RESUMEN
Recent findings indicate that histidine triad nucleotide-binding protein 1 (HINT1) is implicated in the pathophysiology of certain psychiatric disorders and also exhibits tumor suppressor properties. However, the authentic functions of HINT1 in cellular physiology and especially its role in Ca(2+) signaling remain unclear. Here, we studied Ca(2+) signaling in cultured embryonic fibroblasts derived from wild-type control and HINT1 knockout (KO) mice. The resting cytosolic Ca(2+) level (measured with fura-2) was not altered in fibroblasts lacking HINT1. The stored Ca(2+) evaluated by measuring peak amplitude of ATP (10 µM)-induced Ca(2+) transients in Ca(2+)-free medium was significantly larger in HINT1 KO fibroblasts than in wild-type cells. Ca(2+) influx after external Ca(2+) restoration, likely via store- and receptor-operated channels (SOCs and ROCs, respectively), was greatly (by 2-fold) reduced in HINT1 KO fibroblasts. This correlated with a downregulated expression of Orai1 and stromal interacting molecule 1 (STIM1), essential components of store-operated Ca(2+) entry pathway. Expression of canonical transient receptor potential (TRPC)3 and TRPC6, which function as ROCs, was not altered in HINT1 KO fibroblasts. Immunoblots also revealed that Orai1 was downregulated by twofold in brain lysates of HINT1 KO mice compared with the wild-type littermates. Importantly, silencer RNA knockdown of HINT1 in Neuro-2A cells markedly downregulated Orai1 and STIM1 protein expression and significantly (by 2.5-fold) reduced ATP-induced Ca(2+) influx, while ATP-evoked Ca(2+) release was not changed. Thus the study demonstrates a novel function of HINT1 that involves the regulation of SOC-mediated Ca(2+) entry pathway (Orai1 and STIM1), essential for regulation of cellular Ca(2+) homeostasis.
Asunto(s)
Señalización del Calcio/fisiología , Fibroblastos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Western Blotting , Canales de Calcio/metabolismo , Células Cultivadas , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Proteína ORAI1 , ARN Interferente Pequeño , Molécula de Interacción Estromal 1RESUMEN
Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 µg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 µM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Cardiotónicos/farmacología , Digoxina/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Ouabaína/farmacología , Familia-src Quinasas/metabolismo , Animales , Aorta/citología , Canales de Calcio/metabolismo , Cardiotónicos/administración & dosificación , Células Cultivadas , Digoxina/administración & dosificación , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Arterias Mesentéricas/citología , Miocitos del Músculo Liso/efectos de los fármacos , Nifedipino/farmacología , Ouabaína/administración & dosificación , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Canales Catiónicos TRPC/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Arterial smooth muscle (ASM) Na(+)/Ca(2+) exchanger type 1 (NCX1) and TRPC/Orai-containing receptor/store-operated cation channels (ROC/SOC) are clustered with α2 Na(+) pumps in plasma membrane microdomains adjacent to the underlying junctional sarcoplasmic reticulum. This arrangement enables these transport proteins to function as integrated units to help regulate local Na(+) metabolism, Ca(2+) signaling, and arterial tone. They thus influence vascular resistance and blood pressure (BP). For instance, upregulation of NCX1 and TRPC6 has been implicated in the pathogenesis of high BP in several models of essential hypertension. The models include ouabain-induced hypertensive rats, Milan hypertensive rats, and Dahl salt-sensitive hypertensive rats, all of which exhibit elevated plasma ouabain levels. We suggest that these molecular mechanisms are key contributors to the increased vascular resistance ("whole body autoregulation") that elevates BP in essential hypertension. Enhanced expression and function of ASM NCX1 and TRPC/Orai1-containing channels in hypertension implies that these proteins are potential targets for pharmacological intervention.
Asunto(s)
Señalización del Calcio , Hipertensión/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Humanos , Hipertensión/genética , Hipertensión/patología , Proteínas Musculares/genética , Músculo Liso Vascular/patología , Ratas , Ratas Endogámicas Dahl , Sodio/metabolismo , Intercambiador de Sodio-Calcio/genética , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6RESUMEN
The mechanisms by which NaCl raises blood pressure (BP) in hypertension are unresolved, but much evidence indicates that endogenous ouabain is involved. In rodents, arterial smooth muscle cell (ASMC) Na(+) pumps with an α(2)-catalytic subunit (ouabain EC(50) ≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na(+) pumps have an α(1)-subunit (ouabain EC(50) ≈ 5 µM). Human α(1)-Na(+) pumps, however, have high ouabain affinity (EC(50) ≈ 10-20 nM). We used immunoblotting, immunocytochemistry, and Ca(2+) imaging (fura-2) to examine the expression, distribution, and function of Na(+) pump α-subunit isoforms in human arteries and primary cultured human ASMCs (hASMCs). hASMCs express α(1)- and α(2)-Na(+) pumps. Further, α(2)-, but not α(1)-, pumps are confined to plasma membrane microdomains adjacent to sarcoplasmic reticulum (SR), where they colocalize with Na/Ca exchanger-1 (NCX1) and C-type transient receptor potential-6 (receptor-operated channels, ROCs). Prolonged inhibition (72 h) with 100 nM ouabain (blocks nearly all α(1)- and α(2)-pumps) was toxic to most cultured hASMCs. Treatment with 10 nM ouabain (72 h), however, increased NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and augmented ATP (10 µM)-induced SR Ca(2+) release in 0 Ca(2+), ouabain-free media, and Ca(2+) influx after external Ca(2+) restoration. The latter was likely mediated primarily by ROCs and store-operated Ca(2+) channels. These hASMC protein expression and Ca(2+) signaling changes are comparable with previous observations on myocytes isolated from arteries of many rat hypertension models. We conclude that the same structurally and functionally coupled mechanisms (α(2)-Na(+) pumps, NCX1, ROCs, and the SR) regulate Ca(2+) homeostasis and signaling in hASMCs and rodent ASMCs. These ouabain/endogenous ouabain-modulated mechanisms underlie the whole body autoregulation associated with increased vascular resistance and elevation of BP in human, salt-sensitive hypertension.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Ouabaína/farmacología , Cloruro de Sodio/farmacología , Intercambiador de Sodio-Calcio/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos , Adolescente , Adulto , Anciano , Animales , Presión Sanguínea/efectos de los fármacos , Western Blotting , Cardenólidos/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Homeostasis , Humanos , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/fisiopatología , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Arterias Mamarias/efectos de los fármacos , Arterias Mamarias/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Persona de Mediana Edad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Ratas , Saponinas/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Cloruro de Sodio/toxicidad , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Canales Catiónicos TRPC/efectos de los fármacos , Canales Catiónicos TRPC/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Adulto JovenRESUMEN
The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 µM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a â¼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.
Asunto(s)
Señalización del Calcio/fisiología , Hipertensión Renal/metabolismo , Arteria Mesentérica Superior/metabolismo , Músculo Liso Vascular/metabolismo , Vasoconstricción/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipertensión Renal/genética , Hipertensión Renal/fisiopatología , Arteria Mesentérica Superior/citología , Arteria Mesentérica Superior/efectos de los fármacos , Músculo Liso Vascular/citología , Ouabaína/farmacología , Ratas , Ratas Mutantes , Retículo Sarcoplasmático/metabolismo , Cloruro de Sodio Dietético/farmacología , España , Resistencia Vascular/efectos de los fármacos , Resistencia Vascular/fisiología , Vasoconstricción/efectos de los fármacosRESUMEN
The relationship between altered metabolism of the amyloid-ß precursor protein (APP) and Alzheimer's disease is well established but the physiological roles of APP still remain unclear. Here, we studied Ca(2+) signaling in primary cultured and freshly dissociated cortical astrocytes from APP knockout (KO) mice and from Tg5469 mice overproducing by five- to sixfold wild-type APP. Resting cytosolic Ca(2+) (measured with fura-2) was not altered in cultured astrocytes from APP KO mice. The stored Ca(2+) evaluated by measuring peak amplitude of cyclopiazonic acid [CPA, endoplasmic reticulum (ER) Ca(2+) ATPase inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium was significantly smaller in APP KO astrocytes than in wild-type cells. Store-operated Ca(2+) entry (SOCE) activated by ER Ca(2+) store depletion with CPA was also greatly reduced in APP KO astrocytes. This reflected a downregulated expression in APP KO astrocytes of TRPC1 (C-type transient receptor potential) and Orai1 proteins, essential components of store-operated channels (SOCs). Indeed, silencer RNA (siRNA) knockdown of Orai1 protein expression in wild-type astrocytes significantly attenuated SOCE. SOCE was also essentially reduced in freshly dissociated APP KO astrocytes. Importantly, knockdown of APP with siRNA in cultured wild-type astrocytes markedly attenuated ATP- and CPA-induced ER Ca(2+) release and extracellular Ca(2+) influx. The latter correlated with downregulation of TRPC1. Overproduction of APP in Tg5469 mice did not alter, however, the stored Ca(2+) level, SOCE, and expression of TRPC1/4/5 in cultured astrocytes from these mice. The data demonstrate that the functional role of APP in astrocytes involves the regulation of TRPC1/Orai1-encoded SOCs critical for Ca(2+) signaling.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Astrocitos/citología , Astrocitos/fisiología , Canales de Calcio/genética , Canales de Calcio/metabolismo , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Fura-2/metabolismo , Homeostasis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína ORAI1 , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca(2+) signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca(2+) signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 +/- 2 vs. 113 +/- 1 mmHg, P < 0.05). Resting cytosolic free Ca(2+) concentration (measured with fura-2) and ATP-induced Ca(2+) transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na(+)/Ca(2+) exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by approximately 13-fold), whereas the expression of plasma membrane Ca(2+)-ATPase was not altered. Ca(2+) entry via the reverse mode of NCX1 evoked by the removal of extracellular Na(+) induced a rapid increase in cytosolic free Ca(2+) concentration that was significantly larger in MHS arterial myocytes. The expression of alpha(1)/alpha(2) Na(+) pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca(2+) signaling. We suggest that the increased Ca(2+) signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats.
Asunto(s)
Arterias/metabolismo , Calcio/metabolismo , Homeostasis/fisiología , Hipertensión/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Catiónicos TRPC/metabolismo , Regulación hacia Arriba/fisiología , Análisis de Varianza , Animales , Western Blotting , Células Cultivadas , Hipertensión/genética , Inmunohistoquímica , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Ratas , Intercambiador de Sodio-Calcio/genética , Canales Catiónicos TRPC/genéticaRESUMEN
Ca(2+) entry through store-operated channels (SOCs) in the plasma membrane plays an important role in regulation of vascular smooth muscle contraction, tone, and cell proliferation. The C-type transient receptor potential (TRPC) channels have been proposed as major candidates for SOCs in vascular smooth muscle. Recently, two families of transmembrane proteins, Orai [also known as Ca(2+) release-activated Ca(2+) channel modulator (CRACM)] and stromal interacting molecule 1 (STIM1), were shown to be essential for the activation of SOCs mainly in nonexcitable cells. Here, using small interfering RNA, we show that Orai1 plays an essential role in activating store-operated Ca(2+) entry (SOCE) in primary cultured proliferating human aortic smooth muscle cells (hASMCs), whereas Orai2 and Orai3 do not contribute to SOCE. Knockdown of Orai1 protein expression significantly attenuated SOCE. Moreover, inhibition of Orai1 downregulated expression of Na(+)/Ca(2+) exchanger type 1 (NCX1) and plasma membrane Ca(2+) pump isoform 1 (PMCA1). The rate of cytosolic free Ca(2+) concentration decay after Ca(2+) transients in Ca(2+)-free medium was also greatly decreased under these conditions. This reduction of Ca(2+) extrusion, presumably via NCX1 and PMCA1, may be a compensation for the reduced SOCE. Immunocytochemical observations indicate that Orai1 and NCX1 are clustered in plasma membrane microdomains. Cell proliferation was attenuated in hASMCs with disrupted Orai1 expression and reduced SOCE. Thus Orai1 appears to be a critical component of SOCE in proliferating vascular smooth muscle cells, and may therefore be a key player during vascular growth and remodeling.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Miocitos del Músculo Liso/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Aorta/metabolismo , Western Blotting , Canales de Calcio/genética , Señalización del Calcio/fisiología , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Músculo Liso Vascular/metabolismo , Proteína ORAI1 , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ARN Interferente Pequeño , Intercambiador de Sodio-Calcio/genéticaRESUMEN
A previous study has demonstrated that the ubiquitous plasma membrane Ca(2+) pump PMCA4 interacted with isoform epsilon of the 14-3-3 protein, whereas the nervous tissue-specific PMCA2 did not. The 14-3-3 proteins are widely expressed small acidic proteins, which modulate cell signaling, intracellular trafficking, transcription and apoptosis. The investigation has been extended to the other tissue-restricted pump (PMCA3) and to the other ubiquitous pump (PMCA1). At variance with PMCA2, PMCA3 interacted with the 14-3-3epsilon protein in a two-hybrid system assay, which could not be used for PMCA1. The 14-3-3epsilon protein immunoprecipitated with both PMCA3 and PMCA1 when expressed in HeLa cells. Pull-down experiments using GST-PMCA1 and GST-PMCA3 fusion products confirmed the interaction of both pumps with the 14-3-3epsilon protein. The binding was phosphorylation-independent with both PMCA3 and PMCA1. The 14-3-3zeta isoform also interacted with PMCA3; however, it did not interact with PMCA1. The effect of the interaction on the activity of the two pumps, and thus on the homeostasis of Ca(2+), was investigated by co-expressing the 14-3-3epsilon protein and PMCA3 or PMCA1 in CHO cells together with the recombinant Ca(2+) indicator aequorin: the ability of cells to re-establish the basal Ca(2+) concentration following a Ca(2+) transient induced by an InsP(3)-producing agonist was substantially decreased with both pumps, indicating that the interaction with the 14-3-3 protein inhibited the activity of both PMCA3 and PMCA1.
Asunto(s)
Proteínas 14-3-3/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Proteínas 14-3-3/efectos de los fármacos , Proteínas 14-3-3/genética , Aequorina , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Agonistas de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Cricetinae , Cricetulus , Células HeLa , Homeostasis/efectos de los fármacos , Homeostasis/genética , Humanos , Indicadores y Reactivos , Datos de Secuencia Molecular , Fosforilación , ATPasas Transportadoras de Calcio de la Membrana Plasmática/efectos de los fármacos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The transmembrane receptor guanylyl cyclase-C (GC-C), expressed on enterocytes along the intestine, is the molecular target of the GC-C agonist peptide linaclotide, an FDA-approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo-2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)-trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase-II (PKG-II) activity assays. Expression and activity of GC-C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo-2BBe cells increased cell surface CFTR levels. Linaclotide-induced activation of the GC-C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASPser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide-induced CFTR trafficking to the apical membrane. Inhibition of protein kinase-A (PKA) also attenuated linaclotide-induced CFTR cell surface trafficking, implying cGMP-dependent cross-activation of PKA pathway. Together, these findings support linaclotide-induced activation of the GC-C/cGMP/PKG-II/CFTR pathway as the major pathway of linaclotide-mediated intestinal fluid secretion, and that linaclotide-dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Agonistas de la Guanilato Ciclasa C/farmacología , Mucosa Intestinal/metabolismo , Péptidos/farmacología , Transducción de Señal , Animales , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Humanos , Mucosa Intestinal/efectos de los fármacos , Masculino , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a la Guanilato-Ciclasa/metabolismoRESUMEN
Central nervous system (CNS) administration of angiotensin II (Ang II) raises blood pressure (BP). The rise in BP reflects increased sympathetic outflow and a slower neuromodulatory pressor mechanism mediated by CNS mineralocorticoid receptors (MR). We investigated the hypothesis that the sustained phase of hypertension is associated also with elevated circulating levels of endogenous ouabain (EO), and chronic stimulation of arterial calcium transport proteins including the sodium-calcium exchanger (NCX1), the type 6 canonical transient receptor potential protein (TRPC6), and the sarcoplasmic reticulum calcium ATPase (SERCA2). Wistar rats received a chronic intra-cerebroventricular infusion of vehicle (C) or Ang II (A, 2.5 ng/min, for 14 days) alone or combined with the MR blocker, eplerenone (A+E, 5 µg/day), or the aldosterone synthase inhibitor, FAD286 (A+F, 25 µg/day). Conscious mean BP increased (P<0.05) in A (123 ± 4 mm Hg) vs all other groups. Blood, pituitary and adrenal samples were taken for EO radioimmunoassay (RIA), and aortas for NCX1, TRPC6 and SERCA2 immunoblotting. Central infusion of Ang II raised plasma EO (0.58 ± 0.08 vs C 0.34 ± 0.07 nM (P<0.05), but not in A + E and A + F groups as confirmed by off-line liquid chromatography (LC)-RIA and LC-multistage mass spectrometry. Two novel isomers of EO were elevated by Ang II; the second less polar isomer increased >50-fold in the A+F group. Central Ang II increased arterial expression of NCX1, TRPC6 and SERCA2 (2.6, 1.75 and 3.7-fold, respectively; P<0.01)) but not when co-infused with E or F. Adrenal and pituitary EO were unchanged. We conclude that brain Ang II activates a CNS-humoral axis involving plasma EO. The elevated EO reprograms peripheral ion transport pathways known to control arterial Na(+) and Ca(2+) homeostasis; this increases contractility and augments sympathetic effects. The new axis likely contributes to the chronic pressor effect of brain Ang II.