The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study

The Malpighian tubules of Tenebrio molitor provide a model system for interpreting the actions of endogenous diuretic and antidiuretic peptides. The effects of diuretic (Tenmo-DH(37)) and antidiuretic (Tenmo-ADFa) peptides and their respective second messengers (cyclic AMP and cyclic GMP) on basolateral (V(bl)) and transepithelial (V(te)) potentials of Tenebrio Malpighian tubules were determined using conventional microelectrodes. In the presence of 6 mmol l(-1) Ba(2+), Tenmo-DH(37) (100 nmol l(-1)) reversibly hyperpolarized V(bl) and depolarized V(te). A similar response was seen with the addition of 1 mmol l(-1) cyclic AMP; however, the apical membrane potential (V(ap)) then showed a hyperpolarization, whereas a depolarization of V(ap) was observed with Tenmo-DH(37). Bafilomycin A(1) (5 micromol l(-1)) inhibited fluid secretion of stimulated tubules and reversed the hyperpolarization of V(bl) in response to Tenmo-DH(37). In response to 100 nmol l(-1) Tenmo-ADFa or 1 mmol l(-1) cyclic GMP, V(bl) and V(te) depolarized, although cyclic GMP affected membrane potentials somewhat differently by causing an initial hyperpolarization of V(bl) and V(te). In high [K(+)]-low [Na(+)] Ringer, 1 mmol l(-1) amiloride decreased fluid secretion rates, and depolarized both V(bl) and V(te). Amiloride significantly decreased luminal pH in paired experiments, indicating the presence of a K(+)/nH(+) exchanger in tubule cells of Tenebrio. The results suggest that the endogenous factors and their second messengers stimulate/inhibit fluid secretion by acting on the apical V-ATPase, basolateral K(+) transport, and possibly Cl(-) transport.     

Immunocytochemical localization of a diuretic hormone of the beetle Tenebrio molitor,Tenmo-DH(37), in nervous system and midgut

Although the mealworm Tenebrio molitor inhabits very dry environments, it has at least two diuretic peptides, which increase fluid secretion by the free portions of the Malpighian tubules. Unlike other insect corticotropin-releasing factor (CRF)-related peptides isolated to date, these are non-amidated peptides. The immunocytochemical localization of Tenmo-DH(37) was investigated using antisera raised against this hormone. Immunoreactive neurosecretory cells were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH(37) were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-DH(37) is widely distributed and its staining pattern resembles that found for other, amidated CRF-related diuretic peptides.     

Isolation,identification and localization of a second beetle antidiuretic peptide

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr-Asp-Asp-Gly-Ser-Tyr-Lys-Pro-His-Ile-Tyr-Gly-Phe-OH with an EC(50) of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

EXPRESSION OF cfos IN THE MEDULLA OBLONGATA AFTER CAROTID BARORECEPTOR ACTIVATION BYELEVATED INTRASINUS PRESSURE AND ADENOSINE

在１４只隔离灌流颈动脉窦区的大鼠,观察了窦内压（ＩＳＰ）升高和灌流腺苷（ａｄｅｎｏｓｉｎｅ,Ａｄｏ）激活压力感受器时延髓内ｃｆｏｓ蛋白的表达。结果显示：在孤束核、最后区、延髓腹外侧头端区和中缝苍白核可见Ｆｏｓ蛋白样免疫阳性反应（ＦＬＩ）神经元分布,且其数量随ＩＳＰ升高而增多。在给定ＩＳＰ下,颈动脉窦内灌流Ａｄｏ,可使上述区域中ＦＬＩ表达明显增多。根据以上结果,得出如下结论：ｃｆｏｓ在压力感受器反射延髓通路中的表达,可由ＩＳＰ增高和灌流Ａｄｏ而增强,表明Ａｄｏ对压力感受器反射有易化作用。     

Body extracts from the forest ant and honeybee: chromatographic comparison of factors affecting the ant Malpighian tubule

Abstract.  Crude trifluoroacetic acid extracts were prepared from different body parts of the forest ant, Formica polyctena Foerster, Hymenoptera, Formicidae, and the honeybee, Apis mellifera carnica Pollmann, Hymenoptera, Apidae. Extracts were prepurified by means of solid phase extraction over reversed-phase cartridges. The effects of the resulting fractions on fluid secretion rates were tested on single preparations of ant Malpighian tubules. Diuretic and antidiuretic factors were shown to be present. High-performance liquid chromatography allowed for the identification of a diuretic factor from the bee-head extract which had a similar elution profile to a previously purified diuretic peptide from ant-head extracts. An antidiuretic factor was also identified from the bee abdominal extract. Similar to an antidiuretic peptide, which was purified from ant abdominal extracts, this antidiuretic factor inhibited fluid secretion and depolarized the transepithelial potential of ant Malpighian tubules.     

小鼠胚胎干细胞分化心肌细胞血管紧张素Ⅱ 1型和2型受体的表达特征

胚胎干细胞(ESC)具有无限增殖和分化为体内3个胚层来源的各种类型组织细胞的潜能,经过体外诱导能够分化为心肌细胞,亦称为胚胎干细胞分化心肌细胞(ESCM).本研究探讨了ESC诱导分化心肌细胞过程中血管紧张素Ⅱ受体(ATR)的亚型AT1R和AT2R的表达特征.10-4mol/L维生素C体外诱导小鼠R1胚胎干细胞分化为自发搏动的心肌细胞,用免疫荧光法检测分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞在诱导分化为心肌细胞以后,逆转录聚合酶链反应(RT-PCR)和实时定量RT-PCR(Real-timeRT-PCR)方法检测到ESCM表达AT1R,并且呈时间依赖性逐渐增加的特点,在第14d达到高峰.Western印记法检测AT1R表达特征与RT-PCR结果相符.Western印记法的结果显示,血管紧张素Ⅱ(10-6mol/L)可作为AT1R激动剂激活AT1R,并使其下游的细胞外信号调节激酶(ERK1/2)磷酸化水平上调,预孵育AT1R抑制剂Losartan(10-6mol/L),此作用被抑制.RT-PCR方法显示,与新生小鼠心室肌细胞相比,ESCM的AT2R表达水平较低.     

Expression of a novel neuropeptide, NVGTLARDFQLPIPNamide, in the larval and adult brain of Drosophila melanogaster

Advances in mass spectrometry and the availability of genomic databases made it possible to determine the peptidome or peptide content of a specific tissue. Peptidomics by nanoflow capillary liquid chromatography tandem mass spectrometry of an extract of 50 larval Drosophila brains, yielded 28 neuropeptides. Eight were entirely novel and encoded by five not yet annotated genes; only two genes had a homologue in the Anopheles gambiae genome. Seven of the eight peptides did not show relevant sequence homology to any known peptide. Therefore, no evidence towards the physiological role of these 'orphan' peptides was available. We identified one of the eight peptides, IPNamide, in an extract of the Drosophila adult brain as well. Next, specific antisera were raised to reveal the distribution pattern of IPNamide and other peptides from the same precursor, in larval and adult brains by means of whole-mount immunocytochemistry and confocal microscopy. IPNamide immunoreactivity is abundantly present in both stages and a striking similarity was found between the distribution patterns of IPNamide and TPAEDFMRFamide, a member of the FMRFamide peptide family. Based on this distribution pattern, IPNamide might be involved in phototransduction, in processing sensory stimuli, as well as in controlling the activity of the oesophagus.