Isoform specificity of cardiac glycosides binding to human Na,K-ATPase α1β1, α2β1 and α3β1

Cardiac glycosides inhibit the Na⁺,K⁺-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na⁺,K⁺-ATPase are expressed: α₁β₁, α₂β₁ and α₃β₁. It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na⁺,K⁺-ATPase isoforms α₁β₁, α₂β₁ and α₃β₁ were expressed in yeast which has no endogenous Na⁺,K⁺-ATPase. Isoform specific affinities of digoxin, digitoxin, β-acetyldigoxin, methyldigoxin and ouabain were assessed in [³H]-ouabain binding assays in the absence or presence of K⁺ (each n = 5). The subcellular localizations of the Na⁺,K⁺-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K⁺, methyldigoxin (α₁ > α₃ > α₂) and ouabain (α₁ = α₃ > α₂) showed distinct isoform specific affinities, while for digoxin, digitoxin and β-acetyldigoxin no differences were found. In the presence of K⁺, also digoxin (α₂ = α₃ > α₁) and β-acetyldigoxin (α₁ > α₃) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only α₁β₁ and α₂β₁ are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na⁺,K⁺-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.     

A threonine residue in the M2 region of the β1 subunit is needed for expression of functional α1β1 GABAA receptors

Although there is a high degree of homology in the M2 transmembrane segments of α₁ and β₁ subunits, subunit-specific effects were observed in α₁β₁ GABA_A receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13′ threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13′) was mutated to alanine in the β₁ subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13′) was mutated to alanine in the α₁ subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

Synthesis of [2H3]‐dextromethorphan and its major urinary metabolites [2H3]‐dextrorphan and [2H3]‐dextrorphan‐β‐glucuronide

Starting from dextromethorphan, [²H₃]‐dextrorphan‐β‐glucuronide was synthesized in four steps with [²H₃]‐dextromethorphan and [²H₃]‐dextrorphan as intermediates with an overall yield of 11%. Copyright © 2002 John Wiley & Sons, Ltd.     

α-Methyl-5-HT, a 5-HT2 receptor agonist, stimulates β2-adrenoceptors in guinea pig airway smooth muscle

Alpha-methyl-5-HT is widely used as a high-affinity 5-HT(2) receptors agonist, though some studies have postulated that this drug also activates other serotonergic receptors. In the present work, we found that a wide range of concentrations of alpha-methyl-5-HT induced biphasic responses (contraction followed by relaxation) in guinea pig tracheal rings. The relaxing phase caused by 32microM alpha-methyl-5-HT was blocked by 0.1microM propranolol. Furthermore, during an ongoing histamine-induced contraction, alpha-methyl-5-HT (0.1-100microM) produced a concentration-dependent relaxation starting at 10microM. This relaxation was fully abolished by 0.1microM propranolol or 1microM ICI 118,551 (a selective beta(2)-adrenoceptor antagonist). Additionally, in electrophysiological recordings, 32microM alpha-methyl-5-HT also enhanced the membrane K(+) currents of single tracheal myocytes, an effect reverted by propranolol and ICI 118,551, and mimicked by 0.1microM salbutamol. Thus, we concluded that alpha-methyl-5-HT activates beta(2)-adrenoceptors in guinea pig tracheal smooth muscle at concentrations >or=10microM. This effect must be taken into account when this drug is used in airway smooth muscle and in other tissues expressing beta(2)-adrenoceptors.     

Central effects of prostaglandins E2, A1 and F2α in adult fowls

Adult fowls (Gallus domesticus) with cannulae chronically implanted into the 3rd cerebral ventricle, the anterior or posterior hypothalamus and various other sites of the brain received infusions of prostaglandins (PG) E₂, A₁ or F_2α. The effects of these drugs on behaviour, posture, electrocortical activity and body temperature were studied.Prostaglandin E₂ and PGA₁ infused into the 3rd cerebral ventricle and into the hypothalamus induced sedation and/or sleep and increased body temperature. Prostaglandin F_2α lowered body temperature but did not affect behaviour.Behavioural, electrocortical and body temperature changes were dose-dependent. Prostaglandin E₂ was more potent than FGA₁Infusion of PGE₂, PGA₁ and PGF_2α into other cerebral areas e.g. paleostriatum augmentatum, telencephalon and mesencephalon lacked effects on behaviour, electrocortical activity and body temperature.The site through which behavioural, electrocortical and body temperature effects of prostaglandins E₂ and A₁ were mediated appears to be the hypothalamus.     

Effects of the novel antidepressant S-adenosyl-methionine on α1- and β-adrenoceptors in rat brain

α₁- and β-adrenoceptors were studied ex vivo in the brains of rats receiving repeated daily treatment with the standard antidepressant imipramine or the atypical antidepressant S-adenosyl-L-methionine (SAM), which has minimal effects on monoamine reuptake or turnover. Consistent with past studies, a decrease in the density of β receptors at three weeks and an increase in the affinity of α₁ receptors for the agonist phenylephrine at one week of treatment was observed with imipramine. By comparison, an increase in the density of β receptors and a decrease in the affinity of α₁ receptors for phenylephrine was observed at one week of treatment with SAM. These changes were no longer apparent at three weeks of treatment. The results suggest that treatment with SAM does lead to changes in adrenergic neurotransmission, but that down regulation of β receptors or increased agonist affinity of α₁ receptors may not be necessary for the production of antidepressant effects.     

Block of human NaV1.5 sodium channels by novel α-hydroxyphenylamide analogues of phenytoin

Voltage-gated sodium (Na) channels are a critical component of electrically excitable cells. Phenytoin (diphenylhydantoin, DPH) is an established sodium channel blocker and is a useful anticonvulsant and class 1b antiarrhythmic, and has been effectively used in the treatment of neuropathic pain. In this study, we have synthesized novel alpha-hydroxyphenylamide analogues of diphenylhydantoin and examined their ability to inhibit human Na(V)1.5 sodium channels expressed in Chinese Hamster Ovary (CHO-K1) cells. Phenyl ring substitutions were examined including para-methyl, para-fluoro, para-chloro, ortho-chloro and meta-chloro. We have found that phenyl ring substitutions with electron withdrawing properties resulted in compounds with greater activity. In comparison to diphenylhydantoin, the novel chloro-substituted alpha-hydroxyphenylamide compounds produced as much as a 20-fold greater tonic and frequency-dependent blockade of Na(V)1.5 channels with an IC(50) value of 14.5 microM. In addition, the chloro-substitutions have position specific state dependent blocking properties. The ortho-, meta- and para-chloro substitutions have an 8-, 13- and 3-fold increased affinity for the inactivated state, respectively. Molecular modeling suggests that these differences in affinity are due to a direct interaction with the receptor. Comparing models of diphenylhydantoin to the novel alpha-hydroxyphenlyamide compound suggests that the increased activity may be due to an optimized phenyl ring position and increased molecular volume. This information may be useful in the development of more potent sodium channel blockers.     

Characterization of [3H]brevetoxin binding to voltage-dependent sodium channels in adrenal medullary cells

We have previously reported that in bovine adrenal chromaffin cells Ptychodiscus brevis toxin-3 (PbTx-3) does not alter the veratridine-induced ²²Na influx when given alone, but increases the influx of ²²Na when co-applied with either - or -scorpion venom (Wada et al. 1992). In the present study, we characterized [³H]PbTx-3 binding in bovine adrenal chromaffin cells. [³H]PbTx-3 binding was saturable, reversible and of high-affinity with an equilibrium dissociation constant (K_d) of 32.0±4.9 nmol/1 and a maximum binding capacity B_max of 6.2 ± 1.2 pmol/4 × 10⁶ cells (4.5 ± 0.9 pmol/mg cell protein). A Hill plot revealed the lack of cooperative interaction among the binding sites. Unlabelled PbTx-3 inhibited [³H]PbTx-3 binding with an IC₅₀ of 31 nmol/l. However, tetrodotoxin, veratridine, - and -scorpion venom, or veratridine in combination with either - or -scorpion venom did not alter [³H]PbTx-3 binding. All these results suggest that PbTx-3 binds to a site (site 5) distinct from the previously known four toxin binding sites, which does not gate voltage-dependent Na channels by itself, but is specifically involved in the allosteric modulation of Na channels in adrenal medullary cells. Correspondence to: A. Wada at the above address     

Pharmacological characterization of α2D-adrenergic receptor-mediated [S]GTPγS binding in rat cerebral cortical membranes

Using the rat cerebral cortical membranes that had once been frozen and stored at -80 degrees C, the stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding mediated through alpha(2)-adrenergic receptors (alpha(2)-ARs) was pharmacologically characterized. The stimulatory effects of (-)-noepinephrine ((-)-NE) and 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK-14,304) at maximally effective concentrations were prominent only in the presence of micromolar to submillimolar levels of GDP, with the greatest signal/noise ratio achieved at 100 and 30-100 microM GDP for (-)-NE and UK-14,304, respectively. The alpha(2)-AR-mediated [(35)S]GTPgammaS binding was also critically dependent on the presence of millimolar concentrations of MgCl(2) in assay medium. The maximum stimulation induced by UK-14,304 was equivalent to, or even greater than, that of an endogenous ligand (-)-NE at lower concentrations of NaCl, while it became a partial agonist with higher concentrations of NaCl. Concentration-response curves for several agonists revealed that the omission of NaCl from incubation buffer generally shifted the curves leftward and increased the relative efficacies as compared to the endogenous ligand. The pharmacological profile characterized with a series of adrenergic agonists and antagonists indicated that this response was derived from activation of G proteins coupled with alpha(2)-ARs, in particular alpha(2D)-AR subtype, though the possible involvement of alpha(2C)-ARs was not completely excluded. However, the stimulatory effects of several adrenergic compounds, such as rauwolscine and yohimbine, were ascribed to their agonist properties at 5-HT(1A) receptors. This method serves as a simple but useful strategy for investigating the functional interaction between alpha(2D)-ARs and their coupled G proteins in rat native cerebral cortical membranes, especially in the field of psychopharmacology and biological psychiatry.     

Multiple structural elements contribute to voltage-dependent facilitation of neuronal α1C (CaV1.2) L-type calcium channels

Voltage- and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that α_1C L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal α_IC L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032–5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between «facilitable» α_1C and «non facilitable» α_1E calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of α_1C contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of α_1C is a complex process which involves multiple structural elements were domains I and III play the major role.     

Pharmacological blockade of brain α1-adrenoceptors as measured by ex vivo [H]prazosin binding is correlated with behavioral immobility

The present studies examined the relationship between the blockade of central α₁-adrenoceptors, as measured by ex vivo binding of [³H]prazosin in the cerebral cortex and the inhibition of behavioral activation to a mildly novel environment (cage change). It was found that intraventricular (i.v.t.) terazosin, a saline-soluble α₁-adrenoceptor antagonist, dose dependently inhibited both ex vivo cortical binding and behavioral activation and that there was a highly significant positive correlation between the two with a slope near unity. Prazosin, a nonsaline soluble antagonist which had to be given intraperitoneally (i.p.), was much less potent at blocking both behavioral activity and cortical ex vivo binding, although it blocked ex vivo binding in the lung, indicating that it was effective peripherally but did not readily enter the brain. Despite this, however, the inhibition of cortical binding and behavioral activation that i.p. prazosin did produce were highly correlated with each other and had a slope near unity as with terazosin, whereas the more potent inhibition of lung binding was less well correlated with behavioral inhibition and had a slope significantly less than one. These results confirm our earlier studies, which have shown that α₁-adrenoceptor activity is essential for gross and fine motor behavior in the mouse and that prazosin, which is used extensively in behavioral research, has difficulty entering the mouse brain.     

Behavioral and receptor binding analysis of the α2-adrenergic agonist, 5-bromo-6 [2-imidazoline-2-YL amino] quinoxaline (UK-14304): evidence for cognitive enhancement at an α2-adrenoceptor subtype

The ability of the α₂-agonists clonidine, B-HT920 (6-allyl-2-amino-5,6,7,8-tetrohydro-4H-thi-azolo-[4,5-d]-azepine) and guanfacine to improve memory in aged monkeys has been related to their affinity to bind at a proposed rauwolscine-insensitive (Ri) subtype of α₂-adrenergic receptor, while their hypotensive and sedating effects have been related to affinity at a rauwolscine-sensitive site (Rs) (Arnsten et al., 1988). The present study examined the α₂-agonist UK-14304 (5-bromo-6 [2-imidazoline-2-yl amino] quinoxaline) for its binding characteristics in tissue from the brain of the rat and for its behavioral effects in aged monkeys. The drug UK-14304 was found to have slightly higher affinity for the Ri than the Rs site (K_i values of 138 and 245 nM, respectively), but was not as selective as the α₂-agonist guanfacine (K_i values of 23 and 340 nM, respectively). Consistent with this binding profile, very small doses of UK-14304 (0.00017–0.17 μg/kg) produced a reliable but modest improvement in memory in the aged monkeys (average improvement of 16.7% ± 2.6% following an optimal dose). No hypotensive or sedating side effects were observed at these small doses. However, hypotension and sedation emerged rapidly when the dose was raised above 1.7 μg/kg and at the largest doses tested (50.0–100.0 μg/kg), hypotension was severe (systolic pressure below 70 mm Hg) and the animals were too sedated to complete cognitive testing. The separation between doses that improved memory and those that produced hypotension and sedation was not as great for UK-14304 as it was for guanfacine, consistent with the greater selectivity of guanfacine for the Ri site. These results offer a fourth example whereby the ability of an α₂-agonist to improve cognitive function, without side effects, could be related to the relative affinities for the Ri and Rs sites.     

Liver toxicity and apoptosis: role of TGF-β1, cytochrome c and the apoptosome

Transforming growth factor-β₁ (TGF-β₁), is involved in controlling liver size, by inducing apoptotic cell death in hepatocytes. However the mechanism by which TGF-β₁ induces caspase activation and cell death is unknown. Apoptosis can be initiated either by receptor-mediated (e.g. Fas/CD95) or non-receptor chemically mediated (stress-induced) processes. With Fas/CD95 receptor mediated cell death, a multi-protein complex (DISC) is assembled at the plasma membrane, which activates the downstream caspases and cell death. In stress-mediated apoptosis, a cytosolic DISC equivalent, the apoptosome is formed that activates the effector caspases. We have characterised this complex in THP.1 cells, and shown that this is a cytochrome c dependent process that induces the formation of an 700 kDa apoptosome caspase processing complex. This is formed by oligomerisation of apoptotic protease-activating factor 1 (Apaf-1), and recruitment and processing of caspase-9. We have now shown that TGF-β₁-induced apoptosis also occurs via the release of cytochrome c and the subsequent oligomerisation of Apaf-1 into an 700 kDa apoptosome complex. Our studies show that, even though TGF-β₁ induction of apoptosis is a receptor-mediated event, it operates through the mitochondrial/Apaf-1 caspase activation pathway that appears to act as a common execution pathway for many diverse apoptotic stimuli.     

Modification of reserpine-induced emetic response in pigeons by α2-adrenoceptors

In the present study, an attempt has been made to elucidate the role of α₂-adrenoceptors in reserpine-induced emesis in pigeons. Reserpine was found to induce dose-dependent emesis and a 500 jug kg⁻¹ dose was found to be the 100% emetic dose. a2-adrenoceptor agonists clonidine and a-methylnoradrenaline inhibited the reserpine induced emesis. Out of the two selective a2-adrenoceptor antagonists idazoxan and yohimbine, only the latter induced a dose-dependent emesis. However, both the drugs potentiated reserpine-induced emesis and antagonised its inhibition by clonidine. Prior depletion of monoamines by reserpine also blocked the emetic response of reserpine. These observations indicate that release of monoamines is responsible for its emetic response in pigeons which is modulated by presynaptic a,-adrenoceptors in a predictable manner.     

A simple and efficient synthesis of [3α‐3H]5α‐androst‐16‐en‐3β‐ol

An efficient one step synthesis of [3α‐³H]5α‐androst‐16‐en‐3β‐ol by NaBT₄ reduction of a ketone precursor is described. The specific activity of the product was 21.6 Ci/mmol with a radiochemical purity >99%. Synthesis of the precursor, 5α‐androst‐16‐en‐3‐one, from commercially available 5α‐androst‐16‐en‐3α‐ol is also presented. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of tracer labelled [11,12‐3H]‐β‐carotene

The synthesis of tracer labelled [11,12‐³H]‐β‐carotene is described. The procedure uses Wittig condensation of tracer labelled ³H‐retinal (retinal spiked with [11,12‐³H]‐retinal) with retinyl triphenylphosphonium bromide. The preparation of tracer labelled[³H]‐β‐carotene is suitable for studies involving bioavailability and bioconversion of β‐carotene to vitamin A. Copyright © 2003 John Wiley & Sons, Ltd.     

Mn3[Co (CN)6]2@SiO2对鼠C6脑胶质瘤模型3T MR成像及生长规律的研究

目的 利用Mn₃[Co（CN）₆]₂@SiO₂对大鼠C6脑胶质瘤模型进行3T MR成像,研究肿瘤的生长规律。方法 收集对数生长期鼠C6胶质瘤细胞,建立鼠C6脑胶质瘤模型,于细胞接种后第7 d行MR扫描,然后鼠尾静脉注射0.5 mL Mn₃[Co（CN）₆]₂@SiO₂,于注射后5 min、24 h再行MR扫描,扫描结束后取出肿瘤组织行HE染色。随机抽取脑内成功接种C6胶质瘤细胞的荷瘤鼠10只,接种后第 7、9、11、13、15、17、19天行MR扫描,测量肿瘤体积,绘制出肿瘤生长曲线。结果 成功建立了鼠C6脑胶质瘤模型,并且鼠尾静脉注射Mn₃[Co（CN）₆]₂@SiO₂后5 min、24 h肿瘤均强化,24 h肿瘤强化较前明显;MRI检测出胶质瘤大小与时间呈正相关关系（r_s=0.9944,P=0.000）。结论 应用Mn₃[Co（CN）₆]₂@SiO₂对鼠C6脑胶质瘤活体示踪MR成像,可以反映肿瘤的生物学特性,适用于肿瘤生长规律的观测。