Alcohol metabolite acetic acid activates BK channels in a pH-dependent manner and decreases calcium oscillations and exocytosis of secretory granules in rat pituitary GH3 cells

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.

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pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules

Biochemical Journal ◽

10.1042/bj3210065 ◽

1997 ◽

Vol 321 (1) ◽

pp. 65-74 ◽

Cited By ~ 40

Author(s):

Sylvie URBÉ ◽

Andrea S. DITTIÉ ◽

Sharon A. TOOZE

Keyword(s):

Membrane Fraction ◽

Secretory Granules ◽

Dependent Manner ◽

Ph Indicator ◽

Standard Curve ◽

Ph Values ◽

Secretogranin Ii ◽

Ph Dependent ◽

Golgi Network ◽

Immature Secretory Granule

We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H+-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3±0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0–5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.

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Effects of adamantane derivatives on the stability and assembly of bacteriophage PM2

Canadian Journal of Microbiology ◽

10.1139/m82-134 ◽

1982 ◽

Vol 28 (7) ◽

pp. 897-900 ◽

Cited By ~ 1

Author(s):

Soo-Young Choo ◽

Kathryn Vollherbst ◽

Alec Keith ◽

Wallace Snipes

Keyword(s):

Acetic Acid ◽

Virus Production ◽

Adamantane Derivatives ◽

Dependent Manner ◽

Ph Dependent ◽

The Stability ◽

Related Compounds

1-Adamantanamine and several related compounds were tested for their effect on the lipid-containing bacteriophage PM2. 2-Adamantanol and 1-adamantane methanol were virucidal, while 1-adamantane acetic acid inhibited virus production in a highly pH-dependent manner.

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Calcium dynamics and chromatin remodelling underlie heterogeneity in prolactin transcription

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0223 ◽

2021 ◽

Vol 66 (1) ◽

pp. 59-69

Author(s):

Claire V Harper ◽

Anne V McNamara ◽

David G Spiller ◽

Jayne C Charnock ◽

Michael R H White ◽

...

Keyword(s):

Histone Acetylation ◽

Transcriptional Response ◽

Time Lapse ◽

Calcium Oscillations ◽

Gh3 Cells ◽

Pituitary Cells ◽

Calcium Activity ◽

Calcium Indicator ◽

Rat Pituitary ◽

Measured Activity

Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.

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Sapecin B, a novel fly toxin, blocks macroscopic K+ currents in the GH3 rat pituitary cell line

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.1.c289 ◽

1997 ◽

Vol 273 (1) ◽

pp. C289-C296 ◽

Cited By ~ 92

Author(s):

N. Suzuki ◽

M. Hirono ◽

K. Kawahara ◽

T. Yoshioka

Keyword(s):

Inhibitory Concentration ◽

Cell Voltage ◽

Scorpion Venom ◽

Bk Channels ◽

Gh3 Cells ◽

Pituitary Cells ◽

Dependent Manner ◽

20 Nm ◽

Dose Dependent ◽

K Currents

Sapecin B is structurally homologous to charybdotoxin (CTX), which is found in scorpion venom. This study investigated the effects of sapecin B on the Ca(2+)-activated K+ currents [IK(Ca)] and the rapidly inactivating K+ currents in clonal rat GH3 pituitary cells with whole cell voltage-clamp methods. Sapecin B (20 nM) reversibly blocked the CTX-sensitive Ix(Ca) (the BK current) in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 0.9 nM, comparable to that of 0.08-0.4 nM for CTX. The Ca2+ currents in GH3 cells, however, were not affected by sapecin B (40 nM), indicating that the blockade of IK(Ca) by sapecin B is not a secondary effect of Ca2+ current inhibition. The effect of sapecin B on IK(Ca) resembled that of CTX, as expected from the structural similarities shared by CTX and sapecin B. We also found that sapecin B largely inhibited the 4-aminopyridine-sensitive, rapidly inactivating K+ currents in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 40 nM, whereas CTX had little effect on this current in GH3 cells. Sapecin B may thus provide a useful tool, complementary to CTX, for probing the functional role of molecular domains in the BK channels and the structural similarities common to the BK and the rapidly inactivating A-type K+ channels.

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Pituitary Follicular Cells: Their Origin, Possible Function and Fate

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050184 ◽

1974 ◽

Vol 32 ◽

pp. 34-35

Author(s):

E. Horvath ◽

K. Kovacs ◽

G. Penz ◽

C. Ezrin

Keyword(s):

Fine Structure ◽

Secretory Granules ◽

Follicular Cells ◽

Human Pituitary ◽

Rat Pituitary ◽

Pituitary Glands ◽

Junctional Complexes

Follicular structures, in the rat pituitary, composed of cells joined by junctional complexes and possessing few organelles and few, if any, secretory granules, were first described by Farquhar in 1957. Cells of the same description have since been observed in several species including man. The importance of these cells, however, remains obscure. While studying human pituitary glands, we have observed wide variations in the fine structure of follicular cells which may lead to a better understanding of their morphogenesis and significance.

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Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

10.26434/chemrxiv.7985618.v1 ◽

2019 ◽

Author(s):

Adrian Roitberg ◽

Pancham Lal Gupta

Keyword(s):

Transfer Reaction ◽

Catalytic Mechanism ◽

Ph Dependence ◽

Ph Effects ◽

Dependent Manner ◽

E Coli ◽

Gar Tfase ◽

Activity Curve ◽

Ph Dependent ◽

Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

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Correlation of membrane protein conformational and functional dynamics

Nature Communications ◽

10.1038/s41467-021-24660-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Raghavendar Reddy Sanganna Gari ◽

Joel José Montalvo‐Acosta ◽

George R. Heath ◽

Yining Jiang ◽

Xiaolong Gao ◽

...

Keyword(s):

Membrane Protein ◽

Conformational Changes ◽

Single Channel ◽

Conformational Dynamics ◽

Md Simulations ◽

High Temporal Resolution ◽

Dependent Manner ◽

Functional Dynamics ◽

Ph Dependent ◽

Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

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Volatile 1-octanol of tea (Camellia sinensis L.) fuels cell division and indole-3-acetic acid production in phylloplane isolate Pseudomonas sp. NEEL19

Scientific Reports ◽

10.1038/s41598-021-82442-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Poovarasan Neelakandan ◽

Chiu-Chung Young ◽

Asif Hameed ◽

Yu-Ning Wang ◽

Kui-Nuo Chen ◽

...

Keyword(s):

Acetic Acid ◽

Cell Division ◽

Petri Dish ◽

Gas Chromatography Mass Spectrometry ◽

Dependent Manner ◽

Tea Leaves ◽

Acetic Acid Production ◽

Iaa Production ◽

Solvent Tolerant ◽

Indole 3 Acetic Acid

AbstractTea leaves possess numerous volatile organic compounds (VOC) that contribute to tea’s characteristic aroma. Some components of tea VOC were known to exhibit antimicrobial activity; however, their impact on bacteria remains elusive. Here, we showed that the VOC of fresh aqueous tea leaf extract, recovered through hydrodistillation, promoted cell division and tryptophan-dependent indole-3-acetic acid (IAA) production in Pseudomonas sp. NEEL19, a solvent-tolerant isolate of the tea phylloplane. 1-octanol was identified as one of the responsible volatiles stimulating cell division, metabolic change, swimming motility, putative pili/nanowire formation and IAA production, through gas chromatography-mass spectrometry, microscopy and partition petri dish culture analyses. The bacterial metabolic responses including IAA production increased under 1-octanol vapor in a dose-dependent manner, whereas direct-contact in liquid culture failed to elicit such response. Thus, volatile 1-octanol emitting from tea leaves is a potential modulator of cell division, colonization and phytohormone production in NEEL19, possibly influencing the tea aroma.

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pH-responsive antibodies for therapeutic applications

Journal of Biomedical Science ◽

10.1186/s12929-021-00709-7 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Tomasz Klaus ◽

Sameer Deshmukh

Keyword(s):

Drug Delivery ◽

Treatment Outcome ◽

Tumor Microenvironment ◽

Dependent Manner ◽

Therapeutic Antibodies ◽

Ph Responsive ◽

Biologic Drugs ◽

Car T ◽

Ph Dependent ◽

The Brain

AbstractTherapeutic antibodies are instrumental in improving the treatment outcome for certain disease conditions. However, to enhance their efficacy and specificity, many efforts are continuously made. One of the approaches that are increasingly explored in this field are pH-responsive antibodies capable of binding target antigens in a pH-dependent manner. We reviewed suitability and examples of these antibodies that are functionally modulated by the tumor microenvironment. Provided in this review is an update about antigens targeted by pH-responsive, sweeping, and recycling antibodies. Applicability of the pH-responsive antibodies in the engineering of chimeric antigen receptor T-cells (CAR-T) and in improving drug delivery to the brain by the enhanced crossing of the blood–brain barrier is also discussed. The pH-responsive antibodies possess strong treatment potential. They emerge as next-generation programmable engineered biologic drugs that are active only within the targeted biological space. Thus, they are valuable in targeting acidified tumor microenvironment because of improved spatial persistence and reduced on-target off-tumor toxicities. We predict that the programmable pH-dependent antibodies become powerful tools in therapies of cancer.

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