Platelet-derived growth factor is a potent inhibitor of angiotensin II-induced aldosterone synthesis.

Platelet-derived growth factor (PDGF) is a potent mitogen for several cell types. In addition, PDGF has vasoconstrictive action and shares some signal transduction mechanisms with angiotensin II (AII). In the present study, we have examined the effects of PDGF on basal and AII-induced aldosterone synthesis in freshly isolated rat adrenal glomerulosa cells. Recombinant human PDGF-BB caused a dose-dependent inhibition of AII-induced aldosterone synthesis being effective at concentrations as low as 10(-12) M. We also investigated possible mechanisms of action of PDGF. We have previously reported that the 12-lipoxygenase (LO) pathway of arachidonic acid plays a key role in AII-induced aldosterone synthesis. We thus examined whether PDGF action is mediated by changes in 12-LO activation. PDGF, at the same doses that blocked AII-induced synthesis also significantly inhibited AII-induced increases in the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE) formation. Further, the addition of 12-HETE completely restored the stimulatory effect of AII during inhibition by PDGF. These results suggest that PDGF could act, at least in part, by inhibition of AII-induced 12-HETE formation. We also examined the role of diacylglycerol (DG) formation since we have previously reported that DG is the source of arachidonic acid for 12-HETE formation. We observed that both AII and PDGF stimulated [3H]arachidonic acid-labeled DG formation. However, PDGF did not alter AII-induced DG formation suggesting that PDGF action is not mediated by affecting AII-induced increases in DG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of angiotensin-II-mediated aldosterone secretion by 5-hydroxyeicosatetraenoic acid

We have previously demonstrated that the 12-lipoxygenase (12-LO) pathway plays a key role in angiotensin-II (AII)-dependent aldosterone production. In the present study we examined the role of the 5LO pathway on AII-induced aldosterone secretion in rat glomerulosa cells in vitro. The 5LO product 5-hydroxyeicosatetraenoic acid (5HETE) and its unstable precursor 5-hydroxyperoxyeicosatetraenoic acid did not significantly alter basal aldosterone secretion in concentrations from 10(-9)-10(-7) M. In contrast, 5HETE reduced peak AII-induced aldosterone production from 59.1 +/- 9.0 to 37.96 +/- 7.2 ng/10(6) cells (P less than 0.01). This was accompanied by inhibition of the AII-stimulated rise in 12HETE production (10(-9)M AII, 160 +/- 4% of control; 10(-9) M AII plus 10(-7) M 5HETE, 90 +/- 1% of control production). However, 5HETE had no effect on the aldosterone response to potassium or ACTH, secretagogues that cause no activation of the 12LO pathway. These results suggest that the 5LO product 5HETE can selectively modulate AII-dependent aldosterone secretion. Further, the selective inhibitory effect of 5HETE on the AII effect in rat glomerulosa cells may be exerted by blockade of arachidonate metabolism via the 12LO pathway. These results suggest that the 5LO pathway may negatively modulate AII action in the adrenal zona glomerulosa.     

Angiotensin feedback inhibition on renin is expressed via the lipoxygenase pathway

Angiotensin II (AII) action on adrenal and smooth muscle cells is mediated via mechanisms that include changes in calcium flux and phosphoinositide hydrolysis. Phosphoinositide metabolism results in the release of arachidonic acid, a precursor of both the cyclooxygenase (CO) and lipoxygenase (LO) pathway. The effects of both LO and CO inhibitors on AII action were studied using both static incubations and perifusions of rat renal cortical slices. 12-Hydroperoxyeicosatetraenoic acid and its stable metabolite 12-hydroxyacid mimicked the inhibitory actions of AII on renin. A specific CO blocker did not alter AII inhibition of renin and a 5-LO blocker U60,257 was also ineffective, whereas the LO blockers BW755c, phenidone, and baicalein all eliminated or interfered with the action of AII on renin. All inhibition in the presence of a LO blocker was restored by adding nanomolar concentrations of 12-hydroperoxyeicosatetraenoic acid. LO inhibitors were specific for blocking AII, as they did not interfere with potassium (K+)-induced renin inhibition. These results imply that 12 and/or 15 products of the LO pathway are involved in AII action.     

Tumor necrosis factor and interleukin-1 are potent inhibitors of angiotensin-II-induced aldosterone synthesis

Cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), mediate many inflammatory and cellular responses. However, the effects of TNF and IL-1 on basal and angiotensin-II (AII)-stimulated aldosterone synthesis are not known. We studied the effect of recombinant and purified TNF and IL-1 on basal as well as AII-, ACTH-, and K+-induced aldosterone synthesis in isolated rat adrenal glomerulosa cells. Since we have previously shown that AII action is mediated by activation of the 12-lipoxygenase (12LO) pathway of arachidonic acid, we also evaluated the effects of these cytokines on the 12LO product 12-hydroxyeicosatetraenoic acid (12HETE) using a validated RIA technique. TNF at 2.5 and 5.0 ng/ml produced a dose-dependent inhibition of AII-induced aldosterone synthesis [AII, 39.0 +/- 3.3 ng/10(6) cells.h; AII plus TNF (5.0 ng/ml), 14.3 +/- 1.6; P less than 0.001 vs. AII; AII plus TNF (2.5 ng/ml), 24.7 +/- 3.2; P less than 0.01 vs. AII]. Similarly, TNF at 5.0 ng/ml also attenuated the stimulatory effect of ACTH (10(-9) M). However, K+-induced aldosterone synthesis was not altered. TNF also did not alter basal aldosterone levels. AII, as previously shown, stimulates 12HETE synthesis (basal, 608 +/- 114 pg/10(5) cells.h; versus AII, 1268 +/- 197; P less than 0.02). TNF at concentrations of 1.0-5.0 ng/ml produced a dose-dependent inhibition of AII stimulatory action on 12HETE synthesis [AII plus TNF (1.0 ng/ml), 650 +/- 26 pg, P less than 0.03 vs. AII; AII plus TNF (5.0 ng/ml), 390 +/- 46; P less than 0.01 vs. AII plus TNF (1.0 ng/ml)]. In addition, 12HETE at 10(-8) M completely restored the effects of AII during blockage by TNF. Purified human IL-1 (75% beta, 25% alpha) as well as recombinant human IL-1 beta at concentrations as low as 50 pg/ml inhibited AII-induced aldosterone synthesis. IL-1 beta did not alter ACTH- or K+-induced aldosterone synthesis and, in fact, had a tendency to potentiate ACTH effects. These results suggest that the cytokines TNF and IL-1 are potent inhibitors, particularly of AII action in the adrenal glomerulosa cell. Therefore, local or systemically produced TNF or IL-1 may be important negative modulators of aldosterone synthesis.     

Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells.

The peptide hormone angiotensin-II (AII) is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AII also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AII receptor antagonists and the role of protein kinase-C (PKC). AII-induced DNA synthesis was significantly attenuated by two structurally dissimilar LO inhibitors, baicalein and phenidone. In addition, the LO product 12-hydroxyeicosatetraenoic acid (12-HETE) itself caused a significant increase in DNA synthesis, suggesting that the 12-LO pathway in part plays a role in AII-mediated mitogenesis. AII-induced proliferative responses were blocked by the type 1 AII receptor antagonist. Both AII- and 12-HETE-induced increases in DNA synthesis were markedly inhibited by two PKC blockers, staurosporine and sangivamycin. Further, both AII and 12-HETE could activate PKC by translocating it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation.     

Tumor necrosis factor and interleukin-1 may regulate renin secretion

Cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) are not only immunoregulatory polypeptides, but may have endocrine functions. We have studied the direct effects of recombinant and purified TNF and IL-1 on renin secretion using both static incubations and perifusions of rat renal cortical slices. Ultrapure human IL-1 (hIL-1) at concentrations as low as 5 U/ml (3 X 10(-12) M) significantly stimulated renin secretion (control, 98 +/- 4%; hIL-1, 153 +/- 13%; P less than 0.01). TNF similarly induced renin release [control, 97 +/- 6%; TNF (10 U/ml), 151 +/- 13%; P less than 0.005]. TNF and recombinant human IL-1 beta (rhIL-i beta) also blocked the inhibitory actions of angiotensin-II (AII) on renin release [control, 100 +/- 3%; AII (2 X 10(-7) M), 80 +/- 5%; AII plus TNF (20 U/ml), 102 +/- 7%; AII plus rhIL beta (10 U/ml), 106 +/- 6%; both P less than 0.02 vs. AII]. A cyclooxygenase (CO) blocker, meclofenamate (M), which does not significantly alter basal renin release, attenuated the TNF- and rhIL-1 beta-induced renin secretion [TNF (20 U/ml), 132 +/- 11%; TNF plus M (5 X 10(-5) M), 100 +/- 3% (P less than 0.01); rhIL-1 beta (10 U/ml), 135 +/- 9%; rhIL-1 beta plus M, 105 +/- 10% (P less than 0.05)]. The stimulatory effects of TNF and IL-1 on renin were reversible. These results suggest that IL-1 and TNF are renin secretagogues and can also block the inhibitory actions of AII on renin. Since the effect of TNF and IL-1 on renin can be blocked by a (CO) inhibitor, the studies indicate a role of prostaglandins in their action. Therefore, locally produced TNF and IL-1 may play an important paracrine role in regulation of the renin-angiotensin system.     

Angiotensin II-induced aldosterone synthesis is potentiated by epidermal growth factor.

Epidermal growth factor (EGF) is a potent mitogen and has effects in several endocrine systems. We examined the effects of EGF on basal and angiotensin II (AII)-induced aldosterone synthesis in freshly isolated rat and cultured human adrenal glomerulosa cells. EGF alone caused a significant increase in basal aldosterone synthesis in both the rat and human cells. In addition, EGF caused a significant increase in AII-induced aldosterone secretion in both rat and cultured human cells during short and long term incubations. Further, we observed that the effect of EGF was highly specific to AII since it did not alter either potassium (8.7 mM) or ACTH (10(-10) M) mediated increases in aldosterone synthesis. We also investigated possible mechanisms of action of EGF. Since earlier studies showed that the lipoxygenase pathway of arachidonic acid plays a key role in mediating AII-induced aldosterone synthesis, we studied the effect of lipoxygenase inhibition in EGF action. We observed that the nonselective lipoxygenase blocker BW755c, which blocked AII-induced aldosterone synthesis, also inhibited EGF mediated increase in aldosterone synthesis. We also examined the effects of EGF on diacylglycerol (DG) formation since DG is an important second messenger in AII action. We found that EGF stimulated basal DG levels and also potentiated AII-induced DG formation, suggesting that EGF may potentiate AII-induced aldosterone synthesis via increases in DG. These results suggest that EGF may play an important role in aldosterone synthesis by acting as a specific positive modulator of AII action in the adrenal.     

5-Hydroxyeicosatetraenoic acid increases prolactin release from rat anterior pituitary cells

The enzymatic breakdown of phospholipids to form arachidonic acid and its subsequent conversion to metabolites produced via the lipoxygenase pathway in anterior pituitary cells may contribute to the process of PRL release. The incubation of primary cultures of pituitary cells from female rats with the lipoxygenase product 5-hydroxyeicosatetraenoic acid (5-HETE; 5-100 microM) significantly increased PRL release in a concentration-dependent manner. The release of PRL induced by 45 microM 5-HETE was completely blocked by 1 microM dopamine. Penfluridol, an agent that binds to and inactivates several Ca+2-binding proteins, including calmodulin, decreased (P less than 0.01) basal and 5-HETE-stimulated PRL release. Similarly, 50 microM D-600, a Ca+2 channel antagonist, significantly (P less than 0.01) reduced basal and 5-HETE-induced PRL release. BW755c or RHC 80267, both of which reduce the production of arachidonic acid metabolites, including 5-HETE, significantly reduced basal PRL release. The inhibitory effects of BW755c and RHC 80267 on PRL release, however, could be overcome by the addition of 5-HETE. In conclusion, 5-HETE or similar lipoxygenase metabolites may be important cellular components in the process of PRL release, and the inhibitory action of dopamine on PRL would seem to be mediated at some step after stimulation by these metabolites.     

12-lipoxygenase in opioid-induced delayed cardioprotection: gene array,mass spectrometric,and pharmacological analyses

12-lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a delta-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic-mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (P<0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50+/-4% and 42+/-3% versus 29+/-2%, P<0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.     

Key role of diacylglycerol-mediated 12-lipoxygenase product formation in angiotensin II-induced aldosterone synthesis

We have shown earlier that the 12-lipoxygenase product of arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE), plays an important role in mediating angiotensin II (AII)-induced aldosterone secretion (J. Clin. Invest. (1987) 80, 1763). In the present study, we have evaluated whether diacylglycerol (DG) is the source of arachidonic acid giving rise to this 12-HETE. Treatment of rat adrenal glomerulosa cells with a DG lipase inhibitor, RHC 80267, which prevents conversion of DG to AA and HETEs, blocked AII-induced aldosterone and 12-HETE formation. In contrast, a DG kinase inhibitor, R59022, which prevents conversion of DG to phosphatidic acid, potentiated AII-induced aldosterone and 12-HETE formation. These two inhibitors block DG metabolism which would be expected to lead to increased DG levels and protein kinase C activity and AII-induced steroidogenesis. However, only R59022 potentiated AII action while RHC 80267 was inhibitory. This suggests that conversion of DG to AA and 12-HETE is important for AII action. Further proof for this was obtained by measuring [3H]AA-labeled DG levels. The combination of the inhibitors significantly potentiated AII-induced DG formation even though this same combination was inhibitory on AII-induced aldosterone and 12-HETE. Thus, the inhibitory effect of RHC 80267 is due to blockade of AA release and not of DG formation. These results suggest that DG plays a dual role in AII action, both as an activator of protein kinase C and as a source of AA for 12-HETE formation.     

Increased levels of 12(S)-HETE in patients with essential hypertension

The platelet-type 12-lipoxygenase (12-LO) catalyzes the transformation of arachidonic acid into 12-hydroperoxyeicosatetraenoic acid [12-(S)HPETE], which is reduced to 12-hydroxyeicosatetraenoic acid [12-(S)HETE]. These metabolites exhibit a variety of biological activities such as mediation of angiotensin II-induced intracellular calcium transients in cultured rat vascular smooth muscle cells. It has recently been reported that platelet 12(S)-HETE production is enhanced in the spontaneously hypertensive rat. The pronounced hypotensive effect of LO inhibition in SHR suggests that LO activity may play a role in this form of hypertension. The aim of this study was to determine the basal and thrombin-induced platelet 12(S)-HETE production and the urinary 12(S)-HETE excretion in essential hypertension. We studied 19 patients with this disease (57+/-2 years of age) and 9 normotensive control subjects (48+/-5 years of age) (P:=0.074). 12(S)-HETE was measured in Sep-Pack-extracted samples with specific ELISA and high-performance liquid chromatography. The platelet basal level of 12(S)-HETE was significantly higher in patients than in control subjects (3.56+/-1.22 versus 0.64+/-0.13 ng/10(6) platelets, P:<0.025). In contrast, there were no differences in thrombin-stimulated (1 U/mL) 12(S)-HETE generation: 7.66+/-2.14 in patients versus 4.87+/-1.46 in control subjects (P:=0.61). Platelet 12-LO protein levels, measured by Western blotting with a polyclonal antibody, were higher in the patients than in the control subjects. The urinary excretion of 12(S)-HETE was higher in patients than in control subjects: 36.8+/-7.24 versus 17.1+/-3.14 ng/mg creatinine (P:<0.01). These results indicate that 12(S)-HETE levels and 12-LO protein are increased in patients with essential hypertension, suggesting a role for this metabolite in human hypertension.     

Effect of atrial natriuretic peptide on renin release in a superfusion system of kidney slices and dispersed juxtaglomerular cells

The effect of atrial natriuretic peptide (ANP) on renin release is controversial. Several reports state that ANP inhibits renin secretion, while others have shown no effect. We investigated the effect of synthetic rat ANP with 24 amino acids (atriopeptin III) on renin release in vitro in a dynamic superfusion system of renal cortical slices as well as collagenase-dispersed juxtaglomerular cells. In the superfusion system of kidney slices, isoproterenol (5 x 10(-8) M) clearly stimulated renin release from kidney slices, while angiotensin II (AII; 10(-5) M) suppressed renin release. ANP (10(-10)-10(-6) M) did not inhibit basal renin release or blunt the stimulatory effect of isoproterenol. The suppression of renin secretion by AII was never modified in the presence of ANP. The superfusion system of juxtaglomerular cells demonstrated greater sensitivity of renin release in responses to isoproterenol and AII. In this system, ANP (10(-6) M) did not alter renin release from the cells stimulated by isoproterenol (5 x 10(-8) M) or inhibited by AII (10(-8) M). However, basal renin release was slightly stimulated in the late phase of ANP superfusion and for 20 min after the ANP perfusion was stopped. Similarly, 8 bromo-cGMP (10(-6) M) did not inhibit, but, rather, stimulated basal renin release slightly. These results suggest that ANP does not inhibit renin release by a direct effect on the juxtaglomerular cell in the rat.     

The contrasting influence of two lipoxygenase inhibitors on hypoxic pulmonary vasoconstriction in anesthetized pigs

Because leukotrienes may mediate hypoxic pulmonary vasoconstriction (HPV), we examined the influence of two lipoxygenase inhibitors on HPV in anesthetized pigs. HPV was induced by ventilation with a hypoxic gas mixture (FIO2 at 0.095), resulting in a fall in PaO2 to 23 +/- 2 mm Hg and a rise in pulmonary vascular resistance from 285 +/- 15 to 595 +/- 30 dyne/s/cm-5. After infusion of either U-60,257B (50 mg/kg, n = 13) or BW 755c (20 mg/kg, n = 8), the responses to repeated hypoxic challenges were recorded. After U-60,257B infusion the hypoxic pressor response was eliminated at 10 and 30 min and remained significantly (p less than 0.01) attenuated at 50 min. The pulmonary pressor response to angiotensin II infusion (0.2 micrograms/kg/min) was also ablated, whereas the systemic response was unchanged. In contrast, after BW 755c infusion there was a modest but sustained augmentation of HPV, maximum at 30 min (pulmonary vascular resistance, 158 +/- 23% control, p less than 0.01), and no alteration of the responses to angiotensin II. BW 755c inhibited the A23187-induced release of leukotrienes, but not histamine, from isolated porcine lung cells (IC50, 6.3 x 10(-5) M), whereas U-60,257B inhibited the release of both leukotriene (IC50, 1.1 x 10(-4) M) and histamine. These findings indicate that reduction of HPV by lipoxygenase inhibitors is not necessarily a consequence of inhibition of leukotriene synthesis.     

Role of atrial natriuretic factor in renin release

Atrial natriuretic factor (ANF) was studied for its effect on renin release by rat renal cortical slices. ANF (rat) alone (10(-9)-10(-6) M) had no effect on basal renin release, but significantly (P less than 0.001) potentiated angiotensin II (AII) inhibition of renin secretion in doses as low as 10(-9) M. ANF also potentiated the inhibitory effect of AII on 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (which alters intracellular calcium) and calmidazolium (a calmodulin blocker) effects on renin release. ANF did not inhibit the action of isoproterenol, which probably acts through cAMP, at doses below micromolar concentrations. Large, probably pharmacological amounts (greater than 10(-6) M) produce some inhibition. Since AII action on renin is associated with increases in intracellular calcium, our studies suggest that ANF acts by altering the intracellular calcium-calmodulin-mediated steps of AII action on this tissue and not via cAMP.     

Thromboxane mediation of the pressor response to infused angiotensin II

The role of thromboxane A2(Tx) in mediating the pressor response to angiotensin II (AII) was studied in anesthetized rats. Intravenous AII (500 ng/kg/min) increased mean arterial pressure (MAP) by 35 +/- 3 mm Hg and increased the excretion of prostaglandin PGE2, the metabolites of prostacyclin (6kPGF1 alpha) and Tx (TxB2) (P less than .05). A similar pressor infusion of the alpha 1-adrenoreceptor agonist phenylephrine (PE) increased the excretion of PGE2 and 6kPGF1 alpha but not TxB2. The increases in MAP and prostaglandin excretion produced by AII were reversed by the AII-receptor antagonist saralasin (10 micrograms/kg/min) while those produced by PE were reversed by the alpha-adrenoreceptor antagonist phenoxybenzamine (250 micrograms/kg). The Tx receptor antagonist, SQ-29,548 (8 mg/kg) attenuated (P less than .0001) the AII-induced rise in MAP (13 +/- 1 mm Hg) but did not modify the pressor response to PE. The Tx synthetase inhibitor, UK-38,485 (50 mg/kg/d) given for 3 days, reduced basal TxB2 excretion by 75% and also attenuated (P less than .001) the AII-induced rise in MAP (11 +/- 2 mm Hg). However, when given 40 min before the AII infusion, UK-38,485 did not attenuate the pressor response. In separate groups of rats, the log dose-response curve for bolus intravenous injection of AII was shifted to the right by SQ-29,548 while that for PE was unaffected. In conclusion: 1) AII releases Tx; 2) Tx release is not secondary to hypertension; and 3) Tx can mediate up to two-thirds of the short-term pressor response to high-dose AII infusion.     

The role of calcium in the control of renin release

The effects of removing external calcium and inhibiting entry of calcium into the cell by treatment with D-600 on renin release from renal cortical slices of male Sprague-Dawley rats were examined. Baseline renin release, angiotensin II (AII)-induced inhibition, and isoproterenol-induced stimulation of renin release were studied. Removal of external calcium by chelation with 5 mM EGTA inhibited basal renin release while treatment with 1 mM EGTA stimulated basal renin release slightly. Incubation of slices with zero calcium medium containing 1 mM EGTA had no effect on isoproterenol-induced stimulation of renin release. In contrast, similar treatment reduced the inhibitory effect of AII from 58.7% of baseline to 85.3% (p less than 0.001). Similarly, blockage of calcium entry into cells with D-600 had no effect on isoproterenol-induced stimulation of renin release but abolished AII-induced inhibition. Replacement of sodium in the bathing medium with choline had no effect on baseline renin release or on AII-induced inhibition of renin release, ruling out the possibility that D-600 altered renin release via an effect on sodium influx. Taken together, the data strongly suggest that AII-induced inhibition of renin release is partially dependent on the presence of external calcium but that isoproterenol-induced stimulation of renin release is not.     

Effects of atrial natriuretic factor on angiotensin-II-and phorbol ester-stimulated protein kinase-C and prostacyclin production in cultured rat aortic smooth muscle cells

The vasoconstrictor hormone angiotensin-II (AII) raises cytosolic free calcium and stimulates protein kinase-C (PKC) activity in vascular smooth muscle cells (VSMC). Phorbol-12-myristate-13-acetate (PMA) directly activates PKC in these cells. Both of these agonists stimulate prostacyclin production. Several studies have shown that atrial natriuretic factor (ANF) does not interfere with the AII-induced early calcium response. We, therefore, examined the effects of ANF on PKC activity and prostacyclin production in cultured rat aortic VSMC. PKC activity was determined in the membranous and cytosolic fractions after anion exchange chromatography. ANF (10(-7) M) inhibited by 44 +/- 3% and 39 +/- 8% the increase in membranous PKC activity induced by AII and PMA, respectively. ANF (10(-7) M) inhibited PMA-stimulated prostacyclin production, whereas AII-induced prostacyclin production remained unaffected. Thus, our results suggest that some biological effects induced by ANF in VSMC are mediated by an inhibition of membranous PKC activity.     

Lipoxygenase-derived metabolites are regulators of peroxisome proliferator-activated receptor gamma-2 expression in human vascular smooth muscle cells

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor family that has been implicated in cell differentiation and proliferation, glucose metabolism, macrophage development, and inflammatory responses. PPAR-gamma can be activated by a range of synthetic substances and also by products of lipid oxidation such as oxidized low-density lipoprotein, 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Since 12- and 15-lipoxygenase (12- and 15-LO) are expressed in human vascular smooth muscle cells (VSMCs), we set out to investigate the possible relation between PPAR-gamma and LO system in these cells. METHODS: In vitro experiments in human VSMC using standard methods. RESULTS: The LO products, 12-HETE (10(-7) mol/l), 15-HETE (10(-7) mol/l) and 13-HODE (10(-7) mol//l) increased the expression of PPAR-gamma-2 messenger RNA (mRNA) in VSMC (by 100, 50, and 100%, respectively. Rosiglitazone (1-10 micromol/l) was found to upregulate both the mRNA expression of two LO enzymes, platelet-type 12-lipoxygenase (12-LO; +70%) and 15-lipoxygenase type 2 (15-LO; +60%), and the secretion of their eicosanoid products 12- and 15-HETE. In addition, rosiglitazone-induced a threefold increase in PPAR-gamma-2 mRNA expressions and modest 50% rise in PPAR-gamma-1 mRNA expression. The effect of rosiglitazone on PPAR-gamma-2 could be entirely blocked by the LO inhibitor baicalein and restored by the addition of exogenous 12-HETE. CONCLUSIONS: These results suggest a novel amplification cycle in which PPAR-gamma activation induces production of 12- and 15-LO-derived metabolites which in turn feed back to upregulate PPAR-gamma-2's own expression. The implications of this link in VSMC pathophysiology remain to be elucidated.     

Beta-endorphin and adrenocorticotropin release from rat adenohypophysis in vitro: evidence for local modulation by arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathway

This study was performed to examine an involvement of adenohypophysial arachidonic acid metabolites in the local mechanisms controlling the release of peptide hormones from the corticotrope cells of the anterior pituitary gland. Therefore, we investigated the effect of blockers of the lipoxygenase (nordihydroguaiaretic acid, NDGA), cyclooxygenase (indomethacin) or both of these enzyme systems (BW755C; eicosatetraynoic acid, ETYA) on the release of beta-endorphin-like (beta-E-IR) and adrenocorticotropin-like immunoreactivity (ACTH-IR) from rat anterior pituitary quarters incubated in vitro. NDGA and ETYA did not influence the basal release of beta-E- and ACTH-IR. However, upon stimulation by arginine-vasopressin (AVP) or synthetic ovine corticotropin-releasing factor (CRF(1-41], NDGA inhibited beta-E-IR release by 40%. ETYA inhibited AVP-induced release of beta-E- and ACTH-IR by 75%. Indomethacin and BW755C (lower concentration) enhanced beta-E-IR release, induced by AVP, by about 100%, whereas BW755C (higher concentration) had no effect. When indomethacin was present, NDGA, ETYA and BW755C (higher concentration) inhibited AVP-induced release of beta-E- and ACTH-IR. Prostaglandin E2 (PGE2) inhibited beta-E-IR release in response to AVP but failed to do so in the presence of NDGA. 12-OH-5,8,10,14-eicosatetraenoic acid (12-HETE) had no effect. When anterior pituitary quarters were incubated with 3H-arachidonic acid (3H-AA), NDGA and BW755C (higher concentration) but not indomethacin and BW755C (lower concentration) blocked the formation of a metabolite which co-migrated with 12-HETE on thin-layer chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.