Cultures Of Adrenocortical Cells Research Articles

Insulin-like growth factors enhance steroidogenic enzyme and corticotropin receptor messenger ribonucleic acid levels and corticotropin steroidogenic responsiveness in cultured human adrenocortical cells.

In several species, including the human fetus, insulin-like growth factors (IGF-I and IGF-II) have been reported to modulate adrenal steroidogenesis, thus contributing to adrenal cortical differentiation. In the present study, we examined the long term effects of IGF-I and -II on human adult adrenal fasciculata-reticularis cells cultured in a chemically defined medium and compared them to the effects of insulin, human GH, and ACTH. Treatment for 3 days with IGF-I or -II at nanomolar concentrations or with insulin at micromolar concentrations slightly increased the production of androstenedione, cortisol, and dehydroepiandrosterone about 1.5-fold over that by control cells. Moreover, the acute steroidogenic response to ACTH of cells pretreated with IGF-I, IGF-II, or insulin was 3- to 6-fold higher than that of control cells. For each hormone, these effects of IGF-I and -II were dose dependent between 0.1-26 nmol/L (1-200 ng/mL). The secretion of androstenedione was more potently stimulated than that of dehydroepiandrosterone and cortisol, and this effect was more clearly yielded by pretreatment with IGF-II than with IGF-I or insulin. Human GH had no effect on these cells. In cells treated with IGF-I or -II, the messenger ribonucleic acid (mRNA) levels of cytochrome P450 17 alpha-hydroxylase and of 3 beta-hydroxysteroid dehydrogenase were increased, and the abundance of ACTH receptor mRNA was also slightly enhanced, but the mRNA of cytochrome P450 cholesterol side-chain cleavage enzyme was unchanged. In conclusion, IGFs enhance the steroidogenesis and ACTH responsiveness of human adrenocortical cells in culture. We speculate, that by this mechanism, IGFs may contribute to clinical states with hyperandrogenemia.

Opposite regulation of thrombospondin‐1 and corticotropin‐induced secreted protein/thrombospondin‐2 expression by adrenocorticotropic hormone in adrenocortical cells

Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenocortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10−7 M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by ten-fold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not. © 1996 Wiley-Liss, Inc.

Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells.

Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10(-7) M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by tenfold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not.

Differential toxicity of an inhibitor of mitochondrial respiration in canine hepatocytes and adrenocortical cell cultures

PD132301-2, a novel inhibitor of acyl-CoA: cholesterol acyltransferase, was previously shown to be an inhibitor of mitochondrial respiration and an adrenal toxicant in several species. To investigate potential mechanisms of tissue-specific toxicity in vivo, dog adrenocortical and hepatocyte cell cultures were exposed to 0.01–30 μm PD132301-2 for 0–24 hr. Cell viability was assessed by neutral red uptake or release assays. Cytotoxicity was observed in adrenocortical cells at 0.01 μ m or above after 2hr of exposure, while 30 μm was not toxic to hepatocytes after 24 hr of exposure. Decreases in adrenocortical cell viability were attenuated in the presence of 20 m m fructose, a glycolytic substrate, and fructose protection was in turn blocked by the glycolytic inhibitor NaF (1 m m). In contrast, PD132301-2-induced hepatocellular toxicity was evident only following pretreatment with NaF or metyrapone, a broad-spectrum cytochrome P-450 inhibitor. With either co-treatment, hepatocyte viability was reduced 50% after 6 hr at 1 μ m or more PD132301-2. These data indicate that the relative sensitivity of adrenocortical cells to the cytotoxic effects of PD132301-2 may be due, in part, to their relative lack of the metabolic detoxification and glycolytic reserve capacities that appear to protect hepatocytes from PD132301-2 toxicity.

Ouabain production by cultured adrenal cells.

The adrenal cortex releases a sodium pump inhibitor. The present studies tested whether this material was endogenous and identical to ouabain by 1) studying the production of ouabain in long term cultures of adrenocortical cells, 2) seeking evidence that ouabain might be taken up from exogenous sources by adrenocortical cells, 3) examining the release of adrenocortical cells loaded with exogenous ouabain, 4) attempting to stimulate ouabain steroidogenesis in cultured adrenocortical cells, and 5) performing further chemical analysis on ouabain immunoreactivity released by cultured adrenocortical cells. Our results indicate that ouabain immunoreactivity is present in conditioned medium from both murine Y-1 adrenocortical cultures and primary bovine adrenocortical cell (BAC) cultures. We also found that BACs bind and internalize [3H]ouabain. Bound [3H]ouabain is released from BACs by both receptor dissociation and cytoplasmic release of internalized [3H]ouabain. Only one isoform of membrane sodium, potassium-adenosine triphosphatase, alpha 1, was expressed in the adrenal. Authentic ouabain was not metabolized during membrane binding or while present intracellularly. Stimulation of steroidogenesis in Y-1 and BAC with 22R-hydroxycholesterol and 25-hydroxycholesterol was performed and confirmed increased steroidogenesis; however, there was no effect on ouabain immunoreactivity content or release. Comparison of the ouabain binding density in cultured BAC, hepatoma cells, and 3T3 fibroblasts indicated that adrenocortical cells have a high ouabain-binding capacity. HPLC studies of the ouabain immunoreactivity released by bovine adrenocortical cells indicated that essentially no authentic ouabain was secreted. The present studies confirm that both BAC and Y-1 cultures release a ouabain-like material that differs in structure from authentic plant ouabain and is not a product of cholesterol side-chain cleavage.

Alpha 2-macroglobulin: a binding protein for transforming growth factor-beta and various cytokines.

alpha 2-Macroglobulin (alpha 2M) is a large plasma glycoprotein that has long been known as an irreversible inhibitor of a variety of proteinases. More recently, it has been reported that numerous growth factors, cytokines and hormones bind to alpha 2M through diverse mechanisms. We review here a series of observations from our laboratory that support the concept that alpha 2M is a carrier protein for transforming growth factor-beta (TGF-beta) and allows this factor to act as an autocrine regulator of adrenocortical steroidogenic functions. alpha 2M was found to by synthesized and secreted by primary cultures of bovine adrenocortical cells in fairly large amounts (1 microgram/10(6) cells/24 h). TGF-beta is also secreted by this cell type, although under a latent form. Two distinct latent TGF-beta complexes have been characterized in adrenocortical cell conditioned medium, one of which is a complex between alpha 2M and TGF-beta. Although alpha 2M prevents the binding of TGF-beta to its membrane receptors, long-term incubation of alpha 2M with adrenocortical cells results in inhibition of cortisol production similar to that observed in the presence of TGF-beta alone. Taken together, these observations suggest that adrenocortical cells can release active TGF-beta from its latent complex with alpha 2M through an unknown mechanism. alpha 2M can therefore be considered as a TGF-beta carrier protein.

Evidence for the Involvement of an Anion Exchanger in Cortisol Release from Bovine Adrenocortical Cells

In this study the influence of probenecid as inhibitor of the renal excretory system for organic anions was tested on the release of cortisol from primary cultures of bovine adrenocortical cells. ACTH-stimulated cortisol release (30 min to 24 h) was inhibited by probenecid (Ki = 0.093 mM). Analysis of steroid synthesis with ³H-pregnenolone as precursor indicated that part of the inhibition resulted from an attenuation of steroid synthesis by probenecid. The increased intracellular cortisol levels in the presence of probenecid however indicate an inhibitory effect on an efflux mechanism. Cortisol efflux was trans-stimulated by <i>p</i>-aminohippurate (PAH) and tetrafluorosuccinate, substrates of the renal PAH/anion exchanger. Further evidence for the PAH exchanger provided the expression of a probenecid-sensitive PAH transporter after injection of mRNA from adrenal cells into <i>Xenopus laevis </i>oocytes.

Specific inhibition of the last steps of aldosterone biosynthesis by 18-vinylprogesterone in bovine adrenocortical cells

18-Vinylprogesterone (18-VP), designed for mechanism-based specific inhibition of the last steps of the aldosterone biosynthesis, was used to characterize the mechanism of the 11- and 18-hydroxylase activities of bovine cytochrome P450 11 β . In the present work, its action was studied by observations on a primary culture of bovine adrenocortical cells. First, we investigated the effects of 18-VP on the different enzymatic steps of the biosynthesis of cortisol and aldosterone. The production of cortisol, baseline or hormone-stimulated (ACTH or AII), was inhibited by 18-VP in a dose-dependent manner with a maximal inhibition at 5 μM. Supply of different exogenous substrates to support steroidogenesis revealed an inhibition of the last step of cortisol or corticosterone biosynthesis. We then used specific blockers to measure individual activities and conclude that 11β-hydroxylation was the only enzymatic activity affected. Aldosterone, as well as 18-hydroxycorticosterone, was also measured following addition of corticosterone. The 18-hydroxylation of corticosterone was inhibited by 18-VP, with 50% inhibition occurring at 0.04 μM compared with the 50% inhibition value of 0.3 μM obtained for 11-hydroxylation. Surprisingly, 18-ethynyl-progesterone (18-EP), which has a structure very similar to 18-VP, only weakly inhibits 11β-hydroxylation. The inhibition of aldosterone formation was also much lower with 18-EP than with 18-VP. These studies demonstrate that 18-VP inhibits only the later steps of aldosterone biosynthesis and more specifically 18- than 11-hydroxylation activity.

Secretion of Endogenous Ouabain from Bovine Adrenocortical Cells: Role of the Zona Glomerulosa and Zona Fasciculata

Recent experiments have demonstrated the secretion of a ouabain-like compound from primary cultures of bovine adrenocortical cells (1). To determine the relative contribution of the outer zones of the adrenal cortex to the secretion of endogenous ouabain, we measured the content of this steroid in zona glomerulosa and zona fasciculata tissue freshly excised from bovine adrenals and in the secretion media of primary cell cultures derived from these zones. The tissue content of endogenous ouabain was 5.7-fold higher in the zona glomerulosa than in the zona fasciculata. The concentration of endogenous ouabain was 4.1-fold higher in 24-hour conditioned media from zona glomerulosa than in zona fasciculata cultures. Angiotensin II and ACTH stimulated the secretion of endogenous ouabain from zona glomerulosa cultures, but had no effect on zona fasciculata cultures. These findings suggest that endogenous ouabain is primarily synthesized and secreted from the adrenal zona glomerulosa.

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines. In steroidogenic cell line Y1, the expression of ferredoxin was stimulated by cAMP and repressed slightly by angiotensin II and phorbol ester PMA. These drugs exhibited the same effect on the basal promoter of the ferredoxin gene, which includes one TATA box and an SP1 site. In human adrenocortical cell line H295, the stimulation of the ferredoxin gene by cAMP was blocked by cycloheximide, as observed in bovine adrenocortical cell culture. In nonsteroidogenic cell lines such as HeLa and COS-1, the stimulation of ferredoxin gene expression by cAMP was not observed, although basal expression was strong. Transfection studies showed that the ferredoxin promoter could not be stimulated by cAMP in nonsteroidogenic cells. Therefore the steroidogenic cell-specific regulation and the general expression pattern appears to be a property unique to the ferredoxin gene.

Contribution of apoptosis to the phenotypic changes of adrenocortical cells in primary culture

The aim of this study was to evaluate the occurrence and physiological consequences of apoptosis in primary cultures of bovine adrenocortical cells (of fasciculata-reticularis origin). Under ACTH-free culture conditions, we observed apoptotic cells in the cell layer and the accumulation of apoptotic bodies in the culture medium. These were hardly detectable in ACTH-supplemented cultures. Under ACTH-free conditions, the DNA content of apoptotic bodies collected over 48 h represented up to 10–15% of that of the cell layer at the onset of the culture (as compared to 3% in ACTH-supplemented cultures). Past the fourth day of culture in the absence of ACTH, most cells lacked several markers of their originating fasciculata-reticularis phenotype and progressively evolved to an undifferentiated phenotype. The vast majority of the apoptotic bodies released during the first 4 days of culture were immunoreactive for P450 17α. Inversely, during the same period of time, the proliferating cells (PCNA-positive) did not appear to express P450 17α. Therefore, apoptosis could contribute, together with dedifferentiation, to the phenotype shift observed in ACTH-depleted cultures of adrenal fasciculata-reticularis cells. These observations also characterize this endocrine cell system as an in vitro model for the study of hormone-repressed apoptosis.

Steroidogenic adrenocortical cells synthesize α2-macroglobulin in vitro, not in vivo

We previously identified α 2-macroglobulin as the major protein secreted by primary cultures of adrenocortical cells. We report here that in the adrenal gland, the distribution of α 2-macroglobulin in the adrenocortical tissue is restricted to the endothelium of blood vessels and that no immunoreactivity is found in Steroidogenic cells. A time course study revealed that freshly dissociated bovine adrenocortical cells were void of α 2-macroglobulin immunoreactivity whereas the proportion of α 2-croglobulin-positive cells reached more than two-thirds of the population between day 4 and day 7 of culture. Double immunoenzymatic labeling of 6-day-old cultures revealed a co-localization of α 2-macroglobulin and the Steroidogenic enzyme P-450 scc. Treatment of 5-day-old cultures (expressing α 2-macroglobulin) for 24 h by either ACTH (10 −9-10 −6 M) or α 2-macroglobulin (2.5 mg/ml) resulted in a marked decrease of the expression of α 2-macroglobulin. These data indicate that ACTH and plasmatic α 2-macroglobulin could physiologically repress α 2-macroglobulin expression in the adrenal cortex in vivo.

Effect of age on cholesterol uptake and utilization by rat adrenals: I. Internalization of lipoprotein-derived cholesteryl esters

Previous studies from this laboratory have documented a progressive age-related decline in trophic hormone (or second messenger cAMP) stimulated corticosterone production in isolated adrenocortical cells. In the current study, we examined the possibility that the aging process exerts this effect by interfering with an early step in the delivery of lipoprotein-derived cholesteryl esters to the cell. As such, we monitored the ability of two different rat adrenocortical cell model systems (intact perfused adrenal glands and primary cultures of adrenocortical cells from 5- and 18- to 20-month-old rats) to internalize lipoprotein cholesteryl esters, and to convert the newly internalized cholesteryl esters to corticosterone production. The results indicate that lipoprotein (hHDL 3 and rHDL) cholesteryl ester internalization (by both the endocytic and ‘selective’ pathways) is comparable in adrenocortical cells of the young and old rats. However, despite this, both the mass of corticosterone produced and the ratio of newly internalized (radiolabeled) cholesteryl ester incorporated into corticosterone is dramatically reduced in cells of the older animals. Thus, the lipoprotein uptake pathway appears to be intact in adrenals of older rats, but the intracellular processing of internalized cholesteryl ester is defective.

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine 5'-triphosphate, adenosine 5'- diphosphate, and uridine 5'-triphosphate via a nucleotide receptor that may be coupled to two signal generation systems [corrected and republished with original paging, article originally printed in Endocrinology 1994 Mar;134(3):1553-60

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine 5'-triphosphate, adenosine 5'- diphosphate, and uridine 5'-triphosphate via a nucleotide receptor that may be coupled to two signal generation systems [corrected and republished with original paging, article originally printed in Endocrinology 1994 Mar;134(3):1553-60

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine 5'-triphosphate, adenosine 5'-diphosphate, and uridine 5'-triphosphate via a nucleotide receptor that may be coupled to two signal generation systems.

Cultured inner zone cells isolated from bovine adrenal cortex secreted cortisol in a dose-dependent fashion in response to ATP and ADP. The threshold response was at 10(-6) M ATP, reaching a maximum by 10(-4) M ATP, at which concentration the n-fold increase relative to basal was 43.8 +/- 22.3 (mean +/- SD; n = 3). Cells were also responsive to the pyrimidine nucleotide UTP. EC50 values for ATP, ADP, and UTP were 5.83 +/- 3.98 x 10(-6), 13.7 +/- 5.67 x 10(-6), and 7.33 +/- 4.52 x 10(-7) M, respectively (mean +/- SD; n = 3). The response to 10(-4) M ATP was linear for at least 60 min, and the cells appeared morphologically normal after removal of the stimulus. The purinergic antagonist suramin was relatively ineffective. The potency order of a range of purines was as follows: ATP = UTP > ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Stimulation of cortisol secretion by ATP was evident after 24 h in primary culture and reached a maximum after 48-72 h, thereafter declining. No response was detected in static incubations of freshly isolated cells. The possibility that added ATP was degraded over the course of the incubation was investigated by separating ATP, ADP, AMP, and adenosine by high resolution anion exchange chromatography after different times of exposure to the cells. Although there was degradation, largely to ADP, about 50% of the ATP remained at 1 h. Cells grown in the presence of [3H]inositol (10 microCi/ml) for 48 h (to prelabel the membrane phosphoinositide pool to isotopic equilibrium) showed a time- and dose-dependent increase in [3H]inositol-labeled total phosphoinositols to ATP or ADP; the response was linear for at least 20 min. Cells labeled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium to 10(-4) M ATP on days 3 and 4 of culture. The basal intracellular Ca2+ concentration was 57.3 +/- 39.3 nmol/liter (mean +/- SD; n = 12 cell suspensions), rising to 171 +/- 84 nmol/liter (mean +/- SD; n = 12 cell suspensions) after the addition of ATP (10(-4) M). Bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min of stimulation with ATP. It was not possible to account for the cAMP response on the basis of conversion of ATP to adenosine, which then acted at an A2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine triphosphate, adenosine diphosphate, and uridine triphosphate via a nucleotide receptor which may be coupled to two signal generation systems.

Cultured inner zone cells isolated from bovine adrenal cortex secreted cortisol in a dose-dependent fashion in response to ATP and ADP. The threshold response was at 10(-6) M ATP, reaching a maximum by 10(-4) M ATP, at which concentration the n-fold relative to basal was 43.8 +/- 22.3 (mean +/- SD, n = 3). The response to 10(-4) M ATP remained linear for up to 2 h, and the cells appeared morphologically normal after removal of the stimulus. Stimulation of cortisol secretion by ATP was evident after 24 h in primary culture and reached a maximum after 48-72 h, thereafter declining. No response was detected in freshly isolated cells. The possibility that added ATP was degraded over the course of the incubation was investigated by separating ATP, ADP, AMP, and adenosine by high resolution anion exchange chromatography after different times of exposure to the cells. Although there was degradation--largely to ADP--about 50% of the ATP remained at 1 h. The potency order of a range of purines was as follows: ATP = ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Cells were also responsive to the pyrimidine nucleotide uridine 5'-triphosphate, which was equipotent with ATP. The purinergic antagonist suramin was relatively ineffective. Cells grown in the presence of [3H]inositol (10 microCi/ml) for 48 h (to prelabel the membrane phosphoinositide pool to isotopic equilibrium) showed a time- and dose-dependent increase in [3H]inositol-labeled total phosphoinositols in response to ATP or ADP; the response was linear for at least 60 min. Cells labelled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium in response to 10(-4) M ATP on days 3 and 4 of culture. Basal intracellular Ca2+ was found to be 57.3 +/- 39.3 nmol/liter (mean +/- SD, n = 12 cell suspensions) rising to 171 +/- 84 nmol/liter (mean +/- SD, n = 12 cell suspensions) in response to ATP (10(-4) M). In response to ATP, bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min stimulation. It was not possible to account for the cAMP response on the basis of conversion of ATP to adenosine, which then acted at an A2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine triphosphate, adenosine diphosphate, and uridine triphosphate via a nucleotide receptor which may be coupled to two signal generation systems [corrected and republished in Endocrinology 1994 Oct;135(4):1553-60

Cultured inner zone cells isolated from bovine adrenal cortex secreted cortisol in a dose-dependent fashion in response to ATP and ADP. The threshold response was at 10(-6) M ATP, reaching a maximum by 10(-4) M ATP, at which concentration the n-fold relative to basal was 43.8 +/- 22.3 (mean +/- SD, n = 3). The response to 10(-4) M ATP remained linear for up to 2 h, and the cells appeared morphologically normal after removal of the stimulus. Stimulation of cortisol secretion by ATP was evident after 24 h in primary culture and reached a maximum after 48-72 h, thereafter declining. No response was detected in freshly isolated cells. The possibility that added ATP was degraded over the course of the incubation was investigated by separating ATP, ADP, AMP, and adenosine by high resolution anion exchange chromatography after different times of exposure to the cells. Although there was degradation—largely to ADP—about 50% of the ATP remained at 1 h. The potency order of a range of purines was as follows: ATP = ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Cells were also responsive to the pyrimidine nucleotide uridine 5'-triphosphate, which was equipotent with ATP. The purinergic antagonist suramin was relatively ineffective. Cells grown in the presence of [3H]inositol (10 microCi/ml) for 48 h (to prelabel the membrane phosphoinositide pool to isotopic equilibrium) showed a time- and dose-dependent increase in [3H]inositol-labeled total phosphoinositols in response to ATP or ADP; the response was linear for at least 60 min. Cells labelled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium in response to 10(-4) M ATP on days 3 and 4 of culture. Basal intracellular Ca2+ was found to be 57.3 +/- 39.3 nmol/liter (mean +/- SD, n = 12 cell suspensions) rising to 171 +/- 84 nmol/liter (mean +/- SD, n = 12 cell suspensions) in response to ATP (10(-4) M). In response to ATP, bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min stimulation. It was not possible to account for the cAMP response on the basis of conversion of ATP to adenosine, which then acted at an A2 receptor.

Oligomeric Casein Kinase II Isoforms Are Expressed in Bovine Tissues and Adrenocortical Cells in Culture

Casein kinase II is a ubiquitous serine-threonine kinase made of two different (α and β) subunits, with an α2β2 stoichiometry, α bearing the catalytic site of the enzyme. Two different genes encode two different (α and α′) catalytic subunits. The present work shows that at least two forms of CKII (CKIIa and CKIIb) can be isolated from bovine tissues and adrenocortical cells in culture. The use of specific antibodies developed against the α and α′ subunits disclosed that while CKIIa contains both α and α′, the α subunit was highly prominent in CKIIb. This suggests that CKIIb may be an α2β2 oligomer whereas CKIIa may be an αα′β2 moiety or an αα′β2 and α2β2 mixture. Transition from quiescence to proliferation was concomitant with an average two fold increase in total cell CKII activity involving both CKIIa and CKIIb isoforms. The two isoforms were found in cytosolic and particulate adrenocortical subcellular preparations and they similarly increased their association to intracellular organelles in response to growth stimulatory conditions.

The new adrenal steroid hormone ouabain

It has been proposed that an unknown circulating factor inhibits the plasma membrane sodium-potassium pumps and thereby plays an important role as a natriuretic or vasoconstrictive agent in the regulation of volume and pathogenesis of low-renin hypertension. The sodium pumps have specific high-affinity receptors for the digitalis glyrosides. With the analogy of endogenous opioids and nitric oxide, this has fostered speculation that an endogenous ligand exists that is “digitalis-like.” Recently, an endogenous factor has been extracted from human plasma, has been purified, and has been structurally identified by mass spectroscopy. This factor binds with high affinity to the sodium pump receptor and is indistinguishable from the cardiotonic steroid ouabain. Considerable evidence suggests that ouabain is synthesized in the adrenal cortex. It has a basic steroid nucleus, it is detected in high concentration in adrenocortical tissue, a continuous venous-arterial gradient across the adrenal can be demonstrated in the dog, and after bilateral adrenalectomy, plasma ouabain levels fall significantly. Additionally, adrenocortical cells in culture synthesize ouabain de novo. A modest elevation in plasma ouabain has been demonstrated in various forms of hypertension in humans, and the chronic infusion of ouabain to raise the plasma level to a comparably modest degree produces reversible normal renin hypertension in the rat. Circulating ouabain may also prove to be important in heart failure and in the pathogenesis and evolution of primary aldosteronism. To date, the precise site in the adrenal cortex and pathways for the biosynthesis of ouabain have not been determined. This information and the clarification of the pressor mechanism and the significance of ouabain and other possible sodium transport inhibitors in disorders involving volume regulation are major areas for future research. Nevertheless, the presence of ouabain in the circulation and tissues suggests that it could prove to be of widespread biologic significance.

High-performance liquid chromatographic study of the regulation of phospholipid metabolism in cultured adrenocortical cells

A rapid high-performance liquid chromatographic (HPLC) method for the separation of phospholipids was developed for minute samples of total lipids ( ca. 200 μg). The method was applied to the study of the phospholipid metabolism in adrenocortical cell cultures. A complete separation of the different cellular phospholipid classes was achieved in 40 min. Good resolution of the phospholipid peaks was obtained, which allowed the collection of each individual class of phospholipids for further analysis of radioactivity and fatty acid composition by gas chromatography. When cells were incubated with [U- 14C]glycerol or [U- 14C]palmitate the bulk of the radioactivity was found in cellular phosphatidylcholines. Exogenous phospholipids were incorporated into cellular lipids to a large extent, however without an increase in the cellular phospholipid content. 12-O-Tetradecanoyl-phorbol-13-acetate induced a 20% increase in the polyunsaturated fatty acid content of the cellular phosphatidylethanolamines, but no change was detected in the cellular phosphatidylcholines. The developed method is well-suited to the study of the phospholipid metabolism in adrenocortical cells where the phospholipid metabolism is closely linked to the specialized functions of the cells.