Yi-Jia Liu

Origin of slow and fast oscillations of Ca2+ in mouse pancreatic islets.

1 Pancreatic islets exposed to 11 mM glucose exhibited complex variations of cytoplasmic Ca2+ concentration ([Ca2+]i) with slow (0.3‐0.9 min−1) or fast (2‐7 min−1) oscillations or with a mixed pattern. 2 Using digital imaging and confocal microscopy we demonstrated that the mixed pattern with slow and superimposed fast oscillations was due to separate cell populations with the respective responses. 3 In islets with mixed [Ca2+]i oscillations, exposure to the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (DTBHQ) resulted in a selective disappearance of the fast pattern and amplification of the slow pattern. 4 In addition, the protein kinase A inhibitor RP‐cyclic adenosine 3′,5′‐monophosphorothioate sodium salt transformed the mixed [Ca2+]i oscillations into slow oscillations with larger amplitude. 5 Islets exhibiting only slow oscillations reacted to low concentrations of glucagon with induction of the fast or the mixed pattern. In this case the fast oscillations were also counteracted by DTBHQ. 6 The spontaneously occurring fast oscillations seemed to require the presence of cAMP‐elevating glucagon, since they were more common in large islets and suppressed during culture. 7 Image analysis revealed [Ca2+]i spikes occurring irregularly in time and space within an islet. These spikes were preferentially observed together with fast [Ca2+]i oscillations, and they became more common after exposure to glucagon. 8 Both the slow and fast oscillations of [Ca2+]i in pancreatic islets rely on periodic entry of Ca2+. However, the fast oscillations also depend in some way on paracrine factors promoting mobilization of Ca2+ from intracellular stores. It is proposed that such a mobilization in different cells within a tightly coupled islet syncytium generates spikes which co‐ordinate the regular bursts of action potentials underlying the fast oscillations.

Storemoperated Ca2+ entry in insulin-releasing pancreatic β-cells

Abstract The fluorescent indicator Fura-2 was used to characterize the store-operated Ca2+ entry in insulin-releasing pancreatic β-cells. To avoid interference with voltage-dependent Ca2+ entry, the cells were hyperpolarized with 400 μM diazoxide and the channel blocker methoxyverapamil was also present in some experiments. The cytoplasmic Ca2+ concentration ([Ca2+]i) of hyperpolarized mouse β-cells was strikingly resistant to changes in external Ca2+. In cells exposed to 20 mM glucose, stimulation with 100 μM carbachol induced an initial [Ca2+]i peak followed by a sustained increase due to store-operated influx of the cation. Store-operated influx was also induced by the intracellular Ca2+-ATPase inhibitor thapsigargin. In the presence of store-operated influx, [Ca2+]i became markedly sensitive to variations in external Ca2+, but this sensitivity was blocked by La3+. In β-cells exposed to both Ca2+ and Mn2+ there was slow Mn2+ quenching of the Fura-2 fluorescence, which was accelerated upon stimulation of store-operated influx. This acceleration was reversed by glucose-stimulated filling of the internal Ca2+ stores. The store-operated Ca2+ entry increased markedly during culture of the β-cells. Activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13 acetate, inhibition of serine/threonine phosphatase by okadaic acid and inhibition of tyrosine kinase by genistein had little effect on the store-operated influx of Ca2+. In β-cells equilibrated in 5 mM Sr2+, carbachol exposure resulted in a pronounced cytoplasmic Sr2+ ([Sr2+]i) peak due to intracellular mobilization, but little or no sustained elevation. Moreover, after activating the store-operated pathway by exposure to thapsigargin, variations in extracellular Sr2+ between 0–20 mM had only marginal effects on [Sr2+]i. Although the store-operated influx apparently accounts for a minor fraction of the Ca2+ entry, its depolarizing influence may under certain conditions be up-regulated with resulting distortion of the β-cell function.

Involvement of α1 and β-adrenoceptors in adrenaline stimulation of the glucagon-secreting mouse α-cell

Stimulation of glucagon release and inhibition of insulin secretion from the islets of Langerhans are important for the blood-glucose-elevating effect of adrenaline. The mechanisms by which adrenaline accomplishes these actions may involve direct effects and indirect ones mediated by altered release of other islet hormones. In the present study we investigated how adrenaline affects the cytoplasmic Ca2+ concentration, which controls glucagon secretion from the pancreatic α-cell. The studies were performed on isolated mouse α-cells, which were identified by immunocytochemistry.The adrenaline effects consisted of initial mobilisation of intracellular Ca2+, accompanied by voltage-dependent influx of the ion. Part of the effect could be attributed to β-adrenoceptor activation, as it was mimicked by the rise in cAMP and inhibited by the antagonist propranolol as well as the protein kinase A inhibitor adenosine 3’,5’-cyclic monophosphorothioate Rp-isomer. α1-Adrenoceptors were also involved, since the antagonists phentolamine and prazosin completely abolished the effects of adrenaline. Experiments with clonidine and yohimbine gave little evidence of a role of α2-adrenoceptors. The results indicate that α1- and β-adrenoceptors on the α-cells mediate adrenaline-stimulated glucagon secretion. The complete inhibition of the adrenaline response after blocking α1-adrenoceptors indicates an interaction with the β-adrenergic pathway.

Oxytocin-induced oscillations of cytoplasmic Ca2+ in human myometrial cells.

Background. To investigate the mechanisms of oxytocin (OT) induced oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) in cultured human myometrial cells.

Ca2+ Signaling in Mouse Pancreatic Polypeptide Cells

Ca2+signaling was studied in pancreatic polypeptide (PP)-secreting cells isolated from mouse islets of Langerhans. After measuring the cytoplasmic Ca2+ concentration ([Ca2+]i), the cells were identified by immunocytochemistry. Most PP-cells reacted to carbachol and epinephrine with prompt and reversible elevation of[ Ca2+]i, often manifested as slow oscillations. The carbachol effect was muscarinic, because it was inhibited by atropine. β-adrenergic elevation of cAMP explains the epinephrine stimulation, which was mimicked by an activator of adenylate cyclase and blocked by an inhibitor of protein kinase A. The responses to carbachol and epinephrine apparently involve depolarization with opening of voltage-dependent Ca2+ channels, because the effects were prevented by the Ca2+ channel antagonist methoxyverapamil and by diazoxide, which activates ATP-dependent K+ (KATP) channels. Being equipped with KATP channels, the PP-cells often responded to tolbutamide or high concentrations of glucose with elevation ...

Oscillatory Ca2+ signaling in somatostatin-producing cells from the human pancreas

Oscillatory Ca2+ signaling was studied in human somatostatin-releasing pancreatic delta cells identified by immunostaining. A ratiometric fura-2 technique was used for measuring cytoplasmic concentrations of Ca2+ and Sr2+ in delta cells exposed to the respective cation. Rhythmic activity in terms of slow (frequency, 0.1 to 0.4 per minute) oscillations from close to the basal level was seen in the presence of 3 to 20 mmol/L glucose during superfusion with medium containing 2.6 to 5 mmol/L Ca2+ or 5 mmol/L Sr2. These oscillations could be transformed into a sustained increase by decreasing extracellular Ca2+ or adding 1 mmol/L tolbutamide or 20 nmol/L glucagon. Addition of glucagon to a medium containing 20 mmol/L glucose resulted in the generation of short (< 30 seconds) transients, which disappeared upon exposure to 100 nmol/L of the intracellular Ca(2+)-adenosine triphosphatase (ATPase) inhibitor thapsigargin. When analyzing small aggregates of islet cells, it became evident that oscillatory activity in delta cells can be synchronous with that in adjacent non-delta cells. It is concluded that secretion of pancreatic somatostatin in man involves Ca2+ signaling similar to that regulating the pulsatile release of insulin.

Calcium sensing by human medullary thyroid carcinoma cells

Regulation of the cytoplasmic calcium concentration ([Ca2+]i) was studied in fura‐2‐loaded C‐cells from two human medullary thyroid carcinomas (MTC). K+ depolarization induced sustained rise of [Ca2+]i reversed by verapamil. Elevation of external Ca2+ from 0.5 to 3.0 mM triggered regular oscillations or steady‐state increases of [Ca2+]i. In Ca2+‐deficient medium Sr2+ caused steady‐state increase or oscillations of the 340/380 nm fluorescence ratio. The Ca2+ and Sr2+ actions were partially reversible by verapamil. La3+ and Ce3+ elicited transient [Ca2+]i peaks independent of external Ca+2+, but no oscillations. The results indicate that human MTC cells express a parathyroid‐like Ca2+ sensor coupled to intracellular mobilization and influx of Ca2+. A voltage‐dependent Ca2+ influx may be of importance for the oscillations of [Ca2+]i.

OSCILLATORY SIGNALING AND INSULIN RELEASE IN HUMAN PANCREATIC BETA -CELLS EXPOSED TO STRONTIUM

Oscillatory signaling and insulin release were studied in isolated pancreatic islets and β-cells obtained from human cadaveric organ donors. Taking advantage of Sr2+ as an analog for Ca2+, it was possible to demonstrate glucose-induced rhythmic activity in individual β-cells identified by immunostaining. Glucose-induced slow oscillations of Sr2+ (frequency, 0.1–1.0/min) were sometimes seen at a sugar concentration as low as 3 mm. Addition of 20 nm glucagon resulted in a broadening of the oscillations or in their transformation into sustained elevation. Moreover, the presence of glucagon resulted in the appearance of short transients of Sr2+, which disappeared after exposure to the intracellular Ca2+-adenosine triphosphatase inhibitor thapsigargin. Digital image analyses indicated that slow oscillations can be synchronized among cells in small aggregates and intact islets. The rhythmic activity in the glucose-stimulated β-cell had its counterpart in pulsatile insulin release when single islets were perifus...