Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+) handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c), sarcolemmal Na+/Ca2+ exchanger (NCX), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), ryanodine receptor (RyR2), and phospholamban (PLB) mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control) or high-fat diet (obese) for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05) but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

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 &#946;2-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 &#946;2- and &#946;3-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.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-&#945;-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 &#945; (TNF-&#945;). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-&#945; (100 &#956;g/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, CaMKII&#948;B 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-&#945; induced a significant increase in protein content in a dose-dependent manner from 10 ��g/L (53.56 ��g protein/well) to 100 &#956;g/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-&#945; (100 &#956;g/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-&#945; increased the expression of CaMKII&#948;B 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-&#945; induces increases in [Ca2+]i, CaMKII&#948;B 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-&#945;.

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.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

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.

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.

Nutrientes na solu����o do solo em floresta de terra firme na Amaz��nia Central submetida �� extra����o seletiva de madeira

Os solos de terra firme da Amaz��nia Central, na sua maioria, s��o ��cidos, pobres em nutrientes e a manuten����o da floresta sobre esses solos �� garantida pela ciclagem de nutrientes, praticamente fechada. A substitui����o de floresta por pastagens ou outras atividades agr��colas leva �� diminui����o de nutrientes do compartimento biomassa, podendo comprometer os processos de ciclagem no solo, pois plantas absorvem nutrientes que presentes na solu����o do solo. Para entender o efeito de retirada de ��rvores, foi realizado um estudo em uma ��rea de floresta de terra firme na Amaz��nia Central submetida �� extra����o seletiva de madeira (6-10 ��rvores, ou 34 m�� ha-1 de madeira) localizada 80 km ao norte de Manaus, foram determinados os teores NO3-, NH4+, K+, Ca2+, Mg2+ e Na+ na solu����o do solo na camada de 0-30 cm. O experimento constou de tr��s blocos, cada um contendo uma parcela controle e uma que sofreu o corte seletivo de ��rvores, todos sobre um Latossolo Amarelo ��lico de textura muito argilosa. As medidas foram realizadas durante 13 meses, em cinco tratamentos em cada bloco: controle (floresta intacta), centro de clareira, borda de clareira, borda da floresta remanescente e floresta remanescente. Os teores de pot��ssio, c��lcio, magn��sio e s��dio mostraram diferen��as significativas entre os tratamentos. As quantidades dos ��ons am��nio e nitrato foram as menos afetadas. Os valores mais elevados foram geralmente encontrados nos tratamentos centro de clareira e borda de clareira. A maior diferen��a ocorreu na quantidade de s��dio na solu����o do solo, que chegou a mais de 5 kg ha-1, no centro de clareira de dois blocos, praticamente o dobro da encontrada nas suas respectivas parcelas controles. As concentra����es mais baixas dos nutrientes na solu����o do solo da floresta intacta (controle) e da floresta remanescente, confirmam a efici��ncia da floresta na ciclagem de nutrientes. Por��m, no centro de clareira, al��m da remo����o de ��rvores, a disponibilidade de materiais de f��cil decomposi����o, como ra��zes mortas e a liteira acumulada, podem ter contribu��do para uma maior concentra����o de nutrientes na solu����o do solo.

Efeitos de diferentes usos da terra sobre as caracter��sticas qu��micas de um latossolo amarelo do estado do Par��

Estudou-se o efeito de diferentes tipos de cobertura vegetal e manejo sobre algumas caracter��sticas qu��micas de um Latossolo Amarelo, textura m��dia, da Esta����o Experimental "Jos�� Haroldo", da CEPLAC, Marituba-PA. Foram amostradas parcelas sob floresta secund��ria (FS) explorada seletivamente, de aproximadamente 40 anos; de derruba e queima (DQ) da mesma floresta secund��ria; sob cultura de cacau (CC); e, sob ��rea de pastagem abandonada (PA). Em cada ��rea estudada foram abertos tr��s perfis de 2 m de profundidade, coletando-se amostras de solo dos horizontes Ap, AB, BA e B21, totalizando 48 amostras compostas, que foram analisadas para pH, carbono org��nico, nitrog��nio total (N), f��sforo dispon��vel (P) e bases troc��veis (K+, Ca2+, Mg2+ e Na+). Foram utilizadas an��lises de regress��o para verificar as correla����es existentes entre pH, nitrog��nio total (N), f��sforo dispon��vel (P), mat��ria org��nica (MO) e soma de bases (SB). Houve aumento no pH, na soma de bases e no f��sforo dispon��vel e diminui����o no conte��do de mat��ria org��nica em ��rea de derruba e queima; aumento no conte��do de mat��ria org��nica em ��rea de pastagem abandonada; aumento do conte��do de P do solo em ��rea de cultivo de cacau. N��o houve efeito do tipo de cobertura vegetal no N-total do solo. Houve correla����es positivas entre o pH x mat��ria org��nica do solo e desta com os conte��dos de N e P do solo.

Influ��ncia de um aterro sanit��rio e de efluentes dom��sticos nas ��guas superficiais do rio Tarum�� e afluentes - AM

O rio Tarum�� e seus afluentes, os igarap��s Matrinch�� e da Bol��via (Manaus-AM) est��o impactados por res��duos provenientes de um aterro sanit��rio e secundariamente por esgotos dom��sticos. Com o intuito de determinar as conseq����ncias desse impacto foram estudadas as caracter��sticas f��sicas e qu��micas e quantificados os metais potencialmente t��xicos nas ��guas em tr��s per��odos pluviom��tricos. Foi constatado que h�� altera����es consider��veis nas caracter��sticas qu��micas, especialmente em NH4+, Ni, Pb, Cu e Zn, quando comparados ��s ��guas naturais da regi��o. A condutividade, dureza, alcalinidade, PO4(3-), NH4+, K+, Na+, Ca2+, Fe3+ e Mg+ que foram os ��ndices que melhor qualificaram as ��guas estudadas, indicam que sua qualidade tende a ser um pouco melhor no in��cio das chuvas no igarap�� da Bol��via, mas n��o h�� diferen��as marcantes �� sua montante. Essas varia����es mostram que o per��odo de amostragem afeta as caracter��sticas da ��gua, mas n��o por igual e que apesar da tend��ncia de dilui����o ser maior no final do per��odo chuvoso, n��o h�� significativa melhora na qualidade da ��gua nesse per��odo. N��o foram constatadas varia����es qu��micas na composi����o das ��guas em fun����o da contamina����o ser proveniente do aterro ou diretamente dos esgotos.