Calcium channels in excitable cells: divergent genotypic and phenotypic expression of alpha 1-subunits

1994 ◽  
Vol 267 (2) ◽  
pp. C411-C424 ◽  
Author(s):  
A. Lievano ◽  
A. Bolden ◽  
R. Horn
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Phenotypic Expression ◽  
Gh3 Cells ◽  
Mrna Levels ◽  
Excitable Cells ◽  
Patch Clamp Recording ◽  
Whole Cell Patch Clamp ◽  
Ribonuclease Protection ◽  
P Type

The Ba2+ currents and mRNA levels of four members of the rat brain family of alpha 1-subunit Ca2+ channel genes were examined and compared in the rat cell lines GH3 and PC-12 and in the mouse lines NIE-115 and AtT-20. The RNA was measured with ribonuclease protection assays using probes derived from rat brain (rb) Ca2+ channel cDNAs (rbA, rbB, rbC, and rbD), and the Ba2+ currents were studied by whole cell patch-clamp recording. L-, N-, P-, and T-type currents were discriminated by the voltage dependence and pharmacological properties of Ba2+ currents. All cell lines expressed all four rat brain Ca2+ channel genes, except GH3 cells, which lacked rbB. The functional diversity of Ba2+ currents, however, was quite different among the cell lines. GH3 cells showed evidence of L- and T-type currents, undifferentiated PC-12 cells of L-type currents, AtT-20 cells of L-, N-, and P-type currents, and undifferentiated NIE-115 cells of a T-type current that was partially blocked by both nifedipine and BAY K 8644. Dimethyl sulfoxide-differentiated NIE-115 cells also had an L-type current. Differentiation of NIE-115 cells caused an increase in the levels of rbB, rbC, and rbD RNAs. Differentiation by nerve growth factor caused an increase in levels of all four genes in PC-12. Our data give further support for the assignment of rbA, rbB, and rbC/rbD gene products as components of P-, N-, and L-type Ca2+ channels, respectively.

Protein kinase activator 1-oleoyl-2-acetyl-sn-glycerol inhibits two types of calcium currents in GH3 cells

1988 ◽  
Vol 254 (1) ◽  
pp. C206-C210 ◽  
Author(s):  
C. Marchetti ◽  
A. M. Brown
Keyword(s):  
Protein Kinase ◽  
Chick Embryo ◽  
Calcium Currents ◽  
Gh3 Cells ◽  
High Threshold ◽  
Pituitary Cells ◽  
Excitable Cells ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Low Threshold

Two types of Ca2+ currents, high-threshold, long-lasting, or L currents and low-threshold, transient, or T currents, are present in many excitable cells. L-type Ca2+ current is modulated by, among others, beta- and alpha-adrenoreceptors and intracellular Ca2+, but modulation of T-type Ca2+ current is less well established. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic activator of protein kinase C (PKC), modulates whole cell Ca2+ currents in a variety of excitable cells. Whether activators of PKC affect preferentially L and T types of Ca2+ currents is unknown. We tested OAGs effects on whole cell Ca2+ currents in the clonal GH3 line of anterior pituitary cells. The currents were measured using the whole cell patch-clamp method. Four to 60 microM OAG reversibly reduced Ca2+ currents produced by test potentials to 10 mV, and the inhibition was half maximal at approximately 25 microM. Such concentrations depress Ca2+ currents in chick embryo dorsal root ganglion (DRG) cells and clonal AtT-20 pituitary cells. To test whether OAG acted preferentially on L or T current, we separated the two using depolarizing prepulses to inactivate T current. OAG (40 microM) attenuated T currents by 60% and L currents by 50%. The current waveforms were not changed and were simply scaled, and the effects on both occurred approximately 15 s after OAG was applied. In chick embryo DRGs OAG inhibited the T current by 30% and the L current by 50%. We conclude that PKC modulates Ca2+ currents by acting on both L and T Ca2+ channels.

scholarly journals Effects of Carboxypeptidase E Overexpression on Insulin mRNA Levels, Regulated Insulin Secretion, and Proinsulin Processing of Pituitary GH3 Cells Transfected with a Furin-Cleavable Human Proinsulin cDNA

2002 ◽  
Vol 11 (8) ◽  
pp. 803-811 ◽  
Author(s):  
Luca Polastri ◽  
Francesca Galbiati ◽  
Franco Folli ◽  
Alberto M. Davalli
Keyword(s):  
Insulin Secretion ◽  
Cell Lines ◽  
Secretory Granules ◽  
Gh3 Cells ◽  
Soluble Form ◽  
Mrna Levels ◽  
Carboxypeptidase E ◽  
Proinsulin Processing ◽  
Membrane Bound ◽  
Overexpressing Cell

We recently developed two rat pituitary GH3 cell clones engineered to secrete human insulin (InsGH3). InsGH3 cells convert proinsulin into mature insulin, which is partially stored into a readily releasable pool of secretory granules. The efficiency of these processes, however, is relatively low in these cells, either in vitro or in vivo. This study was aimed at determining whether carboxypeptidase E (Cpe) overexpression can increase proinsulin processing and regulated secretion by InsGH3 clones. Indeed, in its membrane-bound form Cpe works as sorting receptor for the regulated secretory pathway of many hormones while, in its soluble form, Cpe takes part to the late step of insulin maturation. We obtained two Cpe-overexpressing cell lines from two different InsGH3 clones (InsGH3/C1 and C7). In the Cpe-overexpressing cell lines, derived from InsGH3 of clone 1 (InsGH3/C1-HACpe), in which the membrane-bound form of exogenous Cpe is accounted for by 90% of total Cpe immunoreactivity, we observed an increase in proinsulin gene expression, and in basal and stimulated insulin secretion compared with the original clone. In contrast, in the Cpe-overexpressing cell line derived from InsGH3 of clone 7 (InsGH3/C7-HACpe), where the exogenous membrane-bound form was only 60% of total Cpe, we detected a decrease in basal insulin release and a modest, albeit significant, increase in intracellular proinsulin processing. In conclusion, Cpe overexpression can increase regulated insulin secretion and proinsulin processing in InsGH3 cells; however, such improvements appear quantitatively and qualitatively modest.

Dihydropyridine- and Neurotoxin-Sensitive and -Insensitive Calcium Currents in Acutely Dissociated Neurons of the Rat Central Amygdala

1997 ◽  
Vol 77 (2) ◽  
pp. 690-701 ◽  
Author(s):  
Baojian Yu ◽  
Patricia Shinnick-Gallagher
Keyword(s):  
Calcium Current ◽  
Calcium Currents ◽  
Similar Proportion ◽  
Central Amygdala ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Moderate Rate ◽  
Rate Of Decay ◽  
P Type

Yu, Baojian and Patricia Shinnick-Gallagher. Dihydropyridine- and neurotoxin-sensitive and -insensitive calcium currents in acutely dissociated neurons of the rat central amygdala. J. Neurophysiol. 77: 690–701, 1997. The central amygdala (CeA) is an area involved in emotional learning and stress, and identification of Ca2+ currents is essential to understanding interneuronal communication through this nucleus. The purpose of this study was to separate and characterize dihydropyridine (DHP)- and neurotoxin-sensitive and -resistant components of the whole cell Ca2+ current ( I Ca) in acutely dissociated rat CeA neurons with the use of whole cell patch-clamp recording. Saturating concentrations of nimodipine (NIM, 5 μM), a DHP antagonist, blocked 22% of I Ca; this NIM-sensitive (L-type) current was recorded in 68% of CeA neurons. The DHP agonist Bay K 8644 (5 μM) produced a 36% increase in I Ca in a similar proportion of CeA neurons (70%). ω-Conotoxin GVIA (CgTx GVIA, 1 μM) in saturating concentrations inhibited 30% of I Ca, whereas ω-agatoxin IVA (Aga IVA, 100 nM), in concentrations known to block P-type currents, did not affect I Ca. Higher concentrations of Aga IVA (1 μM) alone reduced I Ca by 34%, but in the presence of NIM (5 μM) and CgTx GVIA (1 μM) blocked only 18% of I Ca. ω-Conotoxin MVIIC (CgTx MVIIC, 250 nM) reduced I Ca by 13% in the presence of CgTx GVIA (1 μM). Application of NIM (5 mM), CgTx GVIA (1 μM), and Aga IVA (1 μM) blocked ∼67% of I Ca. A similar portion (63%) of Ca2+ current was blocked with CgTx MVIIC (250 nM) in the presence of NIM (5 μM) and CgTx GVIA (1 μM). The current resistant to NIM and the neurotoxins represented 37% of I Ca, whereas in neurons not having L-type currents the resistant current made up ∼53% of I Ca (49 ± 2%, mean ± SE). The resistant current activated at around −40 mV and peaked at ∼0 mV with half-activation and -inactivation potentials of −17 and −58 mV and slopes for activation and inactivation of −5 and 13 mV, respectively. The resistant current was sensitive to Cd2+ (IC50 = 2.5 μM) and Ni2+ (IC50 = 86 μM), was larger in Ca2+ than in Ba2+ (ratio = 1.31:1), and showed a moderate rate of decay. In summary, our results show that the high-voltage-activated calcium current in rat CeA neurons is composed of at least four pharmacologically distinct components: L-type current (NIM sensitive, 22%), N-type current (CgTx GVIA sensitive, 30%), Q-type current [Aga IVA (1 μM) and CgTx MVIIC sensitive, ∼13–18%], and a resistant current (Non-L, -N, and -Q current, 33 ∼ 37%), amounting to 37–53% of the total current. The resistant current has some electrophysiological and pharmacological characteristics in common with doe-1, α1E, and R-type calcium currents, but remains unclassified.

scholarly journals Estradiol Alters Only GAD67 mRNA Levels in Ischemic Rat Brain with No Consequent Effects on GABA

2005 ◽  
Vol 26 (4) ◽  
pp. 518-526 ◽  
Author(s):  
Hung-Dong Joh ◽  
Robin V Searles ◽  
Michael Selmanoff ◽  
Nabil J Alkayed ◽  
Raymond C Koehler ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Rat Brain ◽  
Glutamic Acid Decarboxylase ◽  
Brain Regions ◽  
Artery Occlusion ◽  
Acid Decarboxylase ◽  
Ribonuclease Protection Assay ◽  
Mrna Levels ◽  
Gaba Transporters ◽  
Ribonuclease Protection

The present study tested the hypothesis that estradiol reduces tissue infarction after middle cerebral artery occlusion (MCAO) in estradiol-deficient females by augmenting glutamic acid decarboxylase (GAD) expression and thus activity, leading to increases in γ-amino-butyric acid (GABA) tissue levels. Glutamic acid decarboxylase is the principal enzyme for GABA synthesis and has two isoforms, GAD65 and GAD67, which differ in size and cellular distribution. Rats were ovariectomized 7 to 8 days before receiving no hormone, placebo, or 25 μg estradiol via subcutaneous implant 7 to 10 days before harvesting tissue in either ischemic cohorts after 2 h of MCAO (end-ischemia) or in nonischemic cohorts. Selected cortical and striatal regions were microdissected from harvested brains. GAD65/67 mRNA levels were determined by microlysate ribonuclease protection assay. End-ischemic GABA concentrations were determined by HPLC. Steroid treatment selectively decreased ischemic cortical GAD67 mRNA levels. In most brain regions evaluated, regional GABA concentrations increased with ischemia regardless of treatment. Estradiol blocked MCAO-induced increases in GABA concentration only in dorsomedial cortex. These data suggest that estradiol repletion in ischemic rat brain selectively decreases GAD67 mRNA levels but does not alter steady-state GABA concentrations. It may be that estradiol under ischemic conditions is attenuating GABA metabolism rather than enhancing synthesis or is augmenting other aspects of GABAergic transmission such as GABA transporters and receptors.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

2021 ◽  
Vol 22 (13) ◽  
pp. 7226
Author(s):  
Violeta Stojanovska ◽  
Aneri Shah ◽  
Katja Woidacki ◽  
Florence Fischer ◽  
Mario Bauer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Cell Function ◽  
Trophoblast Cell ◽  
Mrna Levels ◽  
Trophoblast Cells ◽  
Invasion And Migration ◽  
And Migration ◽  
Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

scholarly journals Characterization of Direct Perturbations on Voltage-Gated Sodium Current by Esaxerenone, a Nonsteroidal Mineralocorticoid Receptor Blocker

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 549
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Mineralocorticoid Receptor ◽  
Sodium Current ◽  
Ionic Currents ◽  
Antagonistic Effect ◽  
Gh3 Cells ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Ic50 Value ◽  
Low Threshold

Esaxerenone (ESAX; CS-3150, Minnebro®) is known to be a newly non-steroidal mineralocorticoid receptor (MR) antagonist. However, its modulatory actions on different types of ionic currents in electrically excitable cells remain largely unanswered. The present investigations were undertaken to explore the possible perturbations of ESAX on the transient, late and persistent components of voltage-gated Na+ current (INa) identified from pituitary GH3 or MMQ cells. GH3-cell exposure to ESAX depressed the transient and late components of INa with varying potencies. The IC50 value of ESAX required for its differential reduction in peak or late INa in GH3 cells was estimated to be 13.2 or 3.2 μM, respectively. The steady-state activation curve of peak INa remained unchanged during exposure to ESAX; however, recovery of peak INa block was prolonged in the presence 3 μM ESAX. In continued presence of aldosterone (10 μM), further addition of 3 μM ESAX remained effective at inhibiting INa. ESAX (3 μM) potently reversed Tef-induced augmentation of INa. By using isosceles-triangular ramp pulse with varying durations, the amplitude of persistent INa measured at high or low threshold was enhanced by the presence of tefluthrin (Tef), in combination with the appearance of the figure-of-eight hysteretic loop; moreover, hysteretic strength of the current was attenuated by subsequent addition of ESAX. Likewise, in MMQ lactotrophs, the addition of ESAX also effectively decreased the peak amplitude of INa along with the increased current inactivation rate. Taken together, the present results provide a noticeable yet unidentified finding disclosing that, apart from its antagonistic effect on MR receptor, ESAX may directly and concertedly modify the amplitude, gating properties and hysteresis of INa in electrically excitable cells.

scholarly journals CircTP63 promotes hepatocellular carcinoma progression by sponging miR-155-5p and upregulating ZBTB18

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiantao Wang ◽  
Jinbiao Che
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Circular Rnas ◽  
Epithelial Cell Line ◽  
High Morbidity ◽  
Mrna Levels ◽  
Promoting Effect ◽  
Tumor Stages ◽  
Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear. Methods qRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18. Results circTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression. Conclusion Our data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

scholarly journals Orexin depolarizes rat hypothalamic paraventricular nucleus neurons

2001 ◽  
Vol 281 (4) ◽  
pp. R1114-R1118 ◽  
Author(s):  
Tetsuro Shirasaka ◽  
Satoshi Miyahara ◽  
Takato Kunitake ◽  
Qing-Hua Jin ◽  
Kazuo Kato ◽  
...  
Keyword(s):  
Paraventricular Nucleus ◽  
Brain Slices ◽  
Hypothalamic Paraventricular Nucleus ◽  
Dependent Manner ◽  
Patch Clamp Recording ◽  
Concentration Dependent Manner ◽  
Whole Cell Patch Clamp ◽  
Orexin Receptors ◽  
Bath Application

Orexins, also called hypocretins, are newly discovered hypothalamic peptides that are thought to be involved in various physiological functions. In spite of the fact that orexin receptors, especially orexin receptor 2, are abundant in the hypothalamic paraventricular nucleus (PVN), the effects of orexins on PVN neurons remain unknown. Using a whole cell patch-clamp recording technique, we investigated the effects of orexin-B on PVN neurons of rat brain slices. Bath application of orexin-B (0.01–1.0 μM) depolarized 80.8% of type 1 ( n = 26) and 79.2% of type 2 neurons tested ( n = 24) in the PVN in a concentration-dependent manner. The effects of orexin-B persisted in the presence of TTX (1 μM), indicating that these depolarizing effects were generated postsynaptically. Addition of Cd2+(1 mM) to artificial cerebrospinal fluid containing TTX (1 μM) significantly reduced the depolarizing effect in type 2 neurons. These results suggest that orexin-B has excitatory effects on the PVN neurons mediated via a depolarization of the membrane potential.

Error analysis of Cm measurement under the whole-cell patch-clamp recording

2010 ◽  
Vol 185 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Hao Zhang ◽  
Anlian Qu ◽  
Jie Luo ◽  
Jie Luo
Keyword(s):  
Patch Clamp ◽  
Error Analysis ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp
Sign in / Sign up

Export Citation Format

Share Document