scholarly journals Calmodulin Antagonist W-7 Enhances Intermediate Conductance Ca2+- Sensitive Basolateral Potassium Channel (IKCa) Activity in Human Colonic Crypts

2021 ◽  
Author(s):  
Kate A. Bowley ◽  
Geoffrey I. Sandle
Keyword(s):  
Colonic Crypt ◽  
Channel Activity ◽  
Calmodulin Antagonist ◽  
Colonic Crypts ◽  
Intracellular Ca ◽  
Normal Human ◽  
Channel Dependent ◽  
High Level ◽  
Carboxyl Tail ◽  
Colonic Biopsies

AbstractIntermediate conductance potassium (IKCa) channels are exquisitively Ca2+ sensitive, intracellular Ca2+ regulating channel activity by complexing with calmodulin (CaM), which is bound to the cytosolic carboxyl tail. Although CaM antagonists might be expected to decrease IKCa channel activity, the effect of W-7 in human T lymphocytes are conflicting. We therefore evaluated the effect of W-7 on basolateral IKCa channels in human colonic crypt cells. Intact crypts obtained from normal human colonic biopsies by Ca2+ chelation were used for patch clamp studies of basolateral IKCa channels in the cell-attached configuration. IKCa channel activity was studied when the bath Ca2+ concentration was changed from 1.2 mmol/L to 100 μmol/L and back to 1.2 mmol/L, as well as from 100 μmol/L to 1.2 mmol/L and back to 100 μmol/L, both in the absence and presence of 25 μmol/L W-7. Decreasing bath Ca2+ from 1.2 mmol/L to 100 μmol/L decreased IKCa channel activity reversibly in the absence of W-7, whereas there was a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. In separate experiments, increasing bath Ca2+ from 100 μmol/L to 1.2 mmol/L increased IKCa channel activity reversibly in the absence of W-7, whereas there was again a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. We, therefore, propose that W-7 has a specific stimulatory effect on basolateral IKCa channel activity, despite its ability to inhibit Ca2+/CaM-mediated, IKCa channel-dependent Cl− secretion in human colonic epithelial cells. Graphic Abstract

Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts

1999 ◽  
Vol 277 (5) ◽  
pp. G967-G975 ◽  
Author(s):  
Geoffrey I. Sandle ◽  
Geoffrey Warhurst ◽  
Ian Butterfield ◽  
Norman B. Higgs ◽  
Richard B. Lomax
Keyword(s):  
G Protein ◽  
Basolateral Membrane ◽  
Secretory Process ◽  
Synthetic Analog ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Channel Inhibition ◽  
Colonic Crypts ◽  
Intracellular Ca ◽  
Dependent Mechanism

Somatostatin is a powerful inhibitor of intestinal Cl− secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K+ channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl− secretory process. Somatostatin (2 μM) elicited a >80% decrease in “spontaneous” K+ channel activity in cell-attached patches in nonstimulated crypts (50% inhibition =∼8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100–200 μM dibutyryl cAMP, 2 μM somatostatin and its synthetic analog octreotide (2 μM) both produced similar degrees of K+ channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl− (5 mM) conditions. In addition, 2 μM somatostatin abolished the increase in K+ channel activity stimulated by 2 μM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca2+. These results indicate that somatostatin peptides inhibit 23-pS basolateral K+ channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca2+sensitivity.

scholarly journals Role of KCa3.1 Channels in Modulating Ca2+ Oscillations during Glioblastoma Cell Migration and Invasion

2018 ◽  
Vol 19 (10) ◽  
pp. 2970 ◽  
Author(s):  
Luigi Catacuzzeno ◽  
Fabio Franciolini
Keyword(s):  
Cell Migration ◽  
Theoretical Model ◽  
Migration And Invasion ◽  
Channel Activity ◽  
Cell Migration And Invasion ◽  
Channel Modulation ◽  
Invasion Mechanisms ◽  
Migratory Cycle ◽  
Channel Dependent

Cell migration and invasion in glioblastoma (GBM), the most lethal form of primary brain tumors, are critically dependent on Ca2+ signaling. Increases of [Ca2+]i in GBM cells often result from Ca2+ release from the endoplasmic reticulum (ER), promoted by a variety of agents present in the tumor microenvironment and able to activate the phospholipase C/inositol 1,4,5-trisphosphate PLC/IP3 pathway. The Ca2+ signaling is further strengthened by the Ca2+ influx from the extracellular space through Ca2+ release-activated Ca2+ (CRAC) currents sustained by Orai/STIM channels, meant to replenish the partially depleted ER. Notably, the elevated cytosolic [Ca2+]i activates the intermediate conductance Ca2+-activated K (KCa3.1) channels highly expressed in the plasma membrane of GBM cells, and the resulting K+ efflux hyperpolarizes the cell membrane. This translates to an enhancement of Ca2+ entry through Orai/STIM channels as a result of the increased electromotive (driving) force on Ca2+ influx, ending with the establishment of a recurrent cycle reinforcing the Ca2+ signal. Ca2+ signaling in migrating GBM cells often emerges in the form of intracellular Ca2+ oscillations, instrumental to promote key processes in the migratory cycle. This has suggested that KCa3.1 channels may promote GBM cell migration by inducing or modulating the shape of Ca2+ oscillations. In accordance, we recently built a theoretical model of Ca2+ oscillations incorporating the KCa3.1 channel-dependent dynamics of the membrane potential, and found that the KCa3.1 channel activity could significantly affect the IP3 driven Ca2+ oscillations. Here we review our new theoretical model of Ca2+ oscillations in GBM, upgraded in the light of better knowledge of the KCa3.1 channel kinetics and Ca2+ sensitivity, the dynamics of the Orai/STIM channel modulation, the migration and invasion mechanisms of GBM cells, and their regulation by Ca2+ signals.

Cerebral artery KATP- and KCa-channel activity and contractility: changes with development

2000 ◽  
Vol 279 (6) ◽  
pp. R2004-R2014 ◽  
Author(s):  
Wen Long ◽  
Lubo Zhang ◽  
Lawrence D. Longo
Keyword(s):  
Channel Blocker ◽  
Cerebral Arteries ◽  
Dependent Manner ◽  
Channel Activity ◽  
Channel Opener ◽  
Channel Inhibition ◽  
Middle Cerebral Arteries ◽  
Intracellular Ca ◽  
Near Term ◽  
Nonpregnant Adult

The present study was designed to test the hypothesis that in cerebral arteries of the fetus, ATP-sensitive (KATP) and Ca2+-activated K+channels (KCa) play an important role in the regulation of intracellular Ca2+ concentration ([Ca2+]i) and that this differs significantly from that of the adult. In main branch middle cerebral arteries (MCA) from near-term fetal (∼140 days) and nonpregnant adult sheep, simultaneously we measured norepinephrine (NE)-induced responses of vascular tension and [Ca2+]i in the absence and presence of selective K+-channel openers/blockers. In fetal MCA, in a dose-dependent manner, both the KATP-channel opener pinacidil and the KCa-channel opener NS 1619 significantly inhibited NE-induced tension [negative logarithm of the half-maximal inhibitory concentration (pIC50) = 5.0 ± 0.1 and 8.2 ± 0.1, respectively], with a modest decrease of [Ca2+]i. In the adult MCA, in contrast, both pinacidil and NS 1619 produced a significant tension decrease (pIC50 = 5.1 ± 0.1 and 7.6 ± 0.1, respectively) with no change in [Ca2+]i. In addition, the KCa-channel blocker iberiotoxin (10−7 to 10−6 M) resulted in increased tension and [Ca2+]i in both adult and fetal MCA, although the KATP-channel blocker glibenclamide (10−7 to 3 × 10−5 M) failed to do so. Of interest, administration of 10−7 M iberiotoxin totally eliminated vascular contraction and increase in [Ca2+]i seen in response to 10−5M ryanodine. In precontracted fetal cerebral arteries, activation of the KATP and KCa channels significantly decreased both tension and [Ca2+]i, suggesting that both K+ channels play an important role in regulating L-type channel Ca2+ flux and therefore vascular tone in these vessels. In the adult, KATP and the KCa channels also appear to play an important role in this regard; however, in the adult vessel, activation of these channels with resultant vasorelaxation can occur with no significant change in [Ca2+]i. These channels show differing responses to inhibition, e.g., KCa-channel inhibition, resulting in increased tension and [Ca2+]i, whereas KATP-channel inhibition showed no such effect. In addition, the KCa channel appears to be coupled to the sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma membrane K+-channel activity may account, in part, for the differences in the regulation of contractility of fetal and adult cerebral arteries.

Apical Na+/H+ exchange near the base of mouse colonic crypts

2002 ◽  
Vol 283 (1) ◽  
pp. C358-C372 ◽  
Author(s):  
Jingsong Chu ◽  
Shaoyou Chu ◽  
Marshall H. Montrose
Keyword(s):  
Epithelial Cells ◽  
Lucifer Yellow ◽  
Extracellular Fluid ◽  
Colonic Crypt ◽  
Fluid Flux ◽  
Functional Attributes ◽  
Colonic Crypts ◽  
Vectorial Transport ◽  
Crypt Base ◽  
Exchange Activity

Colonic crypts can absorb fluid, but the identity of the absorptive transporters remains speculative. Near the crypt base, the epithelial cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only Cl−secretion. We have applied confocal microscopy in combination with an extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive dye (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to study mouse colonic crypt epithelial cells directly adjacent to the crypt base within an intact mucosal sheet. Measurements of intracellular pH report activation of colonocyte Na+/H+ exchange in response to luminal or serosal Na+. Studies with LY demonstrate the presence of a paracellular fluid flux, but luminal Na+ does not activate Na+/H+ exchange in the nonepithelial cells of the lamina propria, and studies with LY suggest that the fluid bathing colonocyte basolateral membranes is rapidly refreshed by serosal perfusates. The apical Na+/H+ exchange in crypt colonocytes is inhibited equivalently by luminal 20 μM ethylisopropylamiloride and 20 μM HOE-694 but is not inhibited by luminal 20 μM S-1611. Immunostaining reveals the presence of epitopes from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of colonic crypts. Comparison of apical Na+/H+exchange activity in the presence of Cl− with that in the absence of Cl− (substitution by gluconate or nitrate) revealed no evidence of the Cl−-dependent Na+/H+ exchange that had been previously reported as the sole apical Na+/H+ exchange activity in the colonic crypt. Results suggest the presence of an apical Na+/H+ exchanger near the base of crypts with functional attributes similar to those of the cloned NHE2 isoform.

PGE2, Ca2+, and cAMP mediate ATP activation of Cl− channels in pigmented ciliary epithelial cells

2001 ◽  
Vol 281 (5) ◽  
pp. C1614-C1623 ◽  
Author(s):  
Johannes C. Fleischhauer ◽  
Claire H. Mitchell ◽  
Kim Peterson-Yantorno ◽  
Miguel Coca-Prados ◽  
Mortimer M. Civan
Keyword(s):  
Intraocular Pressure ◽  
Epithelial Cells ◽  
Aqueous Humor ◽  
Second Messengers ◽  
Atp Release ◽  
Channel Activity ◽  
Atp Assay ◽  
Intracellular Ca ◽  
Cl Channels ◽  
Nonpigmented Ciliary Epithelial

Purines regulate intraocular pressure. Adenosine activates Cl− channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl− channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl− channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl−channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 μM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2, and cAMP activated Cl− channels without enhancing effects of 100 μM ATP. Buffering intracellular Ca2+activity with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid or blocking PGE2 formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3′,5′-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3′,5′-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca2+, PGE2, and cAMP. cAMP acts directly on Cl−channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump

1993 ◽  
Vol 264 (3) ◽  
pp. F557-F564 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Feedback Regulation ◽  
Peak Level ◽  
Na Channels ◽  
Channel Activity ◽  
Electrical Measurements ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
The Mean

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Human myeloid alpha 3-fucosyltransferase is involved in the expression of the sialyl-Lewis(x) determinant, a ligand for E- and P-selectin

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2978-2986 ◽  
Author(s):  
EW Easton ◽  
WE Schiphorst ◽  
E van Drunen ◽  
CE van der Schoot ◽  
DH van den Eijnden
Keyword(s):  
Human Chromosome ◽  
Leukemic Cell ◽  
Sialyl Lewis X ◽  
Chromosome 11 ◽  
Sialyl Lewis ◽  
Sialyl Lex ◽  
Structure Flow ◽  
Normal Human ◽  
High Level

Abstract The sialyl-Lex determinant (NeuAc alpha 2-->3Gal beta 1-->4[Fuc alpha- 1-->3]GlcNAc) has been identified as a major ligand in the selectin- mediated adhesion of neutrophils and monocytes to activated endothelium or platelets. This carbohydrate epitope is formed by the sequential action of alpha 3-sialyltransferase and alpha 3-fucosyltransferase on N- acetyllactosamine (Gal beta 1-->4GlcNAc) disaccharide termini of glycoconjugates. We have addressed the role of the human myeloid alpha 3-fucosyltransferase in the expression of this epitope at the leucocyte surface by determining its activity in human-mouse leukemic cell hybrids (WEGLI), normal human granulocytes and chronic myeloid leukemia (CML) cells using sialylated and desialylated glycoproteins and oligosaccharides as acceptor substrates. In contrast to what has been reported for the myeloid-type enzyme, we found that the alpha 3- fucosyltransferase of the cells studied can use sialylated acceptors be it that the activity is several times lower than with asialo- substrates. Characterization of the product obtained with a sialylated oligosaccharide indicated that the enzyme can catalyze the formation of the sialyl-Le(x) structure. Flow cytometry of the WEGLI cells using a sialyl-Le(x)-specific monoclonal antibody (MoAb) showed that these cells indeed express sialyl-Lex at their surface, provided that they contain human chromosome 11. Earlier the presence of this chromosome had been correlated with the expression of alpha 3-fucosyltransferase activity. In addition to sialyl-Le(x), WEGLI cells containing chromosome 11 showed high-expression levels of related structures recognized by antibodies VIM-2 and VIM-8, suggesting that fucose addition can occur at both distal and proximal GlcNAc residues in poly- N-acetyl-lactosaminoglycan sequences. Based on the human chromosome contents it could be ruled out that the alpha 3-fucosyltransferase of WEGLI cells is a Lewis-type alpha 3/4- or plasma-type alpha 3- fucosyltransferase, the genes of which have been mapped to chromosome 19. It is concluded that the enzyme studied is of the myeloid-type and indeed is involved in the synthesis of sialyl-Le(x) (and also VIM-2 and VIM-8 structures) in leukocytes provided that its expression is at a sufficiently high level.

scholarly journals Enhanced Factor H Binding to Sialylated Gonococci Is Restricted to the Sialylated Lacto-N-Neotetraose Lipooligosaccharide Species: Implications for Serum Resistance and Evidence for a Bifunctional Lipooligosaccharide Sialyltransferase in Gonococci

2005 ◽  
Vol 73 (11) ◽  
pp. 7390-7397 ◽  
Author(s):  
Sunita Gulati ◽  
Andrew Cox ◽  
Lisa A. Lewis ◽  
Frank St. Michael ◽  
Jianjun Li ◽  
...  
Keyword(s):  
Variable Region ◽  
Factor H ◽  
Serum Resistance ◽  
Nuclear Magnetic Resonance Analysis ◽  
Sex Partners ◽  
Resonance Analysis ◽  
Regulatory Molecule ◽  
Insertional Inactivation ◽  
Normal Human ◽  
High Level

ABSTRACT We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Galα1 → 3Galβ1 → 4Glcβ1 → 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an α2 → 6 linkage and, as expected, to the terminal Gal of LNT LOS via an α2→ 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.

Neutrophil activation in response to monomeric myeloperoxidase

2018 ◽  
Vol 96 (5) ◽  
pp. 592-601 ◽  
Author(s):  
Irina V. Gorudko ◽  
Daria V. Grigorieva ◽  
Alexey V. Sokolov ◽  
Ekaterina V. Shamova ◽  
Valeria A. Kostevich ◽  
...  
Keyword(s):  
Acute Inflammation ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Calcium Stores ◽  
Cellular Functions ◽  
Leukocyte Accumulation ◽  
Cytoskeleton Reorganization ◽  
Neutrophil Degranulation ◽  
Intracellular Ca ◽  
Normal Human

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Monoclonality of normal human colonic crypts

1995 ◽  
Vol 45 (8) ◽  
pp. 602-604 ◽  
Author(s):  
Yutaka Endo ◽  
Haruhiko Sugimura ◽  
Isamu Kino
Keyword(s):  
Colonic Crypts ◽  
Normal Human
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