Spotlight from the American Society for Preventive Cardiology on Key Features of the 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guidelines on the Management of Blood Cholesterol

The 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol retains focus on recommendations for statin treatment in the original four statin-eligible groups [those with atherosclerotic cardiovascular disease (ASCVD), diabetes, low-density lipoprotein cholesterol (LDL-C) ≥ 190 mg/dL, and higher risk primary prevention] without the use of treatment initiation or target LDL-C levels from the earlier 2013 American College of Cardiology/American Heart Association (ACC/AHA) guideline, but has several new features. First, patients with primary prevention are divided into those who are at low (< 5%), borderline (5% to < 7.5%), intermediate (7.5% to < 20%), and high (≥ 20%) risk based on the ASCVD risk estimator. Moreover, the new guideline goes further to consider a wider range of factors [now called “risk enhancers”—premature family history of ASCVD, persistently high LDL-C, chronic kidney disease (CKD), metabolic syndrome, conditions specific to women, inflammatory diseases, and high-risk ethnicities] that can be used to better inform the treatment decision. Moreover, more detailed recommendations on how the results of coronary calcium scanning can be used to inform the treatment decision are provided, including how it may be used to “de-risk” certain patients for delaying or avoiding the use of statin therapy. There are also specific sections for cholesterol management in other patient subgroups including women, children, certain ethnic groups, those with CKD, chronic inflammatory disorders and HIV, as well as discussion on the management of hypertriglyceridemia. Importantly, for persons with known ASCVD, a distinction is made for those who are at “very high risk” based on having had two major ASCVD events or one major event and two or more other high risk conditions, such as diabetes or other major risk factors, or bypass surgery or percutaneous intervention. Finally, the concept of a threshold LDL-C for initiating a non-statin therapy (after considering highest tolerated statin dosage) is provided, with ezetimibe recommended as the key non-statin to be added if the LDL-C still remains ≥ 70 mg/dL for all ASCVD patients, and in those who are at “very high risk”, further consideration for using a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. While the new guideline does have greater detail (and arguably, complexity), the refinements provide a strategy for guiding the clinician to target both statin and non-statin therapy to those most likely to derive benefit.     

Conversion of the N-O-glucuronide and N-O-sulfate conjugates of N-hydroxyphenacetin to reactive intermediates

The N-O-glucuronide of [¹⁴C]acetyl-N-hydroxyphenacetin is sufficiently stable to purify, but slowly breaks down in aqueous solutions to a reactive intermediate that can covalently bind to protein. When the pure compound was incubated in Tris buffer, pH 7.4, at 37°, it decomposed with a half-life of about 8.7 hr to the following compounds: phenacetin, 2-hydroxyphenacetin glucuronide, acetamide and acetaminophen. On addition of glulathione to the systems and allowing the reactions to go to completion, a glutathione-acetaminophen conjugate was formed at the expense of acetamide and acetaminophen: the fraction converted to phenacetin or to the 2-hydroxyphenacetin glucuronide was unchanged. On addition of ascorbic acid to the system and allowing the reactions to go to completion, the fraction converted to acetaminophen was increased at the expense of acetamide: the fractions converted to phenacetin and 2-hydroxyphenacetin glucuronide, however, were again unchanged. When the glucuronide was incubated with bovine serum albumin, covalent binding to the protein occurred at the expense of acetaminophen and acetamide; again, the fraction of the glucuronide converted to phenacetin and 2-hydroxyphenacetin glucuronide was unchanged. Moreover, the covalent binding could be partially prevented by addition of ascorbic acid or glutathione. Since there is formation of covalently bound material, the glutathione conjugate and acetaminophen appear to be interrelated; it seems likely that they are formed from a common intermediate, possibly acetylimidoquinone. However, the data suggest that the formation of phenacetin and 2-hydroxyphenacetin glucuronide occurs by different mechanisms. The N-O-sulfate of [¹⁴C]acetyl-N-hydroxyphenacetin also breaks down to a reactive intermediate that has properties similar to those of the reactive intermediate formed from the N-O-glucuronide and thus may also be N-acetylimidoquinone. By contrast, the relative ability of various nucleophiles to prevent the covalent binding of the reactive intermediate formed from the N-O-sulfate of 2-acetylaminofluorene to protein differs from the relative ability of the nucleophiles to prevent the covalent binding of the reactive intermediate of either the N-O-sulfate or the N-O-glucuronide of phenacetin, suggesting that the relative rates at which these intermediates combine with the different macromolecules may differ markedly.     

Effects on the rat oxyntic mucosa of the histamine2-antagonist loxtidine and the H+, K+-ATPase inhibitor omepvazole

The present study examined whether histamine could affect the growth of the enterochromaffin-like (ECL) cell and the parietal cell. The effects of the unsurmountable histamine H2-receptor antagonist loxtidine (80 mg/kg) and the H+, K(+)-ATPase inhibitor omeprazole (100 mumol/kg) were compared in female Sprague-Dawley rats. Both drugs were given by gavage once daily for 3 months. Omeprazole induced a more pronounced and sustained hypergastrinaemia than loxtidine. In spite of marked hypergastrinaemia during most of the day, even in the loxtidine-treated rats, the weights of the stomach and oxyntic mucosa were elevated only in the omeprazole-treated rats. The ECL cell density was slightly higher in the loxtidine- than in the omeprazole-treated rats. Both treatments elevated the gastrin-stimulated histamine release from the vascularly perfused stomach. The parietal cell density was unaffected by omeprazole treatment, whereas it tended to be reduced in the loxtidine-treated rats. Simultaneous administration of loxtidine and omeprazole reduced the sustained hypergastrinaemia induced by omeprazole given alone. The present study may indicate that histamine inhibits the growth of the ECL cell, but further studies are needed to elucidate if histamine has any trophic effect on the parietal cells.     

Comparison of the diabetes guidelines from the ADA/EASD and the AACE/ACE

Objective

To compare recent diabetes guideline updates from the American Diabetes Association–European Association for the Study of Diabetes (ADA/EASD) and the American Association of Clinical Endocrinologists–American College of Endocrinology (AACE/ACE).

Background to the ADI/TDI/PTWI

International scientific committees such as the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Joint FAO/WHO Meeting on Pesticide Residues (JMPR), regional scientific committees such as those of the European Union, and national regulatory agencies generally use the safety factor approach for establishing acceptable or tolerable intakes of substances that exhibit thresholds of toxicity. The acceptable daily intake (ADI) is used widely to describe “safe” levels of intake; other terms that are used are the reference dose (RfD) and tolerable intakes that are expressed on either a daily (TDI or tolerable daily intake) or weekly basis. JECFA uses the term PTWI, or provisional tolerable daily intake, for contaminants that may accumulate in the body. The weekly designation is used to stress the importance of limiting intake over a period of time for such substances. When using this approach no-observed-effect levels (NOELs) or no-observed-adverse-effect levels (NOAELs) are identified in the critical studies, to which appropriate safety or uncertainty factors are applied. Although the value of safety factors varies depending upon a number of factors, 100 is most often used, which is designed to account for interspecies and intraspecies variations. Within the framework of the IPCS project on harmonization of approaches to the assessment of risk from exposure to chemicals, issues relating to uncertainty and variability are being addressed with the aim of relying, whenever appropriate, on data-derived safety/uncertainty factors. The ILSI Europe ADI Task Force has, for the past few years, been considering the scientific basis for the safety factor, which will be discussed by other speakers in the workshop. The value of the NOAEL is dependent on the design of the study. Because of the expense and time required conduct many studies, doses are usually spread over wide intervals. Thus, the no-observed-adverse-effect level may be considerably less than a marginally effective dose. In addition, use traditionally has not been made of the dose–response relationship when establishing ADIs. Newer approaches such as the benchmark dose may provide ways of making use of dose–response information. It is unlikely that consumption at the level of the ADI will result in significant risk to the consumer because of the conservatisms that are built into it. It usually is based on long-term studies that are intended to mimic consumption over the lifetime of humans. The ADI is applied to “discretionary” chemicals (food additives, veterinary drugs, and pesticides) by JECFA and JMPR, which are relatively easy to control if safety problems are identified. On the other hand, when tolerable intakes are derived for contaminants that are present in the environment at high levels, the use of standard safety factors could result in discarding large portions of the food supply. Thus, it is very important that the basis for the tolerable intake is fully described so that informed judgements can be made about the health consequences of exceeding it.     

地尔硫卓中间体的合成工艺研究

目的 合成地尔硫卓的重要中间体d-cis -2-(4-甲氧基苯基)-3-羟基-2,3-二氢-1,5-苯并硫氮杂 。 方法 以对甲氧基苯甲醛为原料,经Darzens缩合、硫烷基化、水解、拆分、环合5步反应得到地尔硫卓的重要手性中间体。结果与结论 目标化合物的总收率为34.0 %,本法提高了收率,降低了成本,简化了操作,适于工业化生产。     

LC/MS/MS法测定人血浆中罗红霉素的血药浓度

目的建立LC/MS/MS法测定人血浆中罗红霉素的浓度。方法采用Diamonsil C18色谱柱,流动相甲醇-水-甲酸(75∶25∶0.5),流速为0.5mL·min-1。质谱检测方式:SIM。克拉霉素为内标,血浆样品用乙醚提取浓集,进行LC/MS/MS分析。结果本方法在0.1～40.0μg·mL-1内线性关系良好(r=0.9997),平均回收率87.6%,日内和日间RSD<10%。结论本方法快速、准确,适用于罗红霉素的临床药动学研究和血药浓度检测。     

Effect of camphor/cyclodextrin complexation on the stability of O/W/O multiple emulsions

Camphor (CA) encapsulation in oil/water/oil multiple emulsions prepared with cyclodextrin disturbs the emulsifier potential of alpha- and beta-natural cyclodextrins (CD). It was suggested that the size and geometrical fit between the CD cavity and CA could induce CD/CA complex formation in place of emulsifier formation leading to perturbation of emulsion stability. The complexation between CA and alpha-, beta- or gamma-CD in solution in the presence of oil phase are confirmed by phase-solubility diagrams, circular dichroism and 1H NMR. Furthermore, in order to mimic the emulsion system, CD/CA/soybean oil ternary dispersions were prepared to observe the complexation behavior of alpha-, beta- or gamma-CD/CA by circular dichroism. X-ray diffraction on emulsion samples prepared with alpha- and beta-CD confirms that the precipitates observed in emulsions are probably composed of crystals of CD/CA complexes. A preliminary study of the interaction between drug and CD before the formulation seems indispensable to prevent the risk of incompatibility.     

Involvement of the GABA/benzodiazepine receptor in the axiolytic-like effect of nociceptin/orphanin FQ

We investigated the mechanism underlying the anxiolytic actions of the neuropeptide nociceptin/orphanin FQ (N/OFQ) with an elevated plus-maze test. In mice, intracerebroventricular (i.c.v.) infusions of N/OFQ (0.1 and 0.32 nmol) led to an increase in time spent in the open arms (anxiolytic-like effects). A non-peptidyl N/OFQ receptor (NOP) antagonist, J-113397(1-{(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl}-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), (1.0 and 3.2 mg/kg, s.c.) blocked the increase induced by N/OFQ. On the other hand, a benzodiazepine receptor antagonist, flumazenil, (10 mg/kg, i.p.) and a GABAA receptor antagonist, (+)-bicuculline, (5.6 mg/kg, i.p.) also inhibited the increase induced by N/OFQ. In rats, microinfusions of N/OFQ (10 and 32 pmol) into the amygdala led to an increase in time spent in the open arms. However, intracranial infusions of N/OFQ (10-100 pmol) into the dorsal hippocampus did not affect the time spent in the open arms. These findings suggest that the anxiolytic-like effects of N/OFQ may be related to the GABA/benzodiazepine system in the amygdala.     

Role of α1-and α2-adrenoceptors in the modulation of the baroreflex vagal bradycardia

Yohimbine (100 μg/kg), an α₂-adrenoceptor blocking agent when injected into the vertebral artery of anaesthetized dogs decreased the vagally mediated bradycardia induced by carotid sinus nerve stimulation. Intracisternal administration of phenylephrine (30 μg/kg) an α₁-adrenpceptor agonist decreased, whereas AR-C 239 (5 μg/kg) and prazosin (5 μg/kg) two potent α₁-adrenoceptor antagonists injected into the vertebral artery, potentiated the bradycardic response. These results suggest the presence of two types of α-adrenoceptors to modulate the baroreceptor pathway: α₁-adrenoceptors inhibit and α₂-adrenoceptors facilitate the transmission of baroreceptor impulses.     

Increase in force of contraction by activation of the Na+'ox/Ca2+'ox-exchangerin human myocardium

Aims The aim of the present study was to investigate whether agents which enhance force of contraction via increasing intracellular Na⁺, i.e. cAMP-independently, remain effective in failing human myocardium.Methods Cumulative concentration-response curves with (±)BDF 9148 (0.01–10 μmol l⁻¹ ), a Na⁺-channel activator, and ouabain (0.01–0.1 μmol l⁻¹ ), a Na⁺/K⁺-ATPase inhibitor, were performed on electrically driven left ventricular human papillary muscle strips (1 Hz, 37° C; dilative cardiomyopathy, NYHA IV, heart transplantation, n=16; nonfailing, donor hearts, n=5). The β-adrenoceptor agonist isoprenaline (0.001–1 μmol l⁻¹ ) and Ca²⁺ (1.8–15 mmol l⁻¹ ) were studied for control. In addition, Ca²⁺ response curves were obtained on skinned fibre preparations from left ventricular myocardium (NYHA IV, n=7) in the presence of BDF 9148 (1 μmol l⁻¹ ) or a high Na⁺ concentration (50 mmol l⁻¹ ) to investigate a possible direct or indirect interaction of (±)BDF 9148 with the myofilaments.Results While isoprenaline was significantly less effective in increasing force of contraction in failing human myocardium than in nonfailing myocardium (P<0.01), in NYHA IV, (±)BDF 9148 and ouabain were as effective as in nonfailing human tissue. In failing and nonfailing myocardium, (±)BDF 9148 and ouabain exerted positive inotropic effects similar to those of Ca²⁺. However, the potency for (±)BDF 9148 to increase force of contraction was higher in NYHA IV than in nonfailing human myocardium (P<0.05). Neither (±)BDF 9148 (1 μmol l⁻¹ ) nor an increased concentration of Na⁺ (50 mmol l⁻¹ ) altered the Ca²⁺ sensitivity or maximal developed tension of the contractile apparatus in experiments on chemically skinned left ventricular fibres.Conclusions The enhanced sensitivity of the failing human myocardium towards Na⁺-channel modulation is not due to a direct or indirect interaction of (±)BDF 9148 with cardiac myofilaments but may be due to an altered Na⁺-homeostasis in human heart failure.     

头孢他啶/阿维巴坦耐药肺炎克雷伯菌的体外抗菌药物治疗方案研究

目的 探索对头孢他啶/阿维巴坦耐药的肺炎克雷伯菌体外抗菌药物治疗方案，为临床治疗方案选择提供理论依据。方法 采用PCR对2株肺炎克雷伯菌株可能耐药基因进行鉴定，并对扩增产物进行测序。采用体外时间杀菌试验评估不同抗菌药物单药或联合用药对耐药菌株生长的影响。结果 膜孔蛋白严重缺失和产金属酶分别介导了两菌株对头孢他啶/阿维巴坦耐药；多黏菌素B、亚胺培南、美罗培南和磷霉素单药或两药合并不能显示足够抗菌效果，而多黏菌素B-美罗培南-磷霉素或多黏菌素B-头孢他啶-阿维巴坦三药联合方案可分别对这2株耐药株产生强大持续的杀菌作用。结论 多黏菌素B为基础三药联合可能是对抗具有不同机制介导的头孢他啶/阿维巴坦耐药肺炎克雷伯菌的有效方案。     

N-oxidation of N,N-dimethylaniline in the rabbit and rat lung

We have reported previously that chlorpromazine (CPZ) and imipramine (IMP) are metabolized via N-oxidation by the rat lung, while they are not appreciably metabolized by the rabbit lung. Indeed, marked species differences exist in the pulmonary N-oxidation of these pneumophilic drugs. In the present studies, the isolated, ventilated and perfused lung (IPL) preparations as well as in vitro preparations of the rabbit and rat lungs were used to examine the pulmonary disposition of [¹⁴C]-N,N-dimethylaniline (DMA) which has been used frequently as a substrate for N-oxidation. Although the IPLs of both species were active in DMA N-oxidation, the rabbit lung was more active in DMA N-oxidation than the rat lung on the basis of per g lung. The gradual decline in radiolabel concentration in the perfusate was more marked in the rat than in the rabbit IPL. This decline was not due to the drug accumulation in the lung, but to its volatility. There was no dose dependency in the tissue/medium DMA concentration ratios (approximately 1.60), indicating uptake by simple diffusion and low affinity for the lung tissue. In vitro lung preparations showed higher DMA N-oxidase activity in the rabbit than in the rat, regardless of whether whole homogenate, post-mitochondrial supernatant fraction or microsomal fraction was used, or how the activities were expressed (per mg protein or per g tissue). These results suggest that, although DMA is not highly concentrated in the lung, it is N-oxidized by the lung and that DMA N-oxidase is different from CPZ or IMP N-oxidase reported previously.     

Estimation of the ethanol/water solubility profile from the octanol/water partition coefficient

While the ethanol/water solubility profiles of very polar and very non-polar drugs are monotonic, many semi-polar drugs show a maximum solubility at an ethanol volume fraction (f(max)) between 0 and 1. A sigmoidal relationship was observed between the value of f(max) and the log of the octanol/water partition coefficient (logK(ow)) of the solute. This relationship reasonably predicts the value of the volume fraction of ethanol that gives maximum solubility (f(max)). Combining this sigmoidal relationship with the previously reported linear relationship between the logK(ow) and the initial slope of the plot of log solubility versus ethanol composition [Li, A., Yalkowsky, S.H., 1994. Solubility of organic solutes in ethanol/water mixtures. J. Pharm. Sci. 83, 1735-1740] enables the estimation of the total ethanol/water solubility profile.     

rhBMP-2/hTGF-β1/胶原复合材料的生物相容性研究

目的评价rhBMP-2/hTGF-β1/胶原复合材料的生物相容性。方法通过进行溶血试验、结膜刺激试验以及皮内刺激试验来研究rhBMP-2/hTGF-β1/胶原复合材料的生物相容性。结果此复合材料不引起溶血反应,无结膜刺激性并且对皮肤无刺激作用。结论rhBMP-2/hTGF-β1/胶原复合材料具有良好的生物相容性,可望成为一种良好的骨再生诱导材料,具有很好的临床应用前景,使待进一步深入研究。     

Characterization and clinical evaluation of live influenza a vaccine prepared from a recombinant of the A/USSR/92/77 (H1N1) and the cold-adapted A/Ann Arbor/6/60 (H2N2) strains

Live influenza vaccine was prepared after genetic recombination of the A/USSR/92/77 (H₁N₁) strain with the cold-adapted A/Ann Arbor/6/60 (H₂N₂) strain. The recombinant contains the genes coding for the HA and NA proteins from the A/USSR/92/77 (H₁N₁) strain and the genes coding for the P₁, P₂, P₃, NP, M and NS proteins from the A/Ann Arbor/6/60 (H₂N₂) strain. To assess the properties of this vaccine, it was administered under double-blind conditions to 14 healthy volunteers, while another 14 healthy volunteers received placebo.The vaccine virus appeared to be sufficiently attenuated. No febrile reactions were observed. The vaccinees showed an increase in mean serum haemagglutination-inhibiting antibody level from 19 to 73 after two vaccinations. From nasal swabs and antibody responses, it was concluded that the vaccine virus showed no transmission to the placebo group under conditions of close contact. Also, the vaccine virus was found to be genetically stable.It is concluded that this live influenza virus vaccine meets the requirements for safe use in humans. However, several problems still exist which may impede a general use of live influenza vaccines.     

Evidence for the active renal secretion of s-pentachlorophenyl-N-acetyl-L-cysteine by female rats

Male and female rats received 50 μmoles of pentachloronitrobenzene/kg by oral intubation daily for seven days. The final excreta were hydrolysed and analysed by electron capture GLC for the presence of pentachlorobenzenethiol and tetrachloro-1,4-benzenedithiol (derived from the equivalent N-acetylcysteine conjugates). No differences were found between the sexes for faeces and bile but the urinary excretion of both thiols by females was more than 10-fold greater than males. A similar result for urine was obtained following i.p. administration of a single 20 μmoles/kg dose of S-pentachlorophenyl -N-acetyl-L-cysteine (pentachlorophenyl mercapturate); in addition co-treatment with probenecid did not greatly change excretion by the males but considerably reduced excretion by females. The sex difference in the urinary levels of pentachlorophenyl N-acetylcysteine after 40 and 100 μmoles/kg doses of pentachloronitrobenzene was confirmed by h.p.l.c. of the mercapturate and again probenecid inhibited the excretion. Analysis of urine by TLC following a dose of [¹⁴C]hexachlorobenzene (8 μCi/kg; 0.67 μmoles/kg) showed that more radioactivity was associated with the mercapturate from female rats than males. The results suggest that S-pentachlorophenyl-N-acetyl-L-cysteine, a metabolite of hexachlorobenzene and pentachloronitrobenzene, may be excreted by an active renal secretion which is particularly developed in female F344 rats.     

Quantitative evaluation of the metabolic formation of methylhydrazine from acetaldehyde-N-methyl-N-formylhydrazone,the main poisonous compound of Gyromitra esculenta

The amounts of methylhydrazine in the peritoneal fluids of mice treated orally with acetaldehyde-N-methyl-N-formylhydrazone, the main poisonous compound of the mushroom Gyromitra esculenta Fr. ex. Pers., were determined in order to estimate quantitatively the metabolic formation of methylhydrazine from this compound. For the experiments 2.0 ml of saline was injected into the peritoneal cavities of experimental animals immediately after the dosing. The animals were sacrificed at different time points and their peritoneal cavities flushed with saline. From this fluid methylhydrazine was determined as benzaldehyde-N-methylhydrazone by gas liquid chromatography. For comparison, an identical test procedure was carried out on mice treated orally with methylhydrazine. The results indicate that more than 25%, or a toxicologically significant amount of acetaldehyde-N-methyl-N-formylhydrazone is metabolically hydrolysed to methylhydrazine.     

肾上腺髓质素对肾肿瘤细胞 PI3 K／Akt／eNOS／VEGF信号通路的作用

目的：研究肾上腺髓质素（ADM）对肾肿瘤细胞磷脂酰肌醇-3激酶（PI3K）／蛋白激酶B（Akt）／内皮型一氧化氮合酶（ eNOS）／血管内皮生长因子（ VEGF）信号通路的影响。方法将肾肿瘤细胞分为对照组、ADM组、VEGF组,ADM＋VEGF受体抑制剂组,ADM＋ADM shRNA组,每组设置24 h、48 h、72 h 3个时间梯度,western-blot检测对照组和ADM组细胞总蛋白中PI3K、Akt、eNOS、VEGF表达;检测VEGF组,ADM＋VEGF受体抑制剂组和ADM＋ADM shRNA组细胞总蛋白中PI3K、Akt、eNOS的表达。结果 PI3K、Akt、eNOS、VEGF在ADM组各时间梯度组肾肿瘤细胞中表达明显高于其在对照组细胞的表达（ P ＜0．05）。 PI3K、Akt、eNOS在VEGF组和ADM＋VEGF受体抑制剂组各时间组肾肿瘤细胞中表达明显高于其在对照组细胞中表达（ P ＜0．05）。 PI3K、Akt 、eNOS在ADM＋ADM shRNA组各时间组肾肿瘤细胞中表达与其在对照组细胞中表达差异无统计学意义（ P ＞0．05）。结论 ADM可激活PI3K／Akt／eNOS／VEGF信号通路;VEGF 受体抑制剂不能阻断 ADM 激活 PI3K／Akt／eNOS 信号通路。 ADM 一方面通过VEGF发挥肾肿瘤血管生成作用,另一方面通过其受体激活PI3K／Akt／eNOS信号通路参与肾肿瘤血管生成作用。ADM可能成为抗血管治疗肾肿瘤新的靶点。     

Distinct properties of amlodipine and nicardipine block of the voltage-dependent Ca channels Cav1.2 and Cav2.1 and the mutant channels Cav1.2/Dihydropyridine insensitive and Cav2.1/Dihydropyridine sensitive

The binding site within the L-type Ca²⁺ channel Ca_v1.2 for neutral dihydropyridines is well characterized. However, the contributions of the alkylamino side chains of charged dihydropyridines such as amlodipine and nicardipine to channel block are not clear. We tested the hypothesis that the distinct locations of the charged side chains on amlodipine and nicardipine would confer distinct properties of channel block by these two drugs. Using whole-cell voltage clamp, we investigated block of wild type Ca_v 2.1, wild type Ca_v1.2, and Ca_v1.2/Dihydropyridine insensitive, a mutant channel insensitive to neutral DHPs, by amlodipine and nicardipine. The potency of nicardipine and amlodipine for block of closed (stimulation frequency of 0.05 Hz) Ca_v1.2 channels was not different (IC₅₀ values of 60 nM and 57 nM, respectively), but only nicardipine block was enhanced by increasing the stimulation frequency to 1 Hz. The frequency-dependent block of Ca_v1.2 by nicardipine is the result of a strong interaction of nicardipine with the inactivated state of Ca_v1.2. However, nicardipine block of Ca_v1.2/Dihydropyridine insensitive was much more potent than block by amlodipine (IC₅₀ values of 2.0 μM and 26 μM, respectively). A mutant Ca_v2.1 channel containing the neutral DHP binding site (Ca_v2.1/Dihydropyridine sensitive) was more potently blocked by amlodipine (IC₅₀ = 41 nM) and nicardipine (IC₅₀ = 175 nM) than the parent Ca_v2.1 channel. These data suggest that the alkylamino group of nicardipine and amlodipine project into distinct regions of Ca_v1.2 such that the side chain of nicardipine, but not amlodipine, contributes to the potency of closed-channel block, and confers frequency-dependent block.