栉孔扇贝血细胞的形态和功能研究

栉孔扇贝Chlamys farreri血细胞可分为三大类:非颗粒细胞(约34.75%)、颗粒细胞(约61.25%)和桑葚胚样细胞.非颗粒细胞可进一步分为透明细胞和成血细胞;颗粒细胞可分为嗜中性、嗜碱性和嗜酸性颗粒细胞.颗粒细胞能够吞噬酵母菌,其中嗜中性颗粒细胞吞噬能力最强.     

海湾扇贝血细胞的表面结构及超微结构

通过光镜、扫描电镜和透射电镜的观察对海湾扇贝血细胞的形态、表面结构及超微结构进行了研究。根据细胞的大小、形态结构可将血细胞分成四种类型:Ⅰ型小透明细胞,大小约(2·38±0·08)μm,约占比例30%-35%;Ⅰ型大透明细胞,大小约(4·41±0·33)μm,约占比例15%-25%;Ⅱ型小颗粒细胞,大小约(4·15±0·26)μm,约占比例20%-25%;Ⅱ型大颗粒细胞,大小约(8·26±0·52)μm,约占比例25%-30%。血细胞在血淋巴中的平均密度为(3·75±0·65)×107cell/ml。其中Ⅰ型透明细胞占55·3%,Ⅱ型颗粒细胞占44·7%。表面结构观察结果显示有5种形态:圆形血细胞,梨形或梭形血细胞,松果形血细胞,阿米巴样细胞,大型细胞,表面结构与功能密切相关。透射电镜观察结果表明血细胞主要归属于两大类型:Ⅰ型透明细胞和Ⅱ型颗粒细胞。超微结构显示颗粒细胞的细胞质颗粒可区分成三种类型:Ⅰ型高电子密度颗粒,Ⅱ型低电子密度颗粒和Ⅲ型中等电子密度颗粒,并推测Ⅰ型高电子密度颗粒是细胞吞噬的异物(微生物等)或细胞内的废物(沉积颗粒,衰老的胞器或碎片);Ⅱ型低电子密度颗粒是溶酶体类的胞内分泌颗粒,来源于高尔基复合体或内质网;Ⅲ型中等电子密度颗粒可能是次级溶酶体,由Ⅰ型颗粒向Ⅱ型颗粒融合并注入裂解酶类而形成[动物学报51(3):486-494,2005]。     

贝类血细胞及其免疫功能研究进展

贝类由于是开放性系统,不存在特异性体液免疫,贝类的宿主免疫防御包括以血细胞为基础的细胞和体液系统。血细胞能够在体内或体外吞噬各种有机和无机颗粒,清除病原生物和自身损伤或死亡细胞,而且血细胞能够产生各种非特异性体液因子来参与宿主的免疫防御过程。所以贝类的血细胞在宿主免疫防御机制中起着重要作用。近年来,随着贝类养殖规模的扩大,贝类病害日益严重,给中国的贝类养殖业造成了重大经济损失[1,2]。所以研究贝类免疫学,特别是贝类血细胞的免疫防御功能和机制,以解决贝类养殖中发生的病害问题已成为研究的热点。本文依据国内外有关文献,就贝类血细胞的分类、结构及免疫功能方面的研究作一综述,为国内外研究提供理论基础及资料。1贝类血细胞结构、分类贝类血细胞的分类一直分歧很大,有人将其分为三类:无颗粒细胞、小颗粒细胞、大颗粒细胞,也有人将其分为四类:大颗粒细胞、小颗粒细胞、透明细胞、浆细胞,还有人将其分为八类,甚至几十类,至今无统一命名[3]。关于血细胞的分类,Moore等[4]根据血细胞超微结构和对染色反应的不同,将紫贻贝(Mytilus galloprovincialis)血细胞分为两大类,无颗粒的嗜碱性细胞和有颗粒的嗜酸性颗粒细胞...     

不同壳色福寿螺血细胞的比较研究

为探究不同壳色福寿螺在免疫学上的差异,通过瑞士染色法,比较了不同壳色螺的血细胞分类、血细胞形态、数目和比例的差异。结果表明:不同壳色福寿螺的血细胞均可分为透明细胞和颗粒细胞,根据其嗜性又将颗粒细胞分为嗜酸性和嗜碱性颗粒细胞。透明细胞多呈圆形或椭圆形,且黑壳福寿螺的细胞核及细胞形态较黄壳福寿螺更为多样;黑壳和黄壳福寿螺的血细胞总数(THC)分别为(1.31±0.29)×106个/mL和(1.37±0.28)×106个/mL,统计学上无显著性差异(P>0.05);血细胞的比例大小均为嗜碱性颗粒细胞>透明细胞>嗜酸性颗粒细胞;但黑壳福寿螺的嗜酸性颗粒细胞比例显著比黄壳福寿螺低3.08%(P<0.05);由此可以推测,黑壳福寿螺较黄壳福寿螺分布数量差异的免疫学上的原因之一,可能是由其血细胞核形态及比例的差异造成其免疫力强弱和存活能力的差异引起的。     

池蝶蚌(贝)血细胞显微观察

本文对池蝶蚌血细胞的形态结构及其动态变化进行了研究。根据血细胞形态、大小和密度等特征,把血细胞分为六类：颗粒细胞、透明细胞、浆液细胞、类淋巴细胞、梭形细胞和血栓细胞;并对各种血细胞显微结构予以描述,统计了血细胞胞体大小、细胞核大小、核质比、密度及所占比例,其中颗粒细胞所占比例最大,透明细胞次之,类淋巴细胞最少,颗粒细胞和透明细胞是两种主要的细胞类型,约占总数的82%,担负着最基本的代谢和免疫功能;通过对池蝶蚌血细胞形态的连续观察,发现血细胞存在形态变化现象,推测细胞间可能存在相互转化的情况。     

长毛对虾血细胞与血淋巴液的生化特性初步研究

长毛对虾(Fenneropenaeus penicillatus)是我国东南沿海一带一种主要的水产经济动物.为了解其血淋巴液的基本特性,借助显微观察、生化实验等方法对长毛对虾的血细胞和血浆的细胞化学特征进行了初步研究.结果表明,长毛对虾血细胞密度为(0.43±0.11)×107个/ml;可明显分无颗粒细胞:长径(9.7±1.4)μm、短径(5.8±0.8)μm,占细胞总数的16.2%±1.8%;大颗粒细胞:长径(11.3±1.3)μm、短径(6.7±1.7)μm,占细胞总数的46.6%±3.2%;小颗粒细胞:长径(7.1±0.4)μm、短径(5.4±0.4)μm,占细胞总数的35.7%±4.5%.在16～18℃下,60 min的反应时间内,血细胞对金黄色葡萄球菌(Staphylococcusaureus)菌体细胞悬液(106～107个/ml)的吞噬率为67%±5%.长毛对虾血浆蛋白浓度为11.78 mg/ml,对3%的鸡血红细胞悬液的溶血相对活性为2.6,对5%的鸡血红细胞悬液的凝集效价为15～18.血浆平板抑菌实验表明,长毛对虾血浆对金黄色葡萄球菌有明显的抗菌效果,抑菌圈大小为(10±1)mm,对大肠杆菌(Escherichia coli)的抑菌圈大小为(6±2)mm.本工作为长毛对虾血浆中诸多活性多肽的后续研究奠定了基础.     

罗氏沼虾与克氏原螯虾血细胞的比较研究

对罗氏沼虾与克氏原螫虾血细胞的分类与组成进行了染色观察比较研究。根据染色后光镜下血细胞的核质比、颗粒的大小和数量等来对血细胞进行分类,罗氏沼虾与克氏原螯虾的血细胞均可分为透明细胞、半颗粒细胞和颗粒细胞三类;其血细胞浓度分别为1.02±0.21×10⁷ Ind·ml^-1和0.85±0.15×10⁷Ind·ml^-1。2种虾血细胞的颗粒形态存在显著差异;透明细胞、半颗粒细胞和颗粒细胞占血细胞总量的百分比在罗氏沼虾为21.3±6.3%,45.7±2.5%,33.0±6.8%:在克氏原螯虾为12.0±5.8%、49.5±5.1%和38.5±9.5%。     

栉孔扇贝血液细胞的免疫功能

利用光镜、扫描电镜和透射电镜技术对栉孔扇贝(Chlamysferreri)血细胞与细胞免疫功能相关的几个因素进行了初步研究。对血细胞的数量和不同功能细胞的比例研究结果表明,健康血淋巴中血细胞的平均密度为3.03±0.11×107cell/ml,其中颗粒细胞占42.6%,透明细胞占57.4%;病贝血淋巴中血细胞的平均密度为2.78±0.34×107cell/ml,其中颗粒细胞占40.2%,透明细胞占59.8%。扫描电镜观察表明,血细胞的表面结构主要有表面光滑型,表面松果型和表面褶皱阿米巴型3类。透射电镜观察表明,颗粒细胞吞噬外源性颗粒(Ⅰ型颗粒)通过溶酶体(Ⅱ型颗粒)进行降解。并观察到同心片层结构出现在吞噬泡的降解过程中。利用APIZYM试剂盒对栉孔扇贝血细胞及血清中的19种酶进行检测,结果在血清中检测到了13种酶,在血细胞中检测到10种酶,健康血淋巴中酶的含量高于病贝。对血细胞吞噬活性的研究结果表明,血细胞对大肠杆菌和对类立克次体(RLO)的吞噬率分别为25.4%和21.7%。颗粒细胞的吞噬活性(30%-40%)远远大于透明细胞(4.8%-14%)。环境胁迫对血细胞吞噬活性的影响的研究结果表明,病原菌感染和温度、盐度等环境胁迫因素对血细胞的吞噬活性均有不同程度的影响,其中高温因素影响较大,但未发现贝龄有显著影响     

中华绒螯蟹血细胞的显微、亚显微形态结构及其分类

通过相差显微镜和电镜观察,根据中华绒螯蟹血细胞胞质内有无颗粒以及颗粒大小、染色反应、折光性和形成方式的特点,血细胞分为胞质内无颗粒的无颗粒细胞、胞质内只有具折光性和呈淡红色反应大颗粒的大颗粒细胞、胞质内只有无折光性和呈淡蓝色染色反应小颗粒的小颗粒细胞以及胞质内同时具有大颗粒和小颗粒二种颗粒特性的大小颗粒中间型细胞.小颗粒的形成方式是高尔基体成熟面小泡脱离后直接成为小颗粒,而大颗粒的形成方式是高尔基体成熟面小泡脱离后,数个小泡逐渐聚集成蜂窝状大颗粒,进一步发育成熟为均质大颗粒.实验结果表明:三种有颗粒的细胞是互相独立的,可能分别由无颗粒细胞分化而成.       

黑水虻幼虫血细胞类型的研究

黑水虻Hermetia illucens(L.)是一种重要的资源昆虫。本文旨在筛选出适合黑水虻血细胞观察的染色方法,明确黑水虻血细胞类型、数量及组成,为黑水虻血细胞免疫研究奠定基础。采用Giemsa和Giemsa-Wright's染色方法和血球计数板法,对黑水虻血细胞染色方法和血细胞数量及形态进行研究。结果表明,甲醇固定4 min,Giemsa-Wright's染液染色9 min、pH 7.2磷酸盐缓冲液分色10 min是黑水虻幼虫血细胞最佳染色方法;黑水虻幼虫血细胞包括原血细胞、浆血细胞、粒血细胞、类绛血细胞、珠血细胞5类;4龄黑水虻幼虫血细胞数量大约为2917个/μL,其中浆血细胞占53.20%±2.78%,粒血细胞占37.49%±3.96%,原血细胞占7.97%±1.51%,类绛血细胞占1.02%±0.24%,珠血细胞占0.62%±0.08%。Giemsa-Wright's染色法为黑水虻幼虫血细胞最佳染色方法,黑水虻幼虫血细胞可分为5类10种。     

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

贝类血细胞硫代黄素T荧光染色方法

硫代黄素T( thioflavin T,TFT)是一种用于组织学的苯并噻唑荧光染料,因其对淀粉样蛋白有高亲和性而主要被用于淀粉样病变的荧光显微检测.本研究分别以软体动物门双壳纲的栉孔扇贝(Chlamys farreri)和中国蛤蜊(Mactra chinensis)、腹足纲的拟紫口玉螺(Natica janthosto...     

不同地理种群两针松光合和生长特性的差异

为认识两针松中的赤松（Pinus densiflora）、长白松（Pinus sylvestris var. sylvestriformis）和樟子松（Pinus sylvestris var. mongolica）光合作用对环境变化的响应和适应特征,在其自然分布区内选择地理和气候差异显著的9个地理种群,采集成熟种子并播种于东北林业大学温室,2 a后,测定针叶的光合能力及其相关因子,并同时测定幼苗的株高和基径,比较种间和地理种群间差异。结果表明：赤松、长白松和樟子松种间最大光合速率（p=0.34）、呼吸速率（p=0.15）和表观量子效率（p=0.18）的差异均不显著;地理种群间表观量子效率（AQY）差异显著(p=0.08),其中兴凯湖种群表观量子效率最高,为0.084 5±0.002 4 mol CO₂·mol^-1 photons,较其他种群高13.10%~159.23%。地理种群间呼吸速率(R_d)差异显著(p=0.01),黑河和兴凯湖种群的呼吸速率最高（分别为1.62±0.18 μmol CO₂·m^-2·s^-1,1.52±0.30 μmol CO₂·m^-2·s^-1）,安图和东宁种群的呼吸速率最低,分别为0.40±0.01 μmol CO₂·m^-2·s^-1,0.34±0.03 μmol CO₂·m^-2·s^-1。地理种群间最大净光合速率(P_max)差异显著(p=0.02),其中兴凯湖、东宁、韩国、鸡东、二道白河、红花尔基种群的最大光合速率差异不显著,均值为18.36±1.81 μmol CO₂·m^-2·s^-1,高于安图、漠河、黑河种群。安图、漠河、黑河种群间最大光合速率差异不显著,均值为12.57±0.86 μmol CO₂·m^-2·s^-1。地理种群间的株高和基径差异均显著,其中韩国种群株高最高,黑河种群最低;基径兴凯湖种群最高,安图种群最低。株高和基径最大值约为最小值的3倍。两针松针叶的光合能力及其一些相关因子的地理种群间差异可能是其光合机构对种源地环境条件长期生理适应的结果。     

Excitable Active Electrogenic Ion-Transport Units in the Plasmalemma of Nitellopsis obtusa

With the use of voltage clamp and current clamp techniques thesupposition was proved that during the hyperpolarizing response(HR) N. obtusa cells generate active electromotive force (emf)at the expense of metabolic energy. Threshold inward currentsent through the plasmalemma of the cell which was depolarizedwith 100 mol m^–3 KG resulted in the HR with the transferof the membrane's excitable units from the high-conductive stateto the low-conductive state. During the HR the membrane potentialV_m increased from –135±10 mV to –290±15mV, the membrane resistance increased from 3.3±1.5 kOhmcm² to 5.8±1.2 kOhm cm² and the membrane emf E_m increasedfrom –20±4 mV to –93± 15 mV. Changesin the external concentration of K^–, Na⁺, Cl^– andH^– did not affect the patterns of HR. Cells which weredepolarized by light also generated HR (in normal medium) whichwas accompanied with the increase of V_m, R_m and E_m. The highvalue of Em generated during the HR can be explained only withthe involvement of active electrogenic charge transfer acrossthe membrane. 0.05 mol m^–3 DCCD added to the externalmedium inhibited the HR in both cases. Key words: Active ion transport, Hyperpolarizing response, Nitellopsis obtusa     

Open-vacuole method for measuring membrane potential and membrane resistance of Characeae cells

A simple method called the open-vacuole (o.v.) method was developedto measure the vacuolar potential (E_vo) and membrane resistance(R_m) of Characeae cells without inserting a microelectrode intothe vacuole. Values of E_vo and R_m measured by this method arehigher than those measured by the microelectrode method. Usingthe o.v. method we can measure E_vo and R_m exactly in cells withinternal media of extremely low ionic concentrations. In respect to E_vo and R_m, the tonoplast is less sensitive thanthe plasmalemma to a change in ionic concentration. The existenceof a significant amount of E_vo (–40 mV), even when boththe internal and external media are isotonic artificial pondwater with a high Ca^2$content, may be accounted for by the differencein sensitivity to ionic species between these two membranes. Irrespective of the presence or absence of Ca^2$in the vacuole,practically the same values of E_vo and R_m were measured withthe open-vacuole method, when measurements were carried outwithin 20 min after the end of perfusion. The discrepanciesbetween the present and previous results (16) may be accountedfor by the difference in methods. ¹Present address: Sanki Engineering Ltd., Nagaokakyo, Kyoto. (Received January 31, 1975; )     

Estrogen increases the permeability of the cultured human cervical epithelium by modulating cell deformability

Estrogens increase secretion of cervical mucusin females. The objective of this research was to study the mechanismsof estrogen action. The experimental models were human CaSki(endocervical) and hECE (ectocervical) epithelial cells cultured onfilters. Incubation in steroid-free medium increased transepithelialelectrical resistance(R_TE) anddecreased epithelial permeability to the cell-impermeant acid pyranine.Estrogen treatment reversed the effects, indicating estrogen decreasesepithelial paracellular resistance. The estrogen effect was time anddose related (EC₅₀ ~1 nM) andspecific (estradiol = diethylstilbestrol > estrone, estriol; noeffect by progesterone, testosterone, or cortisol) and was blocked byprogesterone, tamoxifen, and ICI-182780 (an estrogen receptorantagonist). Estrogen treatment did not modulate dilution potential orchanges in R_TE inresponse to diC8 or to low extracellularCa²⁺ (modulators of tightjunctional resistance). In contrast, estrogen augmented decreases inR_TE in responseto hydrostatic and hypertonic gradients [modulators of resistanceof lateral intercellular space (R_LIS)],suggesting estrogen decreasesR_LIS. Estrogendecreased cervical cell size, shortened response time relative tochanges in cell size after hypertonic challenge, and augmented thedecrease in cell size in response to hypertonic and hydrostaticgradients. Lowering luminal NaCl had no significant effect onR_TE, and the Cl channel blockerdiphenylamine-2-carboxylate attenuated the hypertonicity-induced decrease in cell size to the same degree in control andestrogen-treated cells, suggesting estrogen effects on permeability andcell size are not mediated by modulatingNa⁺ orCl transport. In contrast,estrogen increased cellular G-actin levels, suggesting estrogens shiftactin steady-state toward G-actin and the cervical cell cytoskeletontoward a more flexible structure. We suggest that the mechanism bywhich estrogens decreaseR_LIS and increasepermeability is by fragmenting the cytoskeleton and facilitatingdeformability and decreases in cervical cell size.

     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations

Effects of basolateral monovalent cation replacements(Na⁺ byLi⁺,K⁺,Cs⁺, methylammonium, andguanidinium) on permeability to⁸⁶Rb of volume-sensitive cationchannels (VSCC) in the basolateral membrane and on regulatory volumedecrease (RVD), elicited by a hyposmotic shock, were studied in A6epithelia in the absence of apicalNa⁺ uptake. A complete and quickRVD occurred only when the cells were perfused withNa⁺ orLi⁺ saline. With both cations,hypotonicity increased basolateral ⁸⁶Rb release(R^bl_Rb), which reached a maximum after 15 min and declined back to control level. When the major cation wasK⁺,Cs⁺, methylammonium, orguanidinium, the RVD was abolished. Methylammonium induced a biphasictime course of cell thickness(T_c), with an initial decline ofT_c followed by a gradual increase.With K⁺,Cs⁺, or guanidinium,T_c increased monotonously afterthe rapid initial rise evoked by the hypotonic challenge. In thepresence of K⁺,Cs⁺, or methylammonium,R^bl_Rb remained high during most of thehypotonic period, whereas with guanidinium blockage of R^bl_Rb was initiated after 6 min ofhypotonicity, suggesting an intracellular location of the site ofaction. With all cations, 0.5 mM basolateralGd³⁺ completely blocked RVD andfully abolished the R^bl_Rb increaseinduced by the hypotonic shock. The lanthanide also blocked theadditional volume increase induced byCs⁺,K⁺, guanidinium, ormethylammonium. When pH was lowered from 7.4 to 6.0, RVD andR^bl_Rb were markedly inhibited. This studydemonstrates that the VSCCs in the basolateral membrane of A6 cells arepermeable to K⁺,Rb⁺,Cs⁺, methylammonium, andguanidinium, whereas a marked inhibitory effect is exerted byGd³⁺, protons, and possiblyintracellular guanidinium.

     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.Key words: Carcinus aestuarii, crab, haemocytes, light microscopy, cytochemical assays, morphological characterisation.