Estrogen-receptor interaction

The interaction of estradiol with uterine cells involves the association of the hormone with an extranuclear receptor protein, followed by temperature dependent translocation of the resulting complex to the nucleus. During this process, the steroid binding unit of the protein undergoes an alteration, called "receptor transformation," that can be recognized by an increase in its sedimentation rate from 3.8S to 5.2S, and by its acquisition of the ability to bind to isolated uterine nuclei and to alleviate a tissue specific deficiency in the RNA synthesizing capacity of such nuclei. Receptor transformation can be effected in the absence of nuclei by warming uterine cytosol with estradiol. This preparation of transformed complex resembles that extracted from nuclei both in its sedimentation rate (5.3S) and in its ability to bind to uterine nuclei and augment RNA synthesis, properties that are not shown by the native complex. It is proposed that receptor transformation is an important step in estrogen action and that a principal role of the hormone is to induce conversion of the receptor protein to a biochemically functional form.     

Effects of tetrodotoxin on excitability of squid giant axons in sodium-free media

The effect of tetrodotoxin on excitability of internally perfused squid giant axons immersed in various sodium-free media was examined. Action potentials were found to be suppressed by this substance, with or without sodium ion in the external medium. Tetrodotoxin showed a strong suppressive effect upon action potentials produced in media containing salts of only divalent cations (CaCl(2), CaBr(2), SrCl(2), or BaCl(2)). Our findings concerning the action of tetrodotoxin do not support the separate-channel hypothesis for excitable membranes.     

Phosphorylase a activity in uterine muscle: stimulation by ethylenediaminetetraacetic acid

Injection of ethylenediaminetetraacetic acid into spayed rats or its use in phosphorylase extraction increases the activity of glycogen-phosphorylase a in uterine smooth muscle tissue. Since the total phosphorylase activity is not increased, the increase in phosphorylase a appears to result from a conversion of inactive phosphorylase b to the active form of the enzyme.     

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.     

Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes

Primary mouse oocytes contain untranslated stable messenger RNA for tissue plasminogen activator (t-PA). During meiotic maturation, this maternal mRNA undergoes a 3'-polyadenylation, is translated, and is degraded. Injections of maturing oocytes with different antisense RNA's complementary to both coding and noncoding portions of t-PA mRNA all selectively blocked t-PA synthesis. RNA blot analysis of t-PA mRNA in injected, matured oocytes suggested a cleavage of the RNA.RNA hybrid region, yielding a stable 5' portion, and an unstable 3' portion. In primary oocytes, the 3' noncoding region was susceptible to cleavage, while the other portions of the mRNA were blocked from hybrid formation until maturation occurred. Injection of antisense RNA complementary to 103 nucleotides of its extreme 3' untranslated region was sufficient to prevent the polyadenylation, translational activation, and destabilization of t-PA mRNA. These results demonstrate a critical role for the 3' noncoding region of a dormant mRNA in its translational recruitment during meiotic maturation of mouse oocytes.     

Ontogeny of the estrogen receptor during early uterine development

The number of estrogen binding sites in uterine cytoplasm on a per cell basis reaches a maximum by day 10 of life in both intact and castrate female rats. After this peak is reached, the number of binding sites per cell decreases, and the ratio remains constant until days 22 to 23 of life. Thus, the ontogeny of the estrogen binding protein is not dependent upon estrogen from the ovary and is probably an autonomous property of uterine cells. Sedimentation values and dissociation constants of the protein when the animals are 5 to 10 days of age are similar to those of the 22-day-old animal, indicating that the same protein is present throughout postnatal development.     

Crayfish muscle fiber: ionic requirements for depolarizing synaptic electrogenesis

Presence of sodium in the bathing medium is not essential for the electrically excitable depolarizing electrogenesis of crayfish muscle fibers, production of action potentials being dependent on calcium. The depolarizing electrogenesis of the excitatory synaptic membrane component does require sodium, however, and this ion cannot be replaced by lithium as it can in spike electrogenesis of many cells. Ionophoretic applications of glutamate, which in the presence of sodium depolarize the cell by activating the excitatory synaptic membrane, are without effect in the absence of sodium. Not only is there no depolarization, but the membrane conductance also remains unchanged. Thus, in the absence of inward movement of sodium across the synaptic membrane there is also no outward movement of potassium. Accordingly, it seems that increased conductance for potassium is not an independent process in the synaptic membrane, whereas it is independent of sodium activation in spike electrogenesis. Chloride activation is independent, however; increase in conductance and the electrogenesis of the inhibitory synaptic component are not affected by the absence of sodium. Implications of these findings regarding the structure of differently excitable membrane components are discussed.     

Estrogenic induction of ornithine decarboxylase in vivo and in vitro

Injection of estrogens (17beta-estradiol or diethylstilbestrol) into immature chicks results in a marked (30- to 50-fold) increase in the ornithine decarboxylase activity of oviductal homogenates within a 4-hour period. Similar stimulations were obtained when estrogen was injected into hypophysectomized or castrated rats and the uterus was examined for decarboxylase activity. An elevation of decarboxylase activity was obtained in vitro when oviducts from immature chicks were incubated in the presence of estrogen. These data indicate a direct action of estrogen on oviduct tissue to promote a rapid increase in the activity of a specific enzyme and represent the first example of a completely in vitro enzyme response to estrogen.     

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.     

Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes

Oligonucleotides complementary to regions of U1 and U2 small nuclear RNAs (snRNAs), when injected into Xenopus laevis oocytes, rapidly induced the specific degradation of U1 and U2 snRNAs, respectively, and then themselves were degraded. After such treatment, splicing of simian virus 40 (SV40) late pre-mRNA transcribed from microinjected viral DNA was blocked in oocytes. If before introduction of SV40 DNA into oocytes HeLa cell U1 or U2 snRNAs were injected and allowed to assemble into small nuclear ribonucleoprotein particle (snRNP)-like complexes, SV40 late RNA was as efficiently spliced as in oocytes that did not receive U1 or U2 oligonucleotides. This demonstrates that oocytes can form fully functional hybrid U1 and U2 snRNPs consisting of human snRNA and amphibian proteins.     

Collagen synthesis in Xenopus oocytes after injection of nuclear RNA of frog embryos

A messenger RNA fraction from polysomes of frog larvae or RNA preparations from isolated nuclei of developing frog embryos were injected into growing Xenopus laevis oocytes that were incubated with labeled proline. Column chromatography of protein hydrolyzates revealed labeled hydroxyproline after injection of the messenger RNA fraction and neurula nuclear RNA, indicating that the injected material had promoted collagen synthesis.     

Modulation of calcium channels in cardiac and neuronal cells by an endogenous peptide

Calcium channels mediate the generation of action potentials, pacemaking, excitation-contraction coupling, and secretion and signal integration in muscle, secretory, and neuronal cells. The physiological regulation of the L-type calcium channel is thought to be mediated primarily by guanine nucleotide-binding proteins (G proteins). A low molecular weight endogenous peptide has been isolated and purified from rat brain. This peptide regulates up and down the cardiac and neuronal calcium channels, respectively. In cardiac myocytes, the peptide-induced enhancement of the L-type calcium current had a slow onset (half-time approximately 75 seconds), occurred via a G protein-independent mechanism, and could not be inhibited by alpha 1-adrenergic, beta-adrenergic, or angiotensin II blockers. In neuronal cells, on the other hand, the negative effect had a rapid onset (half-time less than 500 milliseconds) and was observed on both T-type and L-type calcium channels.     

Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes

Receptors for N-methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.     

Extracellular potassium ions mediate specific neuronal interaction

The giant interneurons from the nerve system of the cockroach Periplaneta americana exhibit a peculiar reciprocal synaptic interaction. The synaptic potentials are not blocked by addition of 5 millimolar cobalt chloride and have an extrapolated reversal potential close to 0 millivolt. Hyperpolarizing current injected into one cell does not spread to the other. Intracellular injection of tetraethylammonium ions into one giant interneuron increases the duration of the action potential of the injected cell to 30 milliseconds and reduces the rise time and amplitude of the postsynaptic response recorded in the other giant interneuron. These results indicate that the interaction between the interneurons is not mediated by conventional chemical or electrotonic synapses.. All evidence points to generation of the potentials by localized increases in extracellular potassium concentrations as a consequence of firing of one neuron.     

Command neurons in Pleurobranchaea receive synaptic feedback from the motor network they excite

Command neurons that cause rhythmic feeding behavior in the marine mollusc Pleurobranchaea californica have been identified in the cerebropleural ganglion (brain). Intracellular stimulation of single command neurons in isolated nervous systems, semi-intact prepartions, and restrained whole animals causes the same rhythmic motor output pattern as occurs during feeding. During this motor output pattern, action potentials recorded intracellularly from the command neurons occur in cyclic bursts that are phase-locked with the feeding rhythm. This modulation results from repetitive, alternating bursts of excitatory and inhibitory postsynaptic potentials, which are caused at least in part by synaptic feedback to the command neurons from identified classes of neurons in the feeding network. Central feedback to command neurons from the motor network they excite provides a possible general physiological mechanism for the sustained oscillation of neural networks controlling cyclic behavior.     

Stimulation of RNA and protein synthesis by intracellular insulin

Like insulin-sensitive somatic cells, stage IV oocytes from Xenopus laevis increase their synthesis of RNA, protein, and glycogen in response to extracellular insulin. Synthesis of RNA and protein are also increased when oocytes are maintained under paraffin oil and insulin is microinjected into the cytoplasm. The effects of external and intracellular insulin are additive, suggesting separate mechanisms of action. Experiments with nuclei isolated under oil show that RNA synthesis can be stimulated by applying insulin to the nucleus directly. Thus, the nucleus appears to be one intracellular site of hormone action.     

Prolonged Ca2+-dependent afterhyperpolarizations in hippocampal neurons of aged rats

The possibility that calcium is elevated in brain neurons during aging was examined by quantifying afterhyperpolarizations induced by spike bursts in CAl neurons of hippocampal slices from young and aged rats. The afterhyperpolarizations result from Ca2+-dependent K+ conductance increases and are blocked in medium low in Ca2+ and prolonged in medium high in Ca2+. The afterhyperpolarization and associated conductance increases were considerably prolonged in cells from aged rats, although inhibitory postsynaptic potentials did not differ with age. Since elevated intracellular Ca2+ can exert deleterious effects on neurons, the data suggest that altered Ca2+ homeostasis may play a significant role in normal brain aging.     

急性心肌缺血大鼠左心室流出道自发性慢反应电位去极离子流变化分析

目的探讨急性心肌缺血大鼠左室流出道自发性慢反应电位去极离子流的变化。方法常规玻璃微电极细胞内方法观测急性心肌缺血大鼠的离体心脏最大舒张电位(MDP)、0相除极幅度(APA)、0相最大除极速度(Vmax)、4相自动除极速度(VDD)、复极50%(APD50)和90%(APD90)的时间以及自发放电频率(RPF)。结果与给药前相比①1.2mmol.L-1河豚毒(TTX)使APA和Vmax有所减小(P<0.05),VDD和RPF明显减慢(P<0.01);②1.0μmol.L-1维拉帕米(VER)可使该慢电位的APA、Vmax、VDD明显减小,RPF减慢(P<0.01);APD、APD90延长(P<0.05);③2mmol.L-1 4-氨基吡啶(4-AP)使该慢电位的MDP的绝对值、APA、Vmax减小,VDD和RPF加快(P<0.01);④1.5mmol.L-1 CsCl作用6min时,VDD和RPF明显降低(P<0.05),10min时恢复。结论①左心室流出道的自发慢电位0相主要去极离子流除Ca2+内流外,还有少量Na+内流。②4相去极离子流中,除Ca2+、Na+的内流和Ik衰减外,If电流可能也起部分作用。     

Expression of a cloned rat brain potassium channel in Xenopus oocytes

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.