Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism.

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavß2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gßγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Gißγ-AKT-Cavß2 pathway, suggesting a new mechanism for regulation of cardiac function.

Ca²âº signals promote GLUT4 exocytosis and reduce its endocytosis in muscle cells.

Elevating cytosolic Ca(2+) stimulates glucose uptake in skeletal muscle, but how Ca(2+) affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca(2+) leading to contraction preclude analysis of the impact of individual, Ca(2+)-derived signals. In L6 muscle cells stably expressing GLUT4myc, the Ca(2+) ionophore ionomycin raised cytosolic Ca(2+) and caused a gain in cell surface GLUT4myc. Extra- and intracellular Ca(2+) chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4myc. Ionomycin stimulated GLUT4myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4myc endocytosis triggered by ionomycin. In summary, rapid Ca(2+) influx into muscle cells accelerates GLUT4myc exocytosis while slowing GLUT4myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca(2+)-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

Regulation of autophagy by the inositol trisphosphate receptor.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.

The STIM1 inhibitor ML9 disrupts basal autophagy in cardiomyocytes by decreasing lysosome content.

Stromal-interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo. Moreover, there is growing support for the contribution of SOCE to the Ca2+ overload associated with ischemia/reperfusion injury. Therefore, STIM1 inhibition is proposed as a novel target for controlling both hypertrophy and ischemia/reperfusion-induced Ca2+ overload. Our aim was to evaluate the effect of ML9, a STIM1 inhibitor, on cardiomyocyte viability. ML9 was found to induce cell death in cultured neonatal rat cardiomyocytes. Caspase-3 activation, apoptotic index and release of the necrosis marker lactate dehydrogenase to the extracellular medium were evaluated. ML9-induced cardiomyocyte death was not associated with increased intracellular ROS or decreased ATP levels. Moreover, treatment with ML9 significantly increased levels of the autophagy marker LC3-II, without altering Beclin1 or p62 protein levels. However, treatment with ML9 followed by bafilomycin-A1 did not produce further increases in LC3-II content. Furthermore, treatment with ML9 resulted in decreased LysoTracker® Green staining. Collectively, these data suggest that ML9-induced cardiomyocyte death is triggered by a ML9-dependent disruption of autophagic flux due to lysosomal dysfunction.

Changes in cyclic AMP dependent protein kinase and active stiffness in the rat volume overload model of heart hypertrophy.

OBJECTIVE: The aim was to clarify the role of cyclic AMP dependent protein kinase (PKA) and changes in mechanical heart function during development of cardiac hypertrophy induced by volume overload. METHODS: Protein and DNA contents, PKA activity, and peak systolic stress-strain relationships in hearts from animals submitted to aortocaval shunt were assessed as a function of time. Sham operated (control) rats were used as controls. RESULTS: Heart weight to body weight ratio and cardiac protein content per heart increased from d 7 (p < 0.005 and p < 0.01, respectively) reaching their highest values by d 56; the same occurred with cardiac DNA content. PKA activity.g-1 tissue in soluble extracts of hearts from rats with aortocaval shunt increased by 2.7-fold on d 2 (p < 0.005), reached a ninefold peak increase by d 7 (p < 0.0001) and declined to fourfold by d 56 with respect to control values. The end peak systolic stress-strain relation slopes were: control, 368(SEM 14) g.cm-2 (n = 16); aortocaval shunt values: 2 d, 514(28) g.cm-2 (n = 6); 7 d, 579(10) g.cm-2 (n = 7); and 56 d, 554(28) g.cm-2 (n = 7). The force generating capacity at 0% strain was also significantly higher in the shunt groups as compared to sham operated controls (p < 0.01). Early activation of PKA was also confirmed through endogenous cardiac protein phosphorylation. SDS-PAGE gel electrophoretogram and autoradiography showed more heavily phosphorylated bands in aortocaval shunt hearts than in the control group. CONCLUSIONS: PKA activity and the slope of systolic stress-strain regression line followed a similar trend throughout the study, with an early increase in both variables by d 2 in the shunt group, reaching a peak at d 7, and decreasing thereafter but remaining higher than in controls. PKA activity appears to be related to increased force generating capacity rather than to hypertrophy or increased cardiac protein content. Thus PKA activation is an early biochemical event after aortocaval shunt, followed later by cardiac hypertrophy. Changes in PKA activity showed a similar trend to mechanical heart function over time. These findings help to explain the changes in the mechanical properties of the heart preceding the development of cardiac hypertrophy in the rat model of volume overload.

In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development.

Basic fibroblast growth factor (bFGF) stimulated [3H]thymidine incorporation at all stages of development, although the magnitude of this effect was the greatest in cells derived from pregnant mice. Cells primed with insulin and bFGF synthesized more casein than cells not exposed to either hormone. bFGF inhibited casein synthesis and decreased the amounts of beta-casein and alpha-lactalbumin transcripts in cells from pregnant animals simultaneously incubated with insulin, hydrocortisone and prolactin. bFGF content in mammary gland increased with puberty and pregnancy, but decreased markedly in lactation; the number of bFGF receptors in epithelial cells changed in parallel. These data suggest that bFGF may have a physiological role both in stimulating growth and in inhibiting functional differentiation of normal mouse mammary epithelial cells.

Effect of inhibitors of signal transduction on IGF-1-induced protein synthesis associated with hypertrophy in cultured neonatal rat ventricular myocytes.

IGF-1 increased 2-fold protein synthesis in cardiac myocytes. Genistein, whether added during preincubation or with IGF-1 at the start of incubation, significantly inhibited the IGF-1-induced stimulation of protein synthesis, autophosphorylation of the beta-subunit of IGF-1 receptor and inhibition of ERK. When added 1 or 6 h after IGF-1, however, genistein was without effect. IGF-1-stimulated protein synthesis was also significantly inhibited by PD-098059, staurosporine, and rapamycin, but not by wortmannin, in cardiac myocytes. Some inhibitors produced a reduction in cell size. Activation of the ERK cascade by IGF-1 may be responsible for some of the features associated with cardiac myocyte hypertrophy.

A role for adrenaline and calmodulin in modulating cyclic AMP levels during the lactogenic cycle.

The effect on lactose production of several external modulators of intracellular cyclic AMP was studied in rat mammary gland tissue slices and explants. Adrenaline, a beta-adrenergic receptor effector, forskolin, a direct adenylate cyclase activator and fluphenazine, a calmodulin inhibitor, all produced an increase in the intracellular level of cyclic AMP and a concomitant inhibition of lactose production. These results suggest a role for adrenaline and calmodulin in modulating cyclic AMP levels in mammary tissue during the lactogenic cycle.

Use of octadecyl silica as an alternative non-conventional solid phase in immunoradiometric assays.

The use of chemically modified silica (octadecyl silica or Si-C18), a liquid chromatography stationary reverse phase, is proposed as an alternative solid phase for immunoradiometric assays (IRMAs). Si-C18 and polyvinylchloride solid phases were compared in their efficiency to detect a murine plasma protein (sex-limited protein or Slp) in a two site-two antibody IRMA. Si-C18 is at least as sensitive as PVC since Slp could be detected in plasmas diluted 1/128,000 and 1/64,000 respectively. The fact that 1 mg of Si-C18 can adsorb 5-14 times more protein than a PVC well is in agreement with the larger relative surface of Si-C18 beads (approximately 50 cm2/mg), compared with the PVC (approximately 1 cm2/well). These results strongly suggest that the Si-C18 matrix, saturated with specific antibodies, could be an exceptional probe to rescue and detect antigenic moieties diluted in large volumes of complex biological fluids.

Prevalence of the angiotensin I converting enzyme insertion/deletion polymorphism, plasma angiotensin converting enzyme activity, and left ventricular mass in a normotensive Chilean population.

The aim of this study was to estimate the prevalence of the different alleles of the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and associated plasma ACE activity, as well as cardiac echocardiographic structure, in a healthy Chilean population. We selected 117 healthy normotensive subjects (aged 45 to 60 years, middle socioeconomic status, nonobese, and nondiabetic) from a population-based study concerning the prevalence of risk factors for chronic diseases (Conjunto de Acciones Para la Reducción Multifactorial de las Enfermedades no Transmisibles [CARMEN]). The frequencies of the I and D alleles were 0.57 and 0.43, respectively. Mean plasma ACE activity was 15.3 +/- 3.9 U/mL. Compared with subjects with the II genotype, plasma ACE activity was significantly higher in subjects with the ID and DD genotypes with no difference between them. No correlation was observed between blood pressure and plasma ACE activity. Among the three different genotypes there was no difference in left ventricular (LV) dimensions or in LV mass. No correlation between plasma ACE activity and LV mass was observed for either gender or different genotypes. Multivariate linear regression analysis using LV mass and LV mass index as dependent variables showed independent effects (P < .05) for gender (higher LV mass in men) and diastolic blood pressure, but not for the DD genotype. In conclusion, in this population, the presence of the D allele on the ACE gene determined higher circulating ACE activity. However, in this normotensive healthy population, male gender and diastolic blood pressure, but not the presence of the D allele, were associated with increased LV mass.

An immunogenetically defined and immunodominant Trypanosoma cruzi antigen.

Two strains of mice, A. SW (H-2s) and A.CA (H-2f), were immunized with live trypomastigotes or epimastigotes of the Tulahuen strain of Trypanosoma cruzi or with their sonicates. By immunowestern blotting, sera from A.SW mice, but not from A.CA, recognized, in an immunodominant fashion, a 45 kDal polypeptide (Tc45) present in both epimastigotes and trypomastigotes. Since A.SW and A.CA strains are congenic for the major histocompatibility H-2 complex, recognition of Tc45 seems to be controlled by this genetic region or by gene(s) located in its immediate vicinity. Subcellular fractionation revealed that Tc45 is mainly present at the cytoplasmic compartment.

Neutral endopeptidase and angiotensin I converting enzyme insertion/deletion gene polymorphism in humans.

Neutral endopeptidase (NEP) hydrolyses angiotensins (Ang) I and II and generates angiotensin-(1-7) [Ang-(1-7)]. In humans, the insertion/deletion (I/D) angiotensin-I converting enzyme (ACE) gene polymorphism determined plasma ACE levels by 40%. In rats, a similar polymorphism determines ACE levels which are inversely associated to NEP activity. The objective of this study is to evaluate the relationship between ACE expression and plasma NEP activity in normotensive subjects and in hypertensive patients. In total, 58 consecutive patients with hypertension, evaluated in our Hypertension Clinic, were compared according to their ACE I/D genotypes with 54 control subjects in terms of both plasma ACE activity and NEP activities. Plasma ACE activity was elevated 51 and 70% in both DD ACE groups (normotensives and hypertensives) compared with their respective ID and II ACE groups (P<0.001). A significant effect of the ACE polymorphism and of the hypertensive status on ACE activity was observed (P<0.001). In normotensive DD ACE subjects, NEP activity was 0.30+/-0.02 U/ml, whereas in the normotensive II ACE and in the normotensive ID ACE subjects NEP activity was increased 65 and 48%, respectively (P<0.001). In the hypertensive DD ACE patients, NEP activity was 0.47+/-0.03 U/mg. An effect of the I/D ACE genotypes on NEP activity (P<0.04) and an interaction effect between the I/D ACE genotype and the hypertensive status were also observed (P<0.001). These results are consistent with a normal and inverse relationship between the ACE polymorphism and NEP activity in normotensive humans (as is also observed in rats). This normal relationship is not observed in hypertensive patients.

Effects of antihypertensive treatment on cardiac IGF-1 during prevention of ventricular hypertrophy in the rat.

There is some evidence that cardiac rather than circulating insulin-like growth factor-1 (IGF-1) levels contribute to the development of renovascular hypertensive left ventricular hypertrophy (LVH), remaining unknown the effects of antihypertensive drugs on IGF-1 levels. We have assessed here the preventive effects of enalapril, losartan, propanolol and alpha-methyldopa on left ventricle (LV) and circulating IGF-1 levels in a rat model of hypertension and LVH (Goldblatt, GB). Our results show that relative LV mass and the LV content of IGF-1 were significantly lower with all antihypertensive drugs in GB rats (p<0.001). Serum concentrations of IGF-1 were lower in GB rats treated with enalapril, alpha-methyldopa and propanolol (p<0.01), but not in those treated with losartan. These results support the hypothesis that local rather than seric IGF-1 contributes to the development of left ventricular hypertrophy induced by pressure overload in the rat.

Omeprazole, a specific gastric secretion inhibitor on oxynticopeptic cells, reduces gizzard erosion in broiler chicks fed with toxic fish meals.

The relation between gizzard erosion-black vomit (GE-BV) and gastric secretion is not completely understood. A pharmacological approach to reduce the presence of GE-BV in chicks due to fish meal in diets is also unknown. In this study the use of omeprazole, a H+/K+ ATPase inhibitor, and fish meals of different biotoxicological characteristics, showed that: 1) Omeprazole decreased total gastric acid content, GE scores and severe GE (SGE) cases, in a dose-dependent manner. This reduction was significant at levels higher than 20 mg omeprazole/Kg body weight (BW)/day (p < 0.01). The addition of 50 mg omeprazole/kg BW/day almost completely prevented the incidence of SGE cases and reduced in 50% GE score in chicks (p < 0.01). 2) A significant reduction in specific mortality, near 90%, was also seen with all toxic fish meals when omeprazole (50 mg/Kg BW/day) was added to experimental diets in comparison to control groups. However, no mortality was observed when omeprazole was added to diets containing non-toxic fish meals. 3) In chicks fed with toxic fish meals, addition of different amounts of omeprazole to diets changed the relative weight of proventriculus (p < 0.01) and gizzard (p < 0.05). Maximum effect was obtained with omeprazole concentration higher than 50 mg/Kg BW/day. 4) Omeprazole did not change feed intake in chicks fed with toxic fish meal. However, in some fish meal a reduction on weight gain was observed with the addition of omeprazole.

[Early prevention of experimental left ventricular hypertrophy in experimental hypertension and angiotensin II levels]. / Prevención precoz de hipertrofia ventricular izquierda en la hipertensión experimental y concentraciones de angiotensina II.

INTRODUCTION: Angiotensin II levels can be partially inhibited during chronic administration of angiotensin converting enzyme (ACE) inhibitors, limiting from a clinical point of view its efficacy in the treatment of hypertension. There are few studies relating ACE activity directly with early prevention of left ventricular hypertrophy (LVH) in systemic hypertension during the administration of an ACE inhibitor (ACEI). AIM: To evaluate the effects of early ACE inhibition with perindopril on the development of hypertension, LVH and levels of angiotensin II (Ang II) in plasma as well as in LV in the rat Goldblatt model (Gb; 2 kidneys-1 clip), 2 weeks after surgery. RESULTS: Systolic blood pressure and relative LV mass increased by 42% and 20% respectively, in the Gb group (p < 0.001). Plasma and LV ACE activities were significantly higher in the Gb rats compared with the control rats. Plasma and LV Ang II levels also increased by 129% and 800%, respectively. Perindorpil prevented hypertension and LVH development by inhibiting plasma ACE (and also LV ACE), and also circulation Ang II in plasma and in the LV. CONCLUSIONS: In this experimental model of hypertensive LVH, there is an early activation of plasma and cardiac ACE. Early administration of an ACE inhibitor prevents the development of hypertension and LVH by inhibiting the increases of plasma and LV Ang II.

[Evaluation of the effect of enalapril, a converting enzyme inhibitor, on lymphocytic beta-adrenergic receptors of patients with chronic cardiac insufficiency]. / Evaluación del efecto de enalapril, inhibidor de la enzima conversiva, en los receptores betaadrenérgicos de linfocitos de pacientes con insuficiencia cardíaca crónica.

We have previously corroborated that lymphocyte beta-adrenergic receptor density is significantly reduced in patients with chronic heart failure. It is well known that angiotensin converting enzyme inhibitors normalize the function of sympathetic nervous system. We have assessed the effect of enalapril on lymphocyte beta-adrenergic receptor system from patients with chronic heart failure (n = 14) using a random, cross and double blind protocol. Our results show that the improvement in clinical score and ventricular function were not related with changes in the number and affinity of beta-adrenergic receptor nor cyclic AMP content in lymphocytes obtained from these patients.

Research note: ability of fenthion to increase gizzard erosion in broiler chicks.

Fenthion, an irreversible cholinesterase inhibitor, was used to study the role of the cholinergic system on the development of gizzard erosion. Fenthion increases the gizzard erosion score in a dose-dependent manner and this effect became significant at levels higher than .1 ppm (p less than .05). An inverse relationship between plasma cholinesterase activity and pesticide concentration was also observed at doses higher than 1 ppm (P less than .05). These results show the necessity to evaluate organophosphate pesticide levels during the selection of fish meals in poultry.

Calcium signaling in insulin action on striated muscle.

Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca(2+) mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca(2+) into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R). In skeletal muscle cells, a rapid, insulin-triggered Ca(2+) release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca(2+)]i trough L-type Ca(2+) channels activation. In both cell types, a relatively slower Ca(2+) release also occurs through IP3R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca(2+) released from IP3R of skeletal muscle also promotes mitochondrial Ca(2+) uptake. We review here these actions of insulin on intracellular Ca(2+) channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca(2+) release from the SER.

4-Phenylbutyric acid prevent cytotoxicity induced by thapsigargin in rat cardiac fibroblast.

Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail. Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot. In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA. Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.