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1.
Plant Biol (Stuttg) ; 23(4): 556-563, 2021 Jul.
Article in English | MEDLINE | ID: mdl-33550673

ABSTRACT

Floral structures, such as stamen appendages, play crucial roles in pollinator attraction, pollen release dynamics and, ultimately, the reproductive success of plants. The pollen-rewarding, bee buzz-pollinated flowers of Melastomataceae often bear conspicuous staminal appendages. Surprisingly, their functional role in the pollination process remains largely unclear. We use Huberia bradeana Bochorny & R. Goldenb. (Melastomataceae) with conspicuously elongated, twisted stamen appendages to investigate their functional role in the pollination process. We studied the effect of stamen appendages on pollinator behaviour and reproductive success by comparing manipulated flowers (appendages removed) with unmanipulated flowers. To assess bee pollinator behaviour, we measured three properties of buzzes (vibrations) produced by bees on Huberia flowers: frequency, duration and number of buzzes per flower visit. We measured male and female reproductive success by monitoring pollen release and deposition after single bee visits. Finally, we used artificial vibrations and laser vibrometry to assess how flower vibrational properties change with the removal of stamen appendages. Our results show that the absence of staminal appendages does not modify bee buzzing behaviour. Pollen release was higher in unmanipulated flowers, but stigmatic pollen loads differ only marginally between the two treatments. We also detected lower vibration amplitudes in intact flowers as compared to manipulated flowers in artificial vibration experiments. The presence of connective appendages are crucial in transmitting vibrations and assuring optimal pollen release. Therefore, we propose that the high diversity of colours, shapes and sizes of connective appendages in buzz-pollinated flowers may have evolved by selection through male fitness.


Subject(s)
Melastomataceae , Pollination , Animals , Bees , Flowers , Pollen , Reproduction
2.
Int J Microbiol ; 2020: 8823764, 2020.
Article in English | MEDLINE | ID: mdl-32802074

ABSTRACT

Global control and elimination of tuberculosis are hindered by the high prevalence of drug-resistant strains, making the development of new drugs to fight tuberculosis a public health priority. In this study, we evaluated 118 extracts from 58 Venezuelan plant species for their ability to inhibit the growth of Mycobacterium tuberculosis mc26020, using the agar dilution method. Additionally, we determined the ability of these extracts to inhibit the activity of PknB protein, an essential M. tuberculosis serine/threonine kinase, using a high-throughput luminescent assay. Of the 118 extracts tested, 14 inhibited bacterial growth with a minimum inhibitory concentration ≤500 µg/ml, and 36 inhibited the kinase activity with a half-maximal inhibitory concentration <200 µg/ml. Five extracts inhibited M. tuberculosis growth and inhibited the activity of the kinase protein, suggesting that this could be the basis of their growth inhibition.

3.
J Ethnopharmacol ; 150(3): 1154-62, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24212077

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria guianensis (Aublet) Gmell (Rubiaceae) is a medicinal plant from the jungles of South and Central America, used to treat cancer, arthritis, diabetes, and inflammation. Evaluate the anti-inflammatory and anti-tumor effects of Uncaria guianensis preparations. MATERIALS AND METHODS: Bio-guided fractionation of a hydroethanolic extract of Uncaria guianensis was performed, evaluating the fractions and subfractions for their effect on inflammatory mediators, tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) by ELISA and nitric oxide (NO) by the Griess reaction in cultured supernatant from RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). The expression of cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and inhibitor of κB (IκB) were investigated in RAW 264.7 macrophages by flow cytometry. The activity of NF-κB in HeLa cells transfected with a luciferase reporter system was determined. The effect of Uncaria guianensis on the inflammatory response in vivo was assessed in BALB/c mice stimulated with LPS, on rat paw oedema induced by carrageenan, and on tumour growth and lung metastasis in BALB/c mice inoculated with 4T1 mammary tumour cells. Immune cell infiltrates and inflammatory mediators were evaluated in the tumour by immunohistochemistry. RESULTS: Sub-fraction Ug AIV inhibited, to varying degrees, NO, TNF-α, IL-6 and PGE2 production by macrophages in vitro (30 µg/ml) and in the serum of LPS-challenged mice (5 mg/kg). Macrophage expression of Cox-2 was inhibited (35%), IκB degradation was completely inhibited and NF-κB activation was inhibited (70%) by Ug AIV at 30 µg/ml. Ug AIV decreased paw oedema by 86% (5 mg/kg) and serum NO and TNF-α by 45% and 65% respectively. Ug AIV reduced 4T1 mammary tumour growth by 91% on day 33 post-inoculation as well as the levels of serum NO, IL-6 and TNF-α in the same animals. Ug AIV decreased the number of tumour-infiltrating T lymphocytes, macrophages and neutrophils as well as the number of cells positive for COX-2, iNOS, IL-6, TNF-α and p65. CONCLUSIONS: As Ug AIV was not cytotoxic for tumour cells or macrophages, its anti-tumour effect may be due to a reduction in pro-tumoural inflammatory processes in the tumour microenvironment, possibly mediated through NF-κB.


Subject(s)
Anti-Inflammatory Agents , Antineoplastic Agents , Plant Extracts , Uncaria , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Carrageenan , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Dinoprostone/metabolism , Edema/chemically induced , Edema/drug therapy , Female , Humans , I-kappa B Proteins/metabolism , Inflammation/chemically induced , Inflammation/drug therapy , Interleukin-6/metabolism , Lipopolysaccharides , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Male , Mice , Mice, Inbred BALB C , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type II/metabolism , Phytotherapy , Plant Bark , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/metabolism
4.
Cell Death Dis ; 4: e720, 2013 Jul 11.
Article in English | MEDLINE | ID: mdl-23846222

ABSTRACT

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase pump, leading to autophagy induction through the activation of the Ca(2+)/calmodulin-dependent kinase kinase-AMP-activated protein kinase-mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Oleanolic Acid/analogs & derivatives , Saponins/pharmacology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Adenylate Kinase/metabolism , Animals , Apoptosis , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 7 , Beclin-1 , Binding Sites , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Cell Line, Tumor , Class III Phosphatidylinositol 3-Kinases/metabolism , Membrane Proteins/metabolism , Mice , Models, Molecular , Oleanolic Acid/pharmacology , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/metabolism , Ubiquitin-Activating Enzymes/metabolism , Unfolded Protein Response/drug effects
5.
Nat Prod Res ; 25(16): 1565-9, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21827337

ABSTRACT

The primary objective of this study was to search for natural products capable of inhibiting hepatitis B virus (HBV) replication. The research design, methods and procedures included testing hydro-alcoholic extracts (n = 66) of 31 species from the Venezuelan Amazonian rain forest on the cell line HepG2 2.2.15, which constitutively produces HBV. The main outcomes and results were as follows: the species Euterpe precatoria, Jacaranda copaia, Jacaranda obtusifolia, Senna silvestris, Warscewiczia coccinea and Vochysia glaberrima exerted some degree of inhibition on HBV replication. The leaves of W. coccinea showed a significant antiviral activity: 80% inhibition with 100 µg mL⁻¹ of extract. This extract also exerted inhibition on covalently closed circular deoxyribonucleic acid (cccDNA) production and on HIV-1 replication in MT4 cells (more than 90% inhibition with 50 µg mL⁻¹ of extract). Initial fractionation using organic solvents of increasing polarity and water showed that the ethanol fraction was responsible for most of the antiviral inhibitory activities of both the viruses. It was concluded that Warscewiczia coccinea extract showed inhibition of HBV and HIV-1 replication. Bioassay-guided purification of this fraction may allow the isolation of an antiviral compound with inhibitory activity against both viruses.


Subject(s)
Antiviral Agents/pharmacology , HIV-1/drug effects , Hepatitis B virus/drug effects , Plant Extracts/pharmacology , Rubiaceae/chemistry , Virus Replication/drug effects , Cell Line, Transformed , Ethanol , Hep G2 Cells , Humans , Time Factors
6.
Soc Reprod Fertil Suppl ; 63: 237-55, 2007.
Article in English | MEDLINE | ID: mdl-17566277

ABSTRACT

Sperm dysfunction is the single most common defined cause of infertility. One in 15 men is sub-fertile and the condition is increasing in frequency. However, the diagnosis is poor and, excluding assisted conception, there is no treatment. The reason for this is our limited understanding of the biochemical, molecular and genetic functions of the spermatozoon. The underlying premise of our research programme is to establish a rudimentary understanding of the processes necessary for successful fertilisation. In this manuscript, we detail advances in our understanding of calcium signalling in the cell and outline genetic and proteomic technologies that are being used to improve the diagnosis of the condition.


Subject(s)
Calcium Signaling/physiology , Infertility, Male/diagnosis , Spermatozoa/physiology , Calcium/metabolism , Humans , Infertility, Male/pathology , Infertility, Male/therapy , Male , Proteomics , Sodium-Calcium Exchanger/metabolism
7.
Hum Reprod Update ; 12(3): 253-67, 2006.
Article in English | MEDLINE | ID: mdl-16338990

ABSTRACT

Ca(2+) is a ubiquitous intracellular messenger which encodes information by temporal and spatial patterns of concentration. In spermatozoa, several key functions, including acrosome reaction and motility, are regulated by cytoplasmic Ca(2+) concentration. Despite the very small size and apparent structural simplicity of spermatozoa, evidence is accumulating that they possess sophisticated mechanisms for regulation of cytoplasmic Ca(2+) concentration and generation of complex Ca(2+) signals. In this review, we consider the various components of the Ca(2+)-signalling 'toolkit' that have been characterized in somatic cells and summarize the evidence for their presence and activity in spermatozoa. In particular, data accumulated over the last few years show that spermatozoa possess one (and probably two) Ca(2+) stores as well as a range of plasma membrane pumps and channels. Selective regulation of the various components of the 'toolkit' by agonists probably allows spermatozoa to generate localized Ca(2+) signals despite their very small cytoplasmic volume, permitting the discrete and selective activation of cell functions.


Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Calcium/metabolism , Spermatozoa/metabolism , Calcium/agonists , Calcium-Transporting ATPases/metabolism , Humans , Male
8.
Cell Calcium ; 31(1): 45-52, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11990299

ABSTRACT

Curcumin (diferuoylmethane or 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6-hepatadiene-3,5-dione) is the active ingredient of the spice turmeric. Curcumin has been shown to have a number of pharmacological and therapeutic uses. This study shows that curcumin is a potent inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (InsP3 receptor). In porcine cerebellar microsomes, the extent of InsP3-induced Ca2+ release (IICR) is almost completely inhibited by 50 microM curcumin (IC50 = 10 microM). As the extent of IICR cannot be restored back to control levels by the addition of excess InsP3 and since it has little effect on [3H]InsP3 binding to cerebellar microsomes, this inhibition is likely to be non-competitive in nature. IICR in cerebellar microsomes is biphasic consisting of a fast and slow component. The rate constants for the two components are both reduced by curcumin to similar extents (by about 70% of control values at 40 microM curcumin). In addition, curcumin also reduces agonist (ATP)-stimulated Ca2+ mobilization from intact HL-60 cells, indicating that curcumin is cell permeant. However, since it also affects intracellular Ca2+ pumps and possibly ryanodine receptors, it may lead to complex Ca2+ transient responses within cells, which may well explain some of its putative therapeutic properties.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Calcium/metabolism , Curcumin/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Calcium Channels/metabolism , Cerebellum/metabolism , Curcumin/metabolism , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors , Kinetics , Microsomes/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Swine , Tritium
9.
Eur J Biochem ; 268(23): 6318-27, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11733029

ABSTRACT

Curcumin is a compound derived from the spice, tumeric. It is a potent inhibitor of the SERCA Ca2+ pumps (all isoforms), inhibiting Ca2+-dependent ATPase activity with IC50 values of between 7 and 15 microm. It also inhibits ATP-dependent Ca2+-uptake in a variety of microsomal membranes, although for cerebellar and platelet microsomes, a stimulation in Ca2+ uptake is observed at low curcumin concentrations (<10 microm). For the skeletal muscle isoform of the Ca2+ pump (SERCA1), the inhibition of curcumin is noncompetitive with respect to Ca2+, and competitive with respect to ATP at high curcumin concentrations ( approximately 10-25 microm). This was confirmed by ATP binding studies that showed inhibition in the presence of curcumin: ATP-dependent phosphorylation was also reduced. Experiments with fluorescein 5'-isothiocyanate (FITC)-labelled ATPase also suggest that curcumin stabilizes the E1 conformational state. The fact that FITC labels the nucleotide binding site of the ATPase (precluding ATP from binding), and the fact that curcumin affects FITC fluorescence indicate that curcumin must be binding to another site within the ATPase that induces a conformational change to prevent ATP from binding. This observation is interpreted, with the aid of recent structural information, as curcumin stabilizing the interaction between the nucleotide-binding and phosphorylation domains, precluding ATP binding.


Subject(s)
Calcium-Transporting ATPases/antagonists & inhibitors , Curcumin/pharmacology , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Calcium-Transporting ATPases/chemistry , Calcium-Transporting ATPases/metabolism , Enzyme Stability/drug effects , In Vitro Techniques , Kinetics , Liposomes , Permeability , Phosphorylation , Protein Conformation/drug effects , Protein Structure, Tertiary , Rabbits , Sarcoplasmic Reticulum Calcium-Transporting ATPases
10.
J Physiol ; 537(Pt 3): 735-45, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11744751

ABSTRACT

1. Activation of H(+) secretion in the intracellular canaliculi of parietal cells occurs on an unknown time scale with ill-defined kinetics for the coupling of H(+) secretion and the elevation of intracellular calcium ([Ca(2+)](i)) stimulated by secretagogues. 2. We developed a method to determine H(+) secretion in isolated rabbit gastric glands with spatio-temporal resolution, using the fluorescent indicator Lysosensor Yellow-Blue (LYB). Glands accumulated the dye exclusively in the intracellular canaliculi of parietal cells and the gland lumen. Dye fluorescence in the acid spaces of the glands increased upon stimulation of acid secretion by carbachol, histamine and forskolin. Simultaneous fluorescence measurements of acid secretion and [Ca(2+)](i) at 1 s resolution were made by joint loading of LYB and Fluo-3. 3. Carbachol-stimulated H(+) secretion was detected in the gland lumen as early as 3 s after the onset of the [Ca(2+)](i) spike. H(+) accumulation appeared to be transient and paralleled the release component of the [Ca(2+)](i) spike. Short and repetitive stimulations with carbachol elicited repetitive responses in [Ca(2+)](i) and H(+) secretion. 4. Histamine or forskolin stimulated H(+) secretion with a delayed onset (around 2 min) and a sustained response. Acid secretion was temporally unrelated to the oscillatory Ca(2+) responses. 5. The striking difference in the kinetics of activation of H(+) secretion by cholinergic and cAMP-dependent secretagogues indicates that two distinct mechanisms are operating in the final stimulation of the pump, in spite of both eliciting a [Ca(2+)](i) response.


Subject(s)
Calcium/metabolism , Hydrogen/metabolism , Intracellular Membranes/metabolism , Parietal Cells, Gastric/metabolism , Animals , Biological Transport , Carbachol/pharmacology , Colforsin/pharmacology , Gastric Acid/metabolism , Histamine/pharmacology , In Vitro Techniques , Osmolar Concentration , Oxazoles , Parietal Cells, Gastric/drug effects , Rabbits
11.
Cell Calcium ; 30(4): 245-50, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11587548

ABSTRACT

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel which upon activation initiates many cellular functions. Multiple InsP3R subtypes are expressed in most cell types but the physiological significance of this heterogeneity is poorly understood. This study has directly compared the functional properties of the three different InsP3R isoforms by analyzing their InsP3-induced Ca2+ release (IICR) properties in cell lines which predominantly express each isoform subtype. The InsP3-dependence of the amount or extent of IICR was InsP3R isoform-specific, with the type III isoform having the lowest affinity with respect to Ca2+ release. The transient kinetics of IICR, measured using stopped-flow spectrofluorimetry, however, were similar for all three InsP3R isoforms. At maximal InsP3 concentrations (20 microM) the rate constants where between 0.8 and 1.0 s(-1) for the fast phase and 0.25-0.45 s(-1) for the slow phase. The concentration of InsP3 required to induce half-maximal rates of Ca2+ release (EC50) were also similar for the three isoforms (0.2-0.4 microM for the fast phase and 0.75-0.95 microM for the slow phase). These results indicate the InsP3R channel does not significantly differ functionally in terms of Ca2+ release rates between isoforms. The temporal and spatial features of intracellular Ca2+ signals are thus probably achieved through InsP3R isoform-specific regulation or localization rather than their intrinsic Ca2+ efflux properties.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Calcium Channels/chemistry , Calcium Channels/drug effects , Humans , Inositol 1,4,5-Trisphosphate Receptors , Kinetics , Protein Isoforms/drug effects , Protein Isoforms/metabolism , Rats , Receptors, Cytoplasmic and Nuclear/chemistry , Tumor Cells, Cultured
12.
Cell Signal ; 13(1): 57-63, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11257448

ABSTRACT

This study describes the effects of a number of calmodulin antagonists on the cerebellar type 1 inositol 1,4,5-trisphosphate (InsP3) receptor. All the antagonists tested (trifluoperazine, fluphenazine, chlorpromazine and calmidazolium) inhibited the extent of InsP3-induced Ca2+ release (IICR) with similar IC(50) values (between 60 and 85 microM). They did not affect the efficacy of InsP3 to release Ca2+, since the concentrations of InsP3 required to cause half-maximal release was little affected in the presence of these agents. In addition, these agents did not affect InsP3 binding to its receptor. Stopped-flow studies to determine the rate constants of IICR showed this process to be biphasic with a fast and slow component. All the calmodulin antagonists appeared to reduce the rate constants for Ca2+ release in a phase-specific manner, preferentially reducing the fast phase component. Chlorpromazine (75 microM) appeared to have the most potent effect on the fast phase rate constant, reducing it from 1.0 to 0.08 s(-1), while only reducing the rate constant for the slow phase about twofold (0.2-0.08 s(-1)). The fact that calmodulin itself inhibits both IICR and InsP3 binding, while these calmodulin antagonists also reduce Ca2+ release and do not affect InsP3 binding, suggests that the mechanism of action of these agents is unlikely to be due to the reversal of the modulatory action of calmodulin on this receptor.


Subject(s)
Calcium Channels/drug effects , Calmodulin/antagonists & inhibitors , Cerebellum/cytology , Phenothiazines/administration & dosage , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Animals , Chlorpromazine , Dose-Response Relationship, Drug , Fluphenazine , Imidazoles , In Vitro Techniques , Inositol 1,4,5-Trisphosphate Receptors , Microsomes/drug effects , Microsomes/metabolism , Swine , Trifluoperazine
13.
Biochem Pharmacol ; 60(12): 1797-806, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11108794

ABSTRACT

The effects of a number of phenothiazines and other calmodulin antagonists on the Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) were investigated. The drugs used in this study were trifluoperazine, calmidazolium, fluphenazine, chlorpromazine, W-7, and calmodulin-binding peptide. Our results showed that calmidazolium and calmodulin-binding peptide were the most potent inhibitors of skeletal muscle SR Ca(2+)-ATPase activity (isoform SERCA 1) (IC(50) values of 0.5 and 7 microM, respectively), while W-7 was the least potent inhibitor (IC(50), 125 microM). All of the antagonists had little effect on the cerebellar ER Ca(2+)-ATPase activity (isoform SERCA 2b), except for trifluoperazine, which had a biphasic effect, causing stimulation at low concentrations and inhibition at higher concentrations. Our results suggest that the effects of these calmodulin antagonists are independent of calmodulin and that they inhibit the Ca(2+)-ATPase in an isoform-specific manner. It was found that these antagonists inhibit the skeletal muscle isoform of the Ca(2+) pump by altering the Ca(2+) affinity and the associated Ca(2+)-binding steps, as well as possibly stabilising the E1 conformational state of the enzyme.


Subject(s)
Calcium-Transporting ATPases/drug effects , Calmodulin/antagonists & inhibitors , Endoplasmic Reticulum/drug effects , Phenothiazines/pharmacology , Sarcoplasmic Reticulum/drug effects , Adenosine Triphosphatases/metabolism , Animals , Binding Sites , Calcium-Transporting ATPases/metabolism , Calmodulin/metabolism , Endoplasmic Reticulum/metabolism , Fluphenazine/pharmacology , Imidazoles/pharmacology , In Vitro Techniques , Phosphorylation , Rabbits , Sarcoplasmic Reticulum/metabolism , Swine
14.
Eur J Pharmacol ; 408(3): 219-25, 2000 Nov 24.
Article in English | MEDLINE | ID: mdl-11090637

ABSTRACT

Paxilline, a tremorgenic alkaloid mycotoxin produced by Penicillium paxilline, is a reversible inhibitor of the cerebellar inositol 1,4, 5-trisphophate (InsP(3)) receptor. It inhibits the amount or extent of InsP(3)-induced Ca(2+) release (IICR), at sub-maximal concentrations of InsP(3), in a biphasic manner consistent with two inhibition constants (K(i)'s 6.7 and > or =400 microM). As paxilline does not affect InsP(3) binding to the receptor, it can be considered a non-competitive inhibitor. The fact that IICR is biphasic has been interpreted as there being two populations of InsP(3)-sensitive Ca(2+) stores, which release Ca(2+) in either a fast or slow fashion. This study has shown that the rate constants for Ca(2+) release from both the fast and slow populations are reduced by paxilline (100 microM) by about 70% and 60%, respectively. Detailed analysis of the way different concentrations of paxilline inhibit the rate constants for Ca(2+) release indicates that the population of Ca(2+) stores that contribute to the slower phase of Ca(2+) release is more sensitive to the inhibitory action of paxilline.


Subject(s)
Cerebellum/drug effects , Indoles/pharmacology , Mycotoxins/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Animals , Binding, Competitive , Calcium/metabolism , Calcium Channels/metabolism , Cerebellum/metabolism , Dose-Response Relationship, Drug , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate/pharmacology , Inositol 1,4,5-Trisphosphate Receptors , Microsomes/drug effects , Microsomes/metabolism , Rats , Receptors, Cytoplasmic and Nuclear/metabolism
15.
Pharmacol Res ; 42(6): 581-90, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11058412

ABSTRACT

One of the major mechanisms by which hormones elevate intracellular Ca(2+)levels is by generating the second messenger inositol 1,4, 5-trisphosphate (InsP(3)), which activates a Ca(2+)channel (InsP(3)receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP(3)receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP(3)receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP(3)receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [(3)H]InsP(3)and InsP(3)-induced Ca(2+)release studies using Fluo-3 as the Ca(2+)sensing dye. All three isoforms of the InsP(3)receptor were identified using RT-PCR and immunochemical analyses. [(3)H]InsP(3)binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP(3)receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP(3)binding to the receptor was also different in the three tissues. In cerebellum the K(d)was 60 nM, while aorta had a much higher K(d)of 220 nM. Heart microsomes, appeared to show two classes of binding affinity with K(d)s of 150 nM and 60 nM. Furthermore, the effects of free [Ca(2+)] on [(3)H]InsP(3)binding levels were also different for the three tissues. InsP(3)binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca(2+)], while InsP(3)binding to heart was relatively insensitive to changes in [Ca(2+)]. At maximal InsP(3)concentrations, aorta microsomes were able to release about 5% of the accumulated Ca(2+), compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP(3)-induced Ca(2+)release ( <0.5%). The EC(50)concentration for InsP(3)-induced Ca(2+)release was 1.2 micro M for aorta while that for cerebellum was 0.3 micro M. Known agonists of the cerebellar InsP(3)receptor such as 3-deoxy InsP(3)and adenophostin A were also able to mobilize Ca(2+)from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP(3)receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP(3)-induced Ca(2+)release from aorta microsomes.


Subject(s)
Aorta/chemistry , Calcium Channels/classification , Myocardium/chemistry , Receptors, Cytoplasmic and Nuclear/classification , Animals , Calcium/metabolism , Calcium Channels/physiology , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors , Protein Isoforms/analysis , Rats , Receptors, Cytoplasmic and Nuclear/physiology , Reverse Transcriptase Polymerase Chain Reaction , Swine
16.
Biochem Biophys Res Commun ; 277(3): 568-74, 2000 Nov 02.
Article in English | MEDLINE | ID: mdl-11061995

ABSTRACT

Industrial alkylphenols in the environment may act as "xenoestrogens" to disrupt testicular development and decrease male fertility. Amongst possible targets for these compounds are testicular Sertoli cells, which nurture the developing sperm cells. We demonstrate that SERCA 2 and 3 Ca(2+) pumps are relatively abundant in rat testis microsomal membranes, and also in Sertoli, myoid, and TM4 cells (a Sertoli cell line). A number of estrogenic alkylphenols such as nonylphenol, octylphenol, bisphenol A, and butylated hydroxytoluene all inhibit testicular Ca(2+) ATPase in the low micromolar concentration range. These agents also mobilize intracellular Ca(2+) in intact TM4 cells in a manner consistent with the inhibition of ER Ca(2+) pumps. Alkylphenols dramatically decrease the viability of TM4 cells, an effect that is reversed by either a caspase inhibitor or by BAPTA, and is therefore consistent with Ca(2+)-dependent cell death via apoptosis. We postulate that alkylphenols disrupt testicular development by inhibiting ER Ca(2+) pumps, thus disturbing testicular Ca(2+) homeostasis.


Subject(s)
Apoptosis , Calcium-Transporting ATPases/antagonists & inhibitors , Endoplasmic Reticulum/drug effects , Phenols/pharmacology , Testis/drug effects , Animals , Calcium-Transporting ATPases/metabolism , Endoplasmic Reticulum/metabolism , Estradiol Congeners/pharmacology , In Vitro Techniques , Male , Rats , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sertoli Cells/cytology , Sertoli Cells/drug effects , Testis/cytology , Testis/metabolism
17.
Cell Calcium ; 28(3): 137-49, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11020376

ABSTRACT

Ca2+ plays a key role in many pathological processes, including viral infections. Rotavirus, the major etiological agent of viral gastroenteritis in children and young animals, provides a useful model to study a number of Ca2+ dependent virus-cell interactions. Rotavirus entry, activation of transcription, morphogenesis, cell lysis, particle release, and the distant action of viral proteins are Ca2+ dependent processes. In the extracellular medium, Ca2+ stabilizes the structure of the viral capsid. During entry into the cell the low cytoplasmic Ca2+ concentration induced the solubilization of the outer protein layer of the capsid and transcriptase activation. Viral protein synthesis modifies Ca2+ homeostasis which, in turn, favours viral morphogenesis and induces cell death. The generation of diarrhea is a multifactorial process involving Ca2+ dependent secretory processes of mediators and water and electrolytes, as well as the induction of cell death in the different cell types that compose the intestinal epithelium. The discovery of the non-structural viral protein NSP4 as a viral enterotoxin and the possible participation of the enteric nervous system in the pathogenesis of diarrhea represent significant advances in its understanding. Ca2+ also plays a role in the replication cycles and pathogenesis of other viral diseases such as poliovirus, Coxsackie virus, cytomegalovirus, vaccinia and measles virus and HIV.


Subject(s)
Calcium , Rotavirus Infections/virology , Rotavirus/physiology , Rotavirus/pathogenicity , Animals , Calcium/metabolism , Capsid/metabolism , Cell Death , Diarrhea/virology , Endocytosis , Glycoproteins/metabolism , Homeostasis , Humans , Toxins, Biological , Viral Nonstructural Proteins/metabolism , Virus Replication
18.
J Enzyme Inhib ; 15(2): 163-70, 2000.
Article in English | MEDLINE | ID: mdl-10938541

ABSTRACT

Intracellular free calcium is regulated by Ca(++)-ATPase, one form present on the plasma membrane (PM Ca(++)-ATPase) and the other on sarcoplasmic (endoplasmic) reticulum (SR/ER Ca(++)-ATPase). An endogenous inhibitor of SR Ca(++)-ATPase from human placenta was shown to be present in normal placenta and the activity was not detectable in placenta from preeclamptic patients. The inhibitor was distributed in cytosol and microsomes. The inhibition of Ca(++)-ATPase by this inhibitor was concentration- and time-dependent. The inhibitor neither bound to DEAE- nor CM-sepharose resins at pH 7.5 and 8.5. Furthermore, it was heat stable for 15 min up to 55 degrees C and completely destroyed at 80 degrees C in a few minutes. It was also observed to be stable at room temperature for at least 3 months. The purification and characterization of this inhibitor would be valuable in achieving an understanding of the normal regulation of Ca(++)-ATPase in the placenta during pregnancy.


Subject(s)
Calcium-Transporting ATPases/antagonists & inhibitors , Enzyme Inhibitors/metabolism , Placenta/metabolism , Animals , Cytosol/metabolism , Female , Humans , Microsomes/metabolism , Pregnancy , Rabbits , Temperature
19.
Lab Invest ; 80(1): 37-45, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10653001

ABSTRACT

Early reports indicated that ECV304 was a spontaneously-transformed line derived from a Japanese human umbilical vein endothelial cells (HUVEC) culture. Many morphological, immunochemical, and genetic studies provided further evidence that ECV304 was a valuable biomedical research tool and could be used to study processes that include angiogenesis in vitro and signal transduction by a variety of G protein-coupled receptors. However, several distinct differences between ECV304 and HUVEC are now apparent and recent reports have indicated genetic similarity between ECV304 and T24/83, a human bladder cancer cell line. To further assess the utility of ECV304 as a human endothelial cell model, we compared the functional responses of ECV304 and T24/83 to a range of G protein-coupled receptor agonists. We also used DNA fingerprinting to karyotype both ECV304 and T24/83. Both ATP and uridine triphosphate (UTP) stimulated inositol phosphate metabolism in ECV304 without alteration of cAMP levels. Comparative data using selective P2Y receptor agonists indicated that this response, leading to calcium mobilization from intracellular stores, was predominantly mediated by the activation of P2Y2 receptors. Similar responses were recorded from both ECV304 and T24/83 cells. ECV304 expressed a relatively high basal activity of NOS that was reduced by L-NAME and stimulated by P2Y2 receptor agonists. In contrast, P2Y2 receptor activation did not induce prostaglandin synthesis in ECV304. Both ECV304 and T24/83 express receptors for adenosine, adrenaline, and calcitonin, which stimulate adenylate cyclase. Proliferation of ECV304 and T24/83 cells, measured by the incorporation of [3H]thymidine into DNA, was largely serum-independent. This was in contrast to parallel experiments with porcine and bovine aortic endothelial cells that indicated a marked serum-dependent increase in DNA synthesis. Genetic analysis confirmed that ECV304 and T24/83 are identical. ECV304 displays some endothelial characteristics and is useful for the study of receptor pharmacology. However, ECV304 is not of HUVEC origin and is therefore an inappropriate cell line to study endothelial cell biology.


Subject(s)
Endothelium, Vascular/cytology , Urinary Bladder Neoplasms/pathology , Adenylyl Cyclases/metabolism , Blood , Calcium/metabolism , DNA Fingerprinting , DNA Replication , Evaluation Studies as Topic , Humans , Models, Biological , Receptors, Purinergic P2/drug effects , Receptors, Purinergic P2/physiology , Tumor Cells, Cultured
20.
Biochem Mol Biol Int ; 47(4): 631-8, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10319415

ABSTRACT

The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the co-chaperone, GroES, and ATP. In this study it is shown that GroEL alone substantially reduces the inactivation of purified Ca(++)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum. In the absence of GroEL, the enzyme became completely inactive in about 45-60 hours when kept at 25 degrees C, while in the presence of an equimolar amount of GroEL, the enzyme remained approximately 80% active even after 75 hours. Equimolar amounts of BSA or lysozyme were unable to protect the enzyme from inactivation under identical conditions. Analysis by SDS-PAGE showed GroEL was acting by blocking the aggregation of ATPase at 25 degrees C. GroEL was not as effective in protection at -20 degrees C or 4 degrees C. These results are discussed in the context of current models of the GroEL mechanism.


Subject(s)
Calcium-Transporting ATPases/metabolism , Chaperonin 60/metabolism , Sarcoplasmic Reticulum/enzymology , Animals , Enzyme Activation , Rabbits
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