was used contains arachidonic acid. [Ca(2+)](i) oscillations could be induced by low lipid albumin with arachidonic acid added. The albumin-bound lipids were also important for osteoblast growth since DNA synthesis and the total cell protein content was higher in hOB cells exposed to high-lipid BSA. The effect of arachidonic acid on hOB cell proliferation was bone-donor dependent; both stimulatory and inhibitory effects were observed. The physiological importance of albumin-bound lipids is unclear; given that albumin has only minimal contact with osteoblasts under normal conditions. Only when bone capillaries are disrupted, e.g. during a fracture, would significant amounts of albumin reach osteoblasts. Albumin-bound lipids could therefore contribute to stimulation of osteoblast proliferation during fracture healing.     

Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons

Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2-based ratiometric Ca(2+)-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 μM, 100 μM and 300 μM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca(2+) imaging protocols revealed that HiK(+) (30 mM)-induced intracellular free Ca(2+) ([Ca(2+)](i)) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 μM, 100 μM and 300 μM LEV (respectively), without any significant effect on basal levels of [Ca(2+)](i). This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca(2+), providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.     

Intracellular Ca(2+) signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin

Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 microg/ml) and angiostatin (5 microg/ml) elicited transient, approximately threefold increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca(2+)](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca(2+) signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP(3)) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca(2+)](i), and endostatin rapidly elevated endothelial cell IP(3) levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca(2+) signal. Removal of extracellular Ca(2+) inhibited the endostatin-induced rise in [Ca(2+)](i), as did a subset of Ca(2+)-entry inhibitors. Peak Ca(2+) responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca(2+)](i) in response to vascular endothelial growth factor or to fibroblast growth factor by approximately 70%. Intracellular Ca(2+) signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.     

Fluid flow induced calcium response in osteoblasts: mathematical modeling

Fluid flow in the bone lacuno-canalicular network can induce dynamic fluctuation of intracellular calcium concentration ([Ca(2+)](i)) in osteoblasts, which plays an important role in bone remodeling. There has been limited progress in the mathematical modeling of this process probably due to its complexity, which is controlled by various factors such as Ca(2+) channels and extracellular messengers. In this study we developed a mathematical model to describe [Ca(2+)](i) response induced by fluid shear stress (SS) by integrating the major factors involved and analyzed the effects of different experimental setups (e.g. [Ca(2+)](i) baseline, pretreatment with ATP). In this model we considered the ATP release process and the activities of multiple ion channels and purinergic receptors. The model was further verified quantitatively by comparing the simulation results with experimental data reported in literature. The results showed that: (i) extracellular ATP concentration has more significant effect on [Ca(2+)](i) baseline (73% increase in [Ca(2+)](i) with extracellular ATP concentration varying between 0 and 10 μM), as compared to that induced by SS (25% variation in [Ca(2+)](i) with SS varying from 0 to 3.5 Pa); (ii) Pretreatment with ATP-medium results in different [Ca(2+)](i) response as compared to the control group (ATP-free medium) under SS; (iii) Relative [Ca(2+)](i) fluctuation over baseline is more reliable to show the [Ca(2+)](i) response process than the absolute [Ca(2+)](i) response peak. The developed model may improve the experimental design and facilitate our understanding of the mechanotransduction process in osteoblasts.     

Endothelin-stimulated Ca(2+)signaling and endothelin receptor expression are decreased by parathyroid hormone treatment in UMR-106 osteoblastic osteosarcoma cells

Modulation of endothelin (ET-1)-induced [Ca(2+)](i)transients and receptor expression by parathyroid hormone (PTH) was studied in UMR-106 osteoblastic osteosarcoma cells. Ca(2+)signaling was assessed with Fura-2, and ET receptor mRNA expression was determined using ET(A)- and ET(B)-specific primers and RT-PCR amplification. ET-1 binding in UMR-106 cell membranes was also measured. PTH pretreatment for 8 h decreased the [Ca(2+)](i)transients elicited by ET-1 and by the ET(B)-selective agonist sarafotoxin 6c (S6c). When ET(B)receptors were desensitized by pretreatment with S6c or blocked with the ET(B)-selective antagonist BQ-788, the remaining ET(A)component of the signal was also decreased by PTH pretreatment. In contrast, [Ca(2+)](i)transients elicited by PGF(2alpha)and ionomycin were increased following PTH pretreatment, indicating that the effect of PTH to decrease ET-1-stimulated transients was selective. PTH pretreatment also decreased [(125)I]ET-1 binding and ET(A)and ET(B)mRNA, with maximal effects at approximately 8 h. ET-1 was not detectable in medium from either control or PTH treated UMR-106 cultures, suggesting that the decreased expression of ET receptors was not due to enhanced ET production and subsequent homologous desensitization. The downregulation of ET receptors in osteoblasts by PTH pretreatment may serve as a homeostatic mechanism in bone.     

Calcium elevation elicited by reverse mode Na+/Ca2+ exchange activity is facilitated by intracellular calcium stores in bovine chromaffin cells

The Na(+)/Ca(2+) exchanger (NCX) in plasma membranes either moves Ca(2+) out of (forward mode) or into (reverse mode) cells depending on the electrochemical gradient of these ions across the membrane. In this report, we characterize the sources responsible for the elevation in [Ca(2+)](i) elicited by reverse mode NCX activity. The elevation in [Ca(2+)](i) elicited by reverse mode NCX activity was significantly diminished by thapsigargin. KB-R7943 could only partially suppress the [Ca(2+)](i) change. Measurement of the [Ca(2+)](i) concurrent with reverse mode NCX current by perforated whole-cell patch showed that elevation in [Ca(2+)](i), but not the current, was inhibited by thapsigargin. The change in [Ca(2+)](i) response elicited by nicotinic acetylcholine receptor agonist was inhibited by thapsigargin. These suggest the importance of intracellular Ca(2+) stores in facilitating the [Ca(2+)](i) elevation elicited by reverse mode NCX activity under physiological condition.     

TRPC channels and diacylglycerol dependent calcium signaling in rat sensory neurons

Transient receptor potential (TRP) channels of the TRPV, TRPA, and TRPM subfamilies play important roles in somatosensation including nociception. While particularly the Thermo TRPs have been extensively investigated in sensory neurons, the relevance of the subclass of "canonical" TRPC channels in primary afferents is yet elusive. In the present study, we investigated the presence and contribution to Ca(2+) transients of TRPC channels in dorsal root ganglion neurons. We found that six of the seven known TRPC subtypes were expressed in lumbar DRG, with TRPC1, C3, and C6 being the most abundant. Microfluorimetric calcium measurements showed Ca(2+) influx induced by oleylacylglycerol (OAG), an activator of the TRPC3/C6/C7 subgroup. Furthermore, OAG induced rises in [Ca(2+)](i) were inhibited by SKF96365, an inhibitor of receptor and store operated calcium channel. OAG induced calcium transients were also inhibited by blockers of diacylglycerol (DAG) lipase, lipoxygenase or cyclooxygenase and, intriguingly, by inhibitors of the capsaicin receptor TRPV1. Notably, SKF96365 did not affect capsaicin-induced calcium transients. Taken together, our findings suggest that TRPC are functionally expressed in subpopulations of DRG neurons. These channels, along with TRPV1, contribute to calcium homeostasis in rat sensory neurons.     

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²⁺

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.