This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Increases or decreases in the contractile response of smooth muscle underlie important pathologic... more Increases or decreases in the contractile response of smooth muscle underlie important pathological conditions such as hypertension, incontinence and altered gastrointestinal transit. These disorders are also frequently encountered in the aged population. Oxidative stress and inflammation are key features in the initiation, progression, and clinical manifestations of smooth muscle disorders. Melatonin, the major secretory product of the pineal gland, has free radical scavenging and antioxidative properties and protects against oxidative insult. Recently, widespread interest has grown regarding the apparent protective effects of melatonin on smooth muscle dysfunction. "In vitro" studies have shown that melatonin decreased vascular tone of vascular beds from control, hypertensive or aged animals, through the reduction of adrenergic contraction and the increase in acetylcholine-induced relaxation. "In vivo", melatonin also attenuates sympathetic tone by direct activation of melatonin receptors, scavenging free radicals or increasing NO availability in the central nervous system. In the gastrointestinal tract, melatonin treatment improves age-related impairments in gallbladder contractility and prevents deleterious effects of cholecystitis on smooth muscle and the enteric nervous system through suppression of oxidative stress. In addition, melatonin improves colonic transit time in constipation-predominant IBS patients. Melatonin is also able to restore impaired contractility of the detrusor muscle from old animals through normalization of Ca(2+) dependent and independent contraction, mitochondrial polarity, neuromuscular function and oxidative stress, which would explain the effects of melatonin counteracting cystometric changes in senescent animals. It also reverses bladder damage following ischemia/reperfusion. In conclusion, melatonin may be a promising candidate for future research of agents that modulate smooth muscle motility.
Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Jun 1, 2021
The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced ... more The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.
In anaesthetized rats, interaction between either vagal stimulation or acetylcholine (ACh) and th... more In anaesthetized rats, interaction between either vagal stimulation or acetylcholine (ACh) and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) and staurosporine on pancreatic juice secretion have been investigated in vivo. In vitro, rat cytosolic free calcium concentration (Ca2+)i in pancreatic acini loaded with the fluorescent dye, Fura-2 acetoxymethyl ester (AM) have been analysed after the same modifications. In vivo, vagotomy caused a marked reduction in the rate of pancreatic juice flow, total protein output and amylase secretion compared to basal secretory parameters. Furthermore, bolus injection of either saline, TPA (10(-8) mol/kg b wt), staurosporine (10(-8) mol/kg b wt) or a combination of TPA and staurosporine (all 10(-8) mol/kg b wt) had no statistically significant effect on pancreatic juice flow, protein output and amylase secretion compared to vagotomy values. Electrical stimulation (E.S.) of vagues nerves resulted in marked and statistically significant (P < 0.05) increases in the rate of pancreatic juice flow, total protein output and amylase secretion in rats injected with either saline or staurosporine, compared to control values. In contrast, E.S. of the vagus nerves failed to enhance all secretory parameters in the presence of either TPA or a combination of TPA and staurosporine. In vitro, on isolated acini, ACh evoked dose dependent increases in (Ca2+)i. Pretreatment these acini with either TPA, staurosporine or a combination of TPA and staurosporine had no significant effect on the ACh-induced (Ca2+)i. These results indicate that TPA can decrease the secretory responses evoked by E.S. of the vagus nerves in the anaesthetized rat. This attenuation is not associated with either protein kinase C inhibition or the mobilization of the second messenger Ca2+ but possibly through activation of protein kinase C by TPA.
In many tissues the cellular responses mediated through different intracellular messenger systems... more In many tissues the cellular responses mediated through different intracellular messenger systems are mutually interactive. In the exocrine pancreas the secretagogues acting via adenosine cyclic monophosphate (cAMP) and those acting via calcium-phosphoinositides can potentiate one another. On the other hand, protein kinase C (PK-C) modulates receptor-induced responses in exocrine pancreatic cells and other cell types. Recording total protein output, monitored on-line at 280 nm, from superfused rat pancreatic segments, we demonstrate that secretin (a cAMP-acting hormone) reduces the efficacy of the calcium-mediated secretagogue cholecystokinin-octapeptide (CCK-8). Likewise, the PK-C activator 12,O,tetradecanoyl phorbol 13 acetate (TPA) reduces both the efficacy of secretin and the potency of cholecystokinin. Thus, the hypothesis of potentiation between different stimulus-secretion coupling mechanisms must be revised, and receptor-activated responses in the exocrine pancreas must be considered a complex model with multiple inhibitory and stimulatory interactions.
Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion... more Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internaliza...
The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-acti... more The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plas...
La secrecion pancreatica exocrina es el resultado final de la accion simultanea de multiples mens... more La secrecion pancreatica exocrina es el resultado final de la accion simultanea de multiples mensajeros extracelularessobre las celulas acinares y ductulares. El proceso global esta mediado por un grupo de mensajeros intracelularescomo cAMP, IP3, IP4 o diacilglicerol.Hormonas como histamina e insulina pueden participar tambien como agentes reguladores moduladores de lasecrecion pancreatica. Se ha demostrado que la histamina ejerce un efecto secretagogo moderado a traves de lamodulacion de la respuesta secretora inducida por los secretagogos clasicos. La insulina puede potenciar acusadamentela respuesta secretora a acetilcolina y colecistoquinina. Ademas, otros peptidos liberados de los islotes de Langerhanscomo glucagon, somatostatina y polipeptido pancreatico, pueden atenuar las respuestas secretoras inducidas porcolecistoquinina y acetilcolina.Los factores de crecimiento participan en el control del crecimiento, replicacion y diferenciacion celular. Tambienestan implicados en la s...
1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from... more 1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from pancreatic acinar cells with preserved intracellular environments. The calcium dependence of calcium extrusion indicated a strong co‐operativity (Hill coefficient, 3). The half‐maximal rate of calcium extrusion occurred at an intracellular free calcium concentration ([Ca2+]i) of approximately 200 nM. At [Ca2+]i levels higher than 400 nM the calcium extrusion mechanism was almost completely saturated. 2. The rate of [Ca2+]i recovery was measured with the same cells under conditions where both calcium extrusion and calcium reuptake occurred simultaneously and under conditions when calcium reuptake was prevented and recovery depended entirely upon calcium extrusion. The rate of [Ca2+]i recovery due to calcium reuptake displayed a very sharp dependence on [Ca2+]i. The rate of [Ca2+]i recovery due to reuptake increased approximately 10 times (from 4.3 to 44.1 nM s‐1) for an increase of [Ca2+...
1. In the present time-course study, we have examined the interactions between the phorbol ester,... more 1. In the present time-course study, we have examined the interactions between the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic gut hormones, cholecystokinin-octapeptide (CCK-8) and secretin on pancreatic juice secretion in anaesthetized rat. 2. Administration of either TPA (10(-8) mol kg-1 hr-1), secretin (100 pmol kg-1 hr-1) or CCK-8 (150 pmol kg-1 hr-1) in the anaesthetized rat resulted in marked time-course increases in pancreatic juice flow, amylase secretion and total protein output compared to saline controls. The effect of secretin on juice flow was more pronounced and sustained compared to the smaller responses obtained with either CCK-8 or TPA. Similarly, CCK-8 evoked increases in protein output and amylase secretion compared to the responses obtained with either secretin or TPA. 3. Simultaneous infusion of TPA with either CCK-8 or secretin resulted in a marked reduction in pancreatic juice flow, total protein output and amylase secretion compared to the responses obtained with either CCK-8 or secretin alone. 4. Administration of polymyxin B (10(-8) mol kg-1 hr-1), a protein kinase C inhibitor with either TPA and CCK-8 or TPA and secretin caused a partial reduction of the inhibitory effect of TPA on CCK-8 and secretin-evoked secretory responses. 5. The present study further implicates the involvement of protein kinase C in the modulation of CCK-8 and secretin-induced pancreatic juice secretion in the anaesthetized rat.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Increases or decreases in the contractile response of smooth muscle underlie important pathologic... more Increases or decreases in the contractile response of smooth muscle underlie important pathological conditions such as hypertension, incontinence and altered gastrointestinal transit. These disorders are also frequently encountered in the aged population. Oxidative stress and inflammation are key features in the initiation, progression, and clinical manifestations of smooth muscle disorders. Melatonin, the major secretory product of the pineal gland, has free radical scavenging and antioxidative properties and protects against oxidative insult. Recently, widespread interest has grown regarding the apparent protective effects of melatonin on smooth muscle dysfunction. "In vitro" studies have shown that melatonin decreased vascular tone of vascular beds from control, hypertensive or aged animals, through the reduction of adrenergic contraction and the increase in acetylcholine-induced relaxation. "In vivo", melatonin also attenuates sympathetic tone by direct activation of melatonin receptors, scavenging free radicals or increasing NO availability in the central nervous system. In the gastrointestinal tract, melatonin treatment improves age-related impairments in gallbladder contractility and prevents deleterious effects of cholecystitis on smooth muscle and the enteric nervous system through suppression of oxidative stress. In addition, melatonin improves colonic transit time in constipation-predominant IBS patients. Melatonin is also able to restore impaired contractility of the detrusor muscle from old animals through normalization of Ca(2+) dependent and independent contraction, mitochondrial polarity, neuromuscular function and oxidative stress, which would explain the effects of melatonin counteracting cystometric changes in senescent animals. It also reverses bladder damage following ischemia/reperfusion. In conclusion, melatonin may be a promising candidate for future research of agents that modulate smooth muscle motility.
Biochimica Et Biophysica Acta - Molecular And Cell Biology Of Lipids, Jun 1, 2021
The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced ... more The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.
In anaesthetized rats, interaction between either vagal stimulation or acetylcholine (ACh) and th... more In anaesthetized rats, interaction between either vagal stimulation or acetylcholine (ACh) and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) and staurosporine on pancreatic juice secretion have been investigated in vivo. In vitro, rat cytosolic free calcium concentration (Ca2+)i in pancreatic acini loaded with the fluorescent dye, Fura-2 acetoxymethyl ester (AM) have been analysed after the same modifications. In vivo, vagotomy caused a marked reduction in the rate of pancreatic juice flow, total protein output and amylase secretion compared to basal secretory parameters. Furthermore, bolus injection of either saline, TPA (10(-8) mol/kg b wt), staurosporine (10(-8) mol/kg b wt) or a combination of TPA and staurosporine (all 10(-8) mol/kg b wt) had no statistically significant effect on pancreatic juice flow, protein output and amylase secretion compared to vagotomy values. Electrical stimulation (E.S.) of vagues nerves resulted in marked and statistically significant (P < 0.05) increases in the rate of pancreatic juice flow, total protein output and amylase secretion in rats injected with either saline or staurosporine, compared to control values. In contrast, E.S. of the vagus nerves failed to enhance all secretory parameters in the presence of either TPA or a combination of TPA and staurosporine. In vitro, on isolated acini, ACh evoked dose dependent increases in (Ca2+)i. Pretreatment these acini with either TPA, staurosporine or a combination of TPA and staurosporine had no significant effect on the ACh-induced (Ca2+)i. These results indicate that TPA can decrease the secretory responses evoked by E.S. of the vagus nerves in the anaesthetized rat. This attenuation is not associated with either protein kinase C inhibition or the mobilization of the second messenger Ca2+ but possibly through activation of protein kinase C by TPA.
In many tissues the cellular responses mediated through different intracellular messenger systems... more In many tissues the cellular responses mediated through different intracellular messenger systems are mutually interactive. In the exocrine pancreas the secretagogues acting via adenosine cyclic monophosphate (cAMP) and those acting via calcium-phosphoinositides can potentiate one another. On the other hand, protein kinase C (PK-C) modulates receptor-induced responses in exocrine pancreatic cells and other cell types. Recording total protein output, monitored on-line at 280 nm, from superfused rat pancreatic segments, we demonstrate that secretin (a cAMP-acting hormone) reduces the efficacy of the calcium-mediated secretagogue cholecystokinin-octapeptide (CCK-8). Likewise, the PK-C activator 12,O,tetradecanoyl phorbol 13 acetate (TPA) reduces both the efficacy of secretin and the potency of cholecystokinin. Thus, the hypothesis of potentiation between different stimulus-secretion coupling mechanisms must be revised, and receptor-activated responses in the exocrine pancreas must be considered a complex model with multiple inhibitory and stimulatory interactions.
Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion... more Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internaliza...
The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-acti... more The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plas...
La secrecion pancreatica exocrina es el resultado final de la accion simultanea de multiples mens... more La secrecion pancreatica exocrina es el resultado final de la accion simultanea de multiples mensajeros extracelularessobre las celulas acinares y ductulares. El proceso global esta mediado por un grupo de mensajeros intracelularescomo cAMP, IP3, IP4 o diacilglicerol.Hormonas como histamina e insulina pueden participar tambien como agentes reguladores moduladores de lasecrecion pancreatica. Se ha demostrado que la histamina ejerce un efecto secretagogo moderado a traves de lamodulacion de la respuesta secretora inducida por los secretagogos clasicos. La insulina puede potenciar acusadamentela respuesta secretora a acetilcolina y colecistoquinina. Ademas, otros peptidos liberados de los islotes de Langerhanscomo glucagon, somatostatina y polipeptido pancreatico, pueden atenuar las respuestas secretoras inducidas porcolecistoquinina y acetilcolina.Los factores de crecimiento participan en el control del crecimiento, replicacion y diferenciacion celular. Tambienestan implicados en la s...
1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from... more 1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from pancreatic acinar cells with preserved intracellular environments. The calcium dependence of calcium extrusion indicated a strong co‐operativity (Hill coefficient, 3). The half‐maximal rate of calcium extrusion occurred at an intracellular free calcium concentration ([Ca2+]i) of approximately 200 nM. At [Ca2+]i levels higher than 400 nM the calcium extrusion mechanism was almost completely saturated. 2. The rate of [Ca2+]i recovery was measured with the same cells under conditions where both calcium extrusion and calcium reuptake occurred simultaneously and under conditions when calcium reuptake was prevented and recovery depended entirely upon calcium extrusion. The rate of [Ca2+]i recovery due to calcium reuptake displayed a very sharp dependence on [Ca2+]i. The rate of [Ca2+]i recovery due to reuptake increased approximately 10 times (from 4.3 to 44.1 nM s‐1) for an increase of [Ca2+...
1. In the present time-course study, we have examined the interactions between the phorbol ester,... more 1. In the present time-course study, we have examined the interactions between the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic gut hormones, cholecystokinin-octapeptide (CCK-8) and secretin on pancreatic juice secretion in anaesthetized rat. 2. Administration of either TPA (10(-8) mol kg-1 hr-1), secretin (100 pmol kg-1 hr-1) or CCK-8 (150 pmol kg-1 hr-1) in the anaesthetized rat resulted in marked time-course increases in pancreatic juice flow, amylase secretion and total protein output compared to saline controls. The effect of secretin on juice flow was more pronounced and sustained compared to the smaller responses obtained with either CCK-8 or TPA. Similarly, CCK-8 evoked increases in protein output and amylase secretion compared to the responses obtained with either secretin or TPA. 3. Simultaneous infusion of TPA with either CCK-8 or secretin resulted in a marked reduction in pancreatic juice flow, total protein output and amylase secretion compared to the responses obtained with either CCK-8 or secretin alone. 4. Administration of polymyxin B (10(-8) mol kg-1 hr-1), a protein kinase C inhibitor with either TPA and CCK-8 or TPA and secretin caused a partial reduction of the inhibitory effect of TPA on CCK-8 and secretin-evoked secretory responses. 5. The present study further implicates the involvement of protein kinase C in the modulation of CCK-8 and secretin-induced pancreatic juice secretion in the anaesthetized rat.
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