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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract: The effect of the endogenous cannabinoid anandamide on cytosolic free Ca2+ concentration ([Ca2+]i) and proliferation is largely unknown. This study examined whether anandamide altered Ca2+ levels and caused Ca2+-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 78% by removing extracellular Ca2+. The anandamide-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and the cannabinoid receptor antagonist AM 251, but was inhibited differently by aristolochic acid, WIN 55,212-2 (a cannabinoid receptor agonist), phorbol ester, GF 109203X and forskolin. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), anandamide-induced Ca2+ release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca2+ release. At concentrations of 100 μM and 200 μM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 μM anandamide was completely reversed by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca2+]i rises by causing Ca2+ release from endoplasmic reticulum and Ca2+ influx from extracellular space. Furthermore, anandamide can cause Ca2+-dependent cytotoxicity in a concentration-dependent manner.

Anandamide (N-arachidonoyl-ethanolamine) is the first endogenous ligand of cannabinoid receptors to be discovered. Yet, anandamide can also act in a cannabinoid receptor-independent manner (Di Marzo et al. 2002). Anandamide is present in central and peripheral tissues, and can reach different organs once injected intravenously (Willoughby et al. 1997). Some actions of anandamide result from activation of G proteins, particularly those of the Gi/o family. Signal transduction pathways that are regulated by these G proteins include inhibition of adenylyl cyclase, inhibition of voltage-gated Ca2+-currents (Chemin et al. 2001), activation of K+ currents, activation of kinases and induction of early genes. Effects of anandamide that are not mediated via cannabinoid receptors include inhibition of Ca2+ channels, stimulation of VR1 vanilloid receptors (Ross 2003), transient changes in intracellular Ca2+, and disruption of gap junction function (Howlett & Mukhopadhyay 2000).

Kidneys possess both the amidase that degrades anandamide and transcripts for anandamide receptors; thus, an anandamide signaling system exists in the kidney where it exerts vasorelaxant and neuromodulatory effects (Deutsch et al. 1997). Anandamide is metabolized into prostaglandin, arachidonic acid, thromboxane, prostacyclin, glycerol esters and ethanolamides (Kozak et al. 2002). In kidneys, anandamide has been shown to decrease glomerular filtration rate via both cannabinoid receptors and non-cannabinoid receptors (Koura et al. 2004); however, the underlying mechanism is largely unclear.

A rise in intracellular free Ca2+ levels ([Ca2+]i) is a crucial message for diverse pathophysiological events in all cell types (Berridge 2005). However, an unregulated [Ca2+]i rise is cytotoxic and can lead to necrosis, dysfunction of proteins, interference of ion movement, etc. (Annunziato et al. 2003). Anandamide has been shown to induce transient [Ca2+]i rises via CB2 cannabinoid receptors linked to phospholipase C in calf pulmonary endothelial cells (Zoratti et al. 2003); and also to increase [Ca2+]i in human arterial endothelial cells (Fimiani et al. 1999). The effect of anandamide on[Ca2+]i in renal tubular cells has not been explored previously. Given the action of anandamide on kidney function, the purpose of this study was to investigate the effect of this ligand on Ca2+ signaling in renal tubular cells. The Madin Darby canine kidney (MDCK) cell line is a useful model for renal cell research. It has been shown that in this cell , [Ca2+]i can be increased in response to the stimulation of various endogenous and exogenous compounds, such as ATP (Jan et al. 1998a), linoleamide (Huang & Jan 2001), and oestrogens (Chen et al. 2002) etc.

Using fura-2 as a fluorescent Ca2+ indicator, this study shows that anandamide induces [Ca2+]i rises in a concentration-dependent manner in MDCK cells. The time course and the concentration-response relationship, the Ca2+ sources of the Ca2+ signal, the role of phospholipase C, protein kinase C and cAMP in the signal were explored. The effects of anandamide on cell growth and its Ca2+-dependence were also examined by using the fluorescent dye WST-1.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture. MDCK cells obtained from American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated foetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

Solutions. Ca2+-containing medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, and 5 mM glucose. In Ca2+-free medium, Ca2+ was substituted with 0.1 mM EGTA and 2 mM MgCl2. Anandamide was dissolved in ethanol as a stock solution. The other agents were dissolved in water, ethanol or dimethyl sulfoxide as stock solutions. The concentration of organic solvents in the solution used in experiments did not exceed 0.1%, and did not alter basal [Ca2+]i.

[Ca2+]i measurements. Trypsinized cells (106/ml) were loaded with 2 μM of the acetoxymethyl ester form of fura-2, fura-2/AM, for 30 min. at 25 ° in culture medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 °) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer by recording excitation signals at 340 nm and 380 nm and emission signal at 510 nm at 1 sec. intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 (plus 5 mM CaCl2) and 10 mM EGTA sequentially at the end of each experiment. [Ca2+]i was calculated as previously described (Grynkiewicz et al. 1985).

Cell proliferation assay. The measurement of cell proliferation was based on the ability of viable cells to cleave tetrazolium salts by mitochondrial dehydrogenases. Augmentation in the amount of developed color directly correlated with the number of live cells. Assays were performed according to manufacturer's instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at 10,000 cells/well in culture medium for 24 hr in the presence of zero or different concentrations of anandamide. The cell proliferation reagent WST-1 (4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (10 μl pure solution) was added to samples after anandamide treatment, and cells were incubated for 30 min. in a humidified atmosphere. In experiments using BAPTA to chelate intracellular Ca2+, fura-2-loaded cells were treated with 20 μM BAPTA/AM for 1 hr prior to anandamide incubation. The cells were washed once with Ca2+-containing medium and incubated with or without 100 μM anandamide for 24 hr. The absorbance of samples (A450) was determined using enzyme-linked immunosorbent assay (ELISA) reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value. Experiments were repeated three times in six replicates.

Chemicals. The reagents for cell culture were from Gibco. Fura-2/AM (cat.# F1201) and BAPTA/AM (cat.# A-4926) were from Molecular Probes. Anandamide (cat.# A-176), AM 251 (cat.# CR-108), AM-404 (cat.# CR-106), GF 109203X (cat.# EI-246), phorbol ester (cat/# PE-160), forskolin (cat.# RBIF-105), WIN 55,212-2 (cat.# CR-105), aristolochic acid (cat.# EI-175), U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (cat.# ST-391) and U73343 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione) (cat.# ST-392) were from Biomol. Other agents were from Sigma-Aldrich.

Statistics. Data are reported as means±S.E.M. of five experiments. Data were analyzed by one-way or two-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS®, SAS Institute Inc., Cary, NC) on a personal computer powered by Intel Pentium IV CPU (Santa Clara, CA, USA) at 1.8 GHz. Multiple comparisons between group means were performed by post-hoc analysis using the Tukey's HSD (honestly significant difference) procedure. A P-value less than 0.05 was designated as statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Anandamide at concentrations above 5 μM increased [Ca2+]i in a concentration-dependent manner in Ca2+-containing medium. Fig. 1A shows the effects of 5–100 μM. At a concentration of 1 μM, the agent had no effect (=baseline). The [Ca2+]i increase induced by 50 μM anandamide (trace b) expressed a rapid initial rise that reached a net (baseline subtracted) sustained phase of 301±3 nM. The Ca2+ signal did not saturate at 100 μM anandamide since 200 μM anandamide induced dramatic cell death as observed under the microscope. Fig. 1D (filled circles) shows the concentration-response curve of the anandamide response.

image

Figure 1. Effect of anandamide on [Ca2+]i in MDCK cells. A. Effect in Ca2+-containing medium. Anandamide was added at 30 sec. The concentration of anandamide was indicated. B. Effect of extracellular Ca2+ removal on anandamide-induced [Ca2+]i increases. The experiments were performed in Ca2+-free medium. Anandamide (100 μM) was added at 40 sec. C. Effect of anandamide on 340 and 380 excitation wavelengths of fura-2 fluorescence. Y axis is arbitrary fluorescence unit. Anandamide (100 μM) was added as indicated in Ca2+-containing medium. D. Concentration-response plots of anandamide-induced Ca2+ signals in the presence (solid circles) and absence (open circles) of extracellular Ca2+. Y axis is the percentage of control which is the net (baseline subtracted) area under the curve of the [Ca2+]i response induced by 100 μM anandamide in Ca2+-containing medium. Data are mean±S.E.M. of five experiments. *P<0.05.

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Further experiments were performed to determine the relative contribution of extracellular Ca2+ influx and intracellular Ca2+ release in anandamide-induced [Ca2+]i increases. The [Ca2+]i increases induced by 100 μM anandamide in Ca2+-free medium are shown in fig. 1B. Removal of extracellular Ca2+ did not alter the baseline, suggesting that the amount of leaked fura-2 from the cells was insignificant. Anandamide (100 μM) increased [Ca2+]i by 72±2 nM above baseline (n=5). The concentration-response curve of anandamide-induced [Ca2+]i increases in Ca2+-free medium is shown in Fig. 1D (open circles), expressed as the percentage of the area under the curve of the 100 μM anandamide-induced [Ca2+]i increases. Fig. 1C shows that addition of 100 μM anandamide induced an increase in 340 nm excitation signal and an decrease in 380 nm excitation signal, suggesting that the anandamide induced increases in 340/380 ratio of fura-2 fluorescence reported [Ca2+]i increases.

Efforts were made to examine the Ca2+ stores involved in anandamide-induced [Ca2+]i increases. Previous reports show that the endoplasmic reticulum is a major Ca2+ store in MDCK cells (Huang & Jan 2001; Chen et al. 2002), while mitochondria in some cases may also play a role. Fig. 2A shows that, in Ca2+-free medium, after 100 μM anandamide-induced [Ca2+]i increases, addition of 1 μM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor that increases [Ca2+]i by passively depleting the endoplasmic reticulum Ca2+ store (Thastrup et al. 1990), failed to induce a [Ca2+]i increase (n=5). Conversely, fig. 2B shows that addition of thapsigargin induced a [Ca2+]i increase of 112±2 nM (n=4). After depletion of Ca2+ stores in the endoplasmic reticulum for 12 min., addition of 100 μM anandamide evoked a [Ca2+]i increase of 35±2 nM, which was smaller than control anandamide (fig. 2A; 70±2 nM) by 50% (n=4). Due to the incomplete depletion of anandamide-sensitive Ca2+ stores by thapsigargin, the role of mitochondrial Ca2+ store was examined. Fig. 2C shows that addition of the proton ionophore carbonyl cyanide-m-chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler that can release Ca2+ from mitochondria, induced a transient [Ca2+]i increase of 45±2 nM (n=5). Addition of 1 μM thapsigargin 4 min. after addition of CCCP induced a [Ca2+]i increase similar to that shown in fig. 2B. Subsequently added anandamide (100 μM) still induced a [Ca2+]i increase of 30±2 nM (n=4).

image

Figure 2. Experiments were performed in Ca2+-free medium. (A)-(C) Anandamide (100 μM), thapsigargin (1 μM), and CCCP (carbonyl cyanide-m-chlorophenylhydrazone; 2 μM) were added at time points indicated. Data are mean±S.E.M. of five experiments.

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Because anandamide was able to release Ca2+ from the endoplasmic reticulum, the role of phospholipase C in this releasing process was examined. As demonstrated previously, MDCK cells do not have ryanodine-sensitive Ca2+ stores (Jan et al. 1998b). U73122, a phospholipase C inhibitor (Thompson et al. 1991), was applied to see whether phospholipase C was involved in anandamide-induced Ca2+ release. Fig. 3A shows that ATP (10 μM) induced a [Ca2+]i increase of 169±3 nM. ATP is a well-established phospholipase C-dependent agonist in many cells (Nishizaki 2004). Fig. 3B shows that incubation with 2 μM U73122 did not alter basal [Ca2+]i but abolished ATP-induced [Ca2+]i increases. U73343, a biologically inactive analogue of U73122 (Thompson et al. 1991), did not affect ATP-induced [Ca2+]i increase (n=4; not shown). This suggests that U73122 effectively suppressed phospholipase C activity. Fig. 3B also shows that addition of anandamide (100 μM) after U73122 and ATP treatments caused a [Ca2+]i increase indistinguishable from the control response shown in Fig. 1B (n=4).

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Figure 3. Lack of effect of U73122 on anandamide-induced Ca2+ release. Experiments were performed in Ca2+-free medium. (A) ATP (10 μM) was added at 30 sec. (B) U73122 (2 μM), ATP (10 μM), and anandamide (100 μM) were added at time points indicated. Data are mean±S.E.M. of five experiments.

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The next set of experiments were performed to examine the modulation of anandamide-induced [Ca2+]i increases. The data are shown in fig. 4A and 4B. L-Type Ca2+ channel blockers (1 μM) nifedipine, nimodipine, verapamil and diltiazem, and the cannabinoid CB1 antagonist AM 251 (50 μM) failed to inhibit the anandamide response. The response was inhibited to different degrees by aristolochic acid (a phospholipase A2 inhibitor; 20 μM), WIN 55,212-2 (a cannabinoid receptor agonist; 1 μM), phorbol myristate acetate (1 nM; a protein kinase C activator), GF 109203X (2 μM; a protein kinase C inhibitor) and forskolin (to increase cAMP levels; 10 nM).

image

Figure 4. Modulation of anandamide-induced [Ca2+]i increases. (A) The bar graph of the effects of various agents on anandamdie-induced [Ca2+]i increases. Control: The maximum value of anandamide (100 μM)-induced [Ca2+]i increases. Y axis: percentage of control. The agents were added 100 sec. before anandamide. The concentration was 1 μM for nifedipine, nimodipine, verapamil and diltiazem; 20 μM for aristolochic acid; 1 μM for WIN 55,212-2; 50 μM for AM 251; 10 μM for AM-404; 1 nM for PMA, 2 μM for GF 109203X; 10 nM for forskolin. Data are mean±S.E.M. of 5 experiments. *P<0.05. (B) Traces demonstrating the effects of forskolin, PMA and GF 109203X on anandamide-induced [Ca2+]i increases as shown in (A).

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Given that acute incubation with anandamide induced a substantial and lasting [Ca2+]i increase, and that unregulated [Ca2+]i increases were often linked to cytotoxicity, experiments were performed to examine the effect of overnight incubation with anandamide on the proliferation of MDCK cells. Cells were treated with 0–200 μM anandamide overnight, and the tetrazolium assay was performed. In the presence of 1–50 μM anandamide, cell viability did not decrease, whereas in the presence of 100 and 200 μM anandamide, cell viability decreased to 50±8% and 7±2%, respectively (fig. 5A; n=5; P<0.05). The next issue is whether the anandamide-induced cytotoxicity is caused by preceding [Ca2+]i increases. The intracellular Ca2+ chelator BAPTA was used to prevent [Ca2+]i increases during anandamide pretreatment. Fig. 5B shows that BAPTA (20 μM) loading did not significantly alter control cell viability. In the presence of 100 μM anandamide, cell viability was reduced to 50±10% (n=18). In the presence of BAPTA, the anandamide-induced decrease in cell viability was completely reversed (P<0.05).

image

Figure 5. A tetrazolium assay of effect of anandamide on proliferation of MDCK cells. (A) Cells were treated with 0–200 μM of anandamide overnight, and the tetrazolium assay was performed as described in Methods. Data are mean±S.E.M. of three experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control that was the increase in cell number in anandamide-free groups. Control had 10,256±897 cells/well before experiments, and had 13,754±852 cells/well after overnight incubation. *P<0.05 compared to control (the first column). (B) Dependence of anandamide-induced cell death on preceding [Ca2+]i increases. The Ca2+ chelator BAPTA was added to fura-2-loaded cells as described in Methods. *P<0.05 compared with control. #P<0.05 compared with the third column (anandamide; 100 μM).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our study is the first to demonstrate that the endogenous ligand anandamide induced [Ca2+]i increases and Ca2+-dependent cell death in renal tubular cells. Anandamide increased [Ca2+]i by depleting intracellular Ca2+ stores and causing Ca2+ influx from extracellular milieu because removing extracellular Ca2+ reduced the major portion of anandamide-induced [Ca2+]i increases. Removal of extracellular Ca2+ reduced the anandamide-induced [Ca2+]i increases throughout the measurement period, suggesting that Ca2+ influx occurred during the whole stimulation period of 220 sec. The anandamide-induced [Ca2+]i increases were not affected by dihydropyridines, verapamil and diltiazem, consistent with previous data that MDCK cells do not have L-type Ca2+ channels (Lang & Paulmichl 1995). A possible Ca2+ channel that can be recruited by anandamide is the assumed transient receptor potential V1 (TRPV1) receptors (Myrdal & Steyger 2005). These receptors are typically Ca2+-permeant cation channels that transduce environmental stimuli and are thought to exist in kidney epithelial cells. However, so far, no specific blockers are available for these receptors.

Regarding the intracellular Ca2+ stores involved in anandamide-induced Ca2+ release, it appears that the endoplasmic reticulum plays a major role because anandamide completely depleted the thapsigargin-sensitive endoplasmic reticulum stores, and conversely thapsigargin pretreatment considerably decreased anandamide-induced Ca2+ response. Although mitochondria can release Ca2+ upon the stimulation of an uncoupler, it seems that anandamide did not utilize mitochondrial Ca2+ stores because CCCP pretreatment failed to enhance thapsigargin-evoked decrease of anandamide-induced Ca2+ response. Other stores that may be involved, but of smaller influence, include lysosomes, nuclei, cytoskeleton, etc. (Bootman et al. 2001; Berridge 2005); however, the role of these stores is more difficult to explore because no blockers as selective as thapsigargin for endoplasmic reticulum stores are available for these stores. The question arose regarding the mechanism underlying the Ca2+ release. It seems that phospholipase C-dependent elevation of cytosolic inositol-1,4,5-trisphosphate did not play a role in the Ca2+ release, since the release was not changed when phospholipase C activity was suppressed. MDCK cells do not have ryanodine-sensitive Ca2+ stores (Jan et al. 1998b); thus, how anandamide releases intracellular Ca2+ is unknown. Whether anandamide can inhibit endoplasmic reticulum Ca2+ pumps similarly to thapsigargin awaits evidence.

Among the various actions that anandamide exerts, some are mediated via cannabinoid receptors, whereas the others are not. The latter include inhibition of Ca2+ channels, stimulation of vanilloid receptors (Ross 2003), transient changes in [Ca2+]i, and disruption of gap junction function (Howlett & Mukhopadhyay 2003). Consistently, our data suggest that the anandamide-induced [Ca2+]i increases were not altered by the cannabinoid receptor antagonist AM 251. The data that the cannabinoid receptor agonist WIN 55,212-2 inhibited anandamide-induced [Ca2+]i increases by 85% suggest that these two cannabinoids may compete for the same acting site. Anandamide-induced effects appear to be regulated by different second messengers. In rat brain, anandamide is thought to modulate protein kinase C (De Petrocellis et al. 1995). Anandamide-induced depolarization of guinea-pig isolated vagus nerve (Kagaya et al. 2002) and gating of the vanilloid receptor VR1 (Vellani et al. 2001) are potentiated by modulation of protein kinase C activity. In MDCK cells, anandamide-induced [Ca2+]i increases were also regulated by protein kinase C since activation or inhibition of protein kinase C decreases the Ca2+ signal. Increasing cAMP levels exerts an inhibitory effect on anandamide-induced [Ca2+]i increases. It has been shown that some effects of anandamide are altered by the cAMP-dependent protein kinase (De Petrocellis et al. 1995). In human breast cancer cells, the cAMP/protein kinase A pathway is thought to play a regulatory role in the antiproliferative effects of anandamide (Melck et al. 1999). Lastly, our data show that aristolochic acid pretreatment inhibited anandamide-induced Ca2+ signal by 85%. The phytotoxin aristolochic acid has been widely used as a phospholipase A2 inhibitor (Caro & Cederbaum 2003). Phospholipase A2 is responsible for endogenous generation of anandamide (Sun et al. 2004). Because in our study anandamide was externally added, the inhibitory effect of aristolochic acid on anandamide-induced Ca2+ signal could not be mediated by inhibition of anandamide synthesis. One possibility is that phospholipase A2 activity is required for the formation of a Ca2+ response. Indeed, phospholipase A2 was shown to play a role in the regulation of Ca2+ signaling in MDCK cells (Huang & Jan 1999).

Anandamide has been reported to cause cell death in several other types of cells (Maccarrone & Finazzi-Agro 2003; Bari et al. 2005; Siegmund et al. 2005) including VR1-lacking human embryonic kidney (HEK) cells (Sarker & Maruyama 2003). Our study is the first to show that anandamide is toxic to renal tubular cells. Ca2+ overloading is known to initiate processes leading to cell death (Annunziato et al. 2003). Whether anandamide induces cell death by evoking [Ca2+]i increases is an important issue. Our findings showed that 100 μM anandamide-induced 50% decrease in cell viability was totally reversed by prechelating cytosolic Ca2+ with BAPTA. This suggests that anandamide-induced cell death depends on a pre-elevation in [Ca2+]i. It has been shown that in many cell types, cell death could be induced in a Ca2+-dependent or independent manner, depending on the stimulating agent (Matuszyk et al. 1998; Das et al. 1999). Taken together, the results show that anandamide can induce Ca2+ release from stores and Ca2+ influx across plasma membrane leading to cell death in MDCK renal tubular cells. A [Ca2+]i signal can affect various cellular processes, thus, caution should be taken in using anandamide as an agonist of cannabinoid or vanilloid receptors in other studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS94G-11) (VGHKS94-054) (NSC94-2320-B-075B-006) to C. R. Jan, and VGHKS94-044 to J. H. Yeh.

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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