RESUMEN
BACKGROUND: Blockers of angiotensin II type 1 receptor (AT1R) and the voltage gated calcium channel 1.2 (CaV1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT1R signaling and whether there is a reciprocal regulation of AT1R signaling by CaV1.2. METHODS: To elucidate these questions, we have studied the Ca2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of CaV1.2 blockers. Semi-quantitative imaging methods were used to assess the plasma membrane expression of AT1R and G-protein activation. RESULTS: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca2+ response. The up-regulation of the Ca2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT1R plasma membrane expression. The Ca2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the CaV1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT1R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT1R is sensitive to calcium influx. CONCLUSIONS: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca2+ channel blockers as mono-therapy in hypertension.
Asunto(s)
Angiotensina II/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Nifedipino/farmacología , Receptor de Angiotensina Tipo 1/agonistas , Verapamilo/farmacología , Animales , Animales Recién Nacidos , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Músculo Liso Vascular/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/metabolismo , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/metabolismo , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
OBJECTIVES: The use of primary human ß-cells for studying Ca signaling is limited by the scarcity of human pancreatic islets. Rodent insulinoma cell lines are widely used, but it is difficult to extrapolate results obtained from rodent cells to human. Recently, a genetically engineered human ß-cell line EndoC-BH1 has been developed. We have examined whether the EndoC-BH1 cells could be used as a model for studying Ca signaling in the ß-cells. METHODS: We used microscope-based fluorometry to measure cytoplasmic-free Ca concentration from fura-2-loaded single EndoC-BH1 cells cultured on glass cover slips. Ca responses to different agonists of insulin secretion were studied. Insulin secretion was measured by radioimmunoassay. RESULTS: EndoC-BH1 cells secreted insulin in response to glucose in a dose-dependent manner, and the secretion was enhanced by GLP-1 (glucagon-like peptide 1). Glucose, potassium chloride, carbachol, L-arginine, and tolbutamide increased cytoplasmic-free Ca concentration in the EndoC-BH1 cells. We found that GLP-1 was essential for Ca response to glucose and tolbutamide. CONCLUSIONS: We concluded that the EndoC-BH1 cells can be used as model cells to study Ca signaling and stimulus-secretion coupling in the human ß-cells.
Asunto(s)
Señalización del Calcio , Ingeniería Genética , Células Secretoras de Insulina/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes , Fluorometría , Fura-2 , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Cinética , Potenciales de la MembranaRESUMEN
OBJECTIVE: Several studies have reported that the transient receptor potential melastatin-like subtype 5 (TRPM5) channel, a Ca(2+)-activated monovalent cation channel, is involved in the stimulus-secretion coupling in the mouse pancreatic ß-cells. We have studied the role of the TRPM5 channel in regulating insulin secretion and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in the rat ß-cells by using triphenylphosphine oxide, a selective inhibitor of the channel. METHODS: Insulin secretion from islets from Sprague-Dawley rats was measured in batch incubations. Cytoplasmic free Ca(2+) concentration was measured from single ß-cells by fura-2-based microfluorometry. RESULTS: Triphenylphosphine oxide did not alter insulin secretion and [Ca(2+)](i) response triggered by KCl or fructose. It inhibited insulin secretion in response to glucose, L-arginine, and glucagon-like peptide 1. It also inhibited glucose-induced insulin secretion by mechanisms that are independent of the adenosine triphosphate-sensitive potassium channels and [Ca(2+)](i) increase. CONCLUSIONS: Our results suggest that in the rat islets, TRPM5 is involved in mediating insulin secretion by glucose and l-arginine and in potentiating the glucose-induced insulin secretion by glucagon-like peptide 1.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Arginina/metabolismo , Calcio/metabolismo , Células Cultivadas , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Potenciales de la Membrana , Compuestos Organofosforados/farmacología , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Canales Catiónicos TRPM/antagonistas & inhibidores , Factores de TiempoRESUMEN
The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca(2+) concentration ([Ca(2+)](i)) remains unknown. We measured [Ca(2+)](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary ß-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca(2+)](i) in the form of a peak followed by a plateau dependent on extracellular Ca(2+). NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca(2+)](i). The ADPr-induced [Ca(2+)](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP(3) receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPr-induced [Ca(2+)](i) increase. ADPr increased [Ca(2+)](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors.