Ca Agonist Research Articles

Pharmacological characterization of novel small molecule agonists and antagonists for the orphan receptor GPR139

The orphan G protein-coupled receptor GPR139 is predominantly expressed in the central nervous system and has attracted considerable interest as a therapeutic target. However, the biological role of this receptor remains somewhat elusive, in part due to the lack of quality pharmacological tools to investigate GPR139 function. In an effort to understand GPR139 signaling and to identify improved compounds, in this study we performed virtual screening and analog searches, in combination with multiple pharmacological assays. We characterized GPR139-dependent signaling using previously published reference agonists in Ca2+ mobilization and inositol monophosphate accumulation assays, as well as a novel real-time GPR139 internalization assay. For the four reference agonists tested, the rank order of potency was conserved across signaling and internalization assays: JNJ-63533054 > Compound 1a » Takeda > AC4 > DL43, consistent with previously reported values. We noted an increased efficacy of JNJ-63533054-mediated inositol monophosphate signaling and internalization, relative to Compound 1a. We then performed virtual screening for GPR139 agonist and antagonist compounds that were screened and validated in GPR139 functional assays. We identified four GPR139 agonists that were active in all assays, with similar or reduced potency relative to known compounds. Likewise, compound analogs selected based on GPR139 agonist and antagonist substructure searches behaved similarly to their parent compounds. Thus, we have characterized GPR139 signaling for multiple new ligands using G protein-dependent assays and a new real-time internalization assay. These data add to the GPR139 tool compound repertoire, which could be optimized in future medical chemistry campaigns.

Catecholaminergic Modulation of Semantic Processing in Sentence Comprehension.

Catecholamine (CA) function has been widely implicated in cognitive functions that are tied to the prefrontal cortex and striatal areas. The present study investigated the effects of methylphenidate, which is a CA agonist, on the electroencephalogram (EEG) response related to semantic processing using a double-blind, placebo-controlled, randomized, crossover, within-subject design. Forty-eight healthy participants read semantically congruent or incongruent sentences after receiving 20-mg methylphenidate or a placebo while their brain activity was monitored with EEG. To probe whether the catecholaminergic modulation is task-dependent, in one condition participants had to focus on comprehending the sentences, while in the other condition, they only had to attend to the font size of the sentence. The results demonstrate that methylphenidate has a task-dependent effect on semantic processing. Compared to placebo, when semantic processing was task-irrelevant, methylphenidate enhanced the detection of semantic incongruence as indexed by a larger N400 amplitude in the incongruent sentences; when semantic processing was task-relevant, methylphenidate induced a larger N400 amplitude in the semantically congruent condition, which was followed by a larger late positive complex effect. These results suggest that CA-related neurotransmitters influence language processing, possibly through the projections between the prefrontal cortex and the striatum, which contain many CA receptors.

The Effects of Ion Channel Inhibitors on the Generation of Electrical Impulses in Right Atrial Pacemaker Cells of 10-Day-Old Chicken Embryos

The cellular mechanisms of pacemaker-cell activity and the contribution of various ion channels of the embryonic heart are not yet fully understood. This paper presents the results of an experiment to study the effects of specific ion-channel inhibitors on the generation of action potentials in pacemaker cells of the right atrium in chicken embryos. It has been found that the exclusion of calcium ions from the extracellular solution or the strip exposure to 10 μM nifedipine (an L-type Ca2+-channel blocker) did not inhibit the generation of action potentials but led to an increase in the frequency of electrical impulses by 45%. Ryanodine, an agonist of Ca2+-channels of the sarcoplasmic reticulum, also had a positive chronotropic effect. The application of lidocaine led to a negative chronotropic effect and inhibited generation of elecrical impulses. We concluded that the role of ryanodine-sensitive Ca2+-channels (RyR-channels) of the sarcoplasmic reticulum and L-type Ca2+-channels is not crucial for maintaining the pacemaker activity in the right atrial cells of 10-day-old chicken embryos with a four-chambered heart, a not fully formed sinoatrial node, and incomplete morphofunctional maturation of the central nervous system. The demonstrated inhibition of action potential generation by lidocaine and the absence of such an effect from nifedipine indicated the substantial contribution of the Na+ current to the pacemaker activity.

Propofol inhibits invasion and proliferation of C6 glioma cells by regulating the Ca2+ permeable AMPA receptor-system xc− pathway

Anesthetics are documented to affect tumors; therefore, we studied the antiglioma effect of propofol on proliferation and invasiveness of glioma cells and explored the underlying mechanism. C6 glioma cells were cultured and treated with propofol, and cell viability, invasiveness, and migration were measured. Glutamate release was measured using an enzyme-catalyzed kinetic reaction. xCT protein and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR2 subunit protein expression was assessed with Western blot analysis and immunofluorescent staining. We observed that propofol significantly inhibited C6 glioma cell viability, invasiveness, and migration and decreased glutamate release. An agonist of the cystine/glutamate antiporter system (system xc−), N-acetylcysteine (NAC), reversed propofol's effects, and propofol could inhibit C6 glioma cell proliferation by adding excess exogenous glutamate (100μM). Finally, propofol increased the surface expression of GluR2, but decreased surface expression of xCT. The effects of propofol on surface expression of GluR2 and xCT could be rescued by (R, S)-AMPA, an agonist of Ca2+ permeable AMPA receptor (CPAR). Thus, propofol can inhibit cell viability, invasiveness, and migration of C6 glioma cells, and the CPAR-system xc− pathway contributes to these events.

High-energy compounds mobilize intracellular Ca2+ and activate calpain in cultured cells: Is calpain an energy-dependent protease?

Deficiency in energy metabolisms is perhaps the earliest modifiable defect in brain aging and sporadic Alzheimer's disease (sAD). Several high-energy compounds (HECs) such as ATP, phosphoenolpyruvate, phosphocreatine and acetyl coenzyme A have been shown to exhibit neuroprotective effects. To understand their mechanism of actions, we tested the effects of these HECs on intracellular Ca(2+), a central regulator in brain function. Our data showed that the HECs robustly and dose-dependently mobilized intracellular Ca(2+) in cultured SH-SY5Y cells, and the actions were sensitive to intracellular Ca(2+) chelator BAPTA-AM or energy metabolism blocker rotenone. The Ca(2+) influx triggered by the HECs was from both extracellular medium and intracellular stores and the HECs also induced repetitive Ca(2+) oscillations. As these actions were similar to those of classical Ca(2+) agonists, the HECs may be viewed as a new group of physiological Ca(2+) agonists. We also found that the HECs promoted the intracellular activity of calpain, a Ca(2+)-dependent protease, and the enzyme activity fluctuated in concert with cellular energy levels, suggesting that calpain activity may also be energy-driven or energy-dependent. These findings may add to current knowledge for the regulatory mechanisms of Ca(2+) and calpain. Since Ca(2+) and calpain undergo critical dysfunction in brain aging but the underlying mechanisms remain elusive, our work may provide a new perspective for clarifying some controversies. More importantly, the HECs, as key intermediates in glucose catabolism, the primary source of energy supply in the brain, may be used as potential drugs for rational prevention of sAD.

Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium

Calcium (Ca(2+)) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca(2+) level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca(2+) triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca(2+) agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.

Evidence supporting the role of calpain in the α-processing of amyloid-β precursor protein

Amyloid plaques are a hallmark of the aging and senile dementia brains, yet their mechanism of origins has remained elusive. A central issue is the regulatory mechanism and identity of α-secretase, a protease responsible for α-processing of amyloid-β precursor protein (APP). A remarkable feature of this enzyme is its high sensitivity to a wide range of cellular stimulators, many of which are agonists for Ca2+ signaling. This feature, together with previous work in our laboratory, has suggested that calpain, a Ca2+-dependent protease, plays a key role in APP α-processing. In this study we report that overexpression of the μ-calpain gene in HEK293 cells resulted in a 2.7-fold increase of the protein levels. Measurements of intracellular calpain enzymatic activity revealed that the calpain overexpressing cells displayed a prominent elevation of the activity compared to wild-type cells. When the cells were stimulated by nicotine, glutamate or phorbol 12,13-dibutylester, the activity increase was even more remarkable and sensitive to calpeptin, a calpain inhibitor. Meanwhile, APP secretion from the calpain overexpressing cells was robustly increased under both resting and stimulated conditions over wild-type cells. Furthermore, cell surface biotinylation experiments showed that μ-calpain was clearly detected among the cell surface proteins. These data together support our view that calpain should be a reasonable candidate for α-secretase for further study. This model is discussed with an interesting fact that three other deposited proteins (tau, spectrin and crystalline) are also the known substrates of calpain. Finally we discuss some current misconceptions in senile dementia research.

Perspective on the role of P2X‐purinoceptor activation in human vas deferens contractility

The contractile actions of α,β-methylene ATP (α,β-meATP) and ATP and the effects of K(+) channel blockers in longitudinal and circular muscles of human vas deferens were investigated with a view to clarifying the functional importance of P2X(1)-purinoceptor activation and K(+) channels in modulating contractility of the tissues. The results provide an experiment-based perspective for resolving differing reports on purinergic activation of the tissues and uncertain roles of large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated delayed rectifier K(+) (K(V)) channels. α,β-Methylene ATP (3-100 μm) evoked suramin-sensitive contractions of longitudinal muscle but rarely of circular muscle. ATP (0.1-3 mm) less reliably activated only longitudinal muscle contractions. These were enhanced by ARL 67156 (100 μm), but a different ectonucleotidase inhibitor, POM 1, was ineffective. Both muscle types were unresponsive to ADP-βS (100 μm), a P2Y-purinoceptor agonist. Longitudinal muscle contractions in response to α,β-meATP were enhanced by FPL 64176 (1 μm), an L-type Ca(2+) agonist, TEA (1 mm), a non-specific K(+) channel blocker, 4-aminopyridine (0.3 mm), a selective blocker of K(V) channels, and iberiotoxin (0.1 μm), a selective blocker of BK(Ca) channels. Quiescent circular muscles responded to α,β-meATP reliably in the presence of FPL 64176 or iberiotoxin. Apamin (0.1 μm), a selective blocker of small conductance Ca(2+)-activated K(+) (SK(Ca)) channels had no effect in both muscle types. Y-27632 (1-10 μm) reduced longitudinal muscle contractions in response to α,β-meATP, suggesting involvement of Rho-kinase-dependent contractile mechanisms. The results indicate that P2X(1)-purinoceptor stimulation elicits excitatory effects that: (a) lead to longitudinal muscle contraction and secondary activation of 4-aminopyridine-sensitive (K(V)) and iberiotoxin-sensitive (BK(Ca)) K(+) channels; and (b) are subcontractile in circular muscle due to ancillary activation of BK(Ca) channels. The novel finding of differential action by P2X(1)-purinoceptor agonists in the muscle types has functional implication in terms of the purinergic contribution to overall contractile function of human vas deferens. The modulatory effects of K(V) and BK(Ca) channels following P2X(1)-purinoceptor activation may be pivotal in providing the crucial physiological mechanism that ensures temporal co-ordination of longitudinal and circular muscle contractility.

Hydrogen Sulfide in the Rostral Ventrolateral Medulla Inhibits Sympathetic Vasomotor Tone through ATP-Sensitive K+ Channels

Hydrogen sulfide (H(2)S) acts as an endogenous gaseous transmitter in the central nervous system and plays important roles in regulating cardiovascular function. The rostral ventrolateral medulla (RVLM) is a putative critical central region in the control of sympathetic vasomotor tone and plays an important role in the baroreflex by integrating the inputs from a variety of visceral and somatic stimuli. In this study, we tested the hypothesis that H(2)S decreases sympathetic vasomotor tone through ATP-sensitive potassium channels (K(ATP)) in the RVLM. The arterial blood pressure (ABP), heart rate (HR), and renal sympathetic nerve activity (RSNA) of anesthetized rats were recorded. Bilateral microinjections of sodium hydrosulfide (NaHS; 4, 8, and 16 mM, 50 nl), an H(2)S donor, into the RVLM decreased ABP, HR, and RSNA in a dose-dependent manner. Preinjection of glibenclamide (40 μM, 50 nl), a K(ATP) channel blocker, abolished the sympathoinhibitory effects of NaHS (8 mM, 50 nl). Preinjection of a nitric-oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester (200 μM, 50 nl) partially inhibited the sympathoinhibitory effects of NaHS. Prior microinjection of 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)pyridine-3-carboxylic acid methyl ester (Bay K8644) (1 μM, 50 nl), an agonist of Ca(2+) channels, did not alter the effects of NaHS. Infusion of hydroxylamine (30 mM, 50 nl), a cystathionine β-synthase inhibitor, increased ABP, HR, and RSNA. Taken together, these findings suggest that exogenous H(2)S in the RVLM inhibits sympathetic vasomotor tone by opening K(ATP) channels. Nitric-oxide signaling may partially be involved in the sympathoinhibitory effect of H(2)S in the RVLM.

Comparative effects of T‐type and L‐type Ca2+‐antagonists against noradrenaline‐induced contractions of human vas deferens

To investigate the effects of the relatively selective T-type Ca(2+)-antagonists, mibefradil and flunarizine, and the L-type Ca(2+)-antagonist, nifedipine, on the contractions of longitudinal and circular muscles of human vas deferens, to elucidate the possible involvement of T-type voltage-gated Ca(2+) channels (VOCs) in the contractile function of the tissue. Human vas deferens specimens from elective vasectomies were cut into strips of longitudinal muscle or transversely into rings of circular muscle. These were set up for tension recording and superfused with Krebs' medium (36 degrees C). Contractions were evoked by noradrenaline or high [K(+)](o) (in the presence of the L-type Ca(2+) agonist, FPL 64176) and the effects of Ca(2+) antagonists were determined. Noradrenaline (0.1-100 micromol/L) evoked rhythmic and tonic contractions of longitudinal and circular muscles, which were potently inhibited by nifedipine (<or=0.1 micromol/L). Mibefradil (1-10 micromol/L) inhibited the contractions but was comparatively more effective in longitudinal than circular muscle. Flunarizine was ineffective except against contractions to low concentrations of noradrenaline. The drugs' potencies as antagonists of L-type VOCs were determined against contractions to high K(+) (120 mmol/L in the presence of FPL 64176, 1 micromol/L). The contractions in longitudinal and circular muscle had different times to peak and decline but were inhibited comparably by nifedipine (50% inhibitory concentration, IC(50), longitudinal and circular muscle, approximately 2 nmol/L) or by mibefradil (IC(50) longitudinal muscle, 1.1 micromol/L; circular muscle, 2.4 micromol/L) and were less sensitive to flunarizine (up to 30 micromol/L). These results indicate that noradrenaline-induced contractions of human vas deferens depend primarily on nifedipine-sensitive L-type VOCs, as opposed to mibefradil/flunarizine-sensitive T-type VOCs. The effects of mibefradil and flunarizine, at concentrations found to be effective against noradrenaline-induced contractions, involve the blockade of L-type VOCs. The modest differential effect of mibefradil in longitudinal and circular muscle is discussed in relation to factors that modulate activation and drug-sensitivity of L-type VOCs.

Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels.

RhoA kinase (ROCK) participates in K(+) depolarization (KCl)-induced Ca(2+) sensitization of contraction. Whether constitutive, depolarization- or Ca(2+)-activated ROCK plays the major role in this signalling system remains to be determined. Here, we determined whether Bay K 8644, a dihydropyridine that promotes Ca(2+) channel clusters to operate in a persistent Ca(2+) influx mode, could cause ROCK-dependent Ca(2+) sensitization. Renal and femoral artery rings from New Zealand white rabbits were contracted with Bay K 8644. Tissues were frozen and processed to measure active RhoA and ROCK substrate (myosin phosphatase targeting subunit, MYPT1) and myosin light chain (MLC) phosphorylation, or loaded with fura-2 to measure intracellular free Ca(2+) ([Ca(2+)](i)). Effects of selective inhibitors of contraction were assessed in resting (basal) tissues and those contracted with Bay K 8644. Bay K 8644 produced strong increases in [Ca(2+)](i), MLC phosphorylation and tension, but not in MYPT1 phosphorylation. ROCK inhibition by H-1152 abolished basal MYPT1-pT853, diminished basal MLC phosphorylation and inhibited Bay K 8644-induced increases in MLC phosphorylation and tension. MLC kinase inhibition by wortmannin abolished Bay K 8644-induced contraction and increase in MLC phosphorylation but did not inhibit basal MYPT1-pT853. H-1152 and wortmannin had no effect on MYPT1-pT696, but 1 microM staurosporine inhibited basal MYPT1-pT853, MYPT1-pT696 and MLC phosphorylation. These data suggest that the constitutive activities of ROCK and a staurosporine-sensitive kinase regulate basal phosphorylation of MYPT1, which participates along with activation of MLC kinase in determining the strength of contraction induced by the Ca(2+) agonist, Bay K 8644.

Modulation of redox status and calcium handling by extremely low frequency electromagnetic fields in C2C12 muscle cells: A real-time, single-cell approach

The biological effects of electric and magnetic fields, which are ubiquitous in modern society, remain poorly understood. Here, we applied a single-cell approach to study the effects of short-term exposure to extremely low frequency electromagnetic fields (ELF-EMFs) on muscle cell differentiation and function using C2C12 cells as an in vitro model of the skeletal muscle phenotype. Our focus was on markers of oxidative stress and calcium (Ca 2+) handling, two interrelated cellular processes previously shown to be affected by such radiation in other cell models. Collectively, our data reveal that ELF-EMFs (1) induced reactive oxygen species production in myoblasts and myotubes with a concomitant decrease in mitochondrial membrane potential; (2) activated the cellular detoxification system, increasing catalase and glutathione peroxidase activities; and (3) altered intracellular Ca 2+homeostasis, increasing the spontaneous activity of myotubes and enhancing cellular reactivity to a depolarizing agent (KCl) or an agonist (caffeine) of intracellular store Ca 2+channels. In conclusion, our data support a possible link between exposure to ELF-EMFs and modification of the cellular redox state, which could, in turn, increase the level of intracellular Ca 2+and thus modulate the metabolic activity of C2C12 cells.

Mitochondrial-Driven Ubiquinone Enhances Extracellular Calcium-Dependent Nitric Oxide Production and Reduces Glycochenodeoxycholic Acid-Induced Cell Death in Hepatocytes

Ca(2+) mobilization, nitric oxide (NO), and oxidative stress have been involved in cell death induced by hydrophobic bile acid in hepatocytes. The aim of the study was the elucidation of the effect of the antioxidant mitochondrial-driven ubiquinone (Mito Q) on the intracellular Ca(2+) concentration, NO production, and cell death in glycochenodeoxycholic acid (GCDCA)-treated HepG2 cells. The role of the regulation of the intracellular Ca(2+) concentration by Ca(2+) chelators (EGTA or BAPTA-AM), agonist of Ca(2+) entrance (A23187) or NO (L-NAME or NO donor), was assessed during Mito Q cytoprotection in GCDCA-treated HepG2 cells. Cell death, NO synthase (NOS)-1, -2, and -3 expression, Ca(2+) mobilization, and NO production were evaluated. GCDCA reduced the intracellular Ca(2+) concentration and NOS-3 expression and enhanced cell death in HepG2. NO donor prevented and L-NAME enhanced GCDCA-induced cell death. The reduction of Ca(2+) entry by EGTA, but not its release from intracellular stores by BAPTA-AM, reduced the expression of NOS-3 and enhanced cell death in control and GCDCA-treated cells. Mito Q prevented the reduction of intracellular Ca(2+) concentration, NOS-3 expression, NO production, and cell death in GCDCA-treated HepG2 cells. The conclusion is that the recovery of Ca(2+)-dependent NOS-3 expression by Mito Q may be considered an additional cytoprotective property of an antioxidant.

Dual effects of lipophilic extract of Salvia miltiorrhiza (Danshen) on catecholamine secretion in cultured bovine adrenal medullary cells

Ethnopharmacological relevance Salvia miltiorrhiza (Danshen) is a well known traditional Chinese herb, which has been used widely in China for the treatment of cardiovascular diseases in clinic. Aim of this study The aim of the present study is to clarify the effects of lipophilic extract of Salvia miltiorrhiza (LESM) on catecholamine (CA) secretion, a traditional Chinese medicine used widely for the treatment of cardiovascular diseases in China. Materials and methods LESM was evaluated for its effects on CA secretion using HPLC-ECD method. The effects of LESM on 22Na + influx and intracellular calcium ([Ca 2+] i) were also investigated. Results Our results showed that LEMS directly stimulated basal CA secretion in an extracellular Ca 2+-dependent manner. And the stimulation was not affected by combination of hexamethonium (Hex), an inhibitor of nAChR. LESM also directly elevated [Ca 2+] i. In addition, using selective blockers of voltage-dependent Ca 2+ channels, such as nitrendipine (for L-type), ω-agatoxin-IVA (for P-type) and ω-conotoxin-GVIA (for N-type), it was found that nitrendipine suppressed the elevation of [Ca 2+] i induced by LESM, but not ω-agatoxin-IVA or ω-conotoxin-GVIA. Compared with acetylcholine (ACh) only, however, combination of LESM with ACh inhibited the raise of CA secretion, 22Na + influx and [Ca 2+] i in a concentration-dependent manner. Furthermore, LESM also inhibited CA secretion induced by veratridine (Ver), and 56 mM K + at concentrations similar to those for [Ca 2+] i rise. One of the lipophilic active compounds, cryptotanshione (Cryp), also had the same effects on CA secretion with LESM. Conclusions All these findings suggest that LESM exerts dual effects on CA secretion in cultured bovine adrenal medullary cells. LESM exerts antagonistic effects on nAChR, voltage-dependent Na + and Ca 2+ channels, whereas it is an agonist of L-type Ca 2+ channel when it used alone.

Different effects of K Ca and K ATP agonists on brain tumor permeability between syngeneic and allogeneic rat models

The blood–brain tumor barrier (BTB) significantly limits delivery of effective concentrations of chemotherapeutic drugs to brain tumors. Previous studies suggest that BTB permeability may be modulated via alteration in the activity of potassium channels. In this study, we studied the relationship of BTB permeability increase mediated by potassium channel agonists to channel expression in two rat brain tumor models. Intravenous infusion of KCO912 (K ATP agonist), minoxidil sulfate (K ATP agonist) or NS1619 (K Ca agonist) increased tumor permeability more in the 9L allogeneic brain tumor model than in the syngeneic brain tumor model. Consistently, expression of both K ATP and K Ca channels in 9L tumors was increased to a significantly greater extent in Wistar rats (allogeneic) as compared to Fischer rats (syngeneic). Furthermore, as a preliminary effort to understand clinical implication of potassium channels in brain tumor treatment, we determined the expression of K ATP in surgical specimens. K ATP mRNA was detected in glioblastoma multiforme (GBM) from nineteen patients examined, with a wide range of expression levels. Interestingly, in paired GBM tissues from seven patients before and after vaccination therapy, increased levels of K ATP were detected in five patients after vaccination that had positive response to chemotherapy after vaccination. The present study indicates that the effects of potassium channel agonists on BTB permeability are different between syngeneic and allogeneic models which have different expression levels of potassium channels. The expression of potassium channels in brain tumors is variable, which may be associated with different tumor permeability to therapeutic agents among patients.

Tamoxifen Stimulates Calcium Entry Into Human Platelets

The anti-estrogenic drug tamoxifen, which is used therapeutically for treatment and prevention of breast cancer, can lead to the development of thrombosis. We found that tamoxifen rapidly increased intracellular free calcium [Ca2+]i in human platelets from both male and female donors. Thus 10 microM tamoxifen increased [Ca2+]i above the resting level by 197 +/- 19%. Tamoxifen acted synergistically with thrombin, ADP, and vasopressin to increase [Ca2+]i. The anti-estrogen ICI 182780 did not attenuate the effects of tamoxifen to increase [Ca2+]i; however, phospholipase C inhibitor U-73122 blocked this effect. 4-hydroxytamoxifen, a major metabolite of tamoxifen, also increased [Ca2+]i, but other tamoxifen metabolites and synthetic derivatives did not. Three hydroxylated derivatives of triphenylethylene (corresponding to the hydrophobic core of tamoxifen) which are transitional structures between tamoxifen (Ca agonist) and diethylstilbestrol (Ca antagonist) increased [Ca2+]i slightly (6% to 24%) and partially inhibited thrombin-induced [Ca2+]i elevation (68% to 79%). Therefore the dimethylaminoethyl moiety is responsible for tamoxifen being a Ca agonist rather than antagonist. 4-Hydroxytamoxifen and polymer-conjugated derivatives of 4-hydroxytamoxifen increased [Ca2+]i, with similar efficacy. The ability of tamoxifen to increase [Ca2+]i in platelets, leading to platelet activation, and its ability to act synergistically with other platelet agonists may contribute to development of tamoxifen-induced thrombosis.

Cyclic AMP Regulates Bicarbonate Secretion in Cholangiocytes Through Release of ATP Into Bile

Bicarbonate secretion is a primary function of cholangiocytes. Either adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca(2+) can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion because this is the only intracellular Ca(2+) release channel in cholangiocytes. Intrahepatic bile duct units (IBDUs) were microdissected from rat liver then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca(2+) was increased using acetylcholine (ACh) or adenosine triphosphate (ATP). Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments, IBDUs were pretreated with short interfering RNA (siRNA) to silence expression of specific InsP3R isoforms. Both cAMP and Ca(2+) agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH. Cyclic AMP-induced ductular bicarbonate secretion depends on an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate.

Intracellular Ca2+and Zn2+signals during monochloramine-induced oxidative stress in isolated rat colon crypts

During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) and Zn(2+) accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to Ca(2+) and Zn(2+) in the cytoplasm. Responses to different concentrations of NH(2)Cl (50, 100, and 200 microM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH(2)Cl-induced increases in Ca(2+) and Zn(2+) in the cytoplasm. Exposure to NH(2)Cl led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the range of 200-400 nM above baseline levels. Further studies indicated that NH(2)Cl-induced accumulation of Ca(2+) in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca(2+) through store-operated channels. In addition, exposure to NH(2)Cl resulted in dose-dependent and sustained increases in intracellular Zn(2+) concentration ([Zn(2+)](i)) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca(2+)- and Zn(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca(2+)](i) and [Zn(2+)](i). Under certain conditions, NH(2)Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.

Toosendanin, a triterpenoid derivative, acts as a novel agonist of L-type Ca 2+ channels in neonatal rat ventricular cells

Toosendanin, a triterpenoid derivative extracted from Melia toosendan Sieb et Zucc, was demonstrated to be potentially useful in medical and scientific researches. Here, we investigated the effects of toosendanin on L-type voltage-dependent Ca 2+ channels in cultured neonatal rat ventricular cells, using whole-cell patch-clamp method. Toosendanin irreversibly increased L-type Ca 2+ current ( I Ca(L)) in a concentration-dependent manner and shifted the maximum of the current/voltage relationship from 8.3±3.7 to 1.7±3.7 mV, without modifying the threshold potential of the current. Toosendanin shifted the steady-state activation and inactivation curves to the left. The deactivation kinetics of the I Ca(L) was significantly slowed by toosendanin while the activation kinetics was not affected. The cells pretreated with 100 nM 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester ( S(−)-BayK8644) still respond to further addition of 87 μM toosendanin, and vice versa. These results prove toosendanin to be a novel L-type Ca 2+ channel agonist, which possesses a distinct binding site from BayK8644.

Ca2+-mediated Potentiation of the Swelling-induced Taurine Efflux from HeLa Cells: On the Role of Calmodulin and Novel Protein Kinase C Isoforms

The present work sets out to investigate how Ca(2+) regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca(2+)-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca(2+)-free or high-K(+) media. Addition of Ca(2+)-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca(2+)-activated K(+) channels. The taurine release from control cells and cells exposed to Ca(2+) agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca(2+)-augmented taurine release. Exposure to Ca(2+)-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca(2+)-concentration has returned to the unstimulated level. The Ca(2+)-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca(2+)-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca(2+) regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).