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1.
J Cell Biol ; 138(6): 1333-41, 1997 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-9298988

RESUMEN

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661-671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 microM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 microM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.


Asunto(s)
Calcio/metabolismo , Calcio/farmacocinética , Canales Iónicos/genética , Angiotensina II/farmacología , Animales , Células CHO/química , Células CHO/fisiología , Canales de Calcio/fisiología , Cationes/metabolismo , Clonación Molecular , Cricetinae , Expresión Génica/fisiología , Activación del Canal Iónico/fisiología , Ionomicina/farmacología , Ionóforos/farmacología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Canales Catiónicos TRPC , Vasodilatadores/farmacología
2.
Neuron ; 29(3): 645-55, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11301024

RESUMEN

TRP proteins are cation channels responding to receptor-dependent activation of phospholipase C. Mammalian (TRPC) channels can form hetero-oligomeric channels in vitro, but native TRPC channel complexes have not been identified to date. We demonstrate here that TRPC1 and TRPC5 are subunits of a heteromeric neuronal channel. Both TRPC proteins have overlapping distributions in the hippocampus. Coexpression of TRPC1 and TRPC5 in HEK293 cells resulted in a novel nonselective cation channel with a voltage dependence similar to NMDA receptor channels, but unlike that of any reported TRPC channel. TRPC1/TRPC5 heteromers were activated by G(q)-coupled receptors but not by depletion of intracellular Ca(2+) stores. In contrast to the more common view of the TRP family as comprising store-operated channels, we propose that many TRPC heteromers form diverse receptor-regulated nonselective cation channels in the mammalian brain.


Asunto(s)
Química Encefálica , Canales de Calcio/química , Proteínas de Transporte de Catión , Canales Iónicos/química , Animales , Axones/química , Calcio/análisis , Canales de Calcio/análisis , Canales de Calcio/genética , Cationes , Línea Celular , Dendritas/química , Conductividad Eléctrica , Embrión de Mamíferos , Expresión Génica , Hipocampo/química , Humanos , Riñón , Sustancias Macromoleculares , Neuronas/química , Neuronas/ultraestructura , Ratas , Receptores de N-Metil-D-Aspartato/fisiología , Canales Catiónicos TRPC , Transfección
3.
J Gen Physiol ; 113(2): 239-48, 1999 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9925822

RESUMEN

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (approximately 10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.


Asunto(s)
Calcio/metabolismo , Núcleo Celular/metabolismo , Línea Celular , Quelantes/farmacología , Citoplasma/metabolismo , Dexametasona/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Glucocorticoides/farmacología , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Receptores de Glucocorticoides/metabolismo
4.
Mech Dev ; 53(2): 275-87, 1995 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8562428

RESUMEN

Embryonic stem (ES) cells represent a suitable model to analyze cell differentiation processes in vitro. Here, we report that pluripotent ES cells of the line BLC 6 differentiate in vitro into neuronal cells possessing the complex electrophysiological and immunocytochemical properties of postmitotic nerve cells. In the course of differentiation BLC 6-derived neurons differentially express voltage-dependent (K+, Na+, Ca2+) and receptor-operated (GABAA, glycine, AMPA, NMDA receptors) ionic channels. They generate fast Na(+)-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents. Moreover, BLC 6-derived neurons express neuron-specific cytoskeletal, cell adhesion and synaptic vesicle proteins and exhibit a Ca(2+)-dependent GABA secretion. Thus, the ES cell model enables the investigation of cell lineage determination and signaling mechanisms in the developing nervous system from a pluripotential stem cell to a differentiated postmitotic neuron. The in vitro differentiation of neurons from ES cells may be an excellent approach to study by targeted gene disruption a variety of neuronal functions.


Asunto(s)
Blastocisto/citología , Neuronas/citología , Células Madre/citología , Potenciales de Acción/fisiología , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Senescencia Celular/fisiología , Canales Iónicos/fisiología , Técnicas de Placa-Clamp , Receptores de Superficie Celular/fisiología , Sinapsis/fisiología
5.
Cell Signal ; 5(6): 727-34, 1993 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8130076

RESUMEN

Undifferentiated P19 embryonal carcinoma cells (ECC P19), the P19-derived clonal cell lines END-2 (visceral endoderm-like), EPI-7 (epithelioid ectoderm-like), MES-1 (mesoderm-like) and a parietal yolk sac cell line (PYS-2) were used as cellular models to examine the functional expression of voltage-dependent Ca channels and other Ca-permeable cation channels at various stages of early embryonic development. Whole-cell currents were recorded by means of the patch clamp technique. Whereas more than 75% of MES-1 cells possessed Ca channel currents, neither P19, END-2, EPI-7 nor PYS-2 cells had detectable voltage-dependent inward currents. Ca channel currents of MES-1 cells were highly sensitive towards 1,4-dihydropyridines and blocked by cadmium. Adrenaline (10 microM) caused Ca channel stimulation in only 14% of MES-1 cells examined. However, in 62% of the cells adrenaline activated a linear current component which under physiological conditions reversed close to 0 mV. Removal of extracellular Na+ suppressed the adrenaline-induced inward current, while reducing extracellular Cl- had no significant effect. These findings suggest that the adrenaline-induced current is carried through non-selective cation channels which were found to be permeable for Na+, K+, Cs+ >> Ca2+. Remarkably, the intracellular signalling pathway for activation of the non-selective cation current involved the cascade of reactions leading to cAMP-dependent phosphorylation, a regulatory pathway well known for cardiac Ca channels. A possible functional role of adrenaline-induced non-selective cation currents and Ca channels in embryonal development is discussed.


Asunto(s)
Canales de Calcio/metabolismo , Carcinoma/fisiopatología , AMP Cíclico/metabolismo , Mesodermo/fisiología , Canales de Calcio/efectos de los fármacos , Epinefrina/farmacología , Humanos , Potenciales de la Membrana/fisiología , Fosforilación , Células Tumorales Cultivadas
6.
Br J Pharmacol ; 172(14): 3650-60, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25847402

RESUMEN

BACKGROUND AND PURPOSE: The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents. EXPERIMENTAL APPROACH: A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca(2+) levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated. KEY RESULTS: SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca(2+) influx into cells with IC50 s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca(2+) entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR). CONCLUSIONS AND IMPLICATIONS: Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.


Asunto(s)
Diglicéridos/farmacología , Descubrimiento de Drogas , Indanos/farmacología , Canales Catiónicos TRPC/antagonistas & inhibidores , Células Cultivadas , Diglicéridos/síntesis química , Diglicéridos/química , Relación Dosis-Respuesta a Droga , Humanos , Indanos/síntesis química , Indanos/química , Datos de Secuencia Molecular , Estructura Molecular , Relación Estructura-Actividad , Canales Catiónicos TRPC/metabolismo
7.
Br J Pharmacol ; 108(4): 884-91, 1993 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-7683566

RESUMEN

1. The location of the binding domain for agonist dihydropyridines (DHP) has been studied by comparing the action of (+)-202,791 and (-)-Bay K 8644 on Ba2+ currents (IBa) in whole cell patch clamp experiments. Drug effects were examined upon internal and external (extracellular) application in A7r5 smooth muscle cells and BC3H1 cells, a cell line expressing Ca channels of the skeletal muscle type. 2. Efficiency of internal drug application in the whole cell studies was demonstrated by inhibition of potassium currents and barium currents (IBa) upon internal perfusion with tetraethylammonium (TEA+) (10 mM) and the permanently charged phenylalkylamine, D 890 (100 microM) respectively. The uncharged DHP, (-)-STBODIPY-DHP (2 microM) was used to estimate the time course of internal perfusion by monitoring its fluorescence. 3. Intracellular application of (+)-202,791 and (-)-Bay K 8644 (5 microM) in patch clamp experiments was ineffective in stimulating Ca2+ channel currents in both cell lines. In contrast a 50 fold lower agonist concentration (0.1 microM (-)-Bay K 8644) applied to the external face of the membrane induced typical changes in tail currents and a current increase under conditions when up to 10 microM of the agonist was present in the intracellular perfusion solution. 4. In cell-attached patches in A7r5 cells, (-)-Bay K 8644 increased and (+)-PN 200,110 inhibited single channel activity when applied via the bath solution. This suggests partitioning and lateral diffusion of the DHPs in the lipid of the plasma membrane. 5. We conclude that the binding site for agonist DHPs on Ca2+ channels in A7r5 and BC3H1 cells is located close to the external surface of the membrane. The DHP binding domain can be reached by agonists and antagonists from the extracellular but not from the intracellular face of the membrane.


Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Músculo Liso/metabolismo , Músculos/metabolismo , Ácidos Nicotínicos/farmacología , Oxadiazoles , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Bario/metabolismo , Canales de Calcio/efectos de los fármacos , Línea Celular , Difusión , Electrofisiología , Canales Iónicos/efectos de los fármacos , Ratones , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Espectrometría de Fluorescencia
8.
Naunyn Schmiedebergs Arch Pharmacol ; 370(3): 183-92, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15340774

RESUMEN

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC(50) values of 5.4+/-0.7 microM and 6.2+/-0.4 microM respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC(50) of 1.1+/-0.2 microM. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation ( tau(inact)), and the integral current was inhibited with an IC(50) of 3.4+/-0.5 microM. At 10 microM AVE0118 tau(inact) decreased from 9.3+/-0.6 ms ( n=8, control) to 3.0+/-0.3 ms ( n=8). The K(ACh) current was investigated in isolated pig atrial myocytes by application of 10 microM carbachol. At a clamp potential of -100 mV the I(KACh) was half-maximally blocked by 4.5+/-1.6 microM AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at -100 mV. Effects on the I(Kr) current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 microM. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I(Kr) tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I(Ks), I(KATP) (recorded in guinea pig ventricular myocytes), and L-type Ca(2+) (recorded in pig atrial myocytes) were blocked by 10 microM AVE0118 by 10+/-3% ( n=6), 28+/-7% ( n=4), and 22+/-13% ( n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K(+) channels I(Kur), I(to) and I(KACH). This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.


Asunto(s)
Antiarrítmicos/farmacología , Carbacol/farmacología , Cardiotónicos/farmacología , Canales Iónicos/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Animales , Células CHO , Proteínas de Unión al Calcio/antagonistas & inhibidores , Células Cultivadas , Cricetinae , Cricetulus , Electrofisiología , Humanos , Proteínas de Interacción con los Canales Kv , Canal de Potasio Kv1.5 , Biología Molecular , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio Shal , Porcinos , Xenopus
9.
Biophys J ; 70(4): 1662-8, 1996 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-8785324

RESUMEN

Under physiological conditions, nonselective cation (NSC) channels mediate the entry of cations into cells, the most important being Na+ and Ca2+. In contrast to the Ca(2+)-dependent signaling mechanisms, little is known about the consequences and the spatial distribution of intracellular [Na+] elevation. In this study we demonstrate that Na+ entry, during the opening of ATP-activated NSC channels, leads to an inhibition of voltage-dependent K+ currents (IK) in cromaffin-like undifferentiated PC-12 cells. The effect was dependent on the charge carrier as well as on the density of the ATP-activated current. Extracellular alkali cations (Na+, Li+) were more efficient than NH4+ in suppressing IK. Intracellular infusion of Na+ had the same effect as Na+ influx through ATP-activated NSC channels. The inhibition of IK persisted when the total ATP-induced Na+ entry was reduced by membrane depolarization, suggesting a spatial restriction of the required Na+ accumulation. Our results indicate that NSC channels influence the function of other ion channels by changing local intracellular ion concentrations.


Asunto(s)
Canales Iónicos/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Cationes/metabolismo , Electroquímica , Transporte Iónico , Cinética , Potenciales de la Membrana , Células PC12 , Ratas
10.
Nat Rev Neurosci ; 2(6): 387-96, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11389472

RESUMEN

Mammalian homologues of the Drosophila transient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathways. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of the cell cycle.


Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Animales , Humanos , Transducción de Señal/fisiología , Canales Catiónicos TRPC
11.
Eur J Neurosci ; 9(4): 824-32, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9153590

RESUMEN

Besides other mechanisms, the influx of Ca2+ into embryonic neurons controls growth and differentiation processes. To study the expression and regulation of voltage-gated Ca2+ channels during early neurogenesis, we measured whole-cell Ca2+ currents (I(Ca)) in neurons developing from pluripotent embryonic stem cells. Various receptor agonists, including somatostatin and baclofen, reversibly inhibited I(Ca) in embryonic stem cell-derived neurons. The effects of somatostatin and baclofen were abolished by pretreatment of cells with pertussis toxin and mimicked by intracellular infusion of guanosine 5'-O-(3-thiotriphosphate), suggesting the involvement of pertussis toxin-sensitive G proteins in I(Ca) inhibition. Investigations at different stages of neuronal differentiation showed that somatostatin efficiently suppressed L- and N-type Ca2+ channels in immature as well as mature neurons. In contrast, inhibition of L- and N-type channels by baclofen was rarely observed at the early stage. In terminally differentiated neurons, responses to baclofen were as prominent as those to somatostatin but were confined to N-type Ca2+ channels. The stage-dependent sensitivity of voltage-gated Ca2+ channels to somatostatin and baclofen was not due to differential expression of G alpha(o) isoforms, as revealed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. These findings demonstrate that specific neurotransmitters such as somatostatin regulate voltage-gated Ca2+ channels via G proteins during the early stages of neurogenesis, thus providing a mechanism for the epigenetic control of neuronal differentiation.


Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Proteínas de Unión al GTP/fisiología , Neuronas/fisiología , Células Madre/fisiología , omega-Conotoxinas , Animales , Baclofeno/farmacología , Canales de Calcio/biosíntesis , Canales de Calcio/efectos de los fármacos , Diferenciación Celular , Línea Celular , Conductividad Eléctrica , Embrión de Mamíferos , Regulación de la Expresión Génica/efectos de los fármacos , Homeostasis , Isradipino/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Neuronas/citología , Péptidos/farmacología , Somatostatina/farmacología , Venenos de Araña/farmacología , Células Madre/citología , omega-Agatoxina IVA , omega-Conotoxina GVIA
12.
Mol Pharmacol ; 43(5): 820-6, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-8388993

RESUMEN

To determine which side of L-type Ca2+ channels forms the benzothiazepine binding domain, we tested the effects of a membrane-impermeable, diltiazem-like, Ca2+ antagonist, SQ32,428 [(cis)-1,3,4,5-tetrahydro-4-(4-methoxyphenyl)-3-methyl-6- (trifluoromethyl)-1-[2-trimethylammonio)ethyl]-2H-1-benzazepin-2-o ne], on Ca2+ channels in smooth muscle-like cells (A7r5 cells) and skeletal muscle-like cells (differentiated BC3H1 cells). This permanently charged, quaternary benzazepine bound to the benzothiazepine-selective domain of skeletal muscle Ca2+ channels with a Ki of 1.2 +/- 0.1 microM. Extracellular application of SQ32,428 reversibly blocked whole-cell barium currents through L-type Ca2+ channels in A7r5 and BC3H1 cells with similar potencies (A7r5, IC50 = 86 microM; BC3H1, IC50 = microM). Block was fully reversible, was independent of stimulation frequency, and did not affect steady state inactivation of the channel in A7r5 cells. Intracellular dialysis of the cells with 100 microM SQ32,428 was without effect, but the same concentration of the quaternary phenylalkylamine D890 blocked channel activity from the cytoplasmic side. Our data demonstrate that the benzothiazepine binding domain of L-type Ca2+ channels binds diltiazem-like benzazepine Ca2+ antagonists and is formed by amino acid residues exposed to the extracellular channel surface.


Asunto(s)
Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio/metabolismo , Tiazepinas/metabolismo , Animales , Benzazepinas/metabolismo , Benzazepinas/farmacología , Sitios de Unión , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Línea Celular , Electrofisiología , Técnicas In Vitro , Ratones , Conejos , Ensayo de Unión Radioligante , Ratas
13.
Gastroenterology ; 113(1): 90-100, 1997 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9207266

RESUMEN

BACKGROUND & AIMS: Because of their diffuse distribution, neuroendocrine cells of the gut have not been isolated successfully for electrophysiological characterization. We therefore established primary cell cultures from surgically resected human carcinoids and investigated them electrophysiologically. METHODS: The neuroendocrine identity of the isolated gut tumor cells was determined immunocytochemically. The electrophysiological properties of the cells were studied by the patch-clamp technique. RESULTS: The primary cell cultures expressed neurofilament proteins, cytokeratins, and key proteins of the secretion machinery. Spontaneous action potentials were observed in most cells. Using the whole-cell mode of the patch-clamp technique, tetrodotoxin-sensitive voltage-gated sodium currents as well as voltage-gated calcium currents were identified. Calcium channel currents were carried mainly by dihydropyridine-sensitive, L-type calcium channels. The L-type calcium channel currents were also partially blocked by the omega-conotoxins GVIA and MVIIC. Moreover, omega-agatoxin IVA reversibly reduced a component of the calcium channel currents, indicating that neuroendocrine gut tumor cells express different types of voltage-gated calcium channels. In addition, somatostatin was found to inhibit partially the voltage-dependent calcium channel currents and thus calcium-dependent hormone release. CONCLUSIONS: Carcinoid cells of the human gut are electrically excitable cells. They express voltage-dependent sodium and calcium channels as well as somatostatin receptors.


Asunto(s)
Canales de Calcio/fisiología , Tumor Carcinoide/fisiopatología , Neoplasias del Íleon/fisiopatología , Receptores de Somatostatina/fisiología , Canales de Sodio/fisiología , Potenciales de Acción/fisiología , Tumor Carcinoide/patología , Femenino , Humanos , Neoplasias del Íleon/patología , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Técnicas de Placa-Clamp , Células Tumorales Cultivadas
14.
EMBO J ; 13(11): 2502-7, 1994 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-8013450

RESUMEN

Frequent strong depolarizations facilitate Ca2+ channels in various cell types by shifting their gating behavior towards mode 2, which is characterized by long openings and high probability of being open. In cardiac cells, the same type of gating behavior is potentiated by beta-adrenoceptors presumably acting via phosphorylation of a protein identical to or associated with the channel. Voltage-dependent phosphorylation has also been reported to underlie Ca2+ channel facilitation in chromaffin adrenal medulla and in skeletal muscle cells. We studied a possible voltage-dependent facilitation of the principal channel forming alpha 1-subunit of the dihydropyridine-sensitive smooth muscle Ca2+ channel. Single channel and whole-cell Ca2+ currents were recorded in Chinese hamster ovary cells stably expressing the class Cb Ca2+ channel alpha 1-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca2+ channel activity and induction of mode 2-like gating behavior. Accordingly we observed a significant potentiation of the whole-cell current by approximately 50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C and other protein kinases utilizing ATP gamma S) in the control and facilitated current. The data demonstrate that the L-type Ca2+ channel alpha 1-subunit solely expressed in Chinese hamster ovary cells is subject to a voltage-dependent facilitation but not to phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Canales de Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Activación del Canal Iónico/fisiología , Músculo Liso/química , Animales , Células CHO , Bloqueadores de los Canales de Calcio , Canales de Calcio/genética , Proteínas Portadoras/farmacología , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario , Dihidropiridinas/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Potenciales de la Membrana/fisiología , Fosforilación , Proteína Quinasa C/metabolismo , Tionucleótidos/farmacología
15.
Gastroenterology ; 110(5): 1595-604, 1996 May.
Artículo en Inglés | MEDLINE | ID: mdl-8613067

RESUMEN

BACKGROUND & AIMS: Neuroendocrine cells and tumors derived therefrom contain hormone-storing large dense core vesicles and neuron-like small synaptic vesicle analogues with unknown function. The aim of this study was to characterize the small synaptic vesicle pathway in detail. METHODS: In human pancreatic neuroendocrine tumors and corresponding mammalian cell lines, the expression of key proteins if regulated secretion were detected by immunofluorescence microscopy. Using 3H-gamma-aminobutyric acid (GABA), uptake and release by small synaptic vesicle analogues were studied. RESULTS: Tumor tissues obtained from 14 patients expressed key proteins of neurosecretion such as synaptobrevin, syntaxins, and SNAP 25. These proteins were also found in the cell lines AR42J, BON, RIN, and INR. The cell lines specifically transported GABA by low-affinity plasma membrane transporter and showed an adenosine triphosphate-sensitive GABA uptake into an intracellular compartment. Stored GABA was released upon stimulation by regulated exocytosis. Electrophysiological analyses suggested that calcium-dependent secretion was mediated by activation of voltage-dependent calcium channels of mainly the L type, but also of the N and probably the T type. CONCLUSIONS: Small synaptic vesicle analogues in neuroendocrine cells and tumors can store and secrete GABA and probably other amino acid transmitters by regulated exocytosis comparable with neurons.


Asunto(s)
Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Adulto , Canales de Calcio/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Qa-SNARE , Proteínas R-SNARE , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas , Células Tumorales Cultivadas/metabolismo
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