Stimulation of calmodulin by cadmium ion

Cd²⁺, a serious environmental pollutant in certain industrial regions, accumulates in mammalian tissues with a very slow turnover. Using various criteria, we studied the ability of Cd²⁺ to substitute for Ca²⁺ in calmodulin (CaM), a ubiquitious Ca²⁺-binding protein that mediates many of the Ca²⁺ effects. CaM bound Cd²⁺ with a Kd of 4.5 M, presumably to the Ca²⁺-binding sites. Binding of Cd²⁺ allowed CaM to bind 2 moles chlorpromazine, or to form a complex with skeletal muscle troponin-I, troponin-T, or phosphodiesterase. Complex formation with phosphodiesterase led to its activation, which was observed even in the presence of glutathione or cysteine, agents known to chelate Cd²⁺. This raises the possibility that one manifestation of Cd²⁺ toxicity may be through its activation of CaM, thus upsetting its normal regulation by a cellular flux of Ca²⁺.This work was supported by grants CA 21765, NS 08059 and GM 28178 from the NIH and by American Lebanese Syrian Associated Charities. J. R. Z. was a recipient of a National Research Service Award, CA 09346, USA     

Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel

Summary Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca²⁺ regulated events, we have studied its effects on the compartmentation of free Ca²⁺ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca²⁺ concentration ([Ca²⁺]_i) to micromolar levels. The increase in [Ca²⁺]_i was not transient and a steady rise was observed with time. Omission of Ca²⁺ from the external medium prevented the AM- and DEA-induced rise in [Ca²⁺]_i suggesting that AM and DEA increased the intracellular [Ca²⁺]_i due to increased influx of Ca²⁺ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca²⁺]_i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na⁺ channel blocker, tetrodotoxin. However, the blockade of [Ca²⁺]_i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca²⁺]_i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca²⁺]_i through receptor mediated channel. ⁴⁵Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca²⁺ release from these intracellular organelles. Also, both these drugs did not interfere with inositol 1,4,5-trisphosphate induced Ca²⁺ release from microsomes even at 10 M concentration. These results clearly indicate that both AM and DEA increase intrasynaptosomal [Ca²⁺]_i by an action on receptor mediated channel in plasma membrane, but not due to the release of Ca²⁺ from intracellular storage sites. This initial rise in [Ca²⁺]_i, together with other changes in Ca²⁺ homeostasis, might be responsible for AM and DEA-induced neurotoxicity. Send offprint requests to D. Desaiah at the above address     

Influence of kaempferol,a flavonoid compound,on membrane-bound ATPases in streptozotocin-induced diabetic rats

Abstract

Context: Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities.

Objective: The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats.

Materials and methods: Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40?mg/kg body weight (BW)). Kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) was administered orally once daily for 45?d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined.

Results: In our study, diabetic rats had significantly (p?<?0.05) decreased activities of total ATPases, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase in erythrocytes and tissues. Oral administration of kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) for a period of 45?d resulted in significant (p?<?0.05) reversal of these enzymes' activities to near normal in erythrocytes and tissues when compared with diabetic control rats.

Discussion and conclusion: Thus, obtained results indicate that administration of kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol’s action mechanism.     

赛庚啶对离体犬心肌肌质网Ca2+,Mg2+—ATP酶活性和45Ca2+摄取功能的影响

当赛庚啶浓度在8×10^-6mol/L～2×10^-4mol/L之间时,该药对正常犬心肌肌质网Ca²⁺,Mg²⁺—ATP酶活性几乎没有影响,仅在10^-3mol/L时对该酶活性才有一定的抑制作用(抑制率为39.85%,P<0.01)。正常犬心肌肌质网的⁴⁵Ca²⁺摄取过程有明显的时间依赖性,至第30 min,其⁴⁵Ca²⁺摄取量可达312.79±22.25 nmol/mg protein.赛庚啶对心肌肌质网的~(45)Ca²⁺摄取有一定的抑制作用,其IC₅₀为1.94×10^-4mol/L。     

Synergism of ochratoxin B and calcium-channel antagonist verapamil caused mitochondrial dysfunction

We examined the mechanism by which the ochratoxin B induced interaction with calcium-channel antagonist verapamil and mitochondrial dysfunction of the rat trachea in vitro experiment. The tracheas were cut into 2–3?mm wide rings and suspended in a tissue bath. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Verapamil (1?×?10^?6?M) produced a concentration-dependent contraction response in rat’s tracheal rings pre-contracted by acetylcholine. Incubation of rat’s tracheal rings with the ochratoxin B significantly potentiated the contraction responses of verapamil. Verapamil and OTB accelerate the overloading of Ca²⁺ in tracheal smooth muscle contributes the tissue toxicity as shown in electron microscopy and mitochondrial enzymes, through a mechanism that could involve perturbations of Ca²⁺ homeostasis. These results proved that ochratoxin B is a potential vasoconstrictor mycotoxin with the presence of calcium-channel antagonist. In conclusion, disturbance of Ca²⁺ homeostasis caused by OTA and plays a significant role in produces toxicity through mitochondrial enzyme inhibition.     

Evidence for a distinct Ca2+ antagonist receptor for the novel benzothiazinone compound HOE 166

Summary The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3, 4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-trimethoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1, 4-benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl^– but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC₅₀-values of 70 nM (5–11 times less potent than nifedipine, 2–16 times more potent than verapamil), the respective S-(–)-enantiomer being 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(–)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4–15 times more potent inhibitor of reversible (+)-[³H]PN200-110, (–)-[³H]desmethoxyverapamil and d-cis [³H]diltiazem binding compared to its pharmacologically less active (S)-(–)-enantiomer, with IC₅₀ values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca²⁺-antagonistic effect by binding to a Ca²⁺-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca²⁺ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.Send offprint requests to H. Glossmann     

Effect of calmodulin on ginseng saponin-induced Ca2+-Activated CI-channel activation inXenopus laevis oocytes

We previously demonstrated the ability of ginseng saponins (active ingredients of Panax ginseng) to enhance Ca2+-activated Cl- current. The mechanism for this ginseng saponin-induced enhancement was proposed to be the release of Ca2+ from IP3-sensitive intracellular stores through the activation of PTX-insensitive Galpha(q/11) proteins and PLC pathway. Recent studies have shown that calmodulin (CaM) regulates IP3 receptor-mediated Ca2+ release in both Ca2+-dependent and -independent manner. In the present study, we have investigated the effects of CaM on ginseng saponin-induced Ca2+-activated Cl- current responses in Xenopus oocytes. Intraoocyte injection of CaM inhibited ginseng saponin-induced Ca2+-activated Cl- current enhancement, whereas co-injection of calmidazolium, a CaM antagonist, with CaM blocked CaM action. The inhibitory effect of CaM on ginseng saponin-induced Ca2+-activated Cl- current enhancement was dose- and time-dependent, with an IC50 of 14.9 +/- 3.5 microM. The inhibitory effect of CaM on saponin's activity was maximal after 6 h of intraoocyte injection of CaM, and after 48 h the activity of saponin recovered to control level. The half-recovery time was calculated to be 16.7 +/- 4.3 h. Intraoocyte injection of CaM inhibited Ca2+-induced Ca2+-activated Cl- current enhancement and also attenuated IP3-induced Ca2+-activated Cl- current enhancement. Ca2+/CaM kinase II inhibitor did not inhibit CaM-caused attenuation of ginseng saponin-induced Ca2+-activated Cl- current enhancement. These results suggest that CaM regulates ginseng saponin effect on Ca2+-activated Cl current enhancement via Ca2+-independent manner.     

Methanol-Induced Contraction of Canine Cerebral Artery and Its Possible Mechanism of Action

In the present report, we investigated the effects of methanol on canine basilar cerebral arterial rings. Our data indicate that acute methanol exposure (5–675 mM) induces potent contractile responses of cerebral arteries in a concentration-dependent manner. Pharmacological antagonists, such as propranolol, phentolamine, haloperidol, methysergide, naloxone, diphenhydramine, and cimetidine, did not exert any effects on these methanol-induced contractions. Likewise, a potent antagonist of cyclo-oxygenase, and subsequent synthesis of prostanoids (i.e., indomethacin), failed to exert any effect on methanol-induced contractions. No differences in responsiveness to methanol in canine cerebral arteries were found in vessel segments with or without endothelial cells. Removal of extracellular Ca²⁺([Ca²⁺]_o) partially attenuated methanol-induced contractions, while withdrawal of extracellular Mg²⁺([Mg²⁺]_o) potentiated the contractions. In the complete absence of [Ca²⁺]_o, 10 mM caffeine and 400 mM methanol induced similar, transient contractions followed by relaxation in K⁺-depolarized cerebral vascular tissues. Methanol-induced contractions were, however, completely abolished by pretreatment of tissue with 10 mM caffeine. Our results indicate that (1) methanol causes contractile responses of cerebral arterial smooth muscle (independent of amine, prostanoid, or opioid mediation; (2) in addition to a need for [Ca²⁺]_o, an intracellular release of Ca²⁺is required for methanol-induced contractions; and (3) Mg deficiency potentiates the contractile responses of methanol on these brain vessels. The data presented in the study suggest that methanol-induced contractions occur via an sarcoplasmic reticulum-releasable store of [Ca²⁺]_i; via mediation of either ryanodine–caffeine type receptors or a caffeine-releasable intracellular store of Ca²⁺.     

Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides

Background and purpose:

Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).

Experimental approach:

RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.

Key results:

With10 µmol·L⁻¹ cytosolic Ca²⁺, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L⁻¹–10 µmol·L⁻¹. High Po values were observed at >10 µmol·L⁻¹ Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC₅₀ of 16 µmol·L⁻¹ (Ap5A EC₅₀ = 140 µmol·L⁻¹). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.

Conclusions and implications:

The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.     

Modulation of P2X3 receptors by Mg2+ on rat DRG neurons in culture

On nociceptive neurons the commonest response to ATP is a rapidly desensitizing current mediated by P2X(3) receptors and believed to be involved in certain forms of pain. P2X(3) receptor recovery from desensitization is a slow process. We studied whether Mg(2+) might modulate such ATP-evoked currents on rat cultured DRG neurons, and thus account for its analgesic action in vivo. Transient increases in extracellular Mg(2+) strongly and reversibly depressed ATP currents which had not recovered from desensitization. Ca(2+)-free solution had the same action as Mg(2+). High Mg(2+) or Ca(2+)-free modulation depended on exposure length to modified divalent cation solutions, whereas it was independent from membrane potential or intracellular Ca(2+) buffering. Paired-pulse protocols showed that high Mg(2+) or Ca(2+)-free medium delayed ATP receptor recovery from desensitization, while leaving desensitization onset apparently unchanged. Tests with various concentrations of Ca(2+) and Mg(2+) showed that the depressant action by Mg(2+) was primarily due to functional antagonism of a facilitatory effect of Ca(2+) on ATP receptor function. The present results suggest that, on sensory neurons, P2X(3) receptors could be inhibited by high Mg(2+) or lack of Ca(2+), representing a negative feedback process to limit ATP-mediated nociception.     

Stellettamide-A,a novel inhibitor of calmodulin,isolated from a marine sponge

1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K⁺(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC₅₀ of 88 μM.
2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca²⁺ (3 and 10 μM)-induced contractions with an IC₅₀ of 46 μM for 3 μM Ca²⁺ and 105 μM for 10 μM Ca²⁺. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca²⁺. ST-A had no effect on this contraction.
3. ST-A inhibited Mg²⁺-ATPase activity in native actomyosin prepared from chicken gizzard with an IC₅₀ of 25 μM.
4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC₅₀ of 152 μM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin.
5. ST-A inhibited calmodulin activity, assessed by Ca²⁺/calmodulin-dependent enzymes, (Ca²⁺-Mg²⁺)-ATPase of erythrocyte membrane, with an IC₅₀ of 100 μM and phosphodiesterase prepared from bovine cardiac muscle with an IC₅₀ of 52 μM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration.
6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3–300 μM). Similar results were obtained in the presence or absence of Ca²⁺ suggesting that ST-A binds to calmodulin and that Ca²⁺ is not essential for the binding of ST-A to calmodulin.
7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

     

The influence of divalent cations on the analgesic effect of opioid and non-opioid drugs

It is generally accepted that divalent cations are involved in the nociceptive pathway. The effect of systemic co-administration of magnesium sulfate and calcium channel blockers (nifedipine, verapamil) on the analgesic effect of opioid (mixed mu/kappa: butorphanol) and non-opioid drugs (paracetamol) was investigated. Albino mice and rats were used as experimental animals. Magnesium sulfate and calcium channel blockers were given i.p., 30 min before the administration of butorphanol tartrate and paracetamol. Analgesia was measured using "hot-plate" ( 52.5( composite function)C), "tail-flick" (radiant heat source), "writhing" (acetic acid, 1%, i.p.) and "tail-clip" tests. The pain threshold was evaluated before and after the administration (i.p.) of the different agents. The effect of the combined administration of different agents on behavior, blood pressure and heart rate, was also determined. Nifedipine (5 mg kg(-1), i.p.) and verapamil (10 mg kg(-1), i.p.) potentiated the analgesic effect of butorphanol tartrate (0.25-2 mg kg(-1), i.p.) in all tests (synergism) and enhanced analgesic effect of paracetamol (50-125 mg kg(-1), i.p.), only in acetic acid writhing and tail-clip tests. Magensium sulfate (2.5 mg kg(-1), i.p.) potentiated the analgesic effect of butorphanol, but not that of paracetamol. Co-administration of nifedipine and verapamil with either of butorphanol (0.25-2 mg kg(-1)) or paracetamol (50-125 mg kg(-1), i.p.) produced no significant effects on motor coordination, motor performance, locomotor activity, long-term memory or on the blood pressure and heart rate of experimental animals. Co-administration of magnesium sulfate, however, significantly induced sedation, inhibition of locomotor activity, motor performance and coordination, as well as impairing of long-term memory, as compared with butorphanol and paracetamol, administered alone. We conclude that the systemic co-administration of calcium channel blockers potentiated the analgesic effect of butorphanol against thermal, mechanical and chemical pain but enhanced that of paracetamol only against mechanical and chemical pain. Magensium sulfate enhanced the analgesic effect of butorphanol, but not that of paracetamol. These findings, merit further studies in animals and humans to evaluate the potential therapeutic benefits of such interactions.     

阿托伐他汀的肝毒性机制研究

目的 研究阿托伐他汀肝毒性损伤作用及机制。方法 将24只Wistar han雄鼠分为对照组和阿托伐他汀低（68.5mg/kg）、高剂量组（205.5 mg/kg）,按照10 mL/kg的药液体积给药,溶媒对照组ig等体积5% CMC-Na,连续ig 28 d。检测血清中天门冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）、尿素氮（BUN）和血肌酐（CRE）的含量,HE染色观察肝组织病理。在体外,HepG2细胞经传代培养后,给予阿托伐他汀干预24 h,检测细胞存活率,丙二醛（MDA）水平、Na⁺-K⁺-ATP酶和Ca²⁺-Mg²⁺-ATP酶活性及线粒体膜电位。结果 与对照组比较,阿托伐他汀高剂量组大鼠肝细胞弥漫性肿胀,核分裂多见,部分肝细胞极性消失,排列紊乱（P<0.05）。与对照组比较,阿托伐他汀高剂量组给药后血清中ALT和AST显著升高（P<0.05、0.01）。在体外,与对照组比较,阿托伐他汀125、250、500 μmol/L能明显抑制细胞存活率（P<0.05、0.001）。与对照组比较,阿托伐他汀500 μmol/L HepG2细胞MDA含量明显升高（P<0.01）。与对照组比较,阿托伐他汀125 μmol/L能使Na⁺-K⁺-ATP酶活性增强,500 μmol/L使Na⁺-K⁺-ATP酶活性降低（P<0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能使能使Ca²⁺-Mg²⁺-ATP酶活性降低（P<0.01,0.001）。与对照组比较,阿托伐他汀125、250、500 μmol/L均能降低线粒体膜电位（P<0.001）。结论 阿托伐他汀高剂量可导致肝组织损伤,其毒性作用通过破坏细胞的线粒体膜电位,抑制Na⁺-K⁺-ATP酶、Ca²⁺-Mg²⁺-ATP酶活性,细胞膜脂质过氧化,从而破坏细胞内微环境的平衡,导致细胞凋亡和坏死。     

Influence of Haplophyllum tuberculatum on the Cardiovascular System

The aqueous extract of H. tuberculatum significantly decreased the contractility and the heart rate but did not affect the flow rate of isolated perfused rabbit heart. This effect was not blocked by atropine; however, the muscarinic antagonist blocked the fall in blood pressure seen when the extract was administered to anaesthetized cats. The extract also stimulated rabbit aortic strip, rat vas deferens, and rat anococcygeus muscles. These adrenergic effects were largely reduced by phentolamine. The extract may contain a mixture of pharmacologically active ingredients that necessiate its separation.     

SERCA2a stimulation by istaroxime: a novel mechanism of action with translational implications

Sarcoplasmic reticular (SR) Ca²⁺-ATPase (SERCA2a) is central to cardiac electrophysiological and mechanical function. It ensures full diastolic relaxation minimizing delayed after-potentials that would otherwise compromise membrane electrophysiological stability, and optimizes SR Ca²⁺ refilling and systolic contraction. Previous studies demonstrated that the small molecule agent istaroxime stimulates SERCA2a-ATPase activity, restoring its function in failing hearts, and enhancing indices of mechanical, and SR Ca²⁺ release and re-uptake, activity. Ferrandi et al (2013) now elegantly demonstrate its ability to dissociate the phospholamdan (PB) bound to cardiac SERCA2a, thereby removing the inhibitory effect of PB on SERCA2a. This effect was independent of the cAMP/PKA system and modified a specific SERCA2a reaction step. They used SERCA-enriched SR preparations from a rigorously validated and realistic physiological, canine model of cardiac failure with established Na⁺-K⁺-ATPase sensitivity to cardiac glycosides and SR Ca²⁺ handling features. These findings potentially translate into a novel management of the major and increasingly important public health challenge of chronic cardiac failure.

Linked Article

This article is a commentary on Ferrandi et al., pp. 1849–1861 of volume 169 issue 8. To view this paper visit http://dx.doi.org/10.1111/bph.12278.     

Long-term increase of hippocampal excitability by histamine and cyclic AMP

The action of histamine (HA) on rat hippocampal CA1 pyramidal cells in vitro was investigated in slices perfused with solution containing 0.2 mM Ca²⁺/4.0 mM Mg²⁺. Extracellular recordings of the spontaneous discharges occurring under these conditions revealed that HA caused a long-lasting increase in cell firing. The HA-effects were dose-dependent, in that low concentrations of HA (0.1–0.5 μM) exhibited an initial transient depression of cell firing and practically no long-lasting action, whereas higher concentrations of HA (1–10 μM) exerted strong, non-declining increases. The H₁-receptor antagonist mepyramine (1 μM) blocked the initial depression of firing and attenuated the long-lasting HA-mediated excitation. Pure H₁-receptor activation, tested with the H₁-receptor agonist 2-(3-fluorphenyl)histamine (1–10 μM) depressed cell firing, similar to the low dose effects of HA. HA-induced excitations were prevented by the H₂-receptor antagonist cimetidine (10–50 μM), and mimicked by the very potent H₂-receptor agonist impromidine (1 or 3 μM) which was, however, less effective compared to equal concentrations of HA. H₃-receptor activation by R-α-methylhistamine had no significant effect on cell firing. Thus, histamine H₁ and H₂ receptors seem to cooperate in producing this long-lasting augmentation of excitability. 8-Bromo-cyclic AMP monophosphate (8-Br-cAMP, 50–100 μM) mimicked the long-term excitation, whereas the adenylyl-cyclase inhibitor 9-tetrahydro-2-furyladenine (THFA, 100–500 μM) or the PKA-inhibitor Rp-adenosine-3′5′-cyclic monophosphate (Rp-cAMPS, 10 μM) blocked it, indicating that the HA-mediated increase of excitability in the hippocampus is dependent on the adenylate cyclase/PKA-signal transduction cascade. -2-Amino-5-phosphonopentanoic acid (APV, 50 μM) significantly attenuated the magnitude of the HA-induced enhancement, indicating an NMDA receptor-dependent component. Other biogenic amines, acting through receptors positively coupled to adenylyl cyclase, elicited similar responses as HA, indicating common mechanisms by which these substances modulate excitability in CA1 pyramidal cells.     

Sildenafil and T-1032, phosphodiesterase type 5 inhibitors, showed a different vasorelaxant property in the isolated rat aorta

The vasorelaxant effects of sildenafil and T-1032 [methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate], two phosphodiesterase type 5 inhibitors, were examined in the isolated rat aorta. Sildenafil and T-1032, both of which have almost the same potency and selectivity regarding phosphodiesterase type 5 inhibitory activity, produced a similar, moderate, relaxation at 10(-10) to 10(-7) M (sildenafil: 66.8 +/- 13.7%; T-1032: 77.9 +/- 10.8% at 10(-7) M). However, sildenafil, but not T-1032, produced further relaxation at the higher concentrations (sildenafil: 102.0 +/- 0.6%; T-1032: 81.0 +/- 7.2% at 10(-4) M, P < 0.05). Sildenafil also produced a more potent relaxation than did T-1032 at the high concentrations (10(-5) and 10(-4) M) in endothelium-denuded aortic rings and in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (3 x 10(-4) M). Moreover, the high concentrations of sildenafil, but not of T-1032, caused a rightward shift of the concentration-response curve for calcium chloride in K(+)-depolarized endothelium-denuded preparations. In the ligand binding assay for the L-type Ca(2+) channels, the affinities of sildenafil at 10(-5) M for binding sites of nitrendipine and (--)-desmethoxyverapamil [(--)- D888] (35.2 +/- 3.3% and 35.8 +/- 1.9%, respectively) were higher than those of T-1032 (11.8 +/- 4.0% and -13.1 +/- 1.3%, respectively, P < 0.05). Regarding cyclic nucleotide levels, both phosphodiesterase type 5 inhibitors increased cGMP levels at 10(-6) M. However, sildenafil, but not T-1032, further increased cGMP levels at the higher concentrations (sildenafil: 15.7 +/- 2.7 pmol/mg protein; T-1032: 5.6 +/- 0.6 pmol/mg protein at 10(-4) M, P < 0.05). These results suggested that high concentrations of sildenafil had additional vasorelaxant properties through mechanisms other than phosphodiesterase type 5 inhibition. Sildenafil-induced relaxation appears to be due to inhibition of the external Ca(2+)-dependent cascade for contraction and/or to an increase in cGMP levels. In contrast, T-1032 only showed a vasorelaxant property due to phosphodiesterase type 5 inhibition. In conclusion, T-1032 appears to be a specific phosphodiesterase type 5 inhibitor compared with sildenafil and a useful compound to examine the physiological function of phosphodiesterase type 5.     

Forskolin inhibits potassium-evoked release of vasopressin from rat neurohypophyses

Summary The effects of forskolin, an activator of adenylate cyclase, on the release of vasopressin from isolated rat neurointermediate lobes during a 10 min period of potassium stimulation were investigated. Forskolin was added 5 min after the onset of potassium stimulation. A concentration-dependent reduction in the amount of hormone released during the remaining 5 min period of stimulation was observed. The results suggest that an increase in cyclic AMP following depolarisation inhibits hormone release. This contrasts with several other reports showing that an increase in cyclic AMP prior to stimulation enhances release. We therefore propose that cyclic AMP plays a dual role in stimulus-secretion coupling in the neurohypophysis.     

Mechanisms underlying the slow onset of action of a new dihydropyridine,NZ-105, on a cultured smooth muscle cell line

Summary The inhibitory effect of a new dihydropyridine derivative, (±)-2-[benzyl(phenyl)amino]ethyl-1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinan-2-yl)-4-(3-nitrophenyl)-3-pyridinecarboxylate hydrochloride (NZ-105), on whole cell Ca²⁺ current (I_Ca) in cultured vascular smooth muscle cells was investigated with the patch clamp technique. NZ-105 blocked I_Ca in a concentration-dependent manner when the command pulse ranged from +10 mV to –50 mV. The inhibitory effect of NZ-105 appeared at concentrations higher than 10 mol/l and it blocked I_Ca completely at a concentration of 1 nmol/l. The concentration which produced the half-maximal inhibitory effect was estimated to be around 20 mol/l. NZ-105 (500 pmol/l) completely blocked I_Ca elicited by depolarization to + 10 mV at a holding potential of –40 mV, whereas it blocked I_Ca by only 67% at a holding potential of –90 mV. NZ-105 (100 mol/l) shifted the steady-state inactivation curve by 40 mV to more negative potentials without affecting its slope factor. The blocking time constant of 500 mol/l NZ-105 was 57.6 + 9.9 s at a holding potential of –70 mV. These results indicate that NZ-105 has characteristics typical of dihydropyridines and binds to Ca²⁺ channels of vascular smooth muscle cells with a high affinity. They also suggested that the slow onset of its action is due to the slow binding of the drug to Ca²⁺ channels. Send offprint requests to S. Kokubun at the above address     

Brief low [Mg2+]o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

Reducing [Mg²⁺]_o to 0.1 mM can evoke repetitive [Ca²⁺]_i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg²⁺]_o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca²⁺ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg²⁺]_o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca²⁺ channel antagonist nimodipine, which blocked 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca²⁺]_i spikes. The intracellular Ca²⁺ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca²⁺]_i spikes. While Gö6976, a specific inhibitor of PKCα had no effect on the tolerance, both the PKCε translocation inhibitor and the PKCζ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca²⁺]_i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg²⁺]_o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca²⁺]_i spike-induced activation of PKCε and PKCξ, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.