inositol-1-4-5-trisphosphate and Adenoma--Islet-Cell

In this investigation we studied pancreastatin (PST) secretion from a human PST producing cell line (QGP-1N) in response to various secretagogues. Immunocytochemical study revealed the immunoreactivity of PST and somatostatin (SMT) in the same cells of a monolayer culture. Ki-ras DNA point mutation on codon 12 was found. Carbachol stimulated secretion of PST and SMT and intracellular Ca2+ mobilization in the range of 10(-6)-10(-4) M. The secretion and Ca2+ mobilization were inhibited by atropine, a muscarinic receptor antagonist. Phorbol ester and calcium ionophore (A23187) stimulated secretion of PST and SMT. The removal of extracellular calcium suppressed both secretions throughout stimulation with 10(-5) M carbachol. Fluoride, a well-known activator of guanine nucleotide binding (G) protein, stimulated intracellular Ca2+ mobilization and secretion of PST and SMT in a dose-dependent manner in the range of 5-40 mM. Also, 10(-5) M carbachol and 20 mM fluoride stimulated inositol 1,4,5-triphosphate production. However, cholecystokinin and gastrin-releasing peptide did not stimulate Ca2+ mobilization or secretion of the two peptides. These results suggest that secretion of PST and SMT from QGP-1N cells is regulated mainly by acetylcholine in a parallel fashion through muscarinic receptors coupled to the activation of polyphosphoinositide breakdown by a G-protein and that increases in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

Topics: Adenoma, Islet Cell; Calcium; Carbachol; Chromogranin A; Genes, ras; Humans; Inositol 1,4,5-Trisphosphate; Pancreatic Hormones; Pancreatic Neoplasms; Sincalide; Somatostatin; Tumor Cells, Cultured

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.

Topics: Adenoma, Islet Cell; Animals; Calcimycin; Calcium; Cell Line; Cell Membrane Permeability; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insulinoma; Kinetics; Mitochondria; Pancreatic Neoplasms; Rats; Subcellular Fractions; Sugar Phosphates

An early event associated with the stimulation of various secretory cells is the breakdown of phosphatidylinositol 4,5-bisphosphate and the mobilization of cellular calcium. Hydrolysis of this inositol lipid by a phosphodiesterase produces inositol trisphosphate (InsP3), a small water-soluble molecule which may serve a messenger function to release Ca2+ from internal stores. In order to assess the role of inositol lipid breakdown in the stimulation of insulin secretion we have examined the effect of InsP3 on Ca2+ fluxes in three different insulin-secreting tumor cells permeabilized by the addition of saponin. A rapid, transient release of Ca2+ from a non-mitochondrial pool occurred upon addition of InsP3 to all three cell types. Half-maximal Ca2+ release from the RIN-1046-38 and RIN-m5F cells was obtained in the concentration range 0.1-0.2 microM. However, the cells obtained from a transplantable tumor of the Syrian hamster were far more sensitive to InsP3 with half-maximal release being observed at 0.025 microM. A partially purified preparation of vesicles was isolated from this tumor which retained its responsiveness to InsP3. Half-maximal Ca2+ release from the vesicles was obtained at 0.2 microM InsP3. Our data are consistent with a role for InsP3 in mediating the increase in cytosolic free Ca2+ which occurs in response to a number of stimuli that promote the secretion of insulin.

Topics: Adenoma, Islet Cell; Animals; Calcium; Calcium-Transporting ATPases; Cell Line; Cricetinae; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insulinoma; Kinetics; Mesocricetus; Microsomes; Microsomes, Liver; Pancreatic Neoplasms; Sugar Phosphates

A possible role in secretory processes is proposed for inositol 1,4,5-triphosphate (IP3), based upon investigations of the Ca2+ steady state maintained by "leaky', insulin-secreting RINm5F cells. These cells had been treated with digitonin to permeabilize their plasma membranes and thereby ensure that only intracellular Ca2+ buffering mechanisms were active. When placed in a medium with a cation composition resembling that of the cytosol, cells rapidly took up Ca2+ as measured by a Ca2+-specific minielectrode. Two Ca2+ steady states were observed. A lower level of around 120nM required ATP-dependent Ca2+ uptake and was probably determined by the endoplasmic reticulum. The higher steady state (approx. 800 nM), seen only in the absence of ATP, was shown to be due to mitochondrial activity. IP3 specifically released Ca2+ accumulated in the ATP-dependent pool, but not from mitochondria, since Ca2+ release was demonstrated in the presence of the respiratory poison antimycin. The IP3-induced Ca2+ release was rapid, with 50% of the response being seen within 15s. The apparent Km was 0.5 microM and maximal concentrations of IP3 (2.5 microM) produced a peak Ca2+ release of 10 nmol/mg of cell protein, which was followed by re-uptake. A full Ca2+ response was seen if sequential pulses of 2.5 microM-IP3 were added at 20 min intervals, although there was a slight (less than 20%) attenuation if the intervening period was decreased to 10 min. These observations could be related to the rate of IP3 degradation which, in this system, corresponded to a 25% loss of added 32P label within 2 min, and a 75% loss within 20 min. The results suggest that IP3 might act as a link between metabolic, cationic and secretory events during the stimulation of insulin release.

Topics: Adenoma, Islet Cell; Calcium; Cell Line; Cell Membrane Permeability; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insulin; Insulin Secretion; Insulinoma; Pancreatic Neoplasms; Sugar Phosphates