Hans-Peter Bode

Glucagon-Like Peptide 1 Elevates Cytosolic Calcium in Pancreaticβ -Cells Independently of Protein Kinase A1

Glucagon-like peptide 1 (7–36)amide (GLP-1) is an insulinotropic intestinal peptide hormone with a potential role as antidiabetogenic therapeutic agent. It mediates a potentiation of glucose-induced insulin secretion, by activation of adenylate cyclase and subsequent elevation of cytosolic free calcium,[ Ca2+]cyt. We investigated the role of protein kinase A (PKA) in GLP-1 signal transduction, using isolated mouse islets as well as the differentiated β-cell line INS-1. Two specific inhibitors of PKA, (Rp)-adenosine cyclic 3′,5′-phosporothioate (Rp-cAMPS, up to 3 mm) and KT5720 (up to 10 μm), did not inhibit the GLP-1-induced[ Ca2+]cyt elevation. Another PKA inhibitor, H-89, reduced the [Ca2+]cyt elevation only when applied at high concentrations (10–40 μm), higher than sufficient for PKA inhibition in many cell types. Furthermore, at these concentrations, H-89 also inhibited presumably PKA-independent processes such as glucose-induced [Ca2+]cyt elevations and intracellular calcium storage. This suggests a...

Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F

This study examines the calcium store‐regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage‐dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin‐producing cells.

Agonist-releasable intracellular calcium stores and the phenomenon of store-dependent calcium entry: A novel hypothesis based on calcium stores in organelles of the endo- and exocytotic apparatus

Store-dependent calcium entry represents a little characterized calcium permeation pathway that is present in a variety of cell types. It is activated in an unknown way by depletion of intracellular calcium stores, for example in the course of phospholipase C stimulation. Current hypotheses propose that depleted calcium stores signal their filling state to this permeation pathway either by direct, protein-mediated interaction or by release of a small, diffusible messenger. The further characterization of store-dependent calcium entry will benefit from progress in the identification of the intracellular calcium storing compartments. Recent findings reviewed here suggest that these compartments include parts of the organelle system that is involved in endo- and exocytosis. This commentary describes a novel model of store-dependent calcium entry based on calcium stores belonging to the endo- and exocytotic organelle system. Such calcium stores could establish a tubule-like connection with the extracellular space, in analogy to the cellular compartments that contain the insulin-sensitive glucose transporter or the gastric proton pump. This connection will provide a pathway for store-dependent calcium entry. Under store depletion, extracellular calcium will permeate through the tubule-like connection into the store lumen and from there into the cytosol. The consequences of this model for the development of drugs modulating store-dependent calcium entry are discussed.

Evidence for vacuolar-type proton pumps in nonmitochondrial and inositol 1,4,5- trisphosphate-sensitive calcium stores of insulin-secreting cells

This study examines whether acidic, vacuolar-type, proton-pump-carrying organelles of insulinsecreting cells (clonal endocrine pancreatic cell line INS-1) function as rapidly exchanging, inositol 1,4,5-trisphosphate-sensitive calcium stores. Calcium uptake into calcium stores will be modulated by the proton concentration within the stores, since calcium pumps in general appear to mediate a countertransport of calcium with protons. We therefore tested for sensitivity of calcium sequestration by nonmitochondrial stores (inhibition of mitochondrial calcium uptake by 2 μM ruthenium red) in saponin-permeabilized cells to proton-conducting ionophores and proton pump inhibition, using this as a marker for involvement of acidic organelles. Calcium sequestration was partially inhibited by the protonophores nigericin (10–50 μM) and carbonylcyanide m-chlorophenylhydrazone (CCCP; 20-50 μM), as well as by inclusion of 30 mM NH4C1. Bafilomycin A1 a potent and selective inhibitor of vacuolar-type proton pumps, alone (1 – 500 nM) had no effect on calcium sequestration, however, it induced an inhibitory effect in the presence of nigericin or CCCP, even at low concentrations (5 μM) of these ionophores, lacking itself an inhibitory action on calcium sequestration. Bafilomycin A, then was already maximally active at a concentration as low as 10 nM. Corresponding to inhibition of total nonmitochondrial calcium sequestration, filling of inositol 1,4,5-trisphosphate-sensitive stores was decreased or even abolished by the protonophores alone or the protonophores combined with bafilomycin A1. We conclude that vacuolartype proton pumps are present in at least a part of nonmitochondrial and inositol 1,4,5-trisphosphate-sensitive calcium stores in INS-1 cells. This assigns these stores to organelles such as secretory granules, the trans Golgi network, or endosomes. Luminal acidity of these stores will stimulate calcium sequestration by providing more protons for countertransport of calcium by calcium pumps. High concentrations of protonophores may be required for inhibitory effects because otherwise the proton pumps may be able to compensate sufficiently for ionophore-mediated proton loss. The lack of effect of bafilomycin A, without protonophores may be due to a sufficient luminal buffering capacity or to preceding inhibition of the pump by an inside-positive transmembrane potential.

Vanadium uptake by yeast cells

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.

A novel membrane associated carbonyl reducing enzyme is present in smooth endoplasmic reticulum of mouse liver

Evidence supporting the existence of two distinct carbonyl (metyrapone) reducing enzymes which differ in subcellular localization and immunological homology has been provided. A soluble enzyme, designated as carbonyl reductase (EC 1.1.1.184) is located in the cytosol. The distribution of the second, membrane associated, MPON-reductase shows an excellent linear correlation to NADPH-cytochrome c reductase and, on the other hand, is reciprocal to the RNA/protein ratio of submicrosomal preparations. This indicates that the membrane associated MPON-reductase is exclusively located in the smooth endoplasmic reticulum. Using antibodies against the purified membrane associated enzyme the extent of immunological crossreaction corresponds well to the specific activities of MPON-reductase in the granular fractions, thus further confirming the localization of this enzyme within this organelle. The absence of antigenic crossreaction to cytosolic MPON-reductase indicates differences also in terms of the immunological relationship between the two enzymes.