Kenji Mizuno

Local generation and release of angiotensin II in peripheral vascular tissue.

Isolated rat hindlegs were perfused with Krebs-Ringer solution, and immunoreactive angiotensin II (irAng II) released into the perfusate was directly determined using a Sep-Pak C18 cartridge connected to the perfusion system. High performance liquid chromatography clearly demonstrated the presence of angiotensin I (Ang I), angiotensin II (Ang II), and a small amount of angiotensin III. The spontaneous release of irAng II was as high as about 600 pg/30 min, which was stable up to 3 hours. Captopril added to the perfusion medium (10(-9) to 10(-6) M) suppressed irAng II release in a dose-dependent manner (p less than 0.001), and it (10(-6)M) caused a reciprocal increase of irAng I release (p less than 0.05). Oral pretreatment of captopril (50 mg/kg/day) for 1 week suppressed the irAng II release by 31% (p less than 0.02). The same treatment with SA 446, a highly lipophilic angiotensin converting enzyme inhibitor, inhibited the irAng II release by 63% (p less than 0.001). On the other hand, the two inhibitors suppressed the plasma irAng II to very similar extents. Pretreatment with SA 446 plus nephrectomy did not cause any further change in irAng II release as compared with that with SA 446 alone. These results provide direct proof for local generation and subsequent secretion of Ang II by peripheral vascular tissue.

Suppression of angiotensin II release by prostaglandin synthesis inhibitors in hind legs.

Previously we reported that immunoreactive angiotensin II (Ang II) release from isolated perfused rat mesenteric arteries was mediated by β-adrenergic receptor activation. However, the precise mechanism of regulation of vascular renin-angiotensin is not completely understood. In this study, we examined the effect of indomethacin and medofenamate on immunoreactive angiotensin I (Ang I) and Immunoreactive Ang II release from perfused rat hind leg vasculature to delineate the possible relevance of prostaglandins to the vascular renin-angiotensin system in vitro. We also examined the effects of isoproterenol and propranolol on the immunoreactive Ang I and II release. Isolated rat hind legs were perfused with Krebs-Ringer solution, and immunoreactive Ang I and II released into the perfusate were measured directly by using a Sep-Pak CSs18 cartridge connected to the perfusion system. Indomethacin and meclofenamate (10−8 to 2 × 10−6 M) added to the perfusion medium suppressed immunoreactive Ang I and II release to similar extents in a dose-dependent manner (p < 0.001); the maximal percent inhibition of immunoreactive Ang II release evoked by these Inhibitors (2 × 10−6 M) was 60 ± 6% {p < 0.001) for indomethacin and 50 ± 4% (p < 0.001) for meclofenamate. Isoproterenol (10−6 M) failed to cause a change in the release of both pep tides, but propranolol (10−6 M) slightly decreased the release of immunoreactive Ang I and II by 28 ± 4% (p < 0.001) and 32 ± 4% (p < 0.001), respectively. There was a highly significant positive correlation between the released amount of immunoreactive Ang I and that of immunoreactive Ang II altered by indomethacin (r = 0.91), meclofenamate (r = 0.94), or propranolol administration (r = 0.90). These results suggest that the renin-angiotensin in the hind legs is modulated by prostaglandins and that a difference exists in the β-adrenergk receptor-mediated release of Ang II among diverse vascular beds.

The renin-angiotensin system: an overview of its intracellular function.

SummaryThe enzyme reiin has been purified and characterized by structural analysis. Pure renin protein was used to produce a specific antibody to renin, which was useful in demonstrating the presence of a specific renin in many tissues other than kidney. Further, in these cells angiotensins I and II and converting enzyme all were found to coexist with renin by immunohistochemical studies, indicating the local production of renin, angiotensinogen and angiotensins in these cells. Angiotensisn II produced in the cultured cells was secreted to the outside of the cells. Secretion of angiotensin II from the angiotensin-producing cells was demonstrated with perfused mesenteric artery. The secretion of angiotensin II from the vascular beds was inhibited by converting enzyme inhibitors, and was stimulated by the adrenergic β-agonist isoproterenol. These studies demonstrate local production and controlled secretion of angiotensin II and define its physiologic role.

RE-EVALUATION OF THE PLASMA RENIN-ANGIOTENSIN SYSTEM IN ANEPHRIC PATIENTS

In view of recent observations that a number of extrarenal tissues have the potential to produce angiotensin II and release it in a regulated fashion, we made measurements of immunoreactive angiotensin I (irAng I) and angiotensin II (irAng II), along with active and inactive renin, and angiotensinogen in plasma of seven anephric patients and of 16 normal healthy volunteers to gain insight into possible sources of plasma Ang II. High performance liquid chromatography clearly demonstrated that the predominant component of irAng II in anephric plasma is the biologically active octapeptide Ang II. Plasma renin activity (PRA), and active and inactive renin all were detected in all of the anephrics but their levels were decreased to 33% for PRA, 12% for active renin, and 18% for inactive renin when compared with those in healthy subjects. While plasma angiotensinogen was significantly but only slightly increased in anephric patients (+28% over the mean value for normal subjects), irAng I and irAng II both were present in quantities almost comparable with those in normals. These results suggest that local angiotensin production contributes, in part at least, to the circulating plasma Ang II. Vascular tissue seems to be the best candidate responsible for such a mechanism, on the basis of recent demonstrations of unequivocal, regulated release of Ang II from diverse vascular beds.

Direct release of angiotensins I and II from isolated rat kidney perfused with angiotensinogen-free medium

A direct measurement of both angiotensins I and II immunoreactive substances was made in the perfusate from isolated rat kidney perfused with Krebs-Ringer solution which was free of any component of the renin-angiotensin system. The identity of the immunoreactive peptides was confirmed as angiotensin I and angiotensin II by high-pressure liquid chromatography in reference to standard compounds. The rate of release of angiotensins was as high as 1313.5 +/- 184.5 and 772.4 +/- 82.5 pg for angiotensins I and II, respectively, during the first perfusion period of 20 min, and it remained stable at least for 2 hours. There was a good relationship between the angiotensin I secretion rate and renin secretion rate simultaneously determined in the perfusate, and also between the angiotensin I secretion rate and angiotensin II secretion rate. These results taken together with the previous observations of the coexistence of renin and angiotensins I and II in juxtaglomerular cells of the kidney provide evidence for intrarenal formation and release of angiotensin II. It does not agree with the notion that these peptides are internalized from circulation. Angiotensin II secreted from the kidney may play diverse functions in intrarenal regulation.

Conceptual Evolution of Renin Research

Research on renin in the last 90 years has undergone numerous conceptual evolution since its discovery by Tigerstedt and Bergman in 1898. This paper briefly reviews the evolutionary steps leading up to the recently discovered intracellular function of renin and generation of angiotensin II in renin containing cells and molecular biological studies approaching the regulation of renin expression in various tissues.

Exaggerated Response to Electrical Nerve Stimulation of Angiotensin II Release in Isolated Perfused Hind Legs of Spontaneously Hypertensive Rats

Previously we reported that a large amount of immunoreactive angiotensin II (Ang II) was released from isolated perfused rat hind legs at steady rates for several hours. In view of a recent intriguing hypothesis that the vascular renin-angiotensin system plays an important role in the maintenance of high blood pressure in certain forms of experimental hypertensive models, the release of immunoreactive Ang II from isolated hind legs of spontaneously hypertensive rats (SHR) was examined in comparison with normotensive rats of Wistar-Kyoto strain (WKY) by using a Sep-Pak C18 cartridge directly connected to the perfusion system. We also examined effect of electrically-induced nerve stimulation on the release of immunoreactive Ang II in the two strains. High performance liquid chromatography demonstrated the presence of Ang II in the perfusate. The spontaneous release of immunoreactive Ang II was as high as about 300 to 500 pg/30 min, tended to be higher in SHR rats (435.0 +/- 68.2 pg/30 min) than in WKY rats (342.1 +/- 65.1 pg/30 min), and stable up to 3 hours of perfusion for both strains. Periarterial nerve stimulation elicited a significant increment in the release of immunoreactive Ang II in either SHR (p less than 0.02) or WKY rats (p less than 0.05); however, the amount of released immunoreactive Ang II evoked by nerve stimulation was significantly greater in SHR than in WKY rats (781.3 +/- 89.6 vs 498.8 +/- 54.6 pg/30 min, p less than 0.05). These results further provide evidence for local generation and release of Ang II in peripheral vascular tissues, and are consistent with the hypothesis that the vascular renin-angiotensin system is one of important factors responsible for the maintenance of blood pressure.