Creating tissue on chip constructs in microtitre plates for drug discovery.

We report upon a novel coplanar dielectrophoresis (DEP) based cell patterning system for generating transferrable hepatic cell constructs, resembling a liver-lobule, in culture. The use of paper reinforced gel substrates provided sufficient strength to enable these constructs to be transfered into 96-well plates for long term functional studies, including in the future, drug development studies. Experimental results showed that hepatic cells formed DEP field-induced structures corresponding to an array of lobule-mimetic patterns. Hepatic viability was observed over a period of 3 days by the use of a fluorescent cell staining technique, whilst the liver specific functionality of albumin secretion showed a significant enhancement due to the layer patterning of cell lines (HepG2/C3A), compared to 2D patterned cells and un-patterned control. This "build and transfer" concept could, in future, also be adapted for the layer-by-layer construction of organs-on-chip in microtitre formats.

Dielectrophoretic tweezer for isolating and manipulating target cells.

The ability to isolate and accurately position single cells in three dimensions is becoming increasingly important in many areas of biological research. The authors describe the design, theoretical modelling and testing of a novel dielectrophoretic (DEP) tweezer for picking out and relocating single target cells. The device is constructed using facilities available in most electrophysiology laboratories, without the requirement of sophisticated and expensive microfabrication technology, and offers improved practical features over previously reported DEP tweezer designs. The DEP tweezer has been tested using transfected HEI-193 human schwannoma cells, with visual identification of the target cells being aided by labelling the incorporated gene product with a green fluorescent protein.

Dielectrophoretic assembly of insulinoma cells and fluorescent nanosensors into three-dimensional pseudo-islet constructs.

Dielectrophoretic forces, generated by radio-frequency voltages applied to micromachined, transparent, indium tin oxide electrodes, have been used to condense suspensions of insulinoma cells (BETA-TC-6 and INS-1) into a 10 x 10 array of three-dimensional cell constructs. Some of these constructs, measuring approximately 150 microm in diameter, 120 microm in height and containing around 1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the dielectrophoretic force maintained, these engineered cell constructs were able to withstand mechanical shock and fluid flow forces. Reproducibility of the process required knowledge of cellular dielectric properties, in terms of membrane capacitance and membrane conductance, which were obtained by electrorotation measurements. The ability to incorporate fluorescent nanosensors, as probes of cellular oxygen and pH levels, into these 'pseudo-islets' was also demonstrated. The footprint of the 10 x 10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment.

Controlling cell destruction using dielectrophoretic forces.

Measurements are reported of the main factors, namely the AC voltage frequency and magnitude, that were observed to influence the number of cells destroyed during dielectrophoresis (DEP) experiments on Jurkat T cells and HL60 leukemia cells. Microelectrodes of interdigitated and quadrupolar geometries were used. A field-frequency window has been identified that should be either avoided or utilised, depending on whether or not cell damage is to be minimised or is a desired objective. The width and location of this frequency window depends on the cell type, as defined by cell size, morphology and dielectric properties, and is bounded by two characteristic frequencies. These frequencies are the DEP cross-over frequency, where a cell makes the transition from negative to positive DEP, and a frequency determined by the time constant that controls the frequency dependence of the field induced across the cell membrane. When operating in this frequency window, and for the microelectrode designs used in this work, cell destruction can be minimised by ensuring that cells are not directed by positive DEP to electrode edges where fields exceeding 30-40 kV/m are generated. Alternatively, this field-frequency window can be exploited to selectively destroy specific cell types in a cell mixture.

Effect of progesterone on aldosterone secretion in rats.

In both human and animal studies high progesterone states are associated with elevated aldosterone production but variable changes in PRA. These experiments were designed to test the hypothesis that progesterone has an effect similar to a low sodium diet on the glomerulosa cell: increasing aldosterone synthase messenger RNA activity and aldosterone production. Ovariectomized (OVX) rats were injected with progesterone (1 mg/100 g) or vehicle (SHAM) for 5 days. In a separate study, intact rats were placed on a low (0.02%) or high (1.6%) sodium diet for 5 days. On the day of death, rats were decapitated and blood collected for serum hormone determinations. Isolated adrenal glomerulosa cells were incubated +/- 10 nM angiotensin II (A II), after which aldosterone and corticosterone were measured. Early (conversion of cholesterol to pregnenolone) and late (conversion of corticosterone to aldosterone) aldosterone pathway activity was assessed in parallel incubates by adding cyanoketone and excess corticosterone with subsequent measurement of pregnenolone and aldosterone. In vivo, progesterone administration, like dietary sodium restriction, caused a significant increase in PRA (p < or = 0.043) and plasma aldosterone (p < or = 0.009), with no change in plasma corticosterone. Additionally, both treatments caused a significant increase in baseline (P < or = 0.01) and A II-stimulated (p < or = 0.027) aldosterone secretion in vitro. This increased responsiveness was secondary to activation of late pathway activity (p < or = 0.022) as determined by both an increased conversion of corticosterone to aldosterone and by an increase in messenger RNA levels of the late pathway enzyme aldosterone synthase. Thus, chronic progesterone administration apparently does not directly influence aldosterone secretion, but rather acts indirectly to increase aldosterone by mechanisms similar to sodium restriction.

Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase.

Bovine adrenal fasciculata cells, exposed to either ACTH or AII, synthesize glucocorticoids at an enhanced rate. It is generally accepted that the signaling pathways triggered by these two peptides are not identical. ACTH presumably acts via a cAMP-dependent protein kinase (PKA) and AII, via a calcium-dependent protein kinase. We have found that either peptide hormone stimulates synthesis of a mitochondrial phosphoprotein pp37, leading to accumulation of its proteolytically processed products pp30 and pp29. On the basis of a number of criteria, this 37 kDa protein is the bovine homolog of the 37 kDa protein that we have characterized in rodent steroidogenic tissue (Epstein L. F. and Orme-Johnson N. R.: J. Biol. Chem 266 (1991) 19,739-19,745). Further, bovine pp37 is phosphorylated when PKA or protein kinase C (PKC) is activated directly by (Bu)2 cAMP or PMA, respectively. These studies indicate that either pp37 is a common substrate for PKA and PKC in these cells or there is a common downstream kinase, which is activated by exposure to either ACTH or AII. Rat adrenal glomerulosa cells, exposed to either ACTH or AII, show an enhanced rate of mineralocorticoid synthesis. As for bovine fasciculata cells, it is thought that the signaling pathway triggered by ACTH differs from that triggered by AII. As we found for bovine fasciculata, pp37 is phosphorylated when the rat cells are exposed to either peptide hormone. However, in contrast to the finding for bovine fasciculata, while exposure of the rat glomerulosa cells to (Bu)2cAMP does cause the synthesis of pp37, exposure of the cells to PMA does not. Taken together, these findings provide further evidence that the subcellular signaling events, triggered by the action of AII on bovine adrenal fasciculata and rat adrenal glomerulosa cells, differ. Further, the fact, that pp37 is phosphorylated only when the rate of steroidogenesis is enhanced, reaffirms its potential involvement in the signaling pathway that causes stimulation of steroid hormone biosynthesis.

Sodium restriction increases aldosterone biosynthesis by increasing late pathway, but not early pathway, messenger ribonucleic acid levels and enzyme activity in normotensive rats.

To determine whether changes in dietary sodium intake modify the early and/or late pathways of aldosterone biosynthesis, we studied in Sprague-Dawley rats the effect of sodium restriction on early (conversion of cholesterol to pregnenolone) and late (conversion of corticosterone to aldosterone) pathway activity and on the mRNA levels for the enzymes regulating these steps. Sodium restriction increased basal and angiotensin-II-stimulated aldosterone output from isolated zona glomerulosa cells by 5- to 9-fold. This increase in aldosterone output did not appear to be due to changes in the conversion of cholesterol to pregnenolone or in the mRNA levels of the early pathway enzyme, cholesterol side-chain cleavage cytochrome P-450. In contrast, sodium restriction increased the conversion of corticosterone to aldosterone 10-fold and increased by over 10-fold the mRNA levels of the late pathway enzyme aldosterone synthase. Sodium restriction had no effect on zona glomerulosa levels of 11 beta-hydroxylase mRNA. In two other normotensive rats, Dahl salt-resistant and Wistar Kyoto, sodium restriction again specifically increased aldosterone synthase mRNA without altering 11 beta-hydroxylase or cholesterol side-chain cleavage cytochrome P-450 mRNA levels. Thus, it appears that sodium restriction specifically increases late pathway aldosterone synthase mRNA levels, resulting in an increase in enzyme levels, followed by an increase in late pathway activity and an increase in aldosterone output.

Red cell sodium-proton exchange is increased in Dahl salt-sensitive hypertensive rats.

To investigate the relationship between red blood cell Na+/H+ exchange (EXC) and genetic factors in hypertension, we studied the maximal rate of the antiporter (mmol/liter cell x hr; flux units = FU) in three strains of genetically hypertensive rats. Salt-resistant Dahl rats (DR) were normotensive under low (0.02%) and high (8%) NaCl diets, while salt-sensitive Dahl rats (DS) became markedly hypertensive after four weeks on the high-NaCl diet. Na+/H+ exchange did not differ between DR and DS rats when both were fed with the low-NaCl diet (mean +/- SE, 31 +/- 3, N = 15, vs. 29 +/- 3 FU, N = 14). On the high-NaCl diet, the DR strain did not exhibit significant changes in blood pressure and antiporter activity, but the DS rats significantly increased their blood pressure and Na+/H+ exchange (57 +/- 4 FU, N = 13) versus DR rats (38 +/- 3 FU, N = 15, P < 0.02). DS rats also significantly increased blood pressure and antiporter activity when fed with high-NaCl diet for one week. These data indicate that high NaCl intake per se does not increase Na+/H+ EXC because the control DR strain did not exhibit transport and blood pressure alterations as observed in the DS strain. Milan hypertensive and spontaneously hypertensive rats (Charles River substrain) had higher blood pressures than Milan and Wistar-Kyoto normotensive rats when they were maintained for four weeks on a 1.5% NaCl diet; however, no differences were seen among normotensive and hypertensive strains in Na+/H+ exchange activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal norepinephrine and aldosterone responses to upright posture in nonmodulating hypertension.

The subgroup of patients with nonmodulating hypertension demonstrates a number of abnormalities of the renin-angiotensin-aldosterone axis. We previously identified abnormalities in plasma and urinary dopamine in nonmodulators and posited that this may be in part due to a generalized defect in sympathetic nervous system activity. In the present study we assessed the state of activation of the renin-angiotensin system and the sympathetic nervous system in normal subjects and patients with modulating, nonmodulating, and low renin essential hypertension during sodium depletion and change from supine to upright posture. Levels of plasma norepinephrine were higher in non-modulators during the posture study (P < 0.05). PRA rose with upright posture in all groups, but low renin subjects had a blunted response. Nonmodulators and low renin subjects had lower aldosterone levels both supine (P< 0.05) and upright (P< 0.01). However, the aldosterone/PRA increment ratio was increased in low renin subjects (P< 0.01), whereas it was decreased in nonmodulators. Twenty-four-hour urine collections for catecholamine determinations were obtained in a subgroup of the subjects, with nonmodulators showing higher levels of norepinephrine excretion which approached significance (P = 0.08). In vitro experiments using rat and human adrenal glomerulosa cells showed that norepinephrine does not affect aldosterone secretion per se. These observations extend the series of abnormalities observed in nonmodulating hypertension. However, it is likely that the alterations in norepinephrine levels during sodium depetion and upright posture are a secondary event and not linked to the altered aldosterone production in these patients.

Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cells: in vitro correlate of hyperreninemic hypoaldosteronism.

In some critically ill patients, aldosterone secretion is diminished despite hyperreninemia. These same patients demonstrate appropriately elevated plasma ACTH and cortisol levels. In addition, infusion of ACTH or angiotensin-II (AII) fails to elicit the normal aldosterone response, implying that the defect is at the level of the zona glomerulosa (ZG) cell. To test the hypothesis that elevated ACTH levels induce this defect, Percoll-purified bovine ZG cells were plated in serum-free defined medium and cultured for 5 days. On days 1-4, cells were exposed to various concentrations of ACTH for 1 h. On the fifth day of culture, half of the wells pretreated with ACTH were treated for 1 h with AII (10(-7) M); the other half of the wells received another dose of ACTH for 1 h. Additionally, cells were exposed to daily 1-h pulses of AII (10(-7) M) alone or in combination with ACTH (10(-8) M) for 5 days. Acutely dispersed bovine ZG cells showed dose-dependent increases in aldosterone when incubated with ACTH, potassium, or AII, with minimal cortisol production. Acutely dispersed bovine fasciculata cells produced no aldosterone, but demonstrated a dose-dependent cortisol response to ACTH and AII, but not potassium. On day 1 of culture, the ZG cells demonstrated a significant (P less than 0.001 in all cases) dose-related increase in aldosterone secretion in response to ACTH. However, continued daily pulsation with ACTH resulted in a dose-dependent decrease in aldosterone secretion, with a concomitant dose- and time-related rise in cortisol production. Indeed, ACTH-induced cortisol production in ZG became similar to ACTH-induced cortisol production in zona fasciculata cells. The addition of AII to the daily ACTH pulse did not significantly alter the aldosterone or cortisol response patterns to ACTH alone. In contrast, ZG cells treated with AII alone for 5 days showed a minimal change in cortisol production and no reduction in aldosterone production until day 4. Northern blot analysis of total RNA isolated from ZG cells pulsed with ACTH for 5 days demonstrated a parallel dose-dependent increase in 17 alpha-hydroxylase mRNA, which did not occur in cells pulsed with AII alone. These in vitro results suggest that elevated ACTH levels over time induce 17 alpha-hydroxylase activity in ZG cells, thereby shifting steroid biosynthesis from an aldosterone-producing to a cortisol-producing pathway. It is likely that the chronically elevated ACTH levels in critically ill patients induce a similar change in ZG cell biosynthesis, resulting in their hyperreninemic hypoaldosterone state.

Dissociation in plasma renin and adrenal ANG II and aldosterone responses to sodium restriction in rats.

In rats, plasma renin activity (PRA) increases sharply, reaching a plateau within hours of sodium restriction. Plasma aldosterone increases gradually, not reaching a plateau for 1-2 days. To determine whether this dissociation is secondary to the time needed to modify adrenal sensitivity to angiotensin II (ANG II) and to assess the role of locally produced ANG II in this process, rats were salt restricted for 0-120 h. Plasma hormone levels were assessed, adrenal ANG II was measured, and basal and ANG II (1 x 10(-8) M)-stimulated steroidogenesis were determined in vitro. Although PRA attained an elevated plateau within 8 h, plasma aldosterone did not peak until after 48 h of sodium depletion. The in vitro aldosterone sensitivity to exogenous ANG II was not apparent until rats had been salt restricted for 16 h. A plateau (4-fold increase above the ANG II response on high salt) was achieved between 24 and 48 h. Adrenal ANG II also exhibited a similar delayed response that correlates significantly with changes in aldosterone biosynthesis and late pathway activity. Thus the dissociation between PRA and plasma aldosterone may be secondary to a lag in the zona glomerulosa's (ZG) steroidogenic response to ANG II as well as a parallel lag in tissue ANG II production, suggesting that changes in tissue ANG II may mediate ZG sensitivity to ANG II during sodium deprivation.

Potassium-stimulated angiotensin release from superfused adrenal capsules and enzymatically dispersed cells of the zona glomerulosa.

The cells of the adrenal cortex contain angiotensin-II (AII), but whether this peptide is synthesized there (vs. internalized from the systemic circulation), whether it is secreted, and whether it is important in aldosterone production remain uncertain. To address these issues, we studied AI and AII release from superfused rat adrenal capsules and dispersed glomerulosa cells. Superfused adrenal capsules released 7-fold more AII in 270 min than the capsules originally contained (495 +/- 101 fmol AII/rat released vs. 66 +/- 8 fmol AII/rat tissue content). The amount of AI released in the same period only slightly exceeded the tissue content. In response to higher potassium concentrations in the medium (9 vs. 3.6 mM K+), adrenal capsules and dispersed glomerulosa cells both released significantly more AI and AII into the superfusate. This release of AI and AII was oscillatory. The oscillations occurred in each of 15 experiments, with a period of 45-90 min. Decapsulated adrenal glands (the zona faciculata/reticularis plus medulla) also contained and released AII, but did not respond to potassium stimulation. There was a highly significant correlation between AII and aldosterone release. This was especially apparent if aldosterone secretion was examined during oscillations of AII release (r = 0.97; P less than 0.0001). We conclude that AII is synthesized in the zona glomerulosa and can be released in response to stimuli. The close correlation between AII and aldosterone secretion suggests that locally produced AII may play an important role in aldosterone biosynthesis.

The effect of sodium intake on angiotensin content of the rat adrenal gland.

To determine whether dietary sodium intake modifies the generation of adrenal-produced angiotensins and/or their relative proportions, Sprague-Dawley rats were maintained on a low (0.02%), normal (0.4%), or high (1.5%) sodium intake for 5 days. The animals were then killed by decapitation at 0900 h, and their adrenal glands were removed and dissected into two parts: capsular tissue, containing the zona glomerulosa (ZG), and the decapsulated adrenal gland. The tissue was frozen in liquid nitrogen and extracted, and the individual angiotensins [angiotensin-II (AII), angiotensin-III (AIII), angiotensin-I (AI), and Des-Asp-angiotensin-I (Des-Asp-AI)] were separated by HPLC and quantitated by RIA. On a normal sodium intake, the molar contents of the four angiotensins were similar in ZG, ranging from 3.1-6.6 pmol/g, although AII was present in a 60-70% higher concentration than AIII. In the decapsulated adrenal, the concentrations of the various angiotensins were again similar, but the absolute levels (per g tissue) were significantly (P less than 0.02) less than those in the ZG layer. With sodium restriction, the AII content increased more than 2-fold in the ZG, but not in the decapsulated adrenal tissue. In contrast, both AI and Des-Asp-AI significantly (P less than 0.01) decreased with sodium restriction, so that their contents on the low salt diet were only 15-20% of those observed on the high sodium diet. Thus, there was an inverse correlation (P less than 0.001) between the salt content of rat chow and the AII content of the ZG. The correlation between salt intake and AI as well as Des-Asp-AI levels was direct and significant (P less than 0.02). The AIII level in the ZG was similar on all diets. After a lag period, ZG AII increased sharply between 16-48 h of sodium restriction. These data document that sodium intake has a profound effect on the angiotensin content of the ZG, with sodium restriction substantially increasing the levels of AII while reducing the level of its substrate, AI. This also appears to be unique for glomerulosa cells, as in the decapsulated adrenal gland there is little if any change with sodium restriction. We conclude that these sodium-mediated changes in tissue AII production may be involved in the increased responsiveness of glomerulosa cells to aldosterone secretagogues during sodium restriction.

Sodium-mediated modulation of aldosterone secretion: impact of converting enzyme inhibition on rat glomerulosa cell response to angiotensin-II.

Sodium restriction enhances the aldosterone response to angiotensin-II (AII) in normal rats, but not in spontaneously hypertensive rats (SHR). To determine whether a change and/or abnormality in the circulating or adrenal renin-angiotensin systems are responsible for these observations, three groups of animals were studied on a low sodium diet with and without the administration of a converting enzyme inhibitor (enalapril). Sprague-Dawley and Wistar-Kyoto (normotensive rat strains) and SHR were placed on low sodium (0.1%) for 9 days, the last 4 days of which enalapril was administered to half of the animals. In all groups enalapril treatment resulted in a significant (P less than 0.001) reduction in blood pressure, an increase in renin activity, and a reduction in plasma aldosterone when all of the animals were considered together, although the change in blood pressure achieved statistical significance only in the Wistar-Kyoto rats. Additionally, basal aldosterone output from isolated glomerulosa cells was lower in the normotensive animals pretreated with enalapril. However, despite the evidence for inhibition of converting enzyme, there was no change in the hypertensive animals. Thus, neither locally nor systemically generated AII appear to participate in the maintenance of the increased aldosterone responsiveness to AII with sodium restriction. Furthermore, they do not appear to contribute to the altered adrenal responsiveness to AII with sodium restriction in SHR. These data provide further support for the hypothesis that as yet undefined glomerulosa intracellular mechanisms are altered by dietary sodium restriction in normotensive, but not hypertensive, rats.

Effect of sodium intake on phosphoinositides and inositol trisphosphate response to angiotensin II, K+ and ACTH in rat glomerulosa cells.

To assess the impact of sodium intake on the adrenal phosphoinositide system, rats were maintained on a low or normal salt diet for 5 days, and glomerulosa cell preparations (2 x 10(5) cells) were stimulated by angiotensin II (AII; 10 nmol/l), potassium (K+; 8.7 mmol/l) or ACTH (0.1 nmol/l) for 0, 2, 4, 6, 12, 15 and 60 s. Levels of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns 4-P), phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) + inositol 1,3,4-trisphosphate (Ins 1,3,4-P3) were assayed by a microspectrophotometric procedure. Non-stimulated levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2 and Ins 1,4,5-P3 (+ Ins 1,3,4-P3) (means +/- S.E.M.; n = 36) in cells from rats on the low Na+ intake were 580 +/- 6.5, 187 +/- 2.6, 82 +/- 3 and 95 +/- 1.2 pmol per incubate respectively, indistinguishable from those observed in rats on a normal Na+ intake, except for the significantly (P less than 0.025) greater PtdIns 4,5-P2 level. In response to AII stimulation, all four compounds showed an earlier and greater peak response when cells were obtained from animals on a low rather than a high sodium intake. All values has returned to control levels by 12-15 s, regardless of the level of sodium intake. In contrast, with K+ stimulation there were no differences in the peak response of cells from rats on the two dietary intakes, but there was a shift of the peak to a longer time-interval (6 versus 8 s) in animals maintained on a low sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Rate of activation of renin-angiotensin-aldosterone axis and sodium intake in rats.

When sodium intake in the rat is reduced abruptly from the typical high level to a very low level (0.02%), sodium excretion falls exponentially, with a half time of 2-3 h. The result is that the rat achieves external sodium balance, in which intake equals excretion, on the new low intake within a few hours. In this study, we assessed the rate of activation of the renin-angiotensin-aldosterone axis and its contribution to blood pressure during that interval. Plasma renin activity and angiotensin II concentration had risen sharply within 8 h and did not change over the next 40 h. Plasma aldosterone concentration, on the other hand, continued to rise over 48 h. Within 8 h, blood pressure dependency on angiotensin II had increased sharply, as assessed by depressor responses to an angiotensin antagonist (Sar1-Ala8-angiotensin II) and to converting-enzyme inhibition (captopril). The depressor response to neither agent changed over the next 40 h. The pressor response to angiotensin II was blunted significantly by 8 h and also did not change over the next 40 h. The findings indicate that the rapid tempo of sodium homeostasis in the rat is matched by an equally rapid tempo of activation of the renin-angiotensin system, although the factors responsible for aldosterone release are probably more complex. Experiments to assess the renin-angiotensin system in the rat must be designed with this rapid tempo in mind.

Mass determination of polyphosphoinositides and inositol triphosphate in rat adrenal glomerulosa cells with a microspectrophotometric method.

A method is described for the assay of subnanogram amounts of phosphorus in phospholipids and organic phosphates. The formation of a complex with a high molar absorption coefficient at 600 nm when malachite green is added to phosphomolybdate at low pH and the adaptation of a microspectrophotometer to quantify the color in 10 microliters solution have made it possible for a dose-response curve from 0.1-1.2 ng phosphorus to be developed. The method has been applied to the assay of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns 4-P), phosphatidylinositol-4,5-diphosphate (PtdIns 4,5-P2), and inositol-1,4,5-triphosphate (Ins 1,4,5-P3) in rat adrenal glomerulosa cells after stimulation with angiotensin II (AII), K+, and ACTH for 0, 2, 4, 6, 8, 10, 12, 15, and 60 sec. A control (nonstimulated) sample was incubated concomitantly for every time period. Nonstimulated cell levels (mean +/- SEM; n = 216) were: PtdIns, 577 +/- 6.4; PtdIns 4-P, 183 +/- 3.1; PtdIns 4,5-P2, 59 +/- 1.8; and Ins 1,4,5-P3, 94 +/- 1.3 pmol/incubate. Maximum increase in levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2, and Ins 1,4,5-P3 above control values was obtained after 8 sec with AII (10(-8) M) and after 6 sec with K+ (8.7 mM) stimulation. The values (picomoles per 2 X 10(5) cell incubate; n = 4) were: PtdIns, 808 +/- 28; PtdIns 4-P, 263 +/- 20; PtdIns 4,5-P2, 112 +/- 10; and Ins 1,4,5-P3, 136 +/- 4 for AII stimulation, and PtdIns, 925 +/- 76, PtdIns 4-P, 308 +/- 11; PtdIns 4,5-P2 146 +/- 28; and Ins 1,4,5-P3, 149 +/- 5 for K+ stimulation. No increase above control levels could be found at any incubation time after ACTH stimulation. Thus, both AII and K+ stimulate a short-lived increase in the mass of several elements of the phosphatidylinositol pathway. The discrepancy between these mass determinations and isotope study suggests that only some, but not all, pools are labeled by currently available techniques.

Comparative effect of angiotensin II, potassium, adrenocorticotropin, and cyclic adenosine 3',5'-monophosphate on cytosolic calcium in rat adrenal cells.

The present study compares changes in cytosolic calcium and steroidogenesis when rat adrenal cells are stimulated with potassium (K+), angiotensin II (AII), ACTH, and (Bu)2cAMP (cAMP). The calcium-sensitive fluorescent dye, quin 2, was used to determine cytosolic calcium concentrations. K+ and AII both induced parallel increases in cytosolic calcium and aldosterone output. Removal of external calcium from the incubation media or addition of nifedipine inhibited the rise in cytosolic calcium in response to these two secretagogues. Inhibition of release of intracellularly-bound calcium by incubating the cells with 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or dantrolene sodium reduced the rise in cytosolic calcium in response to these two secretagogues by 40-50%. In contrast, neither ACTH nor cAMP altered cytosolic calcium levels in the glomerulosa cells, even though quin 2-loaded cells showed a normal steroidogenic response to these agents. Thus, there was a dissociation between the cytosolic calcium response and steroidogenesis during cAMP stimulation of glomerulosa cells. Fasciculata cells incubated in the presence of increasing concentrations of cAMP, ACTH, K+, or AII failed to demonstrate an increase in cytosolic calcium, although the cells had a normal steroidogenic response to ACTH and cAMP. These results suggest that the responses of fasciculata and glomerulosa cells to secretagogues have different dependencies on calcium. The fasciculata cell has little calcium dependency while the glomerulosa cell has a variable dependency. In the glomerulosa cell, both AII and K+ induced similar responses in steroid output and cytosolic calcium, suggesting an important role for cytosolic calcium as a mediator of the steroidogenic effect of these secretagogues. Furthermore, part of the increase in cytosolic calcium induced by these agents is due to release of intracellularly bound calcium and part from increased calcium flux across the cell membrane. The absence of such dependency with cAMP suggests that an increase in intracellular calcium levels is not required for increased steroidogenesis in glomerulosa cells.

Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat.

This study compared the extracellular calcium dependency and the enzymatic locus of that dependency for N6 O2'-dibutyryl cyclic AMP (dbcAMP)-, angiotensin II- and potassium-stimulated aldosterone secretion in dispersed rat glomerulosa cells. The need for extracellular calcium, calcium influx, and specifically for calcium influx through the calcium channel was examined. dbcAMP, angiotensin II and potassium, in the presence of calcium (3.5 mmol/l), significantly (P less than 0.01) increased aldosterone output by at least 1.5-fold. Yet in the absence of extracellular calcium or in the presence of lanthanum (an inhibitor of calcium influx by most mechanisms) all three stimuli failed to increase aldosterone secretion. Nifedipine, a dihydropyridine calcium channel antagonist, significantly (P less than 0.01) reduced angiotensin II- and potassium-stimulated aldosterone secretion, but had no effect on dbcAMP-stimulated aldosterone secretion (100 +/- 14 vs 105 +/- 19 pmol/10(6) cells). Likewise nitrendipine failed to inhibit ACTH-stimulated aldosterone secretion. Angiotension II and potassium activation of both the early aldosterone biosynthetic pathway (as reflected by pregnenolone production in the presence of cyanoketone) and also its late pathway (as reflected by the conversion of exogenous corticosterone to aldosterone in the presence of cyanoketone) were significantly (P less than 0.01) inhibited by lanthanum, nifedipine and by reducing the extracellular calcium concentration. However, with dbcAMP stimulation, none of these manipulations modified pregnenolone production. Late pathway activation by dbcAMP was inhibited by lanthanum and a reduction in extracellular calcium, but not by nifedipine. These observations suggest that: the extracellular calcium dependency of dbcAMP-, angiotensin II- and potassium-stimulated aldosterone secretion reflects a need for calcium influx; with dbcAMP stimulation, activation of the late pathway is dependent on calcium influx by a calcium channel-independent mechanism, whereas activation of the early pathway is not dependent on extracellular calcium or calcium influx and activation of both the early and late pathway by angiotensin II and potassium is dependent on calcium influx by a calcium channel-dependent mechanism. Therefore, we conclude that the mechanism of activation of the early aldosterone biosynthetic pathway by dbcAMP is different from angiotensin II or potassium and early pathway activation is distinct from that of late pathway activation with dbcAMP stimulation.

Calcium, a "third messenger" of cAMP-stimulated adrenal steroid secretion.

This study examines the role of extracellular calcium and calcium mobilization from intracellular stores in mediating cAMP-stimulated steroid secretion by rat adrenal glomerulosa cells (GC) and fasciculata cells (FC). When GC were incubated acutely in a calcium-deficient buffer, cAMP failed to significantly increase aldosterone secretion above base line. Aldosterone secretion, however, rose from 17 +/- 2 to 32 +/- 4 ng/10(6) cells (P less than 0.01) as calcium in the medium was increased from 0 to 3.5 mM. In contrast, cAMP-stimulated corticosterone production by FC was not influenced by changes in the external calcium concentration. Lanthanum (10(-4) M), an inhibitor of calcium influx, reduced cAMP-stimulated aldosterone secretion from 69 +/- 10 to 42 +/- 5 ng/10(6) cells (P less than 0.01) but failed to alter cAMP-stimulated fasciculata steroidogenesis. Depletion of intracellular calcium stores, achieved by incubating with EGTA, markedly blunted cAMP-stimulated corticosterone secretion in GC from 666 +/- 126 to 32 +/- 6 ng/10(6) cells (P less than 0.01), and cAMP-stimulated corticosterone secretion in FC from 2,223 +/- 407 to 414 +/- 58 ng/10(6) cells (P less than 0.01). TMB-8, a putative inhibitor of intracellular calcium mobilization, markedly inhibited (P less than 0.01) cAMP-stimulated aldosterone secretion by GC from 469 +/- 31 to 48 +/- 8 ng/10(6) cells and corticosterone secretion by FC from 9,867 +/- 1,821 to 2,832 +/- 586 ng/10(6) cells. These observations suggest that cAMP activation of adrenal steroidogenesis requires the release of calcium from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)