Absence Of External Calcium Research Articles

Inhibition of uterine contractility by guanine-based purines in non-pregnant rats.

Growing evidence pointed out that guanine-based purines are able to modulate smooth muscle contractile activity of blood vessels and gastrointestinal tract. Since, so far, possible guanine-based purine modulation of uterine musculature is unknown, the aim of the present study was to investigate in vitro, using organ bath technique, guanosine and guanine effects on spontaneous uterine contraction, and uterine contraction induced by K+-depolarization and oxytocin in a non-pregnant rat. Guanosine, but not guanine, reduced the amplitude of spontaneous contraction of the uterine muscle in a dose-dependent manner. The inhibitory response was antagonized by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a membrane nucleoside transporter inhibitor, but persisted in the presence of theophylline, a nonselective adenosine receptor antagonist, or propanolol, β1/β2 adrenoreceptor antagonist or blockers of a nitrergic pathway. In addition, potassium channel blockers did not influence guanosine-induced effects. Guanosine was able to inhibit the external calcium (Ca2+) influx-induced contraction, but it did not affect the contraction induced by high-KCl solution, indicating that guanosine does not interact with L-type voltage-gated calcium channel. Guanosine prevented/reduced uterine contractions induced by oxytocin, even in the absence of external calcium. In conclusion, guanosine is able to reduce both spontaneous and oxytocin-induced contractions of rat myometrium, likely subsequently to its intracellular intake. The blockade of extracellular Ca2+ influx and reduction of Ca2+ release from the intracellular store are the mechanisms involved in the guanosine-induced tocolytic effects.

TRPV1 channels regulate the automaticity of embryonic stem cell-derived cardiomyocytes through stimulating the Na+ /Ca2+ exchanger current.

Calcium controls the excitation-contraction coupling in cardiomyocytes. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) are an important cardiomyocyte source for regenerative medicine and drug screening. Transient receptor potential vanilloid 1 (TRPV1) channels are nonselective cation channels that permeate sodium and calcium. This study aimed to investigate whether TRPV1 channels regulate the electrophysiological characteristics of ESC-CMs. If yes, what is the mechanism behind? By immunostaining and subcellular fractionation, followed by western blotting, TRPV1 was found to locate intracellularly. The staining pattern of TRPV1 was found to largely overlap with that of the sarco/endoplasmic reticulum Ca2+ -ATPase, the sarcoplasmic reticulum (SR) marker. By electrophysiology and calcium imaging, pharmacological blocker of TRPV1 and the molecular tool TRPV1β (which could functionally knockdown TRPV1) were found to decrease the rate and diastolic depolarization slope of spontaneous action potentials, and the amplitude and frequency of global calcium transients. By calcium imaging, in the absence of external calcium, TRPV1-specific opener increased intracellular calcium; this increase was abolished by preincubation with caffeine, which could deplete SR calcium store. The results suggest that TRPV1 controls calcium release from the SR. By electrophysiology, TRPV1 blockade and functional knockdown of TRPV1 decreased the Na+ /Ca2+ exchanger (NCX) currents from both the forward and reverse modes, suggesting that sodium and calcium through TRPV1 stimulate the NCX activity. Our novel findings suggest that TRPV1 activity is important for regulating the spontaneous activity of ESC-CMs and reveal a novel interplay between TRPV1 and NCX in regulating the physiological functions of ESC-CMs.

Voltage‐induced Ca2+release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation-secretion coupling is assumed to be dependent only on extracellular calcium ([Ca2+ ]o ). Ryanodine receptor (RyR)-sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca2+ ]i ) that are voltage-dependent. We hypothesised that voltage-elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage-gated calcium channels and/or from such voltage-sensitive ryanodine-mediated calcium stores. Increases in [Ca2+ ]i upon depolarisation in the presence of voltage-gated calcium channel blockers, or in the absence of [Ca2+ ]o , still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca2+ ]o , there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Furthermore, such depolarisations lead to increases in [Ca2+ ]i . Pre-incubation with BAPTA-AM resulted in >50% inhibition of peptide secretion elicited by high K+ in 0 [Ca2+ ]o . Nifedipine but not nicardipine inhibited both the high K+ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage-induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation-induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine-sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.

Synthesis and biological activities of 2,3-dihydro-1,3,4-oxadiazole compounds and its derivatives as potential activator of ryanodine receptors

A series of novel 2,3-dihydro-1,3,4-oxadiazoles containing N-pyridylpyrazole carboxamides moieties were obtained by applying a new synthetic route. Their insecticidal tests against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella) indicated that most of the compounds showed moderate to excellent activities at the testing concentrations. In particular, compound 6a showed 40% larvicidal activities against oriental armyworm at 1mg/L, while 7a against diamondback was 100% at 0.01mg/L. Calcium imaging results demonstrated that 6a, 6d and 7a stimulated a transient elevation in [Ca2+]i in the absence of external calcium after the central neurons dye loading with fluo-3 AM, implying that these novel compounds were potential activators of the ligand-gated calcium channel on the endoplasmic reticulum.

DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain β (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer

Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

Synthesis and insecticidal evaluation of novel N-pyridylpyrazolecarboxamides containing cyano substituent in the ortho-position

In an attempt to search for potent insecticides targeting the ryanodine receptor (RyR), a series of novel N-pyridylpyrazolecarboxamides containing cyano substituent in the ortho-position were designed and synthesized. Their insecticidal activities of target compounds against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella) indicated that most of the compounds showed moderate to high activities at the tested concentrations. In particular, compound 6l and 6o showed 86% larvicidal activities against Plutella xylostella at the concentration of 0.1mg/L, while the activity of compound 6h against Mythimna separate was 80% at 1mg/L. The calcium imaging technique was applied to investigate the effects of some title compounds on the intracellular calcium ion concentration ([Ca2+]i), experimental results demonstrated that compound 6h stimulates a transient elevation in [Ca2+]i in the absence of external calcium after the central neurons dye loading with fluo-3 AM. However, when the central neurons were dyed with fluo-5N and incubated with 2-APB, [Ca2+]i decreased transiently by treated of compound 6h. All of the calcium imaging technique experiments demonstrated that these novel compounds deliver calcium from endoplasmic reticulum to cytoplasm, which proved that the title compounds were the possible activators of insect RyR.

Sarcoplasmic Reticulum Calcium Leak in Normal and Dystrophic Skeletal Muscle

Under resting conditions, external calcium is known to enter skeletal muscle cells while calcium stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the resting sarcolemmal calcium entry and in the SR calcium leak are still debated but several lines of evidence suggest that an up-regulation of these calcium fluxes occurs in Duchenne Muscular Dystrophy (DMD). We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the murine model of DMD. Using the cytosolic calcium dye Fura2, we first demonstrated that the rate of calcium increase induced by CPA (cyclopiazonic acid) inhibition of SR Ca2+-ATPases at resting potential is significantly higher in mdx fibers suggesting an elevated SR passive calcium leak. However, in these experiments, sarcolemmal calcium influx may contribute to the CPA-induced calcium increase and another series of experiments indicated that CPA-induced SR calcium leak was deeply modified in the absence of external calcium. Fibers were then loaded with the low affinity calcium dye Fluo5N-AM and dialyzed with 50 mM EGTA to measure intraluminal SR calcium changes. Depolarization pulses evoked voltage-dependent Fluo5N fluorescence decreases followed by a recovery phase which was inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR calcium changes. Voltage-dependence and magnitude of depolarization-induced SR calcium depletion were found to be unchanged in mdx fibers but the rate of the recovery phase that followed depletion was found to be faster, suggesting a higher SR calcium reuptake capacity in mdx fibers. Finally, CPA-induced SR calcium leak at −80 mV was found to be significantly higher in mdx fibers. The elevated SR passive calcium leak may participate to the muscle degenerative process in mdx muscle.

Effect of Cholesterol on the Pore Dilation of TRPV1

The transient receptor potential vanilloid 1 (TRPV1) ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a way of easing pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate how depletion of cholesterol from the cell membrane may modulate ion-permeability of the TRPV1 ion channel expressed in CHO-cells. The ion-permeability properties of TRPV1 measured with whole-cell patch clamp on a Chinese hamster ovary (CHO) cell line expressing TRPV1. Sustained capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole external cation, generated a biphasic current, with a first outward current and a second inward current. Similarly, sustained proton-activation (pH 5.5) of TRPV1 in the absence of external calcium also generated a biphasic current, with a first fast current peak followed by a second larger one. Also, patch clamp recordings of reversal potentials revealed a change of the ion-permeability during prolonged activation of the TRPV1 channel in extremely low extracellular calcium. Our findings show that depletion of cholesterol from the cell membrane inhibited both the second current resulting from sustained agonist-activation, and the change in ion-permeability of the TRPV1 channel. Our results propose a novel mechanism by which cholesterol-depletion may modulate the ion channel function of TRPV1, which may constitute a novel pharmacological based approach for the treatment of pain and neurogenic pain.

Sarcoplasmic Reticulum Calcium Depletion Waves in Vascular Smooth Muscle Cells: An Inside View of Spatiotemporal Calcium Regulation

We present results from a novel study of Ca2+ waves in vascular smooth muscle cells using a sarcoplasmic reticulum (SR)-targeted Ca2+ indicator that specifically binds to the luminal protein calsequestrin.Agonist-stimulated waves of elevated cytoplasmic calcium concentration regulate blood vessel tone, and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic calcium indicators revealed that these calcium waves are generated by a combination of inositol 1,4,5-trisphosphate (IP3) and calcium-induced calcium release (CICR) from the SR. Our findings confirm that these waves are due to regenerative CICR by the receptors for IP3 (IP3R).The main new finding from our bservations is a transient elevation in luminal SR Ca2+ concentration ([Ca2+]_SR) both at the site of wave initiation, just before regenerative Ca2+ release commences, and at the advancing wave front, during wave propagation. This strongly suggests a role of [Ca2+]_SR in activation of IP3R.In addition, we find that these depletion waves are characterized by a decreasing velocity as they progress. We developed a quantitative diffusional model to analyze this finding and conclude that the gradual decrease in the velocity of the SR Ca2+ depletion wave, observed in the absence of external calcium, indicates continuity of the lumen of the sarcoplasmicreticulum network.Finally, our observation that the depletion wave was arrested by the nuclear envelope may have implications for selective Ca2+ signalling.

ATP signalling is crucial for the response of human keratinocytes to mechanical stimulation by hypo-osmotic shock

Touch is detected through receptors located in the skin and the activation of channels in sensory nerve fibres. Epidermal keratinocytes themselves, however, may sense mechanical stimulus and contribute to skin sensation. Here, we showed that the mechanical stimulation of human keratinocytes by hypo-osmotic shock releases adenosine triphosphate (ATP) and increases intracellular calcium. We demonstrated that the release of ATP was found to be calcium independent because emptying the intracellular calcium stores did not cause ATP release; ATP release was still observed in the absence of external calcium and it persisted on chelating cytosolic calcium. On the other hand, the released ATP activated purinergic receptors and mobilized intracellular calcium stores. The resulting depletion of stored calcium led to the activation of capacitative calcium entry. Increase in cytosolic calcium concentration was blocked by the purinergic receptor blocker suramin, phospholipase C inhibitor and apyrase, which hydrolyses ATP. Collectively, our data demonstrate that human keratinocytes are mechanically activated by hypo-osmotic shock, leading first to the release of ATP, which in turn stimulates purinergic receptors, resulting in the mobilization of intracellular calcium and capacitative calcium entry. These results emphasize the crucial role of ATP signalling in the transduction of mechanical stimuli in human keratinocytes.

Properties of Connexin 46 Hemichannels in Dissociated Lens Fiber Cells

To characterize the properties of connexin 46 hemichannels in differentiating fiber cells isolated from mouse lenses. Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of connexin 50 (Cx50) and connexin 46 (Cx46) was assessed by immunofluorescence. Membrane currents were recorded using whole cell patch clamping. Dye uptake was measured using time-lapse imaging. In freshly dissociated fiber cells isolated from knockout Cx50 (KOCx50) mouse lenses, removal of external divalent cations induced a macroscopic current composed of large conductance channels. This current was reduced at a holding potential of -60 mV, activated on depolarization, and had a reversal potential near 0 mV. These properties were very similar to those of Cx46 hemichannel currents recorded in single Xenopus oocytes. If the currents observed in divalent cation-free Ringer's solution were due to Cx46 hemichannel opening, then dye influx by gap junctional/hemichannel permeable dyes should be measurable in the fiber cells. To measure dye influx, the authors used the positively charged dyes, propidium iodide (PrI) and 4'-6-diamidino-2-phenylindole (DAPI). In the absence of external calcium, fiber cells took up both dyes. Furthermore, dye influx could be inhibited by hemichannel blockers. To confirm that this current was due to Cx46 hemichannels, the authors studied fiber cells isolated from the lenses of double knockout (Cx46(-/-); Cx50(-/-)) mice and demonstrated that both the calcium-sensitive conductance and dye influx were absent. These results show that Cx46 can form functional hemichannels in the nonjunctional membrane of fiber cells.

Insulin-releasing properties of the frog skin peptide pseudin-2 and its [Lys18]-substituted analogue

Abstract Pseudin-2 is a cationic alpha-helical peptide that was first isolated from the skin of the paradoxical frog Pseudis paradoxa on the basis of its antimicrobial activity. We have investigated the insulin-releasing properties and cytotoxicity of the peptide, together with selected analogues with increased cationicity and hydrophobicity. At concentrations in the range 10(-9)-10(-6) m, pseudin-2, and its [Lys18], [Phe8], and [d-Lys3,d-Lys10,d-Lys14] derivatives, stimulated insulin release from the BRIN-BD11 clonal beta-cell line without increasing release of lactate dehydrogenase. The [Lys18] analogue was the most potent (46% increase in insulin release at 10(-9) m) and the most effective (215% increase in insulin release at 10(-6) m). The more cationic [Lys3,Lys10,Lys14] and [Lys3,Lys10,Lys14,Lys21] analogues lacked insulinotropic action and the more hydrophobic [Phe16] analogue was cytotoxic at concentrations > or =10(-7) m. Pseudin-2 and [Lys18]-pseudin-2 had no effect on intracellular calcium concentrations and stimulated insulin release in the absence of external calcium. [Lys18]-pseudin-2 (10(-8) m) stimulated insulin release in the presence of diazoxide and verapamil. Our results demonstrate that pseudin-2 stimulates insulin secretion from BRIN-BD11 cells by a mechanism involving Ca2+-independent pathways and identify [Lys18]-pseudin-2 as a peptide that may have potential for development as a therapeutically valuable insulinotropic agent for the treatment of type 2 diabetes.

Do heavy metals play an active role in sponge cell behaviour in the absence of calcium? Consequences in larval settlement

Moderate concentrations of copper and cadmium can have positive effects on sponges by enhancing cell aggregation and larval settlement. The cellular events underlying these effects are not fully understood, but it is known that cell aggregation needs of an extracellular supply of calcium, which in the absence of external calcium may also be provided by the release of internal calcium stores. In order to know the mechanisms by which Cd 2+ and Cu 2+ affected sponge cells and larvae and whether the alteration of calcium homeostasis is involved in such effects, we incubated Scopalina lophyropoda cells for 3 h and larvae for seven days in calcium-free seawater (CFSW), seawater (SW), and CFSW with copper (Cu CFSW) and cadmium (Cd CFSW). Both metals seem to play an active role in calcium dependent processes: sponge cells incubated in Cd CFSW and Cu CFSW, aggregated as much as those in SW control, while aggregates were rare in CFSW. Similarly, Cd and Cu first fastened larval settlement but toxic effects of both metals were evidenced after a longer exposition (five days), leading to settler mortality. Thus, although the effect of these two heavy metals may be positive in the short-term, long term exposition or higher concentrations may alter cell functions and affect sponge viability and fitness.

Modulation of GABAergic synaptic transmission by terminal nicotinic acetylcholine receptors in the central autonomic nucleus of the neonatal rat spinal cord

Using patch clamp recordings from an in vitro spinal cord slice preparation of neonatal rats (9–15 days old), we characterized the GABAergic synaptic transmission in sympathetic preganglionic neurones (SPN) of the central autonomic nucleus (CA) of lamina X. Local applications of isoguvacine (100 μM), a selective agonist at GABA A receptors, induced in all cells tested a chloride current which was abolished by bicuculline, a competitive antagonist at GABA A receptors. In addition, 25% of the recorded cells displayed spontaneous tetrodotoxin-insensitive and bicuculline-sensitive chloride miniature inhibitory postsynaptic currents (mIPSCs). Acetylcholine (100 μM) increased the frequency of GABAergic mIPSCs without affecting their amplitudes or their kinetic properties indicating a presynaptic site of action. The presynaptic effect of ACh was restricted to GABAergic neurones synapsing onto sympathetic preganglionic neurones. The facilitatory effect of ACh was abolished in the absence of external calcium or in the presence of 100 μM cadmium added to the bath solution. Choline 10 mM, an agonist at α7 nicotinic acetylcholine receptors (nAChRs) or muscarine (10 μM), a muscarinic receptor agonist, did not reproduce the presynaptic effect of ACh. The presynaptic effect of ACh was blocked by 1 μM of dihydro-β-erythroidine (DHβE), an antagonist of non-α7 nAChRs but was insensitive to α7 nAChRs antagonists (strychnine, α-bungarotoxin and methyllycaconitine) or to the muscarinic receptor antagonist atropine (10 μM). It was concluded that SPNs of the central autonomic nucleus displayed a functional GABAergic transmission which is facilitated by terminal non α7 nAChRs.

Functional expression of “cardiac-type” Na v1.5 sodium channel in canine intracardiac ganglia

The autonomic nervous system has been implicated in several arrhythmogenic diseases, including long QT syndrome type 3 (LQT3) and Brugada syndrome. Scarce information on the cellular components of the intrinsic cardiac ganglia from higher mammals has limited our understanding of the role of the autonomic nervous system in such diseases. The purpose of this study was to isolate and characterize the electrophysiologic properties of canine intracardiac neurons. Action potentials (APs) and ionic currents were studied in enzymatically dissociated canine intracardiac neurons under current and voltage clamp conditions. Immunohistochemical and reverse transcription-polymerase chain reaction analysis was performed using freshly isolated intracardiac ganglia. APs recorded from intracardiac neurons displayed a tetrodotoxin-resistant (TTX-R) component. TTX-R APs were abolished in the absence of sodium but persisted in the absence of external calcium. Immunohistochemical studies showed the presence of TTX-R sodium channels in these ganglia. Sodium currents were characterized by two components with different affinities for TTX: a tetrodotoxin-sensitive (TTX-S) component and a TTX-R component. TTX-S current inactivation was characteristic of neuronal sodium currents, whereas TTX-R current inactivation time constants were similar to those previously reported for Na(v)1.5 channels. TTX sensitivity (IC(50) = 1.17 microM) of the TTX-R component was in the range reported for Na(v)1.5 channels. Expression of Na(v)1.5 channels in intracardiac ganglia was confirmed by PCR analysis and sequencing. Our results suggest that canine intracardiac neurons functionally express Na(v)1.5 channels. These findings open an exciting new door to our understanding of autonomically modulated arrhythmogenic diseases linked to mutations in Na(v)1.5 channels, including Brugada syndrome and LQT3.

Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin

A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na(+). Li(+) decreased the PTX-evoked rise in [Ca(2+)](c); replacement of Na(+) by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca(2+)](c) increase by PTX was strongly reduced after inhibition of the reverse operation of the Na(+)/Ca(2+) exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca(2+)-ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na(+)-dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na(+)/Ca(2+) exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space.

Vasorelaxant effect of the flavonoid galangin on isolated rat thoracic aorta

Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1–100 μM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 μM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester ( L-NAME, 100 μM), the cyclooxygenase inhibitor indomethacin (10 μM) and the adenylate cyclase inhibitor, SQ 22,536 (100 μM). In another set of experiments, rat aortic rings were incubated with galangin (1–100 μM) and the contractile responses to PE (0.001–3 μM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 μM) on PE (10 μM)-induced contraction in a Ca 2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca 2+] i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca 2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.

Two-photon imaging of spontaneous vesicular release in acute brain slices and its modulation by presynaptic GABA A receptors

Action potential-independent spontaneous vesicular release of γ-aminobutyric acid (GABA) in the CNS mediates miniature inhibitory postsynaptic currents (mIPSCs) and exerts an important control on central excitability. Using dual-photon laser scan microscopy and hyperosmotic loading of the readily releasable vesicle pool with the fluorescent styryl dye FM1-43 in hippocampal slice, we demonstrate action potential-independent release of vesicles (fluorescence destaining) from proximal perisomatic, presumed GABAergic terminals and significant inhibition of this release by the specific GABA A receptor agonist muscimol in the presence of tetrodotoxin and glutamate receptor antagonists CNQX and AP5. These data agree with reduction of mIPSCs by muscimol in whole-cell recordings from CA3 pyramidal neurons. In contrast, rate of vesicle release from distal, presumably glutamatergic terminals, was significantly lower and not changed by muscimol. The effect of muscimol on mIPSCs was not blocked but rather enhanced in the absence of external calcium. Our data directly demonstrate a potent disinhibitory reduction of GABA release by GABA A receptor activation. Those novel methods should be well suited to study pathophysiological changes in inhibition in resections obtained from neurosurgical treatment of epilepsy patients.

Expression and functional properties of group I metabotropic glutamate receptors in bovine chromaffin cells.

We demonstrate the presence and functional properties of Group I metabotropic glutamate receptors (mGluRs) expressed in chromaffin cells. Immunocytochemical techniques revealed that two mGluR subtypes (mGluR1alpha and mGluR5) are expressed in chromaffin cells, located in both the cytoplasmic membrane and the cytosol surrounding the nucleus. These mGluRs are functionally active on catecholamine (CA) secretion in chromaffin cells because both (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and the specific agonist of Group I mGluRs, (S)-3,5-dihydroxyphenylglycine (DHPG), were able to stimulate the release of CAs (adrenaline and noradrenaline) in a dose-response manner. These effects were specifically reversed by L-(+)-2-amino-3-phosphonopropionic acid (L-AP3), a selective antagonist of the Group I metabotropic glutamate receptors. t-ACPD induced an increase in CA secretion in both the presence and absence of extracellular calcium, the former effect being accompanied by cell membrane depolarization. Noradrenaline (NA) release was higher in the presence of extracellular calcium than in its absence, whereas adrenaline release was of the same order under both conditions. These results indicate that different subtypes of Group I mGluRs are present in noradrenergic and adrenergic cells. Fluorescence imaging techniques in single cells showed different t-ACPD-induced increases in intracellular calcium in different chromaffin cells: in chromaffin cells, 67% expressed functional metabotropic glutamate receptors and with nicotinic receptors, whereas the remaining 33% expressed only nicotinic receptors. In the absence of external calcium, only about 25% of cells responded to t-ACPD-increased intracellular calcium by increasing inositol 1,4,5-trisphosphate (IP(3)) concentration and subsequent calcium mobilization from intracellular stores, whereas the remaining 75% increased intracellular calcium by promoting Ca(2+) influx from the extracellular medium through L- and N- but not P/Q voltage-dependent calcium channels.

Polyamine flux in Xenopus oocytes through hemi-gap junctional channels.

Diverse polyamine transport systems have been described in different cells, but the molecular entities that mediate polyamine influx and efflux remain incompletely defined. We have previously demonstrated that spermidine efflux from oocytes is a simple electrodiffusive process, inhibitable by external Ca2+, consistent with permeation through a membrane cation channel. Hemi-gap junctional channels in Xenopus oocytes are formed from connexin 38 (Cx38), and produce a calcium-sensitive (Ic) current that is inhibited by external Ca2+. Spermidine efflux is also calcium sensitive, and removal of external calcium increases both Ic currents and spermidine efflux in Xenopus oocytes. Injection of Cx38 cRNA or Cx38 antisense oligonucleotides (to increase or decrease, respectively, Cx38 expression) also increases or decreases spermidine efflux in parallel. Spermidine efflux has a large voltage-dependent component, which is abolished with injection of Cx38 antisense oligonucleotides. In addition, spermidine uptake is significantly increased in Cx38 cRNA-injected oocytes in the absence of external calcium. The data indicate that hemi-gap junctional channels provide the Ca2+-inhibited pathway for electrodiffusive efflux of polyamines from oocytes, and it is likely that hemi-gap junctional channels provide Ca2+ and metabolism-sensitive polyamine permeation pathways in other cells.