RESUMEN

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

Asunto(s)

Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/citología , Fibras Musculares Esqueléticas/citología , Unión Neuromuscular/metabolismo , Adulto , Antígenos CD34/genética , Antígenos CD34/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Técnicas de Cocultivo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Queratinocitos/citología , Masculino , Neuronas Motoras/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/citología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Colinérgicos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: Pain sensitizes the central nervous system via N-methyl-D-aspartate receptors (NMDARs) leading to an enhancement of pain perception. However, the enhanced responsiveness of pain-processing areas can be suppressed by subanaesthetic doses of the NMDAR antagonist xenon. To analyse the strength of the analgesic effect of low-dose xenon using new economical application methods, we tested xenon applied nasally in an experimental human pain setting. METHODS: We tested 10 healthy volunteers using a multimodal experimental pain testing in a randomized double-blind placebo-controlled repeated measures study. Xenon was administered using a novel low-pressure intranasal application device. Additionally, we measured xenon concentrations in blood samples obtained from intracranial veins of experimental animals to describe the pharmacokinetics of intranasally applied xenon in the cerebral compartment. RESULTS: Intranasal application of xenon at a rate of 1.0 litre h(-1) for 30 min significantly increased pain tolerance of volunteers to ischaemic (+128%), cold (+58%), and mechanical (+40%) stimulation (P<0.01). However, 60 min after terminating the application of xenon, there was no significant alteration of pain tolerance compared with placebo. Cranial blood concentrations of xenon in pigs reached a steady state of approximately 450 nl ml(-1) after 5 min. CONCLUSIONS: In this placebo-controlled experimental human study, we described the increased pain tolerance induced by intranasally applied xenon. On the basis of our results, we conclude that intranasally administered xenon has analgesic properties and suggest that the novel application device presented here offers new possibilities for the administration of NMDAR antagonists within a multimodal analgesia approach.

Asunto(s)

Anestésicos por Inhalación/farmacología , Umbral del Dolor/efectos de los fármacos , Xenón/farmacología , Administración Intranasal , Adulto , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/sangre , Analgésicos no Narcóticos/farmacología , Anestésicos por Inhalación/administración & dosificación , Anestésicos por Inhalación/sangre , Animales , Frío , Modelos Animales de Enfermedad , Método Doble Ciego , Sistemas de Liberación de Medicamentos , Humanos , Isquemia/complicaciones , Masculino , Dolor/etiología , Dolor/prevención & control , Dimensión del Dolor/métodos , Estimulación Física/métodos , Tiempo de Reacción/efectos de los fármacos , Sus scrofa , Xenón/administración & dosificación , Xenón/sangre

RESUMEN

BACKGROUND AND PURPOSE: Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells. EXPERIMENTAL APPROACH: The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique. KEY RESULTS: Glutamate uptake was suppressed by etomidate (3-100 microM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4+/-0.6 microM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V(max) whereas the K(m) of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate. CONCLUSION AND IMPLICATIONS: Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.

Asunto(s)

Anestésicos Intravenosos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Etomidato/farmacología , Ácido Glutámico/metabolismo , Neuroglía/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

BACKGROUND: The anaesthetic, analgesic, and neuroprotective effects of xenon (Xe) are believed to be mediated by a block of the NMDA (N-methyl-D-aspartate) receptor channel. Interestingly, the clinical profile of the noble gas differs markedly from that of specific NMDA receptor antagonists. The aim of this study was, therefore, to investigate whether Xe might be less specific, also inhibiting the two other subtypes of glutamate receptor channels, such as the alpha-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA) and kainate receptors. METHODS: The study was performed on voltage-clamped cortical neurones from embryonic mice and SH-SY5Y cells expressing GluR6 kainate receptors. Drugs were applied by a multi-barreled fast perfusion system. RESULTS: Xe, dissolved at approximately 3.45 mM in aqueous solution, diminished the peak and even more the plateau of AMPA and glutamate induced currents. At the control EC(50) value for AMPA (29 microM) these reductions were by about 40 and 56% and at 3 mM glutamate the reductions were by 45 and 66%, respectively. Currents activated at the control EC(50) value for kainate (57 microM) were inhibited by 42%. Likewise, Xe showed an inhibitory effect on kainate-induced membrane currents of SH-SY5Y cells transfected with the GluR6 subunit of the kainate receptor. Xe reduced kainate-induced currents by between 35 and 60%, depending on the kainate concentration. CONCLUSIONS: Xe blocks not only NMDA receptors, but also AMPA and kainate receptors in cortical neurones as well as GluR6-type receptors expressed in SH-SY5Y cells. Thus, Xe seems to be rather non-specific as a channel blocker and this may contribute to the analgesic and anaesthetic potency of Xe.

Asunto(s)

Anestésicos por Inhalación/farmacología , Corteza Cerebral/efectos de los fármacos , Neuronas/efectos de los fármacos , Xenón/farmacología , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Ácido Kaínico/antagonistas & inhibidores , Ácido Kaínico/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Neuronas/fisiología , Técnicas de Placa-Clamp , Receptores AMPA/efectos de los fármacos , Receptores de Glutamato/efectos de los fármacos , Receptores de Ácido Kaínico/efectos de los fármacos , Receptores de Ácido Kaínico/genética , Receptores de Ácido Kaínico/fisiología , Transfección , Células Tumorales Cultivadas , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/antagonistas & inhibidores , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Receptor de Ácido Kaínico GluK2

RESUMEN

BACKGROUND: Over the past decade hyperpolarized (129)xenon incorporated in lipid emulsions has been studied for the purpose of imaging enhancement in radiology. Xenon (Xe), a NMDA (N-methyl-D-aspartate)-receptor antagonist, has neuroprotective properties even at subanesthetic concentrations. Thus, its intravenous administration for this purpose deserves further evaluation. In this study, we investigated in an in vitro model the effect of Xe, incorporated in a lipid emulsion (Lipofundin MCT(R) 20%), on the NMDA receptor channel of cortical neurons of the mouse. METHODS: Pulses of 50 micro M of NMDA solution were extracellularly applied to the cells for 10 s, and the elicited membrane currents (I) were recorded while the membrane potential (V) was clamped at -80 mV. Either Lipofundin MCT(R) 20% or aqueous solution was loaded with Xe and applied simultaneously with the NMDA pulses by means of a multibarreled pipette attached to a battery of infusion-pumps. RESULTS: Xenon equilibrated in Lipofundin(R) caused a concentration-dependent and reversible inhibition of NMDA-induced currents (maximal Xe content [Xemax]: 190 micro l ml-1). The inhibitory effect was equivalent compared with the effect of Xe dissolved in aqueous solution (Xemax: 89 micro l ml-1) even though the Xe content of the lipid solution was almost doubled. Further enhancement of the Xe content by saturating both the lipid emulsion and the aqueous solutions with Xe (Xemax: 256 micro l ml-1) did not increase the inhibitory action on NMDA-receptors. CONCLUSION: The data demonstrate that Xe dissolved in Lipofundin MCT(R) 20% inhibits NMDA-receptors. Lipid emulsions enriched with Xe may serve as a carrier and a reservoir for Xe.

Asunto(s)

Canales Iónicos/antagonistas & inhibidores , Fosfolípidos/administración & dosificación , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sorbitol/administración & dosificación , Xenón/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C

RESUMEN

The release of Ca2+ in response to caffeine at threshold concentration (5 mM) was studied in mouse skeletal myotubes. Repeated 5-s applications of caffeine, each followed by a 30-s washout, caused Ca2+ releases of consecutively growing amplitude (staircase phenomenon). Each response declined rapidly and had a slow tail. Repeated applications of threshold caffeine lowered the threshold concentration. The interval between threshold applications could be increased to 30 min without loss of potentiation. When threshold caffeine was applied continuously for up to 10 min, the increase in Ca2+ concentration as seen with staircase potentiation did not occur. Depolarization by elevated [K+] or by voltage-clamp steps potentiated caffeine-induced Ca2+ release rapidly as compared to the slow exponential growth of staircase-like potentiation. Gd3+ prevented the depolarization-induced potentiation, but not the staircase phenomenon. Staircase-like potentiation of Ca2+ release was evident even when the voltage sensors were clamped in their resting state; in contrast, potentiated Ca2+ release and its rapid termination apparently require conversion of the voltage sensors to an activated state. Staircase potentiation was blocked when Ca2+ was omitted from the bath, thus pinpointing the source of Ca2+. We suggest that staircase-like potentiation is conditioned by a caffeine-dependent Ca2+ influx across the plasma membrane.

Asunto(s)

Cafeína/farmacología , Calcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Animales , Membrana Celular/metabolismo , Células Cultivadas , Quelantes/farmacología , Ácido Egtácico/farmacología , Espacio Extracelular/metabolismo , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/citología , Técnicas de Placa-Clamp , Canal Liberador de Calcio Receptor de Rianodina/metabolismo

RESUMEN

The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 microM 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41+/-13 to 541+/-319 nM, peaked after 2-5 s and declined within 10 s to nearly resting values (n=36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 microM 4-CmC, the response was small, whereas at 500 microM it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 microM) or ryanodine (10 microM) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems.

Asunto(s)

Calcio/metabolismo , Cresoles/farmacología , Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Animales , Calcio/análisis , Bloqueadores de los Canales de Calcio/farmacología , Colorantes Fluorescentes , Fura-2 , Ratones , Ratones Endogámicos C57BL , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/ultraestructura , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Estimulación Química

RESUMEN

Previously, we provided evidence for the presence of a class of muscarinic receptors on human luteinized granulosa cells (human GC) that is linked to transient increases in intracellular free calcium levels, but not to steroid production. The precise nature of the receptor is not known, and neither its function nor the source of its natural ligand acetylcholine (ACh) is clear. To address these issues we used RT-PCR approaches and isolated complementary DNAs corresponding to the M1 receptor subtype from reverse transcribed human GC messenger ribonucleic acids. M1 receptors were further shown by immunocytochemistry, using a M1 receptor antiserum. Single cell calcium measurements showed that the M1 receptor was functionally active and linked to acute increases in intracellular free calcium, as the M1 receptor specific antagonist pirenzepine blocked the Ca2+-mobilizing effect of oxotremorine M (a muscarinic agonist). An unexpected consequence of M1 receptor activation was evidenced by the ability of muscarinic agonists to stimulate the proliferation of human GC within 24 h. In vivo, ACh, the natural ligand of these receptors is thought to be contained in cholinergic nerve fibers innervating the ovary. Surprisingly, the prerequisite for the synthesis of ACh, the enzyme choline-acetyltransferase (ChAT), is also expressed by human GC, as shown by Western blotting and immunocytochemistry. In addition, these cells express another marker for ACh synthesis, namely the gene for the vesicular acetylcholine transporter, as evidenced by RT-PCR cloning, Western blotting, and immunocytochemistry. In conclusion, our data identify the M1 receptor in human GC and point to a novel, trophic role of the neurotransmitter ACh. Furthermore, the presence of the prerequisites of ACh synthesis in human GC indicate that an autocrine/paracrine regulatory loop also exists in the in vivo counterparts of these cells in the ovary, i.e. in the cells of the preovulatory follicle and/or of the young corpus luteum.

Asunto(s)

Acetilcolina/biosíntesis , Proteínas Portadoras/biosíntesis , Colina O-Acetiltransferasa/biosíntesis , Células de la Granulosa/metabolismo , Fase Luteínica/fisiología , Proteínas de Transporte de Membrana , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Proteínas de Transporte Vesicular , Western Blotting , Calcio/metabolismo , División Celular , Células Cultivadas , Femenino , Líquido Folicular/citología , Humanos , Inmunohistoquímica , Receptor Muscarínico M1 , Receptores Muscarínicos/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Transporte Vesicular de Acetilcolina

RESUMEN

1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability.

Asunto(s)

Calcio/metabolismo , Distrofina/deficiencia , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/fisiopatología , Distrofia Miotónica/fisiopatología , Retículo Sarcoplasmático/metabolismo , Amilorida/farmacología , Animales , Estimulación Eléctrica , Gadolinio/farmacología , Técnicas In Vitro , Lantano/farmacología , Manganeso/farmacocinética , Manganeso/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Mutantes , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/fisiología , Distrofia Muscular Animal/genética , Distrofia Miotónica/genética , Níquel/farmacología

RESUMEN

In cultured tectal neurons of embryonic chicken N-methyl-D-aspartate (NMDA) receptor currents were investigated during two stages of development. Stage I tectal cells were cultivated for 1-5 days from embryonic day 6 or 7, and stage II tectal cells for 10-30 days. Steady state inward currents induced by NMDA were usually several times smaller in tectal cells of stage I than in tectal cells of stage II. At the somata of stage I tectal cells, NMDA-activated single channels with a mean conductance of about 55 pS could be recorded. In tectal cells of stage II, however, NMDA receptor channels were predominantly observed at cellular processes and a mean single channel conductance of about 72 pS was found. Our results suggest changes in regional distribution and function of NMDA receptors during early embryogenesis in the chicken tectum.

Asunto(s)

N-Metilaspartato/farmacología , Receptores de N-Metil-D-Aspartato/fisiología , Techo del Mesencéfalo/fisiología , Animales , Células Cultivadas , Embrión de Pollo , Glicina/farmacología , Técnicas de Placa-Clamp

RESUMEN

Luteal cells are known to possess receptors for LH/hCG and receptors of the beta-adrenergic type. Interactions of specific agonists with either receptor lead to the activation of adenylate cyclase and subsequently to an increase of cAMP. Since in the human there is also evidence for the presence of alpha-adrenergic receptors, we have investigated whether activation of these receptors is linked to calcium as a second messenger and performed measurement of intracellular free calcium (Ca2+) with Fura-2 in single human granulosa-lutein cells. Addition of either hCG (100, 1,000, 25,000 IU/L) or norepinephrine (NE; known to interact with both alpha- and beta-adrenergic receptors), beta-adrenergic receptor agonist isoproterenol (ISO), or alpha-adrenergic receptor agonist phenylephrine (PHE; all at 10 and 100 mumol/L) did not increase free intracellular Ca2+. However, the addition of combinations of NE/hCG, PHE/hCG, but not the combination ISO/hCG, induced a transient increase in cytosolic free Ca2+. The NE/hCG-evoked calcium signal was not abolished in the presence of the beta-adrenergic receptor antagonist propranolol and was not affected by removal of extracellular Ca2+. Furthermore, we tested whether catecholamines affected the release of progesterone in the presence or absence of hCG. As expected, hCG (10,000 IU/L) stimulated progesterone release by cultured granulosa-lutein cells. When these cells were incubated with NE, PHE, or ISO (at 10 mumol/L), production of progesterone by these cells was not affected. However, the combinations of NE and PHE with hCG abolished the hCG-induced progesterone accumulation, but ISO coincubated with hCG did not. Taken together, our results indicate: 1) the presence of functional alpha-adrenergic receptors on human granulosa-lutein cells; 2) simultaneous activation of two different receptors (for hCG and alpha-agonists) are able to evoke intracellular Ca2+ elevation, implicating postreceptor interactions in human granulosa lutein cells; 3) this process occurs even in the absence of extracellular Ca2+, indicating the involvement of intracellular Ca2+ stores, most likely due to activation of phosphoinositide pathway; 4) catecholamines most likely acting via alpha-adrenergic receptors, inhibit the LH/hCG-induced release of progesterone.

Asunto(s)

Calcio/metabolismo , Gonadotropina Coriónica/farmacología , Células de la Granulosa/metabolismo , Células Lúteas/metabolismo , Norepinefrina/farmacología , Receptores Adrenérgicos alfa/fisiología , Células Cultivadas , Femenino , Células de la Granulosa/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Células Lúteas/efectos de los fármacos , Fenilefrina/farmacología , Progesterona/metabolismo , Receptores Adrenérgicos alfa/efectos de los fármacos , Sistemas de Mensajero Secundario , Transducción de Señal/fisiología

Asunto(s)

Calcio , Cationes Bivalentes , Magnesio , Fusión de Membrana , Animales , Tampones (Química) , Ácido Egtácico , Electroquímica/instrumentación , Electroquímica/métodos , Indicadores y Reactivos , Cinética , Ligandos , Matemática , Soluciones

RESUMEN

We investigated whether the stimulation of human granulosa-lutein cells with muscarinic and nicotinic receptor agonists can cause increases in intracellular free calcium (Ca2+), using Fura-2 microfluorimetry. The addition of carbachol (a non-selective muscarinic and nicotinic receptor agonist) to cultured human granulosa-lutein cells increased intracellular free Ca2+ levels. Concentrations as low as 10 nmol/l were effective. In contrast, nicotine did not evoke elevations of intracellular free Ca2+. Basal Ca2+ levels ranged around 70-140 nmol/l and maximal, carbachol-induced peaks reached 1.1 mumol/l. The carbachol-induced Ca2+ signal was abolished after preincubation of the cells with the muscarinic receptor antagonists quinuclidinyl benzilate or atropine, but it was not affected by removal of extracellular Ca2+. Further evidence for the involvement of intracellular Ca2+ stores is provided by experiments in the absence of extracellular Ca2+. While thapsigargin (a blocker of ATP-driven Ca2+ uptake by intracellular stores) and ionomycin (an ionophore by which Ca2+ is released from intracellular stores) evoked small Ca2+ transients, cells pretreated with these agents did not respond to carbachol any more. These data suggest the presence of a functional muscarinic receptor on human granulosa-lutein cells and imply the involvement of intracellular Ca2+ stores during the cellular response. These results also suggest the participation of the nervous system, acting through muscarinic receptors, in the control of the function of human granulosa-lutein cells.

Asunto(s)

Calcio/metabolismo , Carbacol/farmacología , Líquido Intracelular/metabolismo , Células Lúteas/metabolismo , Tapsigargina , Atropina/farmacología , Células Cultivadas , Femenino , Humanos , Líquido Intracelular/efectos de los fármacos , Ionomicina/farmacología , Células Lúteas/efectos de los fármacos , Nicotina/farmacología , Extractos Vegetales/farmacología , Quinuclidinil Bencilato/farmacología , Receptores Muscarínicos/metabolismo

RESUMEN

The potency of oxytocin (OT) in evoking ACTH secretion by isolated, superfused rat adenohypophyseal corticotrophs and its enhancement by CRF and arginine vasopressin (AVP) were analyzed. Each secretagogue effectively released ACTH from adenohypophyseal cells when added separately in pulsatile fashion in physiological concentrations based on hypophyseal portal blood (OT, 10 nM; AVP, 0.5 nM; CRF, 0.1 nM). OT released ACTH at concentrations as low as 1 nM. Moreover, a dose-response relationship up to 10 microM was revealed. Combinations of a constant amount of CRF (0.1 nM) with increasing concentrations of OT exerted a synergistic effect on ACTH release. In contrast, OT given in various concentrations in combination with AVP (0.5 nM) produced an additive effect on ACTH release. To study the mechanism of action of OT on ACTH secretion, cytosolic free calcium levels in single pituitary cells exposed to OT or AVP were measured using the calcium-sensitive fluorescent indicator Fura-2. Corticotrophs among mixed adenohypophyseal cell types in the primary cultures were identified by immunocytochemistry. More than 500 cells were individually stimulated with OT or AVP. Basal cytosolic free calcium levels ranged between 80-130 nM free calcium. The addition of 100 nM OT or 1 microM AVP increased the cytosolic free calcium concentration within 3 sec to values ranging from 500-800 nM. An increase in intracellular calcium ranging from 200-500 nM due to OT could still be observed after extracellular calcium depletion. Taken together, our data demonstrate that physiological concentrations of OT stimulate ACTH secretion, independent of the other ACTH secretagogues, by mobilizing calcium mainly from intracellular stores.

Asunto(s)

Hormona Adrenocorticotrópica/metabolismo , Arginina Vasopresina/farmacología , Calcio/fisiología , Hormona Liberadora de Corticotropina/farmacología , Oxitocina/farmacología , Adenohipófisis/metabolismo , Animales , Sinergismo Farmacológico , Femenino , Técnicas In Vitro , Oxitocina/administración & dosificación , Ratas , Ratas Endogámicas

RESUMEN

Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin permeabilized rat pancreatic acinar cells is specifically inhibited by decavanadate. The Ca2+ release induced with 0.18 microM IP3 is half maximally inhibited with approximately 5 microM decavanadate. Complete inhibition is achieved with around 20 microM decavanadate. Removal of decavanadate from the permeabilized cells fully restores sensitivity towards IP3, indicating the reversibility of the inhibition. Oligovanadate, which inhibits ATP dependent Ca2+ uptake into intracellular stores, does not influence IP3 induced Ca2+ release. In order to reveal the mechanism underlying the effects of the different vanadate species, binding of IP3 to the same cellular preparations was investigated. We found that binding of IP3 to a high affinity receptor site (Kd approx. 1.2 nM) could be abolished by decavanadate but not by oligovanadate. With 0.5 microM decavanadate, IP3 binding was half maximally inhibited. A similar potency of decavanadate was also found with adrenal cortex microsomes which bind IP3 with the same affinity (Kd approx. 1.4 nM) as permeabilized pancreatic acinar cells. Labelled IP3 was displaced from these subcellular membranes with similar kinetics by unlabelled IP3 and decavanadate. The data suggest that the inhibitory action of decavanadate on IP3 induced Ca2+ release is a consequence of its effect on binding of IP3 to its receptor.

Asunto(s)

Canales de Calcio , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Páncreas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares , Vanadatos/farmacología , Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Sitios de Unión , Digitonina/química , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/farmacología , Receptores de Inositol 1,4,5-Trifosfato , Microsomas/efectos de los fármacos , Microsomas/fisiología , Páncreas/efectos de los fármacos , Vanadatos/química

RESUMEN

The inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied using streptolysin O-permeabilized bovine adrenal chromaffin cells. The IP3-induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3-induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 microM GTP fully restored the sensitivity to IP3. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) could not replace GTP but prevented the action of GTP. The effects of GTP and GTP gamma S were reversible. Neither GTP nor GTP gamma S induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 microM free Ca2+, a half-maximal Ca2+ no Ca2+ release was observed with 0.1 microM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 microM) were required to evoke Ca2+ release. At 8 microM free Ca2+, even 0.25 microM IP3 failed to induce release of Ca2+ from the store. The IP3-induced Ca2+ release at constant low (0.2 microM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.

Asunto(s)

Glándulas Suprarrenales/metabolismo , Calcio/metabolismo , Calcio/farmacología , Sistema Cromafín/metabolismo , Guanosina Trifosfato/farmacología , Inositol 1,4,5-Trifosfato/farmacología , Glándulas Suprarrenales/efectos de los fármacos , Animales , Proteínas Bacterianas , Bovinos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sistema Cromafín/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Estreptolisinas/farmacología

RESUMEN

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.

Asunto(s)

Calcio/metabolismo , Guanosina Trifosfato/farmacología , Inositol 1,4,5-Trifosfato/farmacología , Páncreas/metabolismo , Animales , Permeabilidad de la Membrana Celular , Separación Celular , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Masculino , Páncreas/citología , Ratas , Ratas Endogámicas

RESUMEN

The role of Mg2+ during the final steps of exocytosis was investigated using rat pheochromocytoma cells (PC12) permeabilized with bacterial pore forming toxins. Concentrations of free Mg2+ between 0.2 and 2 mM slightly lowered the basal but greatly enhanced the [3H]dopamine release elicited by 8 microM free Ca2+. Maximal effects were obtained at approximately 1 mM free Mg2+. At higher concentrations Mg2+ was less potent. Similar effects of Mg2+ were obtained in cells permeabilized either for small molecules (by alpha-toxin) or for large ones (by streptolysin O). It is concluded that millimolar concentrations of cytoplasmic Mg2+ play an important role in Ca2+ triggered exocytosis.

Asunto(s)

Dopamina/farmacocinética , Exocitosis/efectos de los fármacos , Magnesio/farmacología , Células Tumorales Cultivadas/fisiología , Animales , Feocromocitoma , Ratas , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.

Asunto(s)

Calcio/metabolismo , Hidroximercuribenzoatos/farmacología , Inositol 1,4,5-Trifosfato/farmacología , Vanadatos/farmacología , Adenosina Trifosfato/metabolismo , Neoplasias de las Glándulas Suprarrenales/metabolismo , Animales , Recuento de Células , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Hidroximercuribenzoatos/antagonistas & inhibidores , Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Feocromocitoma/metabolismo , Ratas

Asunto(s)

Toxinas Bacterianas/farmacología , Permeabilidad de la Membrana Celular , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Proteínas Hemolisinas , Orgánulos/ultraestructura , Estreptolisinas/farmacología , Animales , Proteínas Bacterianas , Toxinas Bacterianas/aislamiento & purificación , Calcio/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Indicadores y Reactivos , Cinética , Métodos , Conejos , Staphylococcus aureus , Estreptolisinas/aislamiento & purificación