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1.
Kidney Int ; 83(5): 811-24, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23447069

RESUMO

A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (0.25 h), followed by a significant rise of plasma aldo (0.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about 0.5 h). The functional changes were accompanied by a rapid and sustained (0.25-6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldo-independent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron.


Assuntos
Rim/metabolismo , Potássio na Dieta/sangue , Receptores de Droga/metabolismo , Simportadores/metabolismo , Administração Oral , Aldosterona/sangue , Animais , Transporte Biológico , Citocromo P-450 CYP11B2/deficiência , Citocromo P-450 CYP11B2/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Natriurese , Fosforilação , Potássio na Dieta/administração & dosagem , Potássio na Dieta/urina , Receptores de Droga/deficiência , Receptores de Droga/genética , Membro 3 da Família 12 de Carreador de Soluto , Simportadores/deficiência , Simportadores/genética , Fatores de Tempo , Equilíbrio Hidroeletrolítico
2.
Proc Natl Acad Sci U S A ; 109(33): 13368-73, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22847418

RESUMO

The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema.


Assuntos
Proteínas de Transporte de Ânions/deficiência , Diurese/fisiologia , Receptores de Droga/deficiência , Insuficiência Renal/fisiopatologia , Cloreto de Sódio/metabolismo , Simportadores/deficiência , Alcalose/sangue , Alcalose/complicações , Alcalose/fisiopatologia , Animais , Proteínas de Transporte de Ânions/metabolismo , Peso Corporal , Rim/patologia , Rim/fisiopatologia , Testes de Função Renal , Camundongos , Camundongos Knockout , Modelos Biológicos , Potássio/sangue , Receptores de Droga/metabolismo , Insuficiência Renal/sangue , Insuficiência Renal/complicações , Sódio/sangue , Cloreto de Sódio na Dieta , Membro 3 da Família 12 de Carreador de Soluto , Transportadores de Sulfato , Simportadores/metabolismo , Vasopressinas/administração & dosagem , Vasopressinas/farmacologia , Equilíbrio Hidroeletrolítico/fisiologia
3.
J Thorac Cardiovasc Surg ; 140(5): 1153-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20804990

RESUMO

OBJECTIVE: Animal and human myocytes demonstrate significant swelling and reduced contractility during exposure to stress (metabolic inhibition, hyposmotic stress, or hyperkalemic cardioplegia), and these detrimental consequences may be inhibited by the addition of diazoxide (adenosine triphosphate-sensitive potassium channel opener) via an unknown mechanism. Both SUR1 and SUR2A subunits have been localized to the heart, and mouse sarcolemmal adenosine triphosphate-sensitive potassium channels are composed of SUR2A/Kir6.2 subunits in the ventricle and SUR1/Kir6.2 subunits in the atria. This study was performed to localize the mechanism of diazoxide by direct probing of sarcolemmal adenosine triphosphate-sensitive potassium channel current and by genetic deletion of channel subunits. METHODS: Sarcolemmal adenosine triphosphate-sensitive potassium channel current was recorded in isolated wild-type ventricular mouse myocytes during exposure to Tyrode's solution, Tyrode's + 100 µmol/L diazoxide, hyperkalemic cardioplegia, cardioplegia + diazoxide, cardioplegia + 100 µmol/L pinacidil, or metabolic inhibition using whole-cell voltage clamp (N = 7-12 cells per group). Ventricular myocyte volume was measured from SUR1(-/-) and wild-type mice during exposure to control solution, hyperkalemic cardioplegia, or cardioplegia + 100 µmol/L diazoxide (N = 7-10 cells per group). RESULTS: Diazoxide did not increase sarcolemmal adenosine triphosphate-sensitive potassium current in wild-type myocytes, although they demonstrated significant swelling during exposure to cardioplegia that was prevented by diazoxide. SUR1(-/-) myocytes also demonstrated significant swelling during exposure to cardioplegia, but this was not altered by diazoxide. CONCLUSIONS: Diazoxide does not open the ventricular sarcolemmal adenosine triphosphate-sensitive potassium channel but provides volume homeostasis via an SUR1-dependent pathway in mouse ventricular myocytes, supporting a mechanism of action distinct from sarcolemmal adenosine triphosphate-sensitive potassium channel activation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/agonistas , Cardiotônicos/farmacologia , Tamanho Celular/efeitos dos fármacos , Diazóxido/farmacologia , Canais KATP/agonistas , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Receptores de Droga/agonistas , Sarcolema/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Soluções Cardioplégicas/farmacologia , Feminino , Homeostase , Hiperpotassemia/metabolismo , Canais KATP/deficiência , Canais KATP/genética , Canais KATP/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Pressão Osmótica , Técnicas de Patch-Clamp , Pinacidil/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/deficiência , Receptores de Droga/genética , Receptores de Droga/metabolismo , Sarcolema/metabolismo , Receptores de Sulfonilureias
5.
Sci Transl Med ; 2(28): 28ra29, 2010 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-20410530

RESUMO

Spinal cord injury (SCI) is typically complicated by progressive hemorrhagic necrosis, an autodestructive process of secondary injury characterized by progressive enlargement of a hemorrhagic contusion during the first several hours after trauma. We assessed the role of Abcc8, which encodes sulfonylurea receptor 1 (SUR1), in progressive hemorrhagic necrosis. After SCI, humans and rodents exhibited similar regional and cellular patterns of up-regulation of SUR1 and Abcc8 messenger RNA. Elimination of SUR1 in Abcc8(-/-) mice and in rats given antisense oligodeoxynucleotide against Abcc8 prevented progressive hemorrhagic necrosis, yielded significantly better neurological function, and resulted in lesions that were one-fourth to one-third the size of those in control animals. The beneficial effects of Abcc8 suppression were associated with prevention of oncotic (necrotic) death of capillary endothelial cells. Suppression of Abcc8 with antisense oligodeoxynucleotide after SCI presents an opportunity for reducing the devastating sequelae of SCI.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/prevenção & controle , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glibureto/farmacologia , Hemorragia/complicações , Hemorragia/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Droga/deficiência , Receptores de Droga/genética , Fator de Transcrição Sp1/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Receptores de Sulfonilureias , Resultado do Tratamento
6.
Cell Tissue Res ; 340(2): 335-46, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20383647

RESUMO

Trafficking of pancreatic K(ATP) channels to the plasma membrane critically depends on masking the endoplasmic reticulum (ER) retention signals of the SUR1 and Kir6.2 subunits upon their proper assembly into functional hetero-octamers. When expressed in the absence of the partner protein, each subunit might accumulate in the ER and trigger beta-cell ER stress and oxidative stress. To test this hypothesis, Kir6.2 localisation, ER ultra-structure and ER-stress- and oxidative-stress-response gene mRNA levels were evaluated in pancreatic endocrine cells from adult wild-type (WT) and Sur1 knockout (Sur1 ( -/- )) mice. As previously reported, Kir6.2 was mainly expressed on secretory granules and at the plasma membrane of WT islet cells. In contrast, like the ER chaperone calreticulin, Kir6.2 was primarily localised in the rough endoplasmic reticulum (RER) of Sur1 ( -/- ) islet cells. ER retention of Kir6.2 was demonstrated (electron microscopy) by a significant increase in the length and Kir6.2 density of RER in Sur1 ( -/- ) vs WT islet cells. Despite Kir6.2 retention in RER, Xbp1 mRNA splicing and mRNA levels of preproinsulin and ER-stress-response genes Bip, Edem and Gadd153 were similar in WT and Sur1 ( -/- ) islets. However, mRNA levels of the antioxidant enzymes Sod1, Sod2, Gpx2 and catalase were significantly up-regulated in Sur1 ( -/- ) islets. Sequestration of Kir6.2 in RER of Sur1 ( -/- ) islet cells is thus associated with an increase in RER length and mild oxidative stress without activation of the classical ER stress response.


Assuntos
Retículo Endoplasmático/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/deficiência , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Calreticulina/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Regulação da Expressão Gênica , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Frações Subcelulares/metabolismo , Receptores de Sulfonilureias
7.
J Neurophysiol ; 101(5): 2305-16, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19261705

RESUMO

The mammalian CNS relies on a constant supply of external glucose for its undisturbed operation. However, neurons can readily switch to using fatty acids and ketones as alternative fuels. Here, we show that oleic acid (OA) excites pro-opiomelanocortin (POMC) neurons by inhibition of ATP-activated potassium (K(ATP)) channels. The involvement of K(ATP) channels is further supported by experiments in SUR1 KO animals. Inhibition of beta-oxidation using carnitine palmitoyltransferase-1 inhibitors blocks OA-induced depolarization. The depolarizing effect of OA is specific because it is not mimicked by octanoic acid. Furthermore, OA does not regulate the excitability of agouti-related peptide neurons. High-fat feeding alters POMC neuron excitability, but not its response to OA. Thus beta-oxidation in POMC neurons may mediate the appetite-suppressing (anorexigenic) effects of OA.


Assuntos
Hipotálamo/citologia , Neurônios/efeitos dos fármacos , Ácido Oleico/farmacologia , Pró-Opiomelanocortina/metabolismo , Transportadores de Cassetes de Ligação de ATP , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Trifosfato de Adenosina/farmacologia , Análise de Variância , Animais , Biofísica , Diazóxido/farmacologia , Gorduras na Dieta/administração & dosagem , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Glucose/metabolismo , Glibureto/farmacologia , Proteínas de Fluorescência Verde/genética , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/metabolismo , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Pró-Opiomelanocortina/genética , Receptores de Droga/deficiência , Receptores de Sulfonilureias
8.
Cell Tissue Res ; 335(3): 505-15, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19142666

RESUMO

In congenital hyperinsulinism of infancy (CHI), the loss of K-ATP channels (composed of Kir6.2 and SUR1 subunits) in beta cells induces permanent insulin secretion and severe hypoglycaemia. By contrast, Sur1 ( -/- ) mice do not present such defects. We have investigated the impact of Sur1 gene inactivation on mouse islet cell morphology, structure and basic physiology. Pancreata were collected from young, adult and old wild-type (WT) and Sur1 ( -/- ) mice. After immunostaining for hormone, the total endocrine tissue, cell proportion, cell size and intra-insular distribution, hormone content and Glut-2 expression were quantified by morphometry. Basic physiological parameters were also measured. In young Sur1 ( -/- ) mice, the total endocrine tissue and proportion of beta cells were higher (P<0.05) than in WT mice, whereas the proportion of delta cells was lower (P<0.01). In old Sur1 ( -/- ) mice, alpha cells were frequently located in the central regions of islets (unlike WT islets) and their proportion was increased (P<0.05). Glut-2 protein and mRNA levels were lower in old Sur1 ( -/- ) islets (P<0.02). Insulinaemia, fasting insulin and glucagon contents were equivalent in both groups of pancreata. Thus, the islets of Sur1 ( -/- ) mice present morphological modifications that have not been described in CHI and that might reflect an adaptive mechanism controlling insulin secretion in these mice.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ilhotas Pancreáticas/fisiologia , Pâncreas/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Droga/genética , Fatores Etários , Animais , Glicemia/metabolismo , Feminino , Inativação Gênica , Glucagon/genética , Glucagon/metabolismo , Imuno-Histoquímica , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/anatomia & histologia , Pâncreas/citologia , Pâncreas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Receptores de Droga/deficiência , Receptores de Sulfonilureias
9.
Mol Pharmacol ; 74(3): 736-43, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18511652

RESUMO

ATP-sensitive K+ (KATP) channel openers are vasodilators that activate both plasma membrane and mitochondrial KATP channels. Here, we investigated the molecular mechanisms by which diazoxide and pinacidil induce vasodilation by studying diameter regulation of wild-type [SUR2(+/+)] and sulfonylurea receptor (SUR) 2-deficient [SUR2(-/-)] mouse myogenic mesenteric arteries. Ryanodine (10 microM), a ryanodine-sensitive Ca2+ release (RyR) channel blocker; iberiotoxin (100 nM), a large-conductance Ca2+-activated K+ (KCa) channel blocker; 4-aminopyridine (4-AP; 1 mM), a voltage-gated K+ (KV) channel blocker; manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 100 microM), an antioxidant; and a combination of ryanodine and 4-AP reduced diazoxide (100 microM)-induced dilation in pressurized (60 mm Hg) SUR2(+/+) arteries by 45 to 77%. In contrast, these inhibitors did not alter pinacidil (5 microM)-induced dilation in SUR2(+/+) arteries. Reverse transcription-polymerase chain reaction indicated that SUR2B was the only SUR isoform expressed in SUR2(+/+) mesenteric artery smooth muscle cells, whereas SURs were absent in SUR2(-/-) cells. In SUR2(-/-) arteries, pinacidil-induced vasodilation was 10% of that in SUR2(+/+) arteries, whereas diazoxide-induced vasodilation was similar in SUR2(+/+) and SUR2(-/-) arteries. Atpenin (1 microM), a selective electron transport chain (ETC) complex II inhibitor, dilated arteries similarly to diazoxide, and this effect was attenuated by MnTMPyP and ryanodine + 4-AP. Atpenin also attenuated diazoxide-, but not pinacidil-induced vasodilation. In summary, data indicate that pinacidil-induced vasodilation requires SUR2B, whereas diazoxide-induced vasodilation does not require SURs. Rather, diazoxide-induced vasodilation involves ETCII inhibition; a smooth muscle cell-reactive oxygen species elevation; and RyR, KCa, and KV channel activation. These data indicate that KATP channel openers regulate arterial diameter via SUR-dependent and -independent pathways.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/farmacologia , Moduladores de Transporte de Membrana/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Diazóxido/farmacologia , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Artérias Mesentéricas/citologia , Artérias Mesentéricas/efeitos dos fármacos , Camundongos , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Pinacidil/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Subunidades Proteicas/metabolismo , Receptores de Droga/deficiência , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Receptores de Sulfonilureias
10.
Cell Physiol Biochem ; 20(1-4): 65-74, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17595516

RESUMO

K(ATP) channel activity influences beta cell Ca(2+) homeostasis by regulating Ca(2+) influx through L-type Ca(2+) channels. The present paper demonstrates that loss of K(ATP) channel activity due to pharmacologic or genetic ablation affects Ca(2+) storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca(2+) release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca(2+) ATPases by cyclopiazonic acid abolished the FCCP-induced Ca(2+) transient. In beta cells treated with K(ATP) channel inhibitors FCCP elicited a significantly larger Ca(2+) transient. Cyclopiazonic acid did not abolish this Ca(2+) transient suggesting that non-ER compartments are recruited as additional Ca(2+) stores in beta cells lacking K(ATP) channel activity. Genetic ablation of K(ATP) channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca(2+)-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca(2+) evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K(ATP) channel activity conveys Ca(2+) release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca(2+) it is suggested that mitochondria are the recruited store. The change in Ca(2+) sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Compartimento Celular , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glucose/farmacologia , Técnicas In Vitro , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Canais de Potássio/deficiência , Canais de Potássio/genética , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/deficiência , Receptores de Droga/genética , Receptores de Droga/metabolismo , Receptores de Sulfonilureias , Tolbutamida/farmacologia
11.
J Biol Chem ; 282(5): 3347-56, 2007 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-17138562

RESUMO

Sulfonylurea receptors (SURs) constitute the regulatory subunits of ATP-sensitive K+ channels (K(ATP) channels). SUR binds nucleotides and synthetic K(ATP) channel modulators, e.g. the antidiabetic sulfonylurea glibenclamide, which acts as a channel blocker. However, knowledge about naturally occurring ligands of SUR is very limited. In this study, we show that the plant phenolic compound trans-resveratrol can bind to SUR and displace binding of glibenclamide. Electrophysiological measurements revealed that resveratrol is a blocker of pancreatic SUR1/K(IR)6.2 K(ATP) channels. We further demonstrate that, like glibenclamide, resveratrol induces enhanced apoptosis. This was shown by analyzing different apoptotic parameters (cell detachment, nuclear condensation and fragmentation, and activities of different caspase enzymes). The observed apoptotic effect was specific to cells expressing the SUR1 isoform and was not mediated by the electrical activity of K(ATP) channels, as it was observed in human embryonic kidney 293 cells expressing SUR1 alone. Enhanced susceptibility to resveratrol was not observed in pancreatic beta-cells from SUR1 knock-out mice or in cells expressing the isoform SUR2A or SUR2B or the mutant SUR1(M1289T). Resveratrol was much more potent than glibenclamide in inducing SUR1-specific apoptosis. Treatment with etoposide, a classical inducer of apoptosis, did not result in SUR isoform-specific apoptosis. In conclusion, resveratrol is a natural SUR ligand that can induce apoptosis in a SUR isoform-specific manner. Considering the tissue-specific expression patterns of SUR isoforms and the possible effects of SUR mutations on susceptibility to apoptosis, these observations could be important for diabetes and/or cancer research.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Apoptose/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Canais de Potássio/fisiologia , Receptores de Droga/fisiologia , Estilbenos/farmacologia , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Etoposídeo/farmacologia , Feminino , Humanos , Hipoglicemiantes/farmacologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Rim , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio/deficiência , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Receptores de Droga/deficiência , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/genética , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Resveratrol , Estilbenos/farmacocinética , Receptores de Sulfonilureias , Transfecção
12.
Diabetologia ; 48(5): 913-21, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15830184

RESUMO

AIMS/HYPOTHESIS: Islets or beta cells from Sur1(-/-) mice were used to determine whether changes in plasma membrane potential (V(m)) remain coupled to changes in cytosolic Ca(2+) ([Ca(2+)](i)) in the absence of K(ATP) channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca(2+)](i) influences oscillations in V(m) in sur1(-/-) beta cells. METHODS: Plasma membrane potential and ion currents were measured with microelectrodes and the patch-clamp technique. [Ca(2+)](i) was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations. RESULTS: Membrane depolarisation of Sur1(-/-) islets by arginine or increased extracellular K(+), elevated [Ca(2+)](i) and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1(-/-) islets. Oscillations in V(m) were influenced by [Ca(2+)](i) as follows: (1) elevation of extracellular Ca(2+) lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca(2+)-dependent outward current that was augmented by increased Ca(2+) influx through L-type Ca(2+) channels; (3) Ca(2+) depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1(-/-) islets, elevating [Ca(2+)](i) and insulin secretion; (4) store depletion activated a Ca(2+) influx that was not inhibitable by the L-type Ca(2+) channel blocker D600. CONCLUSIONS/INTERPRETATION: Although V(m) is largely uncoupled from glucose metabolism in the absence of K(ATP) channels, increased electrical activity leads to elevations of [Ca(2+)](i) that are sufficient to stimulate insulin secretion. In Sur1(-/-) beta cells, [Ca(2+)](i) exerts feedback mechanisms on V(m) by activating a hyperpolarising outward current and by depolarising V(m) via store-operated ion channels.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Cálcio/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Potenciais da Membrana/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio/genética , Receptores de Droga/genética , Animais , Arginina/farmacologia , Membrana Celular/fisiologia , Indóis/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Potássio/farmacologia , Canais de Potássio/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Receptores de Droga/deficiência , Receptores de Sulfonilureias
13.
J Neurosci ; 24(28): 6410-5, 2004 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-15254097

RESUMO

Vanilloid receptor 1 (TRPV1) has been proposed to be the principal heat-responsive channel for nociceptive neurons. The skin of both rat and mouse receives major projections from primary sensory afferents that bind the plant lectin isolectin B4 (IB4). The majority of IB4-positive neurons are known to be heat-responsive nociceptors. Previous studies suggested that, unlike rat, mouse IB4-positive cutaneous afferents did not express TRPV1 immunoreactivity. Here, multiple antisera were used to confirm that mouse and rat have different distributions of TRPV1 and that TRPV1 immunoreactivity is absent in heat-sensitive nociceptors. Intracellular recording in TRPV1(-/-) mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1(-/-) mice had more heat-sensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1(-/-) mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.


Assuntos
Canais de Cálcio/deficiência , Gânglios Espinais/citologia , Temperatura Alta , Canais Iônicos/deficiência , Neurônios Aferentes/química , Nociceptores/fisiologia , Receptores de Droga/deficiência , Sequência de Aminoácidos , Animais , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Epiderme/fisiologia , Feminino , Canais Iônicos/genética , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Neurônios Aferentes/fisiologia , Nociceptores/química , Lectinas de Plantas/análise , Ratos , Ratos Sprague-Dawley , Receptores de Droga/genética , Receptores de Droga/fisiologia , Especificidade da Espécie , Canais de Cátion TRPV
14.
Neurosci Lett ; 362(2): 150-3, 2004 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15193774

RESUMO

Sensory irritants initiate respiratory reflexes by stimulating trigeminal sensory nerves. The vanilloid receptor (TRPV1) is expressed on sensory C fibers. The current experiments were aimed at examining the role of this receptor in mediating responses to several airborne irritants including an acidic (acetic acid), electrophilic (acrolein), and lipophilic solvent (styrene) vapor. Wild-type (C57Bl/6J) and VR1 knockout [B6.129S4-VR1(tm1jul)] mice were exposed to these irritants and breathing pattern responses were assessed by plethysmographic techniques; both wild-type and knockout animals responded similarly to the irritants. The TRPV1 antagonist iodoresiniferatoxin was also without effect on the responses to the irritants. Thus, in the C57Bl/6J mouse the TRPV1 receptor does not appear to play a major role in the stimulation of nasal trigeminal central reflex responses by these irritant air pollutants.


Assuntos
Poluentes Atmosféricos/farmacologia , Inalação/efeitos dos fármacos , Receptores de Droga/fisiologia , Ácido Acético/farmacologia , Acroleína/farmacologia , Animais , Feminino , Inalação/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Droga/deficiência , Receptores de Droga/genética , Estireno/farmacologia
15.
Neurosci Lett ; 361(1-3): 155-8, 2004 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-15135917

RESUMO

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC(50) of capsaicin and OLDA on (45)Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 microM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca(2+) transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6-9 degrees C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.


Assuntos
Dopamina/análogos & derivados , Dopamina/farmacologia , Dor/induzido quimicamente , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/deficiência , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Capsaicina/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Temperatura Alta/efeitos adversos , Humanos , Ligantes , Camundongos , Camundongos Knockout , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Dor/genética , Dor/metabolismo , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Ratos , Receptores de Droga/genética , Transfecção
16.
J Neurosci ; 24(18): 4293-9, 2004 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-15128843

RESUMO

Proteinase-activated receptor (PAR) 2 is expressed on a subset of primary afferent neurons and involved in inflammatory nociception. Transient receptor potential vanilloid subfamily 1 (TRPV1) is a sensory neuron-specific cation channel that responds to capsaicin, protons, or heat stimulus. Here, we show that TRPV1 is coexpressed with PAR2 but not with PAR1 or PAR3, and that TRPV1 can functionally interact with PAR2. In human embryonic kidney 293 cells expressing TRPV1 and PAR2, PAR2 agonists increased capsaicin- or proton-evoked TRPV1 currents through a PKC-dependent pathway. After application of PAR2 agonists, temperature threshold for TRPV1 activation was reduced from 42 degrees C to well below the body temperature. PAR2-mediated Fos expression in spinal cord was decreased in TRPV1-deficient mice. The functional interaction was also observed in mouse DRG neurons and proved at a behavioral level. These represent a novel mechanism through which trypsin or tryptase released in response to tissue inflammation might trigger the sensation of pain by PAR2 activation.


Assuntos
Endopeptidases/metabolismo , Inflamação/fisiopatologia , Dor/fisiopatologia , Receptor PAR-2/metabolismo , Receptores de Droga/metabolismo , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Humanos , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Inflamação/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Dor/enzimologia , Técnicas de Patch-Clamp , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Receptores de Droga/deficiência , Receptores de Droga/genética , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Temperatura , Transfecção
17.
Eur J Neurosci ; 19(5): 1421-4, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15016100

RESUMO

Daily body temperature (DBT) rhythm of mice lacking one of the transient receptor potential (TRP) family of proteins, the capsaicin receptor or TRPV1, was recorded by biotelemetry and found to have significantly higher amplitude than that of wild-type mice. Capsaicin-desensitized wild-mice exhibited an even higher DBT amplitude than did TRPV1 deficient mice. A standard heat load (radiant temperature of 36-37 degrees C) resulted in similar rises in body core temperature in wild-type mice and in TRPV1 deficient mice, while capsaicin-desensitized wild-type mice exhibited a robust heat-intolerance. The lack of TRPV1 slightly modifies amplitude of daily body temperature rhythm but does not seem to influence physiological heat defence in mice. In vivo evidence for a TRP protein functioning in the physiological heat-defence range is still lacking.


Assuntos
Temperatura Corporal/fisiologia , Capsaicina/farmacologia , Ritmo Circadiano/fisiologia , Temperatura Alta , Receptores de Droga/deficiência , Animais , Temperatura Corporal/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Droga/agonistas , Receptores de Droga/genética
18.
J Physiol ; 555(Pt 1): 115-23, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14634201

RESUMO

The vanilloid receptor TRPV1 (formerly VR1) has been implicated in the activation of nociceptive sensory nerves by capsaicin, noxious heat, protons, bradykinin, cannabinoids such as anandamide, and certain metabolites of arachidonic acid. Using TRPV1 knockout mouse (TRPV1-/-) we address the question of whether TRPV1 is obligatory for action potential discharge in vagal C-fibre terminals evoked by capsaicin, anandamide, acid and bradykinin. The response of a defined subtype of the vagal afferent bronchopulmonary C-fibres (conduction velocity < 0.7 ms(-1)) to the putative TRPV1 activators was studied in vitro in the mouse isolated/perfused lung-nerve preparation. Capsaicin (1 microm) evoked action potential discharge of approximately 90% (28/31) of C-fibres in the TRPV1+/+ mice, but failed to activate bronchopulmonary C-fibres in TRPV1-/- animals (n = 10). Anandamide (3-100 microm) induced concentration-dependent activation of capsaicin-sensitive TRPV1+/+ C-fibres with a threshold of 3-10 microm, but failed to evoke substantive discharge in TRPV1-/- C-fibres. In the TRPV1+/+ mice, the B2 receptor-mediated activation by bradykinin (1 microm) was restricted to the capsaicin-sensitive C-fibres. Bradykinin was effective in evoking B2 receptor-mediated action potential discharge in TRPV1-/- C-fibres, but the response was significantly (P < 0.05) less persistent than in TRPV1+/+ C-fibres. Exposing the tissue to acid (pH = 5) excited both TRPV1+/+ and TRPV1-/- C-fibres. We conclude that TRPV1 is obligatory for vagal C-fibre activation by capsaicin and anandamide. By contrast, whereas TRPV1 may have a modulatory role in bradykinin and acid-induced activation of bronchopulmonary C-fibres, it is not required for action potential discharge evoked by these stimuli.


Assuntos
Bradicinina/farmacologia , Pulmão/fisiologia , Receptores de Droga/agonistas , Receptores de Droga/deficiência , Nervo Vago/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Relação Dose-Resposta a Droga , Endocanabinoides , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/fisiologia , Alcamidas Poli-Insaturadas , Receptores de Droga/genética , Nervo Vago/efeitos dos fármacos
19.
J Neurosci ; 23(31): 10146-53, 2003 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-14602831

RESUMO

Cell death mechanisms frequently involve the influx of extracellular calcium through voltage- and ligand-gated ion channels, e.g., the NMDA receptor (Greene, 1999). The vanilloid receptor (VR1) is present in regions of the brain (Mezey et al., 2000) that are highly susceptible to neurodegenerative insults, suggesting that this ion channel might contribute to the cellular processes involved in neuronal death. We tested the effects of VR1 ligands in the oxygen glucose deprivation (OGD) model of cell death in organotypic hippocampal slice cultures. The VR1 agonist capsaicin at concentrations that are selective for VR1 did not affect cell viability per se or the extent of neurodegeneration induced by the OGD insult. In contrast, the VR1 antagonist capsazepine (0.1-10 microm) significantly reduced the amount of OGD-induced cell death. However, capsazepine was still neuroprotective in slices prepared from VR1 knock-out mice, which exhibited the same degree of neurodegeneration to that observed in slices prepared from wild-type mice, excluding the possibility that it afforded neuroprotection through inhibition of VR1. Instead, capsazepine inhibited the hyperpolarization-activated nonspecific cation channel generated current I(h) in a concentration range similar to that which was neuroprotective. Furthermore, the specific I(h) blocker ZD-7288 was also neuroprotective, mirroring the effects of capsazepine, in that it was effective at preventing cell death when applied either during or after the OGD insult. These results demonstrate that capsazepine affords neuroprotection through inhibition of I(h) rather than inhibition of VR1.


Assuntos
Capsaicina/análogos & derivados , Capsaicina/farmacologia , Hipóxia Celular/fisiologia , Glucose/metabolismo , Canais Iônicos/antagonistas & inibidores , Proteínas do Tecido Nervoso , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Animais , Morte Celular , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Relação Dose-Resposta a Droga , Glucose/deficiência , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Técnicas In Vitro , Canais Iônicos/metabolismo , Camundongos , Camundongos Knockout , Fármacos Neuroprotetores/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio , Ratos , Ratos Sprague-Dawley , Receptores de Droga/deficiência , Receptores de Droga/genética
20.
J Clin Invest ; 112(3): 423-31, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12897210

RESUMO

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1-/-) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1-/- mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1-/- mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and preproorexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1-/- mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.


Assuntos
Apetite/fisiologia , Canabinoides/metabolismo , Metabolismo Energético , Ácidos Graxos Insaturados/fisiologia , Lipídeos/biossíntese , Receptores de Droga/fisiologia , Adipócitos/metabolismo , Animais , Moduladores de Receptores de Canabinoides , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/fisiologia , Ingestão de Alimentos/fisiologia , Expressão Gênica , Hipotálamo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/fisiologia , Obesidade/fisiopatologia , Obesidade/terapia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Canabinoides , Receptores de Droga/deficiência , Receptores de Droga/genética , Magreza/fisiopatologia
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