Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors.

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.

Molecular cloning and characterization of the rat P2Y4 receptor.

Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full-length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y4 receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2-methylthio-ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y4 receptor but with this significant difference in agonist pharmacology.

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones.

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5'-triphosphate (UTP) or adenosine 5'-triphosphate (ATP) inhibits the N-type voltage-gated calcium current by approximately 65%, with an IC50 of 0.5 microM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

A novel G protein-coupled P2 purinoceptor (P2Y3) activated preferentially by nucleoside diphosphates.

A partial cDNA was isolated by hybridization screening of an embryonic chick brain library for P2Y purinoceptors. After extension to full length, it revealed an open reading frame that encoded a protein, P2Y3, of 328 amino acids that is nearest in sequence identity to the G protein-coupled P2 purinoceptors obtained by DNA cloning. Expression of P2Y3 in cRNA-injected Xenopus oocytes confirmed that this cDNA encodes a member of the metabotropic purinoceptor family, with a novel order for the relative activities of nucleotides. At 100 microM concentrations, ADP gave the highest activity, and UTP and UDP were also strongly active. When expressed in the human T cell line Jurkat, P2Y3 mediated transient increases in intracellular Ca2+ in response to various nucleotides. Again, an unusual agonist rank order was revealed, with uridine nucleotides being more potent than adenosine nucleotides and UDP being the most potent agonist tested (half-maximal concentration, 0.13 microM) and 10-fold more potent than UTP. 2-Methylthlo-ATP was of relatively low activity in both systems. The receptor transcript is expressed in brain, spinal cord, kidney, and lung and is highly abundant in the spleen but not in other peripheral tissues that we tested. The results indicated that P2Y3 is a previously unknown P2 purinoceptor subtype with a preference for nucleoside diphosphates.

Cloning and characterisation of a bovine P2Y receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methylthioadenosine 5' triphosphate = adenosine 5' diphosphate > adenosine 5' triphosphate >> alpha,beta-methyleneadenosine 5' triphosphate and uridine 5' triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

Transient expression of the recombinant chick brain P2y1 purinoceptor and localization of the corresponding mRNA.

Extracellular ATP acts at specific cell surface purinoceptors to elicit a wide range of physiological responses. We have recently isolated a cDNA for a G-protein-coupled P2 purinoceptor (P2y1) from chick brain. It has been defined as a P2Y-like purinoceptor by the rank order of potency of P2 purinoceptor ligands, determined electrophysiologically in the Xenopus oocyte expression system. Here, we examine the ligand selectivity of this recombinant receptor, expressed transiently in COS-7 cells. The regional distribution of the P2y1 purinoceptor transcript within the one-day-post-hatch chick brain was also determined. It is widely expressed in the cerebellum and telencephalon and in specific nuclei of the mesencephalon and diencephalon, suggesting a neuronal localization of the P21 purinoceptor.

Pharmacokinetics relevant to the anti-carcinogenic and anti-tumor activities of glucarate and the synergistic combination of glucarate:retinoid in the rat.

Alone and in synergistic combination with retinoids, dietary glucarate inhibits both the chemical induction and growth of rat mammary tumors. To investigate the pharmacokinetics of glucarate, [14C]glucarate was synthesized, converted to the calcium salt, and administered to rats bearing primary mammary tumors. When given by gavage, [14C]glucarate, as the calcium salt, showed a biphasic response in the blood. After peaking within 1 hr of administration at a level of 0.4 mumol/mL (normal endogenous level is approximately 0.04 mumol/mL), its plasma concentration dropped to 0.1 mumol/mL at 3 hr. In the second phase, there was a semilog increase to 0.6 mumol/mL at 15 hr, followed by a slow rise to 0.75 mumol/mL at 24 hr. Of the 38% of the administered glucarate that was recovered, 38% was excreted in the urine, and 30% remained in the gastrointestinal tract at 24 hr. Glucarate was concentrated 3- to 4-fold in the liver and intestinal mucosa, compared to the level in serum. With minor exception, the pharmacokinetics of [14C]13-cis-retinoic acid administered by gavage to rats was similar or not the semipurified diets were supplemented with 64 mmol/kg of calcium glucarate. During the interval between 5 and 10 hr post-administration of [14C]13-cis-retinoid, there was a transient 35-50% rise in the plasma level in rats on the glucarate-supplemented diet. This rise had no observable effect on the level of retinoid in major organs or in the tumor. A glucarate-binding protein was detected in the tumor cytosol. This potential receptor had a Ka of 1.49 x 10(7) M-1.

Mechanism of growth inhibition of mammary carcinomas by glucarate and the glucarate: retinoid combination.

In synergistic combination 0.75 mmol/kg diet of N-(4-hydroxyphenyl) retinamide and 32 mmol/kg diet of glucarate inhibits the growth of primary rat mammary tumors, but are equally effective as single agents at 1.5 and 128 mmol/kg diet, respectively. Dose-response studies suggest that like retinoids, glucarate acts directly on tumor cells, rather than having an adjuvant effect. Although synergism is maintained down to at least 0.38 mmol/kg diet of the retinoid, experiments using Vitamin A-deficient diets indicates 128 mmol/kg glucarate acts independent of retinoid. Both alone and in combination, glucarate and retinoid inhibited the growth of human mammary tumor cells grown in the athymic mouse, the growth of rat mammary tumors in germfree rats and the hormone-independent MTW 9a/R rat mammary tumor. Like retinoids, glucarate suppresses protein kinase C and induces transforming growth factor-beta, in the mammary tumor cells.

Cloning and functional expression of a brain G-protein-coupled ATP receptor.

A cDNA encoding a novel member of the G-protein-coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly-developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.

Effect of dietary soybean and licorice on the male F344 rat: an integrated study of some parameters relevant to cancer chemoprevention.

The individual and combined effects of dietary toasted soybean meal (3.13-25%) and dietary licorice root extract (0.38-3.0%) on selected liver and intestinal enzyme levels and on clinical chemistry and histopathological parameters were evaluated on male F344 rats. All parameters were measured one and three months after the 50-day-old rats were started on the diets. By use of newly developed high-performance liquid chromatography-based analytic methods, measurable levels of daidzein (2.67 micrograms/ml) and glycyrrhetinic acid (7.87 micrograms/ml) were detected in the sera of rats on the 25% soybean and 3% licorice diets, respectively. Histopathological evaluations of organs and tissues yielded only nonsignificant strain-related changes. At all dosages, there were no significant soybean- or licorice-related anatomic lesions or hematologic changes. In the clinical biochemistry profile, soybean meal caused moderate but significant dose-dependent decreases in serum cholesterol and increases in alkaline phosphatase, blood urea nitrogen, and phosphorus, which remained within the normal range. Liver glutathione transferase, catalase, and protein kinase C showed significant inductions (up to 50%) in response to increasing doses of soybean meal and licorice extract, with evidence for only marginal interaction between the two additives. Their effects on the intestinal mucosa were not significant. Ornithine decarboxylase levels, an indicator of promotional activity, were unchanged or repressed by the additives. The favorable effects of up to 25% toasted soybean meal and 3% licorice root extract on the levels of the four enzymes, without unfavorable changes in clinical parameters, might account in part for the chemopreventive activities of these additives. These effects would be in addition to direct inhibitory effects of known components in these additives on these or other enzymes or modulation of hormone activity that is not evaluated in this study.

Effect of calcium glucarate on beta-glucuronidase activity and glucarate content of certain vegetables and fruits.

Glucarate is normally present in tissues and body fluids and is in equilibrium with D-glucaro-1,4-lactone, a natural inhibitor of beta-glucuronidase activity. Dietary calcium glucarate, a sustained-release from of glucarate, elevates the blood level of D-glucaro-1,4-lactone which suppresses blood and tissue beta-glucuronidase activity. A single dose of CaG (4.5 mmole/kg body weight) inhibited beta-glucuronidase activity in serum and liver, lung, and intestinal microsomes by 57, 44, 37, and 39%, respectively. A chronic administration of calcium glucarate (4% in diet) also decreased beta-glucuronidase activity in intestinal and liver microsomes. Maximal inhibition of beta-glucuronidase activity in serum was observed from 12 noon to 2:00 PM. In contrast, maximum inhibition of beta-glucuronidase activity in intestinal and liver microsomes occurred during mornings, although a secondary depression in intestinal microsomes also occurred around 4 PM. A 4% calcium glucarate supplemented diet also inhibited beta-glucuronidase activity by 70% and 54%, of the bacterial flora obtained from proximal (small intestine) and distal (colon) segments of intestine, respectively. Due to the potential effect of dietary glucarate on net glucuronidation and on other metabolic pathways, glucaric acid levels in various foods were determined. The glucaric acid content varied from a low of 1.12-1.73 mg/100 g for broccoli and potatoes to a high of 4.53 mg/100 g for oranges.

Chemopreventative activity of dietary glucarate on azoxymethane-induced altered hepatic foci in rats.

Previous studies have shown that dietary calcium glucarate, an inhibitor of beta-glucuronidase, is a potent inhibitor of promotion of diethylnitrosamine-induced altered hepatic foci, 7,12-dimethylbenzanthracene-induced mammary tumorigenesis and benzo(a)pyrene-induced lung carcinogenesis. The present study was undertaken to test the chemopreventative activity of calcium glucarate on azoxymethane-induced hepatocarcinogenesis in female Fischer 344 rats. A series of experiments were carried out over 36 weeks to evaluate the effects of calcium glucarate on the initiation and promotion phases separately and also in combination with each other. A calcium gluconate group was included and used as a negative calcium control. Histopathologic evaluation of H&E stained liver sections of all animals in this study showed that a statistically significant inhibition of hepatocarcinogenesis only occurred when dietary calcium glucarate supplementation was provided throughout the combined initiation and promotion phases. This inhibitory effect approximately equaled the summation of that obtained when calcium glucarate was fed only during initiation phase and only during promotion phase.

Synergistic interaction between 13-cis-retinoic acid and glucarate: activity against rat mammary tumor induction and MCF-7 cells.

At high dietary levels in vivo, both 13-cis-retinoic acid and calcium glucarate inhibit the induction of rat mammary tumors by 7,12-dimethylbenz(a)anthracene. The present study shows that sub-optimal dietary levels of each, which individually have no effect on tumor induction, when combined together in the diet, significantly increases tumor latency and suppresses tumor frequency in the rat system. Weight gain of animals was similar in control and experimental groups. Furthermore, ineffective sub-optimal dosages of glucarate and 13-cis-retinoic acid interacted synergistically to inhibit the growth in vitro of the MCF-7 human breast cancer cells. By varying the concentrations of glucarate and 13-cis-retinoic acid independently, evidence was obtained that in combination glucarate may play an adjuvant role, with the retinoid as the effector. Thus, the results of this experimental animal study demonstrate for the first time the potential use in synergistic combination of 2 normal metabolites in non-toxic chemoprevention and chemotherapy.

Effects of the experimental chemopreventative agent, glucarate, on intestinal carcinogenesis in rats.

Dietary calcium glucarate was previously shown to protect effectively against chemically-induced mammary, lung, liver and skin carcinogenesis in rodents, whereas the negative dietary calcium control, calcium gluconate, had no effect. In the present study the chemopreventative activity of dietary calcium glucarate was evaluated in the azoxymethane intestinal carcinogenesis model using the Fischer strain rat. The protocol limited the duration of azoxymethane treatment to 3 weeks to permit the evaluation of the separate effects of glucarate on the initiation and promotion phases. Control rats, treated with azoxymethane and maintained on a low fat chow diet throughout the 32-week experiment had an intestinal adenocarcinoma incidence of 55%, with an equal incidence of 27.7% in the small and large intestines. There was no significant difference between this control group and a negative calcium control group fed 128 mmol/kg chow of calcium as calcium gluconate. In contrast to these two control groups, supplementation of the diet of azoxymethane-treated rats with 128 mmol/kg diet of calcium glucarate during both the initiation and promotion phases significantly inhibited the overall induction of adenocarcinomas in the intestine, the incidence in the entire intestine and in the small and large intestines being 11.8, 5.8 and 5.8%, respectively. When fed only during the initiation phase, the inhibition again was statistically significant, the corresponding values being 11.8%, 5.8 and 5.8%. When calcium glucarate was fed during the promotion phase, a statistically significant inhibition of adenocarcinoma induction was observed only in the colon where the incidence was 5.5%. Weight gain was similar in all groups. These and related data indicate that dietary glucarate exerts a significant inhibitory effect on azoxymethane-induced intestinal and in particular colon carcinogenesis in the rat, decreasing their incidence and size and reducing their metastic potential.

Modulation of chemically initiated and promoted skin tumorigenesis in CD-1 mice by dietary glucarate.

The efficacy of dietary calcium glucarate as a chemopreventative agent has been tested in the mouse skin tumorigenesis system. Skin tumorigenesis was initiated in mice of the CD-1 strain with 7,12-dimethylbenz(a)anthracene (DMBA), then promoted with twice weekly applications of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 13 weeks. The mice were fed a regular chow diet, or a chow diet fortified with calcium glucarate (128 mmol/kg diet), or with equimolar calcium as calcium gluconate (negative calcium control). When mice were fed calcium glucarate throughout both the initiation and promotion phases papilloma formation was inhibited by over 30%. Transfer of these DMBA-initiated, TPA promoted CD-1 mice to chow diet after 13 weeks on the calcium glucarate-supplemented diet, resulted in an increase in the number of skin papillomas within 3 weeks to the level of those seen in control animals maintained exclusively on the chow diet. When calcium glucarate feeding was restricted to either the initiation or promotion phases, papilloma formation was inhibited by 25%. Dietary calcium gluconate had no effect on papilloma formation in the CD-1 mouse system, but increased the calcium concentration in the skin to the same extent as that of calcium glucarate. The data indicate that the elevation of the normally low levels of glucarate in the body through supplementation, results in a marked alteration in the retention, activity and/or metabolism of xenobiotics.

Effects of calcium glucarate on the promotion of diethylnitrosamine-initiated altered hepatic foci in rats.

Calcium glucarate (CGT), an inhibitor of beta-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.

Dietary glucarate as anti-promoter of 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis.

Using as a criterion the inhibition of serum beta-glucuronidase activity, dietary calcium D-glucarate is shown to serve as an efficient slow-release source in vivo of D-glucaro-1,4-lactone, the potent endogenous inhibitor of this enzyme. Using the 7,12-dimethylbenz[a]anthracene model of mammary tumor induction in rats it is shown for the first time that feeding the rats calcium D-glucarate-supplemented diet after treatment with the carcinogen, inhibits tumor development by over 70%. Supportive evidence is presented for the theory that calcium D-glucarate inhibits or delays the promotion phase of mammary carcinogenesis by lowering endogenous levels of estradiol and precursors of 17-ketosteroids. Therefore, dietary glucarate can be used to lower blood and tissue levels of beta-glucuronidase, and in turn of those carcinogens and promoting agents which are excreted, at least in part, as glucuronide conjugates.