U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.     

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.     

U73122 affects the equilibria between the phosphoinositides as well as phospholipase C activity in unstimulated and thrombin-stimulated human and rabbit platelets

The effect of the putative phospholipase C inhibitor U73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione) on platelet phosphoinositide metabolism was examined. In unstimulated rabbit platelets prelabeled with [32P]phosphate and [3H]glycerol, U73122 caused decreases of up to 50% in the amount and labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), but not phosphatidylinositol, within 1 min of addition and inhibited incorporation of [32P]phosphate and [3H]glycerol into PIP2 and PIP during incubations of up to 1 hr. These results point to inhibition by U73122 of the phosphatidylinositol and PIP kinases, although stimulation of the PIP and PIP2 phosphomonoesterases could be involved. In platelets stimulated with thrombin, U73122 blocked the thrombin-induced increases in PIP and phosphatidic acid; most increases in the inositol phosphates were blocked, but significant formation of inositol phosphate was found at 120 sec. The effects on inositol phosphates and phosphatidic acid were consistent with U73122 inhibiting phospholipase C; however, parallel dose-response curves with U73122 for the decreases in PIP2 and inhibition of thrombin-stimulated formation of inositol phosphates indicate that the inhibition of phospholipase C by U73122 may be due to decreased substrate availability rather than direct inhibition. Thrombin-stimulated decreases in PIP2 and PIP, found in the presence of U73122, could be explained by the action of phospholipase C in the absence of resynthesis. Although the changes were not as large, U73122 had a similar effect on PIP2 and PIP in unstimulated and stimulated human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of phospholipase C on human blood platelets

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.     

U73122 blocked the cGMP-induced calcium release in sea urchin eggs

U73122, a phospholipase C inhibitor, dose dependently blocks the cGMP-induced Ca2+ release in sea urchin eggs and homogenates. U73122 inhibition was prevented by cotreatment with dithiothreitol (DTT), but DTT is ineffective when eggs or homogenates were pretreated with U73122. U73122 action is different from the other sulfhydryl reagents, thimerosal and N-ethylmaleimide, which cause Ca2+ release in egg homogenates at high concentration, but at lower concentration have no significant effect on cGMP-induced Ca2+ release. Histone, a reported NAD glycohydrolase (NADase) activator, was found to induce Ca2+ release in egg homogenates via the same pathway as the cGMP response, since histone-induced Ca2+ release is blocked by Rp-8-pCPT-cGMPS, a cGMP-dependent protein kinase (PKG) inhibitor, and nicotinamide, a NADase inhibitor. Histone-induced Ca2+ release is similarly blocked by U73122. The aminosteroid U73122 does not inhibit cADPR-induced Ca2+ release, which is significantly reduced by PKG inhibitors. Furthermore, U73122 has no significant effect on phorbol 12-myristate 13-acetate induced-cytoplasmic alkalinization in intact eggs, which depends on protein kinase C activity. These results suggest that U73122 does not act as a general serine-threonine protein kinase inhibitor, and the aminosteroid inhibition of the cGMP-induced Ca2+ release may interfere with ADP ribosyl cyclase activity.     

Effect of phospholipase C inhibitor U-73122 on antigen-induced airway smooth muscle contraction in guinea pigs

The importance of phospholipase C (PLC) in airway smooth muscle contraction was studied, using an inhibitor of PLC, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122). Tracheas from ovalbumin (OA)-sensitized guinea pigs contracted rapidly after exposure to low concentrations of antigen (OA). However, tracheas treated with U-73122 for 10 min prior to the addition of antigen, demonstrated a 3 log rightward shift in the OA dose-response curve with an IC50 of 7 microM. The analogue of U-73122, 1-[6[[17 beta-3-methoxyestra-1,3,5 trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73433), was approximately 5-fold less active in inhibiting smooth muscle contraction. In addition to the inhibition of antigen-induced smooth muscle contraction, U-73122 inhibited carbachol- and leukotriene D4-induced smooth muscle contraction. Furthermore, U-73122 inhibited in a dose-dependent manner antigen-induced histamine release from guinea pig tracheal tissue. The inhibition of smooth muscle contraction by U-73122 correlated well with the inhibition of polyphosphoinositide mediates smooth muscle contractile responses to muscarinic agonists and leukotrienes as well as antigenic-induced contraction.     

Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.     

Evidence for nitric oxide mediated effects on islet hormone secretory phospholipase C signal transduction mechanisms

We have investigated the putative role of nitric oxide (NO) as a modular of islet hormone release, when stimulated by the muscarinic receptor agonist phospholipase C activator, carbachol, with special regard to whether the IP3-Ca2+ or the diacylglycerol-protein kinase C messenger systems might be involved. It was observed that the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methylester (L-NAME) markedly potentiated insulin release and modestly inhibited glucagon release induced by carbachol. Similarly, insulin release induced by the phorbol ester TPA (protein kinase C activator) was markedly potentiated. Glucagon release, however, was unaffected. Dynamic perifusion experiments with 45C2+ -loaded islets revealed that the inhibitory action of L-NAME on carbachol-stimulated NO-production was reflected in a rapid and sustained increase in insulin secretion above carbachol controls, whereas the 45Ca2+ -efflux pattern was similar in both groups with the exception of a slight elevation of 45C2+ in the L-NAME-carbachol group during the latter part of the perifusion. No difference in either insulin release or 45Ca2+ -efflux pattern between the carbachol group and L-NAME-carbachol group was seen in another series of experiments with identical design but performed in the absence of extracellular Ca2+. However, it should be noted that in the absence of extracellular Ca2+ both 45Ca2+ -efflux and, especially, insulin release were greatly reduced in comparison with experiments in normal Ca2+. Further, in the presence of diazoxide, a potent K+ ATP-channel opener, plus a depolarizing concentration of K+ the NOS-inhibitor L-NAME still markedly potentiated carbachol-induced insulin release and inhibited glucagon release. The enantiomer D-NAME, which is devoid of NOS-inhibitory properties, did not affect carbachol-induced hormone release. TPA-induced hormone release in depolarized islets was not affected by either L-NAME or D-NAME. The pharmacological intracellular NO donor hydroxylamine dose-dependently inhibited insulin release stimulated by TPA. Furthermore, a series of perifusion experiments revealed that hydroxylamine greatly inhibited carbachol-induced insulin release without affecting the 45Ca2+ -efflux pattern. In summary, our results suggest that the inhibitory effect of NO on carbachol-induced insulin release is not to any significant extent exerted on the IP3-Ca2+ messenger system but rather through S-nitrosylation of critical thiol-residues in protein kinase C and/or other secretion-regulatory thiol groups. In contrast, the stimulating action of NO on carbachol-induced glucagon release was, at least partially, connected to the IP3-Ca2+ messenger system. The main effects of NO on both insulin and glucagon release induced by carbachol were apparently exerted independently of membrane depolarization events.     

Inhibition of phospholipase C by U-73122 blocks one component of the receptor current in Limulus photoreceptor

In the ventral nerve photoreceptor of Limulus a short, intense flash evokes a receptor current consisting of three components. In contrast to other hypotheses, we suggested previously that only the second component of the current is activated by the phospholipase C pathway, which releases calcium from intracellular stores by inositol trisphosphate. The present paper gives further evidence to our suggestion. It is demonstrated that U-73122, a specific inhibitor for the phospholipase C, selectively blocks the second-component. The first and third components are moderately affected and could still be activated after the complete block of the second one. Results support the idea that the first and third components are activated by pathways operating independently of phospholipase C.     

Evidence for an increased intracellular free calcium concentration in platelets of bronchial asthma patients

The pathogenesis of bronchial asthma is not yet fully understood. Recently much attention has been given to the hypothesis that intracellular free calcium ([Ca2+]i) metabolism is abnormal in various diseases. In this study we investigated whether [Ca2+]i exists abnormally in subjects with bronchial asthma. The [Ca2+]i in 32 treated or untreated subjects with bronchial asthma were compared with 63 normal subjects. Resting levels of [Ca2+]i were estimated by loading the fluorescent indicator Fura-2 in washed platelets. The [Ca2+]i level in the control subjects was 129.7 +/- 18.0 nM (mean +/- SD). However, in that of the bronchial asthma patients was 152.7 +/- 44.1 nM, significantly higher than that of the control subjects (p < 0.05). It is well recognized that an increase of [Ca2+]i in vascular smooth muscle involves contraction. The findings suggest that the same phenomenon is quite possible in the tracheal smooth muscle and that it plays an important role in the pathogenesis of bronchial asthma.     

Increased expression of phosphatidylinositol-specific phospholipase C resistant prion proteins on the surface of activated platelets

The surface expression of prion protein (PrP(C)) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two-fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrP(C) occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37 degrees C. In comparison, PrP(C) on the surface of platelets, activated at 22 degrees C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrP(C) and P-selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrP(C) was translocated from internal granules to the plasma membrane during activation, as is P-selectin. Platelet PrP(C) was not removed from the surface of platelets by phosphatidylinositol-specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrP(C).     

Serotonin-stimulated calcium responses in human platelets: assay buffer dependency

In Fura-2 labelled human platelets, the observed calcium response to serotonin stimulation is increased if the assay is conducted in a Krebs buffer compared with the HEPES buffer system usually used. The serotonin response in the Krebs buffer system was concentration dependent with an EC50 value of approximately 0.3 microM and was blocked by nanomolar concentrations of the serotonin2-receptor antagonist methiothepin. In a series of samples collected from 11 persons, a reasonable correlation was seen between the response to 1 microM serotonin and the serotonin2A-receptor density measured in corresponding membrane preparations. The calcium response to serotonin was reduced by treatment with hydrogen peroxide. It is concluded that the use of the Krebs buffer gives a more sensitive response of the Fura-2 loaded human platelets to serotonin stimulation than does the use of HEPES buffer and that the increased sensitivity is achieved without affecting the pharmacological properties of the response. For the response to thrombin, the response intensity is affected by the pH of the buffer used but is not sensitive to the buffer composition.     

Receptor-coupled phospholipase C and adenylyl cyclase function with different calcium pools in astrocytes

Astrocytes express phospholipase C (PLC)-coupled metabotropic glutamate receptors (only mGluR5 is detectable) and adenylyl cyclase (AC)-linked beta-adrenergic receptors. Calcium-sensitive effector enzymes are associated with these signal transduction pathways, but the relevant calcium compartments involved were found to be different. mGluR5-linked PLC responded primarily to extracellular Ca2+, suggesting a close spatial relation between the enzyme and Ca2+ entry channels. On the other hand, the calcium-inhibited AC associated with beta-adrenergic receptors was sensitive to intracellular Ca2+ selectively accessible to intracellular Ca2+ chelation. Furthermore, cAMP formation induced by direct activation of AC by forskolin was less responsive to intracellular Ca2+ chelation than that evoked by the receptor-activated AC, raising the possibility of selective access of the receptor to a pool of calcium-inhibited AC and/or the calcium modulation of some components of the coupling pathway.     

Genetic mapping of the human and mouse phospholipase C genes

To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2. Two PLC genes, Plcd1 and Plcg2, mapped to distinct sites on Chr 8. We mapped the human homologs of eight of these genes to six chromosomes by analysis of human x rodent somatic cell hybrids. The map locations of seven of these genes were consistent with previously defined regions of conserved synteny; Plcd1 defines a new region of homology between human Chr 3 and mouse Chr 8.     

Evidence for separate pathways for color and luminance detection mechanisms

We measure threshold versus contrast (TvC) functions for chromatic (red-green) and luminance sine-wave-grating stimuli for (1) the detection of luminance in the presence of color contrast and (2) the detection of color in the presence of luminance contrast. We find that, although these crossed TvC functions both display a dipperlike shape, their facilitation differs from that found for standard uncrossed dipper functions (luminance on luminance or color on color contrast). Their facilitation disappears (cross condition 1) or is reduced (cross condition 2) by randomized presentation of the phase of the test and the mask, and the remaining facilitation (cross condition 2) displays no spatial tuning. We argue that these crossed facilitatory interactions cannot be explained by detection mechanisms with common inputs from color and luminance contrast (a nonindependence of transduction), and we present evidence that instead they reflect the use of local cues in the stimuli. We also measure the luminance-luminance TvC function in the presence of a fixed suprathreshold color contrast. The results demonstrate that, even when the color contrast produces a masking of the luminance thresholds, luminance-luminance facilitation still occurs. Thus the opposing effects of masking and facilitation can occur simultaneously. Furthermore, while luminance-luminance facilitation occurs independently of color contrast, masking can be produced by either contrast. This suggests that masking and facilitation have different underlying origins. Similar results are found for the color detection thresholds in the presence of a luminance pedestal. We conclude that there are separate pathways for the detection of color and luminance contrast, each with no input from the other contrast. We suggest that the cross masking reflects divisive interactions between these pathways that is restricted to high contrasts.     

Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils

In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.     

Density and localization of calcium channels of the L-type in human pulmonary artery

The pharmacological profile and the anatomical localization of Ca2+ channels of the L-type were investigated in the human pulmonary artery to identify possible mechanisms involved in the regulation of the pulmonary vascular tone. Analysis was performed on slide-mounted frozen sections of human pulmonary artery using radioligand binding assay techniques associated with light microscope autoradiography. [3H]-Nicardipine was used as ligand. Human renal and right coronary arteries also were used as systemic reference arteries. Binding of [3H]-nicardipine to sections of human pulmonary artery was time-, temperature- and concentration-dependent, saturable and reversible. In the human pulmonary artery, the apparent equilibrium dissociation constant (Kd) was 0.12+/-0.02 nM and the maximum density of binding sites (Bmax) was 38.15+/-2.25 fmol/mg tissue. Kd values were 0.3+/-0.01 nM and 0.5+/-0.02 in the human renal artery and right coronary artery respectively. Bmax values were 248+/-16 fmol/mg tissue and 173+/-9.5 fmol/mg tissue in the human renal artery and right coronary artery respectively. The pharmacological profile of [3H]-nicardipine binding to sections of human pulmonary artery was consistent with the labeling of Ca2+ channels of the L-type. It was similar in the pulmonary artery and in the human renal and right coronary arteries. Light microscope autoradiography revealed a high density of [3H]-nicardipine binding sites within smooth muscle of the tunica media of human pulmonary artery as well as of human renal and right coronary arteries. A lower accumulation of the radioligand occurred in the tunica adventitia. No specific binding was noticeable in the tunica intima. Our data suggest that human pulmonary artery expresses Ca2+ channels of the L-type sensitive to dihydropyridines. These sites have similar affinity and lower density than those expressed by systemic arteries. The presence of Ca2+ channels of the L-type in human pulmonary artery suggests that their pharmacological manipulation may be considered in the treatment of pulmonary hypertension.     

Effects of divalent cations on calcium levels, aggregation and morphology of human blood platelets in vitro

Some effects of cadmium, nickel, strontium and manganese ions upon human platelets in vitro are reported. Both Mn and Cd induced aggregation, Mn being the most effective. Ni and Sr did not have this effect. Scanning electron microscopic observation showed that Mn and Cd, but not Ni and Sr induced shape changes in the platelets. X-ray microanalysis of pyroantimonate fixed platelets revealed that calcium was displaced from both membrane and cytoplasmic regions by all four cations. The magnitude of displacement occurred in the sequence Mn > Cd > Ni approximately or equal to Sr. Manganese was detected both in the cytoplasm and membrane region of those platelets treated with this ion. It is suggested that the cations are influencing directly certain macromolecular ligands whose condition controls platelet surface properties.