Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.     

Evidence for a role of phosphatidylinositol turnover in stimulus–secretion coupling. Studies with rat peritoneal mast cells

Histamine secretion and phosphatidylinositol turnover were compared in antigen-sensitized rat peritoneal mast cells stimulated with a number of different ligands. A small and variable increase in the incorporation of [(32)P]P(i) and of [(3)H]inositol into phosphatidylinositol was observed when the cells were treated with immunoglobulin E-directed ligands (antigens and concanavalin A), and this was accompanied by a low amount of secretion (<10% of total cell histamine). In the presence of added phosphatidylserine, the addition of immunoglobulin E-directed ligands invariably led to an enhanced rate (approx. 4-fold) of labelling of phosphatidylinositol and, in the presence of Ca(2+), this was accompanied by the secretion of histamine. The labelling of phosphatidylinositol and histamine secretion were also stimulated by chymotrypsin and compound 48/80. Whereas the phosphatidylinositol response did not require the presence of extracellular Ca(2+), the secretion of histamine was either enhanced or dependent on extracellular Ca(2+) (depending on the ligand used). The dependence on ligand concentration for the phosphatidylinositol response and histamine secretion were similar. The increased isotopic incorporation into phosphatidylinositol continued for about 1h although histamine secretion (elicited with concanavalin A) stopped within 2min. These results support the proposition that metabolic events involving phosphatidylinositol play a necessary intermediate role in the regulation of Ca(2+) channels by ligand-activated receptors.     

The stimulus–secretion coupling of amino acid-induced insulin release. Insulinotropic action of l-alanine

Available information on the fate and insulinotropic action of l-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na⁺ co-transport in the insulinotropic action of l-alanine. In the present study conducted in islets prepared from normal adult rats, l-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-¹⁴C]palmitate, (iii) to stimulate ⁴⁵Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or l-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of l-[U-¹⁴C]alanine was unaffected by d-glucose, but inhibited by l-leucine. Inversely, l-alanine decreased the oxidation of d-[U-¹⁴C]glucose, but failed to affect l-[U-¹⁴C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of l-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na⁺ co-transport in the secretory response to l-alanine cannot be ignored.     

Regulation of mucin secretion and inflammation in asthma: a role for MARCKS protein?

BACKGROUND: A major characteristic of asthmatic airways is an increase in mucin (the glycoprotein component of mucus) producing and secreting cells, which leads to increased mucin release that further clogs constricted airways and contributes markedly to airway obstruction and, in the most severe cases, to status asthmaticus. Asthmatic airways show both a hyperplasia and metaplasia of goblet cells, mucin-producing cells in the epithelium; hyperplasia refers to enhanced numbers of goblet cells in larger airways, while metaplasia refers to the appearance of these cells in smaller airways where they normally are not seen. With the number of mucin-producing and secreting cells increased, there is a coincident hypersecretion of mucin which characterizes asthma. On a cellular level, a major regulator of airway mucin secretion in both in vitro and in vivo studies has been shown to be MARCKS (myristoylated alanine-rich C kinase substrate) protein, a ubiquitous substrate of protein kinase C (PKC). GENERAL SIGNIFICANCE: In this review, properties of MARCKS and how the protein may regulate mucin secretion at a cellular level will be discussed. In addition, the roles of MARCKS in airway inflammation related to both influx of inflammatory cells into the lung and release of granules containing inflammatory mediators by these cells will be explored. This article is part of a Special Issue entitled: Biochemistry of Asthma.     

43Ca nuclear magnetic resonance of Ca2+–calmodulin solutions: Effects of trifluoperazine and peptides

By using a conventional FT NMR spectrometer and probe, we first detected ⁴³Ca NMR spectra of the Ca²⁺ (2.9 mM): calmodulin (0.725 mM) (1:1 per binding site) complex in 0.15 M N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid (HEPES)–K⁺ buffer (pH 7.2). The half-band width of the complex was nearly 160 Hz and the signal of the complex was located at the 2.13 ppm (43 Hz) lower field from that of the free Ca²⁺ ion. By adding trifluoperazine, melittin, substance P or glucagon, the half-band widths of the Ca²⁺–calmodulin complex (1:1 per binding site) were remarkably reduced and the chemical shifts of the complex moved back to the upper field. It is suggested that the Ca²⁺ ion may bind to Ca²⁺ low-affinity sites more tightly in the presence of those effectors than in their absence.     

Calcium-dependent Pyk2 activation: a role for calmodulin?

Pyk2 (proline-rich tyrosine kinase 2) and FAK (focal adhesion kinase) are highly related tyrosine kinases. One distinguishing feature is the differential regulation of the two enzymes in response to elevation of cytoplasmic calcium. In the latest issue of the Biochemical Journal, Sasaki and co-workers have provided insight into the calcium-dependent regulation of Pyk2. The findings suggest that calmodulin may bind the FERM (4.1/ezrin/radixin/moesin) domain to promote Pyk2 activation in response to calcium signals triggered by vasopressin. While the molecular details of the protein-protein interaction and mechanism of activation remain to be firmly established, this study is the first to provide mechanistic insight into the regulation of Pyk2 by calcium.     

Distribution and role of calmodulin in tip growing hyphae of Saprolegnia ferax

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Is hypoxia a stimulus for synthesis of oxidative enzymes and myoglobin?

To compare two situations with similar magnitudes of mitochondrial substrate flux but different blood oxygen contents, one-legged training was employed. Ten healthy subjects trained one leg under normobaric conditions and the other under hypobaric conditions. At each session the subjects trained each leg for 30 min. The absolute work intensity was the same for both legs and was chosen to correspond to 65% of the average (right and left) pretraining one-legged maximal work capacity. There were three to four training sessions per week for 4 wk. Muscle biopsies from each leg were taken before and after training and analyzed for fiber types, capillaries, myoglobin, and oxidative and glycolytic enzymes. The most striking finding was a greater increase of citrate synthase activity under hypobaric conditions than under normobaric conditions. In addition, the myoglobin content increased in the leg trained under hypobaric conditions, whereas it tended to decrease in the normobarically trained leg. Because both legs were trained at the same intensity, the oxygen turnover and the substrate flux through the carboxylic acid cycle and the respiratory chain must have been of similar magnitude. Thus a difference in substrate flux is less likely to have caused the differences in enzyme activities and myoglobin content between training under normobaric and hypobaric conditions. Instead, the stimulus seems to be related to the blood oxygen content or tension.     

Autophagy: a broad role in unconventional protein secretion?

Autophagy, a cellular 'self-eating' process in eukaryotic cells, exists in both a basal and in an activated state that is induced in response to starvation. Basal and induced autophagy are associated with the packaging of cellular components, including damaged and/or redundant organelles, into double-membrane vesicles called autophagosomes, followed by autophagosome fusion with lysosomes, in which their contents are degraded and recycled. Recent results highlight a novel role for autophagy that does not involve lysosomal degradation of autophagosomal contents, but instead involves their redirection towards the extracellular delivery of an unconventionally secreted protein. Here, we discuss these findings, evaluate the strength of evidence, consider their implications for the field of protein trafficking, and suggest the next steps required to probe this interesting pathway.     

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca²⁺ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca²⁺ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca²⁺ transients. However, the amplitudes of caffeine-evoked Ca²⁺ transients and the rate of recovery of electrically evoked Ca²⁺ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca²⁺-uptake and Ca²⁺-ATPase activity in a biphasic manner. [³H]-ryanodine binding and passive Ca²⁺ release from SR vesicles were not altered by 10 μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 μg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.     

Endothelial regulation of calmodulin expression and eNOS–calmodulin interaction in vascular smooth muscle

Molecular and Cellular Biochemistry - Calmodulin (CaM) is a Ca2+ sensor protein that is required for numerous vascular smooth muscle cell (VSMC) functions. Since CaM is not expressed enough for its...     

Establishment of variant PC12 subclones deficient in stimulation–secretion coupling

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation–secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K⁺-induced depolarization, but normal release was evoked by the Ca²⁺-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca²⁺ influx was missing, suggesting that PC12-1G2 has defects in L-type Ca²⁺ channel function. PC12-2B3 was not responsive to high-K⁺-induced depolarization and ionomycin, and voltage-dependent Ca²⁺ entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.     

Incidence and mortality of pancreatic cancer on a rapid rise in Taiwan, 1999–2012

BackgroundAccumulating data has revealed a rapidly rising incidence of pancreatic cancer in Western countries, but convincing evidence from the East remains sparse. We aimed to quantify how the incidence and mortality rates of pancreatic malignancy changed over time in Taiwan, and to develop future projection for the next decade.MethodsThis nationwide population-based study analyzed the Taiwan National Cancer Registry and the National Cause of Death Registry to calculate the annual incidence and mortality rates of pancreatic malignancy from 1999 to 2012 in this country. The secular trend of the incidence was also examined by data from the National Health Insurance Research Database.ResultsA total of 21,986 incident cases of pancreatic cancer and 20,720 related deaths occurred during the study period. The age-standardized incidence rate increased from 3.7 per 100,000 in 1999 to 5.0 per 100,000 in 2012, with a significant rising trend (P < 0.01). The increase was nationwide, consistently across subgroups stratified by age, gender, geographic region, and urbanization. Data from the National Health Insurance Research Database corroborated the rise of incident pancreatic cancer. Mortality also increased with time, with the age-standardized rate rising from 3.5 per 100,000 in 1999 to 4.1 per 100,000 in 2012 (P < 0.01). In accordance with the incidence, the mortality trend was consistent in all subgroups. Both the incidence and mortality were projected to further increase by approximately 20% from 2012 to 2027.ConclusionThe incidence and mortality of pancreatic cancer have been rapidly rising and presumably will continue to rise in Taiwan.     

The role of autophagy in pancreatic β-cell and diabetes

Pancreatic β-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic β-cells, the mechanism underlying dynamic protein turnover in β-cells remains largely unknown. We found low-level constitutive autophagy in β-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. β-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with non-diabetic db/misty controls. The functional importance of autophagy was analyzed using β-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of β-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated β-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in β-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in β-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of β-cells. Autophagy also serves as a crucial element of stress responses to protect β-cells under insulin resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes.     

2-Oxocarboxylic acids and function of pancreatic islets in obese–hyperglycaemic mice. Insulin secretion in relation to 45Ca uptake and metabolism

The effects of aliphatic 2-oxocarboxylic acids, at concentrations of up to 40mm, on the function of pancreatic islets from ob/ob (obese–hyperglycaemic) mice were investigated. 1. 2-Oxopentanoate, dl-3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate and 2-oxohexanoate all induced insulin release by isolated incubated islets and a biphasic insulin-secretory pattern in perfused mouse pancreas. The last two substances were similar in potency to glucose. Pyruvate, 2-oxobutyrate, 3-methyl-2-oxobutyrate and 2-oxo-octanoate did not induce insulin release significantly. 2. 2-Oxocarboxylic acids with significant insulin-secretory potency also induced significant ⁴⁵Ca uptake by isolated incubated islets. 3. The rates of decarboxylation of [1-¹⁴C]pyruvate, 3-methyl-2-oxo[1-¹⁴C]butyrate and 4-methyl-2-oxo[1-¹⁴C]pentanoate were twice as high as the rates of oxidation of the corresponding U-¹⁴C-labelled compounds. However, whereas the rates of metabolism of labelled pyruvate and 3-methyl-2-oxobutyrate steadily increased over the concentration range 1–40mm, those of labelled 4-methyl-2-oxopentanoate and d-[U-¹⁴C]glucose levelled off at concentrations above 10mm. 4. Omission of ⁴⁰CaCl₂ from the incubation medium reduced the rate of oxidation of the insulin secretagogue [U-¹⁴C]4-methyl-2-oxopentanoate, but left that of the non-(insulin secretagogue) [U-¹⁴C]3-methyl-2-oxobutyrate unaffected. 5. Only glucose, and not pyruvate, 3-methyl-2-oxobutyrate and 4-methyl-2-oxopentanoate, significantly inhibited oxidation of endogenous fatty acids. 6. It is suggested that stimulus–secretion coupling and the resulting exocytosis of insulin in pancreatic β-cells may modulate both fuel oxidation and ⁴⁵Ca uptake.     

Trypanosomiasis and trypanotolerance in cattle: a role for congopain?

A Trypanosoma congolense cysteine protease (congopain) elicits a high IgG response in trypanotolerant but not in trypanosusceptible cattle during primary infections. As discussed here by Edith Authié, this observation suggests that congopain, like other parasite cysteine proteases, may play a role in pathogenicity and that more efficient immune responses to congopain may contribute to trypanotolerance.     

Effects of exercise training on excitation–contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation–contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation–contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10–11 months following 2–3 months of treadmill exercise training. Shortening, [Ca²⁺]_i and L-type Ca²⁺ current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca²⁺ transients and L-type Ca²⁺ current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca²⁺ transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.     

Resting stages in a submarine canyon: a component of shallow–deep-sea coupling?

The ecological importance of resting stages in shallow waters is well known, but their presence in the deep sea is practically unrecorded. Samples of sinking particles were collected from April 1993 to May 1994 in and around the Foix Canyon (northwest Mediterranean Sea) using PPS3 sediment traps located between –600 m and –1180 m. Dead and viable organisms were collected, and inorganic empty shells constituted most of the biologically-derived matter. Resting stages, considered as POM, had a flux of up to 70 000 items m^–2 d^–1. They were the second most abundant fraction of total POM after tintinnids (mainly represented by empty, chitinous loricas), and first of the viable POM fraction. Most remained unidentified, but 58 morphotypes were referable to coastal species of Dinophyta, Tintinnina and Calanoida. Resting stages were rare in samples collected from the open slope adjacent to the canyon. These preliminary data suggest an important role of submarine canyons in concentrating POM and transferring it from shallow to deep-sea habitats. Due to their resistance to degradation processes, resting stages are probably the only POM component that can return to shallow areas by upwelling currents occurring in the canyon.     

Hybridization and speciation in angiosperms: a role for pollinator shifts?

The majority of convincingly documented cases of hybridization in angiosperms has involved genetic introgression between the parental species or formation of a hybrid species with increased ploidy; however, homoploid (diploid) hybridization may be just as common. Recent studies, including one in BMC Evolutionary Biology, show that pollinator shifts can play a role in both mechanisms of hybrid speciation.