Actions of angiotensin II antagonists upon aldosterone production by isolated adrenal glomerulosa cells.

The biological activities of angiotensin II antagonists upon basal and angiotensin II-stimulated aldosterone production were evaluated in an isolated canine glomerulosa cell preparation. The most potent competitive antagonist of angiotensin II-stimulated aldosterone production was the [Sar1, Ile8]derivative of angiotensin II. However, this peptide was also a partial agonist at concentrations required to inhibit the steroidogenic effect of angiotensin II on dog adrenal cells, and never reduced aldosterone production to basal levels. On a molar basis, the [Sar1, Ala8] and [Sar1, Gly8]derivatives of angiotensin II were relatively less potent as competitive inhibitors of angiotensin II-stimulated aldosterone production. However, the [Ala8] and [Gly8]-analogues did not exhibit significant agonist activity and were therefore more effective antagonists of angiontensin II-stimulated aldosterone production. These results suggest that increased length of the aliphatic side chain at the C-terminus of angiotensin II antagonists is accompanied by enhanced affinity for the receptor site, but also by increased agonist activity upon aldosterone synthesis. The actions of angiotensin II and [Des-Asp1]angiotensin II upon aldosterone production were inhibited identically and completely by [Sar1, Ala8]angiotensin II, and identically, though incompletely, by lower concentrations of [Sar1, Ile8]angiotensin II. The heptapeptide antagonist [Des-Asp1, Ile8]angiotensin II was much less potent than [Sar1, Ile8]angiotensin II as an inhibitor of the actions of both the heptapeptide and octapeptide agonists. The antagonist activity of six angiotensin II analogues at the adrenal level, determined by the concentration required for 50% inhibition of maximum aldosterone secretion, correlated well with their antagonist activity measured upon isolated smooth muscle. These observations demonstrate that the octapeptide antagonists are more effective than the heptapeptide antagonists upon angiotensin II-stimulated aldosterone production, and that angiotensin II receptors in smooth muscle and adrenal cortex exhibit generally similar responses to angiotensin II antagonists. Also, these results do not support the proposal that the [Des-Asp1]heptapeptide is an important intermediate in the action of angiotensin II upon adolesterone production in the adrenal glomerulosa cells. The production of aldosterone by dispersed zona glomerulosa cells in vitro provides a highly sensitive and biologically appropriate response for evaluation of the agonist and antagonist properties of angiotensin II analogues upon the adrenal gland.     

Aldosterone production by isolated glomerulosa cells: modulation of sensitivity to angiotensin II and ACTH by extracellular potassium concentration.

The influence of extracellular potassium concentration on adrenal sensitivity to angiotensin II and ACTH was studied in isolated canine adrenal glomerulosa cells. When potassium was absent from the incubation medium, the aldosterone response to angiotensin II or ACTH was completely abolished. At physiologic angiotensin II concentrations (2.5 x 10(-11) M), aldosterone formation increased 4-fold when potassium concentration was increased from 2.5 to 5.0 mM, and rose 6-fold as potassium was increased from 2.5 to 7.5 mM. In the absence of angiotensin II, the same changes in potassium concentration increased aldosterone production only to 2-fold and 3.5-fold, respectively. The effect of potassium concentration upon the aldosterone response to ACTH was similar but less marked. The concentration and binding affinity of angiotensin II receptor sites in glomerulosa cells were not changed by increasing potassium concentrations from 0 to 7.5 mM. These observations demonstrate that the aldosterone response to the glomerulosa cell to angiotensin II is potassium-dependent within the physiological range for each of these stimuli. Such an interaction suggests that the in vivo effect of potassium upon aldosterone secretion includes a significant modulating action upon adrenal sensitivity to angiotensin II, as well as a direct action of potassium upon the adrenal glomerulosa cell.     

Electrophysiological responses to angiotensin II of isolated rat adrenal glomerulosa cells

The membrane response of isolated rat glomerulosa cells to the application of angiotensin II (A II) has been studied using intracellular voltage measurements. The membrane response is biphasic. The first, brief phase involves an increase in membrane conductance and a hyperpolarization from the resting membrane potential. The second, long-lasting phase is characterized by a large decrease in membrane conductance and a depolarization from the resting membrane potential. The reversal potential for the second phase is -94 +/- 1.2 mV, and a linear relationship between reversal potential and external K+ indicates that the A II-mediated response is predominantly inhibition of K+ permeability. The A II response can be elicited when external Ca2+ is replaced by Sr2+ or Ba2+, but the response is inhibited when Mn2+ is added to the bath or when stimulated in a Ca2+-free solution. A II appears to inhibit at least two conductances, when the cell is stimulated by long current steps. External application of A II inhibited the Ca2+ regenerative response found in glomerulosa cells in a dose-dependent manner. The rate of rise of the regenerative response was greatly attenuated by A II; half-maximal inhibition was produced by about 10(-9) M A II. In addition, rectification, evident at voltages more positive than -60 mV during current stimulation, was also inhibited. In conclusion, A II causes rat glomerulosa cells to depolarize due to the inhibition of resting K+ permeability. Action potential activity is not observed during A II-mediated membrane depolarization; rather, both Ca2+ and K+ conductances appear to be inhibited during A II application.     

Effect of angiotensin II on renin production by rat adrenal glomerulosa cells in culture.

Angiotensin II (Ang II) inhibits renin secretion and production from the kidney, but the effect of Ang II on adrenal renin is not clear. Nephrectomy, via elevated plasma adrenocorticotropic hormone (ACTH) and potassium, is a strong stimulator of adrenal renin production in the rat. This stimulation is inhibited by the infusion of Ang II, suggesting a negative feedback between Ang II and adrenal renin. In the present study, we examined the effect of Ang II on adrenal renin using a primary culture of rat glomerulosa cells. Cells were exposed to ACTH (10(-11) M), high potassium (8 and 12 mM), db-cyclic AMP (db-cAMP), (10(-3) M), or Ang II (10(-11) to 10(-5) M) for 24 hours, and active renin and inactive renin were measured. Active renin was predominant in the cells, whereas inactive renin predominated in the medium. Ang II stimulated renin production in a dose-dependent fashion (cell-active renin, 1.21 +/- 0.20 to 2.39 +/- 0.16; medium-inactive renin, 2.59 +/- 0.40 to 6.14 +/- 0.49 ng Ang I/10(6) cells). Both ACTH and db-cAMP significantly stimulated active renin in the cells (ACTH, 1.73 +/- 0.14 to 9.44 +/- 0.98; db-cAMP, 1.45 +/- 0.16 to 3.96 +/- 0.71 ng Ang I/10(6) cells) and inactive renin in the medium (ACTH, 4.98 +/- 0.38 to 43.7 +/- 5.63; db-cAMP, 3.80 +/- 0.32 to 33.55 +/- 5.62 ng Ang I/10(6) cells). The addition of Ang II (10(-7) M) blunted the stimulation of renin production by both ACTH and db-cAMP by 60%. High potassium-stimulated renin production was not inhibited by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of angiotensin II and saralasin on 18-hydroxy-11-deoxycorticosterone production by isolated human adrenal glomerulosa cells.

To assess the role of angiotensin II (AII) in regulating 18-hydroxy-11-deoxycorticosterone (18-OHDOC) secretion in man, isolated human adrenal glomerulosa cells were incubated with AII and/or its competitive antagonist, saralasin. AII 2.4 X 10(-8) M) elicited an 80% increase in 18-OHDOC levels as well as similar increases in aldosterone, 18-hydroxycorticosterone, and corticosterone (P less than 0.01). Saralasin (10(-8) M) caused a partial but significant inhibition of AII-stimulated 18-OHDOC production, while 10(-6) M saralasin blocked AII-stimulated steroidogenesis completely. In addition, both concentrations of saralasin caused 10--30% decrements in basal steroid levels. The direct AII effect on 18-OHDOC secretion and the antagonistic effect of saralasin on both exogenous and endogenous AII-stimulated steroidogenesis, documented in these experiments, indicate that the increase in 18-OHDOC levels after sodium restriction reported in man is probably mediated by the renin-angiotensin system. Furthermore, because high concentrations of saralasin did not increase aldosterone secretion, the partial agonist properties of saralasin in vivo in man may not be due to a direct effect on the glomerulosa cell.     

Calcium oscillations in single adrenal glomerulosa cells stimulated by angiotensin II.

The cytosolic calcium (Ca2+i) response to angiotensin II (Ang II) was examined in single rat zona glomerulosa cells by monitoring fura-2 fluorescence with microspectrofluorimetry. Ang II concentrations ranged from 5 X 10(-12) to 5 X 10(-8) M. The mean peak Ca2+i increase was similar at all Ang II concentrations (205 +/- 11 nM), with a significant difference (P less than 0.05) found only between 5 X 10(-12) M (151 +/- 16 nM) and 5 X 10(-9) M (236 +/- 24 nM). Striking differences over the range of Ang II concentrations were found in the Ca2+i response kinetics. A dose-dependent delay of the onset of the Ca2+i response was observed ranging from 2.6 +/- 0.3 sec at 5 X 10(-8) M to 181 +/- 27 sec at 5 X 10(-12) M Ang II. After the delay, cells typically responded with an abrupt increase in Ca2+i, complete within 15 sec. At low Ang II concentrations (5 X 10(-11) and 5 X 10(-12) M), a complex response was often observed consisting of Ca2+i oscillations. Higher Ang II concentrations gave some evidence of Ca2+i oscillation, especially at 5 X 10(-10) M where oscillations appeared fused. Above 5 X 10(-10) M Ang II, the initial Ca2+i increase decayed to an apparent steady-state value 38-40% of the peak response within 5 min; 5 X 10(-10) M Ang II produced a smaller decline to 63% of the initial Ca2+i increase. In contrast to cell population studies, assessment of individual glomerulosa cells demonstrates (i) a dose-dependent delay prior to a rapid increase in Ca2+i; (ii) a similar peak increase at most Ang II concentrations; (iii) greater sensitivity of the Ca2+i response; and (iv) a complex oscillating Ca2+i response in the physiological range of Ang II.     

T-type calcium channels in adrenal glomerulosa cells: GTP-dependent modulation by angiotensin II.

With the use of whole-cell and single-channel current recordings, we have examined in more detail the site of action of angiotensin II (AII) on multiple populations of voltage-gated calcium channels in bovine adrenal glomerulosa cells. AII (10 nM) enhances whole-cell T-type calcium channel current and increases the activity of single T-type calcium channels in cell-attached patch recordings. The AII-induced enhancement of whole-cell calcium channel currents is dependent on the presence of internal GTP and can be inhibited by the competitive AII-receptor antagonist saralasin (1 microM). These results show that AII augments the T-type calcium channel current in bovine adrenal glomerulosa cells.     

Cytosolic free calcium oscillates in single bovine adrenal glomerulosa cells in response to angiotensin II stimulation

The characteristics of the change in cytosolic free Ca2+ concentration ([Ca2+]i) in response to agonist stimulation were studied in individual cells of the bovine adrenal zona glomerulosa. Following digestion and dispersion, the cells were loaded with the fluorescent Ca2+ indicator fura-2. The cells adhered to Concanavalin A-coated coverslips and were studied using dual excitation wavelength microfluorimetry. In this procedure individual cells under constant perfusion are visualized by microscopy and excited with light alternating rapidly between 340 and 380 nm. The ratio of fluorescence (F) emitted from the cell (F340/F380) correlates directly with [Ca2+]i. Continuous stimulation with angiotensin II (AII; 10 nmol/l) resulted in a brisk transient rise in [Ca2+]i within 8 s of application of the stimulus. In 50% of cells studied, this initial peak was followed by a series of oscillations in [Ca2+]i lasting up to 13 min, with an average period of 33.0 +/- 5.9 (S.E.M.) seconds. [Ca2+]i did not return to prestimulation levels and, subsequent to the oscillatory phase, the [Ca2+]i remained increased for several minutes. Upon removal of extracellular Ca2+ the oscillations ceased almost immediately although [Ca2+]i remained increased. However, in Ca2+-free medium, a single peak of [Ca2+]i still occurred in response to AII. Cells remained refractory to restimulation over a 15-min period. In contrast, stimulation with K+ (8 mmol/l) rapidly increased [Ca2+]i to a level similar to that induced by AII but without inducing oscillations. Moreover, the effect lasted only while K+ was present and was highly reproducible over multiple stimulations during a 15-min period. These results corroborate, at the single cell level, the known action of AII of causing release of intracellular Ca2+, but reveal a more complex mechanism of action on Ca2+ influx than previously recognized, possibly invoking a role for a putative second messenger-operated membrane Ca2+ channel.     

Aldosterone production and hormone responsiveness in adrenal glomerulosa cells from cows of different ages

Aldosterone production by suspensions of adrenal glomerulosa cells obtained from young and old cows was measured. Basal steroidogenesis was lower in cells from old cows, as were the responses to angiotensin II (AII), potassium, ACTH and dibutyryl cyclic AMP. Receptors for AII and aldosterone production from added progesterone were the same in old and young cells. Synthesis of pregnenolone from endogenous precursor, the 'early pathway' of aldosteronogenesis, was lower in old cells than in young. AII-stimulated incorporation of 32P into phosphatidylinositol and the change in 45Ca2+ flux induced by AII were diminished in old cells. Overall protein synthesis, measured by 3H leucine incorporation, was lower in old cells than young, but was not affected by AII in either. Diminished responsiveness of adrenal glomerulosa cells from old animals results from a change in postreceptor events that affect the early pathway of aldosteronogenesis.     

Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells.

Specific receptors for angiotensin II (A II) were demonstrated in membrane fractions and collagenase-dispersed cells from the zona glomerulosa of the rat adrenal gland. The equilibrium association constant (Ka) of the A II binding sites was similar in particulate fractions (2.0 +/- 0.4 (SE) X 10(9) M-1) and intact glomerulosa cells (1.8 +/- 0.3 X 10(9) M-1). Specific binding of [125I]iodo-A II was enhanced by increasing sodium concentration, and in the presence of dithiothreitol, EDTA, and EGTA. Plasma membrane fractions prepared by density gradient centrifugation showed increased binding of [125I]iodo-A II, and were correspondingly enriched in adenylate cyclase and sodium-potassium-dependent ATPase. Steroid production by collagenase-dispersed adrenal glomerulosa cells was highly responsive to A II and ACTH. Significant increases in aldosterone and corticosterone production were elicited by A II concentrations as low as 3 X 10(-11) M, equivalent to normal blood levels of A II in rats (5 X 10(-11) M). The maximum increase in aldosterone production, of 6--7 times the basal value, was obtained at 10(-9) M A II. Dispersed capsular cells were also highly sensitive to ACTH, responding to concentrations down to 3 X 10(-12) M with increased aldosterone production, reaching a maximum aldosterone response of 20-fold above the basal value. The magnitudes of the aldosterone and corticosterone responses to A II in capsular and fasciculata-reticularis cells were commensurate with the distribution of A II receptors, which were 11-fold more concentrated in capsular cells. The ability of A II to evoke aldosterone production at physiological concentrations, and the correspondence between A II binding and steroidogenesis in capsular cells, demonstrate the functional importance of A II receptor sites in the zona glomerulosa of the rat adrenal cortex.     

In adrenal glomerulosa cells, angiotensin II inhibits proliferation by interfering with fibronectin-integrin signaling

Angiotensin II (Ang II), through the Ang II type 1 receptor subtype, inhibits basal proliferation of adrenal glomerulosa cells by inducing the disruption of actin stress fiber organization. This effect is observed in cells cultured on plastic or on fibronectin. The aim of the present study was to investigate how Ang II may interfere with extracellular matrix/integrin signaling. In cells treated for 3 d with echistatin (EC) (a snake-venom RGD-containing protein that abolishes fibronectin binding to alpha(5)beta(1) or alpha(v)beta(3) integrins), basal proliferation decreased by 38%, whereas Ang II was unable to abolish basal proliferation. In cells grown on fibronectin, Ang II decreased binding of paxillin to focal adhesions and, similarly to EC, induced a rapid dephosphorylation of paxillin (1 min), followed by an increase after 15 min. Fibronectin enhanced RhoA/B and Rac activation induced by Ang II, an effect abolished by EC. Under basal conditions, paxillin was more readily associated with RhoA/B than with Rac. Stimulation with Ang II induced a transient decrease in RhoA/B-associated paxillin (after 5 min), with a return to basal levels after 10 min, while increasing Rac-associated paxillin. Finally, results reveal that glomerulosa cells are able to synthesize and secrete fibronectin, a process by which cells can stimulate their own proliferative activity when cultured on plastic. Together, these results suggest that Ang II acts at the level of integrin-paxillin complexes to disrupt the well- developed microfilament network, a condition necessary for the inhibition of cell proliferation and initiation of steroidogenesis.     

Fate of [125I]angiotensin II in adrenal zona glomerulosa cells

Binding and internalization of [125I]angiotensin II (AII) were studied by morphological and biochemical methods in rats in vivo. Light microscope radioautography demonstrated that [125I]AII binds specifically to adrenal zona glomerulosa (ZG) cells. Ultrastructural radioautographic analysis revealed that [125I]AII binds to the cell surface, clusters in coated pits, is internalized in coated vesicles, and is transported by receptosomes to lysosomes in less than 20 min. Biochemical analysis revealed that as much as 40% of the adrenal radioactive uptake behaves as native [125I]AII as shown by electrophoresis, immunoprecipitation and radioligand binding studies. These results indicate that the effects of AII on the secretion of aldosterone by ZG cells are mediated by cell surface phenomena and not by binding to intracellular organelles involved in steroidogenesis. They also indicate that the half-life of AII bound to receptors and internalized seems to be much longer (min) than in the systemic circulation (sec).     

Na(+)-H+ exchanger kinetics in adrenal glomerulosa cells and its activation by angiotensin II

We have studied the kinetic properties of basal and angiotensin II (ANG II) stimulated Na(+)-H+ exchange in adrenal glomerulosa cells by measuring changes in cytosolic pH (pHi) and initial rates of 22Na uptake in the presence or absence of dimethylamiloride (DMA). The cells were studied 1) under basal conditions, 2) at constant pHi (6.8) with varied external sodium (Na+o), and 3) at varied pHi with constant Na+o (50 mM). In 2,7-biscarboxyethyl-5(6)-carboxyfluorescein loaded cells under basal conditions, pHi rose from 7.09 +/- 0.02 to 7.19 +/- 0.02 (P less than 0.05) with addition of ANG II (100 nM). Similarly, DMA-sensitive Na influx was enhanced from 9.2 +/- 1.3 to 14.8 +/- 2.1 nmol Na+/mg protein x min (P less than 0.01) by ANG II. In cells acid-loaded by preincubation in Na(+)-free media (pHi 6.8), addition of varying Na+o resulted in a rapid H+ efflux that was markedly inhibited by DMA. DMA-sensitive Na+ influx into these acidified cells with varied Na+o exhibited a Michaelis-Menten constant (Km) of 23 mM and a maximum velocity (Vmax) of 43 nmol Na+/mg protein x min. By varying pHi (from pHi 7.1 to 6.2), DMA-sensitive Na+ influx likewise showed activation with cellular acidification with a pK at pHi 7.09. At pHi 6.8, ANG II decreased the Km for Na+o from 23 to 17 mM and increased the Vmax from 43 to 53 nmol Na+/mg protein x min. The pHi dependence of DMA-sensitive Na+ influx was not affected by ANG II (pK at pHi 7.03). DMA also inhibited AII-stimulated aldosterone secretion and Na+ influx similarly. These results indicate that Na(+)-H+ exchange in adrenal glomerulosa cells is 1) functioning under basal conditions, and 2) is modulated by ANG II with enhanced Na+o affinity and Vmax but without a shift in pHi dependence (similar to ANG II effects on vascular smooth muscle cells). These effects suggest an important role for Na(+)-H+ exchange during ANG II stimulation of aldosterone production by glomerulosa cells.     

Inositol trisphosphate isomers in angiotensin II-stimulated adrenal glomerulosa cells

The stimulation of phosphoinositide metabolism by angiotensin II (Ang II) was studied in [3H]inositol-labelled bovine adrenal glomerulosa cells. After separation of the phosphoinositols by ion-exchange high-performance liquid chromatography, it was shown that the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) followed distinct kinetics. The first compound to increase upon stimulation with 10(-7) M Ang II was Ins(1,4,5)P3, which reached a maximum (250% of basal level) within 10 s. At lower concentrations of Ang II, this response was slower. The formation of Ins(1,4,5)P3 depended upon the concentration of Ang II, with an EC50 of 2.4 +/- 1.5 X 10(-9) M Ang II. The potency of Ang II in stimulating the turnover of phosphoinositides and in increasing the biosynthesis of aldosterone was very similar, whereas the peptide was ten times more potent in its ability to mobilize Ca2+. Ang II was also able to stimulate the production of Ins(1,4,5)P3 in permeabilized glomerulosa cells. This effect was mimicked by a non-hydrolysable analog of GTP (GTP gamma S), suggesting that a GTP binding protein is involved in the mechanism coupling the Ang II membrane receptor to phospholipase C. These results strengthen the view that Ins(1,4,5)P3 plays a key role as second messenger in the steroidogenic response to Ang II in adrenal glomerulosa cells.     

Ca channels in adrenal glomerulosa cells: K+ and angiotensin II increase T-type Ca channel current.

Ca channel currents were studied in freshly dispersed bovine adrenal glomerulosa cells to better understand the control of aldosterone secretion by extracellular K concentration (Ko) and angiotensin II (AII). The whole-cell variation of the patch voltage clamp technique was used. Two types of Ca channels were found. One type is similar to the "T-type" Ca channels found in many excitable cells. These channels deactivate slowly (tau approximately equal to 7 ms at -75 mV) and inactivate rapidly during strong depolarizations. The second channel type activates and inactivates at more positive potentials than the T-type Ca channels and deactivates rapidly. These channels are similar to the "L-type" Ca channels found in muscle and nerve. Our studies provide three reasons for concluding that T-type Ca channels have an important role in mediating stimulus-secretion coupling in response to high K+ or AII: (i) aldosterone secretion and steady-state current through T-type Ca channels are biphasic functions of Ko and both increase in parallel for Ko = 2-10 mM; (ii) nitrendipine blocks the T-type Ca channels and the stimulation of aldosterone secretion by high K+ or AII with similar potency; (iii) AII increases Ca entry through the T-type Ca channels.