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Vascular smooth-muscle cells contain AT1 angiotensin receptors coupled to phospholipase D activation.

ABSTRACT: We previously showed that angiotensin II (Ang II) and angiotensin-(2-8)-peptide [Ang-(2-8)] activate a phosphoinositide-specific phospholipase C (PLC) and cause calcium mobilization in rat aortic vascular smooth-muscle cells (VSMC), while Ang II and Ang-(1-7) produce prostaglandins. To define further the signal-transduction mechanisms activated by angiotensin peptides in smooth-muscle cells, we measured diacylglycerol (DAG) accumulation in response to different angiotensin peptides and its inhibition by subtype-selective receptor antagonists. Both an initial (10 s) and secondary (10 min) phase of DAG production in response to 100 nM Ang II were inhibited by 1 microM losartan (DuP 753), an AT1 antagonist, while 1 microM PD 123177, an AT2 antagonist, was ineffective. In contrast, the heptapeptide Ang-(1-7) did not produce DAG in VSMC. Ang II also caused the hydrolysis of phosphatidylinositol and phosphatidylcholine, the formation of phosphatidic acid and the formation of phosphatidylethanol (PEt) in the presence of ethanol, through activation of a PLD and a PLD-induced transphosphatidylation reaction. A similar concentration of Ang-(2-8) also activated PLD; in contrast, Ang-(1-7) was ineffective. PEt production by 100 nM Ang II was significantly attenuated by the AT1 antagonists losartan, its metabolite EXP 3174 or L-158,809 (all at 1 microM), whereas a similar concentration of the AT2 antagonists CGP 42112A or PD 123177 was ineffective. The production of PEt by Ang II was also partially attenuated by the removal of extracellular calcium and potentiated by increasing calcium concentrations, indicating that PLD activity is partially dependent on extracellular calcium. Thus VSMC PLD is coupled to an AT1 receptor and occurs in response to Ang II or Ang-(2-8), but not Ang-(1-7). Since AT1 receptors in VSMC are also coupled to activation of PLC, both PLC and PLD may be coupled to the same or a different AT1 receptor. Alternatively, PLD may be sequentially activated in response to Ang II activation of PLC and a subsequent increase in calcium concentration.

SUBMITTER: Freeman EJ

PROVIDER: S-EPMC1137526 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:Peroxisome proliferator activated receptor γ (PPARγ) has been reported to play a protective role in the vasculature; however, the underlying mechanisms involved are not entirely known. We previously showed that vascular smooth muscle-specific overexpression of a dominant negative human PPARγ mutation in mice (S-P467L) leads to enhanced myogenic tone and increased angiotensin-II-dependent vasoconstriction. S-P467L mice also exhibit increased arterial blood pressure. Here we tested the hypotheses that a) mesenteric smooth muscle cells isolated from S-P467L mice exhibit enhanced angiotensin-II AT1 receptor signaling, and b) the increased arterial pressure of S-P467L mice is angiotensin-II AT1 receptor dependent. Phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) was robustly increased in mesenteric artery smooth muscle cell cultures from S-P467L in response to angiotensin-II. The increase in ERK1/2 activation by angiotensin-II was blocked by losartan, a blocker of AT1 receptors. Angiotensin-II-induced ERK1/2 activation was also blocked by Tempol, a scavenger of reactive oxygen species, and correlated with increased Nox4 protein expression. To investigate whether endogenous renin-angiotensin system activity contributes to the elevated arterial pressure in S-P467L, non-transgenic and S-P467L mice were treated with the AT1 receptor blocker, losartan (30 mg/kg per day), for 14-days and arterial pressure was assessed by radiotelemetry. At baseline S-P467L mice showed a significant increase of systolic arterial pressure (142.0 ± 10.2 vs 129.1 ± 3.0 mmHg, p<0.05). Treatment with losartan lowered systolic arterial pressure in S-P467L (132.2 ± 6.9 mmHg) to a level similar to untreated non-transgenic mice. Losartan also lowered arterial pressure in non-transgenic (113.0 ± 3.9 mmHg) mice, such that there was no difference in the losartan-induced depressor response between groups (-13.53 ± 1.39 in S-P467L vs -16.16 ± 3.14 mmHg in non-transgenic). Our results suggest that interference with PPARγ in smooth muscle: a) causes enhanced angiotensin-II AT1 receptor-mediated ERK1/2 activation in resistance vessels, b) and may elevate arterial pressure through both angiotensin-II AT1 receptor-dependent and -independent mechanisms.

Project description:?-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. Although the functional expression of GABA(B) receptors coupled to the G(i) protein was reported for airway smooth muscle, the role of GABA(B) receptors in airway responsiveness remains unclear. We investigated whether G(i)-coupled GABA(B) receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by G(q)-coupled receptors in human airway smooth muscle cells. Both the GABA(B)-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABA(A) receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i) were blocked by CGP35348 and CGP55845 (selective GABA(B) antagonists), pertussis toxin (PTX, which inactivates the G(i) protein), gallein (a G(??) signaling inhibitor), U73122 (an inhibitor of PLC-?), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i), which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P-induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABA(B) receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca(2+) stores by the synthesis of inositol phosphate via the activation of PLC-?, which is stimulated by G(??) protein liberated from G(i) proteins coupled to GABA(B) receptors. Furthermore, crosstalk between GABA(B) receptors and G(q)-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca(2+)](i), and smooth muscle contraction through G(i) proteins.

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Vascular smooth-muscle cells contain AT1 angiotensin receptors coupled to phospholipase D activation.

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