scholarly journals The internal pH and membrane potential of the insulin-secretory granule

1982 ◽  
Vol 204 (1) ◽  
pp. 171-178 ◽  
Author(s):  
John C. Hutton
Keyword(s):  
Membrane Potential ◽  
Secretory Granule ◽  
Atpase Inhibitor ◽  
Dependent Atpase ◽  
Insulin Granules ◽  
Internal Ph ◽  
Insulin Secretory Granule ◽  
Permeant Anion

The membrane potential (ΔΨ) and the pH gradient (ΔpH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[14C]cyanate or the permeant base [14C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a ΔΨ positive inside. Addition of MgATP2− under these conditions did not alter the ΔpH, but produced a marked increase in the ΔΨ. Conversely, when a permeant anion was also included, ATP produced a marked increase in the ΔpH and a lesser increment in the ΔΨ. NH4+ salts reduced the ΔpH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the ΔΨ. A similar reciprocity was evident when nigericin was added together with K+ or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K+, and by the Mg2+-dependent ATPase inhibitor tributyltin. Uptakes of 14C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg2+-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc–insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.

scholarly journals Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

2021 ◽  
Vol 7 (2) ◽  
pp. 130
Author(s):  
Nathan P. Wiederhold
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Candida Species ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Fungal Infections ◽  
Published Data ◽  
Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

scholarly journals Imaging the transmembrane and transendothelial sodium gradients in gliomas

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Muhammad H. Khan ◽  
John J. Walsh ◽  
Jelena M. Mihailović ◽  
Sandeep K. Mishra ◽  
Daniel Coman ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Magnetic Resonance ◽  
Cell Membrane ◽  
Normal Tissue ◽  
Magnetic Resonance Spectroscopic Imaging ◽  
Biological Factors ◽  
High Sodium ◽  
Intracellular Milieu

AbstractUnder normal conditions, high sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, and these manifest the cell membrane potential (Vm) as well as blood–brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa+mem and ΔNa+end. In vitro 23Na-MRSI established that the 23Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together revealed weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na+end suggesting altered BBB integrity. We anticipate that 23Na-MRSI will allow biomedical explorations of perturbed Na+ homeostasis in vivo.

scholarly journals ELAPOR1 is a secretory granule maturation-promoting factor that is lost during paligenosis

2021 ◽  
Author(s):  
Charles J. Cho ◽  
Dongkook Park ◽  
Jason C. Mills
Keyword(s):  
Transcription Factor ◽  
Secretory Granule ◽  
Cultured Cells ◽  
Tissue Injury ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Spectrometric Analysis ◽  
Granule Maturation

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly-studied MIST1 target, ELAPOR1, is an evolutionarily conserved, novel Mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (ie, to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis-Golgi resident proteins and with the trans-Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1−/− mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1−/− ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantles it during paligenosis.

Temporal constraints in associative synaptic plasticity in hippocampus and neocortex

1995 ◽  
Vol 73 (9) ◽  
pp. 1295-1311 ◽  
Author(s):  
Dominique Debanne ◽  
Daniel E. Shulz ◽  
Yves Frégnac
Keyword(s):  
Visual Cortex ◽  
Membrane Potential ◽  
Temporal Frequency ◽  
Postsynaptic Membrane ◽  
Synaptic Potentiation ◽  
Test Input ◽  
Homosynaptic Depression ◽  
Postsynaptic Cell

We present comparative experimental evidence for the induction of synaptic potentiation and depression in organotypic cultures of hippocampus and in visual cortex in vitro and in vivo. The effects of associative pairings on the efficacy of synaptic transmission are analyzed as a function of the temporal delay between presynaptic activity and post-synaptic changes imposed in membrane potential. Synchronous association at a low temporal frequency (<0.5 Hz) between presynaptic input and postsynaptic depolarization resulted in homosynaptic potentiation of functionally identified postsynaptic potentials in the three types of preparation. Synchronous pairing of afferent activity with hyperpolarization of the postsynaptic cell resulted in homosynaptic depression in visual cortex in vivo and in vitro. An associative form of depression was induced in hippocampus when the test input was followed repeatedly with a fixed-delay postsynaptic depolarization imposed either by intracellular current injection or synaptically. The latter process might play a significant role in heterosynaptic plasticity in visual cortex in vivo and in vitro, if it is assumed that associative depression still operates in visual cortex a few seconds after the initial surge of calcium in the postsynaptic cell. We conclude that the precise timing between presynaptic activity and polarization changes in postsynaptic membrane potential up- and down-regulates the efficacy of active pathways.Key words: synaptic potentiation, synaptic depression, asynchrony, covariance, supervised learning.

scholarly journals Meloxicam, a Selective COX-2 Inhibitor, Mediates Hypoxia-Inducible Factor- (HIF-) 1α Signaling in Hepatocellular Carcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Yinghong Zhou ◽  
Xiaofeng Dong ◽  
Peng Xiu ◽  
Xin Wang ◽  
Jianrong Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Transcriptional Activation ◽  
Mitochondrial Membrane ◽  
Hypoxia Inducible Factor ◽  
Luciferase Assay ◽  
Cox 2

Hepatocellular carcinoma (HCC) is regarded as a leading cause of cancer-related deaths, and its progression is associated with hypoxia and the induction of hypoxia-inducible factor (HIF). Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, induces cell death in various malignancies. However, the underlying mechanism remains to be elucidated in HCC, especially under hypoxic conditions. The alteration of COX-2 and HIF-1α oncogenicity was evaluated in HCC specimens by tissue microarray. Cell viability, angiogenesis assays, and xenografted nude mice were used to evaluate the effects of meloxicam, along with flow cytometry to detect the cell cycle, apoptosis, and mitochondrial membrane potential (ΔΨm) of HCC. qRT-PCR, Western blotting, immunofluorescence, immunohistochemistry, luciferase assay, and RNAi were carried out to determine the HIF-1α signaling affected by meloxicam. In this study, we showed that meloxicam exerts antiproliferative and antiangiogenesis efficacy in vitro and in vivo and causes disruption of mitochondrial membrane potential (ΔΨm), thus leading to caspase-dependent apoptosis under hypoxic environments. Exposure to meloxicam significantly reduced HIF-1α transcriptional activation and expression through sequestering it in the cytoplasm and accelerating degradation via increasing the von Hippel-Lindau tumor suppressor protein (pVHL) in HCC. These data demonstrated that inhibition of HIF-1α by meloxicam could suppress angiogenesis and enhance apoptosis of HCC cells. This discovery highlights that COX-2 specific inhibitors may be a promising therapy in the treatment of HCC.

Milk Ejection Burst-Like Electrical Activity Evoked in Supraoptic Oxytocin Neurons in Slices From Lactating Rats

2004 ◽  
Vol 91 (5) ◽  
pp. 2312-2321 ◽  
Author(s):  
Yu-Feng Wang ◽  
Glenn I. Hatton
Keyword(s):  
Membrane Potential ◽  
Firing Rate ◽  
Membrane Conductance ◽  
Peak Level ◽  
Sudden Increase ◽  
Milk Ejection ◽  
Peak Rate ◽  
Vasopressin Neurons

To examine the mechanisms underlying milk-ejection bursts of oxytocin (OT) neurons during suckling, both in vivo and in vitro studies were performed on supraoptic OT neurons from lactating rats. The bursts were first recorded extracellularly in anesthetized rats. Burst-related electrical parameters were essentially the same as previous reports except for a trend toward transient decreases in basal firing rates immediately preceding the burst. From putative OT neurons in slices with extracellular recordings, bursts that closely mimicked the in vivo bursts were elicited by phenylephrine, an α1-adrenoceptor agonist, in a low-Ca2+ medium. Moreover, in whole cell patch-clamp recordings, the in vitro bursts were recorded from immunocytochemically identified OT neurons. After a transient decrease in the basal firing rate, the in vitro bursts started with a sudden increase in the firing rate, quickly reaching a peak level, then gradually decaying, and ended with a postburst inhibition. A brief depolarization of the membrane potential and an increase in membrane conductance appeared after the onset of the burst. Spikes during a burst were characterized by a significant increase in the duration and decrease in the amplitude around the peak rate firing. These bursts were significantly different from short-lasting burst firing of vasopressin neurons in membrane potential changes, time to reach peak firing rate, spike amplitude and duration during peak rate firing. Our extensive analysis of these results suggests that the in vitro burst is a useful model for further study of mechanisms underlying milk-ejection bursts of OT neurons in vivo.

Evaluation of anticancer activity in vitro and in vivo of iridium(iii) polypyridyl complexes

2019 ◽  
Vol 43 (22) ◽  
pp. 8566-8579 ◽  
Author(s):  
Miao He ◽  
Qiao-Yan Yi ◽  
Wen-Yao Zhang ◽  
Lan Bai ◽  
Fan Du ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Cytotoxic Activity ◽  
Anticancer Activity ◽  
Cell Cycle Arrest ◽  
Mitochondrial Membrane ◽  
Polypyridyl Complexes ◽  
Cycle Arrest

Three new iridium(iii) polypyridyl complexes were synthesized. The cytotoxic activity in vitro and in vivo, apoptosis, cell cycle arrest, mitochondrial membrane potential, ROS and the expression of Bcl-2 family proteins were investigated.

scholarly journals A Method to Estimate Synaptic Conductances From Membrane Potential Fluctuations

2004 ◽  
Vol 91 (6) ◽  
pp. 2884-2896 ◽  
Author(s):  
Michael Rudolph ◽  
Zuzanna Piwkowska ◽  
Mathilde Badoual ◽  
Thierry Bal ◽  
Alain Destexhe
Keyword(s):  
Membrane Potential ◽  
Computational Models ◽  
Network Activity ◽  
Intracellular Recordings ◽  
Neocortical Neurons ◽  
Underlying Network ◽  
Analytic Expression ◽  
Synaptic Conductances

In neocortical neurons, network activity can activate a large number of synaptic inputs, resulting in highly irregular subthreshold membrane potential ( Vm) fluctuations, commonly called “synaptic noise.” This activity contains information about the underlying network dynamics, but it is not easy to extract network properties from such complex and irregular activity. Here, we propose a method to estimate properties of network activity from intracellular recordings and test this method using theoretical and experimental approaches. The method is based on the analytic expression of the subthreshold Vm distribution at steady state in conductance-based models. Fitting this analytic expression to Vm distributions obtained from intracellular recordings provides estimates of the mean and variance of excitatory and inhibitory conductances. We test the accuracy of these estimates against computational models of increasing complexity. We also test the method using dynamic-clamp recordings of neocortical neurons in vitro. By using an on-line analysis procedure, we show that the measured conductances from spontaneous network activity can be used to re-create artificial states equivalent to real network activity. This approach should be applicable to intracellular recordings during different network states in vivo, providing a characterization of the global properties of synaptic conductances and possible insight into the underlying network mechanisms.

Ratiometric measurement of endothelial depolarization in arterioles with a potential-sensitive dye

1996 ◽  
Vol 270 (6) ◽  
pp. H2216-H2227 ◽  
Author(s):  
J. M. Beach ◽  
E. D. McGahren ◽  
J. Xia ◽  
B. R. Duling
Keyword(s):  
Membrane Potential ◽  
Fluorescence Emission ◽  
Cheek Pouch ◽  
Voltage Sensitivity ◽  
Hamster Cheek Pouch ◽  
Endothelial Cell Layer ◽  
Ratiometric Measurement ◽  
Length Constant

A fluorescence ratio technique based on the voltage-sensitive dye 1-(3-sulfonatopropyl)-8-[beta-[2-di-n-butylamino)-6-naphythyl++ +]vinyl] pyridinium betaine (di-8-ANEPPS)has been developed for recording membrane potential changes during vascular responses of arterioles. Perfusion of hamster cheek pouch arterioles with the dye labeled the endothelial cell layer. voltage responses from the endothelium of intact arterioles were determined by analysis of voltage-induced shifts in fluorescence emission wavelengths from dye spectra imaged from the vessel wall. Membrane depolarization caused the dye spectrum to shift toward blue wavelengths, with maximal fluorescence changes near 560 and 620 nm. In isolated nonperfused arterioles, comparison of continuous dual-wavelength recordings with simultaneous microelectrode recordings showed that the ratio of fluorescence intensities (fluorescence at 620 nm to fluorescence at 560 nm) accurately followed changes in membrane potential (6–21 mV) during vasoconstriction. The dye response was linear with respect to potential changes from -56 to -6 mV, with a voltage sensitivity of 9.7% change in the ratio per 100 mV. Membrane potential responses from in vitro and in vivo arterioles after potassium stimulation consisted of rapid ( < 0.5 -s) depolarization followed by slow repolarization over several seconds. Potassium-induced depolarizations were conducted along arterioles, and the values of the electrical length constant for conducted depolarization determined by optical and microelectrode methods were in agreement. We conclude that ratio analysis of di-8-ANEPPS fluorescence emission can be used to accurately record membrane potential changes on the time scale of seconds during vasomotor activity from arterioles.

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