Neuroprotection against ischemic/hypoxic brain damage: blockers of ionotropic glutamate receptor and voltage sensitive calcium channels

The growing number of cellular and molecular pathways believed to be involved in mechanisms of ischemic cell death in the brain has spurred a similar growth in the number of potential neuroprotective modalities, the majority of which are pharmacological in nature. Preventing or minimizing the first few steps in the cascade of events leading to ischemic cell death would have a more profound effect on the postischemic outcome than intervention at later steps in that cascade. This logic is, of course, at the heart of the urgency in providing the stroke or cardiac arrest patient with the earliest possible neuroprotective treatment. For the purpose of assessing potential neuroprotective modalities, the use of a well-established cerebral hypoxic/ischemic model system is a prerequisite. In our studies, we have used two major approaches, in vitro and in vivo. We evaluated both agonists and antagonists of ionotropic glutamate receptor channels (IGRC) and their effects in exacerbating and attenuating, respectively, the posthypoxic/ischemic outcome. Other drugs were tested for their ability to block the L-type voltage-sensitive calcium channels (VSCC), which are responsible for calcium influx and overload upon hypoxia/ischemia. These two membrane protein entities, the IGRC and the VSCC, are believed to be involved in the early stages of the cellular cascade that leads to the demise of neurons posthypoxia/ischemia. Some of the drugs were also tested for possible interaction with each other searching for possible synergy. These and other published studies in the field are reviewed here.     

An antibody to dihydropyridine calcium entry blockers. A comparison with the calcium channel receptor in skeletal muscle

Antibodies that recognize dihydropyridine (DHP) calcium entry blockers were elicited from rabbits. A sensitive and specific radioimmunoassay for dihydropyridines was developed and its specificity compared to the DHP binding site in skeletal muscle membranes. The antibody bound [3H]nitrendipine with a higher affinity (KD = 0.155 nM) than did the DHP receptor of skeletal muscle (KD = 1-3 mM); however, in contrast to the DHP receptor, the antibody recognized only those DHP drugs with meta-nitrophenyl substituents at the 4-position on the DHP ring. Both the antibody and receptor exhibited stereospecificity, with each site recognizing the (+)-isomer of nicardipine as the more potent. This antibody should prove useful in our studies of some potentially irreversible DHP molecules.     

Evidence for an external location of the dihydropyridine agonist receptor site on smooth muscle and skeletal muscle calcium channels.

1. The location of the binding domain for agonist dihydropyridines (DHP) has been studied by comparing the action of (+)-202,791 and (-)-Bay K 8644 on Ba2+ currents (IBa) in whole cell patch clamp experiments. Drug effects were examined upon internal and external (extracellular) application in A7r5 smooth muscle cells and BC3H1 cells, a cell line expressing Ca channels of the skeletal muscle type. 2. Efficiency of internal drug application in the whole cell studies was demonstrated by inhibition of potassium currents and barium currents (IBa) upon internal perfusion with tetraethylammonium (TEA+) (10 mM) and the permanently charged phenylalkylamine, D 890 (100 microM) respectively. The uncharged DHP, (-)-STBODIPY-DHP (2 microM) was used to estimate the time course of internal perfusion by monitoring its fluorescence. 3. Intracellular application of (+)-202,791 and (-)-Bay K 8644 (5 microM) in patch clamp experiments was ineffective in stimulating Ca2+ channel currents in both cell lines. In contrast a 50 fold lower agonist concentration (0.1 microM (-)-Bay K 8644) applied to the external face of the membrane induced typical changes in tail currents and a current increase under conditions when up to 10 microM of the agonist was present in the intracellular perfusion solution. 4. In cell-attached patches in A7r5 cells, (-)-Bay K 8644 increased and (+)-PN 200,110 inhibited single channel activity when applied via the bath solution. This suggests partitioning and lateral diffusion of the DHPs in the lipid of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of calcium channel dihydropyridine receptor binding by purified Mojave toxin

Mojave toxin, the principal toxic component of the venom of the Mojave rattlesnake Crotalus scutulatus scutulatus, is a protein complex of about 22,000 mol. wt. The mechanism of action of this potent (LD50 = 0.039 micrograms/g, mouse, IV) neurotoxin is a matter of conjecture, but physiologic data suggest a presynaptic site of action with disruption of stimulus-secretion coupling and neurotransmitter release. The selectivity of Mojave toxin's effect on several ion channels involved in neurotransmission was assessed in the present study using competitive radioisotopic binding procedures. Synaptic membranes from rat brain were used to assess the toxin's interaction with Ca++ and Cl- channels while membrane fragments from the Torpedo fish electric organ were used to determine toxin interaction with the nicotinic acetylcholine receptor-coupled Na+ channel. Mojave toxin was found to irreversibly inhibit 3H-nitrendipine binding to dihydropyridine receptors associated with Ca++ channels in rat brain, but had no effect on radioligand binding in the Na+ and Cl- channel assays. Saturation analysis of the binding further showed that the effects of MoTX on dihydropyridine binding were noncompetitive, with MoTX producing a decrease in both the affinity and density of 3H-nitrendipine sites. These results are consistent with the hypothesis that MoTX acts selectively on Ca++ channel function and that this interaction occurs via an allosteric mechanism in which MoTX binds to a membrane site that is topologically distinct from the dihydropyridine receptor.     

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

Rationale

Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca^2 + channels and their renowned antioxidant properties.

Methods

We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca^2 + channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca^2 + channel-blocking activity and antioxidant properties. The Ca^2 + channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR.

Results

Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca^2 + channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca^2 + channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties.

Conclusions

Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.     

The interaction of mu-opioid receptors with ion channels and G-proteins

The state-of-the-art in investigations of the mu-opioid receptors (ORs) is reviewed. Published information on the interaction between mu-ORs and various G-proteins is summarized and data on the neutral antagonists and inverse agonists of mu-opioid receptor are generalized. The notions about the spontaneous activation of mu-ORs are considered and numerous reports on the interaction between mu-ORs and GIRK, KATP, and Kv channels are analyzed. The role of L, N, and P/Q types of Ca(2+)-channels in mu-OR-mediated intracellular signaling is discussed.     

去极化作为电压门控性钙通道的调节信号

去极化作为电压门控性钙通道的调节信号ＤａｖｉｄＪＴＲＩＧＧＬＥ（Ｄｅａｎ，ＴｈｅＧｒａｄｕａｔｅＳｃｈｏｏｌ，ＳｔａｔｅＵｎｉｖｅｒｓｉｔｙｏｆＮｅｗＹｏｒｋ，ＢｕｆｆａｌｏＮＹ１４２６０，ＵＳＡ）关键词钙通道；膜电位；神经生理学；电生理学；钙通道激...     

二氢吡啶类钙拮抗剂药物基因组学的研究进展

二氢吡啶类钙拮抗剂是临床常用的一类降压药。临床使用过程中,疗效受到患者基因组学特征的影响。细胞色素P450(CYP3A)酶、P-糖蛋白(P-gp)和乳腺癌耐药蛋白(BCRP)转运体参与了药物体内处置过程,而CACNA1C受体是发挥药效的关键。大量临床研究结果表明,它们的基因多态性对该类药物的药动学和药效学会产生较大影响。本文在查阅近年来国内外文献基础上,就酶、转运体和受体的基因多态性对二氢吡啶类钙拮抗剂药动和药效的影响研究进展进行了综述,为临床用药提供参考。     

二氢吡啶介导的脑定向转释磺胺甲(口恶)唑的合成

目的：合成二氢吡啶介导的脑定向转释磺胺甲口恶唑药物。方法：以磺胺甲口恶唑和烟酸为原料,经酰化,烃化和还原反应制备［ 二氢吡啶磺胺甲口恶唑］ 偶联物。结果：［ 二氢吡啶磺胺甲口恶唑］ 偶联物及其中间体均经波谱鉴定。结论：本文结果为深入开发脑定向转释抗菌药物提供了重要参数     

氨茶碱调节大鼠支气管平滑肌L型钙通道的活动性

目的 探讨氨茶碱对大鼠支气管平滑肌细胞L型钙通道(L-L_(Ca))的作用。方法 将20只大鼠随机分为2组,A组为正常对照组(n=10),B组为氨茶碱组(n=10),观测大鼠支气管平滑肌细胞L-L_(Ca)在正常时及给予氨茶碱治疗后的变化。结果 氨茶碱治疗后大鼠的L-L_(Ca)与正常对照组大鼠通道开放概率相比差异有显著性(P<0.05)。氨茶碱组通道开放时间常数(τ_(O1)和τ_(O2))不变,关闭时间常数(T_(c1)和T_(c2))延长,与正常对照组相比差异有显著性(P<0.05)。大鼠L-钙通道的活动经氨茶碱治疗后活动减弱,表现为通道开放时间常数不变,关闭时间常数延长。结论 氨茶碱通过缩短L-L_(Ca)的开放时间来抑制支气管平滑肌细胞的收缩,可显著抑制大鼠L-L_(Ca)的活动,促进大鼠支气管平滑肌细胞的舒张。     

Functional coupling of a recombinant Human 5-HT5A receptor to G-proteins in HEK-293 cells

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT_5A receptor.
2. We have shown that ligand activation of the Human 5-HT_5A receptor results in functional coupling to G-proteins in HEK-293 cells.
3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [³⁵S]-GTPγS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT_5A receptor in any cell.
4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT_5A receptor cell line, suggesting a functional coupling to a Gαi, G-protein in the HEK-293 cell line.
5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT_5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

     

Role of calcium in mediating the biphasic contraction of the rabbit urinary bladder

• 1.1. The response of the urinary bladder to field stimulation is biphasic in nature consisting of an initial phasic contraction followed by a prolonged tonic phase which lasts for the duration of the stimulation.
• 2.2. The phasic response is mediated by the release of neurohumoral transmitters, primarily acetylcholine (via muscarinic receptor stimulation) and ATP (via purinergic receptor stimulation). The tonic component is mediated entirely via muscarinic receptor stimulation.
• 3.3. The present study investigates the dependence on extracellular calcium of the phasic and tonic contractile responses to field stimulation, bethanechol, and ATP. The results can be summarized as follows:
• 4.4. Field stimulation (2 and 32 Hz) and bethanechol evoke a biphasic contractile response whereas ATP evokes only a phasic response.
• 5.5. There were no significant effects of either calcium channel blockers or calcium fee EGTA medium on either spontaneous contraction or basal tension of muscle strips.
• 6.6. The calcium channel antagonists diltiazem and verapamil inhibited both the phasic and tonic responses induced by field stimulation (both 2 and 32 Hz) in a dose dependent manner.
• 7.7. For both 2 and 32 Hz stimulation, the ED₅₀s for the inhibition of the tonic phases of the responses to field stimulation were significantly lower than the ED₅₀s for the inhibition of the phasic responses.
• 8.8. The tonic phase of the responses to field stimulation were inhibited to a significantly greater degree than the phasic responses by incubation in calcium-free medium containing EGTA.
• 9.9. Both the phasic and tonic components of the response to bethanechol stimulation were inhibited equally, and followed a similar time course as the tonic component of field stimulation.
• 10.10. The response to ATP was relatively insensitive to calcium depletion.
• 11.11. In summary, the tonic response to field stimulation (purely cholinergic) is significantly more dependent on extracellular calcium than is the phasic response (cholinergic + purinergic).

     

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells

Summary In the human T -cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E₂ receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects.Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as G_i2 and G_i3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment.In conclusion, activation of protein kinase C by phorbol esters reveals a G_i-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A₂ receptor stimulated CAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive G_i-protein. Send offprint requests to I. van der Ploeg at the above address     

二氢吡啶介导的脑定向转释磺胺甲口恶唑的合成

目的：合成二氢吡啶介导的脑定向转释磺胺甲恶唑药物。方法：以磺胺甲恶唑和烟酸为原料,经酰化,烃化和还原反应制备「二氢吡啶－磺胺甲恶唑」偶联物。结果：「二氢吡啶－磺胺甲恶唑」偶联物及其中间体均经波谱鉴定。结论：本文结果为深入开发脑定向转释抗菌药物提供了重要参数。     

Cardiovascular effects of a new dihydropyridine calcium antagonist

The effects of methyl 2,6-dimethyl-4-(2-nitrophenyl)-5-(2-oxo- 1,3,2-dioxaphosphorinan-2-yl)-1,4-dihydropyridine-3-carboxylate (DHP-218), a 1,4-dihydropyridine derivative, on the cardiovascular system and myocardial oxygen consumption were investigated. Effects on cardiovascular system: In urethane-alpha-chloralose anesthetized dogs, DHP-218 at a dose of 0.01 mg/kg i.v. decreased blood pressure (BP), and at 0.03 mg/kg i.v., long-lasting BP decrease was accompanied by an increase in heart rate (HR), and an increase in blood flow of the coronary, vertebral and internal carotid arteries. No significant changes in myocardial contractile force (MCF) and blood flow of the common carotid and renal arteries were observed. The duration of action was different for various effects. At doses more than 0.05 mg/kg i.d., blood pressure decreased and HR, MCF and the blood flow of coronary and vertebral arteries increased. The effects of nifedipine on the cardiac and regional blood flow were observed at doses more than 0.001 mg/kg i.v. and 1 mg/kg i.d., but the duration of its action was very short compared to those of DHP-218. Effects on cardiohemodynamics: In urethane-alpha-chloralose anesthetized dogs, DHP-218 at a dose of 0.01 mg/kg i.v. produced a decrease in BP and total peripheral resistance (TPR) and an increase in cardiac output (CO). At a dose of 0.03 mg/kg, a decrease in BP and TPR and increases in HR and CO were observed. The duration of action was different for various parameters. No significant changes in dp/dtmax, stroke volume and cardiac work were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antihypertensive effects of a new dihydropyridine calcium antagonist

Methyl 2,6-dimethyl-4-(2-nitrophenyl)-5-(2-oxo-1,3,2-dioxaphosphorinan-2 -yl)-1,4-dihydropyridine-3-carboxylate (DHP-218) is a new vasodilatory calcium antagonist with pronounced and long-lasting antihypertensive effects. It produced a significant decrease in blood pressure at doses more than 1 mg/kg in normotensive rats, 0.1 mg/kg in spontaneously hypertensive rats (SHR) and desoxycorticosterone acetate (DOCA)-salt hypertensive rats and 0.3 mg/kg in renal hypertensive rats. In SHR, the antihypertensive effect of DHP-218 was approximately 7 times more potent than nifedipine. The antihypertensive effect of DHP-218 appeared very slowly and persisted even after it was injected i.v. No tolerance to the antihypertensive effects of DHP-218 was observed for 5 weeks at daily doses of 0.3 and 1 mg/kg. The antihypertensive effects of DHP-218 in cats and dogs with normal blood pressure was more than 10 and 30 times more potent than those of nifedipine, respectively. At an equivalent antihypertensive dose, the effect of DHP-218 persisted longer than that of nifedipine in all the animal species used.     

Novel interactions of cations with dihydropyridine calcium antagonist binding sites in brain.

The effects of monovalent (Na+, Li+, K+, Rb+) and divalent (Ca2+, Mg2+, Mn2+) cations on dihydropyridine calcium antagonist binding sites in brain and cardiac membranes were investigated using a low ionic strength buffer (5 mM Tris-HCl, pH 7.4), and the dihydropyridine, [3H]-nitrendipine. At 25 degrees C, the monovalent cations Na+, Li+, and K+ (100 mM) but not Rb+ significantly decreased the apparent dissociation constant (KD) but had no effect on the maximum binding site capacity (Bmax) of [3H]-nitrendipine in brain. The divalent cations Ca2+, Mg2+, and Mn2+ (2 mM) significantly increased the Bmax, but did not affect the KD of [3H]-nitrendipine. The effects of cations were concentration-dependent (EC50 monovalent cations 10-25 mM; EC50 divalent cations 50-200 microM) and demonstrated brain region selectivity. The effect of Ca2+, but not Mg2+ or Mn2+ on [3H]-nitrendipine binding was described by a two-site model. At 25 degrees C, neither mono- nor divalent cations altered the characteristics of [3H]-nitrendipine binding to rat cardiac membranes. At 37 degrees C, Na+ (100 mM) but not K+ (100 mM) significantly increased the Bmax of [3H]-nitrendipine in rat brain membranes. Ca2+ (2 mM) significantly increased the Bmax of [3H]-nitrendipine binding to rat brain membranes to a greater extent than at 25 degrees C. Both Na+ and K+ had no effect on [3H]-nitrendipine binding to cardiac membranes, while Ca2+ (2 mM) significantly decreased the KD of [3H]-nitrendipine. It is suggested that the selective effects of mono- and divalent cations on [3H]-nitrendipine binding to rat brain and cardiac membranes may be associated with differences in the calcium current blocking activity of dihydropyridine calcium antagonists in brain and cardiac tissues.