GABA augments basal and electrically stimulated H-norepinephrine release in hypothalamic, preoptic area and cortical slices of female rats

These studies examined the regulation by GABA of norepinephrine release from hypothalamus, preoptic area and frontal cortex. Using superfused brain slicesfrom female rats, we show that 100 μM GABA enhances both basal and electrically stimulated release of ³H-norepinephrine in all three brain regions. The GABA_A agonist muscimol (100 μM) significantly augments ³H-norepinephrine release, but it is somewhat less effective than GABA. The GABA_B agonist baclofen has little or no effect on basal ³H-norepinephrine efflux. GABA also augments both the magnitude and duration of electrically evoked ³H-norepinephrine release in slices from all three brain regions. GABA facilitation of electrically stimulated ³H-norepinephrine release is mediated through GABA_A receptors as evidenced by its blockad by 10 μM bicuculline, a GABA_A antagonist, but not by 200 μM 2-OH-saclofen, a GABA_B antagonist. These data show that the inhibitory amino acid neurotransmitter GABA enhances both basal and evoked release of ³H-norepinephrine in brain slices from female rats. These effects are predominantly mediated by GABA_A receptors. GABA modulation of hypothalamic norepinephrine release may play a role in the regulation of gonadotropin secretion and reproductive behaviors such as lordosis.     

Loss of caveolin-1 expression is associated with disruption of muscarinic cholinergic activities in the urinary bladder

Caveolin-1 (Cav1), a structural protein of caveolae, plays cell- and context-dependent roles in signal transduction pathway regulation. We have generated a knockout mouse homozygous for a null mutation of the Cav1 gene. Cav1 knockout mice exhibited impaired urinary bladder contractions in vivo during cystometry. Contractions of male bladder strips were evoked with electric and pharmacologic stimulation (5–40 Hz, 1–10 μM carbachol, 10 mM ,β-methylene ATP, 100 mM KCl). Acetylcholine (ACh) and norepinephrine (NE) release from bladder strips were measured with a radiochemical method by incubating the strips with ¹⁴C-choline and ³H-NE prior to electric stimulation, whereas ATP release was measured using the luciferin-luciferase assay with a luminometer. A 60–75% decline in contractility was observed when Cav1 knockout muscle strips were stimulated with electric current or carbachol, compared to wildtype muscle strips. No difference in contractility was noted when contractions were evoked either by the purinergic agonist ,β-methylene ATP, or by extracellular potassium. To investigate the relative contribution of non-cholinergic activity to bladder contractility, the amplitude of the electric stimulation-evoked contractions was compared in the presence of the muscarinic antagonist atropine (1 μM). While the non-muscarinic (purinergic) response was unaltered, muscarinic cholinergic response was principally disrupted in Cav1 knockout mice. The loss of Cav1 gene expression was also associated with a 70% reduction in ACh release. NE and ATP release was not altered. It is concluded that the loss of caveolin-1 is associated with disruption of M₃ muscarinic cholinergic activity in the bladder. Both pre-junctional (acetylcholine neurotransmitter release from neuromuscular junctions) and post-junctional (M₃ receptor-mediated signal transduction in bladder smooth muscles) mechanisms are disrupted, resulting in impaired bladder contraction.     

Release of endogenous GABA can occur through Ca-dependent and Ca-independent processes

Release of γ-aminobutyric acid (GABA) can be elicited by electrical field stimulation even in the absence of external Ca²⁺. Indeed, the release of GABA under such conditions is even higher than in the presence of Ca²⁺. To investigate the underlying mechanism of this phenomenon, the release of endogenous GABA from rat striatal slices was measured by high performance liquid chromatography with electro- chemical detection. Electrical stimulation at 2 Hz for 3 min elevated GABA efflux by 4.5-fold. Withdrawing external Ca²⁺ and adding 1 mM EGTA produced a small, transient increase in the basal efflux of GABA and increased electrically-evoked overflow 3-fold. Tetrodotoxin (5 μM) did not affect basal efflux in either normal or Ca²⁺-free conditions, but abolished electrically-evoked release. In the presence of normal Ca²⁺, nipecotic acid (1 mM) enhanced both spontaneous efflux and evoked overflow. Nipecotic acid also increased spontaneous release when external Ca²⁺ was removed. However, in the absence of Ca²⁺, nipecotic acid failed to increase electrically evoked GABA overflow. These results suggest that there exists a Ca²⁺-independent process for GABA release via the same carrier system that is utilized for high affinity GABA uptake.     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 μM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 μM), or the serotonin transporter inhibitor, fluoxetine (10 μM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na⁺ channel blockade by 1 μM tetrototoxin, removal of Ca²⁺ from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 μM) or tetrabenazine (10 and 100 μM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 μM) and CNQX (20 and 50 μM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

Metal–salt co-tolerance and metal removal by indigenous cyanobacterial strains

Chromium and salt tolerance in five indigenous cyanobacterial strains isolated from contaminated sites was investigated along with their metal bioaccumulative potential. All the five species showed significantly better growth when the medium was spiked with salt or chromium. As compared to single metal or salt treatment, the binary metal–salt (MS) treatments had more favorable effect on cyanobacterial growth as indicated by significantly higher concentration of the primary photosynthetic pigment chlorophyll at M₂₀S₂₀₀₀ (9.9–25.3 μg/mL) as compared to that at M₀S₀ (4.0–12.3 μg/mL). Similarly biomass was much higher at M₂₀S₁₀₀₀ and M₂₀S₂₀₀₀ (41.8–86.2 mg/10 mL) as compared to that at control, M₀S₀ (21.5–36.3 mg/10 mL). Accessory pigments like carotenoids and phycobilinproteins too tended to increase significantly in response to both metal and salts in the two species of Lyngbya (L. putealis and L. ceylanica var. constricta) and Gloeocapsa. These species also showed greater potential of chromium bioaccumulation, which increased further as both salt and metal concentration increased. In the two species of Nostoc however, bioaccumulative potential improve at higher metal concentration, but not affected significantly by salt concentration.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and 1/20 of that of bradykinin (BK) on a weight basis. The activity of PGF₁ and PGF₂ was only 1/20 of that of PGE₁ or PGE₂.

In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only 1/3–1/8 of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the anti-histamine, pyrilamine (2.5 mg/kg, i.v.).     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Antiallergic effect of ardisiaquinone A, a potent 5-lipoxygenase inhibitor

The antiallergic effects of ardisiaquinone A, a potent 5-lipoxygenase inhibitor, were examined. Pretreatment with ardisiaquinone A (0.1–10 μM) significantly inhibited compound 48/80-induced production of cysteinyl-leukotrienes (cys-LTs; LTC₄, LTD₄ and LTE₄) in rat peritoneal mast cells, but not histamine release. The IC₅₀ value was 5.56 μM. Pre-administration with ardisiaquinone A (0.1–1 mg/kg, s.c.) dose-dependently inhibited rat homologous passive cutaneous anaphylaxis (PCA) and the maximal inhibitory ratio was 22.3 ± 3.9% at the dose of 1 mg/kg. Ardisiaquinone A (1–5 mg/kg, s.c.) dose-dependently prevented the allergen-induced increase of tracheal pressure in ovalbumin-sensitized guinea pigs, especially during the late phase.

In conclusion, the findings of this study show that 5-lipoxygenase inhibitor ardisiaquinone A partially attenuates the allergen-induced increases of vascular permeability and tracheal pressure via the inhibition of cys-LTs production in mast cells.     

Serotonin and histamine receptor-mediated inhibition of serotonin and noradrenaline release in rat brain cortex under nimodipine treatment

Rat brain cortex slices preincubated with [³H]serotonin or [³H]noradrenaline (25 100 nmol/l each) were superfused and the effects of serotonin and histamine on the electrically (0.3 or 3 Hz) evoked tritium overflow were studied.

In slices preincubated with [³H]serotonin the extent of inhibition of the electrically (3 Hz) evoked tritium overflow produced by histamine was increased when the concentration of [³H]serotonin used for incubation was decreased. The evoked overflow tended to be lower in slices from 2-year-old rats than in slices from 6-month-old animals whereas the inhibitory effect of histamine on the evoked overflow did not differ. Treatment of rats with nimodipine for at least 6 weeks did not significantly affect the evoked overflow in slices from 6-month and 2-year-old rats nor did it significantly alter the serotonin- and histamine-mediated inhibition of the evoked overflow in slices from young adult rats. The extent of histamine-mediated inhibition of the electrically evoked tritium overflow from slices (of young adult rats) preincubated with [³H]noradrenaline did not change when the concentration of [³H]noradrenaline used for incubation was decreased; the degree of inhibition markedly increased when the frequency of stimulation was lowered from 3 to 0.3 Hz. The inhibitory effect of histamine on the electrically (0.3 Hz) evoked overflow was mimicked by the H₃ receptor agonist R-(−)--methylhistamine and antagonized by the H₃ receptor antagonist thioperamide. The electrically evoked overflow and its inhibition by histamine were not affected by nimodipine, irrespective of whether the Ca²⁺ antagonist was administered in vivo (for at least 6 weeks) or added to the superfusion medium in vitro.

It is concluded that (1) the extent of the H₃ receptor-mediated effect in rat brain cortex slices can be markedly increased by lowering the concentration of the tracer in slices preincubated with [³H]serotonin and by lowering the stimulation frequency in slices preincubated with [³H]noradrenaline; (2) the H₃ receptor-mediated inhibition of serotonin release is not changed during aging and (3) nimodipine does not significantly influence serotonin release and noradrenaline release and their serotonin and/or histamine receptor-mediated modulation.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Interaction of ovokinin(2-7) with vascular bradykinin 2 receptors

Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B₂ receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B₁ receptor antagonist [des-Arg¹⁰]-HOE140 (26.3±15.5%). Intracellular Ca²⁺ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg¹⁰]-HOE140. The elevation of intracellular Ca²⁺ by ovokinin(2–7) was dependent on Ca²⁺ entry from extracellular space as it was reduced in a Ca²⁺-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca²⁺ increase by the peptide. PA phosphohydrolase and phospholipase A₂ inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B₂ bradykinin receptors.     

Capsaicin inhibits calmodulin-mediated oxidative burst in rat macrophages

In this study we seek to elucidate the interaction of capsaicin with the calmodulin mediated signal pathways in macrophages, by comparing its action on macrophage functions with a known calmodulin antagonist, fluphenazine. Kinetics of capsaicin uptake by macrophages (10³ cells) revealed that a maximum of 200 μM capsaicin was taken up within 10 min. Ca²⁺ ionophore triggered generation of superoxide anion and hydrogen peroxide by macrophages was inhibited in a dose-dependent manner by fluphenazine (IC₅₀, 20 μM and 12 μM, respectively) and also by capsaicin (IC₅₀, 30 μM and 9 μM, respectively), suggesting an involvement of calmodulin in the regulation of NADPH oxidase. In vitro both fluphenazine and capsaicin inhibited Ca²⁺-Mg²⁺ ATPase and cAMP-phosphodiesterase from macrophages and this inhibition was reversed by exogenous addition of calmodulin. Fluorescence studies revealed a direct Ca²⁺ dependent interaction of capsaicin with calmodulin. From these results we suggest that capsaicin acts via calmodulin to inhibit stimulus-induced macrophage oxidative burst and also that calmodulin regulates the oxidative burst in macrophages.     

Stimulation of platelet serotonin and Rb uptake by N-ethylmaleimide

The effects of N-ethylmaleimide (NEM) on mouse platelet serotonin (5-HT) and ⁸⁶Rb⁺ uptake were studied. The 5-HT transport system showed a biphasic response to increasing concentrations of NEM, with low concentrations (25–50 μM) stimulating and high concentrations (200–400 μM) inhibiting 5-HT transport. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked NEM-induced stimulation of 5-HT transport. The kinetics of 5-HT uptake indicated that NEM (50 μM) markedly increased the maximal rate of 5-HT transport (V_max control = 28.4±1.4 pmol/10⁸ platelets/4 min vs V_max NEM = 64.5±9.5 pmol/10⁸ platelets/4 min but had no significant effect on the K_m value. Platelet Na⁺ K⁺ ATPase activity was determined by measuring ⁸⁶Rb⁺ uptake. Platelet ⁸⁶Rb⁺ uptake showed a biphasic response to NEM, with low concentrations (25–100 μM) significantly stimulating and high concentrations (400 μM) inhibiting uptake. These changes in platelet ⁸⁶Rb⁺ uptake paralleled the biphasic changes in 5-HT transport. In the presence of fluoxetine, 5-HT transport was markedly inhibited but no change in the ability of NEM to stimulate ⁸⁶Rb⁺ uptake was observed. These data suggest that low concentrations of NEM activate plasma membrane Na⁺ K⁺ ATPase which results in a marked stimulation of platelet 5-HT transport.     

Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes

A set of novel heterocyclic ligands (6–27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1–5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M₁, M₂, and M₃ tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC₅₀: 7.40 (M₁), 8.18 (M₂), and 8.14 (M₃)], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M₁ subtype [pK_B: 6.88 (M₁), 5.95 (M₂), 5.53 (M₃)]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M₁ antagonist/M₂ partial agonist/M₃ full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M_1–3 receptors, with an appreciable selectivity for the cardiac M₂ receptors.     

Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H₂O₂ was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1–100 μg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1–10 μg/ml. At a concentration of 100 μg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H₂O₂ at concentrations of 0.1–10 μg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H₂O₂. All the compounds at 10 μg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H₂O₂ in vitro.     

Unlike VIP, the VIP-related peptides PACAP, helodermin and helospectin suppress electrically evoked contractions of rat vas deferens

We have compared the effects of vasoactive intestinal peptide (VIP) and of the VIP-related peptides pituitary adenylate cyclase activating peptide (PACAP) 1–27 and 1–38, helodermin, helospectin I and helospectin II, on the electrically evoked twitches in the isolated vas deferens of the rat. While VIP was virtually without effect, PACAP 1–38 suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC₅₀ value 7.5). The naturally occurring N-terminal fragment PACAP 1–27 was less effective than PACAP 1–38 (I_max values 37.2% suppression compared to 76.5%) and less potent. The C-terminal fragment PACAP 16–38 was virtually inactive. Also helodermin and helospectin I+II suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC₅₀ values 6.9; 7.2; 6.8, respectively). The three peptides produced similar maximum reduction of the twitches (74–80%).

The findings suggest that PACAP, helodermin and helospectin suppress the electrically evoked contractions in the rat vas deferens via receptors distinct from VIP receptors.