Multiple signaling pathways involved in the effect of endothelin type B receptor in rat median eminence

We assessed the possible link between endothelin receptor mediated phosphoinositide breakdown and NO/cGMP signaling pathways in rat arcuate nucleus-median eminence fragments (AN-ME), brain structures known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers, together with densely arranged endothelin ETB-receptors-like immunoreactive fibres. Our data show that ET-1, ET-3 and the ETB-receptors agonist, IRL 1620, increased inositol monophosphate (InsP1) accumulation, NOS activity and cGMP formation, in a similar degree. The stimulatory effect of ETs on InsP1 accumulation and cGMP formation was inhibited by the phospholipase C (PLC) inhibitor, neomycin, and the absence of extracellular calcium, suggesting that calcium is involved in endothelin receptor-induced PLC activation. The L-arginine analog, L-NAME, inhibited ET-1 or IRL1620-stimulated cGMP formation. The ETA receptor antagonists BQ 123, did not alter, while the ETB receptor antagonists BQ788 inhibited ETs-induced increase in the PI metabolism, NOS activity and cGMP generation. Our data indicate that in AN-ME, ETB receptor signals through receptor-mediated calcium dependent-stimulation of phosphoinositide breakdown and activation of NOS/cGMP signaling pathway.     

Comparative studies of phosphoinositide hydrolysis induced by endothelin-related peptides in cultured cerebellar astrocytes, C6-glioma and cerebellar granule cells

Effects of endothelin (ET) homologues (ET-1, 2, 3 and sarafotoxin S6b) and its precursor (big ET-1) on phosphoinositide (PI) turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-1 = ET-2 greater than or equal to S6b greater than ET-3 greater than big ET-1 for granule cell neurons, while it was ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for astrocytes and C6-glioma cells. Short-term pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-1-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and C6-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C6-gliomas, but not in granule cells. Thus, phospholipase C-coupled ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis.     

Inositol phosphate production in response to [Arg8]vasopressin, endothelin 1, and prostaglandin F2 alpha in rat aorta and mesenteric arteries.

Vascular tissues such as rat aorta and mesenteric arteries are extensively used experimentally for the study of cardiovascular diseases. To further our understanding of the signal transduction mechanisms involved in responses to several potent vasoconstrictors, such as [Arg8]vasopressin (AVP), endothelin 1 (ET-1), and prostaglandin F2 alpha (PGF2 alpha), we have investigated the time course for production of inositol monophosphate (InsP1), bisphosphate (InsP2), and trisphosphate (InsP3) in response to these agonists as well as their relative potency for phosphatidylinositol hydrolysis. Time-course studies of production of the different inositol phosphates in response to AVP and PGF2 alpha showed an early increase after 15-30 s of stimulation. Thereafter InsP3 declined towards baseline, with a secondary increase towards steady state after 5-10 min. Rapid turnover of InsP3 was reflected by accumulation of InsP1 and InsP2 in the presence of LiCl (20 mM) to inhibit monophosphatases. After 15-30 min of stimulation, there was accumulation of the Ins(1,3,4)P3 isomer. All three agonists induced greater accumulation of InsP2 in mesenteric arteries than in thoracic aorta, suggesting that turnover of Ins(1,4,5)P3 may be faster in the former than in the latter. The accumulation of total inositol phosphates induced by maximum concentrations of ET-1 was greater than in response to AVP or PGF2 alpha. Dose-response curves showed that the rank order of potency for stimulation of production of inositol phosphates was AVP > ET-1 > PGF2 alpha, similar to the sensitivity of blood vessels to these agents. Comparison of responses to ET-1 and ET-3 showed that the receptors stimulated by endothelins were of the isopeptide selective ETA subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sustained diacylglycerol accumulation resulting from prolonged G protein-coupled receptor agonist-induced phosphoinositide breakdown in hepatocytes

Studies in various cells have led to the idea that agonist-stimulated diacylglycerol (DAG) generation results from an early, transient phospholipase C (PLC)-catalyzed phosphoinositide breakdown, while a more sustained elevation of DAG originates from phosphatidylcholine (PC). We have examined this issue further, using cultured rat hepatocytes, and report here that various G protein-coupled receptor (GPCR) agonists, including vasopressin (VP), angiotensin II (Ang.II), prostaglandin F2alpha, and norepinephrine (NE), may give rise to a prolonged phosphoinositide hydrolysis. Preincubation of hepatocytes with 1-butanol to prevent conversion of phosphatidic acid (PA) did not affect the agonist-induced DAG accumulation, suggesting that phospholipase D-mediated breakdown of PC was not involved. In contrast, the GPCR agonists induced phosphoinositide turnover, assessed by accumulation of inositol phosphates, that was sustained for up to 18 h, even under conditions where PLC was partially desensitized. Pretreatment of hepatocytes with wortmannin, to inhibit synthesis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), prevented agonist-induced inositol phosphate and DAG accumulation. Upon VP stimulation the level of PIP) declined, but only transiently, while increases in inositol 1,4,5-trisphosphate (InsP3) and DAG mass were sustained, suggesting that efficient resynthesis of PIP2 allowed sustained PLC activity. This was confirmed when cells were pretreated with wortmannin to prevent resynthesis of PIP2. Furthermore, metabolism of InsP3 was rapid, compared to that of DAG, with a more than 20-fold difference in half-life. Thus, rapid metabolism of InsP3 and efficient resynthesis of PIP2 may account for the larger amount of DAG generated and the more sustained time course, compared to InsP3. The results suggest that DAG accumulation that is sustained for many hours in response to VP, Ang.II, NE, and prostaglandin F2alpha in hepatocytes is mainly due to phosphoinositide breakdown.     

G-protein-coupled receptor agonists activate endogenous phospholipase Cepsilon and phospholipase Cbeta3 in a temporally distinct manner

Phospholipase Cepsilon (PLCepsilon) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLCbeta isoforms through G(q) family G proteins and Gbetagamma subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLCepsilon and the PLCbeta isoform, PLCbeta3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLCbeta3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLCepsilon, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCepsilon in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCepsilon and PLCbeta3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLCepsilon or PLCbeta3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCepsilon and PLCbeta3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCbeta3 is predominantly involved in acute and PLCepsilon in sustained PI hydrolysis.     

Differential effects of phenylmethanesulfonyl fluoride (PMSF) on carbachol and potassium stimulated phosphoinositide turnover and contraction in longitudinal smooth muscle of guinea pig ileum

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li+ by only 15%-19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown.     

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla.2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP₁) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure.3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [³H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET_B receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP₁ accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET_A receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET_B receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP₁ accumulation.4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET_A receptor, while the latter through the activation of ET_B receptor. These results support the role of endothelins in the regulation of adrenal medulla function.     

ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells

In this study, we analyzed in ratmyometrial cells the signaling pathways involved in the endothelin(ET)-1-induced extracellular signal-regulated kinase (ERK) activationrequired for the induction of DNA synthesis. We found that inhibitionof protein kinase C (PKC) by Ro-31-8220 abolished ERK activation.Inhibition of phospholipase C (PLC) by U-73122 or of phosphoinositide(PI) 3-kinase by wortmannin partially reduced ERK activation. A similarpartial inhibition was observed after treatment with pertussis toxin orPKC downregulation by phorbol ester treatment. The effect of wortmanninwas additive with that produced by PKC downregulation but not with thatdue to pertussis toxin. These results suggest that bothdiacylglycerol-sensitive PKC, activated by PLC products, anddiacylglycerol-insensitive PKC, possibly activated by aG_i-PI 3-kinase-dependent process, are involved inET-1-induced ERK activation. These two pathways were found to beactivated mainly through the ET_A receptor subtype. ET-1 andphorbol ester stimulated Src activity in a PKC-dependent manner, bothresponses being abolished in the presence of Ro-31-8220. Inhibition of Src kinases by PP1 abrogated phorbol ester- and ET-1-induced ERK activation. Finally, ET-1 activated Ras in a PP1-and Ro-31-8220-sensitive manner. Altogether, our results indicatethat ET-1 induces ERK activation in rat myometrial cells through thesequential stimulation of PKC, Src, and Ras.

     

Endothelin- and Sarafotoxin-Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

Abstract: The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphates (IPs) and tracheal smooth muscle contraction. BQ-123, an ET_A receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pK_B values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca²⁺ and was blocked by treatment with EGTA. The addition of Ca²⁺(3–620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca²⁺-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 μM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to down regulation of PKC. Inactive phorbol ester, 4α-phorbol 12, 13-didecanoate at 1 μM, did not inhibit this response. The site of this response was further investigated by examining the effect of PMA on AIF₄^?-induced IP accumulation in canine TSMCs. AIF₄^?-induced IP accumulation was inhibited by PMA treatment, suggesting that G protein(s) can be directly activated by AIF₄^?, which was uncoupled to phospholipase C by PMA treatment. These data conclude that ET-stimulated PI hydrolysis and tracheal smooth muscle contraction are mediated by the activation of ET_Areceptors coupling to a G protein and dependent on the external Ca²⁺. The transduction mechanism of ETs is sensitive to feedback regulation by PKC.     

Sphingosine increases inositol trisphosphate in rat parotid acinar cells by a mechanism that is independent of protein kinase C but dependent on extracellular calcium

In rat parotid acinar cells prelabelled with [3H]-inositol, sphingosine stimulated the accumulation of [3H]-inositol polyphosphates. When the cells were exposed to sphingosine, [3H]-inositol trisphosphate (InsP3) was accumulated in a time- and dose-dependent manner. When the extracellular Ca2+ was chelated by 1 mM EGTA, the effect of sphingosine on InsP3 accumulation was completely inhibited. Ionophores, A23187 and ionomycin, had no significant effect on InsP3 accumulation. An inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), failed to stimulate InsP3 accumulation. In the homogenate of parotid acinar cells, InsP3 3-kinase and 5-phosphomonoesterase activities were not affected by sphingosine. These results suggest that sphingosine activates phosphoinositide turnover by a mechanism dependent upon extracellular Ca2+, but different from that of an ionophore, and independent of protein kinase C.     

P2-purinoceptor-stimulated phosphoinositide turnover in chick myotubes. Calcium mobilization and the role of guanyl nucleotide-binding proteins

ATP, a trigger of P2-purinoceptor-mediated polyphosphoinositide (PI) turnover in cultured myotubes, increased cytosolic calcium levels in a time- and dose-dependent manner (quin2 fluorescence). The calcium was released from intracellular stores, as acute addition of 5 mM EGTA was without significant effect. Adenosine 5'-(3-thiotriphosphate) and 5'-adenylyl imidodiphosphate also increased intracellular levels of inositol phosphates (InsP) and cytosolic calcium levels. Treatment with cholera or pertussis toxin of myotube cultures did not affect the P2-purinoceptor-mediated InsP increase although PI turnover in permeabilized myotubes was stimulated by guanosine 5'-(3-thiotriphosphate). The results suggest that myotube P2-purinoceptors trigger PI turnover and increase intracellular free calcium levels, via a mechanism insensitive to ADP-ribosylation, by cholera or pertussis toxin of guanyl nucleotide-binding (G) proteins. However, the presence of a phospholipase C-coupled G-protein was otherwise demonstrated.     

Effects of nonselective cation channels and PI3K on endothelin-1-induced PYK2 tyrosine phosphorylation in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca²⁺ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca²⁺ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca²⁺ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca²⁺ entry, but not for its maintenance; 3) Ca²⁺ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca²⁺ influx through NSCC-2. endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell     

Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelin(B) receptor (CHO-ET(B)R). These channels can be discriminated using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ET(B)R using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca(2+) influx was partially inhibited in CHO-ET(B)R pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca(2+) influx. The Ca(2+) channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ET(B)R were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca(2+) entry through NSCC-2 but not for its maintenance.     

Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes.

We have assessed the effects of endothelin-1 (ET-1) on transmembrane signaling in adult rat ventricular myocytes. ET-1 stimulates phosphoinositide hydrolysis with an EC50 of 0.3-0.8 nM. This stimulation is linear for up to 30 min in the presence of a protease inhibitor, is additive with the effects of other stimulators of phosphoinositide hydrolysis, is not inhibited by the Ca2+ entry blocker, nifedipine, and is insensitive to pertussis toxin. ET-1 also reduces cyclic AMP production in myocytes in response to isoproterenol and forskolin (EC50, 1 nM). This cyclic AMP-lowering effect of ET-1 is sensitive to pertussis toxin, can be demonstrated directly in assays of adenylate cyclase activity of myocyte membranes, and seems to be mediated by Gi. These data indicate that the effects of endothelin on adult cardiac myocytes involve multiple signaling pathways, including enhanced activity of the inositol phosphate pathway and a decrease in cyclic AMP-mediated responses, neither of which seems likely to account for the positive contractile effects of endothelin.     

The effects of lithium on platelet phosphoinositide metabolism.

The effects on phosphoinositide metabolism of preincubation of platelets for 90 min with 10 mM-Li+ were studied. Measurements were made of [32P]phosphate-labelled phosphoinositides and of [3H]inositol-labelled inositol mono-, bis- and tris-phosphate (InsP, InsP2 and InsP3). Li+ had no effect on the basal radioactivity in the phosphoinositides or in InsP2 or InsP3, but it caused a 1.8-fold increase in the basal radioactivity in InsP. Li+ caused a 4-, 3- and 2-fold enhanced thrombin-induced accumulation of label in InsP, InsP2 and InsP3 respectively. Although the elevated labelling of InsP2 and InsP3 returned to near-basal values within 30-60 min, the high labelling of InsP did not decline over a period of 60 min after addition of thrombin to Li+-treated platelets, consistent with inhibition of InsP phosphatase by Li+. The effect of Li+ was not due to a shift in the thrombin dose-response relationship; increasing concentrations of thrombin enhanced the initial rate of production of radiolabelled inositol phosphates, whereas Li+ affected either a secondary production or the rate of their removal. The only observed effect of Li+ on phosphoinositide metabolism was a thrombin-induced decrease (P less than 0.05) in labelled phosphatidylinositol 4-phosphate in Li+-treated platelets; this suggests an effect on phospholipase C. Li+ enhanced (P less than 0.05) the thrombin-induced increase in labelled lysophosphatidylinositol, suggesting an effect on phospholipase A2. It is concluded that Li+ inhibits InsP phosphatase and has other effects on phosphoinositide metabolism in activated platelets. The observed effects occur too slowly to be the mechanism by which Li+ potentiates agonist-induced platelet activation.     

The effects of the endothelin family peptides on cultured osteoblastic cells from rat calvariae

The activities of three isoforms of the endothelin (ET) family peptides, ET-1, ET-2 and ET-3, were studied in cultured osteoblastic cells from neonatal rat calvariae. All three isoforms induce stimulation of DNA synthesis and reductions in cellular alkaline phosphatase activity in a dose-dependent manner with the rank order of potency: ET-1 congruent to ET-2 greater than ET-3. The 125I-labeled ET binding and affinity-cross linking experiments show the presence of a single class of the ET binding sites with a more than 10-fold higher affinity for ET-1 and ET-2 as compared to ET-3. The endothelins dose-dependently stimulate the production of inositol phosphates and induce mobilization of Ca2+ with the similar relative potency to that for the receptor binding. These results indicate that osteoblastic cells possess the endothelin receptor with a high affinity for ET-1 and ET-2 that is coupled to phospholipase C, and that the endothelins modulate cellular functions via this receptor.     

Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation at 5 microM. Carbachol increased the accumulation of [3H]inositol phosphates only in the presence of added guanine nucleotide. Calcium increased [3H]InsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal [3H]InsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous [3H]InsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited [3H] InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated [3H]InsP3 formation although calcium-stimulated [3H]InsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated [3H]InsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of [3H]InsP3 results from inhibition of the G protein interaction with phospholipase C.     

Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes.

We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.     

Endothelins participate in the central and peripheral regulation of submandibular gland secretion in the rat

We previously reported that endothelins (ETs) are involved in the rat central and peripheral regulation of bile secretion. In this study we sought to establish whether ET-1 and ET-3 modulated submandibular gland secretion when locally or centrally applied. Animals were prepared with gland duct cannulation to collect saliva samples and jugular cannulation to administer sialogogues. ETs were given either into the submandibular gland or brain lateral ventricle. Intraglandularly administered ETs failed to elicit salivation per se. However, ET-1, but not ET-3, potentiated both cholinergic- and adrenergic-evoked salivation through ET(A) receptors. ET-1 decreased cAMP content but increased phosphoinositide hydrolysis, whereas ET-3 attenuated both intracellular pathways. The expression of ET(A) and ET(B) receptor mRNAs as well as that of ETs was revealed in the submandibular gland by RT-PCR. Immunohistochemical studies showed that ET(A) receptor staining was localized around the interlobular ducts and acini, compatible with the myoepithelial cells' location, whereas ET(B) receptor staining was restricted to small blood vessels. When applied to the brain, both ETs induced no salivation but enhanced cholinergic- and adrenergic-evoked salivary secretion through parasympathetic pathways. ET-1 response was mediated by brain ET(A) receptors, whereas that of ET-3 was presumably through nonconventional ET receptors. Present findings show that ETs are involved in the brain regulation of cholinergic- and adrenergic-stimulated submandibular gland secretion through the activation of distinct brain ET receptors and parasympathetic pathways. However, when ETs were administered into the gland, only ET-1 enhanced cholinergic and adrenergic salivation likely through myopithelial cell contraction by activating ET(A) receptors coupled to phospholipase C. The presence of ETs and ET receptors suggests the existence of an endothelinergic system in the submandibular gland.