High-resolution analysis of the cytosolic Ca²⁺ events in β cell collectives in situ

Abstract

The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca2+ concentration ([Ca2+]c). To trigger exocytosis, Ca2+ ions enter the cytosol from intracellular Ca2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca2+]c, were extensively described in isolated single cells from various endocrine glands. Notably less work has been done on endocrine cells in situ to address the heterogeneity of [Ca2+]c events contributing to a collective functional response of a gland. For this beta cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca2+]c dynamics can be simultaneously assessed on both cellular and collective level. Here we measured [Ca2+]c transients across all relevant timescales, from a sub-second to a minute time range, using high-resolution imaging with low-affinity Ca2+ sensor. We quantified the recordings with a novel computational framework for semi-automatic image segmentation and [Ca2+]c event identification. Our results demonstrate that under physiological conditions the duration of [Ca2+]c events is variable, and segregated into 3 reproducible modes, sub-second, second and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in beta cell collectives, and that a subset of [Ca2+]c events could be triggered even in the absence of Ca2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca2+ receptors in shaping the heterogeneity of [Ca2+]c responses in collectives of endocrine cells in situ.

Exendin-4 affects calcium signalling predominantly during activation and activity of beta cell networks in acute mouse pancreas tissue slices

“…Our current study is principally descriptive in nature and was intended to systematically screen for and quantitatively characterize the effects during the different phases of beta cell activity and the mechanisms behind these effects remain to be investigated into detail in future studies. However, among the different possible mechanisms promoting beta cell function during activation and activity in the presence of GLP-1RAs, a plausible contribution to the enhanced [Ca 2+ ] IC dynamics observed in the present study likely stems from potentiating effects on L-type voltage-dependent Ca 2+ channels ( 45 , 52 , 116 , 124 ), together with activation of Ca 2+ induced Ca 2+ release from intracellular stores ( 51 , 52 , 125 – 127 ). However, evidence also exists that activation of hyperpolarization-activated cyclic nucleotide-gated channels by GLP-1RAs plays a role in augmenting beta cell [Ca 2+ ] oscillations ( 2 , 123 ).…”

The effect of forskolin and the role of Epac2A during activation, activity, and deactivation of beta cell networks

“…Next, the plateau phase of response after stimulation with high glucose consists of repetitive fast [Ca 2+ ] IC oscillations that reflect bursts of membrane potential depolarizations ( 46 , 97 , 104 , 105 ) or Ca 2+ release from intracellular Ca 2+ stores ( 106 ). Epac2A ablation increased the beta cell active time to a minor extent ( Figure 3 ) that is most probably biologically irrelevant, since the Epac2 KO animals display normal insulin and glucose levels, as discussed above.…”

The MODY-associatedKCNK16L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults

“…Another possibility is that because TALK-1 channels are functional not only on the plasma membrane but also the ER membrane (27), some of the TALK-1 L114P-mediated reduction in glucose-stimulated [Ca 2+ ] c influx could be due to altered [Ca 2+ ] ER handling. Indeed, glucose-stimulated [Ca 2+ ] c influx has previously been shown to be dependent in part on [Ca 2+ ] ER release and TALK-1 L114P increases β-cell [Ca 2+ ] ER release in response to muscarinic stimulation (56). Thus, future studies are required to determine the contributions of ER membrane localized versus plasma membrane localized TALK-1 L114P to altered glucose-stimulated [Ca 2+ ] c influx.…”