Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels.

Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA.

Effect of normobaric hypoxia on the testis in a murine model.

High-altitude hypoxia generates spermiogram impairment due to germinal epithelium, Leydig cells, sperm and seminal plasma alterations, but precise mechanisms involved are unknown. The objective of this work was to analyse the effect of normobaric hypoxia on the morphology of testicular interstitium and some associated molecular and hormonal factors. Twenty-four mice were exposed to normobaric hypoxia (8.1% inspired oxygen fraction) during 20 days. The effects on body weight, testicular weight, vascularisation, testosterone, HIF1-α and VEGF were analysed at different periods of exposure and compared to controls. Hypoxic mice had lower body weight than mice kept in normoxia. Testicular weight raised significantly the 1st day, but remained normal during the rest of experiment. Number of blood vessels per field and mean diameter of vessels were higher in hypoxic mice. Plasmatic and testicular testosterone raised during first 24 h of hypoxia, but decreased on the 5th day. Vascular/interstitial ratio (proportion of interstice occupied by blood vessels) duplicated at the end of the experiment. Most substantial early effects of hypoxia were testicular oedema, increase in number and diameter of blood vessels and elevation of plasmatic and testicular testosterone. Normobaric hypoxia generates similar effects to those induced by hypobaric hypoxia.

Transgenic mouse model reveals an unsuspected role of the acetylcholine receptor in statin-induced neuromuscular adverse drug reactions.

High cholesterol levels are an established risk factor for cardiovascular disease (CVD), the world's leading cause of death. Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (statins) are prescribed to lower serum cholesterol levels and reduce the risk of CVD. Despite the success of statins, many patients abandon treatment owing to neuromuscular adverse drug reactions (ADRs). Genome-wide association studies have identified the single-nucleotide polymorphism (SNP) rs4149056 in the SLCO1B1 gene as being associated with an increased risk for statin-induced ADRs. By studying slow-channel syndrome transgenic mouse models, we determined that statins trigger ADRs in mice expressing the mutant allele of the rs137852808 SNP in the nicotinic acetylcholine receptor (nAChR) α-subunit gene CHRNA1. Mice expressing this allele show a remarkable contamination of end-plates with caveolin-1 and develop early signs of neuromuscular degeneration upon statin treatment. This study demonstrates that genes coding for nAChR subunits may contain variants associated with statin-induced ADRs.

[Prevalence of hyponutrition in the elderly at admission to the hospital]. / Prevalencia de desnutrición del adulto mayor al ingreso hospitalario.

BACKGROUND: The population older than 60 years in Mexico is growing changing the classic pyramidal demographic structure. This fact is increasing the risk of malnutrition in the elderly, specially under nutrition which is a common problem among elderly people living at home and during hospitalizations, condition that is closely related to the increasing of morbidity, mortality and costs. OBJECTIVE: Describe the prevalence of malnourished elderly who needs hospital admission. DESIGN: Cross-sectional observational study. SETTING: Third-level reference hospital. SUBJECTS: Ninety seven consecutive subjects older than 60 years admitted to hospitalization. INTERVENTIONS: During the first three days of admission all subjects were evaluated to determine their nutritional status using Mini Nutritional Assessment and Subjective Global Assessment; albumin, total lymphocytes, level of income and school grade were also included. RESULTS: Just 48% of patients have finished primary school and 66% had middle economic incomes. According to Mini Nutritional Assessment 69% of patients had risk associated to malnutrition (18% at high risk and 50% at moderate risk). The short form of the Mini Nutritional Assessment described 73% of patients at risk related to malnutrition in correlation with the complete Mini Nutritional Assessment. 46% and 20% of patients were classified at moderate malnutrition and severe malnutrition respectively using the Subjective Global Assessment. Kappa between Mini Nutritional Assessment and Subjective Global Assessment was of 42%. The Nutritional Risk Index mean value was of 85.9 +/- 11, with 80% of patients at risk associated with malnutrition when moderate and severe risk was included. Kappa between Nutritional Risk Index and Mini Nutritional Assessment was 11%. 70% of patients had serum albumin values under 3 g/dl. According to Chang's method 52% had caloric undernutrition, 29% protein undernutrition and 18% mixed undernutrition. CONCLUSIONS: Malnutrition is a common problem in elderly population at hospital admissions according to different methods used. Mini Nutritional Assessment and Subjective Global Assessment are useful low cost and replicable nutritional evaluation tools in elderly population. Mini Nutritional Assessment could have a better value to predict morbidity and mortality in institutionalized and community elderly subjects.

Effects of chronic hypobaric hypoxia on testis histology and round spermatid oxidative metabolism.

In order to evaluate the effects of the exposition to continuous chronic hypobaric hypoxia (CCHH) and intermittent chronic hypobaric hypoxia (ICHH) on testis histology and on oxidative metabolism of spermatogenic cells (SC), male rats were exposed to a 4600-m simulated altitude (PO2: 89.6 mmHg). After 60 days, ICHH and CCHH groups presented a significant decrease in testicular mass, an increase in interstitial space, a decrease in height of the seminiferous epithelium, depletion of cellular elements, vacuolization in epithelial cells and folding of the basal membrane. Round spermatids from animals exposed to CCHH presented a significant decrease in energy-dependent cell shape changes. Round spermatid mitochondria of CCHH rats seem to be limited in their ability to handle reducing equivalents. These mitochondria also appear to be uncoupled under basal conditions. Round spermatids from CCHH rats evidence large oxygen consumption (QO2) insensitive to inhibition by cyanide, a process that could be partly related to lipoperoxidation. Thus, exposure of male rats to CCHH and ICHH induced evident changes in testicular morphology and loss of spermatogenic cells, in all stages of the spermatogenic cycle. This post-meiotic spermatogenic cell loss in the testis correlated well with metabolic changes in round spermatids that evidenced a strong metabolic stress in these cells.

Dynamics of extracellular polymeric substances in UASB and EGSB reactors treating medium and low concentrated wastewaters.

In this work the dynamic study of EPS (Extracellular Polymeric Substances) concentration and distribution during the operation of two different reactor configurations (UASB and EGSB) is presented, treating medium (5 g COD/l) and low-concentrated (0.5 g COD/l) wastewater. Medium-concentrated wastewater was supplied for granules maturation as well as for stabilisation of the process. The effect of substrate change on granule characteristics was followed in both reactors. Total concentration of EPS associated to steady operation, was higher in the UASB reactor. The change to a low-concentrated substrate led to an increased difference, promoting a sharp destabilisation of the EGSB reactor, observing an increment in filamentous structures, causing biomass flotation and wash out. Although total concentration of EPS remained almost constant in the UASB reactor, their composition and distribution presented significant differences. The ratio of protein/polysaccharides as well as acidic-polysaccharides/total (neutral + acidic) polysaccharides decreased drastically in the EGSB reactor, while in the UASB reactor, the decrease was not so important and not enough for destabilisation of granule structure. Moreover, polysaccharides distribution seemed to have an important role in granule stability being enough to maintain granule cohesion only in the case of the UASB reactor. These observations point to composition and distribution of EPS rather than their total concentration as key parameters for granule stability and settleability.

Dynamics of intracellular calcium induced by lactate and glucose in rat pachytene spermatocytes and round spermatids.

Glycolytic metabolism in meiotic and post-meiotic spermatogenic cells shows differentiation-related changes. The developmental and physiological significance of these metabolic changes is not known. The aim of the present study was to test the hypothesis that glucose and lactate metabolism can modulate intracellular calcium [Ca2+](i) in spermatogenic cells in an opposing and dynamic manner. Fluorescent probes were used to measure [Ca2+](i) and pH(i), and HPLC was used to measure intracellular adenine nucleotides and mitochondrial sensing of ATP turnover. [Ca2+](i) in pachytene spermatocytes and round spermatids was modulated by changes in lactate and glucose concentrations in the media. The kinetics and magnitude of the [Ca2+](i) changes induced by lactate and glucose were different in meiotic and post-meiotic spermatogenic cells. The presence of glucose in the medium induced a decrease in pH(i) in spermatogenic cells. This glucose-induced pH(i) decrease occurred later than the changes in [Ca2+](i), which were also observed when the pH(i) decrease was inhibited, indicating that the glucose-induced [Ca2+](i) increase was not a consequence of pH(i) changes. Hexose phosphorylation in glycolysis was part of the mechanism by which glucose metabolism induced a [Ca2+](i) increase in spermatogenic cells. The sensitivity of [Ca2+](i) to carbohydrate metabolism was higher in round spermatids than in pachytene spermatocytes. Thus, differentiation-related changes in carbohydrate metabolism in spermatogenic cells determine a dynamic and differential modulation of their [Ca2+](i) by glucose and lactate, two substrates secreted by the Sertoli cells.

Temperature dependence of intracellular Ca2+ homeostasis in rat meiotic and postmeiotic spermatogenic cells.

The hypothesis that intracellular [Ca2+] is a cell parameter responsive to extreme temperatures in rat meiotic and postmeiotic spermatogenic cells was tested using intracellular fluorescent probes for Ca2+ and pH. In agreement with this hypothesis, extreme temperatures induced a rapid increase of cytosolic [Ca2+] in rat pachytene spermatocytes and round spermatids. Oscillatory changes in temperature can induce oscillations in cytosolic [Ca2+] in these cells. Intracellular [Ca2+] homeostasis in round spermatids was more sensitive to high temperatures compared with pachytene spermatocytes. The calculated activation energies for SERCA ATPase-mediated fluxes in pachytene spermatocytes and round spermatids were 62 and 75 kJ mol(-1), respectively. The activation energies for leak fluxes from intracellular Ca2+ stores were 55 and 68 kJ mol(-1) for pachytene spermatocytes and round spermatids, respectively. Together with changes in cytosolic [Ca2+], round spermatids undergo a decrease in pH(i) at high temperatures. This temperature-induced decrease in pH(i) appears to be partially responsible for the increase in cytosolic [Ca2+] of round spermatids induced by high temperatures. This characteristic of rat meiotic and postmeiotic spermatogenic cells to undergo an increment in cytosolic Ca2+ at temperatures > 33 degrees C can be related to the induction of programmed cell death by high temperatures in these cells.

Enzymatic and osmoregulative alterations in white shrimp Litopenaeus vannamei exposed to pesticides.

Pesticide pollution in coastal ecosystems of Sinaloa, Mexico is considered to be a cause for slow growth, increase of diseases and sometimes massive mortality of shrimp. So it was necessary to develop fast techniques to detect pesticide pollution in shrimp habitats. Enzymatic and osmoregulation tests in shrimp exposed to DDT, Lindane, Chlordane, Lorsban, Gusathion, Folidol, Diazinon and Tamaron were carried out. Activity reductions from 11 to 2 units/ml in acetylcholinesterase and from 1 to 0 units/l in transaminases (GOT and GPT) were detected. Also increases in osmoregulation were observed in shrimp exposed to Folidol, Diazinon and Gusation, whereas decreases with DDT, Lindane and Lorsban at salinity 50/1000. We conclude that pesticides are causing alterations in these biochemical functions and this kind of tests represent a rapid and inexpensive method for pesticide pollution detection.

Energy metabolism and its linkage to intracellular Ca2+ and pH regulation in rat spermatogenic cells.

Energy metabolism and intracellular adenine nucleotides of meiotic and postmeiotic spermatogenic cells are highly dependent on external substrates for oxidative phosphorylation and glycolysis. Using fluorescent probes to measure the changes in cytosolic [Ca2+] ([Ca2+]i) and pH (pHi), we were able to demonstrate that changes in energy metabolism of meiotic and postmeiotic spermatogenic cells were rapidly translated into changes of pHi and [Ca2+]i in the absence or presence of external Ca2+. Under these conditions, mitochondria were gaining cytosolic calcium in these cells. Our results indicate that Ca2+ mobilised by changes in metabolic energy pathways originated in thapsigargin-sensitive intracellular Ca2+ stores. Changes in intracellular adenine nucleotides, measured by HPLC, and a likely colocalization of ATP-producing and ATP-consuming processes in the cells seemed to provide the linkage between metabolic fluxes and the changes in pHi and [Ca2+]i in pachytene spermatocytes and round spermatids. Glucose metabolism produced an increase of [Ca2+]i in round spermatids but not in pachytene spermatocytes, and a decrease in pHi in both cell types. Hence, glucose emerges as a molecule that can differentially modulate [Ca2+]i and pHi in pachytene spermatocytes and round spermatids in rats.

Intracellular Ca2+ homeostasis in rat round spermatids.

Intracellular calcium, [Ca2+]i, can regulate meiotic progression of mammalian oocytes. However, the role of [Ca2+]i in the regulation of the spermatogenic process and its cellular homeostatic mechanisms in spermatogenic cells has not been elucidated. Using intracellular fluorescent probes for Ca2+ and immunodetection of plasma membrane (PM) Ca(2+)-ATPases, we report that: a) rat round spermatids maintain [Ca2+]i levels of 60 +/- 5 nM (SEM), as estimated with fluo-3 in single cells or fura-2 in cells in suspension; b) these cells regulate [Ca2+]i by actively extruding it using a PM Ca(2+)-ATPase; c) rat spermatids also actively transport Ca2+ by sarco-endoplasmic reticulum type ATPases (SERCA); d) rat spermatids possess non-mitochondrial intracellular Ca2+i stores insensitive to thapsigargin but releasable by ionomycin; and e) rat spermatids do not activate Ca2+ entry mechanisms by the release of Ca2+ from SERCA-regulated stores. These results demonstrate that rat round spermatids can generate modulated intracellular Ca2+ signals upon activation of Ca2+ channels or Ca2+ release from intracellular stores.

Intracellular pH regulation in rat round spermatids.

Intracellular pH has been shown to be an important physiological parameter in cell cycle control and differentiation, aspects that are central to the spermatogenic process. However, the pH regulatory mechanisms in spermatogenic cells have not been systematically explored. In this work, measuring intracellular pH (pHi) with a fluorescent probe (BCECF), membrane potential with a fluorescent lipophilic anion (bisoxonol), and net movement of acid using a pH-stat system, we have found that rat round spermatids regulate pHi by means of a V-type H(+)-ATPase, a HCO3- entry pathway, a Na+/HCO3- dependent transport system, and a putative proton conductive pathway. Rat spermatids do not have functional base extruder transport systems. These pH regulatory characteristics seem specially designed to withstand acid challenges, and can generate sustained alkalinization upon acid exit stimulation.

On stage single cell identification of rat spermatogenic cells.

The study of spermatogenic cell physiology has been hindered by the absence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histochemical techniques, digital videoimaging, quantification of chromatin-bound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages of spermatogenesis on which to perform single cell analyses: spermatogonia B/preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. The use of voltage-sensitive dyes and Ca(2+)-sensitive dyes does not interfere with the estimations of DNA content. The estimations of DNA content of spermatogenic cells can be performed both with near-UV excited dyes (H33342) and long wavelength-excited dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to study the spermatogenic process.

c-Jun is a downstream target for ceramide-activated protein phosphatase in A431 cells.

Stimulation of [3H]serine-labeled A431 cells with tumor necrosis factor-alpha (TNFalpha) or bacterial sphingomyelinase (SMase) resulted in a rapid decrease (approximately 50% by 15 min) in cellular [3H]sphingomyelin content and generation of the lipid moiety [3H]ceramide, which remained elevated 60 min later. Sphingomyelin hydrolysis in response to TNFalpha or bacterial SMase resulted in a time-dependent decrease in the phosphorylation state of c-Jun protein, an effect that was also observed in cells treated with the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-ceramide). The rapid dephosphorylation of the c-Jun gene product in response to TNFalpha, SMase, or C6-ceramide was not observed in A431 cells treated with the serine-threonine phosphatase inhibitor okadaic acid. After the initial steps of previously described methods for the purification of a ceramide-activated protein phosphatase termed CAPP (Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530), we obtained a cytosolic fraction from A431 cells that specifically dephosphorylated 32Pi-labeled c-Jun protein used as substrate in an immunocomplex phosphatase assay. Phosphatase activity in vitro was apparent only in the presence of ceramide (5 micro) and was specifically abrogated when okadaic acid (1 n) was included in the immunocomplex phosphatase assay. These results provide strong evidence for c-Jun as a downstream target for CAPP activated in response to post-TNF signaling in A431 cells.

Zinc transport in mammalian cells.

The importance of zinc in cell physiology is related mainly to its intracellular involvement in enzyme catalysis, protein structure, protein-protein interactions, and protein-oligonucleotide interactions. The mechanisms by which Zn2+ enters mammalian cells have been studied in a variety of cell systems. A review of this literature indicates that, in all cells, Zn2+ interacts with extracellular binding sites, which are likely to include binding sites involved in the subsequent translocation of this ion to the cell interior. Inside the cell, Zn2+ binds to cytosolic and organelle binding sites or is taken up by intracellular organelles. Despite these general conclusions, the mechanisms of the different transport and binding steps are, for most cell types, only partially solved. This review critically discusses the literature on mammalian Zn2+ transport and outlines some critical points for future research of the mechanisms of transport of this ion.

Ion dependence of resting membrane potential of rat spermatids.

The membrane potential of rat spermatids was estimated as -22 +/- 2 mV (mean +/- SEM) using three independent methods: using oxonol as a fluorescent membrane potential sensitive probe, from the passive distribution of hydrogen ions and from whole-cell patch-clamp records. The estimated permeability ratios PK+:PCl- and PNa+:PCl- of the plasma membrane of rat spermatids were 1.0 and 0.3, respectively. These data indicate that the high luminal K+ concentration found in seminiferous tubules could partially close voltage-sensitive calcium channels in these cells.

A fluorescence method to determine picomole amounts of Zn(II) in biological systems.

The formation of complexes between n-(6-methoxy-8-quinolyl)-p-toluensulfonamide (TSQ) and Zn(II) in methanol has been used as an analytical procedure for Zn(II) determination in biological systems. Using 1 ml cuvettes, the limit of detection of the method was approximately 20 pmoles of Zn(II). Linearity between fluorescence and zinc concentration was obtained up to approximately 1 microM Zn(II). Common multivalent cations present in biological systems like Al3+, Cu2+, Fe3+, Ca2+, and Mg2+, interfered with the measurement of Zn(II) only when present in excess of 20, 33, 60, 500 and 30,000 times the Zn (II) concentration, respectively. In human serum and semen, deproteinization of the samples permitted a good correlation between the TSQ method and the total Zn content determined by atomic absorption measurements. In rat spermatids, deproteinization and Zn (II) determination using the TSQ method gives approximately a 20% underestimation of the total Zn (II) content of the cells as compared to atomic absorption spectrophotometry. The method gives low resolution of Zn (II) peaks when tested as an analytical procedure to measure Zn (II) binding to protein fractions eluted during column chromatography.

Zn(II) transport and distribution in rat spermatids.

Zn(II) is an essential trace element. In spermatozoa, Zn(II) modulates metabolism and chromatin condensation. The mechanisms of uptake and distribution of this ion in sperm cells have not been explored. In rat spermatids, our results indicate that 1) 65Zn(II) binds with fast kinetics to a labile, presumably extracellular, compartment. This binding is temperature insensitive and not modified by metabolic inhibitors. 2) Entry of 65Zn(II) in the absence of externally added proteins occurs through a mediated transport system that allows exchange to reach steady state in approximately 15 min at 34 degrees C. 3) Upon entering the cells, 65Zn(II) binds tightly to cellular organelles. 4) Exchangeable Zn(II) bound to cytoplasmic proteins plus free intracellular Zn(II) appears to be < 20% of total exchangeable Zn(II). 5) The intracellular exchangeable Zn(II) compartment is decreased by metabolic inhibitors, showing a direct or indirect link between energy metabolism and cellular Zn(II) levels. 6) 65Zn(II) efflux from rat spermatids is a process with a rate constant of 0.16 +/- 0.13 min-1 at 34 degrees C. This exit rate of Zn(II) is likely to be affected by Zn(II) release from cytoplasmic binding sites or organelles.

Glycolytic component of rat spermatid energy and acid-base metabolism.

The impact of glycolysis on rat spermatid energy metabolism is made apparent by the simultaneous occurrence of the following three events upon glucose addition to the extracellular medium of a rat spermatid cell suspension: decrease in ATP content, exit of acid equivalents, and increased lactate production and efflux. In this work, we have studied the interrelations between these three phenomena. By measuring ATP content, net acid transport, lactate exit, oxygen consumption, intracellular pH, CO2 production, and glycolytic intermediates in the presence of glucose and glucose analogues, we conclude that 1) lactate production, decrease in ATP content, and acid equivalent exit are dependent on the metabolism of glucose up to different stages in glycolysis. 2) The decrease in ATP content is not directly related to the exit of acid equivalents from rat spermatids. 3) Glucose metabolism is a net ATP-consuming process at high intracellular ATP content but is a net ATP-producing process at low intracellular ATP concentration in rat spermatids. 4) Acid equivalent production arises from the metabolism of glucose beyond glyceraldehyde 3-phosphate dehydrogenase. 5) Lactic acid diffusion and/or lactate transport and CO2 production and exit could account for the glucose-dependent acid equivalent efflux in rat spermatids.