scholarly journals Determining the pharmacokinetics of nicotinic drugs in the endoplasmic reticulum using biosensors

2019 ◽  
Vol 151 (6) ◽  
pp. 738-757 ◽  
Author(s):  
Amol V. Shivange ◽  
Philip M. Borden ◽  
Anand K. Muthusamy ◽  
Aaron L. Nichols ◽  
Kallol Bera ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Dopaminergic Neurons ◽  
Hippocampal Neurons ◽  
Cell Types ◽  
Early Event ◽  
Pharmacological Chaperone ◽  
Fluorescent Biosensors ◽  
Nicotinic Drugs ◽  
Inside Out

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an “inside-out” pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell–derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker’s day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

scholarly journals Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level.

1994 ◽  
Vol 42 (7) ◽  
pp. 917-929 ◽  
Author(s):  
E Spiess ◽  
A Brüning ◽  
S Gack ◽  
B Ulbricht ◽  
H Spring ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Tumor Cell ◽  
Enzymatic Activity ◽  
Cell Lines ◽  
Cathepsin B ◽  
Lung Tumor ◽  
Cell Types ◽  
Cellular Level ◽  
Tumor Cell Lines

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.

Ultrastructure of the fusion cell in Faucheocolax attenuata Setch. (Rhodophyta, Rhodymeniales)

1984 ◽  
Vol 72 (1) ◽  
pp. 307-319
Author(s):  
S.G. Delivopoulos ◽  
P. Kugrens
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Basal Cell ◽  
Red Algae ◽  
Helical Structure ◽  
Cell Types ◽  
Floridean Starch ◽  
Auxiliary Cell ◽  
Shaped Cell ◽  
Fusion Cell

The fusion cell in Faucheocolax attenuata Setch. is a highly lobed, thick-walled, multinucleate and irregularly shaped cell originating from the basal cell of the auxiliary cell branch. The formation of the fusion cell occurs by an incorporation of vegetative cells into the basal cell, after dissolution of septal plugs between these cell types. Thus the fusion cell is a syncytium containing only haploid nuclei, as well as unusual mitochondria and plastids. Mitochondria lack cristae and instead contain a tubular helical structure. Plastids are atypical with regard to thylakoid organization in red algae, because they lack the peripheral thylakoid and their photosynthetic thylakoids are aggregated to one side. In addition, they contain large osmiophilic bodies. Nuclear envelopes appear to produce large quantities of membrane cisternae. Floridean starch is absent and the cytoplasm contains few ribosomes. The plasma membrane is irregular and endoplasmic reticulum cisternae are situated parallel to it. Bundles of putative microfilaments were commonly found in nuclei and the cytoplasm. Structural evidence does not support any meristematic, nutritive or secretory functions previously ascribed to fusion cells in other genera.

scholarly journals Intracellular labeling with ferritin conjugates. A specificity problem due to the affinity of unconjugated ferritin for selected intracellular sites.

1979 ◽  
Vol 27 (7) ◽  
pp. 1095-1102 ◽  
Author(s):  
E L Parr
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Phosphate Buffer ◽  
Plasma Membranes ◽  
Cell Types ◽  
Purification Procedure ◽  
Nonspecific Binding ◽  
Wide Range ◽  
Antibody Conjugates

Nonspecific binding of ferritin to chromatin and the cytoplasmic aspect of the nuclear envelope was observed when nonantigenic, serum-washed hepatocyte nuclei were incubated in ferritin-antibody conjugates. This labeling was duplicated when nuclei from a wide range of species and cell types were exposed to unconjugated ferritin. Unconjugated ferritin binding to nuclei did not depend on a subpopulation of denatured molecules or on the ferritin purification procedure. Binding occurred equally on unfixed and formaldehyde-fixed nuclei, but no ferritin bound to glutaraldehyde-fixed nuclei. Inconjugated ferritin also bound to the cytoplasmic aspects of the rough endoplasmic reticulum and the plasma membrane. The tracer did not bind to lysosomes, mitochondria, Golgi vesicles, the extracellular surface of plasma membranes, or the intracisternal surfaces of ruptured nuclear envelopes. The addition of 0.4 M KCl or 0.7 M NaCl to ferritin solutions and washing media at neutral pH reduced the binding of conjugated and unconjugated ferritin to nuclei to about 3% of that seen in 0.10 M phosphate buffer alone. The added salts caused little extraction of nuclear contents from formaldehyde-fixed nuclei. The use of one of these salts in ferritin conjugates should considerably improve the specificity of intracellular labeling.

scholarly journals Regulation of neuronal excitation–transcription coupling by Kv2.1-induced clustering of somatic L-type Ca2+ channels at ER-PM junctions

2021 ◽  
Vol 118 (46) ◽  
pp. e2110094118
Author(s):  
Nicholas C. Vierra ◽  
Samantha C. O’Dwyer ◽  
Collin Matsumoto ◽  
L. Fernando Santana ◽  
James S. Trimmer
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Hippocampal Neurons ◽  
Mammalian Brain ◽  
Voltage Gated ◽  
Neuronal Excitation ◽  
C Terminus ◽  
Cav1.2 Channels ◽  
Channel Dependent

In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation–transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation–transcription coupling. The voltage-gated K+ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum–plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation–transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1’s C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum–plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.

scholarly journals PACmn for improved optogenetic control of intracellular cAMP

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shang Yang ◽  
Oana M. Constantin ◽  
Divya Sachidanandan ◽  
Hannes Hofmann ◽  
Tobias C. Kunz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Hippocampal Neurons ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cell Types ◽  
Camp Signaling ◽  
Intracellular Camp ◽  
Extracellular Signals ◽  
Starting Point

Abstract Background Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. Results Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). Conclusions PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.

scholarly journals S-acylation of Orai1 regulates store-operated Ca2+ entry

2021 ◽  
Vol 135 (5) ◽  
Author(s):  
Savannah J. West ◽  
Goutham Kodakandla ◽  
Qioachu Wang ◽  
Ritika Tewari ◽  
Michael X. Zhu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Types ◽  
Central Component ◽  
Channel Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Crac Channel ◽  
Underlying Mechanisms ◽  
Different Cell Types

ABSTRACT Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER–PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER–PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER–PM junctions, subsequent binding to STIM1 and channel activation.

Recruitment of NAADP-sensitive acidic Ca2+ stores by glutamate

2009 ◽  
Vol 422 (3) ◽  
pp. 503-512 ◽  
Author(s):  
Vinita Pandey ◽  
Chia-Chen Chuang ◽  
Alexander M. Lewis ◽  
Parvinder K. Aley ◽  
Eugen Brailoiu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nicotinic Acid ◽  
Hippocampal Neurons ◽  
Second Messenger ◽  
Cell Types ◽  
The Brain ◽  
Acidic Compartments

NAADP (nicotinic acid–adenine dinucleotide phosphate) is an unusual second messenger thought to mobilize acidic Ca2+ stores, such as lysosomes or lysosome-like organelles, that are functionally coupled to the ER (endoplasmic reticulum). Although NAADP-sensitive Ca2+ stores have been described in neurons, the physiological cues that recruit them are not known. Here we show that in both hippocampal neurons and glia, extracellular application of glutamate, in the absence of external Ca2+, evoked cytosolic Ca2+ signals that were inhibited by preventing organelle acidification or following osmotic bursting of lysosomes. The sensitivity of both cell types to glutamate correlated well with lysosomal Ca2+ content. However, interfering with acidic compartments was largely without effect on the Ca2+ content of the ER or Ca2+ signals in response to ATP. Glutamate but not ATP elevated cellular NAADP levels. Our results provide evidence for the agonist-specific recruitment of NAADP-sensitive Ca2+ stores by glutamate. This links the actions of NAADP to a major neurotransmitter in the brain.

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

Prevalence of the pit and antipit in the genus Glycine

1987 ◽  
Vol 45 ◽  
pp. 986-987
Author(s):  
R. W. Yaklich ◽  
E. L. Vigil ◽  
W. P. Wergin
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Seed Coat ◽  
Cell Types ◽  
Abaxial Surface ◽  
Legume Seed ◽  
Initial Report ◽  
Cone Cells ◽  
Glycine Max L

The legume seed coat is the site of sucrose unloading and the metabolism of imported ureides and synthesis of amino acids for the developing embryo. The cell types directly responsible for these functions in the seed coat are not known. We recently described a convex layer of tissue on the inside surface of the soybean (Glycine max L. Merr.) seed coat that was termed “antipit” because it was in direct opposition to the concave pit on the abaxial surface of the cotyledon. Cone cells of the antipit contained numerous hypertrophied Golgi apparatus and laminated rough endoplasmic reticulum common to actively secreting cells. The initial report by Dzikowski (1936) described the morphology of the pit and antipit in G. max and found these structures in only 68 of the 169 seed accessions examined.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

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