Increase In Intracellular Free Ca2 Research Articles

Evidence That 2-Arachidonoylglycerol but Not N-Palmitoylethanolamine or Anandamide Is the Physiological Ligand for the Cannabinoid CB2 Receptor: COMPARISON OF THE AGONISTIC ACTIVITIES OF VARIOUS CANNABINOID RECEPTOR LIGANDS IN HL-60 CELLS

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.

INHIBITION OF PROLIFERATION OF CONTAMINATING FIBROBLASTS BY D-VALINE IN CULTURES OF SMOOTH MUSCLE CELLS FROM HUMAN MYOMETRIUM

Replacement of L-valine with D-valine in a standard culture medium can selectively inhibit fibroblast proliferation. The aim of the present study was to investigate whether human myometrial cells cultured with D-valine instead of L-valine can survive and express their characteristics. Cultured cells (95–98%) maintain expression of the intermediate filament desmin, which is the specific marker for mature muscle cells. By transmission electron microscopy, the cells showed the general morphology of smooth muscle cells in culture. Oxytocin in serum-free culture medium at 37°C (5min) caused a concentration-dependent increase in cellular Na and total Ca, and a decrease in K content as determined by X-ray microanalysis. The percentage of cells cultured with D-valine responding to oxytocin stimulation was larger than that of cells cultured with L-valine, suggesting less contamination of smooth muscle cells by fibroblasts in the presence of D-valine. As shown by measurements with fura-2, D-valine-cultured cells retained the characteristic increase in intracellular free Ca2+ions after oxytocin stimulation.

The effects of angiotensin II, endothelin-1, and protein kinase C inhibitor on DNA synthesis and intracellular calcium mobilization in vascular smooth muscle cells from young normotensive and spontaneously hypertensive rats

Angiotensin II (Ang-II) and endothelin 1 (ET-1) are important peptides that induce a prolonged vasoconstriction and enhance proliferation of vascular smooth muscle cells (VSMC). These substances may have an important role in the development of hypertension and atherosclerosis. Our objectives were to determine whether there are inborn differences in the proliferation patterns of VSMC obtained from spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY) by studying the effects of Ang-II and ET-1 on VSMC from those strains before the onset of hypertension, and to evaluate the roles of protein kinase C (PKC) and intracellular Ca 2+ in the mechanism of action of ET-1 and Ang-II. VSMC from aortas of young (1- to 2-week–old) SHR and WKY rats were grown as primary cultures in plates for 48 h. The cells were incubated with Ang-II (0.1 to 1000 nmol/L) or ET-1 (0.1 to 100 nmol/L). VSMC were also incubated in the presence of various concentrations of a PKC inhibitor, chelerythrine (0.1–10 nmol/L). Thymidine incorporation into DNA was measured as an indicator of DNA synthesis. Intracellular free Ca 2+ was determined by using FURA-2AM. ET-1 and Ang-II caused a marked dose-dependent enhancement of thymidine incorporation into DNA. The responses of VSMC from WKY and SHR to Ang-II and ET-1 were similar. In both strains, chelerythrine caused a dose-dependent suppression in the activity of ET-1 and Ang-II. However, VSMC from SHR incubated in the presence of ET-1 were more susceptible to the inhibitory effect of chelerythrine. Both Ang-II and ET-1 induced an increase of intracellular free Ca 2+. ET-1 induced a larger increase than Ang-II (190% and 100% greater than baseline free Ca 2+ levels, respectively), in spite of a lower concentration of ET-1 (ET-1 = 30 nmol/L; Ang-II = 100 nmol/L). Ang-II and ET-1 exerted a similar mitogenic effect on primary cultures of VSMC obtained from young SHR before the development of hypertension, compared with WKY. The mitogenic activity of Ang-II and ET-1 was accompanied by an increase of intracellular free Ca 2+. The effect of ET-1 upon intracellular Ca 2+ was stronger than that of Ang-II. VSMC cultures of SHR stimulated with ET-1 were more susceptible to PKC inhibition than those of WKY. The similarity of the effects of Ang- II and ET-1 on SHR and WKY does not exclude their role in the pathogenesis of hypertension and atherosclerosis, and further studies should be carried out to determine their role.

Phospholipase C-γ as a Signal-Transducing Element

A ubiquitous signaling event in hormonal responses is the phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the metabolite second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerol. The former provokes a transient increase in intracellular free Ca2+, while the latter serves as a direct activator of protein kinase C. In tyrosine kinase-dependent signaling pathways this reaction is mediated by the PLC-γ isozymes. These are direct substrates of many tyrosine kinases in a wide variety of cell types. The mechanism of PLC-γ activation involves its association with and phosphorylation by receptor and non-receptor tyrosine kinases, as well as interaction with specialized adaptor molecules and, perhaps, other second messenger molecules. However, the biochemistry of PLC-γ is at a more advanced state than a clear understanding of exactly how this signaling element functions in the generation of a mitogenic response.

Carbachol-induced changes in excitability and [Ca2+]i signalling in projection cells of medial entorhinal cortex layers II and III.

The entorhinal cortex (EC) is a major gateway for sensory information into the hippocampus and receives a cholinergic input from the forebrain. Therefore, we studied muscarinic effects on excitability and intracellular Ca2+ signalling in layer II stellate and layer III pyramidal projection neurons of the EC. In both classes of neurons, local pressure-pulse application of carbachol (1 mM) caused small, atropine-sensitive membrane depolarizations that were not accompanied by any detectable changes in [Ca2+]i. At a higher concentration (10 mM), carbachol induced a larger membrane depolarization associated with synaptic oscillations and epileptiform activity in both classes of neurons. In contrast to the intrinsic theta rhythm in stellate cells with one dominant peak frequency at approximately 7 Hz, the synaptically mediated oscillation induced by carbachol showed three characteristic peaks in the theta and gamma frequency range at approximately 11, 23 and 40 Hz. Although carbachol-induced epileptiform activity was associated with increases in intracellular free Ca2+ in both layer II and III cells, the observed [Ca2+]i accumulation was significantly larger in layer III than in layer II cells. Responses to intracellular current injections showed differences in Ca2+ accumulation in layer II and III cells at the same membrane potentials, suggesting a dominant expression of low- and high-voltage-activated Ca2+ channels in these layer II and III cells, respectively. In conclusion, we present evidence for significant differences in the [Ca2+]i regulation between layer II stellate and layer III pyramidal cells of the medial EC.

Evidence that lipoproteins are carriers of bioactive factors.

We recently demonstrated that the mitogenic effect of LDL (100 microg/mL) as well as its early intracellular signaling pathway are mediated by a pertussis-toxin (PTX)-sensitive G(i) protein-coupled receptor that is independent from its classical receptor and involves activation of extracellular response kinases (ERK1/2) (also known as p44(mapk)/p42(mapk)). In the present study we examined whether LDL-adherent factors may be responsible for some of the effects of LDL. The term "signaling activity" is used to characterize fractions that cause an increase in intracellular free Ca(2+) concentration or stimulate ERK1/2 and c-fos mRNA expression. LDL, HDL, and VLDL stimulate ERK1/2 with the following order of potency: LDL>HDL>VLDL. After delipidation of LDL with chloroform/methanol/water mixtures a PTX-sensitive signaling activity was found in one fraction arbitrarily called LDL-F. After further analysis of LDL-F compounds by high pressure liquid chromatography, a PTX-sensitive signaling activity was detected only in the fraction with a retention time of 33 minutes (arbitrarily called LDL-F33). Similarly, after separation of sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) by high pressure liquid chromatography, a PTX-sensitive signaling activity was found in the fractions 33 and 33 to 35, respectively. These findings demonstrate that the effects of LDL-F33 are mimicked by similar fractions collected from SPP/SPC, hence suggesting that these LDL-adherent molecules are possibly closely related to SPP/SPC. A PTX-sensitive signaling activity was also detected in HDL and HDL-F33. Therefore, LDL and other lipoproteins may function as carriers for bioactive phospholipids thereby contributing to the development of coronary artery disease. Our findings support a new research concept that may contribute in elucidating cellular mechanisms promoting coronary artery disease.

Trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets.

1. The phytoestrogenic compound trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. 2. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. 3. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 microM. trans-Resveratrol at 0.1, 1.0 and 10.0 microM produced 20+/-6, 37+/-6 and 57+/-4% inhibition respectively of the effect of thrombin (0.01 u ml(-1)) to increase [Ca2+]i. 4. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 microM trans-resveratrol producing 10+/-5, 30+/-5 and 50+/-7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. 5. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. 6. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. 7. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin.

Regulation of epidermal homeostasis through P2Y2 receptors.

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Decreased intraplatelet Ca2+ release and ATP secretion in pediatric nephrotic syndrome.

Platelets play an important role in the natural history of idiopathic nephrotic syndrome (NS). Although thromboembolic events are rare, the activation of circulating platelets is generally considered an important factor in the prethrombotic state in children with NS. Platelet-activating factor (PAF), a potent endogenous phospholipid mediator of inflammation, stimulates intracellular free calcium (Ca2+) mobilization, aggregation, and release reactions in platelets obtained from normal donors. Platelet-related effects of PAF in children with NS are unknown. We studied PAF-induced intracellular Ca2+ mobilization in washed platelets and ATP secretion in platelet-rich plasma in 34 children with idiopathic NS and in 7 healthy children. There was a significant decrease in ATP secretion: 0.021+/-0.011 microg/10(7) cells with 20 nM PAF and 0.089+/-0.017 microg/10(7) platelets with 200 nM PAF versus control values (0.195+/-0.004 microg/10(7) and 0.813+/-0.09 microg/10(7), respectively). Moreover, PAF-evoked increase in intracellular free Ca2+ concentration was twofold lower in NS patients than in control subjects (230.1+/-22.4 nM versus 455.6+/-15.3 nM). Also, thrombin-induced intracellular free Ca2+ mobilization was diminished in children with NS compared with the control group. Thus, contrary to expectations, a decrease of platelet reactivity in response to PAF in vitro was observed in children with idiopathic NS. We suggest that this decreased platelet reactivity may reflect a period refractory to PAF and may be considered as platelet desensitization to PAF released in vivo in children with NS.

Regulation of gene expression and secretory functions in oxygen-sensing pheochromocytoma cells

The cellular response to hypoxia is complex. Specialized oxygen chemosensitive cells that are excitable respond to reduced O 2 by membrane depolarization, altered gene expression, and neurotransmitter secretion. We have used the O 2-sensitive pheochromocytoma (PC12) cell line to investigate the cellular response to hypoxia. Here, we present evidence that membrane depolarization and increased intracellular free Ca 2+ are major regulatory events in these cells. Membrane depolarization is mediated by the inhibition of a slow-inactivating voltage-dependent potassium (K) channel. Evidence from molecular biology and patch-clamp studies indicate that the O 2-sensitive K channel is a member of the Kv1 family. We also reviewed findings on the regulation of gene expression in PC12 cells during hypoxia. An increase in intracellular free Ca 2+ is required for hypoxia-induced transcription of a number of genes including tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters, and several of the immediate early genes. We also reviewed the role of dopamine (DA) and adenosine (ADO) receptors in regulation of membrane depolarization and gene expression.

Serotonin-induced platelet intracellular Ca2+ responses in untreated depressed patients and imipramine responders in remission

Background: Intracellular Ca2+ metabolism in platelets has been investigated as a peripheral marker of affective disorders.Methods: We investigated the intracellular free Ca2+ concentration in platelets in both untreated depressed patients with no medications and patients in remission who were treated by imipramine (IMI) (IMI responders) using a Ca2+-sensitive fluorescent probe fura-2.Results: The increases in intracellular free Ca2+ concentration in platelets induced by stimulation with serotonin (5-HT) ([Ca2+]Δ) were significantly higher in both the untreated patients and the IMI responders compared with healthy controls; however, there were no significant differences in the basal Ca2+ levels in the platelets ([Ca2+]B) among the three groups. On the other hand, in the IMI responders, we observed positive correlations between the duration of the remission and [Ca2+]B, but not [Ca2+]Δ.Conclusions: Our present data suggest that the enhancement of 5-HT2A-induced Ca2+ responses persisted after remission in depressed patients, and that the duration of the remission is a factor varying the intracellular basal Ca2+ levels.

High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations

We measured glutamate-stimulated increases in intracellular free Ca 2+ concentrations ([Ca 2+] i) in cultured rat forebrain neurons loaded with both a high- and a low-affinity Ca 2+ indicator. In these dual-dye studies, the high-affinity indicators Fluo-3 and Fura-2 gave both qualitatively and quantitatively different results than the low-affinity indicators Mag-fura-2 and Calcium Green-5N. The glutamate-stimulated peak [Ca 2+] i reported using Fluo-3 and Fura-2 were less than 6 μM while the low-affinity indicators Mag-fura-2 and Calcium Green-5N indicated [Ca 2+] i responses were more than 12 μM. The shapes of the responses obtained with the two types of dyes were also different, and only the low-affinity indicators effectively demonstrated that [Ca 2+] i continues to rise during prolonged (5-min) stimulations. These dual-dye studies also revealed that kainate- and depolarization-induced [Ca 2+] i responses could be differentiated from glutamate responses only with the low-affinity indicators. These results suggest that Fluo-3 and Fura-2 underestimate [Ca 2+] i induced by excitotoxic glutamate stimuli and that these responses are greater than have previously been reported. These studies also reveal, in contrast to previous reports, that excitotoxic stimuli do indeed cause increases in [Ca 2+] i that are greater than those produced by non-toxic stimuli.

Adhesion-Dependent Degranulation of Neutrophils Requires the Src Family Kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.

Cytochemical localization of calcium in prefusion myoblasts from the chick embryo myotome.

Myoblast fusion is a Ca2+-dependent process. The aim of this report was to study the localization of Ca2+ in prefusion myoblasts from the brachial somites of chick embryos (51-108 h of incubation), using the potassium pyroantimonate cytochemical method. When observed under a transmission electron microscope, electron-dense precipitates of Ca2+-antimonate were found in the basement membrane of the myotome, which separates the myotome from the adjacent mesenchyma. Within myoblasts, triads and sarcoplasmic reticulum associated with the first newly formed sarcomeres were observed, but a T-tubule network was not found. Moreover, Ca2+-antimonate precipitates were not observed in structures resembling T-tubules or sarcoplasmic reticulum. The results suggest that sarcomerogenesis and sarcoplasmic reticulum development occur simultaneously and that prefusion myoblasts have neither a T-tubule network nor Ca2+ deposits on sarcoplasmic reticulum. Small Ca2+ pools were found in the myoblast nuclei, cytoplasmic vesicles and mitochondrias. Ca2+-antimonate precipitates periodically distributed at the cell periphery, close to the cell membrane, were observed. These precipitates could represent internal Ca2+ stores located in the peripheral couplings and it is proposed that these pools of Ca2+ could be mobilized before fusion, leading to the increase in free intracellular Ca2+ that precedes myoblast fusion.

Evidence That the Cannabinoid CB1 Receptor Is a 2-Arachidonoylglycerol Receptor: STRUCTURE-ACTIVITY RELATIONSHIP OF 2-ARACHIDONOYLGLYCEROL, ETHER-LINKED ANALOGUES, AND RELATED COMPOUNDS

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.

Endothelium-derived nitric oxide and vascular physiology and pathology.

In 1980, Furchgott and Zawadzki demonstrated that the relaxation of vascular smooth muscle cells in response to acetylcholine is dependent on the anatomical integrity of the endothelium. Endothelium-derived relaxing factor was identified 7 years later as the free radical gas nitric oxide (NO). In endothelium, the amino acid L-arginine is converted to L-citrulline and NO by one of the three NO synthases, the endothelial isoform (eNOS). Shear stress and cell proliferation appear to be, quantitatively, the two major regulatory factors of eNOS gene expression. However, eNOS seems to be mainly regulated by modulation of its activity. Stimulation of specific receptors to various agonists (e.g., bradykinin, serotonin, adenosine, ADP/ATP, histamine, thrombin) increases eNOS enzymatic activity at least in part through an increase in intracellular free Ca2+. However, the mechanical stimulus shear stress appears again to be the major stimulus of eNOS activity, although the precise mechanisms activating the enzyme remain to be elucidated. Phosphorylation and subcellular translocation (from plasmalemmal caveolae to the cytoskeleton or cytosol) are probably involved in these regulations. Although eNOS plays a major vasodilatory role in the control of vasomotion, it has not so far been demonstrated that a defect in endothelial NO production could be responsible for high blood pressure in humans. In contrast, a defect in endothelium-dependent vasodilation is known to be promoted by several risk factors (e.g., smoking, diabetes, hypercholesterolemia) and is also the consequence of atheroma (fatty streak infiltration of the neointima). Several mechanisms probably contribute to this decrease in NO bioavailability. Finally, a defect in NO generation contributes to the pathophysiology of pulmonary hypertension. Elucidation of the mechanisms of eNOS enzyme activity and NO bioavailability will contribute to our understanding the physiology of vasomotion and the pathophysiology of endothelial dysfunction, and could provide insights for new therapies, particularly in hypertension and atherosclerosis.

The Antiplatelet Activity of Rutaecarpine, an Alkaloid Isolated from Evodia rutaecarpa, Is Mediated through Inhibition of Phospholipase C

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5×10 8/ml), rutaecarpine (100 and 200 μM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 μM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 μM) dose-dependently inhibited the increase in intracellular free Ca 2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 μM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 μM) significantly inhibited [ 3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Rutaecarpine (100 and 200 μM) did not significantly affect nitrate production in collagen (10 μg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 μM) dose-dependently inhibited [ 3H]inositol monophosphate formation stimulated by collagen (10 μg/ml) in [ 3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A 2 formation, and then inhibition of [Ca 2+]i mobilization of platelet aggregation stimulated by agonists.

Medium change amplifies mitogen-activated protein kinase-mediated prostaglandin E 2 synthesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts, changing the culture medium prior to stimulation resulted in an augmentation of bradykinin-induced prostaglandin E 2 synthesis. The augmentation depended on the duration of the exposure to the fresh medium, with a maximum effect at 1 h. Fetal calf serum in the fresh medium was essential for augmented prostaglandin E 2 synthesis. The medium change slightly augmented the bradykinin-induced increase in intracellular free Ca 2+ concentration and phosphoinositide hydrolysis with a different time course from that for prostaglandin E 2 synthesis. 4′,5,7-Trihydroxyisoflavone (genistein) and 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitors of tyrosine kinases, and 2′-amino-3′-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase (MAPK) kinase, attenuated the increase in prostaglandin E 2 synthesis. Bradykinin caused phosphorylation of cytosolic phospholipase A 2 and p42/p44 MAPK, which was augmented by the medium change. From the results, it is concluded that activation of MAPK and cytosolic phospholipase A 2 is involved in the augmentation of prostaglandin E 2 synthesis produced by the medium change.

Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells.

1. In A7r5 cells loaded with the Ca2+ indicator fura-2, we examined the effect of a Ca2+ channel blocker SK&F 96365 on increases in intracellular free Ca2+ concentrations ([Ca2+]i) and Mn2+ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed. 2. Higher concentrations (> or = 10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca2+]i: removal of extracellular Ca2+ abolished only the latter. A blocker of L-type voltage-operated Ca2+ channel (VOC) nifedipine at 1 microM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 microM). 3. Lower [ET-1] (< or = 1 nM) evoked only a sustained elevation in [Ca2+]i which depends on extracellular Ca2+. The elevation was partly sensitive to nifedipine but not SK&F 96365. 4. In the presence of 1 microM nifedipine, higher [ET-1] increased the rate of Mn2+ quenching but lower [ET-1] had little effect. 5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of -60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca2+ in the bath solution with Mn2+. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365. 6. In a bath solution containing only Ca2+ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn2+, whereas that at higher [ET-1] was partly sensitive to SK&F 96365. 7. These results indicate that in addition to VOC, ET-1 activates two types of Ca2+-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn2+.

Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes: THE ROLES OF Ras AND MITOGEN-ACTIVATED PROTEIN KINASES

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes, i.e. those for c-Fos, skeletal alpha-actin, atrial natriuretic factor, and the alpha3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.