Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells.

CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38- cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.

Effects of intraocular injection of a low concentration of zinc on the rat retina.

The main aim of this study was to investigate whether intraocular injection of low concentrations of zinc (no greater than 10 microM) aid the survival of ganglion cells in the rat retina after excitotoxic (NMDA) and ischemia/reperfusion injuries. We also determined whether low amounts of zinc cause any detectable retinal toxicity. Intraocular injection of NMDA caused substantial reductions in the mRNA levels of the ganglion cell-specific markers Thy-1 and neurofilament light (NF-L). Co-injection of 0.1 or 1 nmol zinc neither diminished nor exacerbated the effect of NMDA on the levels of these mRNAs. Likewise, ischemia/reperfusion caused significant decreases in the levels of Thy-1 and NF-L mRNAs and in the b-wave amplitude of the electroretinogram. These effects were not counteracted by injection of zinc. Intraocular injection of NMDA caused marked toxicological effects in retinal glial cells, including upregulations of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glial fibrial acidic protein (GFAP), basic fibroblast growth factor (FGF-2) and ciliary neurotrophic factor (CNTF). Interestingly, injection of 1 nmol zinc caused no changes in the levels of COX-2 and iNOS, yet produced similar, although quantitatively less pronounced, changes in FGF-2, GFAP and CNTF. The upregulations of FGF-2 and CNTF suggest that increasing zinc intake may benefit injured retinal neurons. However, this was not found to be the case in the present studies, perhaps due to the acute nature of the injury paradigms utilised.

Detection, localization, and sequence analyses of mitochondrial regulatory region RNAs in several mammalian species.

The mitochondrial regulatory region (mrr) located between the tRNAPhe and tRNAPro genes of mitochondrial DNA (mtDNA) is essential for regulation of replication and transcription of the mitochondrial genome. Polyadenylated short RNAs complementary to the L-strand of the mrr in human cells and similar RNAs (polyadenylation status unknown) in rat and mouse cells have been reported. We now report detection of ca. 0.2 kb polyadenylated mrrRNAs in cultured cells of Chinese hamster, African green monkey, mouse, rat, and human. We isolated a cDNA clone to a rat polyadenylated mrrRNA of 158 bp in length excluding the polyadenyl tail, which spans the region from the light strand promoter (LSP) to the origin of heavy strand replication (OriH). This cDNA contains both an open reading frame encoding a 26 amino acid polypeptide and a 12 nucleotide sequence complementary to the 3'-terminus of rat mitochondrial 12S rRNA. A cDNA clone to a human HeLa cell polyadenylated mrrRNA also contains a 12 nucleotide region complementary to the human mitochondrial 12S rRNA. We used a mitochondrial genome-deficient HeLa cell line, rho0 HeLa, and a derived cybrid cell line, HeEB, with a reconstituted mitochondrial genome, to demonstrate that the occurrence of the mrrRNA is dependent on the presence of a mitochondrial genome, and these polyadenylated mrrRNAs are transcribed from the mitochondrial genome. Our results further substantiate the common existence of polyadenylated mrrRNAs among mammals and support previously proposed hypotheses for the multi-functional nature of polyadenylated mrrRNA.

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria.

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.

Comparison of the effects of acarbose and voglibose in healthy subjects.

Acarbose and voglibose are alpha-glucosidase inhibitors. Although the pharmacologic effects and incidence of abdominal adverse events associated with the two drugs have been reported to differ, no study has directly compared acarbose and voglibose. To compare the pharmacologic effects and gastrointestinal adverse events associated with the two drugs, a randomized, placebo-controlled, double-masked, fivefold crossover study was performed in 20 healthy male subjects. To assess the pharmacologic effects, plasma immunoreactive insulin (IRI), plasma glucose, and 24-hour urinary connecting-peptide immunoreactivity (CPR) excretion were measured. Although the postprandial increase in plasma glucose level was reduced significantly with both acarbose and voglibose, the rate of reduction was small. The maximum concentration (Cmax) and area under the plasma concentration-time curve (AUC) of plasma IRI after meals decreased significantly with all treatments except voglibose 0.3 mg compared with placebo. Overall, the Cmax and AUC of plasma IRI decreased more when subjects received acarbose than voglibose. Urinary CPR excretion decreased by 30.6% and 41.7%, respectively, in subjects who received acarbose 50 mg or 100 mg compared with the previous day when no drug was given, whereas the urinary CPR excretion did not decrease significantly with voglibose. There was no significant difference in the frequency of gastrointestinal adverse events between groups, including the placebo group. One-day administration of acarbose and voglibose at currently recommended clinical doses demonstrated that acarbose was more effective in sparing endogenous insulin secretion than was voglibose.

Degradation of c-Fos protein expressed by N-methyl-D-aspartic acid in nuclear fractions of murine hippocampus.

In both nuclear and cytosolic fractions of murine hippocampus, constitutive expression was seen with Fra-2 protein, but not with other Fos family members tested including c-Fos, Fos-B and Fra-1 proteins. Fos-B protein was only detected in nuclear fractions. The systemic administration of N-methyl-D-aspartic acid (NMDA) induced marked and transient expression of c-Fos protein, but not other family members, in both hippocampal fractions 2 h later. In vitro incubation at 30 degrees C led to more rapid degradation of inducible c-Fos protein than constitutive Fra-2 protein in nuclear fractions obtained 2 h after the administration of NMDA, without significantly affecting that of both member proteins in cytosolic fractions. The addition of phosphatase inhibitors significantly delayed the initial degradation rate of inducible c-Fos protein, with concomitant facilitation of that of constitutive Fra-2 protein, in nuclear fractions. The addition of protease inhibitors also delayed the initial degradation of constitutive Fra-2 protein, without markedly altering that of inducible c-Fos protein, in nuclear fractions. Immunoprecipitation analysis revealed that NMDA induced phosphorylation of c-Fos protein on tyrosine residues in nuclear fractions to a lesser extent than that on serine residues 2 h after administration. These results suggest that NMDA signals may be propagated to the nucleus to induce both expression and degradation of c-Fos protein through a molecular mechanism associated with phosphorylation on serine and/or tyrosine residues in murine hippocampus.

Pharmacokinetics and tolerance of pantoprazole, a proton pump inhibitor after single and multiple oral doses in healthy Japanese volunteers.

The pharmacokinetics and tolerance of pantoprazole were investigated after single (20, 40, 80, and 120 mg) and multiple (80 mg once a day for 7 days) oral administration as enteric-coated tablet formulation to healthy male Japanese volunteers. Pantoprazole was well tolerated with no serious adverse events at all doses. Pantoprazole was rapidly absorbed in the fasted state. The mean maximum concentration in serum (Cmax) ranged from 1.77-9.25 micrograms/ml for the 20-120 mg dose and the mean time to reach Cmax (tmax) ranged from 1.92-2.42 h. The half-life (t1/2) ranged from 0.74-1.16 h. A good linear correlation was found between the administered doses (20-120 mg) and the resulting area under the concentration-time curve (AUC) and Cmax with the correlation coefficients of 0.9088 and 0.9263, respectively. Within 24 h, pantoprazole was excreted into urine as the unchanged drug to a negligible extent. In the multiple dose study, 2 apparent poor metabolizers (PMs) of pantoprazole were observed. The means of Cmax, AUC and t1/2 for these 2 PMs were 1.6, 6.7, and 6.8 times higher than those of the extensive metabolizers (EMs). The pharmacokinetic parameters such as Cmax, AUC, and t1/2 after the 7th oral dose were not significantly different from those after the 1st dose both in the PMs and the EMs, which indicated that there was virtually no drug accumulation.

[Spectrophotometric determination of uric acid in serum using a titanium (IV)-porphyrin complex].

Aqueous solution of oxo[5,10,15,20-tetra(4-pyridyl)porphyrinato]titanium (IV) complex, a Ti-TPyP reagent, was found to be very useful for the spectrophotometric determination of hydrogen peroxide. The reagent (lambda max 432 nm) reacts with hydrogen peroxide to form a monoperoxocomplex, resulting in a significant decrease of the absorbance at 432 nm. The decrease (delta A) in absorbance was proportional to the concentration of hydrogen peroxide. The Ti-TPyP reagent was successfully applied to the assay of uric acid in the serum, using uricase to produce hydrogen peroxide through enzymatic oxidation. Using only 5 microliters serum, a linear relationship was obtained between delta A and uric acid concentration in the serum ranging from 5 x 10(-6) to 1 x 10(-3) M. The apparent molar delta A of uric acid was 2.2 x 10(5) M-1 cm-1. The relative standard deviation of repeated runs (n = 8) was 2.8% at 3.77 x 10(-4) M uric acid. The analytical recovery of uric acid (5 x 10(-4) M) added to the serum was 96.8 to 105.0%. No pre-concentration and deproteinization were required to determine uric acid in the serum by the present method because of the high sensitivity and selectivity of the Ti-TPyP reagent for hydrogen peroxide.

The Chinese hamster cell emetine resistance gene. Analysis of cDNA and genomic sequences encoding ribosomal protein S14.

We used an intersecting pool strategy to recognize chimeric plasmids containing Chinese hamster ribosomal protein cDNAs. The screening procedure involved hybridization-selection of messenger RNAs, cell-free translation of selected mRNAs, and electrophoresis of polypeptide products on one- and two-dimensional polyacrylamide gels. The protocol was designed to recognize ribosomal protein S14 cDNAs specifically. Of 500 chimeric plasmids screened, two possessed cDNAs complementary to S14 mRNA and 18 contained sequences complementary to other ribosomal protein messages. Previously we demonstrated that mutations affecting Chinese hamster ovary cell ribosomal protein S14 are responsible for genetic resistance to the translational inhibitor emetine (emt b). Because emetine-resistant mutant and wild type Chinese hamster ovary cells elaborate mRNAs that encode electrophoretically distinguishable forms of S14 protein, we were able to identify S14 cDNA clones unambiguously. The data described here indicate that: 1) clone pCS14-1 contains most, if not all, of the S14 coding sequence as a cDNA; 2) S14 mRNA is approximately 0.01% of a Chinese hamster cell's polyadenylated messenger RNA; and 3) genomic DNA-encoding ribosomal protein S14 is a low, perhaps single, copy sequence with a complex structure, including several, long intervening sequences.

Differential in vitro degradation of particular Fos family members expressed by kainic acid in nuclear and cytosolic fractions of murine hippocampus.

Mice were injected with kainic acid (KA) at a convulsive dose, followed by homogenization of the hippocampus in the presence of different protease and phosphatase inhibitors, and subsequent preparation of nuclear and cytosolic fractions. An intraperitoneal injection of KA resulted in marked expression of particular Fos family members, including c-Fos, Fra-2, and Fos-B, but not Fra-1 proteins, in both fractions 2 to 18 h after administration. These fractions were individually incubated at 30 degrees C for 1 to 18 h for determination of in vitro degradation. Similarly rapid degradation was seen with c-Fos protein between nuclear fractions obtained 2 and 18 h after administration, while no significant degradation was found for c-Fos protein in cytosolic fractions obtained 2 h after administration during incubation. By contrast, in vitro incubation led to rapid degradation of c-Fos protein in cytosolic fractions obtained 18 h after administration. Degradation profiles were peculiar to each member protein in nuclear and cytosolic fractions obtained 2 and 18 h after administration. Dialysis prevented degradation of c-Fos protein in nuclear fractions without markedly affecting that in cytosolic fractions in a manner independent of the time after administration. The addition of inhibitors for phosphatases, but not for proteases, accelerated the degradation of c-Fos protein in nuclear fractions previously dialyzed. These results suggest that in vivo KA signals may modulate heterologous machineries responsible for breakdown of each Fos family member in a unique manner in nuclear fractions, rather than cytosolic fractions, of murine hippocampus.

Detection and cDNA cloning of H-strand mitochondrial regulatory region RNAs in cultured human cells and human tissues.

In the mitochondrion, essential genetic elements for replication and transcription are mostly housed within a shortsegment of its DNA located between tRNA(Phe) and tRNA(Pro) genes, which is called mitochondrial regulatoryregion (mrr). RNAs are known to be transcribed from mrr, thestructures and the functions of which are yet to be fullycharacterized.We detected ca. 1.3 kb H-strand transcripts of mrr (mrrH-RNAs),and 0.2 kb L-strand transcripts of mrr (mrrL-RNAs) in varioushuman cultured cells and tissues using double stranded mrrDNAprobes. The steady state levels of mrrL-RNAs were generally highin cultured cells, while they varied among tissues. On the otherhand, the levels of mrrH-RNAs varied among tissues and amongcultured cells. A tendency was observed in these cells andtissues that a high level of mrrL-RNA is associated with cellproliferation, and a high level of mrrH-RNA withdifferentiation. Several cDNA clones to 1.3 kb mrrH-RNA were obtained from humanskeletal muscle polyadenylated RNAs. The 5' terminus of the 1.3 kb RNA was determined to be at nucleotide position 15953 whichis immediately downstream of tRNA(Thr) sequence.Polyadenylation site for most of the clones was demonstrated tobe at nucleotide position 576 which is immediately upstream oftRNA(Phe) sequence. The longest cDNA insert obtained was 1177 bps long spanning from nucleotide positions 15969 to 576 which could code for a peptide of 76 amino acids. The cDNAs isolatedhere are the first cDNA clones reported to human mrrH-RNAs.These results, together with previous results, furthersubstantiate that polyadenylated mrrH- and mrrL-RNAs are commonly present at varying levels among human tissues andcells. The 3' end sequences of the cloned mrrH-cDNA provideswith insights into the mechanisms of transcription termination.The cDNA clones will provide tools to further the study of thefunction of mrr RNAs.

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast.

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.

Differential stereoselective pharmacokinetics of pantoprazole, a proton pump inhibitor in extensive and poor metabolizers of pantoprazole--a preliminary study.

Pantoprazole (PAN) is a proton pump inhibitor that is administered as a racemic mixture. The pharmacokinetics of PAN enantiomers were investigated in extensive metabolizers (EMs) and apparent poor metabolizers (PMs) of PAN who received a single, 40, 60, or 80 mg oral dose of racemic PAN as enteric-coated formulation. In the EMs, the serum concentrations of (-)-PAN were slightly higher than those of (+)-PAN at each dose level. The (+)/(-) ratios for the area under the concentration-time curve (AUC) and the half-life were 0.58-0.89 and 0.62-0.88, respectively. In the PMs, the serum concentrations and both enantiomers were much higher than those in the EMs at each dose level and significant differences in pharmacokinetics of (+)- and (-)-PAN were observed. The half-lives for (+)-PAN were 2.67-3.77 times longer than those for (-)-PAN. The AUCs for (+)-PAN were 2.65-3.45 times greater than those for (-)-PAN. Therefore, the metabolism of (+)-PAN is impaired to a greater extent than (-)-PAN in the PMs, which resulted in the stereoselective disposition of PAN in the PMs. It has been suggested that the EMs and the PMs of PAN could be differentiated by determining the (+)/(-) enantiomer ratio in serum at one time point, possibly 2-6 h after oral dosing, because the (+)/(-) enantiomer ratios in the PMs were opposite those in the EM subjects.

Structure and expression of integrated hepatitis B virus genes in an HBs antigen producing human cell line (huGK-14).

A human continuous cell line (huGK-14) within a lineage of passaged cultures was investigated in the mode of integration and expression of hepatitis B virus (HBV) genes. HBV DNA was integrated in eight different sites of the cellular DNA, in each of which HBV genome was rearranged, fragmented, and/or partly deleted. Complete HBV genome that may lead to production of infectious virus particles was not detected in the cells nor in the culture medium. Clones of cDNA containing a complete coding frame for small HBs antigen protein (type adr) were obtained from mRNA of the cells. The cells were stable over the period of six months of cultivation and more than 60 population doublings in the mode of HBV integration and HBs mRNA expression.These results provide substantial evidence for the absence of an ability for the integrated DNA to create an infectious product in the cell; for the stable production of HBs mRNA from the cells, and suggest the usefulness of this cell line as a substrate for HBV vaccine production.

A possible function of DNA curvature in a vector system that allows efficient expression of heterologous proteins in CHO cells.

A cis-acting element derived from the nontranscribed spacer region of murine rDNA allows efficient expression of heterologous proteins in transformed Chinese hamster ovary (CHO) cells. Analysis of the structural conformation of this cis-acting element revealed that it has a DNA curvature. Whether the DNA curvature plays any roles in regulating expression of heterologous proteins or not has been studied.

Ribosomal protein gene sequences map to human chromosomes 5, 8, and 17.

DNA sequences complementary to six mammalian ribosomal protein (r-protein) cDNAs are assigned to human chromosomal linkage groups in human-Chinese hamster hybrid cell clones. Ten r-protein DNA fragments map to chromosomes 5, 8 and 17, indicating that these important, housekeeping genes are distributed to multiple sites in the human genome. Each of the chromosome assignments, determined initially by surveying Chinese hamster-human hybrid cell clones with complex karyotypes using Chinese hamster and human cDNA probes, were confirmed in critical minipanels of highly reduced or monochromosomal hybrid cells. As all 10 fragments mapped to only three human chromosomes, r-protein sequences appear to be distributed nonrandomly within human DNA. The r-protein S14 sequence assigned to human chromosome 5 (5q23-5q33) rescues Chinese hamster emetine-resistance mutations (emt b) in interspecific hybrids. Therefore, this sequence corresponds to the transcriptionally active human RPS14 gene. In contrast, other r-protein DNA sequences examined likely are a mixture of functional genes and inactive pseudogenes.

Measles associated with coronary arteritis.

A two-year-old girl with measles virus (MV) and chronic Epstein-Barr virus (EBV) infection developed lethal coronary aneurysmal arteritis accompanied by giant cell pneumonia, systemic lymphadenitis and hepatosplenomegaly. In her coronary arteries, lungs and aorta, cells containing intranuclear and intracytoplasmic inclusions, including syncytial giant cells, were detected, the presence of MV in the organs being proved by electron microscopic and immunofluorescent studies. Immunopathology further demonstrated MV to be disseminated in almost all organs other than lymph nodes. Clinical diagnosis of chronic EBV infection was established on the basis of persistent high titers of antibodies against capsid and early antigens of EBV and viral presence was confirmed by Southern blot hybridization in a mesenterial lymph node obtained at autopsy. To the best of our knowledge, this is the first description of MV association with coronary aneurysmal arteritis, raising the possibility that measles infection can cause severe vasculitis under immuno-suppressive states, such as that caused by chronic EBV infection.

Effect of adrenaline on steroidogenesis in primary cultured bovine adrenocortical cells.

In primary 2-day cultured bovine adrenocortical cells, adrenaline stimulated the steroidogenesis, while the effect of adrenaline did not appear in the freshly isolated cells. Thus the primary 2-day cultured cells were used to study the effect of adrenaline on steroidogenesis. Adrenaline showed the steroidogenesis-stimulating effect at concentrations higher than 10(-9) M, and the maximum effect was obtained between 10(-6) M and 10(-5) M in the primary 2-day cultured cells. The maximum effect of adrenaline was 50-70% of that of adrenocorticotropic hormone (ACTH). Noradrenaline, isoproterenol and phenylephrine also stimulated the steroidogenesis. However, the order of the potency was isoproterenol much greater than adrenaline = noradrenaline much much greater than phenylephrine. Propranolol and alprenolol inhibited the effect of adrenaline, but phenoxybenzamine and phentolamine did not inhibit the effect. Moreover, adrenaline stimulated the cyclic AMP production dose-dependently at concentrations higher than 10(-8) M. These results suggest that there are steroidogenesis-linked adrenergic receptors in primary 2-day cultured bovine adrenocortical cell membrane and that the steroidogenesis-stimulating effect of adrenaline occurs through the beta-adrenergic receptor.

[Effect of Ca(2+)-channel antagonists on the corticoidogenesis in primary cultured bovine adrenocortical cells].

We investigated the effect of Ca(2+)-channel antagonists, Nicardipine(N), Verapamil(V) and Flunarizine(F), on the corticoidogenesis(CG) in primary cultured bovine adrenocortical cells. To examine the effect on receptor operated Ca(2+)-channel(ROC) and voltage operated Ca(2+)-channel(VOC), involved in corticoid synthesis, adrenocorticotropic hormone(ACTH) and high-K+ were used respectively. With or without the antagonists, cells were incubated at 37 degrees C for 1h in the presence of ACTH (100pM) or K+ (30mM). Corticoid was measured fluorometrically using cortisol as the standard. N and V inhibited not only ACTH-induced CG, but high K(+)-induced CG in a concentration-dependent manner. However, F inhibited only high K(+)-induced CG, and did not affect ACTH-induced CG. These inhibitions were observed at the low micromolar concentrations (below 40 microM) of the antagonists. In the regulation of corticoid synthesis, we indicate that F has an inhibitory effect on VOC without ROC; on the other hand, N and V inhibit both ROC and VOC.

Extracellular ATP stimulates steroidogenesis in bovine adrenocortical fasciculata cells via P2 purinoceptors.

Effects of ATP and other purine derivatives on steroidogenesis in primary cultured bovine adrenocortical fasciculata cells were examined. At concentrations higher than 1 microM, ATP showed a potent stimulative effect on the cortisol production of the cells. The potency order of the steroidogenic effect of the tested purine derivatives was ATP greater than ADP much greater than adenosine much greater than AMP. alpha,beta-Methylene ATP had no stimulative effect on the steroidogenesis at concentrations as high as 1 mM. Theophylline did not antagonize the steroidogenic effect of ATP. These results suggest that bovine adrenocortical fasciculata cells possess the P2y purinoceptors that are linked to steroidogenesis.