AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

GnRH相关肽在大鼠垂体前叶的细胞学定位

本研究应用特异性抗GnRH相关肽(GAP)N端11个氨基酸的抗血清和六种垂体前叶激素的抗血清,通过免疫组织化学双重染色技术观察GAP在大鼠垂体前叶细胞的定位。结果发现,GAP样免疫反应性物质存在于LH细胞和FSH细胞,而未见于GH、PRL、TSH和ACTH细胞。本文首次证明GAP存在于正常大鼠垂体促性腺激素细胞,为GAP调节LH和FSH的分泌提供了形态学证据;也支持GAP的功能序列在其分子的N端,或GAP进一步裂解出N端片段而发挥作用。     

THE ENDOCRINE CELLS IN THE EPITHELIUM OF THE GASTROINTESTINAL MUCOSA OF THE RAT : An Electron Microscope Study

The authors of this study examine the question of whether the so-called enterochromaffin or argentaffin cells of the gastrointestinal tract should be considered as a single cell type. The systematic application of purely morphologic methods has led to the conclusion that the epithelium of the gastrointestinal mucosa comprises endocrine cells of several types. This conclusion is primarily based on the uneven and characteristic distribution of the various cell types along the intestinal tract, an observation precluding the interpretation that the different types correspond to diverse functional stages of the same cell. A specific endocrine function may be attributed to each of the given cell types recognized so far on account of their appearance and their localization in characteristic areas of the gastrointestinal tract. It is acknowledged, however, that a purely morphological study leaves room for doubt. The first cell type is probably responsible for the formation of 5-hydroxytryptamine. Cells of type II are morphologically comparable to the pancreatic A cells and may, therefore, be called intestinal A cells. Cell type III comprises intestinal D cells since their appearance corresponds to that of pancreatic D cells. Cell type IV might well be responsible for catecholamine production, whereas gastrin is in all probability produced in endocrine cell type V. As yet, the thorough morphological study of the gastrointestinal epithelium does not provide information as to additional distinct cellular sites of production of the several other hormones isolated from different parts of the gut.     

大鼠垂体前叶中的TH-,ChAT-免疫阳性神经纤维

长期以来,人们一直认为垂体前叶腺细胞没有直接神经支配,前叶内只有自主神经纤维支配垂体前叶的血管。本文在大鼠垂体的相邻切片上,应用免疫组织化学技术,对前叶中的儿茶酚胺的特征性酶——酪氨酸羟化酶（ＴＨ）和乙酰胆碱的特征性酶——胆碱乙酰转移酶（ＣｈＡＴ）进行显色。结果显示,大鼠垂体前叶中存在着ＴＨ－和ＣｈＡＴ－免疫阳性神经终末,两者可同时分布于垂体前叶的同一区域,且有较多终末分布于腺细胞周围。本文提示：儿茶酚胺和胆碱能神经纤维有可能直接调节腺细胞的活动     

抗孕53影响大鼠垂体前叶对GnRH的敏感性反应

本实验应用动情前期大鼠垂体前叶组织块离体培养方法,观察了 A 环失碳类甾体化合物—抗孕53对 LH 基础分泌和动员性分泌的影响。实验分为对照组、GnRH 组、抗孕53组、GnRH-抗孕53组。结果表明,GnRH 可显著促进 LH 分泌并产生自激作用。GnRH 的第一次作用后,LH 分泌增加量由对照组的0.7ng/ml 增加到4.3ng/ml,而当 GnRH 第二次作用后,这一效应显著增强,由对照组的0.5ng/ml 增加到6.8ng/ml,GnRH 的两次作用效应相比,差异极显著。抗孕53可部分抑制垂体对 GnRH 的敏感性反应。抗孕53作用后,LH 分泌增加量由 GnRH 第一次作用后的4.3减至2.5ng/ml,由 GnRH 第二次作用后的6.8降至4.1ng/ml。抗孕53不影响 LH 的基础分泌,与对照组相比,两组的 LH 分泌增加量无显著差异。抗孕53对垂体的这一作用可能是通过直接影响促性腺激素细胞的代谢及调节而实现。     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

THE NATURE OF HYPOTHALAMO-NEUROHYPOPHYSEAL NEUROSECRETION IN THE RAT : A Study by Light- and Electron Microscope Autoradiography

The nature of hypothalamo-neurohypophyseal neurosecretion was examined in the rat by means of intraventricular injections of tritiated amino acids. Quantitation of autoradiographs was used at the light microscope level to study the sites of synthesis of proteins and their time of arrival in the neural lobe. Electron microscope autoradiographs were used to study the labeling of neural lobe tissue. It was concluded that the great majority of the labeled material was translocated inside dense-cored granules and was probably composed mostly of neurophysins. The effect of ether anesthesia was also examined. It was found to remove the dense cores from about 20% of the granules in the neural lobe tissue, a process accompanied by the loss of most of their labeled material. The mechanism of the ether effect is discussed and compared to the normal secretion process.     

INTESTINAL TRIGLYCERIDE ABSORPTION IN THE RAT : An Electron Microscopical Study

This report provides information on the morphology of fat absorption in rat intestinal epithelial cells. Three types of experiments were performed: (a) intubation of corn oil into fasted rats, (b) injection of physiological fatty-chyme prepared from fat-fed donor rats into ligated segments of jejunum of fasted animals, and (c) administration of electron-opaque particles in corn oil and markers given concurrently with the fat. These results support the hypothesis that fat is absorbed by selective diffusion of monoglycerides and fatty acids from micelles rather than by pinocytosis of unhydrolized triglycerides. Evidence is presented that the pits between the microvilli, previously believed to function in the transport of fat, are not involved in this process. Instead they appear to contribute their contents to lysosomes in the apical cytoplasm. Arguments are offered that the monoglycerides and fatty acids diffuse from the micelle while the latter is associated with the microvillous membrane of the absorptive cell. These micellar components penetrate the plasma membrane and diffuse into the cytoplasmic matrix where they encounter the SER. Triglyceride synthesis occurs in the SER and results in the deposition of fat droplets within its lumina. The synthesis of triglycerides and their sequestration into the SER establishes an inward diffusion gradient of monoglycerides and fatty acids.     

白细胞介素—2对大鼠离体垂体前叶细胞增殖的影响

本工作采用大鼠垂体前叶（ＡＰ）细胞原代培养方法,以＾３Ｈ－ＴｄＲ掺入率反映细胞增殖水平,研究了ＩＬ－２对ＡＰ细胞增殖的影响。结果表明：（１）ＩＬ－２（１０￣５００Ｕ／ｍｌ）明显促进性大鼠包括妊娠大鼠的ＡＰ细胞的增殖,而抑制雄性大鼠ＡＰ细胞的增殖。（２）雄性大鼠行卵巢切除（ＯＶＸ）二周后,ＩＬ－２对其ＡＰ细胞增殖的影响反转为抑制效应;若在卵巢切除二周内,每日给予ＯＶＸ大鼠皮下注射５μｇ苯甲酸雌二醇,     

PROTEIN SYNTHESIS IN SYNAPTOSOMAL FRACTIONS : Ultrastructural Radioautographic Study

A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30–50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75–77% of the total processes. 34–45% of the grains were related to processes containing ribosomes which accounted for only 4–7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.     

荷包牡丹碱对垂体前叶组织块释放促肾上腺皮质激素的刺激效应

本实验利用垂体组织块离体灌流技术,观察到－氨基丁酸Ａ受体拮抗剂荷包牡丹碱对切除双侧肾上腺９６ｈ后的大鼠垂体前叶ＡＣＴＨ的分泌具有强烈的刺激作用。但同样浓度的荷包牡丹碱对分离的垂体前叶细胞的ＡＣＴＨ分泌无影响。提示肾上腺切除后,－氨基丁酸在垂体前叶直接或通过间接途径抑制ＡＣＴＨ分泌。     

THE DIFFERENTIATION OF WHITE ADIPOSE CELLS : An Electron Microscope Study

Differentiating white adipose tissue from presumptive and developing fat pads of newborn and young rats was fixed in buffered osmium tetroxide, embedded in Vestopal W, and examined in an electron microscope. Pre-adipose cells were found to be fibroblasts characterized by their spindle shape, long tenuous cytoplasmic extensions, and profuse endoplasmic reticulum. The developmental stages traced from fibroblast to mature adipose cell show a gradual change in cell shape, an accumulation of cytoplasm and non-membrane-bounded lipid, a decrease in the endoplasmic reticulum, and a change in shape of mitochondria. Transitory glycogen appears at mid-differentiation. Numerous smooth-membraned vesicles occur in the cytoplasm throughout differentiation. Pinocytosis is constantly evident. Cells of the multilocular stage are shown to differ from brown fat cells, particularly with respect to cytoplasmic membrane systems and mitochondria. No transport of particulate lipid from the lumen of the capillary to, or within, the adipose cell was detected, nor could any cell organelle be demonstrated to be visibly related to lipid synthesis and/or deposition.     

白细胞介素对大鼠离体垂体前叶细胞增殖的影响

本工作采用大鼠垂体前叶（ＡＰ）细胞原代培养方法,以３ＨＴｄＲ掺入率反映细胞增殖水平,研究了ＩＬ１和ＩＬ６对ＡＰ细胞增殖的影响。结果表明：（１）ＩＬ１（１－１００ｎｇ／ｍｌ）促进雄性大鼠和雌性大鼠ＡＰ细胞的增殖。（２）低浓度的ＩＬ６（０．１ｎｇ／ｍｌ）抑制雄性大鼠的ＡＰ细胞的增殖,而较高浓度的ＩＬ６（１－１０ｎｇ／ｍｌ）则表现为刺激作用。（３）ＩＬ６（０．１－１０ｎｇ／ｍｌ）促进雌性大鼠ＡＰ细胞的增殖。上述结果说明ＩＬ１和ＩＬ６除直接调控ＡＰ细胞的分泌外,也参与调节ＡＰ细胞增殖活动。     

CYTOPLASMIC GRANULE FORMATION IN MYELOCYTES : An Electron Microscope Radioautographic Study on the Mechanism of Formation of Cytoplasmic Granules in Rabbit Heterophilic Myelocytes

The intracellular flow of tritiated lysine as revealed by electron microscope radioautography was studied in heterophilic myelocytes of rabbit marrow. Label over the Golgi complex rose to a maximum of 37% of total cytoplasmic grains 30 min after initial exposure to the tracer and fell to 11% after 3 to 4 hr of incubation. Coincident with decrease in label over the Golgi complex, grain counts over granules rose to 32% after 3 to 4 hr. The time sequence of incorporation and flow of tritiated lysine and the per cent distribution of label was similar in bone marrow myelocytes under in vivo and in vitro conditions. The results demonstrate a function of the Golgi complex in incorporating or packaging certain basic amino acids or proteins into cytoplasmic granules of heterophilic myelocytes.     

电场刺激对垂体前叶ACTH分泌的影响及作用机制的研究

目的和方法：本实验通过切除大鼠肾上腺改变垂体的功能状态,利用垂体组织块离体灌流并施加电场刺激的方法,观察服垂体前叶内神经纤维对促肾上腺皮质激素（ＡＣＴＨ）释放的影响,结果：参数为强度３０ｍＡ,波宽０．５ｍｓ,频率１０Ｈｚ的电场刺激可明显抑制肾上腺切除９６ｈ后垂体前叶组织块释放ＡＣＴＨ,此效应可被预先给予的河豚毒素（ＴＴＸ）所取肖精氨酸加压素（ＡＶＰ）可显著刺激垂体组织块释放ＡＣＴＨ,同样参数的电场     

LYSOSOME FUNCTION IN THE REGULATION OF THE SECRETORY PROCESS IN CELLS OF THE ANTERIOR PITUITARY GLAND

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH¹ was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.     

垂体前叶内神经纤维可能参与ACTH分泌的调节

我们建立了垂体组织块短时温育并施加电场刺激的离体实验体系,运用此方法并结合放射免疫测定激素含量,观察了大鼠垂体前叶内神经纤维对促肾上腺皮质激素（ＡＣＴＨ）释放的影响。结果表明,电场刺激能够促使垂体前叶ＡＣＴＨ释放显著增加,刺激参数为强度３０ｍＡ,波宽０．５ｍｓ,频率１０Ｈｚ。这个效应可为温育液中加入河豚毒素（ＴＴＸ）和藜芦碱所阻断,但ＴＴＸ不能阻断精氨酸加压素（ＡＶＰ）诱发的ＡＣＴＨ分泌。同样参数的电场刺激对分散培养的大鼠垂体前叶细胞ＡＣＴＨ的分泌没有显著作用。以上结果说明,我们所用参数的电场刺激产生的效应是兴奋了垂体前叶内的神经纤维,而非直接刺激腺细胞所致。上述结果提示：垂体前叶激素分泌的调节除了传统的体液途径之外,还可能存在直接的神经控制。