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.     

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.     

Vasopressin stimulates phosphatidylinositol turnover and aldosterone synthesis in rat adrenal glomerulosa cells: comparison with angiotensin II

Vasopressin (VP) action was identified in rat adrenal glomerulosa cells measuring the VP stimulation of phosphatidylinositol breakdown to inositol-1-phosphate (IP). The pKb value (negative log of EC50) for arginine VP (AVP) was 9.1 +/- 0.4 (n = 6). The V2-selective agonist 1-deamino-8-D-arginine VP did not stimulate IP accumulation. Furthermore, the V1-selective antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid) 2-(O-methyl) tyrosine]AVP (10(-7)M) prevented the stimulation by VP. Thus, the VP receptors in adrenal glomerulosa cells appeared to be V1-type similar to those found in liver and vasculature and different from those in kidney. Angiotensin II (AII) also stimulated accumulation of IP but the maximum stimulation achieved was much greater than with VP. In the case of AII, stimulation of phosphatidylinositol breakdown is thought to be the initiating event in the stimulation of aldosterone synthesis. In agreement with this, both VP and AII stimulated aldosterone synthesis, the maximum AII stimulation (6.5- +/- 1.3-fold, n = 7) being much greater than the VP stimulation (1.7 +/- 0.33-fold, n = 7).     

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.     

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.     

Mechanism of angiotensin II-induced phospholipase D activation in bovine adrenal glomerulosa cells

Based on previous data demonstrating activation of phospholipase D (PLD) in response to angiotensin II (AngII), we have hypothesized a role for PLD in mediating aldosterone secretion from bovine adrenal glomerulosa cells. In this study we demonstrate that a PLD-generated signal(s) is required for the AngII-elicited secretory response, since interfering with lipid second messenger formation using a primary alcohol inhibited AngII-induced aldosterone secretion, but not that elicited by incubation with a hydrophilic cholesterol analog, 22(R)-hydroxycholesterol, which bypasses signaling pathways. Three mechanisms for hormonal activation of PLD have been described in other systems: direct receptor coupling, activation through protein kinase C (PKC) and a combination of these two mechanisms. Our results indicate that the PKC activator, phorbol 12-myristic 13-acetate (PMA), is able to activate PLD, and that receptor engagement is apparently not necessary for PLD activation in response to this agent. Maximal doses of AngII and PMA produced no additive effect on PLD activation, suggesting that these two agents function through a common PKC pathway. This interpretation was confirmed by the ability of a PKC inhibitor, G? 6976, to inhibit partially AngII-induced PLD activation. Finally, treatment with the calcium ionophores A23187 or ionomycin or the calcium channel agonist BAY K8644 had no effect on PLD activity. Likewise, inhibiting calcium influx with high-dose nitrendipine affected neither basal PLD activity nor that stimulated by AngII. Thus, our results suggest a role for PKC, independent of calcium influx, in mediating AngII-induced PLD activation in glomerulosa cells.     

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.     

Angiotensin alters 45Ca2+ fluxes in bovine adrenal glomerulosa cells.

Angiotensin II stimulated 45Ca2+ release from bovine adrenal glomerulosa cells. It also decreased the influx of 45Ca2+ into glomerulosa cells. The effects were observed within 2 min of hormone addition and were blocked by Saralasin a competitive inhibitor of angiotensin. Des-Phe8-angiotensin II, a biologically inert analog, was inactive in this system. Angiotensin II also inhibited the influx of 133Ba2+ and 54Mn2+, whereas 51Cr6+ and 57Co2+ were unaffected. Alterations in 45Ca2+ fluxes were seen with concentrations of angiotensin that stimulate aldosterone biosynthesis in bovine glomerulosa cell preparations. These results suggest that calcium plays a key role in angiotensin-stimulated aldosteronogenesis.     

Aldosterone production by isolated adrenal glomerulosa cells: stimulation by physiological concentrations of angiotensin II.

The production of aldosterone by isolated canine zona glomerulosa cells was measured after the incubation of cell suspensions with angiotensin II and ACTH, and during changes in extracellular potassium concentration. Adrenal cell suspensions were prepared by collagenase digestion and physical dispersion of the capsular layer of the dog adrenal cortex, and aldosterone production was determined by direct radioimmunoassay of cell incubation media. The isolated dog adrenal cells were highly responsive to angiotensin II, with aldosterone production significantly stimulated by concentrations of the octapeptide as low as 10(-11)M. Thus, the steroidogenic response of zona glomerulosa cells was consistently observed at peptide concentrations within the physiological range of angiotensin II in dog plasma, i.e., 2.0-5.0 X 5.0 X 10(-11)M. The maximum aldosterone response of 3-8 times the basal level of steroid production was induced by 3 X 10(-10)M angiotensin II, and a decrease in aldosterone production occurred at peptide concentrations above 10(-9)M. The aldosterone response of isolated adrenal cells to ACTH was consistently less sensitive than their response to angiotensin II, by a factor of 10-20 fold. Aldosterone production was significantly increased by 10(-10)M ACTH, and reached a maximum at 10(-8)M ACTH. By contrast with angiotensin II, ACTH usually evoked a higher maximal level of aldosterone production, and did not produce a decline in steroidogenesis at peptide concentrations above the level which caused maximum stimulation of aldosterone formation. Changes in the potassium concentration of cell incubation media were also accompanied by marked effects upon aldosterone synthesis which was abolished in the absence of potassium and became detectable in the presence of 0.5 mM K+. After remaining constant between 2.5 and 4.0 mM K+, aldosterone production rose sharply above 4.5 mM K+ and reached a maximum at 8 mM K+. These observations provide direct evidence that aldosterone production by zona glomerulosa cells is influenced by changes in angiotensin II levels within the normal plasma range.     

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.     

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)     

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).     

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

Aims/hypothesis

There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells.

Methods

We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion in human beta cells.

Results

Extracellular application of ATP evoked small inward currents (3.4?±?0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4?±?2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca²⁺]_i by stimulating Ca²⁺ influx and evoking Ca²⁺ release via InsP₃-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca²⁺ release was sufficient to trigger exocytosis in cells voltage-clamped at ?70 mV. All effects of ATP were mimicked by the P2Y_(1/12/13) agonist ADP and the P2Y₁ agonist MRS-2365, whereas the P2X_(1/3) agonist α,β-methyleneadenosine-5-triphosphate only had a small effect. The P2Y₁ antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca²⁺]_i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca²⁺]_i and exocytosis without affecting Ca²⁺ influx.

Conclusions/interpretation

These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y₁ receptors, stimulating electrical activity and coupling Ca²⁺ influx to Ca²⁺ release from ER stores.     

Angiotensin II-induced activation of Na(+)-H+ exchange in adrenal glomerulosa cells is mediated by protein kinase C.

Modulation of Na(+)-H+ exchange (Na/H EXCH) by hormones and growth factors may be important in cell growth. We have previously shown that Na/H EXCH in adrenal glomerulosa cells is activated by angiotensin II (ANG II), a potent stimulator of aldosterone secretion. In the present paper, we have investigated the role of protein kinase C (PKC) in the activation of Na/H EXCH by ANG II. To accomplish this, we monitored cytosolic pH (pHi) in cells loaded with 2,7-biscarboxyethyl-5 (6)-carboxyflourescein and measured initial rates off 22Na uptake into glomerulosa cells in the presence or absence of dimethylamiloride. Both phorbol 12-myristate 13-acetate (PMA) and ANG II increased the activity of Na/H EXCH similarly when studied under basal conditions (pH 7.1). This was accompanied by alkalinization of pH and an increase in dimethylamiloride-sensitive Na+ influx. Study of kinetics of the antiporter activation showed that both ANG II and PMA led to an increase in the maximal rate (Vmax) and a decrease in the Michaelis-Menten constant (Km) for external Na+. The pHi dependence of Na+ influx was half-minimal (pK) at pHi 7.09 and remained unchanged in the presence of ANG II (pK 7.03) and PMA (pK 7.14). Depleting the cells of PKC by exposing them to PMA (1 microM) for 3 h caused a marked reduction in control and ANG II-stimulated Na+ influx and control pK (7.09 to 6.85, P less than 0.05). However, with PKC depletion, the kinetics of Na/H EXCH (Vmax, Km for external Na+, and pK) were unaffected by ANG II. Thus, Na/H EXCH in adrenal glomerulosa cells functions under basal conditions, and its relatively alkaline pK (7.09) is dependent upon PKC activity. In addition, the ANG II-induced activation of Na/H EXCH is modulated by PKC. These effects suggest an important role for PKC in pHi regulation of adrenal glomerulosa cells, particularly during ANG II stimulation of aldosterone secretion.     

Characterization and phospholipase D mediation of the angiotensin II priming response in adrenal glomerulosa cells

Bovine adrenal glomerulosa cells are primed by an initial treatment with angiotensin II (AngII) to respond with enhanced secretion to a second exposure to AngII or agents that increase calcium influx. We hypothesized that the mechanism of priming involves a persistent increase in diacylglycerol (DAG) generated via sustained activity of phospholipase D (PLD). In this report, we sought to define the time frame of this priming response as well as determine its mechanism using assays of aldosterone secretion, PLD activation, and radiolabeled diacylglycerol levels. We found that in primary cultures priming was observed for up to 50 min after AngII washout, suggesting that the priming window is protracted in these cultures relative to freshly isolated cells. The phorbol ester, phorbol 12,13-dibutyrate (PDBu), was used to investigate the role of sustained PLD activation in the persistent DAG and priming responses. PDBu was able to both prime glomerulosa cells to respond with enhanced secretion to AngII and elicit a persistent increase in DAG after PDBu washout. This persistent increase in DAG levels with an initial exposure to PDBu or AngII was not the result of maintained PLD activity after agent removal because PLD activation returned to basal levels by 30 min after washout. Finally, inhibition of PLD signaling during the initial AngII treatment inhibited the subsequent response to AngII or another agent that increases calcium influx. Thus, our results suggest that persistent DAG resulting from PLD signaling mediates the priming response to AngII or PDBu.     

Transactivation of the insulin-like growth factor-I receptor by angiotensin II mediates downstream signaling from the angiotensin II type 1 receptor to phosphatidylinositol 3-kinase

Angiotensin II (AngII) activates phosphatidylinositol 3-kinase (PI3-kinase), a known effector of receptor tyrosine kinases. Treatment of smooth muscle cells with AngII has also been shown to promote phosphorylation of various tyrosine kinase receptors. We therefore investigated the relationship between AngII and IGF-I receptor activation in smooth muscle cells with a phosphorylation-specific antibody. Our experiments showed that IGF-I receptor phosphorylation was maximally stimulated within 10 min by AngII. Inclusion of an IGF-I-neutralizing antibody in the culture media did not prevent IGF-I receptor phosphorylation after AngII treatment, which argues that a paracrine/autocrine loop is not required. Furthermore, this process was blocked by losartan and 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP-1), indicating stimulation of IGF-I receptor phosphorylation occurs via AngII type 1 receptor-dependent activation of Src kinase. The functional significance of IGF-I receptor transactivation was examined with selective inhibitors of the IGF-I receptor kinase (AG1024, AG538). When AngII-treated cells were incubated with AG1024 or AG538, phosphorylation of the regulatory p85 subunit of PI3-kinase was blocked. Furthermore, phosphorylation of the downstream factor p70(S6K) did not occur. In contrast, AG1024 did not prevent MAPK or Src kinase activation by AngII. AG1024 also did not inhibit AngII-dependent cell migration, although this process was blocked by inhibitors of the epidermal growth factor and platelet-derived growth factor receptors. Transactivation of the IGF-I receptor is therefore a critical mediator of PI3-kinase activation by AngII but is not required for stimulation of the MAPK cascade.     

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.