The existence of a cardiac renin-angiotensin system, independent of the circulating renin-angiotensin system, is still controversial. We compared the tissue levels of reninangiotensin system components in the heart with the levels in blood plasma in healthy pigs and 30 hours after nephrectomy. Angiotensin I (Ang I)-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. We took precautions to prevent the ex vivo generation and breakdown of cardiac angiotensins and made appropriate corrections for any losses of intact Ang I and II during extraction and assay. Tissue levels of renin (n=ll) and Ang I (n=7) and II (n=7) in the left and right atria were higher than in the corresponding ventricles (P< .05). Cardiac renin and Ang I levels (expressed per gram wet weight) were similar to the plasma levels, and Ang II in cardiac tissue was higher than in plasma (P<.05). The presence of these renin-angiotensin system components in cardiac tissue therefore cannot be accounted for by trapped plasma or simple diffusion from plasma into the interstitial fluid. Angiotensinogen levels (n=ll) in cardiac tissue were 10% to 25% of the A ngiotensin I (Ang I) is produced in the circulating / \ blood by the action of renin from the kidney on -Z A . angiotensinogen produced by the liver. Ang I is converted to Ang II, a potent vasoconstrictor, by angiotensin-converting enzyme (ACE) located on the luminal surface of the vascular endothelium. It is now well established that Ang I and II are not only produced in the blood compartment but also locally in tissues. Recent evidence suggests that complete local reninangiotensin systems (RAS) are present in a number of organs, for instance, kidney, adrenal gland, and ovary. 13In such local RAS, the production of Ang I and II is thought to depend on in situ synthesized renin rather than plasma-derived renin.A local cardiac RAS has also been postulated. 4 ' 5 However, direct evidence for Ang I and II production in the heart by in situ synthesized renin is still lacking. Renin mRNA levels in the heart are usually low and can be detected only by polymerase chain reaction. 68 Early studies showed Ang I-generating activity in left ventric-

The bulk of Ang II in the kidney is cell-associated. The high tissue/blood concentration ratio of endogenous Ang II may depend on the same mechanism as demonstrated for 125I-Ang II, that is, AT1 receptor-mediated binding to cells and endocytosis. If so, the results indicate that most renal AT1 receptors are exposed to locally generated Ang II rather than Ang II from the circulation. We propose the existence of a low-Ang II vascular system-related interstitial compartment that is separate from tubular fluid, where, according to micropuncture studies, Ang II levels might be high.

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Background-Angiotensin (Ang) II type 2 (AT 2 ) receptor stimulation results in coronary vasodilation in the rat heart. In contrast, AT 2 receptor-mediated vasodilation could not be observed in large human coronary arteries. We studied Ang II-induced vasodilation of human coronary microarteries (HCMAs). Methods and Results-HCMAs (diameter, 160 to 500 m) were obtained from 49 heart valve donors (age, 3 to 65 years).Ang II constricted HCMAs, mounted in Mulvany myographs, in a concentration-dependent manner (pEC 50 , 8.6Ϯ0.2; maximal effect [E max ], 79Ϯ13% of the contraction to 100 mmol/L K ϩ ). The Ang II type 1 receptor antagonist irbesartan prevented this vasoconstriction, whereas the AT 2 receptor antagonist PD123319 increased E max to 97Ϯ14% (PϽ0.05). The increase in E max was larger in older donors (correlation ⌬E max versus age, rϭ0.47, PϽ0.05). The PD123319-induced potentiation was not observed in the presence of the NO synthase inhibitor L-NAME, the bradykinin type 2 (B 2 ) receptor antagonist Hoe140, or after removal of the endothelium. Ang II relaxed U46619-preconstricted HCMAs in the presence of irbesartan by maximally 49Ϯ16%, and PD123319 prevented this relaxation. Finally, radioligand binding studies and reverse transcription-polymerase chain reaction confirmed the expression of AT 2 receptors in HCMAs. Conclusions-AT 2 receptor-mediated vasodilation in the human heart appears to be limited to coronary microarteries and is mediated by B 2 receptors and NO. Most likely, AT 2 receptors are located on endothelial cells, and their contribution increases with age.

Angiotensin II (Ang II) is internalized by various cell types via receptor-mediated endocytosis. Little is known about the kinetics of this process in the whole animal and about the half-life of intact Ang II after its internalization. We measured the levels of 125I-Ang II and 125I-Ang I that were reached in various tissues and blood plasma during infusions of these peptides into the left cardiac ventricle of pigs. Steady-state concentrations of 125I-Ang II in skeletal muscle, heart, kidney, and adrenal were 8% to 41%, 64% to 150%, 340% to 550%, and 680% to 2100%, respectively, of the 125I-Ang II concentration in arterial blood plasma (ranges of six experiments). The tissue concentrations of 125I-Ang I were less than 5% of the arterial plasma concentrations. 125I-Ang II accumulation seen in heart, kidney, and adrenal was almost completely blocked by a specific Ang II type 1 (AT1) receptor antagonist. Steady-state concentrations of 125I-Ang II were reached within 30 to 60 minutes in the tissues and within 5 minutes in blood plasma. The in vivo half-life of intact 125I-Ang II in heart, kidney, and adrenal was approximately 15 minutes, compared with 0.5 minute in the circulation. Thus, Ang II, but not Ang I, from the circulation is accumulated by some tissues, and this is mediated by AT1 receptors. The time course of this process and the long half-life of the accumulated Ang II support the contention that this Ang II has been internalized after its binding to the AT1 receptor, so that it is protected from rapid degradation by endothelial peptidases. The results of this study are in agreement with growing evidence of an important physiological role for internalized Ang II.

We used a modification of the isolated perfused rat heart, in which coronary effluent and interstitial transudate were separately collected, to investigate the uptake and clearance of exogenous renin, angiotensinogen, and angiotensin I (Ang I) as well as the cardiac production of Ang I. The levels of these compounds in interstitial transudate were considered to be representative of the levels in the cardiac interstitial fluid. During perfusion with renin or angiotensinogen, the steady-state levels (mean +/- SD) in interstitial transudate were 64 +/- 34% (P < .05 for difference from the arterial level, n = 8) and 108 +/- 42% (n = 6) of the arterial level, respectively; the levels in coronary effluent were not significantly different from those in interstitial transudate. Ang I was not detectable in interstitial transudate during perfusion with Tyrode's buffer or angiotensinogen. It was very low in interstitial transudate during perfusion with renin and rose to much higher levels during combined renin and angiotensinogen perfusion. The total production rate of Ang I present in interstitial fluid could be largely explained by the renin-angiotensinogen reaction in the fluid phase of the interstitial compartment. In contrast, the total production rate of Ang I present in coronary effluent and the net ejection rate of Ang I via coronary effluent were, respectively, 4.6 +/- 2.2 and 2.8 +/- 1.3 (P < .01 and P < .05 for difference from 1.0, n = 6) times higher than could be explained by Ang I formation in the fluid phase of the intravascular compartment. Ang I from the interstitial fluid contributed little to the Ang I in the intravascular fluid and vice versa. These data reveal two tissue sites of Ang I production, ie, the interstitial fluid and a site closer to the blood compartment, possibly vascular surface-bound renin. There was no evidence that the release of locally produced Ang I into coronary effluent and interstitial transudate occurred independently of blood-derived renin or angiotensinogen.

Both circulating and locally generated Ang II contribute to remodeling after MI. The rise in tissue Ang II production during angiotensin-converting enzyme inhibition and AT(1) receptor blockade suggests that the antihypertrophic effects of these drugs result not only from diminished AT(1) receptor stimulation but also from increased stimulation of growth-inhibitory Ang II type 2 receptors.

Angiotensin II is a peptide hormone regulator of blood pressure and fluid balance in mammals. Evidence obtained largely in vitro has also suggested that angiotensin II has growth-promoting effects and that it might thereby contribute to such pathological phenomena as cardiac hypertrophy, a major risk factor for cardiovascular mortality. It has been difficult to test for the direct growth-promoting effects of angiotensin II in vivo, however, because of the generalized effects of the peptide on hemodynamics. To overcome this limitation and to test for cardiac-specific functions of angiotensin II, we generated transgenic mice expressing an angiotensin IIproducing fusion protein exclusively in cardiac myocytes. Our findings are the first to distinguish between local and systemic effects of angiotensin II on the heart and introduce a novel technique for studying tissuespecific peptide function.