Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan.

Microorganisms are very powerful tools for the supply of information about the toxic effects of lipophilic compounds, since an impairment of cell growth usually occurs as a result of perturbations related, in most cases, with the partition of toxicants in membranes. The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify - and β-endosulfan interactions with the membrane possibly related with the insecticide toxicity. Two approaches have been pursued: (a) bacterial growth is followed and the effects of endosulfan isomers determined; (b) biophysical studies with the fluorescent fluidity probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to assess the effects of - and β-endosulfan on the organization of the membrane lipid bilayer. The effects on growth were quantitatively evaluated by determination of growth parameters, namely the lag phase, the specific growth rate and the cell density reached by cultures in the stationary phase. Growth inhibition by and β-endosulfan dependent on the concentration is diminished or removed by the addition of 2.5 m Ca²⁺ to bacterial cultures. Fluorescence DPH polarization consistently showed opposite effects of Ca²⁺ and - and β-endosulfan on the physical state of bacterial polar lipid dispersions.     

β1-Adrenoceptor stimulation suppresses endothelial IKCa-channel hyperpolarization and associated dilatation in resistance arteries

Background and Purpose

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK_Ca) and intermediate conductance Ca²⁺-activated K⁺ channels (IK_Ca) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK_Ca channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation.

Experimental Approach

Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]_i, mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording.

Key Results

Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM–10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]_i. The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK_Ca {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK_Ca (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh.

Conclusions and Implications

In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK_Ca channels and may be one consequence of raised circulating catecholamines.     

Positive allosteric modulation of α7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neurons

Background and purpose:

α7-Nicotinic acetylcholine receptors (α7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of α7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca²⁺-induced toxicity through prolonged activation of α7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles.

Experimental approach:

The present studies evaluated cytotoxic effects of type I PAM [N-(4-chlorophenyl)]-α-[(4-chloro-phenyl)-aminomethylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and type II PAM 1-[5-chloro-2,4-dimethoxy-phenyl]-3-[5-methyl-isoxazol-3-yl]-urea (PNU-120596), or 4-[5-(4chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulphonamide (A-867744). The studies used cultures of PC12 cells and primary cultures of rat cortical neuronal cells.

Key results:

Our results showed that neither type I nor type II PAMs had any detrimental effect on cell integrity or cell viability. In particular, type II PAMs did not affect neuron number and neurite outgrowth under conditions when α7 nAChR activity was measured by Ca²⁺ influx and extracellular signal-regulated kinases 1 and 2 phosphorylation, following exposure to α7 nAChR agonists.

Conclusions and implications:

This study demonstrated that both type I and type II α7 nAChR selective PAMs, although exhibiting differential electrophysiological profiles, did not exert cytotoxic effects in cells endogenously expressing α7 nAChRs.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

Omega-3 fatty acids induce Ca2+ mobilization responses in human colon epithelial cell lines endogenously expressing FFA4

Aim:

Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro.

Methods:

HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4.

Results:

Ten to 100 μmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca²⁺]_i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca²⁺]_i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca²⁺]_i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca²⁺ATPase inhibitor thapsigargin, but was not affected by G_i/o protein inhibitor PTX or IP₃R inhibitor 2-APB.

Conclusion:

Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca²⁺ mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive G_i/o protein, followed by Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and Ca²⁺ influx across the plasma membrane.     

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum

1. To characterize increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) associated with discharge of action potentials, membrane potential and [Ca²⁺]_i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques.
2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca²⁺]_i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca²⁺]_i transients with an almost constant amplitude.
3. Nicardipine (1 μM) and La³⁺ (10 μM), blockers of voltage-dependent Ca²⁺ channels (VDCCs), abolished both the action potential discharge and the [Ca²⁺]_i transient.
4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca²⁺-activated K⁺ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca²⁺]_i transients.
5. Thapsigargin (1 μM), an inhibitor of SR Ca²⁺-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca²⁺]_i transients without affecting action potentials. In addition, in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca²⁺ current but exerted a similar effect on its concomitant rise in [Ca²⁺]_i to that on the action potential-triggered [Ca²⁺]_i transient.
6. Similar action potential-triggered [Ca²⁺]_i transients were induced by brief exposures to high-K⁺ solution. They were not decreased, but rather increased, after depletion of intracellular Ca²⁺ stores by a combination of ryanodine (30 μM) and caffeine (10 mM) through an open-lock of Ca²⁺-induced Ca²⁺ release (CICR)-related channels.
7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca²⁺]_i. Intracellular Ca²⁺ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca²⁺]_i transients by replenishing cytosolic Ca²⁺. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca²⁺]_i transient.

     

Liquiritigenin inhibits Aβ25–35-induced neurotoxicity and secretion of Aβ1–40 in rat hippocampal neurons

Aim:

To examine whether liquiritigenin, a newly found agonist of selective estrogen receptor-β, has neuroprotective activity against β-amyloid peptide (Aβ) in rat hippocampal neurons.

Methods:

Primary cultures of rat hippocampal neurons were pretreated with liquiritigenin (0.02, 0.2, and 2 μmol/L) prior to Aβ_25–35 exposure. Following treatment, viability of the cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and by a lactate dehydrogenase activity-based cytotoxicity assay. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) and levels of reactive oxygen species (ROS), as well as apoptotic rates, were determined. Our studies were extended in tests of whether liquiritigenin treatment could inhibit the secretion of Aβ_1–40 as measured using an ELISA method. In order to analyze which genes may be involved, we used a microarray assay to compare gene expression patterns. Finally, the levels of specific proteins related to neurotrophy and neurodenegeration were detected by Western blotting.

Results:

Pretreated neurons with liquiritigenin in the presence of Aβ_25–35 increased cell viability in a concentration-dependent manner. Liquiritigenin treatment also attenuated Aβ_25–35-induced increases in [Ca²⁺]_i and ROS level and decreased the apoptotic rate of neurons. Some genes, including B-cell lymphoma/leukemia-2 (Bcl-2), neurotrophin 3 (Ntf-3) and amyloid β (A4) precursor protein-binding, family B, member 1 (Apbb-1) were regulated by liquiritigenin; similar results were shown at the protein level by Western blotting.

Conclusion:

Our results demonstrate that liquiritigenin exhibits neuroprotective effects against Aβ_25-35-induced neurotoxicity and that it can decrease the secretion of Aβ_1–40. Therefore, liquiritigenin may be useful for further study as a prodrug for treatment of Alzheimer''s disease.     

Montelukast inhibits neutrophil pro-inflammatory activity by a cyclic AMP-dependent mechanism

Background and purpose

The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1–2 µmol·L⁻¹), on Ca²⁺-dependent pro-inflammatory activities, cytosolic Ca²⁺ fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 µmol·L⁻¹) and platelet-activating factor (200 nmol·L⁻¹).

Experimental approach

Generation of reactive oxygen species was measured by lucigenin- and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B₄ and cAMP by competitive binding ELISA procedures, and Ca²⁺ fluxes by fura-2/AM-based spectrofluorimetric and radiometric (⁴⁵Ca²⁺) procedures.

Key results

Pre-incubation of neutrophils with montelukast resulted in dose-related inhibition of the generation of reactive oxygen species and leukotriene B₄ by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 µmol·L⁻¹ (mean percentages of the control values of 30 ± 1, 12 ± 3 and 21 ± 3 respectively; P < 0.05). From a mechanistic perspective, treatment of chemoattractant-activated neutrophils with montelukast resulted in significant reductions in both post-peak cytosolic Ca²⁺ concentrations and store-operated Ca²⁺ influx. These montelukast-mediated alterations in Ca²⁺ handling by the cells were associated with a significant elevation in basal cAMP levels, which resulted from inhibition of cyclic nucleotide phosphodiesterases.

Conclusions and implications

Montelukast, primarily a cysteinyl leukotriene (CysLT₁) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.     

Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

Aim:

To identify the mechanisms underlying the elevation of intracellular Ca²⁺ level ([Ca²⁺]_i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons.

Methods:

Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca²⁺]_i was measured using fura-2 AM. Ca²⁺ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay.

Results:

Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca²⁺]_i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons depended on extracellular Ca²⁺, and was blocked by P/Q-type Ca²⁺channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca²⁺ channel blocker nifedipine (10 μmol/L) or N-type Ca²⁺channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca²⁺ current in ARC neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L).

Conclusion:

Lowering glucose level enhances the activity of P/Q type Ca²⁺channels and elevates [Ca²⁺]_i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.     

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg

1. The sea urchin egg homogenate is an ideal model to characterize Ca²⁺-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP₃- and ryanodine-sensitive Ca²⁺-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist.
2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca²⁺-releasing agent, NAADP, compared to inositol trisphosphate (InsP₃) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors.
3. NAADP-induced Ca²⁺-release was blocked by L-type Ca²⁺-channel blockers and by Bay K 8644, while InsP₃- and cyclic ADPR-induced Ca²⁺-release were insensitive to these agents. L-type Ca²⁺-channel blockers did not displace [³²P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion.
4. Similarly, a number of K⁺-channel antagonists blocked NAADP-induced Ca²⁺-release selectively over InsP₃- and cyclic ADPR-induced Ca²⁺-release. Radioligand studies showed that these agents were not competitive antagonists.
5. As has been shown for InsP₃ and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca²⁺-release mechanisms.
6. The presence of K⁺ was not essential for NAADP-induced Ca²⁺-release, since substitution of K⁺ with other monovalent cations in the experimental media did not significantly alter Ca²⁺ release by NAADP. On the contrary, cyclic ADPR and InsP₃-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K⁺. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca²⁺-release. While the alkaline condition permitted InsP₃ and cyclic ADPR-induced Ca²⁺-release, the acidic condition completely hampered both Ca²⁺-release mechanisms.
7. The present results characterize pharmacologically and biochemically the novel Ca²⁺-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.

     

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca²⁺([Ca²⁺]_i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca²⁺-channel blocker.
2. Midazolam (30 and 100 μM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 μM NA.
3. NA (10 μM) produced a phasic, followed by a tonic increase in both [Ca²⁺]_i and force. Midazolam (10–100 μM) did not modify the resting [Ca²⁺]_i, but attenuated the NA-induced phasic and tonic increases in [Ca²⁺]_i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 μM) attenuated the NA-induced tonic, but not phasic, increases in [Ca²⁺]_i and force.
4. In Ca²⁺-free solution containing 2 mM EGTA, NA (10 μM) transiently increased [Ca²⁺]_i and force. Midazolam (10–100 μM), but not nicardipine (0.3 μM), attenuated this NA-induced increase in [Ca²⁺]_i and force, in a concentration-dependent manner. However, midazolam (10 and 30 μM), had no effect on the increases in [Ca²⁺]_i and force induced by 10 mM caffeine.
5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca²⁺- storage sites, NA slowly increased [Ca²⁺]_i and force. Nicardipine (0.3 μM) did not modify the resting [Ca²⁺]_i but partly attenuated the NA-induced increases in [Ca²⁺]_i and force. In the presence of nicardipine, midazolam (100 μM) lowered the resting [Ca²⁺]_i and further attenuated the remaining NA-induced increases in [Ca²⁺]_i and force.
6. The [Ca²⁺]_i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca²⁺ (0.16–2.6 mM) in Ca²⁺-free solution containing 100 mM K⁺. NA (10 μM) shifted the [Ca²⁺]_i-force relationship to the left and enhanced the maximum Ca²⁺-induced force. Under these conditions, whether in the presence or absence of 10 μM NA, midazolam (10 and 30 μM) attenuated the increases in [Ca²⁺]_i and force induced by Ca²⁺ without changing the [Ca²⁺]_i-force relationship.
7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca²⁺ mobilization. Midazolam attenuates NA-induced Ca²⁺ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca²⁺ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

     

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

1. Effects of adrenomedullin and α-calcitonin gene-related peptide (CGRP) on the contractions and cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α-CGRP were also investigated.
2. Adrenomedullin and α-CGRP caused a concentration-dependent relaxation in the rat aorta contracted with noradrenaline. The IC₅₀ values for adrenomedullin and α-CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, N^G-monomethyl-L-arginine (L-NMMA, 100 μM), but not by a cyclo-oxygenase inhibitor, indomethacin (10 μM).
3. Adrenomedullin and α-CGRP increased the endothelial [Ca²⁺]_i in the rat aorta with endothelium, whereas they did not change [Ca²⁺]_i in the smooth muscle.
4. An antagonist of the CGRP₁ receptor, CGRP (8–37), antagonized the relaxant effects of α-CGRP and the β-isoform of CGRP (β-CGRP) but not those of adrenomedullin in the rat aorta.
5. In the porcine coronary artery contracted with U46619, adrenomedullin and α-CGRP caused a concentration-dependent relaxation with an IC₅₀ of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC₅₀ values nor the maximal relaxations induced by adrenomedullin or α-CGRP. When the artery was contracted with high K⁺ solution (72.7 mM), these peptides caused a small relaxation.
6. Adrenomedullin and α-CGRP increased cyclic AMP content and decreased the smooth muscle [Ca²⁺]_i in the porcine coronary artery.
7. CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α-CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β-isoform of CGRP (β-CGRP).
8. These results suggest that in the rat aorta, adrenomedullin and α-CGRP increase the endothelial [Ca²⁺]_i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α-CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca²⁺]_i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α-CGRP, β-CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α-CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β-CGRP.

     

Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels

BACKGROUND AND PURPOSE

Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well-known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non-junctional connexin43 (Cx43) hemichannels, pore-forming α-haemolysin and channels involved in ATP release under hypotonic conditions.

EXPERIMENTAL APPROACH

Hemichannels were activated by removing extracellular Ca²⁺. The influences of suramin on hemichannel activities were evaluated by its effects on influx of fluorescent dyes and efflux of ATP. The membrane permeability and integrity were assessed through cellular retention of preloaded calcein and LDH release.

KEY RESULTS

Suramin blocked Cx43 hemichannel permeability induced by removal of extracellular Ca²⁺ without much effect on Cx43 expression and gap junctional intercellular communication. This action of suramin was mimicked by its analogue NF023 and NF449 but not by another P2 purinoceptor antagonist PPADS. Besides hemichannels, suramin also significantly blocked intracellular and extracellular exchanges of small molecules caused by α-haemolysin from Staphylococcus aureus and by exposure of cells to hypotonic solution. Furthermore, it prevented α-haemolysin- and hypotonic stress-elicited cell injury.

CONCLUSION AND IMPLICATIONS

Suramin blocked membrane channels and protected cells against toxin- and hypotonic stress-elicited injury. Our finding provides novel mechanistic insights into the pharmacological actions of suramin. Suramin might be therapeutically exploited to protect membrane integrity under certain pathological situations.     

The effect of a secreted form of β-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones

1. The effects of secreted forms of β-amyloid-precursor proteins (APP^Ss) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in rat cultured hippocampal neurones. APP695^S, a secretory form of APP695, attenuated the increase in [Ca²⁺]_i evoked by glutamate. In addition, APP695^S itself evoked an increase in [Ca²⁺]_i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells.
2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695^S with an increase in [Ca²⁺]_i.
3. APP695^S induced a transient rise in [Ca²⁺]_i even in the absence of extracellular Ca²⁺ and produced an elevation in inositol-1,4,5-trisphosphate (IP₃) in a concentration-dependent manner from 100 to 500 ng ml⁻¹. In the presence of extracellular Ca²⁺, APP695^S caused a transient rise in [Ca²⁺]_i followed by a sustained phase at high [Ca²⁺]_i, suggesting Ca²⁺ entry from the extracellular space.
4. The [Ca²⁺]_i elevation was mimicked by amino terminal peptides of APP^S, but not by carboxy terminal peptides.
5. These results taken together suggest that APP695^S induces an increase in [Ca²⁺]_i in hippocampal neurones through an IP₃-dependent mechanism that changes according to the stage of development.

     

Effect of cholesterol on membrane-damaging activity of Naja nigricollis toxin γ toward phospholipid vesicles

Membrane-damaging activity of Naja nigricollis toxin γ on phospholipid vesicles was attenuated by incorporation of cholesterol into phospholipid vesicles. Deprivation of cholesterol from erythrocyte membrane enhanced notably hemolytic activity of toxin γ, while the hemolytic activity of toxin γ on cholesterol-depleted erythrocytes decreased when cholesterol was restored to membrane. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles were modestly affected in the presence of cholesterol. Time-resolved fluorescence and Fourier transform infrared spectra showed that phospholipid-bound toxin γ and cholesterol/phospholipid-bound toxin γ did not adopt the same conformation. Moreover, geometrical arrangement of toxin γ in contact with phospholipid vesicles was different from that with cholesterol/phospholipid vesicles as evidenced by N-(fluorescein-5-thiocarbamoyl)-1, 2-dihexadecanoyl-phosphatidylethanolamine fluorescence enhancement and color transformation of phospholipid/polydiacetylene membrane assay. Taken together, our data show that ordered phospholipid phase arising from incorporation of cholesterol affects conformation and topographical arrangement of toxin γ on water–lipid interface, thus attenuating its membrane-damaging activity against phospholipid vesicles.     

Inotropy and L-type Ca2+ current,activated by β1- and β2-adrenoceptors,are differently controlled by phosphodiesterases 3 and 4 in rat heart

Background and purpose

β₁- and β₂-adrenoceptors coexist in rat heart but β₂-adrenoceptor-mediated inotropic effects are hardly detectable, possibly due to phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide (300 nmol·L⁻¹) and the PDE4 inhibitor rolipram (1 µmol·L⁻¹) on the effects of (−)-catecholamines.

Experimental approach

Cardiostimulation evoked by (−)-noradrenaline (ICI118551 present) and (−)-adrenaline (CGP20712A present) through β₁- and β₂-adrenoceptors, respectively, was compared on sinoatrial beating rate, left atrial and ventricular contractile force in isolated tissues from Wistar rats. L-type Ca²⁺-current (I_Ca-L) was assessed with whole-cell patch clamp.

Key results

Rolipram caused sinoatrial tachycardia. Cilostamide and rolipram did not enhance chronotropic potencies of (−)-noradrenaline and (−)-adrenaline. Rolipram but not cilostamide potentiated atrial and ventricular inotropic effects of (−)-noradrenaline. Cilostamide potentiated the ventricular effects of (−)-adrenaline but not of (−)-noradrenaline. Concurrent cilostamide + rolipram uncovered left atrial effects of (−)-adrenaline. Both rolipram and cilostamide augmented the (−)-noradrenaline (1 µmol·L⁻¹) evoked increase in I_Ca-L. (−)-Adrenaline (10 µmol·L⁻¹) increased I_Ca-L only in the presence of cilostamide but not rolipram.

Conclusions and implications

PDE4 blunts the β₁-adrenoceptor-mediated inotropic effects. PDE4 reduces basal sinoatrial rate in a compartment distinct from compartments controlled by β₁- and β₂-adrenoceptors. PDE3 and PDE4 jointly prevent left atrial β₂-adrenoceptor-mediated inotropy. Both PDE3 and PDE4 reduce I_Ca-L responses through β₁-adrenoceptors but the PDE3 component is unrelated to inotropy. PDE3 blunts both ventricular inotropic and I_Ca-L responses through β₂-adrenoceptors.     

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

1. Two types of Ca²⁺ channel α₁-subunits were co-expressed in Xenopus oocytes with the Ca²⁺ channel α₂- and β₁-subunits. The Ba²⁺ current through the α_1Cα₂β and the α_1Bα₂β channels had electrophysiological and pharmacological properties of L- and N-type Ca²⁺ channels, respectively.
2. Amlodipine had a strong blocking action on both the L-type and N-type Ca²⁺ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca²⁺ channel was comparable to that on the L-type Ca²⁺ channel. At −100 mV holding potential, the IC₅₀ values for amlodipine block on the L-type and N-type Ca²⁺ channel were 2.4 and 5.8 μM, respectively.
3. The blocking action of amlodipine on the N-type Ca²⁺ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca²⁺ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.
4. The time course of block development by amlodipine was similar for L-type and N-type Ca²⁺ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca²⁺ channel.

     

Oestrogen confers cardioprotection by suppressing Ca2+/calmodulin-dependent protein kinase II

Background and purpose:

Oestrogen confers cardioprotection by down-regulating the β₁-adrenoceptor and suppressing the expression and activity of protein kinase A. We hypothesized that oestrogen may also protect the heart by suppressing Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), another signalling messenger activated by the β₁-adrenoceptor, that enhances apoptosis.

Experimental approach:

We first determined the expression of CaMKII in the heart from sham and ovariectomized rats with and without oestrogen replacement. We then determined the effects of CaMKII inhibition (KN93, 2.5 µmol·L⁻¹) in the presence or absence of 10⁻⁷ mol·L⁻¹ isoprenaline, a non-selective β-adrenoceptor agonist. We also determined the percentage apoptosis in myocytes from rats in each group with or without β-adrenoceptor stimulation.

Key results:

Both CaMKIIδ and phosphorylated CaMKII were up-regulated in the hearts from ovariectomized rats, and they were restored to normal by oestrogen replacement. The infarct size and lactate dehydrogenase release were significantly greater after ovariectomy. Similarly, cardiac contractility, the amplitude of the electrically induced intracellular Ca²⁺ transient and the number of apoptotic cells were also greater in ovariectomized rats upon ischaemia/reperfusion in the presence or absence of isoprenaline. Most importantly, the responses to ischaemic insult in ovariectomized rats were reversed not only by oestrogen replacement, but by blockade of CaMKII with KN93.

Conclusions and implications:

Oestrogen confers cardioprotection at least partly by suppressing CaMKIIδ. This effect of oestrogen on CaMKII is independent of the β-adrenoceptor and occurs in addition to down-regulation of the receptor.     

Suitability of the old fowl rectal caecum preparation for investigating the selectivity of β-adrenergic drugs

We tested α- and β-adrenergic drugs on isolated strips of fowl rectal caecum from 14- to 16-week-old Warren hens. Basal tone and spontaneous motility were dose-dependently reduced by isoprenaline and all the selective β-agonists tested (except xamoterol) with the following order of potency: isoprenaline=fenoterol=procaterol=clenbuterol>dobutamine>SR58611A. The results indicate that this tissue preparation consists almost entirely of β₂-adrenoceptors. This preparation may, therefore, be considered a suitable assay for discriminating β₁- from β₂-agonists according to their selectivity.     

Binding of the antiretroviral drug, -aspartate-β-hydroxamate on the NMDA receptor

-Aspartate-β-hydroxamate ( -A β H) exhibits antiretroviral properties in vitro and in vivo. It has glutamate agonist properties at the N-methyl- -aspartate (NMDA) receptor in neuronal cell cultures. This study characterizes its binding properties to the NMDA receptor by measuring its stimulating effect on N-(1-(2-thienyl)[³H]cyclohexyl)piperidine ([³H]TCP) binding to the ionic channel in rat brain membranes. -A β H stimulated [³H]TCP binding in a dose-dependent manner but to a lower extent than glutamate, suggesting only partial glutamate agonist properties. In the presence of antagonists of the different effector sites of the NMDA receptor the affinity of -A β H was competitively decreased by CGS-19755 and 7-chlorokynurenate and unaffected by arcaine. Among several -A β H analogues VHS.125 behaved as a full NMDA agonist, but - or -glutamate γ-monohydroxamate ( -GH or -GH) were without effect. This study shows that -A β H has potential neurotoxic effects due to its direct interaction with the NMDA receptor and that analogues such as -GH or -GH may rather be used in humans.