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PubMed2004

Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro.

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.

Amino Acid Sequence↗

PubMed2004

Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061.

Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K(i), 80 to 90 nM) compared to genotype 1 enzymes (K(i), 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, with K(i) values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.

Amino Acid Sequence↗

PubMed2004

Spontaneous mitochondrial depolarizations are independent of SR Ca2+ release.

The mitochondrial membrane potential (DeltaPsi(m)) underlies many mitochondrial functions, including Ca(2+) influx into the mitochondria, which allows them to serve as buffers of intracellular Ca(2+). Spontaneous depolarizations of DeltaPsi(m), flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca(2+) sparks. We have shown previously that an increase in global Ca(2+) in smooth muscle cells causes an increase in mitochondrial Ca(2+) and depolarization of DeltaPsi(m). Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca(2+) released focally in Ca(2+) sparks. High-speed three-dimensional imaging was used to monitor DeltaPsi(m) in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca(2+) not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca(2+) release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca(2+) release from the SR does not cause flickers in the cells employed here.

Animals↗

PubMed2005

Two-step Ca2+ intracellular release underlies excitation-contraction coupling in mouse urinary bladder myocytes.

The relative contributions of Ca(2+)-induced Ca(2+) release (CICR) versus Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal Ca(2+) images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in several small, discrete areas just beneath the cell membrane. These Ca(2+) "hot spots" then spread slowly through the myoplasm as Ca(2+) waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca(2+) sparks, which declined quickly. The number of Ca(2+) sparks, or hot spots, was closely related to the depolarization duration in the range of approximately 5-20 ms. There was an apparent threshold depolarization duration of approximately 10 ms within which to induce enough Ca(2+) transients to spread globally and then induce a contraction. Application of 100 microM ryanodine to the pipette solution did not change the resting [Ca(2+)](i) or the VDCC current, but it did abolish Ca(2+) hot spots elicited by depolarization. Application of 3 microM xestospongin C reduced ACh-induced Ca(2+) release but did not affect depolarization-induced Ca(2+) events. The addition of 100 microM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca(2+)](i) rise triggered by a single action potential is not due mainly to Ca(2+) influx through VDCCs but is attributable to the subsequent two-step CICR.

Action Potentials↗

PubMed2006

Interleukin-8: novel roles in human airway smooth muscle cell contraction and migration.

In patients with cystic fibrosis (CF) and asthma, elevated levels of interleukin-8 (IL-8) are found in the airways. IL-8 is a CXC chemokine that is a chemoattractant for neutrophils through CXCR1 and CXCR2 G protein-coupled receptors. We hypothesized that IL-8 acts directly on airway smooth muscle cells (ASMC) in a way that may contribute to the enhanced airway responsiveness and airway remodeling observed in CF and asthma. The aim of this study was to determine whether human ASMC (HASMC) express functional IL-8 receptors (CXCR1 and CXCR2) linked to cell contraction and migration. Experiments were conducted on cells harvested from human lung specimens. Real-time PCR and fluorescence-activated cell sorting analysis showed that HASMC expressed mRNA and protein for both CXCR1 and CXCR2. Intracellular Ca(2+) concentration ([Ca(2+)](i)) increased from 115 to 170 nM in response to IL-8 (100 nM) and decreased after inhibition of phospholipase C (PLC) with U-73122. On blocking the receptors with specific neutralizing antibodies, changes in [Ca(2+)](i) were abrogated. IL-8 also contracted the HASMC, decreasing the length of cells by 15%, and induced a 2.5-fold increase in migration. These results indicate that HASMC constitutively express functional CXCR1 and CXCR2 that mediate IL-8-triggered Ca(2+) release, contraction, and migration. These data suggest a potential role for IL-8 in causing abnormal airway structure and function in asthma and CF.

Antibodies↗

PubMed2000

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes.

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to -50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca(2+) with equimolar Mn(2+) caused STOCs to "run down. " Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs" remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels (SK channels). Application of ATP or 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, a P(2) receptor blocker. Similarly, pretreatment of cells with U-73122 (1 microM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P(2Y) receptors results in localized Ca(2+) release via PLC- and IP(3)-dependent mechanisms. Release of Ca(2+) is coupled to STOCs, which are composed of currents mediated by large-conductance Ca(2+)-activated K(+) channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2000

ATP induces dephosphorylation of myosin light chain in endothelial cells.

In cultured porcine aortic endothelial monolayers, the effect of ATP on myosin light chain (MLC) phosphorylation, which controls the endothelial contractile machinery, was studied. ATP (10 microM) reduced MLC phosphorylation but increased cytosolic Ca(2+) concentration ([Ca(2+)](i)). Inhibition of the ATP-evoked [Ca(2+)](i) rise by xestospongin C (10 microM), an inhibitor of the inositol trisphosphate-dependent Ca(2+) release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca(2+)-independent manner. In the presence of calyculin A, MLC phosphorylation was incremented after addition of ATP, an effect that could be abolished when cells were loaded with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid acetoxymethyl ester (10 microM). Thus ATP also activates a Ca(2+)-dependent kinase acting on MLC. In summary, ATP simultaneously stimulates a functional antagonism toward both phosphorylation and dephosphorylation of MLC in which the dephosphorylation prevails. In endothelial cells, ATP is the first physiological mediator identified to activate MLC dephosphorylation by a Ca(2+)-independent mechanism.

Adenosine Triphosphate↗

PubMed2001

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.

Angiogenesis Inhibitors↗

PubMed2001

Regulation of slow wave frequency by IP(3)-sensitive calcium release in the murine small intestine.

Slow waves determine frequency and propagation characteristics of contractions in the small intestine, yet little is known about mechanisms of slow wave regulation. We propose a role for intracellular Ca(2+), inositol 1,4,5,-trisphosphate (IP(3))-sensitive Ca(2+) release, and sarcoplasmic reticulum (SR) Ca(2+) content in the regulation of slow wave frequency because 1) 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, a cytosolic Ca(2+) chelator, reduced the frequency or abolished the slow waves; 2) thapsigargin and cyclopiazonic acid (CPA), inhibitors of SR Ca(2+)-ATPase, decreased slow wave frequency; 3) xestospongin C, a reversible, membrane-permeable blocker of IP(3)-induced Ca(2+) release, abolished slow wave activity; 4) caffeine and phospholipase C inhibitors (U-73122, neomycin, and 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate) inhibited slow wave frequency; 5) in the presence of CPA or thapsigargin, stimulation of IP(3) synthesis with carbachol, norepinephrine, or phenylephrine acting on alpha(1)-adrenoceptors initially increased slow wave frequency but thereafter increased the rate of frequency decline, 6) thimerosal, a sensitizing agent of IP(3) receptors increased slow wave frequency, and 7) ryanodine, a selective modulator of Ca(2+)-induced Ca(2+) release, had no effect on slow wave frequency. In summary, these data are consistent with a role of IP(3)-sensitive Ca(2+) release and the rate of SR Ca(2+) refilling in regulation of intestinal slow wave frequency.

Adrenergic alpha-Agonists↗

PubMed2001

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C.

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.

Adenosine Triphosphate↗

PubMed2005

Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes.

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Selective restoration of calcium coupling to muscarinic M(3) receptors in contractile cultured airway myocytes.

We previously demonstrated that after several days of serum deprivation about one-sixth of confluent cultured canine tracheal myocytes acquire an elongated, structurally and functionally contractile phenotype. These myocytes demonstrated significant shortening on ACh exposure. To evaluate the mechanism by which these myocytes acquire responsiveness to ACh, we assessed receptor-Ca(2+) coupling using fura 2-AM fluorescence imaging and muscarinic receptor expression using Western analysis. Cells were grown to confluence in 10% fetal bovine serum and then maintained for 7-13 days in serum-free medium. A fraction of serum-deprived cells exhibited reproducible intracellular Ca(2+) mobilization in response to ACh that was uniformly absent from airway myocytes before serum deprivation. The Ca(2+) response to 10(-4) M ACh was ablated by inositol 1,4,5-trisphosphate (IP(3)) receptor blockade using 10(-6) M xestospongin C but not by removal of extracellular Ca(2+). Also, 10(-7) M atropine or 10(-7) M 4-diphenylacetoxy-N-methylpiperidine completely blocked the response to ACh, but intracellular Ca(2+) mobilization was not ablated by 10(-6) M pirenzepine or 10(-6) M methoctramine. In contrast, 10(-5) M bradykinin (BK) was without effect in these ACh-responsive myocytes. Interestingly, myocytes that did not respond to ACh demonstrated robust increases in intracellular Ca(2+) on exposure to 10(-5) M BK that were blocked by removal of extracellular Ca(2+) and were only modestly affected by IP(3) receptor blockade. Serum deprivation increased the abundance of M(3) receptor protein and of BK(2) receptor protein by two- to threefold in whole cell lysates within 2 days of serum deprivation, whereas M(2) receptor protein fell by >75%. An increase in M(3) receptor abundance and restoration of M(3) receptor-mediated Ca(2+) mobilization occur concomitant with reacquisition of a contractile phenotype during prolonged serum deprivation. These data demonstrate plasticity in muscarinic surface receptor expression and function in a subpopulation of airway myocytes that show mutually exclusive physiological and pharmacological diversity with other cells in the same culture.

Acetylcholine↗

PubMed2001

cGMP abolishes agonist-induced [Ca(2+)](i) oscillations in human bladder epithelial cells.

Cytosolic calcium oscillations may permit cells to respond to information provided by increases in intracellular Ca(2+) concentration ([Ca(2+)](i) ) while avoiding prolonged exposure to constantly elevated [Ca(2+)](i). In this study, we demonstrated that agonists could induce Ca(2+) oscillations in human bladder epithelial cells. Application of 10 microM acetylcholine or 200 nM bradykinin triggered an initial Ca(2+) transient that was followed by periodic [Ca(2+)](i) oscillations. The oscillations did not depend on extracellular Ca(2+). 8-Bromoguanosine 3',5'-cyclic monophosphate abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [Ca(2+)](i) oscillations. The inhibitory effect of cGMP could be reversed by KT-5823, a highly specific inhibitor of protein kinase G (PKG), suggesting that the action of cGMP was mediated by PKG. Comparison of the effect of cGMP with that of xestospongin C, an inhibitor of the inositol 1,4,5-trisphosphate (IP(3)) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely that cGMP and PKG may target a signal transduction step(s) linked to IP(3) receptor-mediated Ca(2+) release.

Acetylcholine↗

PubMed2001

Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.

Animals↗

PubMed2006

A store-operated Ca(2+) influx pathway in the bag cell neurons of Aplysia.

Although store-operated Ca(2+) influx has been well-studied in nonneuronal cells, an understanding of its nature in neurons remains poor. In the bag cell neurons of Aplysia californica, prior work has suggested that a Ca(2+) entry pathway can be activated by Ca(2+) store depletion. Using fura-based imaging of intracellular Ca(2+) in cultured bag cell neurons, we now characterize this pathway as store-operated Ca(2+) influx. In the absence of extracellular Ca(2+), the endoplasmic reticulum Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) or thapsigargin, depleted intracellular stores and elevated intracellular free Ca(2+). With the subsequent addition of extracellular Ca(2+), a prominent Ca(2+) influx was observed. The ryanodine receptor agonist, chloroethylphenol (CEP), also increased intracellular Ca(2+) but did not initiate store-operated Ca(2+) influx, despite overlap between CEP- and CPA-sensitive stores. Bafilomycin A, a vesicular H(+)-ATPase inhibitor, liberated intracellular Ca(2+) from acidic stores and attenuated subsequent Ca(2+) influx, presumably by replenishing CPA-depleted stores. Store-operated Ca(2+) influx was partially blocked by low concentrations of La(3+) or BTP2, and strongly inhibited by either 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) or a high concentration of Ni(2+). Regarding IP(3) receptor blockers, 2-aminoethyldiphenyl borate, but not xestospongin C, prevented store-operated Ca(2+) influx. However, jasplakinolide, an actin stabilizer reported to inhibit this pathway in smooth muscle cell lines, was ineffective. The bag cell neurons initiate reproductive behavior through a prolonged afterdischarge associated with intracellular Ca(2+) release and neuropeptide secretion. Store-operated Ca(2+) influx may serve to replenish stores depleted during the afterdischarge or participate in the release of peptide that triggers behavior.

Anilides↗

PubMed1998

Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations.

Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations. J. Neurophysiol. 80: 2963-2974, 1998. Cerebellar long-term depression (LTD) is a cellular model system of information storage in which coincident parallel fiber and climbing fiber activation of a Purkinje neuron (PN) gives rise to a sustained attenuation of parallel fiber-PN synaptic strength. Climbing fiber and parallel fiber inputs may be replaced by direct depolarization of the PN and exogenous glutamate pulses, respectively. The parallel fiber-PN synapse has a high-density of mGluR1 receptors that are coupled to phosphoinositide turnover. Several lines of evidence indicated that activation of mGluR1 by parallel fiber stimulation is necessary for the induction of cerebellar LTD. Because phosphoinositide hydrolysis has two initial products, 1, 2-diacylglycerol and inositol-1,4,5-trisphosphate (IP3), we wished to determine whether IP3 signaling via IP3 receptors and consequent Ca mobilization were necessary for the induction of cerebellar LTD. First, ratiometric imaging of free cytosolic Ca was performed on both acutely dissociated and cultured PNs. It was determined that the threshold for glutamate pulses to contribute to LTD induction was below the threshold for producing a Ca transient. Furthermore, the Ca transients produced by depolarization alone and glutamate plus depolarization were not significantly different. Second, the potent and selective IP3 receptor channel blocker xestospongin C was not found to affect the induction of LTD in either acutely dissociated or cultured PNs at a concentration that was sufficient to block mGluR1-evoked Ca mobilization. Third, replacement of mGluR activation by exogenous synthetic diacylglycerol in an LTD induction protocol was successful. Taken together, these results suggest that activation of an IP3 signaling cascade is not required for induction of cerebellar LTD in reduced preparations.

Animals↗

PubMed2002

Nicotinic acid adenine dinucleotide phosphate mediates Ca2+ signals and contraction in arterial smooth muscle via a two-pool mechanism.

Previous studies of arterial smooth muscle have shown that inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose mobilize Ca2+ from the sarcoplasmic reticulum. In contrast, little is known about Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate, a pyridine nucleotide derived from beta-NADP+. We show here that intracellular dialysis of nicotinic acid adenine dinucleotide phosphate (NAADP) induces spatially restricted "bursts" of Ca2+ release that initiate a global Ca2+ wave and contraction in pulmonary artery smooth muscle cells. Depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin and inhibition of ryanodine receptors with ryanodine, respectively, block the global Ca2+ waves by NAADP. Under these conditions, however, localized Ca2+ bursts are still observed. In contrast, xestospongin C, an IP3 receptor antagonist, had no effect on Ca2+ signals by NAADP. We propose that NAADP mobilizes Ca2+ via a 2-pool mechanism, and that initial Ca2+ bursts are amplified by subsequent sarcoplasmic reticulum Ca2+ release via ryanodine receptors but not via IP3 receptors.

Animals↗

PubMed2006

Sex-related differences in the gastrointestinal disposition of ivermectin in the rat: P-glycoprotein involvement and itraconazole modulation.

Ivermectin (IVM), a macrocyclic lactone used as antiparasite agent, has been reported as a P-glycoprotein (P-gp) substrate. The participation of P-gp in the IVM excretion process has been previously demonstrated. Sex-related differences in the kinetic behaviour of some macrocyclic lactone compounds have been observed. The aim of this work was to characterize in-vivo the comparative gastrointestinal disposition of IVM in male and female rats. The sex-related influence on the itraconazole (ITZ) modulation of P-gp-mediated IVM intestinal transport was also assessed. Sixty Wistar rats (30 male, 30 female) received IVM alone or co-administered with ITZ. Rats were killed between 6 and 72 h after treatment and blood, gastrointestinal tissues and lumen contents were collected. IVM concentrations were determined by high performance liquid chromatography. Substantial sex-related differences in the IVM disposition kinetics were observed. Higher IVM systemic availability was observed in female rats. The ITZ-mediated modulation of the IVM disposition kinetics had a differential impact between male and female rats. Co-administration with ITZ resulted in a marked increase in the IVM concentrations in the wall tissue from different portions of the gastrointestinal tract of male rats. The presence of ITZ induced drastic sex-related changes on the P-gp-mediated IVM gastrointestinal disposition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗