Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on Ca-45 efflux, cytoplasmic Ca2+ concentration [Ca2+](i), and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the Ca-45 efflux from isolated islets in a dose-dependant manner. Tetracaine did not affect the increase in Ca-45 efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+](i) in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 mu M D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 mu M thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in Ca-45 efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulurn Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PR,L) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to nonpregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (U). The expression of the sarcoendoplasmic reticulum Ca2+-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in P-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of PI 9 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

Food restriction promotes downregulation of myocardial L-type Ca2+ channels

Food restriction (FR) has been shown to impair myocardial performance. However, the mechanisms behind these changes in myocardial function due to FR remain unknown. Since myocardial L-type Ca2+ channels may contribute to the cardiac dysfunction, we examined the influence of FR on L-type Ca2+ channels. Male 60-day-old Wistar rats were fed a control or a restricted diet (daily intake reduced to 50% of the amount of food consumed by the control group) for 90 days. Myocardial performance was evaluated in isolated left ventricular papillary muscles. The function of myocardial L-type Ca2+ channels was determined by using a pharmacological Ca2+ channel blocker, and changes in the number of channels were evaluated by mRNA and protein expression. FR decreased final body weights, as well as weights of the left and right ventricles. The Ca2+ channel blocker diltiazem promoted a higher blockade on developed tension in FR groups than in controls. The protein content of L-type Ca2+ channels was significantly diminished in FR rats, whereas the mRNA expression was similar between groups. These results suggest that the myocardial dysfunction observed in previous studies with FR animals could be caused by downregulation of L-type Ca2+ channels.

Exercise Training and Caloric Restriction Prevent Reduction in Cardiac Ca2+-Handling Protein Profile in Obese Rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Obesity induces upregulation of genes involved in myocardial Ca2+ handling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison between apo and complexed structures of bothropstoxin-I reveals the role of Lys122 and Ca2+-binding loop region for the catalytically inactive Lys49-PLA(2)s

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystal structure of a myotoxic Asp49-phospholipase A(2) with low catalytic activity: Insights into Ca2+ -independent catalytic mechanism

A myotoxic Asp49-phospholipase A(2) (Asp49-PLA(2)) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA(2)s. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA(2) from B. jararacussu) and other Asp49-PLA(2)s. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA(2)s, making a hydrogen bond with the atom 0 delta 2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA(2)s which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA(2)s indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA(2)s. (c) 2008 Elsevier B.V All rights reserved.

Insights into metal ion binding in phospholipases A(2): ultra high-resolution crystal structures of an acidic phospholipase A(2) in the Ca2+ free and bound states

The electrophile Ca2+ is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca2+ free and bound states at 0.97 and 1.60 angstrom resolutions, respectively. In the Ca2+ bound state, the Ca2+ ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca2+, a water molecule occupies the position of the Ca2+ ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation. (c) 2005 Elsevier SAS. All rights reserved.

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A(2) promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca2+-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca2+ ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)S), substitution of the Asp49 by Lys results in loss of Ca2+ binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA2S cause Ca2+-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca2+-independent membrane damaging activity by similar to4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accomparlied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s. (C) 2004 Published by Elsevier B.V.

Exercício e restrição alimentar aumentam o RNAm de proteínas do trânsito de Ca2+ miocárdico em ratos

FUNDAMENTO: Treinamento físico (TF) aumenta a sensibilidade dos hormônios tireoidianos (HT) e a expressão gênica de estruturas moleculares envolvidas no movimento intracelular de cálcio do miocárdio, enquanto a restrição alimentar (RIA) promove efeitos contrários ao TF. OBJETIVO: Avaliar os efeitos da associação TF e RIA sobre os níveis plasmáticos dos HT e a produção de mRNA dos receptores HT e estruturas moleculares do movimento de cálcio do miocárdio de ratos. MÉTODOS: Utilizaram-se ratos Wistar Kyoto divididos em: controle (C, n = 7), RIA (R50, n = 7), exercício físico (EX, n = 7) e exercício físico + RIA (EX50, n = 7). A RIA foi de 50% e o TF foi natação (1 hora/dia, cinco sessões/semana, 12 semanas consecutivas). Avaliaram-se as concentrações séricas de triiodotironina (T3), tiroxina (T4) e hormônio tireotrófico (TSH). O mRNA da bomba de cálcio do retículo sarcoplasmático (SERCA2a), fosfolamban (PLB), trocador Na+/Ca+2 (NCX), canal lento de cálcio (canal-L), rianodina (RYR), calsequestrina (CQS) e receptor de HT (TRα1 e TRβ1) do miocárdio foram avaliados por reação em cadeia da polimerase (PCR) em tempo real. RESULTADOS: RIA reduziu o T4, TSH e mRNA do TRα1 e aumentou a expressão da PLB, NCX e canal-L. TF aumentou a expressão do TRβ1, canal-L e NCX. A associação TF e RIA reduziu T4 e TSH e aumentou o mRNA do TRβ1, SERCA2a, NCX, PLB e correlação do TRβ1 com a CQS e NCX. CONCLUSÃO: Associação TF e RIA aumentou o mRNA das estruturas moleculares cálcio transiente, porém o eixo HT-receptor não parece participar da transcrição gênica dessas estruturas.

beta(2)-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta(2)-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours.2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective beta(2)-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP.3. The decrease in the rate of total protein degradation induced by 10(-5) M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5) M ICI 118.551, a selective beta(2)-antagonist.4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4) M dibutyryl-cAMP or isobutylmethylxanthine.5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.

Crytallization and high-resolution X-ray diffraction data collection of an Asp49 PLA(2) from Bothrops jararacussu venom both in the presence and absence of Ca2+ ions

Snake venom PLA(2)s have been extensively studied due to their role in mediating and disrupting physiological processes such as coagulation, platelet aggregation and myotoxicity. The Ca2+ ion bound to the putative calcium-binding loop is essential for hydrolytic activity. We report the crystallization in the presence and absence of Ca2+ and X-ray diffraction data collection at 1.60 Angstrom (with Ca2+) and 1.36 Angstrom (without Ca2+) of an Asp49 PLA(2) from Bothrops jararacussu venom. The crystals belong to orthorhombic space group C222(1). Initial refinement and electron density analysis indicate significant conformational. changes upon Ca2+ binding. (C) 2004 Elsevier B.V. All fights reserved.

INGAP-PP up-regulates the expression of genes and proteins related to K-ATP(+) channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K-ATP(+) channel, Ca2+ handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca2+ ([Ca2+](i)), static and dynamic insulin secretion, and Rb-86 efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 mu M tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC50 of 10.0 +/- 0.4 vs. 13.7 +/- 1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 mu M tolbutamide and accelerated the velocity of glucose-induced reduction of Rb-86 efflux in perifused islets. These effects were accompanied by a significant increase in [Ca2+](i) and the maintenance of a considerable degree of [Ca2+](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K-ATP(+) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca2+](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes. (C) 2008 Elsevier B.V. All rights reserved.

The Effects of N Helix Deletion and Mutant F29W on the Ca2+ Binding and Functional Properties of Chicken Skeletal Muscle Troponin C

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L.B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (±Ca2+) of F29W and F29W/ NHdel and titration of the Ca2+-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca2+-activated enzymic activities. These effects are cumulative in the double mutant F29W/ NHdel. On the other hand, maximal isometric tension development in Ca2+-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.

Honey bee lines selected for high propolis production also have superior hygienic behavior and increased honey and pollen stores

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)