Cloning and functional expression of the cytoplasmic form of rat aminopeptidase P.

A rat cytoplasmic aminopeptidase P was purified from liver cytosol with a procedure including an affinity elution step with 3 microM inositol 1,3,4-trisphosphate. Proteolytic fragments were generated, sequenced and the enzyme was cloned from a rat liver cDNA library. The structure shows high (87.8% and 95.5%, respectively) sequence identity at the nucleotide and amino acid levels with the previously described human putative cytoplasmic aminopeptidase P. The cloned rat enzyme was functionally expressed in Escherichia coli and also in COS-1 cells. Western blot analysis, using an antibody generated against the recombinant protein, and Northern blot hybridization showed ubiquitous expression of the protein in different tissues with the highest expression level in the testis.

TASK (TWIK-related acid-sensitive K+ channel) is expressed in glomerulosa cells of rat adrenal cortex and inhibited by angiotensin II.

The present study was conducted to explore the possible contribution of a recently described leak K+ channel, TASK (TWIK-related acid-sensitive K+ channel), to the high resting K+ conductance of adrenal glomerulosa cells. Northern blot analysis showed the strongest TASK message in adrenal glomerulosa (capsular) tissue among the examined tissues including heart and brain. Single-cell PCR demonstrated TASK expression in glomerulosa cells. In patch-clamp experiments performed on isolated glomerulosa cells the inward current at -100 mV in 30 mM [K+] (reflecting mainly potassium conductance) was pH sensitive (17+/-2% reduction when the pH changed from 7.4 to 6.7). In Xenopus oocytes injected with mRNA prepared from adrenal glomerulosa tissue the expressed K+ current at -100 mV was virtually insensitive to tetraethylammonium (3 mM) and 4-aminopyridine (3 mM). Ba2+ (300 microM) and Cs+ (3 mM) induced voltage-dependent block. Lidocaine (1 mM) and extracellular acidification from pH 7.5 to 6.7 inhibited the current (by 28% and 16%, respectively). This inhibitory profile is similar (although it is not identical) to that of TASK expressed by injecting its cRNA. In oocytes injected with adrenal glomerulosa mRNA, TASK antisense oligonucleotide reduced significantly the expression of K+ current at -100 mV, while the sense oligonucleotide failed to have inhibitory effect. Application of angiotensin II (10 nM) both in isolated glomerulosa cells and in oocytes injected with adrenal glomerulosa mRNA inhibited the K+ current at -100 mV. Similarly, in oocytes coexpressing TASK and ATla angiotensin II receptor, angiotensin II inhibited the TASK current. These data together indicate that TASK contributes to the generation of high resting potassium permeability of glomerulosa cells, and this background K+ channel may be a target of hormonal regulation.

Differential sensitivity of TREK-1, TREK-2 and TRAAK background potassium channels to the polycationic dye ruthenium red.

BACKGROUND AND PURPOSE: Pharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2 P 9.1), but not TASK-1 (K2 P 3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2 P channels. EXPERIMENTAL APPROACH: K(+) currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons. KEY RESULTS: RR differentiates between two closely related members of the TREK subfamily. TREK-2 (K2 P 10.1) proved to be highly sensitive to RR (IC50 = 0.2 µM), whereas TREK-1 (K2 P 2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K2 P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K2 P 4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K2 P 18.1) background K(+) channels. We detected the RR-sensitive leak K(+) current component in DRG neurons. CONCLUSIONS AND IMPLICATIONS: We propose that RR may be useful for distinguishing TREK-2 (K2P 10.1) from TREK-1 (K2P 2.1) and other RR-resistant K2 P channels in native cells.

Voltage dependent calcium channels in adrenal glomerulosa cells and in insulin producing cells.

We have examined the structure and function of Ca2+ channels in excitable endocrine cell types, in rat adrenal glomerulosa cells and in two insulin producing cell types, the rat pancreatic beta cell and the INS-1 cell line. In previous studies on glomerulosa cells, we observed low (T-type) and high threshold (L-type) voltage dependent Ca2+ currents in addition to a K+ induced inward rectifying Ca2+ current (Igl). beta cells are known to exhibit T-, L- and N-type currents. We have now found that INS-1 cells also show low threshold (T-type) and high threshold Ca2+ currents. The latter was further resolved by organic inhibitors into L-type and P/Q-type currents and no Igl was detected. The expression of the pore-forming alpha 1 subunit of voltage dependent Ca2+ channels was studied by means of reverse transcription-polymerase chain reaction (RT-PCR), followed by restriction enzyme mapping and/or sequencing. Both in glomerulosa and pancreatic beta cells, the neuroendocrine (D) class of the alpha 1 subunit, known to be responsible for L-type current, represents the majority of the PCR product. Comparable amounts of the neuroendocrine (D) and the neuronal A-type alpha 1 subunits dominate the message in INS-1 cells. Different characteristics of Ca2+ currents in these cell types is discussed in view of the channel repertoire.

Inositol 1,4,5-trisphosphate binding sites copurify with the putative Ca-storage protein calreticulin in rat liver.

Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.

The effects of high extracellular Ca2+ and Mg2+ concentrations on the levels of inositol 1,3,4,5-tetrakisphosphate in bovine parathyroid cells.

We examined the effects of calcium (Ca2+), magnesium (Mg2+), and fluoride ion (F-) on inositol phosphate accumulation in bovine parathyroid cells prelabeled with [3H] inositol to determine whether these agents might modulate cytosolic Ca2+ through accumulation of intracellular inositol 1,3,4,5-tetrakisphosphate (IP4), which has been postulated to be a mediator of the influx of extracellular Ca2+. Both Ca2+ and Mg2+ produced dose-dependent increases in IP4 in the presence of Li+, with maximal 19- and 4-fold increases with 5.0 mM Ca2+ and 20 mM Mg2+, respectively. A 50% rise in IP4 was evident within 1 min in response to 5.0 mM Ca2+. In the absence of Li+, a 2-fold increase in IP4 was seen with 5.0 mM Ca2+ only after 15 min. Fluoride ion generated a dose-dependent increase in IP4, with a 48% rise at 10 mM F-, presumably by activating phospholipase-C through a guanine nucleotide regulatory (G) protein-dependent process. We conclude that inositol 1,4,5-trisphosphate generated in response to high extracellular Ca2+ and Mg2+ concentrations can be converted to IP4 in parathyroid cells. The slow kinetics for the increase in IP4 with high Ca2+ in the absence of Li+, however, do not support a role for IP4 in the early phase of the sustained increase in cytosolic Ca2+ produced by high extracellular Ca2+ concentrations.

Cytosolic calcium and aldosterone response patterns of rat adrenal glomerulosa cells stimulated by vasopressin: comparison with angiotensin II.

Cytosolic calcium (Cai) responses to arginine vasopressin (AVP) and angiotensin-II (Ang II) were examined in single rat adrenal zona glomerulosa (ZG) cells by monitoring fura-2 fluorescence with microspectrofluorimetry. ZG cells displayed dose-dependent Cai responses to a wide range of AVP and Ang II concentrations, starting from a threshold of 1 nM for AVP and less than 5 pM for Ang II. A dose-dependent delay of the onset of the Cai response was observed with both hormones. The response delay for Ang II was consistently briefer than that for the same concentration of AVP, showing a 2-3 log unit separation in the dose-response relations. After the delay, cells typically responded with an abrupt increase in Cai, which peaked within 15 sec. The amplitude of the peak Cai rise showed little dependency on AVP or Ang II concentration. At most AVP concentrations, the response consisted of Cai oscillations, with apparent fusion of these Cai oscillations at the highest AVP concentrations (1-0.1 microM). Similar oscillatory behavior was found with stimulations by much lower Ang II concentrations (0.5 nM to 5 pM). There appeared to be a 2-3 log unit shift in the sensitivity toward AVP and Ang II when Cai responses were compared. Sixty percent of ZG cells were responsive to AVP, while more than 90% displayed an elevation of Cai with Ang II. The Cai and steroid responses to 100 nM AVP and 100 pM Ang II were compared, since these two doses are reported to stimulate the phosphoinositide system to a similar extent. Individual ZG cells tested with both hormones responded with equivalent peak Cai changes, but a slightly longer response delay for AVP. The mean Cai response and aldosterone production for each secretagogue displayed parallel kinetics during 30-min stimulations. After initial oscillations, the Cai response returned to control values within 15 min of 100 nM AVP application. Likewise, the steroid output was transient. In contrast, 100 pM Ang II produced maintained Cai oscillations as well as a sustained and substantially greater aldosterone production for the same period of application. In conclusion, the disparate steroidogenic effects of AVP and Ang II appear to result from distinctly different Cai responses elicited during maintained secretagogue stimulation.

Pressor-type vasopressin receptors in the adrenal cortex: properties of binding, effects on phosphoinositide metabolism and aldosterone secretion.

Specific, high affinity sites that bound tritium-labeled arginine-vasopressin (3H-AVP) were detected in a crude membrane fraction of rat adrenal capsules (chiefly zona glomerulosa). Binding displacement experiments with peptide analogs of AVP suggested that the binding site is a pressor (V1) type receptor for AVP. When added to dispersed rat adrenal glomerulosa cells, vasopressin (10(-8)-10(-6)M) stimulated the incorporation of 32P-phosphate into phosphatidylinositol, and the effect was blocked by the AVP receptor antagonist peptide d(CH2)5Tyr(Me)AVP. Vasopressin also increased the breakdown of phosphatidylinositol-4,5-bisphosphate within 1 min after its addition to the incubation medium. Superfused zona glomerulosa cells responded to AVP (10(-8)-(-6)M) by increasing their aldosterone production. The response could be blocked by the antagonist peptide. These data show that functionally active V1 receptors are present in rat glomerulosa cells, and suggest that vasopressin may regulate the function of the adrenal glomerulosa.

Kinetics of cytosolic calcium and aldosterone responses in rat adrenal glomerulosa cells.

To evaluate the relationship between cytosolic calcium (Cai) and aldosterone production, rat adrenal zona glomerulosa (ZG) cells were studied during long-term stimulation by different secretagogues. Cai was measured in single ZG cells using microspectrofluorimetry, and aldosterone was determined in cell populations using a superfusion system. For external potassium (K+), Cai increases are sustained, with only a slight decrement over time, a feature shared by aldosterone production. The relationship between aldosterone output and Cai is nonlinear, with a Cai value for half-maximal stimulation of approximately 500 nM. Furthermore, the sustained changes in Cai with external K+ indicate that ZG cells can use an amplitude-based Cai signal to stimulate aldosterone production. Cai changes stimulated by angiotensin-II (Ang-II) show a complex dose-response pattern, with high concentrations (greater than or equal to 1 nM) of Ang-II eliciting a peak-plateau signal and lower doses (0.1 nM to 10 pM) producing repeated Cai oscillations. The peak amplitude of the Cai response in individual cells is not dose dependent, with the ZG cell experiencing peak levels repeatedly at the lowest Ang-II concentrations. However, the Cai transients are more frequent with increasing Ang-II concentrations between 0.1 nM and 10 pM. When integrated over time, the mean Cai signal also shows only modest dose-dependency during the sustained phase of Ang-II stimulation. Unlike the integrated Cai signal, aldosterone production increases steeply between 10 pM and 0.1 nM Ang-II, indicating that the Cai signal is likely to be frequency-based. Conversely, the steroid response to high Ang-II closely mirrors the kinetics of the more sustained Cai signals, including the diminished Cai and aldosterone levels during sustained stimulation with the highest Ang-II doses. Arginine vasopressin stimulated Cai and aldosterone responses, which closely resemble those elicited by 0.1 nM Ang-II, except that both Cai and aldosterone return to basal values within 20 min of continuous presentation of arginine vasopressin. Each ZG secretagogue produces a distinct pattern of Cai and aldosterone response. In addition, Cai response patterns can be divided into two general classes: a sustained Cai response, which appears to modulate cell activation by the amplitude of the Cai signal, and an oscillating Cai response, which uses the frequency of the Cai transients to control the magnitude of stimulation.

Inositol 1,4,5-trisphosphate receptor subtypes in adrenal glomerulosa cells.

Expression of the diverse subtypes of inositol 1,4,5-trisphosphate (InsP3) receptor (IP3R) was examined in rat adrenal glomerulosa cells. The polymerase chain reaction products were characterized by means of DNA sequencing and/or restriction enzyme mapping. The predominant subtype expressed is IP3R-1; its alternatively spliced variants containing and lacking segment S1 are present in comparable amounts. The expression level of IP3R-2 is about a quarter that of IP3R-1, whereas IP3R-3 is expressed at a very low level. Sodium depletion, a chronic physiological stimulus of glomerulosa cells, failed to influence the expression of IP3R-1, as measured by competitive polymerase chain reaction, and failed to modify the ratio of the different receptor subtypes, as studied with restriction enzyme mapping.

Thapsigargin-induced increase in cytoplasmic Ca2+ concentration and aldosterone production in rat adrenal glomerulosa cells: interaction with potassium and angiotensin-II.

Thapsigargin (Tg), a microsomal Ca2+ pump inhibitor, dose-dependently increases the cytoplasmic Ca2+ concentration and aldosterone production without having any striking effect on the formation of inositol phosphates in isolated rat adrenal glomerulosa cells. The interaction of Tg with the major Ca2(+)-mediated stimuli of glomerulosa cells on aldosterone production was also examined. The effects of Tg and the Ca2(+)-mobilizing angiotensin-II (AII) were additive. The aldosterone production stimulatory effect of potassium, which induces Ca2+ influx via voltage-operated Ca2+ channels, was potentiated by Tg. The positive interaction between Tg and potassium on aldosterone production raises the possibility that stimuli generating Ca2+ signal by depleting intracellular Ca2+ stores, such as Tg or AII, enhance the response of the cell to depolarization. Such an interaction between AII and potassium may have an important role in the physiological control of aldosterone production.

Angiotensin-II inhibits Na+/K+ pump in rat adrenal glomerulosa cells: possible contribution to stimulation of aldosterone production.

The control of Na+/K+ pump activity was studied in rat adrenal glomerulosa cells. Ninety percent of K+/86Rb accumulation was blocked by ouabain, and the dose-response curve of inhibition by ouabain was monophasic (IC50, approximately 80 microM), suggesting the role of a single type of Na+/K+ pump (alpha-isoenzyme) in 86Rb accumulation by rat glomerulosa cells. The basal activity of the Na+/K+ pump was much higher in glomerulosa cells than in adrenal fasciculata cells or hepatocytes, as judged by the ouabain-sensitive uptake of 86Rb. In contrast to the two other cell types, increasing Na+ influx with the Na+ ionophore monensin failed to significantly affect ouabain-sensitive 86Rb uptake in glomerulosa cells, suggesting that in glomerulosa cells even the resting intracellular Na+ concentration is sufficient for maximal activity of the Na+/K+ pump. Angiotensin-II (AII) inhibited the ouabain-sensitive 86Rb uptake by glomerulosa cells. The effect of AII was abolished by the selective antagonist of the AT1 type of AII receptors (DuP 753), while PD 123177, an AT2 antagonist was ineffective. AT1 receptors of glomerulosa cells coupled to phospholipase-C activation and, thus, to Ca2+ signal. The inhibitory effect of AII was dependent on the extracellular Ca2+ concentration, but an elevation of cytoplasmic Ca2+ by Ca2+ ionophore ionomycin failed to mimic the effect of AII. These data suggest that Ca2+ is required for but does not mediate the inhibitory effect of AII on the Na+/K+pump. Pharmacological activation of protein kinase-C by phorbol ester did not modify 86Rb accumulation by the cells. Ouabain induced a nifedipine-sensitive elevation in the cytoplasmic Ca2+ concentration and exerted a stimulatory effect on aldosterone production, suggesting participation of the inhibition of the Na+/K+ pump in the aldosterone stimulatory action of AII.

Effects of lithium on angiotensin-stimulated phosphatidylinositol turnover and aldosterone production in adrenal glomerulosa cells: a possible causal relationship.

Turnover of 32P-labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P]phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin-stimulated cells. Aldosterone production of superfused cells was inhibited by Li+ when the cells were stimulated with angiotensin II. On the other hand, Li+ did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+ on aldosterone production is related to its effect on PI turnover.

High extracellular Ca2+ and Mg2+ stimulate accumulation of inositol phosphates in bovine parathyroid cells.

We examined the effects of the divalent cations Ca2+ and Mg2+ on inositol phosphate accumulation in bovine parathyroid cells prelabelled with [3H]inositol to determine whether the high extracellular Ca2+ and Mg2+-evoked transients in cytosolic Ca2+ in these cells might result from increases in cellular IP3 levels. In the presence of Li+, both Ca2+ and Mg2+ produced rapid, 2-6-fold increases in IP3 and IP2 and a linear increase in IP of 6-8-fold at 30 min. Smaller (1.5-2-fold) increases in IP2 and IP3 were evident within 7.5-15 s upon exposure to high (3 mM) Ca2+ in the absence of Li+. The relative potencies of Ca2+ and Mg2+ (Ca2+ 3-fold more potent than Mg2+) in elevating inositol phosphates were similar to those for their effects in inhibiting PTH release. Fluoride (5 and 10 mM) also produced similar increases in inositol phosphate accumulation, presumably through activation of phospholipase C by a guanine nucleotide (G) protein-dependent process. Thus, high extracellular Ca2+ and Mg2+-induced spikes in cytosolic Ca2+ in bovine parathyroid cells may be mediated by increases in IP3, perhaps through a receptor-mediated process linked to phospholipase C by a G-protein.

Effect of angiotensin II and arginine vasopressin on aldosterone production and phosphoinositide turnover in rat adrenal glomerulosa cells: a comparative study.

We have previously shown that arginine vasopressin (AVP) stimulates the production of aldosterone in isolated superfused adrenal glomerulosa cells by a mechanism that involves an increased turnover of phosphoinositides. In the present study we compared the characteristics of AVP- and angiotensin II (AII)-induced changes in phosphoinositide turnover and aldosterone production in the rat. Selected concentrations of the two peptides, which were equipotent in terms of the magnitude of changes induced in phosphoinositide turnover, stimulated aldosterone production to the same extent only in the initial phase of the stimulation. A sustained aldosterone response was only observed in AII-stimulated cells. On the other hand, the AVP-induced increase in incorporation of [32P]phosphate into phosphatidylinositol and the stimulation of inositol phosphate production were maintained during incubation. Preincubation of the cells with AVP failed to modify the effects of AII on phosphoinositide breakdown or aldosterone production. These results indicate that desensitization at the level of the receptor or at a post-receptor site is not responsible for the transient character of AVP-induced aldosterone production. Delayed activation of an inhibitory mechanism by AVP can also be excluded. Additivity of the stimulation of the phosphoinositide turnover observed at submaximally, but not maximally, effective concentrations of AII indicates that the two agonists act on the same phosphoinositide pool. We suggest that the sustained steroidogenic effect of AII involves an as yet unidentified mechanism, which is absent when the cells are stimulated with AVP.

Effect of reduced extracellular sodium concentration on the function of adrenal zona glomerulosa: studies on isolated glomerulosa cells from the rat.

Aldosterone production by isolated adrenal glomerulosa cells from the rat was estimated in the presence of varying concentrations of sodium ion. The reduction of sodium concentration by 5-20 mmol/l, with or without osmotic changes, did not influence the rate of aldosterone production. Aldosterone response to angiotensin II was not modified by varying the sodium concentration.

Role of prostaglandins in the control of the function of adrenal glomerulosa cells.

The role of prostaglandins in the control of aldosterone production was studied in isolated rat glomerulosa cells. Exogenous prostaglandin E2 in concentrations above 10(-9) mol/l increased the production rate of aldosterone; this effect was attenuated by the competitive antagonist, 7-oxa-13-prostynoic acid. Prostaglandin F2 alpha (10(-9)--10(-5) mol/l) failed to influence the production rate of aldosterone. The aldosterone-stimulating effect of the prostaglandin precursor, arachidonic acid (5 x 10(-4) mol/l), could not be blocked by inhibitors of prostaglandin synthesis. Basal production rate of aldosterone was not significantly influenced by non-steroidal anti-inflammatory drugs. Glomerulosa cells were stimulated by angiotensin II; this effect was not potentiated by arachidonic acid and was reduced only slightly by indomethacin. The cells were also stimulated by corticotrophin and potassium ions. The effect of these substances was not potentiated by arachidonic acid and was not inhibited by non-steroidal anti-inflammatory drugs. These results do not confirm the presumption that intra-adrenal prostaglandins play an essential role in the control of aldosterone secretion. Some effects of arachidonic acid and its antagonist, eicosatetraynoic acid, on aldosterone production are considered to be independent of changes in prostaglandin synthesis.

Selective inhibition of potassium-stimulated rat adrenal glomerulosa cells by ruthenium red.

The effect of the cationic dye, ruthenium red (RR), on ionic fluxes, Ca2+ signal generation, and stimulation of aldosterone production was studied in isolated rat adrenal glomerulosa cells. In these cells, increased extracellular [K+] as well as angiotensin II (Ang II) elevate cytoplasmic Ca2+ concentration and thereupon activate steroidogenesis. However, the mode of action of the two stimuli are different: while a dihidropyridine-sensitive mechanism contributes to the response to both agonists, Ang II induces Ca2+ release from intracellular stores and causes capacitative Ca2+ influx, whereas K+ was recently shown to activate a plasma membrane Ca2+ current (Igl) independently of membrane depolarization. The difference is reflected in the sensitivity of the response of the cells to RR. The Ang II-induced Ca2+ signal and aldosterone production were not inhibited, but rather slightly potentiated by the dye. This potentiation was probably the consequence of the membrane-depolarizing effect of RR, due to the observed inhibition of the resting K+ conductance. Conversely, Ca2+ signal and aldosterone production were significantly reduced by RR when the cells were stimulated by moderately elevated [K+] (6-8 mM). Our patch clamp studies suggest that this effect was related to the inhibition of different voltage-dependent and -independent inward Ca2+ currents and indicates the functional importance of the latter in the signal transduction of the potassium-stimulated glomerulosa cell.

The effect of angiotensin II on arachidonate metabolism in adrenal glomerulosa cells.

The effect of angiotensin II on arachidonate metabolism was examined in rat adrenal glomerulosa cells. Incorporation of both [3H]arachidonate and [32P]phosphate into phosphatidylinositol (PI) were significantly stimulated by angiotensin II. These effects were abolished by lithium, a cation, which was found suitable to prevent increased synthesis of PI in our previous study (T. Balla et al., FEBS Letters 171, 179, 1984). On the other hand, the phospholipase A2 inhibitor mepacrine failed to inhibit the increased labelling of PI. These observations suggest that the increased 3H labelling of PI occurs via CDP-diacylglycerol, and not via enhanced deacylation-reacylation cycle. The validity of this assumption was further supported, since angiotensin II failed to stimulate the formation of lyso-PI, as examined by both [32P]phosphate incorporation and pulse-chase techniques. Angiotensin II decreased the incorporation of [3H]arachidonate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Considering that we did not find arachidonate release either from phospholipids or from other possible arachidonate sources this decrease may not be due to dilution of the tracer. Thus we assume that angiotensin II may induce a shift in phospholipid synthesis from PC and PE to phosphoinositides. These observations indicate that the enhanced hydrolysis and synthesis of PI in response to angiotensin II is not associated with increased phospholipase A2 activity in adrenal glomerulosa cells.

The mechanism of angiotensin-induced desensitization of adrenal glomerulosa cells.

Superfusion of isolated rat adrenal glomerulosa cells for 6 h with a medium containing 2.5 nM angiotensin II (AII) reduces the aldosterone response to AII, corticotropin and potassium. Here we report that under such conditions there is a decrease in the capacity of the cells to form inositol phosphates in response to a subsequent stimulation with AII. The capacity to convert corticosterone to aldosterone is also reduced by a prior exposure to AII. Superfusion with a high-potassium medium has no such an effect. Reduced phosphoinositide response may be responsible for the decreased aldosterone stimulation by AII, the inhibition of the late stage of aldosterone biosynthesis may account for the heterologous character of desensitization.