thapsigargin and mastoparan

Application of the tetradecapeptide mastoparan to the prothoracic glands (PGs) of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori, resulted in increases in intracellular Ca(2+) ([Ca(2+)](i)). In M. sexta, Gi proteins are involved in the mastoparan-stimulated increase in [Ca(2+)](i). However, there is no involvement of Gi proteins in the mastoparan-stimulated increase in [Ca(2+)](i) in prothoracic gland cells from B. mori. Unlike in M. sexta prothoracic glands, in B. mori prothoracic glands mastoparan increases [Ca(2+)](i) even in the absence of extracellular Ca(2+). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic glands of both insect species suggests that in M. sexta prothoracic glands, mastoparan's first site of action is influx of Ca(2+) through plasma membrane Ca(2+) channels while in B. mori prothoracic glands, mastoparan's first site of action is mobilization of Ca(2+) from intracellular stores. In M. sexta, the combined results indicate the presence of mastoparan-sensitive plasma membrane Ca(2+) channels, distinct from those activated by prothoracicotropic hormone or the IP(3) signalling cascade, that coordinate spatial increases in [Ca(2+)](i) in prothoracic gland cells. We propose that in B. mori, mastoparan stimulates Ca(2+) mobilization from ryanodine-sensitive intracellular Ca(2+) stores in prothoracic gland cells.

Topics: Animals; Bombyx; Boron Compounds; Calcium; Calcium Signaling; Dose-Response Relationship, Drug; Gadolinium; Guanosine Diphosphate; Intercellular Signaling Peptides and Proteins; Manduca; Peptides; Pertussis Toxin; Ryanodine; Thapsigargin; Thionucleotides; Time Factors; Wasp Venoms

Tacrolimus causes posttransplant diabetes mellitus, although the pathogenetic mechanisms remain controversial. We studied the mechanism of tacrolimus-induced impairment of insulin secretion using isolated rat pancreatic islets. Tacrolimus caused reductions in DNA and insulin contents per islet during 7-d culture. Tacrolimus time-dependently suppressed glucose-stimulated insulin secretion, and at a therapeutic concentration of 0.01 micromol/liter, it suppressed glucose-stimulated insulin secretion to 32 +/- 5% of the control value after 7-d incubation. Tacrolimus did not change islet glucose utilization and oxidation, ATP production, insulin mRNA expression, or the capacity for high glucose to increase intracellular Ca(2+), but altered the rapid frequency oscillations of Ca(2+) concentration. Tacrolimus suppressed insulin secretion stimulated by mitochondrial fuel (combination of l-leucine and l-glutamine, and alpha-ketoisocaproate) and glibenclamide, but not by l-arginine. Tacrolimus suppressed insulin secretion induced by carbachol and by a protein kinase C agonist in the presence or absence of extracellular Ca(2+). Under stringent Ca(2+)-free conditions, tacrolimus did not affect mastoparan-induced insulin secretion, but suppressed its glucose augmentation. Our results suggest that tacrolimus impairs glucose-stimulated insulin secretion downstream of the rise in intracellular Ca(2+) at insulin exocytosis, and that protein kinase C-mediated (Ca(2+)-dependent and independent) and Ca(2+)-independent GTP signaling pathways may be involved. However, tacrolimus-induced impaired insulin secretion was reversed 3 d after removal of the drug. Our study demonstrated that tacrolimus impairs insulin secretion at multiple steps in stimulus-secretion coupling.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium-Transporting ATPases; Carbachol; Caspase 3; Caspases; Cell Survival; Cyclic AMP-Dependent Protein Kinases; DNA; Enzyme Inhibitors; Glucose; Glyburide; Immunosuppressive Agents; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Kinetics; Male; Peptides; Protein Kinase C; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tacrolimus; Thapsigargin; Wasp Venoms

Mas-7, a mastoparan derivative, induces elevation of intracellular free Ca2+ concentration ([Ca2+]i) along two independent pathways. The minor contribution occurs via phospholipase C activation and is negatively regulated by treatment with phorbol 12-myristate 13-acetate, a protein kinase C activator. The major contribution involves plasma membrane pores allowing not only Ca2+, Mn2+, and Na+ to enter but also the uptake of ethidium bromide (314 Da) and lucifer yellow (457 Da), but not fura-2 (831 Da), Evans blue (961 Da), and fluorescein-conjugate phalloidin (1,175 Da). Mas-7-induced current, as measured in planar lipid bilayers, reveals that Mas-7-induced pores have two slope conductances, 290 and 94 pS, and that the pores are nonselective for cations. The results also indicate that Mas-7 can produce pores by direct interaction with the plasma membrane without the involvement of membrane proteins and cytosolic factors. Besides in human neuroblastoma cells, similar Mas-7 effects were also observed in other cell lines such as HL-60, 1321N1 human astrocytoma, and bovine chromaffin cells. The data suggest that the Mas-7-induced [Ca2+]i elevation is the combined result of Ca2+ release from stores via phosphoinositide turnover and prolonged Ca2+ influx through membrane pores.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cattle; Cell Membrane; Cell Membrane Permeability; Cytosol; Enzyme Inhibitors; Extracellular Space; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Intercellular Signaling Peptides and Proteins; Ion Channels; Lipid Bilayers; Manganese; Membrane Potentials; Peptides; Phosphatidylinositols; Protein Kinase C; Signal Transduction; Sodium; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Cells, Cultured; Wasp Venoms

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.

Topics: Animals; Calcium; Cell Line; Colforsin; Exocytosis; Hydroquinones; In Vitro Techniques; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Nitrendipine; Peptides; Pertussis Toxin; Rats; Secretory Rate; Terpenes; Thapsigargin; Virulence Factors, Bordetella; Wasp Venoms