Mast cell activation by pedicellarial toxin of sea urchin, Toxopneustes pileolus

AbstractPedicellarial toxin, partially purified from the sea urchin Toxopneustes pileolus, dose-dependently and time-dependently caused histamine release from rat peritoneal mast cells. Pedicellarial toxin induced a rapid initial rise in [Ca2+]i within several seconds which was followed by a further slower increase of [Ca2+] (second rise). The toxin induced a dose-dependent formation of inositol 1,4,5-triphosphate (IP3) as well as the histamine release in mast cells. Furthermore, the toxin stimulated phosphoinositide-specific phospholipase C (PI-PLC) activity in mast cell membranes. 2-Nitro-4-carboxyphenyl-N,N. -diphenylcarbamate (NCDC), a PLC inhibitor, inhibited the activation of PI-PCL induced by pedicellarial toxin. Cholera toxin inhibited pedicellarial toxin-induced histamine release, whereas pretreatment of pertussis toxin failed to inhibit it. These results suggest that pedicellarial toxin from T. pileolus activates PI-PCL and the stimulation of PI turnover may lead to the release of IP3 into the cytoplasm, resulting in histamine release from rat mast cells

G-proteins coupled to phosphoinositide hydrolysis in the cochlear and vestibular sensory epithelia of the rat are insensitive to cholera and pertussis toxins

In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoinositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P2y agonists. Such receptor-mediated activation of PLC is expected to be coupled through guanine nucleotide-binding proteins (G-proteins). Although several classes of G-proteins have been identified in the inner ear, nothing is known about the type of G-proteins associated with the phosphoinositide second messenger system in CSE and VSE. Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs). Ten mM NaF plus 10 [mu]M AlCl3 increased basal InsPs accumulation 2-fold in both CSE and VSE of the rat. Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-[gamma]-S) in saponin-permeabilized tissues. Furthermore, release of InsPs stimulated by both carbamylcholine (CCh) and adenosine 5'-O-[3-thiotriphosphate](ATP-[gamma]-S) was significantly inhibited by 100 [mu]M guanosine 5'-O-[2-thiodiphosphate](GDP-[beta]-S). These results strongly suggest the involvement of G-proteins in the receptor-PLC coupling in CSE and VSE. ADP-ribosylation in membrane fractions of CSE and VSE in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicated the existence of Gs- and Gi-type G-proteins. However, neither CTX nor PTX affected basal or agonist-stimulated release of InsPs. These observations suggest that muscarinic and P2y purinergic receptors are coupled to PLC via CTX- and PTX-insensitive G-proteins in CSE and VSE.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31661/1/0000595.pd

Receptors for insulin interact with G_i-proteins and for epidermal growth factor with G_i- and G_s-proteins in rat pancreatic acinar cells

In rat pancreatic acinar cells epidermal growth factor (EGF) and insulin increase both basal and cholecystokinin (CCK-OP) stimulated amylase release in vitro (1) as a long term function of this tissue. Here we show that preincubation of isolated plasma membranes with EGF or with insulin leads to increased incorporation of the GTP-photoaffinity analogue [alpha-32P]GTP-gamma-azidoanilide into 40/41 kDa proteins and to reduction of pertussis toxin- (PT) catalyzed [alpha-32P]ADP-ribosylation of three 40/41 kDa proteins which had been previously identified as Gi1, Gi2 and Gi3 (2). In the presence of GTP gamma S, EGF- and insulin-induced inhibition of PT-mediated [alpha-32P]ADP-ribosylation of 40/41 kDa proteins is eliminated. EGF enhances cholera toxin- (CT) mediated ADP-ribosylation of all three 40/41 kDa Gi-proteins as well as of five 45 and four 48/50 kDa proteins, which had been previously identified as Gs-proteins (2), whereas insulin has no effect. We conclude from our data that both EGF and insulin interact with the same Gi-proteins as CCK-OP does, whereas EGF additionally interacts with nine Gs-proteins. It is likely that one, two or all three 40/41 kDa Gi-proteins are involved in insulin- and EGF-induced potentiation of CCK-OP-stimulated enzyme secretion. In addition interaction of EGF with Gs-protein could play a role in the potentiation of CCK-OP-induced enzyme secretion from pancreatic acinar cells

Regulation of Signal Transduction by G Proteins in Exocrine Pancreas Cells

The second messenger for hormone-induced Ca2+ release is inositol 1,4,5-triphosph ate (IP3). Following binding of an agonist to its receptor, phospholipase C (PLC) is activated and phosphatidylinositoI4,5-bisphosphate is broken down to IP3 and diacylglycerol (Fig. 1). While IP3 releases Ca2+ from a nonmitochondrial compartment, which is most likely the endoplasmatic reticulum, diacylglycerol activates protein kinase C which in many cells leads to the final cell response by kinase C mediated phosphorylation of target proteins. IP3 can be metabolized by dephosphorylation to inositol 1,4-bisphosphate (IP2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (IP4), which is supposed to be involved in Ca2+ influx into the cell, the mechanism of which is yet not quite clear. The two molecules IP4 and IP3 seem to act together to control Ca2+ influx. A current model is based on the hypothesis that Ca2+ enters the cell through an IP3-sensitive Ca2+ pool in a manner similar to that proposed by Putney, and that IP3 modulates Ca2+ entry into that Ca2+ store. Thus, the Ca2+ pool can be filled from the outside of the cell, and Ca2+ influx takes place only if the pool is emptied due to IP3-induced Ca2+ release. IP4 is dephosphorylated to inositol 1,3,4-trisphosphate of which a second messenger function is not yet known. Evidence suggests that in receptor-mediated activation of PLC GTP-binding proteins (G proteins) are involved

Regulationsmechanismen bei der rezeptorvermittelten Aktivierung der phospholipase C und der Inosit-1,4,5-triphosphate sensitiven Ca²⁺-Freisetzungn und Ca²⁺-Aufnahme in exokrinen Drüsenzellen

The involvement of guanosine triphosphate (GTP)-binding proteins in the receptor-mediated activation of phospholipase C in isolated, permeabilized acinar cells of rat pancreas was studied. Stimulation of phospholipase C (PLC) by agonists such as cholecystokinin (CCK), carbachol (Cch) or GTP-gamma-S, a weakly hydrolysable GTP-analog, induced production of inositol-1,4,5-trisphosphate (IP3) by hydrolysis of its precursor phosphatidylinositol-4,5-bisphosphate (PIP2). Preincubation of permeabilized cells with activated cholera toxin (CT) inhibited cholecystokinin-octapeptide (CCK-OP) and GTP-gamma-S--but not Cch-induced production of IP3. Pertussis toxin had no effect on PLC activity. Neither cyclic adenosine monophosphate (cAMP) nor hormones which activate adenylyl cyclase, inhibited activation of PLC. This indicates that the inhibitory effect of CT is not mediated by stimulation of adenylyl cyclase activity. In isolated plasma membranes of pancreatic acinar cells a 40 kDa protein was adenosine diphosphate (ADP)-ribosylated by CT, which was inhibited by CCK-OP but not by Cch. A 40 kDa protein was also labelled by the photosensitive affinity marker GTP [alpha 32P]-gamma-azidoanilide. Binding of this GTP-analog was enhanced by CCK-OP but not by Cch. It is concluded that CCK- and muscarinic acetylcholine-receptors are functionally coupled by two different G-proteins to phospholipase C. IP3, which is produced by activation of phospholipase C leads to release of Ca2+ from a nonmitochondrial Ca2+ pool, which is likely the endoplasmatic reticulum (ER). Reuptake of Ca2+ by Ca2+ pumps into ER compartments was studied in isolated permeabilized pancreas- and parotid cells as well as in isolated ER vesicle

Acetylcholine and cholecystokinin receptors functionally couple by different G‐proteins to phospholipase C in pancreatic acinar cells

We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin‐octapeptide (CCK‐OP) and GTPγS but not carbachol (CCh)‐induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP‐analogue, 8‐bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist‐induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP‐ribosylated a 40 kDa protein, which was inhibited by CCK‐OP but not by CCh. We conclude from these data that both CCK‐ and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP‐binding proteins

Ca²⁺ signalling in exocrine glands in comparison to that in vascular smooth muscle cells

Intracellular mechanisms involved in the regulation of cytosolic free Ca2+ concentration are very similar in different cell types including exocrine glandular and smooth muscle cells. Since altered calcium metabolism in vascular smooth muscle cells is one important factor involved in the pathogenesis of hypertension, understanding of regulation of cytosolic free Ca2+ concentration is necessary for effective treatment of this disease. In the following, main intracellular pathways in receptor-mediated stimulation of intracellular Ca2+ release, of Ca2+ influx into cells, and of Ca2+- dependent processes in stimulus-response coupling will be discussed. Furthermore, mechanisms involved in the regulation of stimulatory events that lead back to the resting state of the cell will be described. The cell types to be discussed are the acinar cell of the exocrine pancreas and the vascular smooth muscle cell. Although there are basic differences between both types of cells in that smooth muscle cells are excitable, whereas exocrine glandular cells are not, there are also striking similarities in receptor-mediated intracellular events. These pathways, termed “stimulus-secretion coupling” in exocrine glands and “pharmacomechanical coupling” in smooth muscle cells, will be compared in the following