Characterization of Angiotensin II-regulated K+ Conductance in Rat Adrenal Glomerulosa Cells

Nystatin perforated-patch clamp and single-channel recording methods were used to characterize macroscopic and single-channel K⁺ currents and the effects of angiotensin II (AngII) in cultured rat adrenal glomerulosa cells. Two basic patterns of macroscopic current-voltage relationships were observed: type 1 exhibited a rapidly activating, noninactivating, voltage-dependent outward current and type 2 exhibited an inactivating voltage-dependent outward current attributed to charybdotoxin sensitive Ca⁺⁺-dependent K⁺ channels. Most cells exhibited the type 1 pattern and experiments focused on this cell type. Cell-attached and inside-out patches were dominated by a single K⁺ channel class which exhibited an outward conductance of 12 pS (20 mm K⁺ pipette in cell-attached and inside-out configurations, 145 mm K⁺ _in), a mean open time of 2 msec, and a weakly voltage-dependent low open probability that increased with depolarization. Channel open probability was reversibly inhibited by bath stimulation with AngII. At the macroscopic level, type 1 cell macroscopic K⁺ currents appeared comprised of two components: a weakly voltage-dependent current controlling the resting membrane potential (−85 mV) which appeared mediated by the 12 pS K⁺ channel and a rapidly activating, noninactivating voltage-dependent current activated above −50 mV. The presence of the second voltage-dependent K⁺ channel class was suggested by the effects of AngII, the blocking effects of quinidine and Cs⁺, and the properties of the weakly voltage-dependent K⁺ channel described. The K⁺ selectivity of the macroscopic current was demonstrated by the dependence of current reversal potentials on the K⁺ equilibrium potential and by the effects of K⁺ channel blockers, Cs⁺ and quinidine. AngII (10 pm to 1 nm) reversibly inhibited macroscopic K⁺ currents and this effect was blocked by the AT₁ receptor antagonist losartin. Received: 6 August 1996/Revised: 15 November 1996     

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.     

动脉粥样硬化大鼠心肌梗死时钙敏感受体表达的变化及其作用机制

目的:探讨动脉粥样硬化大鼠心肌梗死时钙敏感受体表达的变化及其作用机制。方法:Wistar大鼠随机分为4组:正常对照组(Control);异丙肾上腺组(ISO);动脉硬化组(AS);动脉硬化+异丙肾上腺素组(AS/ISO)。采用腹腔注射VD3和高脂饮食及大剂量异丙肾上腺素(ISO 200 mg/kg)皮下注射复制大鼠在体动脉粥样硬化心肌梗死模型,应用RT-PCR测定心肌组织中Ca SR、Bax、Bcl-2和caspase-3 m RNA的表达变化;TUNEL染色观察心肌细胞凋亡情况;光镜观察心肌形态学变化;紫外分光法检测血清肌酸激酶(CK)、电化学免疫发光法检测肌钙蛋白T(c Tn T)水平。结果:与正常组比较,ISO组CK活性、c Tn T水平以及Ca SR、Bax和caspase-3的表达均增加,同时Bcl-2表达减少,心肌细胞损伤严重,AS/ISO的心肌损伤进一步加重。结论:Ca SR的表达增多参与了动脉硬化大鼠心肌梗死的发生,其机制可能与促进心肌细胞凋亡有关。     

Testosterone-Dependent Interaction between Androgen Receptor and Aryl Hydrocarbon Receptor Induces Liver Receptor Homolog 1 Expression in Rat Granulosa Cells

Androgens play a major role in the regulation of normal ovarian function; however, they are also involved in the development of ovarian pathologies. These contrasting effects may involve a differential response of granulosa cells to the androgens testosterone (T) and dihydrotestosterone (DHT). To determine the molecular pathways that mediate the distinct effects of T and DHT, we studied the expression of the liver receptor homolog 1 (LRH-1) gene, which is differentially regulated by these steroids. We found that although both T and DHT stimulate androgen receptor (AR) binding to the LRH-1 promoter, DHT prevents T-mediated stimulation of LRH-1 expression. T stimulated the expression of aryl hydrocarbon receptor (AHR) and its interaction with the AR. T also promoted the recruitment of the AR/AHR complex to the LRH-1 promoter. These effects were not mimicked by DHT. We also observed that the activation of extracellular regulated kinases by T is required for AR and AHR interaction. In summary, T, but not DHT, stimulates AHR expression and the interaction between AHR and AR, leading to the stimulation of LRH-1 expression. These findings could explain the distinct response of granulosa cells to T and DHT and provide a molecular mechanism by which DHT negatively affects ovarian function.     

Differential Regulation of Action Potentials by Inactivating and Noninactivating BK Channels in Rat Adrenal Chromaffin Cells

Large-conductance Ca²⁺-activated K⁺ (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca²⁺ and membrane potential. In rat chromaffin cells (RCC), inactivating BK_i and noninactivating (BK_s) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK_i and BK_s current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK_i current facilitates repetitive firing of APs, whereas BK_s current does not. However, the key functional difference between BK_i and BK_s current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK_i current at a given [Ca²⁺].     

A Basolateral Chloride Conductance in Rat Lingual Epithelium

We used Ussing chamber measurements and whole-cell recordings to characterize a chloride conductance in rat lingual epithelium. Niflumic acid (NFA) and flufenamic acid (FFA), nonsteroidal anti-inflammatory aromatic compounds known to inhibit Cl⁻ conductances in other tissues, reduced transepithelial short-circuit current (I _sc ) in the intact dorsal anterior rat tongue epithelium when added from the serosal side, and reduced whole-cell currents in rat fungiform taste cells. In both Ussing chamber and patch-clamp experiments, the effect of NFA was mimicked by replacement of bath Cl⁻ with methanesulfonate or gluconate. In low Cl⁻ bath solution, the effect of NFA on whole-cell current was reduced. Replacement of bath Ca²⁺ with Ba²⁺ reduced the whole-cell Cl⁻ current. We conclude that a Ca²⁺-activated Cl⁻ conductance is likely present in the basolateral membrane of the rat lingual epithelium, and is present in the taste receptor cells from fungiform papillae. Further experiments will be required to identify the role of this conductance in taste transduction. Received: 8 September 1997/Revised: 27 March 1998     

肠道味觉受体细胞及其甜味感受

味觉是人和动物的一种基本生理感觉,用来识别食物的性质、调节食欲、控制摄食量.味觉不仅仅存在于口腔中,同样存在于胃肠道中.最近研究表明,动物肠道的粘膜上存在着表达味觉受体和味觉相关因子的细胞,调控着肠道激素如GLP-1和GIP的分泌以及糖转运体SGLT-1和GLUT-2的表达.甜味剂的刺激影响这些激素的分泌及载体的表达,从而影响机体对葡萄糖的吸收和利用.肠道味觉的研究有助于揭示肠道消化吸收功能的调控机理,同时为糖尿病、肥胖、代谢失调及其它饮食相关疾病的治疗提供新的切入点.主要介绍了肠道粘膜上的味觉受体细胞、味觉的信号转导途径以及甜味感受对肠道激素的分泌和葡萄糖吸收的影响,最后讨论了味觉细胞生物学的发展方向.     

A Cyclic Nucleotide-dependent Chloride Conductance in Olfactory Receptor Neurons

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca²⁺ as well as in the presence of 3 mm intracellular Ca²⁺, suggesting that the current did not require either voltage or Ca²⁺ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides. Received: 12 November 1996/Revised: 4 April 1997     

Thrombin causes the Enrichment of Rat Brain Primary Cultures with Ependymal Cells Via Protease-Activated Receptor 1

Ependymal cell culture models from rat have been developed over the last 20 years to facilitate biochemical studies on this least-studied glial cell type. The cell culture protocol calls for the presence of thrombin, which is essential for obtaining a high proportion of multiciliated ependymal cells. The serine protease appears to act via protease-activated receptor 1 to prevent the apoptosis of ependymal precursors and enhance their proliferation without affecting contaminating cells. Unciliated precursors differentiate into polyciliated ependymocytes by passing through a stage of monociliation. The message for protease-activated receptor (PAR) 1 is initially abundant in the cultures, but its level declines as the cells differentiate. Besides PAR 1, signalling through PAR 2 also promotes ciliation in rat brain primary cultures, albeit to a lesser degree than the thrombin receptor. Thrombin and other proteases may be involved in the regulation of ventricular wall development. This action would be mediated mainly by PAR1.     

Rat Macrophage C-Type Lectin Is an Activating Receptor Expressed by Phagocytic Cells

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.     

Serotonergic Agonists Inhibit Calcium-Activated Potassium and Voltage-Dependent Sodium Currents in Rat Taste Receptor Cells

Recently we reported that rat taste receptor cells respond to the neurotransmitter serotonin with an inhibition of a calcium-activated potassium current [17]. In the present study, this observation is confirmed and extended by studying the effects of an array of serotonergic agonists on membrane properties, calcium-activated potassium current, and voltage-dependent sodium current in taste receptor cells using the patch-clamp recording technique in the whole-cell configuration. Serotonergic inhibition of calcium-activated potassium current was mimicked by the agonists N-(3-trifluoromethylphenyl)piperazine and by (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Both produced reversible inhibition of K _Ca as well as significantly increasing the input resistance of the cell. The agonists 1-(1-naphthyl)piperazine and buspirone (both serotonin receptor 1A agonists) were similarly effective in reducing K _Ca . Outward current was unaffected by application of phenylbiguanide, a serotonin receptor 3 agonist, though current was affected by subsequent application of (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Two agonists—N-(3-trifluoromethylphenyl)piperazine and (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene—were also tested on voltage-dependent sodium currents; both were effective and reversible in reducing its magnitude at a variety of applied potentials. These data are consistent with the notion that serotonin effects in rat taste receptor cells are mediated by serotonin 1A receptors, though other receptor subtypes may be additionally expressed. Serotonin may affect the taste cell electrical properties during active stimulation in a paracrine fashion. Received: 10 May 1999/Revised: 27 September 1999     

Early Receptor Potentials of Rods and Cones in Rodents

The second phase (negative peak) of the early receptor potential of cones has been studied in the all-cone eyes of the Mexican and antelope ground squirrels (Citellus mexicanus and Citellus leucurus) and compared with responses from the rod-dominant eyes of the rat and flying squirrel (Glaucomys volans). The responses obtained from the all-cone eyes tended to be smaller in amplitude, to have higher thresholds, and to be considerably more resistant to light adaptation than the responses from the rod-dominant eyes. The wave forms and time courses of the two types of responses were similar, although the cone potential tended to be less sensitive to temperature variations and its time constants tended to be shorter than those of the rod potential. The spectral sensitivity of the second phase of the early receptor potential of the Mexican ground squirrel closely follows the absorption spectrum of a Dartnall nomogram pigment having its absorption maximum at 540 mμ. Moreover, as in the case of the rat, the amplitude of the response appears to be linearly related to the amount of pigment bleached in a flash. Thus, in both all-rod and all-cone systems the early receptor potential appears to arise in the photoexcitation of the respective visual pigment and appears to be closely linked to the initial photochemical events. The similarity of the wave form, time course, and stimulus-response curves in the two systems suggests that the early receptor potential is produced by similar mechanisms in all-rod and all-cone systems.     

不同波段电磁辐射对大鼠Sertoli细胞雄激素受体和卵泡刺激素受体表达的影响

探讨不同波段电磁辐射对大鼠睾丸Sertoli细胞雄激素受体(androgen receptor,AR)和卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)表达的影响。原代培养的Sertoli细胞分别经场强6×104 V/m的电磁脉冲(electromagnetic pulse,EMP)、平均功率密度为100 mW/cm2的S波段微波(S-band high power microwave,S-HPM)和X波段微波(X-bandhigh power microwave,X-HPM)辐射4 min。应用real-time RT-PCR和Western blot方法检测Sertoli细胞AR的表达,结果显示具有不同程度的降低(P<0.05,P<0.01);Wistar大鼠分别经S-HPM、X-HPM和EMP照射20 min,应用免疫组化方法检测睾丸组织AR的表达,发现辐射后7 d有显著降低(P<0.05,P<0.01)。三者比较,AR的表达总体呈EMP>X-HPM>S-HPM的趋势,而FSHR的表达无明显变化。Sertoli细胞AR表达的变化,可能参与了电磁辐射致...     

Tissue-Specific Activation of a Single Gustatory Receptor Produces Opposing Behavioral Responses in Drosophila

Understanding sensory systems that perceive environmental inputs and neural circuits that select appropriate motor outputs is essential for studying how organisms modulate behavior and make decisions necessary for survival. Drosophila melanogaster oviposition is one such important behavior, in which females evaluate their environment and choose to lay eggs on substrates they may find aversive in other contexts. We employed neurogenetic techniques to characterize neurons that influence the choice between repulsive positional and attractive egg-laying responses toward the bitter-tasting compound lobeline. Surprisingly, we found that neurons expressing Gr66a, a gustatory receptor normally involved in avoidance behaviors, receive input for both attractive and aversive preferences. We hypothesized that these opposing responses may result from activation of distinct Gr66a-expressing neurons. Using tissue-specific rescue experiments, we found that Gr66a-expressing neurons on the legs mediate positional aversion. In contrast, pharyngeal taste cells mediate the egg-laying attraction to lobeline, as determined by analysis of mosaic flies in which subsets of Gr66a neurons were silenced. Finally, inactivating mushroom body neurons disrupted both aversive and attractive responses, suggesting that this brain structure is a candidate integration center for decision-making during Drosophila oviposition. We thus define sensory and central neurons critical to the process by which flies decide where to lay an egg. Furthermore, our findings provide insights into the complex nature of gustatory perception in Drosophila. We show that tissue-specific activation of bitter-sensing Gr66a neurons provides one mechanism by which the gustatory system differentially encodes aversive and attractive responses, allowing the female fly to modulate her behavior in a context-dependent manner.     

NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

Embryonic Rat Hippocampal Neurons and GABAA Receptor Subunit-Transfected Non-neuronal Cells Release GABA Tonically

We used patch-clamp recording techniques to investigate the contribution of GABA to baseline membrane properties in cultured embryonic rat hippocampal neurons. Almost all of the neurons recorded with Cl⁻-filled pipettes and clamped at negative potentials exhibited baselines that were noticeably noisy, with microscopic fluctuations superimposed on the macroscopic holding current. A gentle steam of saline applied to the neuronal surface rapidly and reversibly reduced the baseline current and fluctuations, both of which were completely eliminated by bicuculline. Fluctuation analysis showed that the variance in the baseline current signal was exponentially distributed with estimated kinetics comparable to those activated by submicromolar concentrations of exogenous GABA. The kinetics of Cl⁻ channels activated by endogenous GABA displayed a potential sensitivity comparable to those activated by exogenous GABA. Non-neuronal cells stably transfected with α₁ and γ₂ GABA_A receptor subunits exhibited little baseline current variance when recorded with Cl⁻-filled pipettes. Addition of micromolar GABA to the extracellular saline or to the pipette solution induced a saline- and bicuculline-sensitive baseline current signal comparable to that recorded in hippocampal neurons. Thus, both intra- and extracellular sources of GABA could contribute to the baseline properties recorded in these cultured neurons. Received: 13 January 1997/Revised: 16 April, 1988     

Bacteria Associated with Immunoregulatory Cells in Mice

This study examined bacteria-immune interactions in a mouse model possessing microbiota-dependent immune regulatory features similar to those occurring in human atopy, colitis, and immune regulation. Associations between the abundance of several bacterial phylotypes and immunoregulatory target cell types were identified, suggesting that they may play a role in these phenotypes.Bacteria are involved in critical aspects of immune system development and regulation (5, 23, 26, 29). Mice raised under germfree conditions exhibit a variety of abnormalities, including hypoplastic Peyer''s patches, reduced numbers of IgA-producing cells, relatively unstructured spleen and lymph nodes, and hypogammaglobulinemic serum (23). Remarkably, after several weeks of exposure to standard intestinal microbiota, normal immune structure and function are restored. Mechanistic details underlying microbe-immune interactions have been recently elucidated for two common intestinal bacteria. Bacteroides thetaiotaomicron was shown to induce the angiogenin Ang4, a component of innate immunity possessing microbicidal activity against a wide range of intestinal microbes, including both bacterial and fungal pathogens (16). In addition, studies of the Bacteroides fragilis zwitterionic capsular polysaccharide A have established it as a cognate antigen of certain CD4⁺ T cells, which programs immune effector polarization (24) and protection of mice from infection by Helicobacter hepaticus through several immune-mediated mechanisms (25). Resident microbiota also modify the interaction of dendritic cells with regulatory T-cell populations, with resultant susceptibility to chronic inflammatory disease, like colitis (15, 28).Recent work by Braun and colleagues has characterized a mouse model with unique immunologic features linking resident microbiota with levels of regulatory CD8⁺ T cells (13, 17, 39). This model is comprised of two physically isolated colonies of isogenic mice harboring distinct microbial communities: conventional floras (CF) and restricted floras (RF). CF refers to C57BL/6 mice housed in a standard specific-pathogen-free facility, while RF refers to C57BL/6 mice containing a different complement of intestinal microorganisms (13, 30), originally created by transferring several nonpathogenic anaerobic bacteria into antibiotic-treated mice (13). RF mice differ from CF mice in several immunologic phenotypes, including selective reduction of marginal zone (MZ) B cells (39), plasmacytoid dendritic cells (pDC) (13), and invariant natural killer (iNK) T cells (38a), as well as naïve CD4⁺ and CD8⁺ T cells (17). In addition, RF mice were shown to be resistant to colitis under genetic or adoptive transfer conditions that permit disease activity in CF mice (2). RF mice also cleared experimental infections by Campylobacter jejuni more slowly than did their CF counterparts (6). The resulting concept is that certain resident microbiota, which may be more abundant in RF mice than in CF mice, induce invariant Qa-1 T cells, with resultant changes in host immunoregulation and microbial surveillance (2).An important issue raised by the foregoing observations is the identity of resident microbiota responsible for this host immunoregulatory response. The objective of this study was to develop a methodology, based on bacteria-immune interactions in the RF/CF mouse model, to identify candidate microbiota. In this study, we employed a series of experiments examining associations between the population densities of bacterial rRNA genes and several immunologic features that differ between CF and RF mice.     

The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates

To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.