Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 μg/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 ��M), KN-93 (0.2 ��M) and cyclosporin A (CsA, 0.2 ��M). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKIIδB and CaN were evaluated by the Lowry method, [��H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 ��g/L (53.56 ��g protein/well) to 100 μg/L (72.18 ��g protein/well), and in a time-dependent manner from 12 h (37.42 ��g protein/well) to 72 h (42.81 ��g protein/well). TNF-α (100 μg/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [��H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 ��M BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [��H]-leucine incorporation were abolished by 0.2 ��M KN-93 or 0.2 ��M CsA. TNF-α increased the expression of CaMKIIδB by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 ��M BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca2+]i, CaMKIIδB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 ��M) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 ��M) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

Acute exposure to lead increases myocardial contractility independent of hypertension development

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 ��g/kg lead acetateiv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na+,K+-ATPase and myosin Ca2+-ATPase activities. Lead increased left- (control: 112 �� 3.7 vs lead: 129 �� 3.2 mmHg) and right-ventricular systolic pressures (control: 28 �� 1.2vs lead: 34 �� 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 ��M lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 �� 0.07 vs lead: 0.608 �� 0.06 g/mg) and time to peak tension decreased (control: 268 �� 13vs lead: 227 �� 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a &#946;-adrenergic agonist (10 ��M isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 ��M lead acetate presented increased Na+,K+-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (&#8764;115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 ��M). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61��0.26 vs NC=4.79��0.19 per 100 ��m/s) and decreased its amplitude (HC=0.260��0.08 vs NC=0.324��0.10 &#916;F/F0), full width at half-maximum amplitude (HC=1.05��0.08 vs NC=1.26��0.01 ��m), total duration (HC=11.51��0.12 vs NC=14.97��0.24 ms), time to peak (HC=4.84��0.06 vs NC=6.31��0.14 ms), and time constant of decay (HC=8.68��0.12 vs NC=10.21��0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26��0.19 ��m/s, amplitude=0.282��0.10 &#916;F/F0, full width at half-maximum amplitude=1.14��0.01 ��m, total duration=13.34��0.17 ms, time to peak=5.43��0.08 ms, and time constant of decay=9.43��0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.

Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable Paracoccidioides brasiliensis propagules

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6 )viable conidia (Group I), or 6.5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocott��s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.

DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.

Boosting the suppressive effects of Ascaris suum components in IFN-&#947;-deficient mice

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-&#947;-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-&#947;-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-&#947;-independent neutrophils.

Survey of sandfly vectors of leishmaniasis in Marambaia Island, municipality of Mangaratiba, State of Rio de Janeiro, Brazil

IntroductionThe influx and efflux of military personnel in the possible endemic areas of leishmaniasis provided the impetus for research on the sandflies on Marambaia Island.MethodsSandflies were collected with light traps installed in the 3 ecotypes of 3 areas chosen for their particular landscape aspects.ResultsIn 2009, were collected 32,006 specimens of sandflies belonging to 13 species. The species that showed highest density were Nyssomyia intermedia and Migoneimyia migonei.ConclusionsN. intermedia and M. migonei are the principal vectors of American tegumentary leishmaniasis (ATL) in Brazil; thus, extension studies related to the eco-epidemiology of these species of Marambaia Island are essential.

Malaria in the State of Amazonas: a typical Brazilian tropical disease influenced by waves of economic development

In Brazil, more than 99% of malaria cases are reported in the Amazon, and the State of Amazonas accounts for 40% of this total. However, the accumulated experience and challenges in controlling malaria in this region in recent decades have not been reported. Throughout the first economic cycle during the rubber boom (1879 to 1912), malaria was recorded in the entire state, with the highest incidence in the villages near the Madeira River in the Southern part of the State of Amazonas. In the 1970s, during the second economic development cycle, the economy turned to the industrial sector and demanded a large labor force, resulting in a large migratory influx to the capital Manaus. Over time, a gradual increase in malaria transmission was observed in peri-urban areas. In the 1990s, the stimulation of agroforestry, particularly fish farming, led to the formation of permanent Anopheline breeding sites and increased malaria in settlements. The estimation of environmental impacts and the planning of measures to mitigate them, as seen in the construction of the Coari-Manaus gas pipeline, proved effective. Considering the changes occurred since the Amsterdam Conference in 1992, disease control has been based on early diagnosis and treatment, but the development of parasites that are resistant to major antimalarial drugs in Brazilian Amazon has posed a new challenge. Despite the decreased lethality and the gradual decrease in the number of malaria cases, disease elimination, which should be associated with government programs for economic development in the region, continues to be a challenge.

Varia����o temporal de nutrientes na ��gua escorrida pelo caule em floresta prim��ria explorada no nordeste do Par��

Foi enfocada a contribui&#231;&#227;o de nutrientes da &#225;gua escorrida pelo caule (CE), na ciclagem hidroqu&#237;mica em floresta prim&#225;ria explorada, em Benevides, PA. A concentra&#231;&#227;o de nutrientes na CE foi avaliada de dezembro de 1993 a abril de 1995, mediante coletores tipo colarinho, constru&#237;dos com espuma de silicone, acoplados por tubos a recipientes pl&#225;sticos. O monitoramento de CE se restringiu a doze &#225;rvores, nas quais foram coletadas mensalmente amostras para an&#225;lise qu&#237;mica de K+, Na+, Ca2+ e Mg2+ por espectrofotometria de absor&#231;&#227;o at&#244;mica, o N-NH4+, N-NO3 - e P-PO4 3- por colorimetria, em espectofot&#244;metro de fluxo cont&#237;nuo, o N-total por micro Kjeldahl e pH por potenciometria. A quantidade de nutrientes trazidos pela CE foi maior no in&#237;cio da &#233;poca chuvosa, exibindo marcante variabilidade temporal para K+, Na+, Ca2+, Mg2+, N-total, SO4 2-, enquanto que o oposto aconteceu com PO4 3-. A magnitude na concentra&#231;&#227;o dos nutrientes decresceu na seguinte ordem: K+ > Na+ > Ca2+ > N-t > SO4 2- > Mg2+ > PO4 3-. A distribui&#231;&#227;o e a intensidade de chuva n&#227;o influenciam marcantemente o pH na CE.

Contribui����o �� hidroqu��mica de drenagens no Munic��pio de Manaus - AM

O rio Puraquequara, e seu afluente direito, o igarap�� ��gua Branca, localizam-se fora da zona urbana de Manaus e est��o com a bacia ainda protegida por floresta prim��ria. T��m caracter��sticas geol��gicas, pedol��gicas e climatol��gicas de igarap��s naturais de terra-firme da Amaz��nia Central. Foram coletadas amostras de ��gua nos meses de novembro de 1998 (per��odo seco) e abril de 1999 (per��odo chuvoso) e determinados os par��metros temperatura, pH, turbidez, condutividade, alcalinidade, dureza, DQO, Ca2+, Mg2+, Na+, K+, SiO2, NO3-, NO2-, PO4(3-), SO4(2-), Cl-, NH4+ e Fe total. As ��guas apresentaram pH entre 3,8 e 4,1 passando a menos ��cidas na estiagem. A alcalinidade e a turbidez s��o, em geral, mais elevadas na estiagem, enquanto a condutividade, dureza e DQO s��o maiores no per��odo mais chuvoso. SiO2 e o Cl- foram os ��nions mais abundantes, com contribui����o maior do primeiro no Puraquequara e menor no ��gua Branca, enquanto o Cl- tem comportamento oposto. Na+, Fe total e NH4+ s��o os c��tions mais abundantes e predominam, no geral, no per��odo mais seco. O NH4+ �� o ��nico c��tion que tende a aumentar sua contribui����o para jusante do igarap�� no per��odo ��mido. Os teores de K+ s��o mais elevados do que os de Mg2+ e este que os de Ca2+, e s��o todos superados pelo Na+. Os teores da NH4+ e NO3-, acima de 0,2 mg/L e 0,5 mg/L, respectivamente, s��o ind��cios de contamina����o. Essas caracter��sticas definem as ��guas da bacia como muito dilu��das, com predomin��ncia dos ��nions sobre os c��tions e correlacion��veis as ��guas de cor preta.