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Papers by alejandro sandoval

This investigation evaluated the gastroprotective effect of β-lupeol, isolated from Pseudobombax ... more This investigation evaluated the gastroprotective effect of β-lupeol, isolated from Pseudobombax ellipticum. Gastric mucosal damage was induced in rats by intragastric ethanol (1mL/rat). Rats treated orally with β-lupeol suspended in Tween 80 at 3, 10, 30 and 100 mg/kg, showed 21, 60, 79 and 77 % gastroprotection respectively. The gastroprotection observed at 30 mg/kg for this compound was reverted in rats pretreated with indomethacin (10 mg/kg. s.c.) or N-ethylmaleimide (NEM 10 mg/kg, s.c.), suggesting that the gastroprotective mechanism of this triterpene involves, at least in part, the participation of prostaglandins and endogenous sulfhydryls. The gastroprotective effect of β-lupeol was not affected by the pretreatment with L-NAME (70 mg/kg, i.p.), a nitric oxide (NO)-synthase inhibitor. Carbenoxolone was used as gastroprotective model drug and showed dose dependent gastroprotective effect (26, 44 and 88% of gastroprotection, at 1, 10 and 30 mg/kg, respectively). The participation of prostaglandins, sulfhydryls and nitric oxide was observed in the gastroprotective mechanism of carbenoxolone.

International Orthopaedics, 2010

Minimally invasive plate osteosynthesis (MIPO) has been advocated as a safe approach to humeral s... more Minimally invasive plate osteosynthesis (MIPO) has been advocated as a safe approach to humeral shaft fracture management. We evaluated the reproducibility of this technique in a regional hospital. Thirty-five patients underwent MIPO of humerus shaft fractures. Fifteen patients had an open fracture, six a preoperative radial nerve palsy, and nine a concomitant thoracic, musculoskeletal or vascular injury. At an average 12-month follow-up, 91% of fractures healed after a mean of 12 weeks (range, 8–16). Two infections occurred. Final alignment averaged 4° of varus (range, 5° of valgus to 20° of varus). Active elbow ROM averaged 114° (range, 60–135°) and was less than 100° in nine elbows. Five of six preoperative radial nerve injuries recovered spontaneously. Healing and infection rates in this study are consistent with those reported in the literature. Lower elbow ROM and higher fracture angulation at healing were nevertheless found. MIPO is technically demanding and requires adequate intraoperative imaging and surgical experience in order to obtain adequate fracture alignment. Brachialis muscle scarring and inadequate postoperative rehabilitation may be involved in limited elbow range of motion.

International Orthopaedics, 2010

Background Minimally invasive plate osteosynthesis (MIPO) has become a popular option for treatme... more Background Minimally invasive plate osteosynthesis (MIPO) has become a popular option for treatment of humeral shaft fractures. However, indirect reduction might risk unpromising results, with mal-alignment/mal-union or nonunion. The purpose of this study was to describe a reproducible MIPO technique that used an external fixator during the procedure as a tool for reduction and maintenance, and to assess the outcomes in patients with humeral shaft fracture. Methods Of 31 consecutive cases of humeral shaft fracture in 30 patients, 29 were included in this study. There were seven simple (type A) and 22 comminuted (type B or C) fractures. After the insertion of one Schanz pin on each proximal and distal humerus, a provisional reduction was achieved by connecting the pins with a monolateral external fixator. The MIPO procedure was then performed over the anterior aspect of the humerus. To evaluate the efficacy of the provisional reduction by external fixator, coronal and sagittal alignments were assessed. We also assessed bony and functional results, including complications, from this technique.

Biochemical and Biophysical Research Communications, 2011

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in t... more Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K+ channels KV4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the KV4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K+ current and an alteration in the inactivation of the macroscopic outward K+ currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of KV4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.► High-glucose induces expression of neuronal calcium sensor KChIP3, in retinal glia. ► Upregulation of KChIP3 expression is not associated with increased apoptosis. ► KChIP3 interacts with KV4 channels in retinal Müller cells. ► KV4 channel functional expression is also regulated in high glucose-treated cells. ► KChIP3 might regulate the macroscopic K+ current inactivation in these cells.

European Journal of Neuroscience, 2007

By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a v... more By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (α1) and three auxiliary (α2δ, β and γ) subunits. The α2δ subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, α2δ must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular α2 from its transmembrane δ domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature α2δ, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of α2δ was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.

Pflugers Archiv-european Journal of Physiology, 2011

Regulation of presynaptic voltage-gated calcium channels is critical for depolarization-evoked ne... more Regulation of presynaptic voltage-gated calcium channels is critical for depolarization-evoked neurotransmitter release. Various studies attempted to determine the functional implication of Rim1, a component of the vesicle release machinery. Besides to couple voltage-gated Ca2+ channels to the presynaptic vesicle release machinery, it was evidenced that Rim1 also prevents voltage-dependent inactivation of the channels through a direct interaction with the ancillary β-subunits, thus facilitating neurotransmitter release. However, facilitation of synaptic activity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled receptors. Here, we explored the functional implication of Rim1 in G-protein regulation of Cav2.2 channels. Activation of μ-opioid receptors expressed in HEK-293 cells along with Cav2.2 channels produced a drastic current inhibition both in control and Rim1-expressing cells. In contrast, Rim1 considerably promoted the extent of current deinhibition following channel activation, favoring sustained Ca2+ influx under prolonged activity. Our data suggest that Rim1-induced facilitation of neurotransmitter release may come as a consequence of a decrease in the inhibitory pathway carried by G-proteins that contributes, together with the slowing of channel inactivation, to maintain Ca2+ influx under prolonged activity. The present study also furthers functional insights in the importance of proteins from the presynaptic vesicle complex in the regulation of voltage-gated Ca2+ channels by G-proteins.

Cell Calcium, 2000

Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores ga... more Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores gated by depolarization and are essential regulators of the intracellular Ca2+ concentration. By providing a pathway for rapid Ca2+ influx, CaV channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. CaV channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α1 subunit, which typically contains associated β and α2δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α2δ-1 is post-translationally cleaved into an extracellular α2 polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α2δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α2) and Cys1047 (in the extracellular domain of δ) prevented the association of the α2 and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α2δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (CaV2.2α1/β3) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α2δ-1 function.

Cell Calcium, 2009

The auxiliary Ca V ␣ 2 ␦-1 subunit is an important component of voltage-gated Ca 2+ (Ca V ) chann... more The auxiliary Ca V ␣ 2 ␦-1 subunit is an important component of voltage-gated Ca 2+ (Ca V ) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca V ␣ 2 ␦-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca V 1.3 channels in HEK-293 cells. Co-expression of Ca V ␣ 2 ␦-1 significantly increased macroscopic currents and conferred the Ca V 1.3␣ 1 /Ca V ␤ 3 channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca V ␣ 2 ␦-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane ␦ domain of the protein, reduced functionality in terms of enhancement of Ca V 1.3␣ 1 /Ca V ␤ 3 currents. In addition, co-expression of the ␦ domain drastically inhibited macroscopic currents through recombinant Ca V 1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca V ␣ 2 ␦-1 auxiliary subunit may interact with Ca V 1.3 channels and regulate their functional expression.

European Journal of Neuroscience, 2006

Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVbeta3 subunit and provide ... more Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVbeta3 subunit and provide important determinants for neuronal Ca2+ channel activity.

Bioorganic & Medicinal Chemistry, 2005

A facile preparation of 2-aminomethyl-2-tricyclo[3.3.1.11,7]decaneacetic acid hydrochloride 5 (Ad... more A facile preparation of 2-aminomethyl-2-tricyclo[3.3.1.11,7]decaneacetic acid hydrochloride 5 (AdGABA) is described. The synthesis of AdGABA involves the hydrogenation of 2-cyano-2-tricyclo[3.3.1.11,7]decaneacetic acid 11, which was synthesized by two different synthetic routes. AdGABA was found to antagonize the pentylenetetrazole (PTZ) and semicarbazide (SCZ) induced tonic convulsions and exhibits analgesic activity in the hot plate test on mice. Although its mechanism of action is quite similar to that proposed previously for gabapentin (interaction with the α2δ subunit of the voltage gated Ca2+ channels), further studies were undertaken in order to clarify the precise mechanism of the anticonvulsant and analgesic effects of AdGABA on a molecular level.In the current work, we report the design, synthesis (following two different synthetic routes), stability studies, and comprehensive pharmacology of a novel GABA adamantane derivative (AdGABA), which displays a close structure–activity relationship with gabapentin.

Febs Letters, 2004

The CaVα2δ auxiliary subunit is a glycosylated protein that regulates the trafficking and functio... more The CaVα2δ auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltage-gated Ca2+ channels. One of the most prominent roles of CaVα2δ is to increase whole-cell Ca2+ current amplitude. Using N-glycosidase F and truncated forms of CaVα2δ, earlier studies suggested an important role for N-linked glycosylation in current stimulation. Here, we used site-directed mutagenesis and heterologous expression in HEK-293 cells to examine the impact of individual glycosylation sites within the CaVα2δ subunit on the regulation of Ba2+ currents through recombinant Ca2+ channels. We found two N-glycosylation consensus sites (NX(S/T)) in the extracellular α2 domain of the protein that are functional. Substitution of asparagines for glutamines at amino acid positions 136 and 184 rendered these sites non-functional as shown by patch-clamp experiments. These results corroborate that N-glycosylation is required for the CaVα2δ subunit-induced current stimulation and suggest that sites N136 and N184 are directly involved in this action. Likewise, N136Q and N184Q mutations prevented whole-cell current stimulation without altering its kinetic properties, suggesting a regulation on the number of functional channels at the plasma membrane.

Biochemical and Biophysical Research Communications - BIOCHEM BIOPHYS RES COMMUN, 2003

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN)... more In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca2+ channels have. Interestingly, ZD7288 blocked native T-type Ca2+ currents in mouse spermatogenic cells with an IC50 of about 100 μM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca2+ channels in the mouse sperm AR.

Toxicology and Applied Pharmacology, 2011

High-voltage activated Ca 2+ (Ca V ) channels play a key role in the regulation of numerous physi... more High-voltage activated Ca 2+ (Ca V ) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca 2+ concentration. These channels consist of a pore-forming Ca V α 1 protein and three auxiliary subunits (Ca V β, Ca V α 2 δ and Ca V γ). Ca V α 2 δ is an important component of Ca V channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca V α 2 δ and reduces Ca 2+ currents by modulating the expression and/or function of the Ca V α 1 subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca V channels. However, the importance of the interaction of AdGABA with the Ca V α 2 δ subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca 2+ currents acting on Ca V α 2 δ only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca V α 2 δ that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca V α 2 δ ligand that regulates Ca V channels.

Pflügers Archiv - European Journal of Physiology, 2013

The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with... more The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with alterations of surface expression, kinetics, and voltage-dependent properties of the channel complex. Four mammalian genes and several splice α2δ subunit variants have been cloned and described, though very little information concerning the transcriptional mechanisms that regulate their expression is available. Here, we report the identification and characterization of the human α2δ-1 subunit gene promoter and its regulation by specific transcription factor 1 (Sp1). Transient transfection of human neuroblastoma SH-SY5Y cells with a promoter/luciferase reporter construct revealed a ~1.5 kb 5´-UTR fragment of the CACNA2D1 gene that produced high levels of luciferase activity. Deletional analysis of this sequence showed that the minimal promoter was located within a 413-bp region (nt -326 to +98) with respect to the transcription start site. In this region, no canonical TATA box was present, but a high GC content and five potential Sp1 binding sites were found. The ability of two of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Likewise, Sp1 overexpression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of α2δ protein expressed in the SH-SY5Y cells, as well as reduced the amplitude of whole-cell patch clamp Ca(2+) currents in dorsal root ganglion neurons. This study thus represents the first identification of the transcriptional control region in the gene encoding the Ca(2+) channel α2δ-1 auxiliary subunit.

Cellular and Molecular Neurobiology, 2007

(1) Voltage-gated Ca2+ (CaV) channels are multi-subunit membrane complexes that allow depolarizat... more (1) Voltage-gated Ca2+ (CaV) channels are multi-subunit membrane complexes that allow depolarization-induced Ca2+ influx into cells. The skeletal muscle L-type CaV channels consist of an ion-conducting CaV1.1 subunit and auxiliary α2δ−1, β1 and γ1 subunits. This complex serves both as a CaV channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α2δ−1 and β1 subunits regulate CaV channel function, there is far less information on the γ1 subunit. Previously, we characterized the interaction of γ1 with the other components of the skeletal CaV channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca2+ current density in myotubes from γ1 null mice. (3) In the current report, using Western blotting we show that the expression of the CaV1.1 protein is significantly lower when it is heterologously co-expressed with γ1. Consistent with this, patch-clamp recordings showed that transient transfection of γ1 drastically inhibited macroscopic currents through recombinant N-type (CaV2.2/α2δ−1/β3) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ1 subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca2+ current density following γ1 transfection.

Biochemical and Biophysical Research Communications, 2010

Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake an... more Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca 2+ levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca 2+ channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca 2+ channel expression.

Pflugers Archiv-european Journal of Physiology, 2008

Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding ... more Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding for the Cav2.1 subunit of voltage-gated calcium channels. Although various studies attempted to determine biophysical consequences of these mutations on channel activity, it remains unclear exactly how mutations can produce a FHM-1 phenotype. A lower activation threshold of mutated channels resulting in increased channel activity has been proposed. However, hyperactivity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled-receptor activation. The aim of this study is to determine functional consequences of the FHM-1 S218L mutation on direct G-protein regulation of Cav2.1 channels. In HEK 293 cells, DAMGO activation of human μ-opioid receptors induced a 55% Ba2+ current inhibition through both wild-type and S218L mutant Cav2.1 channels. In contrast, this mutation considerably accelerates the kinetic of current deinhibition following channel activation by 1.7- to 2.3-fold depending on membrane potential values. Taken together, these data suggest that the S218L mutation does not affect G-protein association onto the channel in the closed state but promotes its dissociation from the activated channel, thereby decreasing the inhibitory G-protein pathway. Similar results were obtained with the R192Q FHM-1 mutation, although of lesser amplitude, which seems in line with the less severe associated clinical phenotype in patients. Functional consequences of FHM-1 mutations appear thus as the consequence of the alteration of both intrinsic biophysical properties and of the main inhibitory G-protein pathway of Cav2.1 channels. The present study furthers molecular insight in the physiopathology of FHM-1.

Leukemia, 2000

Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leu... more Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leukemia (CLL) and other indolent lymphocytic malignancies. Although the incorporation of fludarabine into DNA is a key event in causing cytotoxicity in proliferating leukemia cells, the precise mechanisms by which fludarabine kills CLL cells remain unclear because of the quiescent nature of this malignancy. The present study demonstrated that inhibition of RNA transcription correlated significantly with the cytotoxic action of fludarabine in CLL cells. In contrast, suppression of the low level of DNA synthesis did not affect the survival of the leukemia cells. In addition, inhibition of fludarabine incorporation into cellular DNA through repair synthesis in CLL cells did not alter the cytotoxicity of this drug. Rather, inhibition of RNA synthesis by fludarabine led to a specific diminishment of certain cellular proteins from CLL cells. The combination of fludarabine with another RNA synthesis inhibitor, actinomycin D, or with the protein synthesis inhibitor, puromycin, substantially enhanced the cytotoxic activity against CLL cells. These results suggest that termination of mRNA transcription and the consequent depletion of proteins required for cell survival may be a novel biochemical mechanism of action of fludarabine in CLL cells. Thus, inhibition of RNA/protein synthesis may provide a new therapeutic strategy for the treatment of CLL patients.

Journal of Neuroscience, 2007

Auxiliary ␥ subunits are an important component of high-voltage-activated calcium (Ca V ) channel... more Auxiliary ␥ subunits are an important component of high-voltage-activated calcium (Ca V ) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the ␥ subunits on neuronal Ca V channel activity and expression. We found that coexpression of ␥ 2 or ␥ 3 subunits drastically inhibited macroscopic currents through recombinant N-type channels (Ca V 2.2/␤ 3 /␣ 2 ␦) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by ␥. Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, ␥ subunit expression altered the voltage dependence of channel activity. Second, ␥ subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal ␥ subunits significantly downregulate Ca V 2.2 channel activity, (2) uncover a role for the ␥ 2 subunit in Ca V 2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the ␥ subunits, supporting the notion that Ca V channels constitute a class of difficult-to-fold proteins.

This investigation evaluated the gastroprotective effect of β-lupeol, isolated from Pseudobombax ... more This investigation evaluated the gastroprotective effect of β-lupeol, isolated from Pseudobombax ellipticum. Gastric mucosal damage was induced in rats by intragastric ethanol (1mL/rat). Rats treated orally with β-lupeol suspended in Tween 80 at 3, 10, 30 and 100 mg/kg, showed 21, 60, 79 and 77 % gastroprotection respectively. The gastroprotection observed at 30 mg/kg for this compound was reverted in rats pretreated with indomethacin (10 mg/kg. s.c.) or N-ethylmaleimide (NEM 10 mg/kg, s.c.), suggesting that the gastroprotective mechanism of this triterpene involves, at least in part, the participation of prostaglandins and endogenous sulfhydryls. The gastroprotective effect of β-lupeol was not affected by the pretreatment with L-NAME (70 mg/kg, i.p.), a nitric oxide (NO)-synthase inhibitor. Carbenoxolone was used as gastroprotective model drug and showed dose dependent gastroprotective effect (26, 44 and 88% of gastroprotection, at 1, 10 and 30 mg/kg, respectively). The participation of prostaglandins, sulfhydryls and nitric oxide was observed in the gastroprotective mechanism of carbenoxolone.

International Orthopaedics, 2010

Minimally invasive plate osteosynthesis (MIPO) has been advocated as a safe approach to humeral s... more Minimally invasive plate osteosynthesis (MIPO) has been advocated as a safe approach to humeral shaft fracture management. We evaluated the reproducibility of this technique in a regional hospital. Thirty-five patients underwent MIPO of humerus shaft fractures. Fifteen patients had an open fracture, six a preoperative radial nerve palsy, and nine a concomitant thoracic, musculoskeletal or vascular injury. At an average 12-month follow-up, 91% of fractures healed after a mean of 12 weeks (range, 8–16). Two infections occurred. Final alignment averaged 4° of varus (range, 5° of valgus to 20° of varus). Active elbow ROM averaged 114° (range, 60–135°) and was less than 100° in nine elbows. Five of six preoperative radial nerve injuries recovered spontaneously. Healing and infection rates in this study are consistent with those reported in the literature. Lower elbow ROM and higher fracture angulation at healing were nevertheless found. MIPO is technically demanding and requires adequate intraoperative imaging and surgical experience in order to obtain adequate fracture alignment. Brachialis muscle scarring and inadequate postoperative rehabilitation may be involved in limited elbow range of motion.

International Orthopaedics, 2010

Background Minimally invasive plate osteosynthesis (MIPO) has become a popular option for treatme... more Background Minimally invasive plate osteosynthesis (MIPO) has become a popular option for treatment of humeral shaft fractures. However, indirect reduction might risk unpromising results, with mal-alignment/mal-union or nonunion. The purpose of this study was to describe a reproducible MIPO technique that used an external fixator during the procedure as a tool for reduction and maintenance, and to assess the outcomes in patients with humeral shaft fracture. Methods Of 31 consecutive cases of humeral shaft fracture in 30 patients, 29 were included in this study. There were seven simple (type A) and 22 comminuted (type B or C) fractures. After the insertion of one Schanz pin on each proximal and distal humerus, a provisional reduction was achieved by connecting the pins with a monolateral external fixator. The MIPO procedure was then performed over the anterior aspect of the humerus. To evaluate the efficacy of the provisional reduction by external fixator, coronal and sagittal alignments were assessed. We also assessed bony and functional results, including complications, from this technique.

Biochemical and Biophysical Research Communications, 2011

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in t... more Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K+ channels KV4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the KV4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K+ current and an alteration in the inactivation of the macroscopic outward K+ currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of KV4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.► High-glucose induces expression of neuronal calcium sensor KChIP3, in retinal glia. ► Upregulation of KChIP3 expression is not associated with increased apoptosis. ► KChIP3 interacts with KV4 channels in retinal Müller cells. ► KV4 channel functional expression is also regulated in high glucose-treated cells. ► KChIP3 might regulate the macroscopic K+ current inactivation in these cells.

European Journal of Neuroscience, 2007

By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a v... more By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (α1) and three auxiliary (α2δ, β and γ) subunits. The α2δ subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, α2δ must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular α2 from its transmembrane δ domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature α2δ, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of α2δ was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane.

Pflugers Archiv-european Journal of Physiology, 2011

Regulation of presynaptic voltage-gated calcium channels is critical for depolarization-evoked ne... more Regulation of presynaptic voltage-gated calcium channels is critical for depolarization-evoked neurotransmitter release. Various studies attempted to determine the functional implication of Rim1, a component of the vesicle release machinery. Besides to couple voltage-gated Ca2+ channels to the presynaptic vesicle release machinery, it was evidenced that Rim1 also prevents voltage-dependent inactivation of the channels through a direct interaction with the ancillary β-subunits, thus facilitating neurotransmitter release. However, facilitation of synaptic activity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled receptors. Here, we explored the functional implication of Rim1 in G-protein regulation of Cav2.2 channels. Activation of μ-opioid receptors expressed in HEK-293 cells along with Cav2.2 channels produced a drastic current inhibition both in control and Rim1-expressing cells. In contrast, Rim1 considerably promoted the extent of current deinhibition following channel activation, favoring sustained Ca2+ influx under prolonged activity. Our data suggest that Rim1-induced facilitation of neurotransmitter release may come as a consequence of a decrease in the inhibitory pathway carried by G-proteins that contributes, together with the slowing of channel inactivation, to maintain Ca2+ influx under prolonged activity. The present study also furthers functional insights in the importance of proteins from the presynaptic vesicle complex in the regulation of voltage-gated Ca2+ channels by G-proteins.

Cell Calcium, 2000

Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores ga... more Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores gated by depolarization and are essential regulators of the intracellular Ca2+ concentration. By providing a pathway for rapid Ca2+ influx, CaV channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. CaV channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α1 subunit, which typically contains associated β and α2δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α2δ-1 is post-translationally cleaved into an extracellular α2 polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α2δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α2) and Cys1047 (in the extracellular domain of δ) prevented the association of the α2 and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α2δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (CaV2.2α1/β3) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α2δ-1 function.

Cell Calcium, 2009

The auxiliary Ca V ␣ 2 ␦-1 subunit is an important component of voltage-gated Ca 2+ (Ca V ) chann... more The auxiliary Ca V ␣ 2 ␦-1 subunit is an important component of voltage-gated Ca 2+ (Ca V ) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca V ␣ 2 ␦-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca V 1.3 channels in HEK-293 cells. Co-expression of Ca V ␣ 2 ␦-1 significantly increased macroscopic currents and conferred the Ca V 1.3␣ 1 /Ca V ␤ 3 channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca V ␣ 2 ␦-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane ␦ domain of the protein, reduced functionality in terms of enhancement of Ca V 1.3␣ 1 /Ca V ␤ 3 currents. In addition, co-expression of the ␦ domain drastically inhibited macroscopic currents through recombinant Ca V 1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca V ␣ 2 ␦-1 auxiliary subunit may interact with Ca V 1.3 channels and regulate their functional expression.

European Journal of Neuroscience, 2006

Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVbeta3 subunit and provide ... more Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVbeta3 subunit and provide important determinants for neuronal Ca2+ channel activity.

Bioorganic & Medicinal Chemistry, 2005

A facile preparation of 2-aminomethyl-2-tricyclo[3.3.1.11,7]decaneacetic acid hydrochloride 5 (Ad... more A facile preparation of 2-aminomethyl-2-tricyclo[3.3.1.11,7]decaneacetic acid hydrochloride 5 (AdGABA) is described. The synthesis of AdGABA involves the hydrogenation of 2-cyano-2-tricyclo[3.3.1.11,7]decaneacetic acid 11, which was synthesized by two different synthetic routes. AdGABA was found to antagonize the pentylenetetrazole (PTZ) and semicarbazide (SCZ) induced tonic convulsions and exhibits analgesic activity in the hot plate test on mice. Although its mechanism of action is quite similar to that proposed previously for gabapentin (interaction with the α2δ subunit of the voltage gated Ca2+ channels), further studies were undertaken in order to clarify the precise mechanism of the anticonvulsant and analgesic effects of AdGABA on a molecular level.In the current work, we report the design, synthesis (following two different synthetic routes), stability studies, and comprehensive pharmacology of a novel GABA adamantane derivative (AdGABA), which displays a close structure–activity relationship with gabapentin.

Febs Letters, 2004

The CaVα2δ auxiliary subunit is a glycosylated protein that regulates the trafficking and functio... more The CaVα2δ auxiliary subunit is a glycosylated protein that regulates the trafficking and function of voltage-gated Ca2+ channels. One of the most prominent roles of CaVα2δ is to increase whole-cell Ca2+ current amplitude. Using N-glycosidase F and truncated forms of CaVα2δ, earlier studies suggested an important role for N-linked glycosylation in current stimulation. Here, we used site-directed mutagenesis and heterologous expression in HEK-293 cells to examine the impact of individual glycosylation sites within the CaVα2δ subunit on the regulation of Ba2+ currents through recombinant Ca2+ channels. We found two N-glycosylation consensus sites (NX(S/T)) in the extracellular α2 domain of the protein that are functional. Substitution of asparagines for glutamines at amino acid positions 136 and 184 rendered these sites non-functional as shown by patch-clamp experiments. These results corroborate that N-glycosylation is required for the CaVα2δ subunit-induced current stimulation and suggest that sites N136 and N184 are directly involved in this action. Likewise, N136Q and N184Q mutations prevented whole-cell current stimulation without altering its kinetic properties, suggesting a regulation on the number of functional channels at the plasma membrane.

Biochemical and Biophysical Research Communications - BIOCHEM BIOPHYS RES COMMUN, 2003

In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN)... more In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca2+ channels have. Interestingly, ZD7288 blocked native T-type Ca2+ currents in mouse spermatogenic cells with an IC50 of about 100 μM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca2+ channels in the mouse sperm AR.

Toxicology and Applied Pharmacology, 2011

High-voltage activated Ca 2+ (Ca V ) channels play a key role in the regulation of numerous physi... more High-voltage activated Ca 2+ (Ca V ) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca 2+ concentration. These channels consist of a pore-forming Ca V α 1 protein and three auxiliary subunits (Ca V β, Ca V α 2 δ and Ca V γ). Ca V α 2 δ is an important component of Ca V channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca V α 2 δ and reduces Ca 2+ currents by modulating the expression and/or function of the Ca V α 1 subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca V channels. However, the importance of the interaction of AdGABA with the Ca V α 2 δ subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca 2+ currents acting on Ca V α 2 δ only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca V α 2 δ that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca V α 2 δ ligand that regulates Ca V channels.

Pflügers Archiv - European Journal of Physiology, 2013

The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with... more The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with alterations of surface expression, kinetics, and voltage-dependent properties of the channel complex. Four mammalian genes and several splice α2δ subunit variants have been cloned and described, though very little information concerning the transcriptional mechanisms that regulate their expression is available. Here, we report the identification and characterization of the human α2δ-1 subunit gene promoter and its regulation by specific transcription factor 1 (Sp1). Transient transfection of human neuroblastoma SH-SY5Y cells with a promoter/luciferase reporter construct revealed a ~1.5 kb 5´-UTR fragment of the CACNA2D1 gene that produced high levels of luciferase activity. Deletional analysis of this sequence showed that the minimal promoter was located within a 413-bp region (nt -326 to +98) with respect to the transcription start site. In this region, no canonical TATA box was present, but a high GC content and five potential Sp1 binding sites were found. The ability of two of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Likewise, Sp1 overexpression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of α2δ protein expressed in the SH-SY5Y cells, as well as reduced the amplitude of whole-cell patch clamp Ca(2+) currents in dorsal root ganglion neurons. This study thus represents the first identification of the transcriptional control region in the gene encoding the Ca(2+) channel α2δ-1 auxiliary subunit.

Cellular and Molecular Neurobiology, 2007

(1) Voltage-gated Ca2+ (CaV) channels are multi-subunit membrane complexes that allow depolarizat... more (1) Voltage-gated Ca2+ (CaV) channels are multi-subunit membrane complexes that allow depolarization-induced Ca2+ influx into cells. The skeletal muscle L-type CaV channels consist of an ion-conducting CaV1.1 subunit and auxiliary α2δ−1, β1 and γ1 subunits. This complex serves both as a CaV channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α2δ−1 and β1 subunits regulate CaV channel function, there is far less information on the γ1 subunit. Previously, we characterized the interaction of γ1 with the other components of the skeletal CaV channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca2+ current density in myotubes from γ1 null mice. (3) In the current report, using Western blotting we show that the expression of the CaV1.1 protein is significantly lower when it is heterologously co-expressed with γ1. Consistent with this, patch-clamp recordings showed that transient transfection of γ1 drastically inhibited macroscopic currents through recombinant N-type (CaV2.2/α2δ−1/β3) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ1 subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca2+ current density following γ1 transfection.

Biochemical and Biophysical Research Communications, 2010

Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake an... more Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca 2+ levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca 2+ channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca 2+ channel expression.

Pflugers Archiv-european Journal of Physiology, 2008

Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding ... more Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding for the Cav2.1 subunit of voltage-gated calcium channels. Although various studies attempted to determine biophysical consequences of these mutations on channel activity, it remains unclear exactly how mutations can produce a FHM-1 phenotype. A lower activation threshold of mutated channels resulting in increased channel activity has been proposed. However, hyperactivity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled-receptor activation. The aim of this study is to determine functional consequences of the FHM-1 S218L mutation on direct G-protein regulation of Cav2.1 channels. In HEK 293 cells, DAMGO activation of human μ-opioid receptors induced a 55% Ba2+ current inhibition through both wild-type and S218L mutant Cav2.1 channels. In contrast, this mutation considerably accelerates the kinetic of current deinhibition following channel activation by 1.7- to 2.3-fold depending on membrane potential values. Taken together, these data suggest that the S218L mutation does not affect G-protein association onto the channel in the closed state but promotes its dissociation from the activated channel, thereby decreasing the inhibitory G-protein pathway. Similar results were obtained with the R192Q FHM-1 mutation, although of lesser amplitude, which seems in line with the less severe associated clinical phenotype in patients. Functional consequences of FHM-1 mutations appear thus as the consequence of the alteration of both intrinsic biophysical properties and of the main inhibitory G-protein pathway of Cav2.1 channels. The present study furthers molecular insight in the physiopathology of FHM-1.

Leukemia, 2000

Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leu... more Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leukemia (CLL) and other indolent lymphocytic malignancies. Although the incorporation of fludarabine into DNA is a key event in causing cytotoxicity in proliferating leukemia cells, the precise mechanisms by which fludarabine kills CLL cells remain unclear because of the quiescent nature of this malignancy. The present study demonstrated that inhibition of RNA transcription correlated significantly with the cytotoxic action of fludarabine in CLL cells. In contrast, suppression of the low level of DNA synthesis did not affect the survival of the leukemia cells. In addition, inhibition of fludarabine incorporation into cellular DNA through repair synthesis in CLL cells did not alter the cytotoxicity of this drug. Rather, inhibition of RNA synthesis by fludarabine led to a specific diminishment of certain cellular proteins from CLL cells. The combination of fludarabine with another RNA synthesis inhibitor, actinomycin D, or with the protein synthesis inhibitor, puromycin, substantially enhanced the cytotoxic activity against CLL cells. These results suggest that termination of mRNA transcription and the consequent depletion of proteins required for cell survival may be a novel biochemical mechanism of action of fludarabine in CLL cells. Thus, inhibition of RNA/protein synthesis may provide a new therapeutic strategy for the treatment of CLL patients.

Journal of Neuroscience, 2007

Auxiliary ␥ subunits are an important component of high-voltage-activated calcium (Ca V ) channel... more Auxiliary ␥ subunits are an important component of high-voltage-activated calcium (Ca V ) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the ␥ subunits on neuronal Ca V channel activity and expression. We found that coexpression of ␥ 2 or ␥ 3 subunits drastically inhibited macroscopic currents through recombinant N-type channels (Ca V 2.2/␤ 3 /␣ 2 ␦) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by ␥. Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, ␥ subunit expression altered the voltage dependence of channel activity. Second, ␥ subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal ␥ subunits significantly downregulate Ca V 2.2 channel activity, (2) uncover a role for the ␥ 2 subunit in Ca V 2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the ␥ subunits, supporting the notion that Ca V channels constitute a class of difficult-to-fold proteins.