Purification of [I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

Vasoactive intestinal peptide (VIP) bound with high affinity (K_d 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His¹]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound ¹²⁵I-VIP whereas VIP(16–28) and VIP(1–22-NH₂) were inactive. The binding of ¹²⁵I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, [pCl-D-Phe⁶,Leu¹⁷]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated ¹²⁵I-VIP was bound to receptors on the cell surface. The internalized ¹²⁵I-VIP was completely degraded to ¹²⁵I-tyrosine which was released from the cells. Degradation of internalized ¹²⁵I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of ¹²⁵I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound ¹²⁵I-VIP, but caused retention of internalized ¹²⁵I-VIP.     

Retinoic acid down-regulates VPAC(1) receptors and TGF-beta 3 but up-regulates TGF-beta 2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific ¹²⁵I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific ¹²⁵I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC₁ receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC₁ receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.     

High affinity binding of VIP to human lung cancer cell lines

The binding of ¹²⁵I-VIP to human lung cancer cell lines was investigated. Radiolabeled VIP bound to adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer (SCLC) cell lines. As SCLC cell line NCI-N592 bound radiolabeled VIP well, its binding was further characterized. ¹²⁵I-VIP bound to membranes in a specific and time dependent manner. ¹²⁵I-VIP bound with high (Kd=0.8 nM) and moderate affinity (Kd=66 nM) to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP PHI > secretin > VIP^10–28. Because VIP receptors are present on human lung cancer cells, VIP may function as a regulatory peptide in lung cancer.     

Vasoactive intestinal polypeptide binds with high affinity to non-small cell lung cancer cells and elevates cyclic AMP levels

Vasoactive intestinal polypeptide (VIP) receptors were characterized on non-small cell lung cancer (NSCLC) cells. ¹²⁵I-VIP bound specifically to membranes derived from 6 NSCLC cell lines. Specific ¹²⁵I-VIP was time dependent and a linear function of EPLC-65H membrane concentration. ¹²⁵I-VIP bound with high (K_d=0.2 nM) and moderate (K_d=39 nM) affinity to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP > rGHRH > PHI = helodermin > secretin > glucagon. Also VIP elevated the cAMP levels 10-fold using cell line ADLC-5M2. These data indicate that functional VIP receptors are present on NSCLC cells.     

Vasoactive intestinal peptide receptor activity in human fetal enterocytes

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind ¹²⁵I-VIP and activate adenylate cyclase) and cyclic AMP-dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half-maximal stimulation of the cyclase and inhibition of ¹²⁵I-VIP binding in membrane preparations were respectively observed at 1.4 and 5 × 10⁻¹⁰ M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10⁻⁷M and above) in the following order of potency: VIP> PHI> GRF> secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin-14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9–12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.     

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and ³H benzo a pyrene (³H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l^-1 and 0.625 μCi l^-1, corresponding to 6 mg kg^-1 body weight and 125 μCi kg^-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of ³H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of ³H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of ³H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 μM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. B_max of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 μM VIP. No significant change was observed in receptor affinity, in B_max of the low-affinity receptor, in ED₅₀, or in ED₁₀₀ of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 μM, 3 h) caused 24% reduction in [¹²⁵I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 μM, 3 h).     

Characterization of VIP- and helodermin-preferring receptors on human small cell lung carcinoma cell lines

We investigated the molecular and pharmacologic characteristics of VIP receptors on two human SCLC cell lines: NCI-N592 and NCI-H345. With NCI-N592 cell, the order of potency of VIP-related peptides in inhibiting ¹²⁵I-VIP binding and in stimulating cAMP production was typical of the human VIP receptor. By covalent cross-linking, a polypeptide of Mr 62,300 was obtained. Conversely, the behavior of NCI-H345 cell line was totally different: helodermin was the most potent peptide, VIP and PHI were equipotent, while hGRF and secretin were totally ineffective. These results suggest that NCI-N592 cells possess a typical VIP receptor while NCI-H345 cells possess a helodermin-preferring receptor, and that the natural target of helodermin might not be the VIP receptor.     

In Vitro Properties of a High Affinity Selective Antagonist of the VIP1 Receptor

Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. In vitro properties of a high affinity selective antagonist of the VIP₁ receptor. Peptides 18(10) 1555–1560, 1997.—A selective high affinity VIP₁ receptor antagonist [Acetyl-His¹, D-Phe², Lys¹⁵, Arg¹⁶, Leu¹⁷] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP₁ receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP₁-, rat VIP₂- or the human VIP₂- recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP₁ receptor. The IC₅₀ values of ¹²⁵I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP₁-, rat VIP₂-, human VIP₁- and human VIP₂ receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP₁ receptor mediated stimulation of adenylate cyclase activity. The K_i values were respectively of 15 ± 5 nM and 2 ± 1 nM for the rat and human VIP₁ receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP₁ receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.     

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 ± 2 μm and, with carbachol treatment, contracted to 89 ± 2 μm. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC₅₀ of 3–7 nM. Galanin (1 μM) and VIP (1 μM) increased cellular cAMP from 118 ± 10 pmol/10⁶ cells in control to 212 ± 14 and 214 ± 12 pmol/10⁶ cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 μM, completely inhibited the relaxant effect of an EC₅₀ concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, -NNA ( ), at 100 μM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, -NNA (100 μM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation. To determine the ability of each peptide to activate nitric oxide, the incorporation of [³H]arginine into [³H]citrulline was determined. Galanin (1 μM) did not cause nitric oxide generation whereas VIP (1 μM) increased nitric oxide generation above the control by 97 ± 14% (p < 0.01). These results demonstrated that with galanin, in contrast to VIP, nitric oxide is not involved in its ability to cause gastric smooth muscle cell relaxation. The relaxant action of galanin can be accounted for completely by its ability to activate protein kinase A and therefore resembles recent results with β-adrenergic agents.     

Vasoactive intestinal peptide (VIP) receptors in the canine gastrointestinal tract

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of ¹²⁵I-VIP receptor binding sites in the canine gastrointestinal tract. While the distribution of VIP binding sites was different for each segment examined, specific VIP binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, lymph nodules, and the circular and longitudinal smooth muscle of the muscularis externa. These results identify putative target tissues of VIP action in the canine gastrointestinal tract. In correlation with physiological data, VIP sites appear to be involved in the regulation of a variety of gastrointestinal functions including epithelial ion transport, gastric secretion, hemodynamic regulation, immune response, esophageal, gastric and intestinal motility.     

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line

R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [¹²⁵I]-PACAP or [¹²⁵I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC₅₀ of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC₅₀ in agreement with the pharmacological profile of the VPAC₁ receptor subtype: PACAP = VIP > [K¹⁵, R^16, L²⁷]VIP(1–7)/GRF(8–27) = [R¹⁶]ChSn (two VPAC₁ agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC₂ receptor was inactive at 1 μM. Dose-response curves of VPAC₁ agonist molecules (PACAP, VIP, [K¹⁵, R¹⁶, L²⁷]VIP(1–7)/GRF(8–27), [R¹⁶]ChSn) were shifted to the right by the VPAC₁ receptor antagonist [AcHis¹, D-Phe², Lys¹⁵, Leu¹⁷]VIP(3–7)/GRF(8–27), with a K_i of 3 ± 1 nM (n = 3). The presence of VPAC₁ receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC₁ receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC₁ receptors undergoing rapid desensitization in HEL cells.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

The synthesis of [26,27-11C]dihydroxyvitamin D(3), a tracer for positron emission tomography (PET).

1,25-Dihydroxyvitamin D₃, an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with ¹¹C for use in biological experiments. The radionuclide was incorporated via the reaction of [¹¹C]methyllithium on a methyl ketone precursor in tetrahydrofuran at −10 °C. Deprotection of the labeled intermediate yielded 2.5–3 GBq [26,27-¹¹C]1,25-dihydroxyvitamin D₃ [¹¹C-1,25(OH)₂ D₃] with specific radioactivity averaging 100 GBq/μmol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; ¹¹C-1,25(OH)₂ D₃ is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.     

Vasoactive intestinal peptide receptor on liver plasma membranes: Solubilization and cross-linking

The hepatic receptor for VIP was solubilized from rat liver plasma membranes with 1.4% digitonin and shown to conserve its ability to bind to the ligand. This solubilized receptor demonstrated the high affinity and specificity for VIP (K_D1 nM, binding preference: VIP > PHI > secretin > thymosin ₁) which were observed with the nonsolubilized VIP receptor on intact liver plasma membranes. ¹²⁵I-VIP was next cross-linked to either the solubilized or nonsolubilized receptor using disuccinimido suberate or disuccinimido dithiobis(propionate), and the resulting complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. A broad autoradiographic band which demonstrated a high affinity for VIP was identified at Mr 56,000 (53,000 in the absence of the reducing agent dithiothreitol) for both the solubilized and nonsolubilized receptors. We have thus been able to solubilize from rat liver plasma membranes a receptor with high affinity and specificity for VIP, and confirmed its structural similarity with the native VIP receptor in nonsolubilized membranes using cross-linking techniques.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.