Debra I. Diz

Subtype 2 angiotensin receptors mediate prostaglandin synthesis in human astrocytes.

We have identified two distinct cellular responses that occur in human astrocytes in the presence of angiotensin (Ang) peptides and are linked to specific receptor subtypes. Ang II and the N-terminal heptapeptide Ang-(1–7) stimulated release of prostaglandin (PG) E2 and PGI2 (measured as the stable metabolite 6-keto-PGFlα). In contrast, only Ang II but not Ang-(1–7) activated phosphoinositide-specific phospholipase C, leading to mobilization of intracellular calcium. The Ang H-induced PGE2 and PGI2 syntheses were attenuated by [Sar1,Ile8] Ang II but not by [Sar1,Thr8]Ang II. Ang-(l–7)-induced PGE2 and PGI2 syntheses were not inhibited by either of these two classical antagonists. DuP 753, a subtype 1-selective Ang receptor antagonist, blocked the Ang II-induced release of PGE2 but not PGI2. In contrast, CGP 42112A, the subtype 2-selective antagonist, totally blocked the Ang II-induced PGI2 release and partially attenuated the PGE2 release. Ang-(l–7)-induced PGE2 and PGI2 release was not altered by DuP 753; however, CGP 42112A totally blocked the effects of Ang-(l–7) on PG stimulation. Calcium mobilization in response to Ang II was blocked by [Sar1,Thr8]Ang II, [Sar1,Ile8]Ang II, and DuP 753 but not by CGP 42112A. These data suggest that human astrocytes contain both Ang receptor subtypes. The subtype 1 Ang receptor participates both in the release of PGs and in the mobilization of calcium, whereas the subtype 2 receptor is coupled to the release of PGs only. In addition, PG release coupled to subtype 2 Ang II receptors occurs through a calcium-independent mechanism and responds uniquely to Ang-(1–7).

Contribution of the vagus nerve to angiotensin II binding sites in the canine medulla

Specific angiotensin II (Ang II) binding sites are present in the dorsal medulla of several species, and dose-related cardiovascular effects are produced by microinjection of the peptide into this region. Because the anatomical location of Ang II binding sites in the area postrema (ap), nucleus tractus solitarii (nTS), and dorsal motor nucleus of the vagus (dmnX) coincides with the topography of vagal afferent fibers and efferent motor neurons, the effect of either nodose ganglionectomy or cervical vagotomy on Ang II binding sites in the dorsomedial medulla was investigated in dogs by in vitro receptor autoradiography. Two weeks after unilateral ganglionectomy, there was a marked reduction in the density of specific Ang II binding sites in the ipsilateral ap, nTS, and dmnX, and an absence of binding sites in the region where vagal afferent fibers course through the rostral medulla. Unilateral cervical vagotomy, which has been shown to spare central processes of afferent fibers, resulted in a loss of binding only in the ipsilateral dmnX. We also show that Ang II binding sites are present in the nodose ganglion and central and peripheral processes of the vagus nerve. The data indicate that medullary Ang II binding sites are associated with both vagal afferent fibers and efferent motor neurons.

Angiotensin II Receptor Subtypes in the Human Renal Cortex and Renal Cell Carcinoma

Selective antagonists were used to determine the presence of angiotensin II (Ang II) receptor subtypes (AT1 and AT2) in normal human renal cortex and renal cell carcinoma. Normal and tumor tissues were obtained from fresh radical nephrectomy specimens in 7 patients. All patients had a patent renal artery, and the mean preoperative serum creatinine level was 1.1 mg./dl. Tissues were snap frozen and sectioned (14 microns.) for in vitro autoradiography, then incubated in 125I-Ang II (0.3 nM.), with or without unlabeled Ang II or subtype selective antagonists (1 nM. to 1 microM.), rinsed, air dried and apposed to SB-5 X-ray film for 3 to 21 days. In normal renal tissue, low densities of diffuse 125I-Ang II binding sites were observed in cortical areas containing tubules. Higher densities of binding sites occurred over glomeruli and large cortical vessels. Specific binding ranged between 60 and 90% depending on the area as determined by displacement with excess unlabeled Ang II. Specific binding in large cortical vessels was displaced by the two AT2 selective antagonists PD123177 (1 microM.) or CGP 42112A (0.01 microM.), whereas these antagonists were less effective competitors for 125I-Ang II binding in glomeruli. In contrast, the AT1 selective antagonists, DuP 753 and L-158,809 (0.1 and .01 microM., respectively), were potent competitors for glomerular, but not extraglomerular, cortical vessel binding. In the normal cortical tubulointerstitium, both AT1 and AT2 antagonists caused partial displacement of specific binding (55 +/- 12% AT1, 39 +/- 12% AT2). Low density 125I-Ang II binding was present in all tumors. Specific binding averaged 59 +/- 10% as defined by displacement with unlabeled Ang II (1 microM.). As in the normal tubulointerstitial area, each of the selective antagonists produced partial displacement of the specific binding (60 +/- 12% AT1, 31 +/- 8% AT2). In conclusion, AT1 receptors predominate in glomeruli, while AT2 binding sites predominate in large preglomerular vessels of the human renal cortex. In the normal tubulointerstitium and renal cell carcinoma, a 60%/40% mixture of AT1 to AT2 receptors exists. These findings provide a pharmacologic framework for the differential effects of Ang II receptor-mediated function in the human kidney.

Pharmacological characterization of angiotensin II binding sites in the canine pancreas.

High affinity 125I-angiotensin II (Ang II) binding sites were characterized in the canine pancreas. Total binding increased with protein concentration and equilibrium was reached within 60-90 min at 22 degrees C. Specific binding was saturable and averaged 70% of total. Scatchard analysis of binding yielded a KD of 0.48 +/- 0.18 nM with a Bmax of 32.8 +/- 6.5 fmol/mg protein (mean +/- SEM, n = 6). The addition of the reducing agent dithiothreitol increased specific binding two-fold. The rank order of displacement of 125I-Ang II binding by native angiotensin peptides was Ang II greater than or equal to Ang III greater than AngI greater than Ang(1-7) much greater than Ang(1-6). The use of the specific Ang II antagonists CGP 42112A, PD 123177, and DuP 753 revealed that the pancreas expresses two receptor subtypes. The majority of Ang II binding sites in the pancreas could be classified as type 2 (AT2), although type 1 (AT1) sites were also detected. In vitro autoradiography revealed binding sites localized over islet cells, acinar and duct cells, as well as the pancreatic vasculature. In addition, the autoradiographic studies confirmed the predominance of the AT2 receptor subtype throughout the pancreas.

Alterations in prostaglandin production in spontaneously hypertensive rat smooth muscle cells.

We have characterized angiotensin binding sites in cultured smooth muscle cells obtained from the aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. In both strains of rats the binding of 125I-angiotensin II (125I-Ang II) in smooth muscle cells was time dependent and reached a maximum at 60 minutes. Scatchard analysis revealed a single binding site in both strains with equilibrium constants (KD) of 5.35 nmol/L in SHR and 3.47 nmol/L in WKY rats. Binding capacities (Bmax) in smooth muscle cells averaged 270 and 150 fmol/mg protein in SHR and WKY rats, respectively. Angiotensin peptides competed for 125I-Ang II binding with an order of potency of Ang II > angiotensin-(1-7) = angiotensin I. In smooth muscle cells of the SHR, basal prostaglandin E2 (PGE2) and prostacyclin (prostaglandin I2 [PGI2]) release were threefold and 15-fold lower than that found in WKY rat smooth muscle cells. Ang II as well as angiotensin-(1-7) stimulated PGE2 and PGI2 release in WKY rat smooth muscle cells. In smooth muscle cells from SHR, Ang II increased the production of both PGE2 and PGI2, whereas angiotensin-(1-7) enhanced only PGE2 but not PGI2 release. There was no significant difference between Ang II-stimulated PGE2 and PGI2 release or angiotensin-(1-7)-stimulated PGE2 production in SHR and WKY rat smooth muscle cells. However, angiotensin-(1-7)-stimulated PGI2 release was significantly lower (p < 0.0005) in SHR compared with WKY smooth muscle cells. Collectively, the data suggest that smooth muscle cells of SHR contain a higher number of angiotensin binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and regulation of angiotensin II receptor subtypes on NG108-15 cells

NG108-15 cells, a neurally derived clonal cell line, express various components of the renin-angiotensin system and thus serve as a model of the cellular action of angiotensin (Ang) II. NG108–15 cells contain a high-affinity binding site for Ang II, with a Kd of 1.1 nM and a Bmax of 6.5 fmol/mg protein. Ang peptides competed for 125I-Ang II binding with an order of potency of Ang II>Ang-(2–8)≫Ang-(l–7). The subtype 1 (or B)-selective Ang II receptor antagonist DuP 753 as well as [Sar1,Ile8]Ang II and [Sar1,Thr8]Ang II competed for Ang II binding with high affinity, whereas the subtype 2 (or A)-selective Ang receptor antagonist CGP 42112A was partially effective only at a 300-fold higher concentration. When NG108-15 cells were induced to differentiate by treatment with dibutyryl cyclic adenosine 3′,5′-monophosphate, the density of Ang II receptors increased dramatically, with little change in affinity (1.1 versus 4.2 nM) or competition by Ang peptides. In marked contrast to undifferentiated cells, CGP 42112A became a potent competitor (IC50, 1 nM) for the majority (90–95%) of Ang II binding, whereas DuP 753 competed for only 5–10% of the binding sites. Ang II caused a dose-dependent mobilization of cytosolic Ca2+; in undifferentiated NG108-15 cells through activation of phospholipase C and the production of Inositol 1,4,5-trisphosphate. In these cells, Ca2+ mobilization was blocked by either DuP 753 or the sarcosine Ang II analogues, whereas CGP 42112A was ineffective. Ang II also mobilized intracellular Ca2+ in differentiated NG108-15 cells. This effect was blocked by either DuP 753 or the sarcosine analogues but not by CGP 42112A. These data show that both undifferentiated and differentiated NG108-15 cells contain a subtype 1 Ang II receptor that activates phospholipase C and mobilizes intracellular Ca2+. However, in differentiated NG108-15 cells, the majority of Ang II receptors are subtype 2, suggesting that neuronal differentiation regulates the expression of subtype 2 receptors.

Human astrocytes contain two distinct angiotensin receptor subtypes.

The ability of angiotensin peptides to stimulate prostaglandin release and raise intracellular calcium levels by activating a phosphoinositide-specific phospholipase C was assessed in three human astrocytoma cell lines (CRTG3, STTG1, and VVITG2). The addition of angiotensin II to CRTG3 cells resulted in a dose-dependent release of prostaglandin E, and prostacyclin, the production of inositol 1,4,5-trisphosphate, and the mobilization of intracellular calcium. Angiotensin-(l-7), previously considered to be an inactive metabolite of angiotensin II, was as potent as angiotensin II for prostaglandin release but did not activate phospholipase C or mobilize intracellular calcium. In contrast, angiotensin-(2-8) caused only a slight increase in prostaglandin release, even though it was as effective as angiotensin II in augmenting inositol 1,4,5-trisphosphate production and calcium mobilization. Moreover, neither the release of prostaglandins in response to angiotensin II or angiotensin-(l-7) nor the mobilization of intracellular calcium in response to angiotensin II required extracellular calcium. Angiotensin II and angiotensin-(l-7) caused the release of prostaglandins from all three human astrocytoma cell lines, but changes in the level of intracellular calcium in response to angiotensin II only occurred in CRTG3 cells. Although previous studies have provided evidence for angiotensin receptor subtypes on the basis of selectivity of antagonists or signal transduction mechanisms, these data suggest that human astrocytes contain multiple angiotensin receptor subtypes on the basis of their response to different angiotensin heptapeptides ‐ angiotensin- (1-7) and angiotensin-(2-8). Furthermore, the data also suggest that preferential production of angiotensin-(1-7) and angiotensin-(2-8) may be one level of regulation whereby a particular signal transduction pathway [i.e., calcium mobilization by angiotensin-(2-8) or prostaglandin release by angiotensin-(l-7)] is selectively activated. {Hypertension 1991;18:32-39

Pressor responses of angiotensin II microinjected into the dorsomedial medulla of the dog.

Angiotensin II (Ang II) was injected into regions of the dorsomedial medulla of dogs where both specific Ang II binding and neural elements containing this peptide are found. Picomole amounts of the peptide were delivered simultaneously from a linear array of 3 micropipettes with tips positioned concurrently in either the area postrema (ap), nucleus tractus solitarii (nTS), dorsal motor nucleus of the vagus (dmnX), or hypoglossal nucleus (nXII). Significant increases in blood pressure occurred with Ang II injections into the medial nTS (+12 +/- 2 mm Hg), the ap(+9 +/- 3 mm Hg), and the nXII (+6 +/- 2 mm Hg). In both the medial nTS and the nXII, the pressor responses were accompanied by significant increases in heart rate (+13 +/- 3 beats/min and +8 +/- 3 beats/min, respectively). Ang II injected into the dmnX did not produce consistent effects on blood pressure or heart rate. These data demonstrate that unilateral injections of picomole amounts of Ang II produce changes in blood pressure and heart rate which involve neural elements in the ap and medial nTS.

Bradykinin and related peptides in central control of the cardiovascular system

The evidence for a brain kallikrein-kinin system and for the possible role for kinins in brain control of the cardiovascular system are reviewed. All components of the kallikrein-kinin system are present in brain and kinins have a variety of cardiovascular actions of central origin following peripheral, intracerebroventricular or brain parenchymal administration. How components of the brain kallikrein-kinin system are regulated or even whether they function as a system remains to be established. However, bradykinin does fulfill several of the criteria necessary for establishing a substance as a neurotransmitter and these are discussed.

Light microscopic localization of angiotensin II binding sites in canine medulla using high resolution autoradiography

Angiotensin II (Ang II) produces dose-related, site-specific cardiovascular effects in the canine and rat dorsal medulla. Our previous studies suggested that Ang II binding sites are associated with presynaptic vagal afferent fibers in the nucleus tractus solitarii (nTS) and vagal efferent neurons in the dorsal motor nucleus of the vagus (dmnX). High resolution autoradiography now establishes the relationship of putative Ang II receptors to the cytoarchitecture of these nuclei. Sections of the canine medulla oblongata were processed for film or emulsion autoradiography with 0.3-1 nM 125I-Ang II. Quantitative densitometry of films before and after processing sections for emulsion coating confirmed no selective alteration in labeling. In emulsion coated sections, dense labeling was seen over the majority of the large perikarya and surrounding neuropil in the ventral dmnX. Bands of label overlaid vagal efferent fibers coursing ventrolaterally to exit the medulla. In the nTS, Ang II binding was restricted to regions with heavy vagal afferent innervation. In the dorsal nTS, label was distributed over both cell bodies and neuropil, with highest density capping the solitary tract. In the medial nTS, label was concentrated over perikarya, with scattered grains over the intervening neuropil. The discrete subnuclear association of Ang II binding sites in the dorsal medulla with vagal cells and fibers documents that Ang II receptors are present on both afferent vagal fibers and intrinsic medullary neurons, and reveals an anatomical substrate for the autonomic effects of Ang II in this region.