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Critical investigation into α-cell biology in health and diabetes has been sparse and at times inconsistent because of the technical difficulties with employing conventional strategies of isolated islets and dispersed single cells. An acute pancreas slice preparation was developed to overcome the enzymatic and mechanical perturbations inherent in conventional islet cell isolation procedures. This preparation preserves intra-islet cellular communication and islet architecture in their in situ native state. α-Cells within tissue slices were directly assessed by patch pipette and electrophysiologically characterized. The identity of the patched cells was confirmed by biocytin dye labelling and immunocytochemistry. α-Cells in mouse pancreas slices exhibited well-described features of I(Na) (excitable at physiological membrane potential), I(KATP), small cell size, low resting membrane conductance, and inducible low and high voltage-activated I(Ca), the latter correlating with exocytosis determined by capacitance measurements. In contrast to previous reports, our large unbiased sampling of α-cells revealed a wide range distribution of all of these parameters, including the amount of K(ATP) conductance, Na+ and Ca2+ current amplitudes, and capacitance changes induced by a train of depolarization pulses. The proposed pancreas slice preparation in combination with standard patch-clamping technique allowed large sampling and rapid assessment of α-cells, which revealed a wide distribution in α-cell ion channel properties. This specific feature explains the apparent inconsistency of previous reports on these α-cell ion channel properties. Our innovative approach will enable future studies into elucidating islet α-cell dysregulation occurring during diabetes.

Cyclic AMP regulates Ca2+-dependent exocytosis through a classical protein kinase A (PKA)-dependent and an alternative cAMP–guanine nucleotide exchange factor (GEF)/Epac-dependent pathway in many secretory cells. Although increased cAMP is believed to double secretory output in isolated pituitary cells, the direct target(s) for cAMP action and a detailed and high-time resolved analysis of the effect of intracellular cAMP levels on the secretory activity in melanotrophs are still lacking. We investigated the effect of 200 μm cAMP on the kinetics of secretory vesicle depletion in mouse melanotrophs from fresh pituitary tissue slices. The whole-cell patch-clamp technique was used to depolarize melanotrophs and increase the cytosolic Ca2+ concentration ([Ca2+]i). Exogenous cAMP elicited an about twofold increase in cumulative membrane capacitance change and ∼34% increase of high-voltage activated Ca2+ channel amplitude. cAMP-dependent mechanisms did not affect [Ca2+]i, since the application of forskolin failed to change [Ca2+]i in melanotrophs, a phenomenon readily observed in anterior lobe. Depolarization-induced secretion resulted in two distinct kinetic components: a linear and a threshold component, both stimulated by cAMP. The linear component (ATP-independent) probably represented the exocytosis of the release-ready vesicles, whereas the threshold component was assigned to the exocytosis of secretory vesicles that required ATP-dependent reaction(s) and > 800 nm[Ca2+]i. The linear component was modulated by 8-pCPT-2Me-cAMP (Epac agonist), while either H-89 (PKA inhibitor) or Rp-cAMPS (the competitive antagonist of cAMP binding to PKA) completely prevented the action of cAMP on the threshold component. In line with this, 6-Phe-cAMP, (PKA agonist), increased the threshold component. From our study, we suggest that the stimulation of cAMP production by application of oestrogen, as found in pregnant mice, increases the efficacy of the hormonal output through both PKA and cAMP–GEFII/Epac2-dependent mechanisms.

Cl− ions are known regulators of Ca2+-dependent secretory activity in many endocrine cells. The suggested mechanisms of Cl− action involve the modulation of GTP-binding proteins, voltage-activated calcium channels or maturation of secretory vesicles. We examined the role of cytosolic Cl− ([Cl−]i) and Cl− currents in the regulation of secretory activity in mouse melanotrophs from fresh pituitary tissue slices by using the whole-cell patch-clamp. We confirmed that elevated [Cl−]i augments Ca2−-dependent exocytosis and showed that Cl− acts on secretory vesicle maturation. The latter process was abolished by a V-type H−-ATPase blocker (bafilomycin), intracellular 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), a Cl− channel blocker, and tolbutamide, a sulphonylurea implicated in secretory vesicle maturation. In a small subset of cells, block of plasmalemmal Cl− current by DIDS reversibly enhanced endocytosis. The direct activation of G-proteins by GTP-γ-S, a non-hydrolysable GTP analogue, did not restore the impaired secretion observed in low [Cl−]i conditions. The amplitude of voltage-activated calcium currents was unaffected by the [Cl−]i. Furthermore, two Cl−-permeable channels, calcium-activated Cl− channels and GABAA receptors, appeared as major regulators of intracellular Cl− homeostasis. In conclusion, the predominant underlying mechanism of Cl− action is mediated by intracellular Cl− fluxes during vesicle maturation, rather than activation of G-proteins or modulation of voltage-activated Ca2+ channels.

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca2+ channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.