Ryanodine receptors (RyR), expressed within the ER or plasma membrane (PM), may contribute to regulated insulin secretion from pancreatic β-cells. Amplification by RyR of the Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals evoked by voltage-gated Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ channels may contribute to the second phase of insulin release, but the role of RyR in the plasma membrane is unresolved. My aim was to define the roles of RyR in pancreatic β-cells. I used INS-1E insulinoma cells, which have been reported to secrete insulin in response to glucose, to characterise the contribution of RyR to Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ entry and release. KCl-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ entry in INS-1E cells was mediated by L-type Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ channels. Both caffeine and carbachol evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ entry and release. Caffeine-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ entry was inhibited by ryanodine and nimodipine. This might be due to direct activation of RyR in the PM, or activation of other Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$-permeable channels in the PM secondary to Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ release via RyR. However, glucose did not evoke insulin secretion from INS-1E cells, and the Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals evoked by caffeine and carbachol became very variable with increasing passage number. It was therefore impossible with INS-1E cells to establish the role of RyR in glucose-evoked insulin release, and preliminary results with primary β-cells suggested that the quantitative analyses required to identify modest contributions from RyR would be impracticable.

Previous work established that in HEK cells expressing type 1 parathyroid hormone receptors (PTH$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$R), PTH(1-34) potentiated carbachol-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ release via a mechanism that required local delivery of cAMP to IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$ receptors (IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$R) within signalling junctions. My aim was to establish whether the interaction between adenylyl cyclase (AC) and IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$R persists during sustained stimulation with PTH, which has been reported to cause internalization of a functional PTH$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$R signalling pathway. In HEK cells expressing type 1 PTH$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$R, stimulation with PTH(1-34) for 1-60 min potentiated carbachol-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals. The potentiation was greater after acute (1 min) relative to sustained (60 min) stimulation. However, intracellular concentrations of cAMP were greater after sustained stimulation. Inhibition of protein kinase A (PKA) or exchange proteins activated by cAMP (EPAC) had no effect on the Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals evoked by carbachol after acute or sustained stimulation with PTH(1-34). Similar results were obtained with acute and sustained stimulation with NKH477 to activate AC or with a membrane-permeant analogue of cAMP, 8-Br-cAMP. Paired combinations of the three stimuli – PTH(1-34), NKH477 and 8-Br-cAMP – had the same maximal effect as each alone during acute and sustained treatments. Neither acute nor sustained treatment with PTH(1-34) alone affected the Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ content of the intracellular stores. These results indicate that sustained increases in cAMP reduce the extent to which cAMP potentiates carbachol-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ release. Analyses of responses to PTH analogues that differ in their abilities to evoke internalization of signalling pathways and/or activate phospholipase C versus AC confirmed that the effects of acute and sustained stimulation with PTH on Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signalling were mediated by cAMP and unlikely to require internalization of PTH$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$R.

Inhibition of AC with SQ22536 and 2′,5′-dideoxyadenosine reduced the amounts of cAMP produced by acute and sustained stimulation with PTH(1-34) by ~80%, but they had no effect on potentiation of carbachol-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals. Inhibition of cyclic nucleotide phosphodiesterases with IBMX had no effect on the Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals evoked by carbachol after acute stimulation with PTH(1-34). However, during sustained stimulation (60 min), when cAMP levels were increased ~15-fold by IBMX, carbachol-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ signals were ~10-fold more sensitive to PTH(1-34), although the maximal potentiation remained smaller than observed during acute stimulation. Using an IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$ biosensor to measure cytosolic IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$ demonstrated that sustained stimulation with PTH(1-34) attenuated the carbachol-evoked increase in cytosolic IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$ concentration. I conclude that acute and sustained responses to PTH(1-34) are mediated by local delivery of saturating concentrations of cAMP to IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$R within signalling junctions. Sustained increases in cAMP, via mechanisms that do not involve internalization of PTH$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$R or activation of PKA, diminish the effectiveness with which this local delivery of cAMP potentiates carbachol-evoked Ca$\mathrm{<mrow\; data-mjx-texclass="ORD">2+</mrow>}$ release, probably by diminishing carbachol-evoked IP$\mathrm{<mrow\; data-mjx-texclass="ORD">3</mrow>}$ formation.