Nifedipine调节白皮松(Pinus bungeana)花粉管蛋白质表达及其对细胞壁构建的影响

花粉管是种子植物受精过程中的雄性生殖单位的载体，由于其生长依赖于胞内Ca2+梯度，并具有典型的顶端极性生长的特点，因而成为近年来研究植物细胞相互识别、胞内和胞外信号传导理想的模式系统。裸子植物花粉与被子植物相比具有萌发时间长、生长缓慢等特点。Ca2+在裸子植物花粉萌发和花粉管生长中的作用机制目前尚不明确。本研究以裸子植物白皮松(Pinus bungeana)的花粉为材料，运用不同浓度钙通道抑制剂Nifedipine（Nif）处理，对其花粉萌发和花粉管生长进行了细胞学研究和蛋白质组学分析，以探讨Ca2+对白皮松花粉生长的调控机制，为进一步揭示裸子植物花粉萌发和花粉管生长机理提供参考。 本论文首先研究了钙通道抑制剂Nif和Ca2+螯合剂EGTA对白皮松花粉萌发和花粉管生长的影响。结果表明，用Nif处理花粉后，花粉萌发和花粉管生长均受到明显抑制。经Ca2+荧光探针Fluo-3AM标记，对Nif处理后Ca2+在花粉管中的分布模式进行了观察，发现Ca2+在正常生长的花粉管中呈梯度分布，并在其顶端的荧光最强。与对照相比，处理后的花粉管荧光强度明显减弱，且顶端Ca2+梯度消失。通过EGTA漂洗后的花粉粒，在正常培养基上萌发率较高，但其生长速率受到抑制。由此说明了细胞壁钙库对花粉管生长的抑制效应显著高于对花粉萌发的影响，是花粉管生长的限速因子。同时外加钙调素还可逆转EGTA对其花粉萌发和花粉管生长的抑制。上述结果表明，白皮松花粉萌发及花粉管生长需要外源Ca2+的内流，以及胞内形成的Ca2+浓度梯度。 用FM4-64探针标记结果发现，正常花粉管中的胞吞作用主要发生在顶端和亚顶端区域，胞吞的小泡也集中分布在这两个区域。经Nif处理后，既不影响花粉管的胞吞过程，同时胞吞发生的位置也与对照相似，只是胞吞的小泡分散于整个花粉管中。电子显微镜观察表明，各种细胞器在白皮松正常生长的花粉管中分布与被子植物存在较大差异，例如前者无明显地分区现象，不具胼胝质塞。白皮松花粉管顶端和亚顶端的壁旁体与质膜融合现象频繁发生。花粉管经Nif处理后，线粒体出现不同程度的解体和液泡化，内质网液泡化和核糖体脱落，液泡大量聚集在花粉管顶端，壁旁体与膜融合现象减少，以及花粉管壁明显变薄等。此外，通过微丝特异性探针鬼笔环肽标记结果表明，正常生长花粉管的微丝呈长轴向排列， Nif处理后微丝断裂，其断裂程度与处理浓度有关。由此可见，外源Ca2+对花粉管的胞吞无明显抑制或促进作用，但对胞吞小泡重回收可能有影响。当胞内Ca2+梯度消失后，则明显抑制了微丝骨架的聚合，进而使胞吐作用减缓，高尔基体分泌小泡聚集，多种细胞器液泡化，引起花粉管顶端膨大，细胞壁变薄，继而抑制花粉管的正常生长。 运用免疫荧光标记技术显示，正常生长的花粉管壁含有纤维素、胼胝质、果胶质和阿拉伯半乳聚糖蛋白（AGPs），其中纤维素和胼胝质在细胞壁上呈均匀分布，而酸性果胶质只存在花粉管两侧壁上，酯化果胶分布于花粉管顶端。经过Nif处理后，胼胝质和酸性果胶质均在花粉管顶端累积，而AGPs和纤维素的分布却无明显变化。另外，傅里叶红外光谱分析结果也同样支持上述结论。通过花粉管壁蛋白的SDS-PAGE分析表明，Nif对细胞壁蛋白的合成也有较大的影响。 在花粉管的钙通道受到抑制后，应用蛋白质组学技术分析其蛋白质的表达图谱，通过双向电泳已分离出约1000个蛋白质斑点，经软件分析发现，除其中50个蛋白斑点外，大部分蛋白质的表达量均未发生变化。上述50个发生变化的蛋白斑点酶切后，再经过ESI－MS/MS鉴定和质谱数据库的搜索，共鉴定出28个蛋白，其中12个为上调蛋白，16个下调蛋白，根据其主要功能分为与代谢、细胞扩展、翻译后修饰以及信号相关的蛋白。经过Nif处理后，花粉管中碳水化合物代谢能力下降，ATP的产生受到抑制，参与细胞壁多糖合成及小泡运输的蛋白，如valosin containing protein（VCP）、reversibly glycolsylated polypeptide(RGP)、UDP-glucose dehydrogenase (UDPGDH)和α-tubulin表达下调。另外，通过上述方法还鉴定出与丝氨酸/苏氨酸激酶保守结构域同源的受体蛋白激酶等。 综上所述，白皮松花粉管钙通道受到抑制后，通过影响花粉管蛋白的表达，抑制微丝微管骨架的组装，致使胞吐速度变慢，花粉管壁酸性果胶质、胼胝质等多糖分布的变化及总多糖含量的减少，最终抑制了花粉管的正常生长。

Assessment of the origin and stability of Nifedipine polymorph IV by differential scanning calorimetry (DSC)

Purpose. To examine the thermal transition(s) between different polymorphic forms of Nifedipine and to define experimental conditions that lead to the generation of polymorph IV. Methods. Experiments were performed using a DSC 823e (Mettler Toledo). Nifedipine exists in four polymorphic forms, as well as an amorphous state. Examination of Nifedipine was conducted using the following method(s): cycle 1: 25ºC to 190ºC, 190ºC to 25ºC (formation of amorphous Nifedipine); cycle 2: 25ºC to X (60,70,80...150ºC), X to 25ºC; cycle 3: 25ºC to 190ºC and holding isothermally for 5 min between cycles (heating/cooling rate of 10ºC/min). Results. The amorphous state Nifedipine can sustain heating up to 90ºC without significant changes in its composition. Cycle 2 of amorphous material heated up to 90ºC shows only the glass transition at ~44ºC. In cycle 3 of the same material, a glass transition has been recorded at ~44ºC, followed by two exotherms (~100 and ~115ºC (crystallisation of polymorph III and II, respectively) and an endotherm (169ºC (melting of polymorphs I/II)). Samples that have been heated to temperatures between 100ºC and 120ºC in the second cycle showed a glass transition at ~44ºC and an additional exotherm at ~95ºC (crystallisation of polymorph III) on cooling a exotherm was observed at ~40ºC (crystallisation of polymorph IV). The same material showed no glass transition in cycle 3 but an endotherm at around 62ºC (melting of polymorph IV) an exotherm (~98ºC) and an endotherm (169ºC) melting of polymorph I/II. Heating the sample to a temperatures greater than 130ºC in cycle two results in a glass transition at ~44ºC, and two exotherms at ~102 and 125ºC (crystallisation of polymorphs III and I, respectively). Conclusions. DSC data suggests that polymorph IV can only be produced from amorphous or polymorph III samples. The presence of polymorph I or II drives the conversion of the less stable polymorphic form IV into the most stable form, I. Although form IV of Nifedipine can easily be created, following defined experimental conditions, it may only coexist with amorphous or polymorph III states. When polymorphs I and II are present in the sample polymorph IV cannot be etected.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing <10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.

A double-blind cross-over study of nifedipine retard in patients with Raynaud's phenomenon

The effectiveness of nifedipine retard as a treatment for Raynaud's phenomenon was assessed in 15 patients in a placebo controlled double blind study. An associated connective tissue disease was evident in 7 patients. Changes in finger and forearm blood flow (venous occlusion plethysmography), digital skin temperature and digital systolic pressure were measured acutely before and after a 2-week treatment period. Subjective assessment of efficacy was based on patient diary data. In addition alpha 2-adrenoceptor density on platelets was measured before and after chronic nifedipine therapy in both the patient group and in an age-and-sex-matched control group. No significant haemodynamic changes were observed. Nifedipine retard significantly reduced the frequency (p less than 0.05) with no change in either the duration or severity of vasospastic attacks. Side effects were common following nifedipine retard. A reduction in alpha 2-adrenoceptor density on platelets was observed in patients compared to a control group (p less than 0.05). Alpha 2-adrenoceptor density was unchanged following a 2-week treatment period with nifedipine retard. This study concludes that nifedipine retard is not effective in the treatment of Raynaud's phenomenon over a short time course. Patients with Raynaud's phenomenon have reduced alpha 2-adrenoceptor densities on their platelets.

Nifedipine-induced gingival hyperplasia

This case study reports on the management of a woman suffering from thrombocytopenic purpura who was referred to the periodontal clinic with gingival swelling and bleeding. This condition was related to nifedipine therapy for hypertension.

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): Histochemical and MMP-1 and -2 and collagen I gene expression analyses

Background: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels.Methods: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies.Results: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group.Conclusion: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

ESTUDO COMPARATIVO DUPLO-CEGO DA EFICACIA E SEGURANCA DO CILAZAPRIL COMPARADAS A NIFEDIPINE 'RETARD' NO TRATAMENTO DA HIPERTENSAO ARTERIAL LEVE E MODERADA

Purpose: To evaluate the antihypertensive efficacy and safety of cilazapril compared to nifedipine retard in mild to moderate hypertension. Methods: Forty randomized out-patients with mild moderate hypertension, diastolic pressure (DP) between 95 and 115 mmg/Hg, with placebo for 15 days were randomized and allocated for treatment, double-blind, once daily with cilazapril 2.5 mg (n = 20) or nifedipine retard 20 mg (20 = n) for four weeks. The non-responders (DP > 90 mmHg) had the dosage increased twice, b.i.d., while responders were maintained up to 10 weeks. Clinical visits were performed before, at baseline and every two weeks and the laboratory test was performed after placebo run-in, 4th and 10th weeks of treatment. Results: The blood pressure (BP) were similar between groups at the end of the placebo (cilazapril 151 ± 14/103 ± 5 - nifedipine 157 ± 17/108 ± 7 mmHg, p > 0.05). DP decreased already at second weeks (cilazapril 95 ± 9 - nifedipine 96 ± 11 mmHg, p < 0.05, compared to week 0) in both groups at the end of study with no differences inter groups. BP normalization was obtained in 58% of the patients with cilazapril and in 61% in the nifedipine group. Adverse biochemical effects were not observed in any group. Six (16%) patients of the cilazapril and 15 (39%) of nifedipine related collateral events, although no difference were observed between groups. Conclusion: Cilazapril 2.5 to 5 mg normalized BP in 58% of mild and moderate hypertension patients, and this efficacy was similar to sustained-release nifedipine 20 to 40 mg. Cilazapril had no adverse effects on the biochemical parameters with low incidence of collateral effects.

Morphological evaluation of combined effects of cyclosporin and nifedipine on gingival overgrowth in rats

It has previously been shown that, while cyclosporin A (CsA) and nifedipine both cause gingival overgrowth in the rat. the combined use of these drugs increases the severity of overgrowth. The aim of this study was to describe the histometry and densities of fibroblasts, collagen fibers and vessels in the gingival tissue of rats that were treated with CsA and nifedipine, either alone or in combination. Rats were treated for 60 days with a daily subcutaneous injection of 10 mg/kg body weight of CsA and/or with 50 mg/kg body weight of nifedipine added to the chow. The results confirmed that CsA causes a more severe overgrowth than nifedipine, and that the combined use of these drugs increases the overgrowth severity. All the rat groups that were studied showed that, as the severity of overgrowth increased, there was a parallel increase in fibroblasts and collagen, and a decrease in vessel content. Therefore, independently of whether the gingival overgrowth was caused by CsA alone, nifedipine alone, or both treatments in combination, the fibroblast and collagen density increased in parallel with the severity of the overgrowth. © Blackwell Munksgaard, 2002.

Combined effects of cyclosporin and nifedipine on gingival overgrowth in rats is not age dependent

Background: Cyclosporin A and nifedipine cause gingival overgrowth in rat, and the combined use of these drugs increases the overgrowth severity. Objective: The purpose of this study was to compare gingival overgrowth of rats of differents ages treated with cyclosporin A and nifedipine alone or given concurrently. Materials and methods: Rats 15, 30, 60 and 90 d old were treated with 10 mg/kg body weight of cyclosporin A and/or 50 mg/kg body weight of nifedipine in the chow. Results: Young rats showed evident gingival overgrowth with nifedipine, cyclosporin A, and cyclosporin A and nifedipine given concurrently. Adult rats did not show significant gingival alterations when treated with cyclosporin A and nifedipine alone. Nevertheless evident gingival overgrowth with alterations of the epithelium and connective tissue were observed when treated simultaneously with cyclosporin A and nifedipine. Conclusion: These results suggest that the combined effects of cyclosporin A and nifedipine on gingival overgrowth in rat is not age dependent.

Influence of age on combined effects of cyclosporin and nifedipine on rat alveolar bone

Background: There is some evidence showing that cyclosporin A (CsA) and nifedipine (NIF) affect bone metabolism. The purpose of this work was to study the effects of CsA and NIF, given alone or concurrently, on alveolar bone of rats of different ages. Methods: Rats 15, 30, 60, and 90 days old were treated daily with 10 mg/kg body weight of CsA subcutaneously injected and/or 50 mg/kg body weight of NIF/day given orally for 60 days. Alveolar bone of the first lower molars was morphologically and stereologically evaluated in serial 5 μm bucco-lingual paraffin sections, stained with hematoxylin and eosin. Serum calcium and alkaline phosphatase levels were measured in all animals at the end of the experimental period. Results: Rats treated with CsA or NIF alone or CsA and NIF concurrently showed decreased alveolar bone density. CsA was more effective than NIF. A significant decrease in serum calcium was found only in animals treated with CsA or CsA/NIF. The results were similar regardless of age. Conclusions: These results indicate that the decrease in the alveolar bone volume in rats caused by CsA and NIF alone or concurrently is not age dependent. Furthermore, NIF (50 mg/kg) did not further increase the loss of alveolar bone volume induced by CsA (10 mg/kg).