The exocytotic signaling pathway induced by nerve growth factor in the presence of lyso-phosphatidylserine in rat peritoneal mast cells involves a type D phospholipase.

Nerve growth factor (NGF) has been previously shown to induce exocytosis in rat peritoneal mast cells (RPMCs) in the presence of lyso-phosphatidylserine (lysoPS) by interacting with high-affinity NGF receptors of the TrkA-type. In RPMCs, type D phosphatidylcholine-selective phospholipases (PLDs) have been postulated to be involved in some exocytotic signaling pathways induced by different agonists. The aim of the present study was to assess a putative functional role of PLD for NGF/lysoPS-induced exocytosis in RPMCs. In 1-[14C]palmitoyl-2-lyso-3-phosphatidylcholine-labelled RPMCs, NGF/lysoPS stimulated the formation of diacylglycerol (DAG) and, in the presence of ethanol (1% [v/v]), phosphatidylethanol (PEtOH). These data indicate PLD-activation by NGF/lysoPS in RPMCs. Preincubation of RPMCs for 2 min with ethanol, an inhibitor of PLD-derived DAG-formation, dose-dependently (IC(50): 0.6% [v/v]) and agonist-selectively inhibited the NGF/lysoPS induced release of [3H]serotonin ([3H]5-HT) in [3H]5-HT-loaded RPMCs, confirming the functional importance of PLD-action. Exocytosis and PEtOH-production was potently inhibited by the broad-spectrum serine/threonine kinase inhibitor staurosporine and activated by the protein kinase C(PKC)-activator PMA (phorbol-12-myristate-13-acetate) suggesting a role for PKC as mediator for NGF/lysoPS-induced activation of PLD.     

The activity of phosphoinositide-specific phospholipase C is required for vegetative growth and cell wall regeneration in Coprinopsis cinerea

Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.     

Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.     

3Beta-hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity

6-Aza steroid analogues were synthesized as PI-PLC inhibitors. The most active compound, 3beta-hydroxy-6-aza-cholestane (1) showed potent PI-PLC inhibition (IC50 = 1.8 microM), similar to that of the commercially available steroid analogue U73122 (IC50 = 1-2.1 microM). Compound 1 exhibited significant growth inhibition effects (IC50 = 1.3 microM in each case) against MCF-7 and HT-29 cancer cells in in vitro cell culture. Compound 1 also inhibited the in vitro adhesion and transmigration of HT-1080 fibrosarcoma cells at 2.5 and 5.0 microM, respectively. In vivo, compound 1, at 1 mg/kg/day, reduced the volume of MCF-7 tumors in xenograft models, without weight loss in mice. Structure activity relationships of this series of compounds revealed that a hydrophobic cholesteryl side chain, 3beta-hydroxy group and a C-6 nitrogen containing a hydrogen atom at position-6 are crucial for activity. N-Maleic amidoacid derivative 11 also exhibited weak inhibition (IC50 = 16.2 microM).     

Characterization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Lilium daviddi pollen

The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.     

U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.

Stimulation of rat pancreatic acinar cells with low concentrations of phosphatidylinositol (PI)-linked secretagogues induces [Ca2+]i oscillations, without measurable changes in the formation of inositol 1,4,5-trisphosphate. Therefore, we tested U73122 a new phospholipase C inhibitor to determine if PI turnover is necessary for the generation of [Ca2+]i oscillations. In acini prelabeled with [3H]inositol, PI hydrolysis on stimulation with either cholecystokinin or carbachol was inhibited dose-dependently by U73122, with a maximal effect seen at 10 microM; the formation of inositol 1,4,5-trisphosphate, measured using a radioreceptor assay, was also similarly inhibited. By contrast secretin- or vasoactive intestinal peptide-stimulated production of cAMP was unaffected by 10 microM U73122. These studies indicate that U73122 is a relatively specific inhibitor of G-protein-mediated phospholipase C activation in pancreatic acini. In fura-2-loaded acini, U73122 inhibited the increases in [Ca2+]i stimulated by these high concentrations of secretagogues which can be demonstrated to elicit PI turnover. The [Ca2+]i signal generated by directly stimulating G-proteins with sodium fluoride was also inhibited by U73122; however, the [Ca2+]i rise induced by thapsigargin was unaffected. These data indicate that the mechanism of inhibition was distal to the occupation of cell surface receptors but did not involve an interference of Ca2+ metabolism in general. When [Ca2+]i oscillations were elicited by low concentrations of cholecystokinin or carbachol, U73122 rapidly inhibited the oscillating [Ca2+]i signal. In contrast, oscillations induced by an analogue of cholecystokinin, JMV-180, which does not stimulate changes in PI metabolism at any concentration, were unaffected. This indicates that cholecystokinin- and carbachol-induced oscillations are probably initiated by small, localized changes in PI metabolism, which are not readily detectable. However, the inability of U73122 to inhibit JMV-180-induced oscillations indicates that PI metabolism may not necessarily be a prerequisite for the generation of [Ca2+]i oscillations.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.     

Is Ca2+ release from internal stores involved in membrane excitation in characean cells?

An action potential in characean cells is accompanied by an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) which subsequently causes cessation of cytoplasmic streaming. Two Ca(2+ )origins are postulated for the increase in [Ca(2+)](c), extracellular and intracellular ones. For the extracellular origin, a Ca(2+) influx through voltage-dependent Ca(2+)-permeable channels is postulated. For the intracellular origin, a chain of reactions is assumed to occur, involving phosphoinositide-specific phospholipase C (PI-PLC) activation, production of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-dependent Ca(2+) release from internal stores [Biskup et al. (1999) FEBS Lett. 453: 72]. The hypothesis of the intracellular Ca(2+) origin was tested in three ways: injection of IP(3) into the streaming endoplasm, application of inhibitors of PI-PLC (U73122 and neomycin) and application of an inhibitor of IP(3)-receptor (2-aminoethoxydiphenyl borate; 2APB). Injection of 1 mM IP(3) into Chara cells did not change the rate of cytoplasmic streaming. Both U73122 (20 micro M) and neomycin (200 micro M) did not affect the generation of the action potential, cessation of cytoplasmic streaming and the increase in [Ca(2+)](c) caused by electric stimulus even 20-30 min after application. 2APB depolarized the membrane and inhibited the excitability of the plasma membrane. The results are not consistent with the data obtained by Biskup et al. (1999) who found inhibition of the excitatory inward current by neomycin and U73122. The hypotheses of internal and external Ca(2+) origins are discussed in the light of the present results.     

ATP receptor. A putative receptor-operated channel in PC-12 cells.

External ATP induces [3H] dopamine [( 3H]DA) release in rat pheochromocytoma cells (PC-12 cells). The ATP-induced release is a saturable process with half-effective concentration of EC50 = 80 microM. ADP is a poor secretagogue of [3H]DA (one-sixth of ATP) and AMP is devoid of secretory capabilities. Adenosine and the non-hydrolyzable analogues of ATP, AppNHp and AppCp are ineffective as inducers of [3H]DA, release, or as inhibitors of the ATP-induced [3H]DA release. The most potent antagonist of ATP-induced release is Coomassie Blue (IC50 = 25 microM), compared to ADP beta S (IC50 = 500 microM). The overall rank order of potency is ATP greater than ADP much greater than AMP greater than adenosine, which is characteristic of the P2-purinergic receptor. ATP-induced secretion is absolutely Ca2+ dependent, indicating an exocytotic process and is independent of Mg2+ (up to 2 mM) suggesting that the active species is not ATP4-. (a) The ATP-induced 45Ca2+ influx into the cells is in good correlation to ATP induction of release (IC50 = 80 and 90 microM, respectively) and is carried over to ADP which has a diminished ability to induce both release and 45Ca2+ influx. (b) Divalent cations (Ba2+ greater than Sr2+ greater than Ln3+ greater than Mn2+) replace Ca2+ and support ATP-induced release similar to their effectiveness in supporting bradykinin- and K+ (50 mM)-induced release in PC-12 cells (Weiss, C., Sela, D., and Atlas, D. (1990) Neurosci. Lett. 119, 241-245). Combined together the absolute requirement of [Ca2+]ex for release, inhibition of release by Gd3+ (IC50 = 100 microM), Ni2+, and Co2+ (IC50 = 1 mM), and support of release by Ba2+, Sr2+, and Mn2+, we suggest that ATP induces Ca2+ entry via ligand-operated Ca2+ channels as previously suggested for ATP in smooth muscle cells (Benham, C.D., Bolton, T.B., Byren, N.G., and Large, W.A. (1987) J. Physiol. (Lond.) 387, 473-488). No significant inhibition by 1 microM verapamil, 10 microM nifedipine, or 2 mM Cd2+ argues against ATP activation of voltage-dependent Ca2+ channels as similarly shown for ATP-induced [3H]noradrenaline release (Inoue, K., Nakazawa, K., Fujimoro, K., and Takanaka, A. (1989) Neurosci. Lett. 106, 294-299). Thus, the widely distributed ATP receptor might play an essential role in Ca2+ homeostasis of the cell by introducing Ca2+ into the cell via specific ligand-gated Ca2+ channels.     

High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C

A nonradioactive spectrometric assay for the evaluation of inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC) is described. l-alpha-Phosphatidylinositol from bovine liver was used as substrate in the presence of the micelle-forming detergent deoxycholic acid. PI-PLC isolated from Bacillus cereus and crude cytosol fractions from porcine brain were used as enzyme sources. PI-PLC activity was determined by measuring the release of 1-stearoyl-2-arachidonoyl-sn-glycerol with reversed-phase HPLC and UV detection at 200 nm. PI-PLC from B. cereus was not inhibited by the putative PI-PLC inhibitors U-73122 and ET-18-OCH(3) at 100 microM, whereas the isobenzofuranone derivative 5 blocked the enzyme with an IC(50) of 75 microM. PI-PLC activity present in porcine brain cytosol was decreased by all three test compounds at 100 microM to approximately 30 to 50%.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Pituitary thyrotropin-releasing hormone receptors: local anesthetic effects on binding and responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel kappa agonist inhibits [3H]nimodipine binding to a Ca++ channel receptor protein in rat brain

The novel kappa agonist U50-488H in vitro produced a concentration-dependent decrease (0.25-25 microM) in [3H]nimodipine binding in neuronal P2 fractions [corrected] from rat brain cortex. Kinetic analysis indicates the decrease in binding results from a reduced Bmax with no change in affinity (Kd). The kappa antagonist, MR2266, blocked the decrease in [3H]nimodipine binding to membrane fractions. At equimolar concentrations (25 microM), morphine in vitro had no effect on [3H]nimodipine binding, while U50-488H demonstrated potent inhibition. Further kinetic analysis indicates that the IC50 for U50-488H is 0.5-0.7 microM with a KI by a Dixon plot of 1.5-1.7 microM [corrected]. These results suggest that kappa opiate receptors may be coupled to dihydropyridine receptors and as a result modulate Ca++ entry and neurotransmitter release in brain neurons.     

Characteristics of collagen-induced Ca2+ mobilization in bovine platelets

We characterized the collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) of bovine platelets loaded with the Ca2+ indicator Fura-PE3/AM. Collagen (10 micrograms/ml)-induced increase in [Ca2+]i was only partially inhibited by aspirin, a cyclooxygenase inhibitor, or adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, a P2Y1 receptor antagonist), while in human platelets it was almost completely suppressed by aspirin. Collagen-induced increase in [Ca2+]i of bovine platelets was inhibited by U73122 (0.3-5 microM), a phospholipase C inhibitor. Collagen (10 micrograms/ml) increased production of inositol 1,4,5-trisphosphate, which was prevented by pretreatment with U73122 (5 microM). Collagen (10 micrograms/ml) accelerated Mn2+ entry, since the rate of Fura-PE3 quenching by Mn2+ was enhanced by 13-fold following stimulation with collagen. U73122 inhibited the acceleration of Mn2+ entry induced by collagen. PGE1 (2.5 microM) partially inhibited the collagen (50 micrograms/ml)-induced increase in [Ca2+]i in bovine platelets but not in human platelets. The data suggest that collagen-induced Ca2+ mobilization in bovine platelets is mediated by phospholipase C. The Ca2+ mobilization in bovine platelets is different from that in human ones as to the dependency on arachidonic acid metabolites and sensitivity to PGE1.     

Dantrolene and azumolene inhibit [3H]PN200-110 binding to porcine skeletal muscle dihydropyridine receptors.

We tested whether the hydantoin muscle relaxants dantrolene, azumolene, or aminodantrolene could alter the binding of [3H]PN200-110 to transverse tubule dihydropyridine receptors or the binding of [3H]ryanodine to junctional sarcoplasmic reticulum Ca2+ release channels. All three drugs inhibited [3H]PN200-110 binding with azumolene (IC50 approximately 20 microM) 3-5 times more potent than dantrolene or aminodantrolene. In contrast, 100 microM azumolene and dantrolene produced a small inhibition of [3H]ryanodine binding (less than 25%) while aminodantrolene was essentially inert. Hence there was a preferential interaction of hydantoins with dihydropyridine receptors instead of ryanodine receptors. Skeletal muscle dihydropyridine receptors may participate in the mechanism of action of dantrolene and azumolene.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

Inhibition of GTPgammaS-dependent L-isoaspartyl protein methylation by tyrosine kinase inhibitors in kidney

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP. GTPgammaS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by L-isoaspartyl-containing peptides. GTPgammaS was the most potent nucleotide tested, whereas GDPbetaS and ATPgammaS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTPgammaS was reached at a physiological pH in the presence of 10 mM MgCl2. Other divalent cations, such as Cu2+, Zn2+ and Co2+ (100 microM), totally inhibited GTPgammaS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPgammaS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 microM, but increasing the vanadate concentration to more than 40 microM resulted in a dose-dependent inhibition of the GTPgammaS effect. The tyrosine kinase inhibitors genistein (IC50 = 4 microM) and tyrphostin (IC50 = 1 microM) abolished GTPgammaS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPgammaS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPgammaS-stimulated PIMT activity in the kidney cytosol.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors.

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.