Synthetic RAB3A Effector Domain Peptide Stir Stimulates Inositol 1,4,5-Trisphosphate Production in Various Permeabilized Cells

1994 ◽  
Vol 203 (2) ◽  
pp. 756-762 ◽  
Author(s):  
A. Piiper ◽  
D. Stryjekkaminska ◽  
S. Zeuzem
Keyword(s):  
Effector Domain ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Domain Peptide

scholarly journals Synthetic peptides containing a BXBXXXB(B) motif activate phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Albrecht PIIPER ◽  
Danuta STRYJEK-KAMINSKA ◽  
Daria ILLENBERGER ◽  
Rolf KLENGEL ◽  
Jürgen M. SCHMIDT ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Synthetic Peptides ◽  
Basic Residue ◽  
Pharmacological Agents ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Effector Domain ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Domain Peptide

We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-β1, -β3, -γ1 and -δ1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-β1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-β1 but not PLC-β3, -γ1 or -δ1. P1 also activated recombinant PLC-β1, indicating direct activation of PLC-β1 by Rab3 effector-domain peptides. To investigate further the structure–function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue) [KVKTVYRH (EC50 of 1 nM to stimulate amylase release) ≈ TVGIDFKVKTVYRH > TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-β1 isoenzyme.

Actions of Rab3 effector domain peptides in chief cells from guinea pig stomach

1995 ◽  
Vol 269 (3) ◽  
pp. G400-G407 ◽  
Author(s):  
G. Singh ◽  
R. D. Raffaniello ◽  
J. Eng ◽  
J. P. Raufman
Keyword(s):  
Binding Proteins ◽  
Secretory Response ◽  
Effector Domain ◽  
Chief Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Pepsinogen Secretion ◽  
Guanine Nucleotide Binding ◽  
Domain Peptide ◽  
Guinea Pig Stomach

Rab3 proteins are low molecular weight guanine nucleotide-binding proteins that belong to the Ras superfamily and are believed to play a role in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secretion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calcium concentration ([Ca2+]) < 1 nM], a maximal concentration (15 microM) of the Rab3 effector domain peptide or Rab3AL peptide, containing alanine and leucine substitutions, stimulated the release of 62 and 66%, respectively, of total pepsinogen from streptolysin O-permeabilized chief cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter secretion from permeabilized cells. An additive secretory response was observed when 5 microM Rab3AL peptide was combined with increasing calcium ([Ca2+] < 1 nM to 3 microM). In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-induced secretion, and Rab3AL peptide did not alter endogenous cAMP production. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 microM guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory response to Rab3AL peptide. This potentiated response indicates that other GTP-binding proteins are involved in calcium-independent secretion. Preincubation of cells with streptolysin O (10-30 min), to allow egress of cytosolic constituents, enhanced the response to Rab3AL peptide, suggesting that the target(s) for this peptide is (are) anchored to chief cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target

1995 ◽  
Vol 309 (2) ◽  
pp. 621-627 ◽  
Author(s):  
A Piiper ◽  
D Stryjek-Kaminska ◽  
R Jahn ◽  
S Zeuzem
Keyword(s):  
Pancreatic Acinar Cells ◽  
Maximal Response ◽  
Cross Linking ◽  
Amylase Secretion ◽  
Potential Candidate ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Effector Domain ◽  
Inositol 1,4,5 Trisphosphate ◽  
Domain Peptide

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.

scholarly journals Effect of sphingosine derivatives on calcium fluxes in thyroid FRTL-5 cells

1994 ◽  
Vol 299 (1) ◽  
pp. 213-218 ◽  
Author(s):  
K Törnquist ◽  
E Ekokoski
Keyword(s):  
Elevated Level ◽  
Channel Blocker ◽  
Thyroid Stimulating Hormone ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Myristate Acetate ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dose Dependent ◽  
In Cells

The effects of sphingosine derivatives on Ca2+ fluxes were investigated in thyroid FRTL-5 cells labelled with Fura 2. Addition of sphingosylphosphocholine (SPC) or sphingosine (SP) increased intracellular free Ca2+ ([Ca2+]i) in a dose-dependent manner. At the highest dose tested (30 microM), the response was biphasic: a rapid transient increase in [Ca2+]i, followed by a new, elevated, level of [Ca2+]i. Both phases of the SPC-evoked increase in [Ca2+]i were dependent on extracellular Ca2+, whereas only the SP-evoked elevated level of [Ca2+]i was dependent on the influx of Ca2+. Both compounds released sequestered Ca2+ from thapsigargin- and inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools. In addition, the increase in [Ca2+]i in response to SPC, but not to SP, was attenuated in cells treated with phorbol myristate acetate or with the putative Ca(2+)-channel blocker SKF 96365, and in cells pretreated with pertussis toxin for 24 h. SPC did not activate the production of IP3. Furthermore, both SPC and SP released sequestered Ca2+ from permeabilized cells. We observed that SPC, but not SP, stimulated release of [3H]arachidonate from cells prelabelled with [3H]arachidonate for 24 h. Both SPC and SP stimulated the incorporation of [3H]thymidine into DNA in cells grown in the absence of thyroid-stimulating hormone (TSH). The results suggest that sphingosine derivatives are putative regulators of Ca2+ fluxes in FRTL-5 cells, and that SP and SPC may act on [Ca2+]i via different mechanisms. Furthermore, both SP and SPC may be of importance in modulating thyroid-cell proliferation.

Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells

1999 ◽  
Vol 276 (1) ◽  
pp. L81-L89 ◽  
Author(s):  
Christina M. Pabelick ◽  
Y. S. Prakash ◽  
Mathur S. Kannan ◽  
Keith A. Jones ◽  
David O. Warner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ryanodine Receptor ◽  
Muscle Cells ◽  
Calcium Oscillations ◽  
Tracheal Smooth Muscle ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Trisphosphate Receptor

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.

Measurement of SR free Ca2+ and Mg2+ in permeabilized smooth muscle cells with use of furaptra

1995 ◽  
Vol 269 (3) ◽  
pp. C698-C705 ◽  
Author(s):  
T. Sugiyama ◽  
W. F. Goldman
Keyword(s):  
Smooth Muscle ◽  
Dissociation Constants ◽  
Time Dependent ◽  
Isosbestic Point ◽  
Half Time ◽  
Permeabilized Cells ◽  
Aortic Smooth Muscle ◽  
A7r5 Cells ◽  
Inositol 1,4,5 Trisphosphate

The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded saponin-permeabilized cultured aortic smooth muscle (A7r5) cells. Ca(2+)-independent fluorescence emitted by furaptra trapped within organelles, excited at 346 nm (isosbestic point), decreased with a half time of 30 min. All Ca2+ measurements appeared to be from SR, because the apparent Ca2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsigargin-induced SR Ca(2+)-adenosinetriphosphatase inhibition caused near-total depletion of Ca2+, and the metabolic poisons oligomycin and rotenone had no effect. Calibration curves relating 370 nm-to-346 nm ratios to [Ca2+] and to [Mg2+] were calculated in situ; dissociation constants for Ca2+ and Mg2+ binding were 49 microM and 6.8 mM, respectively. Resting [Ca2+]SR was 75-130 microM, with a mean of 97.2 +/- 2.2 microM (n = 376), whereas [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-dependent declines in [Ca2+]SR, and pretreatment with guanosine 5'-triphosphate caused a large increase in the rate of inositol 1,4,5-trisphosphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will be a valuable tool with which to directly study [Ca2+]SR and SR function.

scholarly journals Ca2+-sensitivity of inositol 1,4,5-trisphosphate-mediated Ca2+ release in permeabilized pancreatic acinar cells

1990 ◽  
Vol 265 (3) ◽  
pp. 681-687 ◽  
Author(s):  
P H G M Willems ◽  
M D De Jong ◽  
J J H H M De Pont ◽  
C H Van Os
Keyword(s):  
Acinar Cells ◽  
Inhibitory Effect ◽  
Feedback Mechanism ◽  
Pancreatic Acinar Cells ◽  
Release Mechanism ◽  
Concomitant Increase ◽  
Permeabilized Cells ◽  
Negative Feedback Mechanism ◽  
Inositol 1,4,5 Trisphosphate ◽  
Energy Dependent

Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.

Thapsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

1992 ◽  
Vol 262 (5) ◽  
pp. C1258-C1265 ◽  
Author(s):  
Y. T. Xuan ◽  
O. L. Wang ◽  
A. R. Whorton
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate

We have investigated the role of the sarcoplasmic reticulum Ca2+ pool in regulating Ca2+ entry in vascular smooth muscle cells using a receptor-independent means of mobilizing the intracellular Ca2+ pool. Thapsigargin (TG) has been shown to inhibit the endoplasmic reticulum Ca(2+)-ATPase, mobilize intracellular Ca2+, and activate Ca2+ entry in nonmuscle tissues. When smooth muscle cells were treated with 0.2 microM TG, cytosolic Ca2+ concentrations rose gradually over 8 min to a peak value of 365 +/- 18 nM. Cytosolic Ca2+ remained elevated for at least 20 min and was supported by continued entry of extracellular Ca2+. TG also stimulated entry of Mn2+ and 45Ca2+ from outside the cell. Importantly, TG-induced Ca2+ entry and Mn2+ entry were found to occur through mechanisms that were independent of L-type Ca2+ channel activation because influx was not inhibited by concentrations of nicardipine that were found to block either endothelin- or 100 mM extracellular K(+)-induced cation influx. The mechanism through which TG activates cation entry appears to involve mobilization of the inositol 1,4,5-trisphosphate-responsive intracellular Ca2+ pool. In permeabilized cells, TG prevented ATP-stimulated Ca2+ uptake into the sarcoplasmic reticulum and slowly released sequestered Ca2+. The Ca2+ pool involved was responsive to inositol 1,4,5-trisphosphate. However, TG did not initiate the formation of inositol polyphosphates. Thus TG mobilizes the sarcoplasmic reticulum Ca2+ pool and activates Ca2+ entry through a nicardipine-insensitive Ca2+ channel in vascular smooth muscle. The mechanism is independent of inositol polyphosphate formation.

Moving Ca2+ from the endoplasmic reticulum to mitochondria: is spatial intimacy enough?

2006 ◽  
Vol 34 (3) ◽  
pp. 351-355 ◽  
Author(s):  
G.A. Rutter
Keyword(s):  
Endoplasmic Reticulum ◽  
Outer Membrane Proteins ◽  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Interconnected Network ◽  
Voltage Dependent Anion Channel ◽  
Permeabilized Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Voltage Dependent ◽  
Trisphosphate Receptor

A number of studies in recent years have demonstrated that the ER (endoplasmic reticulum) makes intimate contacts with mitochondria, the latter organelles existing both as individual organelles and occasionally as a more extensive interconnected network. Demonstrations that mitochondria take up Ca2+ more avidly upon its mobilization from the ER than when delivered to permeabilized cells as a buffered solution also indicate that a shielded conduit for Ca2+ may exist between the two organelle types, perhaps comprising the inositol 1,4,5-trisphosphate receptor and mitochondrial outer membrane proteins including the VDAC (voltage-dependent anion channel). Although the existence of such intracellular ER–mitochondria ‘synapses’, or of an ER–mitochondria Ca2+ ‘translocon’, is an exciting idea, more definitive experiments are needed to test this possibility.

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