Receptor activated C kinase is down-regulated in the male gonad of the marine bivalve mollusc Mya arenaria exposed to tributyltin (TBT).

This study was aimed at investigating the molecular mechanisms by which tributyltin (TBT) impairs the reproductive processes in the marine bivalve Mya arenaria. The suppression polymerase chain reaction subtractive hybridization (SSH) method was used to identify differentially expressed transcripts in the gonads of adult M. arenaria 72 h after a single injection of 160 ng TBT in the adductor muscle. Subtractive cDNA libraries comprising 322 clones were obtained. These clones were sequenced and corresponded to 55 female and 26 single male non-redundant cDNAs. Following similarity searches in genome databases, some of the transcripts could be assigned to cellular functions including mitochondrial respiration, structural proteins, structure of cytoskeleton, nucleic acid regulation, general metabolism and signal transduction. Among the potentially differentially regulated transcripts, Receptor for activated C kinase 1 (RACK1) represented 6% of the total down-regulated clones in males and the corresponding protein exhibited a high degree of similarity (80%) with the human polypeptide. The RACK1 cDNA from M. arenaria consists of 1085 bp, encoding a 318 deduced polypeptide which contains five internal tryptophan-aspartate (WD) repeats, six putative PKC phosphorylation sites, one tyrosine kinase site, four putative N-myristoylation sites as well as a transmembrane segment spanning amino acid 228-251. A significant down-regulation (by approximately 30% (p<0.05)) of RACK1 expression in male gonads exposed to TBT was confirmed by quantitative real-time RT-PCR. Transcript levels of RACK1 were higher in the female gonads than in the mantle, gills and male gonads. Gene expression as detected by in situ hybridization was strong in mature oocytes comparatively to primary germ cells. RACK1 may be a useful biomarker for TBT exposure in the reproductive system of bivalve molluscs.

Functional analysis of chemically-induced mutations at the flounder TP53 locus, the FACIM assay.

A functional assay was developed in yeast to identify mutations induced by DNA-damaging agents at the flounder TP53 locus. This assay named FACIM for functional analysis of chemically-induced p53 mutations, is based on the assumption that most genotoxin-induced mutations inactivate transcriptional activity of the TP53 protein. The functional status of the protein expressed in yeast was measured using a p53-responsive reporter gene. The FACIM assay was used to evaluate the mutagenesis of the flounder TP53 exposed in vitro to benzo[a]pyrene diol epoxide (BPDE). A dose-dependent increase of p53 mutation rate was observed with increasing concentrations of BPDE and extension of exposure time. Flounder TP53 gene appeared highly sensitive to point mutations since most of those identified targeted different nucleotides. Mutated base-pairs corresponded predominantly to guanines located on the non-transcribed strand of the DNA. The general distribution of mutations along the flounder TP53 protein was different from that identified in the human homologue suggesting species-differences in mutagenesis of the TP53 gene. Most of flounder TP53 mutants were defective for transactivation and cell growth regulation but some maintained a partial wild-type phenotype. This functional assay in yeast could be used for both evaluation of the genotoxic potency of chemicals or environmental samples and screening of p53 mutations in fish tumours.

Molecular cloning of superoxide dismutase (Cu/Zn-SOD) from aquatic molluscs.

The potential of the first line of the active oxygen-scavenging system, partial cDNA encoding Cu/Zn superoxide dismutase (SOD) was isolated in three aquatic mollusc species: Ruditapes decussatus (marine clam), Dreissena polymorpha (continental water mussel) and Bathymodiolus azoricus (hydrothermal vent mussel). These SOD cDNA fragments were amplified by PCR with degenerate oligonucleotide primers derived from the amino acid sequence conserved in the Cu/Zn-SOD from several other organisms. A partial cDNA of CuZn-SOD was obtained for R. decussates (510 bp), D. polymorpha (510 bp) and B. azoricus (195 bp). The deduced amino acid sequence showed high similarity among the three mollusc species (57-63%) and among other species (50-65%). The residues involved in coordinating copper (His-47, 49, 64, 121) and zinc (His-64, 72, 81 and Asp-84) were well conserved among the three Cu/Zn-SOD sequences.

The European flounder (Platichthys flesus) TP53 functions as a temperature-sensitive transcription factor which inhibits cell growth in yeast.

Numerous studies focus on biological roles of the TP53 tumor suppressor gene in mammals but little is known about the actual function of TP53 in lower vertebrates. In this study, we used an in vivo functional assay in yeast to address the transactivation capacity of the flounder TP53 protein. We showed that the flounder TP53 acts as a sequence-specific transcription factor which is able to transactivate various human promoters containing a p53-responsive element (RE). This transcriptional activity was completely abrogated in the Val147Glu TP53 mutant previously identified in two flounder hepatic hyperplasia. In addition, we showed that the wild-type (wt) flounder TP53 but not the Val147Glu mutant inhibits cell growth when expressed in yeast. We finally reported that transcription regulation and growth inhibition by the wild-type flounder TP53 is temperature-dependent. The flounder TP53 optimal temperature appeared lower than those reported for the Xenopus and human homologues.

Identification of differentially expressed genes in Dreissena polymorpha exposed to contaminants.

Development of transcriptome analysis methods such as differential display PCR and construction of subtractive libraries now makes it possible to profile gene expression in response to xenobiotic exposure. As an example of application of these methods, zebra mussels (Dreissena polymorpha) were treated with various contaminants such as Aroclor 1254, 3-methylcholanthrene, chrysene and atrazine. A total of 242 mRNAs were identified as differentially expressed. Analysis of these mRNAs should provide valuable information regarding detoxification mechanisms in this bivalve species. In addition, the use of cDNA array technology applied to these gene products may constitute a multi-marker approach to monitor the effect of contamination on this aquatic species.

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas.

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.

Genetic and immunological characterisation of a multixenobiotic resistance system in the turbot (Scophthalmus maximus).

Pleiotropic resistance driven by transport proteins constitutes a very ubiquitous protection mechanism against natural or synthetic toxic compounds. The multidrug (MDR) or multixenobiotic (MXR) system has been identified in many different species, and may be used as a biomarker for pollution assessment. Here we report the existence of a gene encoding a MXR-related protein in a benthic fish species, the turbot Scophthalmus maximus, and its constitutive expression in several tissues. A 433bp cDNA fragment has been cloned by RT-PCR. The deduced amino-acid sequence shares close to 80% homology with class I or class II mammalian MDR proteins. This cDNA corresponds to a major mRNA of 5.6 kb and encodes a protein having an apparent molecular weight of 83 kDa. Constitutive expression levels assessed by semi-quantitative RT-PCR and Western blot, revealed that the kidney and the brain, and to a lesser extent, the heart, gills and intestine, are the organs which contain the highest amount of both MXR mRNAs or proteins. This tissue specific expression suggests a role for the identified mechanism in protection against endogenous or exogenous toxic compounds.

Validation of an analytical procedure for polychlorinated biphenyls, coplanar polychlorinated biphenyls and polycyclic aromatic hydrocarbons in environmental samples.

This work describes an efficient analytical procedure for the analysis of PCBs (polychlorinated biphenyls), coplanar PCBs and PAHs (polycyclic aromatic hydrocarbons) from the same sample. The method includes a solvent extraction followed by a combined purification-separation step on an alumina-silica column. Coplanar PCBs are isolated from the first fraction (PCBs) by a further high-performance liquid chromatography (HPLC) fractionation on a PYE [2-(1-pyrenyl)ethyldimethylsilylated silica gel] column. PCBs are identified and quantified by gas chromatography (GC) with electron-capture detection whereas GC with flame ionization detection or mass spectrometry are used for PAH determinations. This method allows the measurement of these contaminants in biota and sediment at trace levels as low as 1 pg g(-1) for coplanar PCBs with a precision better than 20%.

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.

Pharmacological profile of the tachykinin receptor involved in the stimulation of corticosteroid secretion in the frog Rana ridibunda.

It has recently been shown that the adrenal gland of the frog Rana ridibunda is densely innervated by a network of fibers containing two novel tachykinins, i.e. ranakinin (the counterpart of substance P) and [Leu3, Ile7]neurokinin A. Both ranakinin and [Leu3, Ile7]neurokinin A stimulate corticosteroid secretion from frog adrenal glands in vitro. In the present study, we have investigated the pharmacological profile of the receptors involved in the stimulatory action of ranakinin on perifused frog adrenal slices. The selective NK-1 receptor antagonists [D-Pro4, D-Trp7,9]substance P 4-11 and CP-96,345, did not affect the stimulatory action of ranakinin. The selective NK-1 agonist substance P 6-11 had no effect on corticosteroid secretion. The non-peptidic NK-1 receptor antagonist RP 67580 significantly reduced the stimulatory effect of ranakinin on corticosterone and aldosterone secretion by 57 and 55%, respectively. In addition, the dual NK-1/NK-2 receptor antagonist FK-224 significantly inhibited the effect of ranakinin on corticosterone (- 80%) and aldosterone secretion (- 95%). Finally, the amphiphilic analogue of substance P, [D-Pro2, D-Phe7, D-Trp9]substance P, had no effect on corticosteroid secretion. These data suggest that in the frog adrenal gland the stimulatory action of ranakinin on steroid secretion is mediated by a novel type of receptor which differs substantially from the mammalian NK-1 receptor subtype.

Effect of ranakinin, a novel tachykinin, on cytosolic free calcium in frog adrenochromaffin cells.

It has recently been shown that two novel tachykinins, ranakinin and [Leu3, Ile7]neurokinin A, are present in fibers innervating the frog adrenal gland, and it has been demonstrated that tachykinins stimulate corticosteroid secretion in vitro through activation of chromaffin cells. The purpose of the present study was to investigate the effect of ranakinin on cytosolic free calcium concentrations ([Ca2+]i) and to determine the source of calcium involved. Cultured adrenal cells were loaded with the fluorescent calcium indicator indo-1, and changes in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Administration of a brief pulse of ranakinin (1 microM; 1 sec) in the vicinity of chromaffin cells caused an immediate and transient increase in [Ca2+]i. Repeated pulses of ranakinin resulted in a gradual decline in the [Ca2+]i response, suggesting the occurrence of a desensitization phenomenon. Preincubation of the cells with the calcium channel blockers nifedipine (10 microM) and omega-conotoxin (1 microM) did not alter the response of chromaffin cells to ranakinin. Chelation of extracellular calcium by EGTA (10 mM) caused a marked decrease in the basal [Ca2+]i, but did not suppress the ranakinin-induced [Ca2+]i increase. Conversely, incubation of the cells with thapsigargin (10 microM), an inhibitor of calcium adenosine triphosphatase activity, abolished the stimulatory effect of ranakinin, indicating that the increase in [Ca2+]i can be ascribed to mobilization of calcium from intracellular stores. Preincubation of adrenal cells with the phospholipase C antagonist U-73122 (1 microM; 18 min) or with pertussis toxin (10 microM; 18 h) totally blocked the ranakinin-induced [Ca2+]i rise. Taken together, these data indicate that in frog adrenochromaffin cells, ranakinin causes mobilization of calcium from intracellular stores. The effect of ranakinin is mediated through activation of a phospholipase C via a pertussis toxin-sensitive G protein.

Evidence for the involvement of chromaffin cells in the stimulatory effect of tachykinins on corticosteroid secretion by the frog adrenal gland.

The adrenal gland of the frog is innervated by a network of fibers containing two tachykinins (ranakinin and [Leu3,Ile7]neurokinin A), which both stimulate corticosteroid secretion from frog adrenal tissue. The aim of the present study was to determine the mode of action of tachykinins on the frog adrenal gland. Double immunolabeling of tissue sections with a monoclonal antibody to tyrosine hydroxylase and an antiserum to substance P showed that tachykinin-containing fibers are preferentially apposed onto chromaffin cells. Immunocytochemical labeling at the electron microscope level revealed that tachykinin-immunoreactive fibers establish close contacts only with adrenochromaffin cells. Ranakinin stimulated corticosterone and aldosterone secretion from perifused adrenal slices, but had no stimulative effect on dispersed adrenal cells. Cytoautoradiographic labeling of frog adrenal cells in primary culture with [3H]substance P revealed the existence of specific binding sites located exclusively on chromaffin cells. Microfluorimetric measurement of cytosolic calcium concentrations ([Ca2+]i) in cultured adrenal cells showed that ranakinin induced a dose-dependent increase in [Ca2+]i in chromaffin cells (ED50 = 2 x 10(-7) M). In contrast, ranakinin did not affect [Ca2+]i in adrenocortical cells. The present results indicate that in the frog adrenal gland, tachykinin-containing fibers make preferential contacts with chromaffin cells, and tachykinins directly activate chromaffin cells. These data suggest that the stimulative effect of tachykinins on corticosteroid secretion is mediated via presynaptic activation of adrenochromaffin cells.

Immunocytochemical localization and biological activity of 3 beta-hydroxysteroid dehydrogenase in the central nervous system of the frog.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes biosynthesis of progesterone (P) and all precursors of glucocorticoids, mineralocorticoids, androgens, and estrogens. Despite the broad interest raised by neurosteroids, the cellular localization of 3 beta-HSD has never been investigated in the brain. We took advantage of the availability of an antiserum raised against human placental 3 beta-HSD to determine the distribution of 3 beta-HSD-immunoreactive structures in the brain of the frog Rana ridibunda by the indirect immunofluorescence technique. Three populations of 3 beta-HSD-immunoreactive cell bodies were observed in the hypothalamus, namely, in the rostral region of the preoptic nucleus, the dorsal infundibular nucleus, and the dorsal part of the ventral infundibular nucleus. A dense network of 3 beta-HSD-immunoreactive nerve fibers was visualized in the dorsal area of the diencephalon, that is, in the lateral neuropil, the corpus geniculatus lateralis, and the nucleus posterolateralis thalami. Reversed-phase HPLC analysis of frog hypothalamic extracts combined with RIA detection showed the presence of substantial amounts of immunoreactive steroids coeluting with P and 17-hydroxyprogesterone (17OH-P). The synthesis of delta 4-3-keto-steroids in the frog hypothalamus was investigated using the pulse-chase technique with 3H-pregnenolone (3H-delta 5P) as a precursor. The formation of five tritiated metabolites of 3H-delta 5P was observed, one of which coeluted with 17OH-P. Conversion of 3H-delta 5P into this radioactive metabolite was significantly reduced by trilostane, a specific inhibitor of 3 beta-HSD. Immunodetection of newly synthesized steroids in HPLC fractions of hypothalamic extracts, using 17OH-P antibodies, revealed the existence of an immunoreactive steroid that exhibited the same retention time as synthetic 17OH-P. The present study provides the first immunocytochemical mapping of 3 beta-HSD, a key enzyme of the steroid biosynthetic pathway, in the CNS of a vertebrate. The data also demonstrate for the first time biosynthesis of neurosteroids in the brain of a nonmammalian vertebrate.

Immunohistochemical distribution, biochemical characterization, and biological action of tachykinins in the frog adrenal gland.

The distribution of tachykinin-like immunoreactivity (LI) was studied in the adrenal gland of the frog Rana ridibunda using the immunofluorescence technique. A dense network of varicose fibers immunoreactive to both substance-P (SP) and neurokinin-A (NKA) was found in the adrenal tissue. In contrast, no positive fibers could be detected using antineurokinin-B (NKB) antibodies. At the electron microscope level, the immunogold technique revealed that tachykinin-LI was sequestered in dense core vesicles of 50-70 nm. Bilateral transection of either splanchnic or vagus nerves or total lesion of celiac sympathetic ganglion did not suppress tachykinin-LI. A combination of HPLC analysis and RIA detection was used to characterize tachykinin-LI in frog adrenal extracts. Two major peaks were resolved, which coeluted, respectively, with synthetic ranakinin, a novel tachykinin previously isolated from the frog brain, and [Leu3,Ile7]NKA previously isolated from the frog gut. No NKB could be detected in the extracts. The effects of various synthetic tachykinins on corticosteroid secretion were studied using perifused frog adrenal slices. For concentrations ranging from 10(-8)-10(-4) M, SP induced a dose-dependent stimulation of corticosterone and aldosterone release. A desensitization phenomenon was observed when iterative or prolonged infusions of SP were administered to the tissue. All mammalian or amphibian tachykinin-related peptides tested in our model also enhanced corticosteroid production. The effectiveness of the tachykinins tested was: [Pro7] NKB > NKA > ranakinin > [Pro9]SP > SP > kassinin > physalaemin > NKB > [Leu3,Ile7]NKA. SP also enhanced prostaglandin E2 and prostacyclin release in the effluent perifusate and the response preceded by 10-15 min the increase in corticosteroid output. Indomethacin (5 x 10(-6) M), a specific blocker of cyclooxygenase activity, totally suppressed SP-evoked steroid secretion. These data indicate that tachykinin-induced stimulation of steroidogenesis was mediated through activation of the arachidonic acid cascade. Taken together, our results show that the frog adrenal gland is innervated by a dense network of peptidergic fibers containing both ranakinin and [Leu3,Ile7]NKA, which, in vitro, stimulates corticosteroid secretion by adrenocortical cells through a prostaglandin-dependent mechanism. The present results support the view that tachykinins released by nerve fibers exert a neuroendocrine control on corticosteroid release in amphibians.

In vitro study of catecholamine release from perifused frog adrenal slices.

The adrenal gland of amphibians is composed of a mixed population of adrenochromaffin and corticosteroid-secreting cells. It has previously been shown that chromaffin cells synthesize several bioactive substances (including biogenic amines and neurotransmitters) which may act locally to regulate corticosteroid secretion. In the present report, we have studied the secretory activity of adrenochromaffin cells in Rana ridibunda. Frozen sections of adrenal gland were immunolabeled with antisera against tyrosine hydroxylase or phenylethanolamine-N-methyltransferase. Comparison of homologous fields on consecutive sections indicated that 77% of catecholaminergic cells produce adrenaline. The concentrations of catecholamines were measured by means of high performance liquid chromatography analysis coupled to electrochemical detection. The concentrations of dopamine, noradrenaline, and adrenaline in fresh adrenal tissue were 24 +/- 4, 763 +/- 68, and 1032 +/- 118 ng/mg wet weight, respectively. After a 12-hr perifusion period, the concentration of adrenaline in the tissue was reduced by 62%, whereas noradrenaline only decreased by 22%. The secretion rates of adrenaline and noradrenaline from perifused adrenal slices significantly diminished during the first 7 hr of the experiment and then remained relatively stable for about 10 hr. Exposure of adrenal tissue to a depolarizing concentration of potassium (55 mM) induced an immediate and substantial rise of adrenaline and noradrenaline release and a delayed increase in corticosterone output. Acetylcholine, which stimulates corticosterone secretion from frog adrenocortical cells, induced a slight but not significant increase of adrenaline and noradrenaline release. Similarly, the selective cholinergic agonists muscarine and nicotine did not significantly affect catecholamine release, while muscarine mimicked the stimulatory action of acetylcholine on corticosterone secretion. This study validates the use of the perifusion model to investigate the mechanism of control of catecholamine release from frog adrenochromaffin tissue. The results presented herein indicate that, in contrast to mammals, the secretion of catecholamines from the amphibian adrenal gland is not regulated by cholinergic inputs.

Characterization of dopamine receptors associated with steroid secretion in frog adrenocortical cells.

We investigated the type of receptors involved in the mechanism of action of dopamine on corticosteroid secretion from the frog interrenal (adrenal) gland, using the in-vitro perifusion technique. Exposure of dispersed interrenal cells to 50 microM dopamine for 20 min had a biphasic effect on corticosterone and aldosterone secretion, i.e. a transient stimulation followed by an inhibitory phase. Repeated administration of equimolar pulses of dopamine, given at 150-min intervals, resulted in an enhancement of corticosteroid secretion followed by a subsequent blockade of the stimulatory phase of the response. In contrast, the dopamine-evoked inhibition of corticosteroid release did not show any sensitization or desensitization phenomena. Infusion of repeated pulses of the D1 receptor agonist SKF38393 (32 microM) stimulated corticosteroid release and mimicked the sensitization-desensitization phenomenon induced by dopamine. Repeated administration of the D2 receptor agonist LY171555 (50 microM) resulted in a reproducible inhibition of corticosterone and aldosterone secretion. These results suggested the presence of two different receptors for dopamine, i.e. D1 and D2, on frog adrenocortical cells, responsible respectively for the stimulatory and inhibitory effects of dopamine on steroid secretion. However, bromocriptine (50 microM) and CV205-502 (50 microM), two other D2 receptor agonists, had no effect on corticosteroid release. In addition, several classical D2 receptor antagonists failed to block the effect of dopamine on steroidogenesis. It was also observed that (-)sulpiride, a specific D2 antagonist, did not alter dopamine-induced inhibition of inositol phosphate formation. On the other hand, dopamine and the selective D1 and D2 antagonists SKF38393 and LY171555 did not affect the formation of cyclic AMP by interrenal tissue. Taken together, these data indicate that dopamine directly regulates corticosteroid secretion from frog adrenocortical cells. The effect of dopamine is not coupled to adenylate cyclase activity but is probably mediated through the phosphoinositide-turnover pathway. The pharmacological characteristics of the receptors involved in the mechanism of action of dopamine clearly differ from those of the D1 and D2 subtypes previously described in mammals.

Self-inhibition of steroid secretion by amphibian adrenocortical cells is not mediated through glucocorticoid receptors.

To investigate a possible direct action of glucocorticoids on adrenal steroidogenesis, the effect of corticosterone on the conversion of pregnenolone into various metabolites by frog adrenal tissue was examined. Frog interrenal slices were incubated with [3H]pregnenolone (1 mCi/ml) and the various labelled metabolites analysed by reverse-phase high-performance liquid chromatography. With the methanol gradient used, five identified steroids were resolved: progesterone, 11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone. Corticosterone (10 micrograms/ml) induced a 45-80% decrease in all steroids synthesized from [3H]pregnenolone. In contrast, the glucocorticoid agonist dexamethasone did not reduce the rate of conversion of pregnenolone into its metabolites. In addition, the inhibitory effect of corticosterone was not reversed by the specific glucocorticoid antagonist RU 43044. These results show that corticosterone exerts a direct inhibitory effect on adrenal steroid secretion. In addition, our data indicate that the ultra-short regulation induced by corticosterone is not mediated through glucocorticoid receptors.

Dopamine inhibits inositol phosphate production, arachidonic acid formation, and corticosteroid release by frog adrenal gland through a pertussis toxin-sensitive G-protein.

We have previously shown that dopamine-evoked inhibition of corticosteroid production from adrenocortical cells is mediated through a decrease in prostaglandin biosynthesis. Since the catecholamine did not alter the stimulatory effect of arachidonic acid, it was proposed that dopamine may inhibit the formation of arachidonate from glycerophospholipids. To test this hypothesis, the effect of dopamine on phosphoinositol lipid metabolism was investigated in frog interrenal (adrenal) tissue. In [3H]myo-inositol-prelabeled frog interrenal slices, a short pulse of dopamine (50 microM) induced a biphasic effect on inositol phosphate production: a transient (1-min) increase, followed by a sustained inhibition. Concurrently, dopamine induced a transient reduction followed by a sustained increase in polyphosphoinositides. A 10-min pulse of the D2 dopamine receptor agonist apomorphine (50 microM) elicited a significant inhibition of basal levels of inositol phosphates (tris-, bis-, and mono-), and an increase in plasma membrane phosphoinositol lipid contents. The inhibitory effect of dopamine on inositol phosphate formation and corticosteroid release was abolished by a 24-h incubation of interrenal slices with pertussis toxin. In [3H]arachidonic acid-prelabeled interrenal slices, dopamine also decreased diacylglycerol (DG) and arachidonic acid (AA) concentrations. A delay of 1 min was observed between inhibition of DG and arachidonate, suggesting that AA is probably generated from DG. We conclude that in the adrenal cortex, activation of dopamine D2 receptors is coupled to a phosphoinositide-specific phospholipase-C mediated via a pertussis toxin-sensitive G-protein. Taken together, our data indicate that inhibition of inositol phosphate and AA formation is one of the mechanisms by which dopamine controls corticosteroid production by adrenocortical cells.