RESUMO
BACKGROUND: A significant proportion of severe familial forms of obesity remain genetically elusive. Taking advantage of our unique cohort of multigenerational obese families, we aimed to assess the contribution of rare mutations in 29 common obesity-associated genes to familial obesity, and to evaluate in these families the putative presence of nine known monogenic forms of obesity. METHODS: Through next-generation sequencing, we sequenced the coding regions of 34 genes involved in polygenic and/or monogenic forms of obesity in 201 participants (75 normal weight individuals, 54 overweight individuals and 72 individuals with obesity class I, II or III) from 13 French families. In vitro functional analyses were performed to investigate the mutation PCSK1-p.Arg80* which was identified in a family. RESULTS: A novel heterozygous nonsense variant in PCSK1 (p.Arg80*), encoding a propeptide truncated to less than two exons (out of 14), was found to co-segregate with obesity in a three-generation family. We demonstrated that this mutation inhibits PCSK1 enzyme activity and that this inhibition most likely does not involve a strong physical interaction. Furthermore, both mutations PCSK1-p.Asn180Ser and POMC-p.Phe144Leu, which had previously been reported to be associated with severe obesity, were also identified in this study, but did not co-segregate with obesity. Finally, we did not identify any rare mutations co-segregating with obesity in common obesity susceptibility genes, except for CADM2 and QPCTL, where we found two novel variants (p.Arg81His and p.Leu98Pro, respectively) in three obese individuals. CONCLUSIONS: We showed for the first time that a nonsense mutation in PCSK1 was likely to cause dominantly inherited human obesity, due to the inhibiting properties of the propeptide fragment encoded by the null allele. Furthermore, the present family sequencing design challenged the contribution of previously reported mutations to monogenic or at least severe obesity.
Assuntos
Códon sem Sentido/genética , Obesidade/genética , Pró-Proteína Convertase 1/genética , População Branca/genética , Feminino , França/epidemiologia , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Obesidade/epidemiologia , LinhagemAssuntos
Diabetes Mellitus/congênito , Diabetes Mellitus/genética , Fator de Transcrição GATA6/genética , Cardiopatias Congênitas/genética , Mutação/genética , Pancreatopatias/congênito , Adolescente , Linhagem da Célula , Códon/genética , Diabetes Mellitus/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Células Secretoras de Insulina/patologia , Pâncreas/anormalidades , Pancreatopatias/genéticaRESUMO
AIM: Membrane stretch due to cell swelling may cause a minute leakage of adenosine triphosphate (ATP) that stimulates endogenous purinergic receptors. The following elevation of the cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) may then participate in cell volume regulation. The aim of the present study was to test if purinergic receptors and large conductance Ca(2+) activated K(+) (BK) channels are activated in response to hypotonic stress in clonal kidney cells (Vero cells). METHODS: The methods used are fura-2 microfluorometry, cell-attached patch clamp and reverse-transcriptase polymerase chain reaction (RT-PCR). METHODS: Subjecting cells to hypotonic stress for 10 s by exposure to a solution with 45% reduced osmolality induced a transient rise in [Ca(2+)](i). This response persisted in virtually Ca(2+)-free extracellular solution, demonstrating that Ca(2+) was mainly released from intracellular stores. The hypotonically induced elevation of [Ca(2+)](i) was completely inhibited by the P2 receptor antagonists suramine (100 microM) and pyridoxalphosphate-6-azophenyl-2'4'-disulphonate (PPADS; 20 microM), indicating that extracellular ATP is crucial for the [Ca(2+)](i) increase. RT-PCR revealed the expression of mRNA for P2Y(1) receptors in Vero cells. The putatively selective P2Y(1) antagonist PPADS did completely block Ca(2+) responses to both ATP and hypotonic stress, suggesting that P2Y(1) receptors are mediating the response. Furthermore, patch clamp recordings in cell-attached configuration revealed that BK channels are activated in response to hypotonic stress. conclusion: Vero cells express functional purinergic receptors, presumably of the P2Y(1) subtype. These receptors are responsible for the elevation of [Ca(2+)](i) evoked by hypotonic stress. The concurrent activation of BK channels permits K(+) efflux that may contribute to regulatory volume decrease.
Assuntos
Cálcio/metabolismo , Rim/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Tamanho Celular/efeitos dos fármacos , Chlorocebus aethiops , Fluorometria , Fura-2 , Humanos , Soluções Hipotônicas/farmacologia , Rim/química , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência , Suramina/farmacologia , Células VeroRESUMO
AIMS: Thyrotropin-releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca(2+) level ([Ca(2+)](i)), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca(2+)-activated K(+) channels (K(Ca)). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH(4) cells was assessed. METHODS: The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca(2+) microfluoromety (fura-2). The involvement of different K(Ca) channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). RESULTS: Application of 100 nM iberiotoxin, 1 microM apamin, and 10 microM clotrimazole reduced the peak value of the outward K(+) current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin-induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. CONCLUSION: All the three main types of K(Ca) channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.
Assuntos
Cálcio/metabolismo , Adeno-Hipófise/citologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Hormônio Liberador de Tireotropina/fisiologia , Animais , Apamina/farmacologia , Linhagem Celular Tumoral , Células Clonais/metabolismo , Clotrimazol/farmacologia , Eletrofisiologia/métodos , Potenciais da Membrana/fisiologia , Peptídeos/farmacologia , Adeno-Hipófise/efeitos dos fármacos , RNA Mensageiro/análise , RatosRESUMO
AIM: Thyrotropin-releasing hormone (TRH) induces biphasic changes in the electrical activity, the cytosolic free Ca2+ concentration ([Ca2+]i), and prolactin secretion from both GH cells and native lactotrophs. It is well established that inhibition of erg channels contributes to the second phase of the TRH response. We have investigated if BK channels are also involved. RESULTS: The BK channels may be active at the resting membrane potential (open probability, Po=0.01) in clonal rat anterior pituitary cells (GH4), which makes it possible that inhibition of these channels may contribute to the reduced K+ conductance during the TRH response. The specific BK channel blocker iberiotoxin (IbTx, 100 nm) had no effect on the resting conductance at holding potentials negative to -40 mV, but significantly reduced the conductance at shallower membrane potentials. This corresponds to the voltage dependency of the sustained [Ca2+]i. Furthermore, IbTx increased the action potential frequency by 36% in spontaneously firing cells. During the second phase of the TRH response, the action potential frequency increased by 34%, concomitantly with 61% reduction of the Po of single BK channels. The protein kinase C (PKC)-activating phorbol ester TPA had no significant effect on BK channel Po within the normal range of the resting potential. CONCLUSION: The BK channels may contribute to the resting membrane conductance, and they are partially inhibited by TRH during the second phase. This modulation seems not to depend on PKC. We propose that inhibition of erg and BK channels acts in concert to enhance the cell excitability during the second phase of the response to TRH.
Assuntos
Adeno-Hipófise/citologia , Canais de Potássio Cálcio-Ativados/fisiologia , Hormônio Liberador de Tireotropina/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Linhagem Celular , Citofotometria/métodos , Ativadores de Enzimas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/efeitos dos fármacos , Peptídeos/farmacologia , Ésteres de Forbol/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Proteína Quinase C/metabolismo , RatosRESUMO
The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately -55 mV and was blocked by 1 mmol l(-1) TEA. The rate of rise of electrically evoked Ca(2+)-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca(2+) concentration. This suggests that the hyperpolarisation may be caused by activation of Ca(2+)-sensitive K(+) channels. The depolarisation was abolished in Ca(2+)-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca(2+) influx through anterior channels, while Ca(2+) released from intracellular stores activates K(+) channels responsible for the delayed hyperpolarisation.
Assuntos
Laranja de Acridina/farmacologia , Corantes Fluorescentes/farmacologia , Luz , Tetrahymena/efeitos dos fármacos , Animais , Cálcio/metabolismo , Eletrofisiologia , Íons , Potenciais da Membrana/efeitos da radiação , Modelos Biológicos , Canais de Potássio/metabolismoRESUMO
1. Simultaneous pre- and postsynaptic patch recordings were obtained from the varicosity synapses formed by Xenopus motoneurons on muscle cells in embryonic cultures, in order to elucidate the contribution of N- and L-type Ca(2+) channels to the varicosity Ca(2+) current (I(Ca)) and evoked transmitter release. 2. Although N-type channels are predominant in the varicosities and generally thought to be responsible for all evoked release, in most synapses a fraction of I(Ca) and release could be reversibly blocked by the L-type channel antagonist nifedipine, and enhanced by the agonist Bay K8644. Up to 50 % (mean, 21 %) of the I(Ca) evoked by a voltage clamp waveform mimicking a normal presynaptic action potential (APWF) is composed of L-type current. 3. Surprisingly, the nifedipine-sensitive (L) channels activated more rapidly (time-constant, 0.46 ms at +30 mV) than the nifedipine-insensitive (N) channels (time constant, 1.42 ms). Thus the L-type current would play a disproportionate role in the I(Ca) linked to a normal action potential. 4. The relationship between I(Ca) and release was the same for nifedipine-sensitive and -resistant components. The N- and L-components of I(Ca) are thus equally potent in evoking release. This may represent an immature stage before N-type channels become predominant. 5. Replacing Ca(2+) in the medium with Ba(2+) strongly enhanced the L-type component, suggesting that L-type channels may be inactivated at Ca(2+) levels close to those at rest. 6. We speculate that populations of L-type channels in different parts of the neuron may be recruited or inactivated by fluctuations of the cytosolic Ca(2+) concentration within the physiological range.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Transmissão Sináptica/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Acetilcolina/metabolismo , Animais , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Células Cultivadas , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Neurônios Motores/citologia , Fibras Musculares Esqueléticas/citologia , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Medula Espinal/citologia , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Xenopus laevisRESUMO
We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP >> UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.
Assuntos
Cálcio/metabolismo , Canais de Potássio Cálcio-Ativados , Canais de Potássio/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Chlorocebus aethiops , Citosol/metabolismo , Ionomicina/farmacologia , Ionóforos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Potássio/farmacocinética , Receptores Purinérgicos P2Y1 , Uridina Trifosfato/farmacologia , Células VeroRESUMO
Fishes have an acute sensitivity to extremely low-frequency linear acceleration, or infrasound, even down to below 1 Hz. The otolith organs are the sensory system responsible for this ability. The hydrodynamic noise generated by swimming fishes is mainly in the infrasound range, and may be important in courtship and prey predator interactions. Intense infrasound has a deterring effect on some species, and has a potential in acoustic barriers. We hypothesize that the pattern of ambient infrasound in the oceans may be used for orientation in migratory fishes, and that pelagic fishes may detect changes in the surface wave pattern associated with altered water depth and distant land formations. We suggest that the acute sensitivity to linear acceleration could be used for inertial guidance, and to detect the relative velocity of layered ocean currents. Sensitivity to infrasound may be a widespread ability among aquatic organisms, and has also been reported in cephalopods and crustaceans.
Assuntos
Percepção Auditiva/fisiologia , Peixes/fisiologia , Audição/fisiologia , Animais , Comportamento Animal/fisiologia , Coração/fisiologia , Membrana dos Otólitos/fisiologiaRESUMO
To study the secretion from endocrine cells in culture, we have developed a cell column perfusion system with a time resolution of 4 s. The core of the system is a perfusion chamber with a cell-supporting matrix of monosized polystyrene beads. The particles are solid and can withstand a high pressure gradient without deformation. The minimum amount of cell material required to obtain detectable levels of secretory products is a function of the assay sensitivity, perfusion flow, fraction volume and time resolution. The volume of the perfusion chamber is therefore adjustable to satisfy varying demands of minimum cell number. The general flow characteristics of the system were characterized using radiolabeled substances of various molecular sizes. Using the system in secretory studies of rat pituitary tumor (GH4C1) cells, we have identified differences in secretion profiles that may be related to the kinetics of the different transmembrane and intracellular mechanisms involved.
Assuntos
Peptídeos/metabolismo , Perfusão/métodos , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Animais , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Células Imobilizadas/ultraestrutura , Cinética , Microscopia Eletrônica de Varredura , Microesferas , Perfusão/instrumentação , Neoplasias Hipofisárias/patologia , Ratos , Reologia , Hormônio Liberador de Tireotropina/farmacologia , Células Tumorais Cultivadas , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
In normal recording solution, the swimming pattern of the freshwater ciliate Coleps hirtus, belonging to the class Prostomatea, consists of alternating periods of nearly linear forward swimming and circular swimming within a small area. Current-clamp recordings were performed to elucidate the mechanism for this behaviour. No members of this class have previously been studied using electrophysiological techniques. The ciliates were maintained in culture and fed on the planctonic alga Rhodomonas minuta. The membrane potential showed spontaneous shifts between a more negative (deep) level of approximately -50 mV and a less negative (shallow) level of approximately -30 mV. The input resistance and capacitance at the more negative level were approximately 400 M capomega and 120 pF respectively. C. hirtus displayed a pronounced inward rectification, which was virtually insensitive to 1 mmol l(-1) Cs(+) and almost completely blocked by 1 mmol l(-1) Ba(2+). Depolarising current injections failed to evoke graded, regenerative Ca(2+) spikes. However, current-induced depolarisations from the more negative potential level (-50 mV) showed a pronounced shoulder during the repolarising phase. Increased current injections prolonged the shoulder, which occasionally stabilised at the shallow membrane potential (-30 mV). The membrane potential could be shifted to the deep level by brief hyperpolarising current injections. Similar biphasic membrane properties have not been reported previously in any ciliate. The bistability of the membrane potential was abolished in Ca(2+)-free solution containing Co(2+) or Mg(2+). In Ca(2+)-free solution containing 1 mmol l(-1) Ba(2+), brief depolarising current injections at the deep potential level evoked all-or-nothing action potentials with a prolonged plateau coinciding with the shallow potential. We conclude that the deep membrane potential in C. hirtus corresponds to the traditional resting potential, whereas the shallow level is a Ca(2+)-dependent plateau potential. In normal solution, the direction of the ciliary beat was backwards at the deep potential level and forwards at the shallow membrane potential, probably reflecting the two main phases of the swimming pattern.
Assuntos
Cilióforos/fisiologia , Potenciais da Membrana , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Potenciais Evocados , Técnicas de Patch-Clamp , NataçãoRESUMO
Activation of large conductance Ca(2+)-activated K+ channels (BK channels) in intact clonal rat pituitary cells (GH4 cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca2+ concentration ([Ca2+]i) in GH cells by IP3-induced release of Ca2+ from intracellular stores. This rise in [Ca2+]i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca2+ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH4 cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca2+]i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH4 cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca2+]i in a wide variety of cell types.
Assuntos
Bradicinina/metabolismo , Cálcio/fisiologia , Citosol/metabolismo , Adeno-Hipófise/metabolismo , Canais de Potássio/metabolismo , Animais , Cálcio/farmacologia , Células Clonais , Citosol/fisiologia , Eletrofisiologia , Corantes Fluorescentes , Fura-2 , Ionomicina/farmacologia , Ionóforos/farmacologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Adeno-Hipófise/citologia , Canais de Potássio/fisiologia , Ratos , Hormônio Liberador de Tireotropina/farmacologiaRESUMO
Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Deltagm, was initially determined. DT-AB induced a approximately 10-fold lower Deltagm than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Deltagm, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Deltagm. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Deltagm and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.
Assuntos
Membrana Celular/efeitos dos fármacos , Toxina Diftérica/farmacologia , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Células Vero/efeitos dos fármacos , Animais , Chlorocebus aethiops , Toxina Diftérica/química , Toxina Diftérica/genética , Concentração de Íons de Hidrogênio , Canais Iônicos/química , Modelos Moleculares , Técnicas de Patch-Clamp , Mutação Puntual , Biossíntese de Proteínas , Conformação Proteica , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The relationships among ischaemic GABA efflux from brain tissue and extracellular and intracellular concentrations of sodium, chloride and potassium ions were investigated by means of 1) transverse hippocampal slices from rat and 2) functional expression of a high affinity GABA transporter in Xenopus oocytes. Brain slices were incubated for 20 min in medium where extracellular sodium and chloride were substituted with impermeant ions. Isethionate (Iseth) substitution for chloride generated a 7-fold increase in GABA efflux. Choline (Chol) but not N-methyl-D-glucamine (NMDG) substitution for sodium likewise increased GABA efflux. Reducing the osmolarity of the medium by decreasing both sodium and chloride concentrations (Hyp) increased GABA efflux 3-fold. This release was blocked by mannitol (Man). Blocking sodium channels with 1 microM of tetrodotoxin (TTX) also increased the release 3-fold. Energy deprivation (ED) increased the GABA release 50-fold. ED/Iseth left the release unchanged, ED/Chol increased the GABA efflux by 23%, whereas ED/NMDG reduced the release by 41%. Adding mannitol did not block the ED-evoked release, whereas TTX reduced it by 52%. Release of preloaded [3H]-GABA from oocytes expressing the GAT-1 GABA transporter was then examined. Depolarisation by current injection or 100 mM extracellular K+ did not increase GABA release. Sodium chloride injection, however, caused membrane depolarisation and a 100-fold increased GABA efflux from the oocytes. This release was blocked when the osmolarity was increased extracellularly by adding mannitol. These results show that 1) TTX releases GABA from brain tissue but blocks release during ED, 2) the high affinity GABA carrier must be altered in order to reverse, 3) ischaemic GABA release is sodium independent, and is modulated by large cations, 4) mannitol blocks the reversal of high affinity carriers in oocytes, but the release from brain slices during ED is unaffected. Taken together, the results suggest that ischaemic release of GABA from brain tissue does not occur by means of reversed high affinity carriers alone, but rather that it is controlled by more complex mechanisms.
Assuntos
Edema Encefálico/fisiopatologia , Isquemia Encefálica/fisiopatologia , Eletrólitos/metabolismo , Proteínas de Membrana Transportadoras , Transportadores de Ânions Orgânicos , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Sobrevivência Celular/fisiologia , Técnicas de Cultura , Metabolismo Energético/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA , Expressão Gênica/fisiologia , Hipocampo/fisiopatologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Oócitos , Ratos , Ratos Wistar , XenopusRESUMO
Optical recordings of membrane depolarization and whole-cell patch-clamp recordings of membrane potentials and currents were obtained from chromaffin cells in slices of rat adrenal medulla. The stimulation of splanchnic nerve fibers caused a discontinuous spread of electrical activity across the slice. Cells in clusters with diameters of about 80 microns were excited simultaneously, suggesting that the adrenal medulla is organized into descrete cell complexes with common innervation. The electrical properties of chromaffin cells in situ were in agreement with previous reports on cultured cells. A fraction of the recorded cells displayed excitatory postsynaptic currents (EPSCs) of 0.2-1 nA upon the stimulation of presynaptic nerve fibers. The EPSC was blocked by hexamethonium, suggesting that nicotinic ACh receptors were involved. The decay phase of the EPSC was well fit by the sum of two exponentials with time constants of 6.3 and 57.3 ms. The relative amplitude of the fast component was 84.1%. These two exponentials may reflect activation of both fast and slow time-constant ACh receptor channels by presynaptic release of ACh. There were multiple peaks in the EPSC amplitude histograms in low-[Ca2+] saline, the first peak was at 37 pA. To resolve the quantal size, miniature EPSCs were recorded in a tetrodotoxin-containing high-[K+] solution. The miniature EPSC amplitude histograms were also multimodal with the first peak at 25 pA, which probably represents the quantal size of the synapse. The second and third peaks were at the integer multiples of the first one.
Assuntos
Medula Suprarrenal/fisiologia , Células Cromafins/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Medula Suprarrenal/anatomia & histologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Comunicação Celular/fisiologia , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Bloqueadores Ganglionares/farmacologia , Hexametônio/farmacologia , Técnicas In Vitro , Cinética , Masculino , Potenciais da Membrana/fisiologia , Óptica e Fotônica , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Receptores Nicotínicos/fisiologia , Análise de Regressão , Nervos Esplâncnicos/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl-. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g = 4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g = 10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na(+)-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na(+)-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.
Assuntos
Canais de Sódio/metabolismo , Glândula Tireoide/metabolismo , Animais , Células Cultivadas , Dinoprostona/farmacologia , Epitélio/metabolismo , Transporte de Íons , SuínosRESUMO
We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.
Assuntos
Toxina Diftérica/metabolismo , Glicosilfosfatidilinositóis , Receptores de Superfície Celular/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Antígenos CD55/química , Membrana Celular/metabolismo , Primers do DNA/química , Receptores ErbB/química , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular , Ativação do Canal Iônico , Camundongos , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Proteínas Recombinantes de Fusão , Relação Estrutura-AtividadeRESUMO
Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and to secrete the anions Cl- and HCO3-. Chloride channels were found in the apical membrane, and displayed a reversal potential close to the resting membrane potential, linear current-voltage relationships, a conductance at physiological temperature of 6.5 pS, and a small but significant permeability to HCO3-. Stimulation of ion transport with prostaglandin E2 or 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate promoted activation of Cl- channels in cell-attached patches, and excised patches were reactivated by exposure of their cytoplasmic surface to protein kinase A and ATP. Physiological temperatures were necessary for activation of Cl- channels in cell-attached patches. The channels exhibited sub-states with a conductance exactly half that of the full unit conductance, suggesting a dual-barrelled channel structure. It is concluded that the apical membrane of thyroid epithelial cells contains cyclic AMP-activated Cl- channels controlling anion transport.
Assuntos
Canais de Cloreto/metabolismo , AMP Cíclico/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Suínos/metabolismo , Glândula Tireoide/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Dinoprostona/farmacologia , Epitélio/metabolismo , Transporte de Íons/efeitos dos fármacos , Temperatura , Tionucleotídeos/farmacologiaRESUMO
The activity and some kinetic parameters of the key enzymes of the glycolysis, the gluconeogenesis and the amino acid catabolism from the liver of male and female mink have been determined and compared to the corresponding activities from rat and cat. The activities of glucose-6-phosphatase and pyruvate kinase are dependent on sex, both being higher in females. Except for pyruvate carboxylase the glycolytic and the gluconeogenic enzyme activities of the mink are higher than those of rat and cat; especially the activities of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are markedly higher. The activities of glutamate dehydrogenase and glutamate oxaloacetate transaminase are smaller than the corresponding activities of rat but higher than those of cat. The results suggest that mink has a high capacity for gluconeogenesis compared to rat.