Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
1.
Mol Ther Methods Clin Dev ; 19: 47-57, 2020 Dec 11.
Article in English | MEDLINE | ID: mdl-32995359

ABSTRACT

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

2.
Pharmacol Res Perspect ; 7(6): e00542, 2019 12.
Article in English | MEDLINE | ID: mdl-31768260

ABSTRACT

The G-protein-coupled receptor GPR132, also known as G2A, is activated by 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized fatty acids. Other suggested GPR132 agonists including lysophosphatidylcholine (LPC) have not been readily reproduced. Here, we identify N-acylamides in particular N-acylglycines, as lipid activators of GPR132 with comparable activity to 9-HODE. The order-of-potency is N-palmitoylglycine > 9-HODE ≈ N-linoleoylglycine > linoleamide > N-oleoylglycine ≈ N-stereoylglycine > N-arachidonoylglycine > N-docosehexanoylglycine. Physiological concentrations of N-acylglycines in tissue are sufficient to activate GPR132. N-linoleoylglycine and 9-HODE also activate rat and mouse GPR132, despite limited sequence conservation to human. We describe pharmacological tools for GPR132, identified through drug screening. SKF-95667 is a novel GPR132 agonist. SB-583831 and SB-583355 are peptidomimetic molecules containing core amino acids (glycine and phenylalanine, respectively), and structurally related to previously described ligands. A telmisartan analog, GSK1820795A, antagonizes the actions of N-acylamides at GPR132. The synthetic cannabinoid CP-55 940 also activates GPR132. Molecular docking to a homology model suggested a site for lipid binding, predicting the acyl side-chain to extend into the membrane bilayer between TM4 and TM5 of GPR132. Small-molecule ligands are envisaged to occupy a "classical" site encapsulated in the 7TM bundle. Structure-directed mutagenesis indicates a critical role for arginine at position 203 in transmembrane domain 5 to mediate GPR132 activation by N-acylamides. Our data suggest distinct modes of binding for small-molecule and lipid agonists to the GPR132 receptor. Antagonists, such as those described here, will be vital to understand the physiological role of this long-studied target.


Subject(s)
Cell Cycle Proteins/agonists , Glycine/analogs & derivatives , Palmitic Acids/pharmacology , Peptidomimetics/pharmacology , Receptors, G-Protein-Coupled/agonists , Animals , CHO Cells , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cricetulus , Cyclohexanols/pharmacology , Drug Antagonism , Fatty Acids, Unsaturated/pharmacology , Glycine/pharmacology , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Structural Homology, Protein , Telmisartan/analogs & derivatives , Telmisartan/pharmacology
3.
Assay Drug Dev Technol ; 16(8): 445-455, 2018 12.
Article in English | MEDLINE | ID: mdl-30481043

ABSTRACT

Mitochondrial permeability transition pore (mPTP) formation is well documented in isolated mitochondria. However, convincing detection of mPTP in whole cells remains elusive. In this study, we describe a high-throughput assay for Ca2+-activated mPTP opening in platelets using HyperCyt flow cytometry. In addition, we demonstrate that in several nucleated cells, using multiple approaches, the detection of cyclophilin D-dependent mPTP opening is highly challenging. Results with the mitochondrial-targeted Ca2+-sensing green fluorescent protein (mito-Case12) suggest the involvement of protein phosphatase 2B (PP2B; calcineurin) in regulating mitochondrial dynamics. Our results highlight the danger of relying on cyclosporine A alone as a pharmacological tool, and the need for comprehensive studies of mPTP in the cell.


Subject(s)
Blood Platelets/cytology , Blood Platelets/enzymology , Calcineurin/metabolism , High-Throughput Screening Assays , Mitochondria, Liver/metabolism , Mitochondrial Dynamics , Mitochondrial Membrane Transport Proteins/metabolism , Animals , HEK293 Cells , Humans , Mitochondrial Permeability Transition Pore , Rats , Tacrolimus/pharmacology
4.
J Biomol Screen ; 20(2): 242-53, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25278498

ABSTRACT

The epithelial sodium channel (ENaC) plays a crucial role in salt and water homeostasis and is primarily involved in sodium reabsorption in the kidney and lung. Modulators of ENaC function, particularly within lung epithelia, could offer potential treatments for a number of diseases. As a constitutively active sodium channel, ENaC expression at the cell membrane is highly regulated through rapid turnover. This short half-life of the channel at the membrane and cytotoxicity from overexpression pose a problem for reagent generation and assay development in drug discovery. We have generated an HEK293 stable cell line expressing ENaC ß and γ subunits containing the PY motif trafficking mutations found in Liddle's syndrome to overcome rapid channel turnover at the membrane. A BacMam virus was used to transiently express the ENaC α subunit to reconstitute channel function to reduce the toxicity associated with long-term overexpression. We have configured a 384-well FLIPR membrane potential antagonist assay for high-throughput screening and an IonWorks Quattro electrophysiology antagonist assay that is predictive of potency values derived from primary lung epithelial cell short-circuit measurements. The triage strategy for compound screening and profiling against this target using these assays has resulted in the discovery of novel chemotypes.


Subject(s)
Drug Evaluation, Preclinical , Epithelial Sodium Channel Agonists/pharmacology , Epithelial Sodium Channel Blockers/pharmacology , Epithelial Sodium Channels/metabolism , High-Throughput Screening Assays/methods , High-Throughput Screening Assays/standards , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Sodium Channels/chemistry , Epithelial Sodium Channels/genetics , Gene Expression , HEK293 Cells , Humans , Membrane Potentials/drug effects , Patch-Clamp Techniques , Respiratory Mucosa/metabolism , Small Molecule Libraries
5.
Eur J Pharmacol ; 735: 68-76, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24747752

ABSTRACT

We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity.


Subject(s)
Large-Conductance Calcium-Activated Potassium Channels/physiology , Prostatic Hyperplasia/physiopathology , Urinary Bladder, Neurogenic/physiopathology , Urinary Bladder, Overactive/physiopathology , Acetic Acid , Adult , Aged , Animals , Carbachol/pharmacology , Electric Stimulation , Female , Humans , Male , Middle Aged , Muscle Contraction/physiology , Rats , Urinary Bladder/physiopathology , Urinary Bladder, Overactive/chemically induced , Young Adult
6.
Assay Drug Dev Technol ; 11(2): 93-100, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23046406

ABSTRACT

Gap junctions (GJs) are intercellular channels which are composed of the connexin family of proteins that allow electrical and chemical communications and synchronization in tissue ensembles. Evidence suggests that pharmaceutical modulators of these channels may have therapeutic potential or carry undesired liability. In this report, we exogenously expressed human connexin 43 (Cx43, GJA1) and demonstrated functionality in a 96-well flow cytometry assay detecting intercellular transfer of the calcein dye. We have designed a 384-well high-throughput method for detecting the transfer of calcium between HeLa cells expressing Cx43. In this assay, donor cells coexpress Cx43 and the α1A adrenergic Gα-coupled receptor, while recipient cells coexpress Cx43 and the cytoplasmic version of the calcium-sensitive luminescent protein aequorin enhanced by codon optimization (cytoAeq). The two cell populations were mixed, dispensed to 384-well plates, and incubated for 3 h to allow the formation of GJs. Activation of α1A by epinephrine in donor cells led to dose-dependent calcium increases in recipient cells, which were detected by measuring the intensity of aequorin luminescence. The response was dependent on the expression of Cx43 and inhibited by the GJ blocker 18α-glycyrrhetinic acid, suggesting Cx43 GJ-mediated activity. In a parallel experiment with capsaicin and the TrpV1 ion channel in place of phenylephrine and α1A, a similar magnitude of difference in the maximal calcium response was detected in both donor and recipient cells, suggesting that calcium is likely the permeant ion through the GJ. This assay may pave the way for high-throughput screening of GJ modulators for drug discovery.


Subject(s)
Aequorin , Biological Assay/instrumentation , Calcium Signaling/physiology , Calcium/metabolism , Connexin 43/analysis , Connexin 43/metabolism , Flow Cytometry/instrumentation , Cell Separation/instrumentation , Codon/genetics , Equipment Design , Equipment Failure Analysis , Flow Injection Analysis/instrumentation , HeLa Cells , Humans , Ion Channel Gating/physiology , Luminescent Agents
7.
Curr Opin Mol Ther ; 11(3): 269-81, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19479660

ABSTRACT

Acquired long QT syndrome is a major cause of drug withdrawals and the failure of compounds during development. Traditionally, in vitro cardiac liability screens have been low throughput and expensive, and have used primary animal cells or tissues that are not necessarily predictive of the human heart. Recent advances in ion channel assay technologies, particularly automated patch clamp, have enabled the early liability screening of individual ion channels at medium-to-high throughput and with acceptable fidelity. The primary aim of this first-tier screening is to aid prioritization of early-stage compounds for progression along the screening cascade. Potential liabilities can be flagged to enable follow-up studies to be initiated if and when compounds approach the costlier stages of development. To date, cardiac safety screening has been focused on hERG channel. This article examines the rationale for the early screening of cardiac channels beyond hERG as part of an integrated strategy for in vitro evaluation of cardiac risk, and reviews recent developments in the relevant technologies.


Subject(s)
Cardiovascular Diseases/diagnosis , Ether-A-Go-Go Potassium Channels/metabolism , Animals , ERG1 Potassium Channel , Humans , Patch-Clamp Techniques , Risk Factors
8.
Comb Chem High Throughput Screen ; 12(1): 96-106, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19149495

ABSTRACT

The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.


Subject(s)
Cell Line/cytology , Drug Discovery/methods , Electrophysiology/methods , Ion Channels , Electrophysiology/instrumentation , Humans , Tissue Array Analysis
9.
BMC Biotechnol ; 7: 93, 2007 Dec 20.
Article in English | MEDLINE | ID: mdl-18096051

ABSTRACT

BACKGROUND: HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance. RESULTS: Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27 degrees C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30 degrees C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37 degrees C, but not at 30 degrees C. CONCLUSION: Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.


Subject(s)
Cell Culture Techniques/methods , Ether-A-Go-Go Potassium Channels/biosynthesis , Protein Transport , Animals , Biological Transport, Active/drug effects , CHO Cells , Ca(2+) Mg(2+)-ATPase/drug effects , Cricetinae , Cricetulus , Ether-A-Go-Go Potassium Channels/drug effects , Humans , Membrane Proteins , Nocodazole/pharmacology , Phenethylamines/pharmacology , Potassium Channel Blockers , Recombinant Proteins , Sulfonamides/pharmacology , Temperature , Thapsigargin/pharmacology , Transport Vesicles/drug effects , Up-Regulation
10.
J Biomol Screen ; 12(1): 50-60, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17092914

ABSTRACT

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.


Subject(s)
Electrophysiology/methods , Patch-Clamp Techniques/methods , Potassium Channels, Calcium-Activated/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Humans , Membrane Transport Modulators/pharmacology , Potassium Channels, Calcium-Activated/antagonists & inhibitors , Reproducibility of Results
11.
J Neurosci ; 25(14): 3489-98, 2005 Apr 06.
Article in English | MEDLINE | ID: mdl-15814779

ABSTRACT

We investigated the role of small-conductance calcium-activated potassium (SK) and intermediate-conductance calcium-activated potassium channels in modulating sensory transmission from peripheral afferents into the rat spinal cord. Subunit-specific antibodies reveal high levels of SK3 immunoreactivity in laminas I, II, and III of the spinal cord. Among dorsal root ganglion neurons, both peripherin-positive (C-type) and peripherin-negative (A-type) cells show intense SK3 immunoreactivity. Furthermore, dorsal root-stimulated sensory responses recorded in vitro are inhibited when SK channel activity is increased with 1-ethyl-2-benzimidazolinone (1-EBIO). In vivo electrophysiological recordings show that neuronal responses to naturally evoked nociceptive and nonnociceptive stimuli increase after application of the selective SK channel blocker 8,14-diaza-1,7(1,4)-diquinolinacyclotetradecaphanedium di-trifluoroacetate (UCL 1848), indicating that SK channels are normally active in moderating afferent input. Conversely, neuronal responses evoked by mechanical stimuli are inhibited when SK channel activity is increased with 1-EBIO. These effects are reversed by the subsequent application of UCL 1848. Our data demonstrate that SK channels have an important role in controlling sensory input into the spinal cord.


Subject(s)
Neurons, Afferent/physiology , Pain/physiopathology , Potassium Channels, Calcium-Activated/physiology , Spinal Cord/cytology , Animals , Animals, Newborn , Benzimidazoles/pharmacology , Calcium Channel Agonists/pharmacology , Cell Line , Dose-Response Relationship, Drug , Electric Stimulation/methods , Ependyma/metabolism , Female , Ganglia, Spinal/cytology , Humans , Immunohistochemistry/methods , In Vitro Techniques , Intermediate Filament Proteins/metabolism , Male , Membrane Glycoproteins/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Nerve Fibers/drug effects , Nerve Fibers/physiology , Nerve Tissue Proteins/metabolism , Neurons, Afferent/drug effects , Patch-Clamp Techniques/methods , Peripherins , Physical Stimulation/methods , Potassium Channel Blockers/pharmacology , Potassium Channels, Calcium-Activated/drug effects , Quinolinium Compounds/pharmacology , Rats , Rats, Sprague-Dawley , Reaction Time/drug effects , Reaction Time/physiology , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Synaptic Transmission/radiation effects , TRPV Cation Channels/metabolism , Time Factors , Transfection
12.
J Neurochem ; 90(6): 1414-22, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15341525

ABSTRACT

Calcium-activated potassium channels are critically important in modulating neuronal cell excitability. One member of the family, the intermediate-conductance potassium (IK) channel, is not thought to play a role in neurones because of its predominant expression in non-excitable cells such as erythrocytes and lymphocytes, in smooth muscle tissues, and its lack of apparent expression in brain. In the present study, we demonstrate that IK channels are localized on specific neurones in the mouse enteric nervous system where they mediate the slow afterhyperpolarization following an action potential. IK channels were localized by immunohistochemistry on intrinsic primary afferent neurones, identified by their characteristic Dogiel type II morphology. The slow afterhyperpolarization recorded from these cells was abolished by the IK channel blocker clotrimazole. RT-PCR and western analysis of extracts from the colon revealed an IK channel transcript and protein identical to the IK channel expressed in other cell types. These results indicate that IK channels are expressed in neurones where they play an important role in modulating firing properties.


Subject(s)
Clotrimazole/pharmacology , Colon/cytology , Gene Expression Regulation , Neurons/metabolism , Potassium Channels, Calcium-Activated , Potassium Channels/metabolism , Animals , Calbindin 2 , Colon/metabolism , Dose-Response Relationship, Drug , Electrophysiology/methods , Immunohistochemistry/methods , In Vitro Techniques , Intermediate-Conductance Calcium-Activated Potassium Channels , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Inbred BALB C , Neurons/classification , Neurons/drug effects , Potassium Channels/drug effects , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , S100 Calcium Binding Protein G/metabolism
13.
Auton Neurosci ; 112(1-2): 93-7, 2004 May 31.
Article in English | MEDLINE | ID: mdl-15233935

ABSTRACT

IK channels, which had been previously found in hemopoetically derived cells (including erythrocytes and lymphocytes) and epithelial cells, where they regulate proliferation, cell volume regulation and secretion, have only recently been discovered in neurons, where they had previously been claimed not to occur. Based on immunohistochemical detection of IK channel-like immunoreactivity, it has been reported that IK channel expression in enteric neurons is suppressed in Crohn's disease. In the present work we have investigated whether authentic IK channels are expressed by enteric neurons. Human and mouse tissue was investigated by immunohistochemistry, Western blot and RT-PCR. Immunohistochemical studies revealed IK channel-like immunoreactivity in large myenteric neurons, but not in other cell types in the external muscle layers. Many of these nerve cells had calbindin immunoreactivity. Western blots from the external muscle revealed an immunoreactive band at the molecular weight of the IK channel. Using RT-PCR, we detected a transcript corresponding to the IK channel gene in extracts from the ganglion containing layer. The sequence obtained from the RT-PCR product was identical to that previously published for the IK channel. We conclude that IK channels are expressed by human enteric neurons, including large smooth surfaced neurons that are possibly the human equivalent of the Dogiel type II neurons that express these channels in small mammals.


Subject(s)
Enteric Nervous System/cytology , Neurons/metabolism , Potassium Channels/metabolism , Adult , Aged , Animals , Blotting, Western , Calbindins , Humans , Immunohistochemistry/methods , Male , Mice , Middle Aged , Myenteric Plexus/cytology , Neurons/classification , Potassium Channels/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , S100 Calcium Binding Protein G/metabolism
14.
Naunyn Schmiedebergs Arch Pharmacol ; 369(6): 602-15, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15127180

ABSTRACT

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.


Subject(s)
Colon/metabolism , Muscle, Smooth, Vascular/metabolism , Potassium Channels, Calcium-Activated/physiology , Adult , Aged , Blotting, Northern , Colon/blood supply , Electric Conductivity , Female , Gene Expression Regulation , Genitalia/blood supply , Genitalia/metabolism , Humans , Immunohistochemistry , Male , Middle Aged , Muscle, Smooth/blood supply , Muscle, Smooth/metabolism , Organ Specificity , Potassium Channels, Calcium-Activated/biosynthesis , Potassium Channels, Calcium-Activated/genetics , RNA, Messenger/analysis , Small-Conductance Calcium-Activated Potassium Channels
15.
Cell Tissue Res ; 314(2): 179-89, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14513356

ABSTRACT

Recent functional evidence suggests that intermediate conductance calcium-activated potassium channels (IK channels) occur in neurons in the small intestine and in mucosal epithelial cells in the colon. This study was undertaken to investigate whether IK channel immunoreactivity occurs at these and at other sites in the gastrointestinal tract of the rat. IK channel immunoreactivity was found in nerve cell bodies throughout the gastrointestinal tract, from the esophagus to the rectum. It was revealed in the initial segments of the axons, but not in axon terminals. The majority of immunoreactive neurons had Dogiel type II morphology and in the myenteric plexus of the ileum all immunoreactive neurons were of this shape. Intrinsic primary afferent neurons in the rat small intestine are Dogiel type II neurons that are immunoreactive for calretinin, and it was found that almost all the IK channel immunoreactive neurons were also calretinin immunoreactive. IK channel immunoreactivity also occurred in calretinin-immunoreactive, Dogiel type II neurons in the caecum. Epithelial cells of the mucosal lining were immunoreactive in the esophagus, stomach, small and large intestines. In the intestines, the immunoreactivity occurred in transporting enterocytes, but not in mucous cells. Immunoreactivity was at both the apical and basolateral surfaces. A small proportion of mucosal endocrine cells was immunoreactive in the duodenum, ileum and caecum, but not in the stomach, proximal colon, distal colon or rectum. There was immunoreactivity of vascular endothelial cells. It is concluded that IK channels are located on cell bodies and proximal parts of axons of intrinsic primary afferent neurons, where, from functional studies, they would be predicted to lower neuronal excitability when opened in response to calcium entry. In the mucosa of the small and large intestine, IK channels are probably involved in control of potassium exchange, and in the esophageal and gastric mucosa they are possibly involved in control of cell volume in response to osmotic challenge.


Subject(s)
Digestive System/cytology , Epithelial Cells/metabolism , Neurons, Afferent/metabolism , Potassium Channels/metabolism , Animals , Blotting, Western , Cell Line , Electric Conductivity , Enteric Nervous System/cytology , Epithelial Cells/cytology , Epithelial Cells/immunology , Female , Humans , Immunohistochemistry , Intestinal Mucosa/cytology , Male , Microscopy, Confocal , Neurons, Afferent/cytology , Neurons, Afferent/immunology , Potassium Channels/immunology , Rats , Rats, Sprague-Dawley , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL
...