K物质对心肌细胞收缩幅度的影响及其机制探讨

目的 :探讨K物质 (SK)对心肌细胞收缩的影响及机制。方法 :原代培养幼鼠心肌细胞 ,利用计算机图像分析系统测定SK处理前后心肌细胞收缩频率和收缩幅度的变化 ,同时观察预先加入速激肽受体拮抗剂DSP、β受体阻断剂心得安、α受体阻断剂酚妥拉明对SK作用的影响。结果 :当加入SK (1 .78× 1 0 - 5mol/L)到培养细胞中时 ,心肌细胞收缩幅度增强 ,但收缩频率变化不显著 ;且在 1 0 - 8～ 1 0 - 5mol/L浓度范围内心肌细胞收缩幅度变化呈剂量 效应关系 ;预先加入DSP可抑制SK对心肌细胞的影响 ,而心得安、酚妥拉明对SK的作用无影响。结论 :SK使心肌细胞的收缩幅度增强 ,其作用是由速激肽受体介导的     

Valproic acid inhibits substance P-induced activation of protein kinase C epsilon and expression of the substance P receptor

The neuropeptide substance P (SP) has been hypothesized to be involved in the etiopathology of affective disorders. This hypothesis is based on the findings that neurokinin-1-receptor antagonists have antidepressant effects in depressed patients and that SP may worsen mood. In this study, we investigated the effect of the mood-stabilizing agents valproic acid (VPA), carbamazepine, and lithium on SP-induced gene expression. As a model system, we used primary rat astrocytes and human astrocytoma cells, which both express functional SP-receptors and, upon stimulation with SP, synthesize interleukin-6 (IL-6), a cytokine which has been shown to be elevated during the acute depressive state. We found that VPA dose-dependently inhibited SP-induced IL-6 synthesis which was seen with pre-incubation periods of 30 min, 3, 7 and 14 days, whereas carbamazepine and lithium showed no inhibitory effect. The inhibitory effect of VPA was not mediated by inhibition of the stress-regulated kinases p38 and p42/44 (Erk1/2) but by inhibition of protein kinase C epsilon activation. Furthermore, VPA down-regulated the expression of the substance P receptor (neurokinin(NK)-1-receptor) as assessed by real-time PCR. Whether both mechanisms contribute to the mood-stabilizing properties of VPA has to be evaluated in further studies.     

Ice binding,recrystallization inhibition,and cryoprotective properties of ice-active substances associated with Antarctic sea ice diatoms

Extracellular macromolecules associated with Antarctic sea ice diatoms were previously shown to have ice-binding activities. The function of these ice-active substances (IASs) has not been identified. Here we show that two of the IASs have a strong ability to inhibit the recrystallization of ice, possibly signifying a cryoprotectant function. To test this possibility, two species of marine diatom (one Antarctic and one temperate) were subjected to a single freeze-thaw cycle (approximately 20h at -4 or -5 degrees C) in the presence or absence of IAS. Viability, based on a double staining technique, was 15-29% higher in the presence of IAS. Etching of single crystal ice hemispheres grown from dilute IAS solutions indicated that the IASs bind to specific faces of ice and are incorporated into the ice lattice. Together, these results suggest that the IASs acts as a cryoprotectant, probably through some ice-binding mechanism.     

Colonic Mechanoreceptor Input to Neurons in Guinea Pig Caudal Mesenteric Ganglion

The background activity of pacemaker-like neurons (PLN) of the guinea-pig caudal mesenteric ganglion (CMG) and their reflex responses to colonic distention were studied on combined isolated preparations including the CMG and a colon segment connected with the lumbar colonic nerves. The results allow us to suggest that the spontaneous and reflex activity of PLN is of a peripheral origin. Synaptic transmission in the peripheral colonofugal nerve pathways is mediated by acetylcholine and substance P (SP). The SP-induced excitation of PLN probably involves activation of the tachykinin NK₃ receptors.     

Neuropeptide Organization of the Nociceptive and Antinociceptive Brain Systems in the Cat

Using radioimmune techniques, we studied in detail the concentrations of -endorphin (-En), met- and leu-enkephalins (mE and lE, respectively), and substance P (SP) in a number of structures of the brainstem and forebrain of the cat. According to the proposed concept, these structures comprise the noci- and antinociceptive brain systems (NS and ANS). The above indices were measured in intact animals and in animals after nociceptive electrocutaneous stimulation (NECS) of the limb, stimulation of the ventrolateral zone of the midbrain central gray (vl SGC, a nociceptive midbrain structure), stimulation of the dorsolateral part of the above region and dorsal raphe nucleus (dl SGC and Rd, antinociceptive midbrain structures), and after combined stimulations (NECS preceded by conditioning stimulation of one of the above midbrain zones). We found that in the norm maximum SP concentrations were observed in the NS structures, while those of -En and mE were the highest in the hypothalamic nuclei belonging to the ANS and in its midbrain centers (dl SGC and Rd). Nociceptive ECS, stimulations of the studied midbrain zones, and combinations of these stimulations could result in specific and, in some cases, very significant (by an order of magnitude and more) shifts in the concentrations of the mentioned neuropeptides in the studied set of the central structures. After NECS and its combination with vl SGC stimulation, SP concentrations in the NS structures considerably increased, while -En and mE concentrations in the ANS components dropped. Stimulations of the dl SGC and Rd were accompanied by increases in the mE and -En levels and simultaneous drops in the SP concentrations in the ANS components; reciprocate shifts were observed within the NS. Changes in the lE level, which were related to the influences used, were less specific and mostly appeared as increases in this index in the structures of both the NS and ANS. Combinations of NECS with conditioning stimulations of the vl SGC, dl SGC, or Rd demonstrated that the latter exert significant modulatory effects on the NECS-induced shifts in the concentrations of the studied neuropeptides. Considering the obtained data, a hypothetical scheme of neuropeptide organization of the cerebral NS and ANS has been proposed. In the examined brain structures, there are neuronal populations belonging to the two main neurochemical systems. One of them is SP-ergic, while another consists of mE- and -En-ergic neurons; these systems are in antagonistic relations. Changes in the levels of mE and -En always induce the attended opposite shifts in the SP levels, and vice versa. The lE-ergic neuronal populations, which co-exist with the above neurochemical systems, are relatively nonspecifically activated by either (noci- and antinociceptive) drives, but, according to the pattern of its responses, the lE-ergic system is closer to the SP-ergic one. It is supposed that pain signals, when coming to the vl SGC, activate SP- and lE-ergic neuronal populations; later on, the posterior and lateral hypothalamic nuclei and preoptic region are involved in the transmission of the above signals. When released by the corresponding neuronal populations in the vl SGC, lE activates the key ANS structures (dl SGC and Rd), and the latter, in turn, activate other components of this system, which form its ascending compartment (ventromedial, dorsomedial, and paraventricular hypothalamic nuclei, septum, basolateral amygdala, hippocampal fields 3 and 4, and cingular cortex). In the ANS,-En and mE function as transmitters.     

New approaches for high-yield purification of Müllerian inhibiting substance improve its bioactivity

We have established a new method to purify Müllerian inhibiting substance (MIS) with higher purity and recovery over existing procedures. Recombinant human MIS was expressed in Chinese hamster ovary cells and secreted into chemically defined serum-free media. The secreted products were concentrated by either precipitation with ammonium sulfate or lectin-affinity chromatography, each of which was followed by anion-exchange chromatography. Further separation of the active carboxy-terminal domain of MIS was achieved after cleavage by plasmin followed by lectin-affinity chromatography. This method may be applicable to other members of the transforming growth factor beta family with which MIS shares sequence homology.     

Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg

A transglutaminase (TGase) cDNA was cloned from carp ovary. It was highly homologous to zebrafish TGase. Immunoblot and enzymatical assay showed that TGase was present on the chorion and in the cytoplasm of carp eggs. Addition of TGase inhibitor, cadaverine or ethylene diaminetetracetic acid (EDTA) to the cortical reaction medium impaired the formation of the outer layer of fertilization envelope (FE(o)), the adhesive structure of carp egg. Fibroin-like substance (FLS), cystatin, cathepsin-like substance (CLS), and FEO-1 were the components of FE(o), wherein the majority of the former three were conjugated to form macromolecules of 90-205 kDa while the latter one was present in monomer of 22 kDa. Cadaverine interfered slightly the discharge of FLS conjugates out of the perivitelline space (PVS) but affected profoundly the recruitment of FLS conjugates to FE, whereas EDTA completely inhibited both the release and the recruitment of FLS conjugates to FE. Both EDTA and cadaverine did not inhibit the discharge of FEO-1 out of PVS but could inhibit the recruitment of FEO-1 to FE. The mechanism was studied. ZP2 and ZP3, the major constituents of inner layer of FE, were cross-linked during cortical reaction, which rendered FE hardened. In the presence of EDTA, the cross-linking of ZP2 and ZP3 were inhibited, thus FE remained soft. The PVS of an egg with a hardened FE was less expanded than an egg with a soft FE. It was assumed that a less expanded PVS would generate a higher fluid pressure than a more expanded PVS did. Therefore, the transportation of the macromolecules such as the FLS-cystatin-CLS conjugates out of PVS was facilitated in control and cadaverine-treated eggs whose FE were hardened but was blocked in EDTA-treated eggs whose FE were unhardened. On the other hand, the transportation of small molecules such as FEO-1 out of FE was not restrained, so they were discharged out of the PVS of the control and TGase inhibitor-treated eggs. In addition, TGase activity was also required for the recruitment of FLS conjugates to FE.     

Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum

Seven cDNA encoding silkworm fibroin homologues were cloned from a carp ovarian cDNA library. The encoded proteins are denoted as carp ovarian fibroin-like substances (FLS). FLS contain a repetitive domain consisting of tandem repeats of dipeptide of Gly-X, where X may be any amino acid. Each FLS has its own unique repeating sequence, such as GQGAGQGS, GQGMGQGM, GRGQGEGHGS, and GFGFGQGS, indicating a family of FLS genes exists in carp. FLS is exclusively expressed in oocytes and is stored in cortical granules. During cortical reaction, FLS is exocytosed to perivitelline space and then gradually added to the outer layer of the fertilization envelope (FEo). The FLS of fertilization envelope is conjugated with cystatin and cathepsin-like substance (CLS) and appears in multiple bands of molecular weights ranging from 40 to 205 kDa. After fertilization or artificial activation, carp eggs adhere firmly to the substratum via FEo. FLS is a major component of FEo. The presence of transglutaminase inhibitor, cadaverine or ethylene diaminetetraacetic acid, in the cortical reaction medium can impair or block the recruitment of FLS and other substances to FEo. As a consequence, FEo is not formed or is greatly reduced, resulting in a great reduction of egg adhesion.     

Selenium and zinc levels in volatile substance abusers

Plasma and erythrocyte levels of selenium (Se) and zinc (Zn) have not been investigated in volatile (inhalant) substance abusers previously, although changes in the activities of antioxidant enzymes resulting from oxidative damage caused by various constituents of volatile substances have been shown in a few animal and human studies. Concentrations of these two elements in erythrocytes and plasma of 37 adolescents with inhalant abuse were measured by atomic absorption spectrophotometry and compared with those of 37 age-matched healthy controls. Erythrocyte and plasma levels of Se and plasma level of Zn were significantly lower in the study group when compared to the control group. Chronic inhalation of volatile substances can decrease the plasma levels of Se and Zn and, thus, may lead to a decrease in the activity of antioxidant enzyme systems in adolescent abusers. The role of Se and Zn supplementation in children with inhalant abuse remains to be determined considering the reduced antioxidant activity resulting from deficiency of these trace elements.