Counterregulatory Actions of Angiotensin-(1-7)
`
`Page 1 of 16
`
`Hypertension
`Volume 30, Issue 3, September 1997, Pages 535-541
`https://doi.org/10.1161/01.HYP.30.3.535
`
`ARTICLE
`Counterregulatory Actions of Angiotensin-(1-7)
`
`Carlos M. Ferrario, Mark C. Chappell, E. Ann Tallant, K. Bridget Brosnihan, and Debra I. Diz
`
`ABSTRACT: Abstract Angiotensin (Ang)-(1-7) is a bioactive component of the renin-
`angiotensin system that is formed endogenously from either Ang I or Ang II. The first
`actions described for Ang-(1-7) indicated that the peptide mimicked some of the effects of
`Ang II, including the release of prostanoids and vasopressin. However, Ang-(1-7) is devoid
`of vasoconstrictor, central pressor, or thirst-stimulating actions. In fact, new findings reveal
`depressor, vasodilator, and antihypertensive actions that may be more apparent in
`hypertensive animals or humans. Thus, the accumulating evidence suggests that Ang-(1-7)
`may oppose the actions of Ang II either directly or by stimulation of prostaglandins and
`nitric oxide. These observations are significant because they may explain the effective
`antihypertensive action of converting enzyme inhibitors in a variety of non–renin-dependent
`models of experimental and genetic hypertension as well as most forms of human
`hypertension. In this context, studies in humans and animals showed that the
`antihypertensive action of converting enzyme inhibitors correlated with increases in plasma
`levels of Ang-(1-7). In this review, we summarize our knowledge of the mechanisms
`accounting for the counterregulatory actions of Ang-(1-7) and elaborate on the emerging
`concept that Ang-(1-7) functions as an antihypertensive peptide within the cascade of the
`renin-angiotensin system.
`Key Words: angiotensin II ◼ angiotensin receptors ◼ blood pressure ◼ hypertension,
`essential ◼ rats, inbred SHR
`t is well recognized that the renin-angiotensin system has an important role in
`cardiovascular physiology, fluid homeostasis, and cell function. Angiotensin (Ang) II has
`long been considered the main biologically active product of an endocrine system that
`contributes significantly to the pathogenesis of arterial hypertension, renal dysfunction, and
`congestive heart failure. Attesting to the importance of this function is the impressive clinical
`therapeutic benefits achieved by angiotensin-converting enzyme (ACE) inhibitors and a new
`class of Ang II receptor antagonists. However, newer studies have revived the possibility
`1
`that other peptide fragments of Ang I may either contribute to or actually oppose the pressor
`and proliferative actions of Ang II, endowing this hormonal system with greater capability for
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`Counterregulatory Actions of Angiotensin-(1-7)
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`the regulation of tissue perfusion. Earlier studies demonstrated selective actions of the
`heptapeptide Ang III [Ang-(2-8)] in the secretion of aldosterone; more recent studies by
`2
`Harding (Swanson et al ) suggest that a smaller carboxyl product of Ang II, Ang IV
`3
`[Ang-(3-8)], is biologically active by virtue of recognizing a binding site that is not competed
`for by selective AT or AT Ang II receptor antagonists.
`1
`2
`The characterization of Ang-(1-7)
` as the first amino-terminal angiotensin peptide
`4 5 6 7
`product possessing biological actions provided a foundation for the pursuit of a new concept
`regarding the regulation of cardiovascular function by the renin-angiotensin system. While
`prostacyclin, bradykinin, and nitric oxide (NO) act as vasodilator hormones limiting the
`pressor and proliferative actions of Ang II, it had not been considered that products of Ang I
`could also function to counterbalance the actions of Ang II. This review updates the
`progress that has been made in the development of this concept since its introduction in
`1993
` and also outlines the areas where further work will be necessary to attain a
`7 8
`mechanistic understanding of how the opposing activities of Ang II and Ang-(1-7) contribute
`to the long-term regulation of blood pressure.
`
`PRINCIPLES OF ANG-(1-7) FORMATION AND FUNCTION
`Synthesis of Ang-(1-7)
`Ang I is the ultimate precursor of both Ang II and Ang-(1-7). This readily indicates a
`common source for the generation of the two active and functionally opposing peptides.
`While ACE cleaves Ang II from Ang I, processing of Ang I into Ang-(1-7) requires the
`participation of tissue-specific endopeptidases found in the plasma membranes of
`neuroepithelial (prolyl-endopeptidase [EC 3.4.24.26]), epithelial (neprilysin [EC 3.4.24.11]),
`vascular endothelial (prolyl-endopeptidase and neprilysin), and smooth muscle cells
`(metalloendopeptidase [EC 3.4.24.15]).
` The processing pathways in these various
`9 10 11
`tissues have been reviewed recently.
` The diversity of the enzymatic pathways by
`10 11 12 13
`which Ang-(1-7) is cleaved from Ang I suggests that the production of the heptapeptide may
`be regulated at the tissue level, an interpretation which favors the possibility that Ang-(1-7)
`functions as a true paracrine hormone.
`Little is known yet about the factors that determine the rate of conversion of Ang I into
`Ang II and Ang-(1-7). We know that any condition that augments plasma or tissue levels of
`Ang I is associated with increased formation of Ang-(1-7). In several experimental
`conditions, Ang-(1-7) is the primary peptide produced from Ang I.
` These findings
`9 14 15 16
`suggest that production of Ang-(1-7) may limit the amount of substrate that is available for
`the generation of Ang II. This theoretical possibility provides a glimpse into the mechanisms
`that may determine the balance of the opposing actions of Ang II and Ang-(1-7) in the
`control of cardiovascular and body fluid functions (see below). In keeping with this
`interpretation, studies in humans and animals
` showed that increased
`17 18 19 20 21 22
`concentrations of Ang I after inhibition of ACE are associated with increases in the
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`Counterregulatory Actions of Angiotensin-(1-7)
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`concentration of Ang-(1-7). While Ang I is a primary substrate for the formation of Ang-(1-7),
`the heptapeptide may be formed from Ang II by the cleavage of the Pro -Phe bond by
`7
`8
`prolyl-endopeptidase
` and a postproline carboxypeptidase.
` The physiological
`14
`23
`24
`significance of this alternate pathway has not been characterized yet; conceivably, it
`provides an additional route for the inactivation of Ang II.
`Fig 1 provides a schematic diagram of the active pathways involved in the production of
`Ang-(1-7) from both Ang I and Ang II. With a more complete understanding of the
`biochemical routes for the processing of Ang I, it becomes apparent that the potential
`involvement of the endopeptidase pathways in the pathogenesis of hypertension may be a
`fruitful area of inquiry. One of the Ang-(1-7)–forming enzymes, neprilysin, converts the atrial
`natriuretic peptide and bradykinin into inactive fragments.
` Potential interactions of these
`25
`enzymes with the various substrates have not been investigated yet, nor have studies been
`undertaken to assess whether polymorphisms in the genes encoding these enzymes might
`be linked to disorders of cardiovascular function.
`
`Physiological Actions of Ang-(1-7)
`The first studies of Ang-(1-7) revealed that the peptide stimulates the activity of
`hypothalamic-neurohypophysial neurons regulating vasopressin release with a potency
`equal to Ang II. Subsequently, we found that Ang-(1-7) releases prostaglandins from
`4
`astrocytes, VSMCs, and endothelial cells in culture.
` Prostaglandin release in
`26 27 28 29
`human astrocytes and porcine smooth muscle cells was mediated by AT receptors,
`2
`whereas a non-AT , non-AT receptor accounted for these actions in rat C6 glioma and
`1
`2
`porcine endothelial cells. Furthermore, Ang-(1-7) elicits prostaglandin production through
`calcium-independent mechanisms in cells in culture and in the vasculature.
` Ang-(1-7) also
`30
`causes a depressor effect when injected into the circulation of the pithed rat, and this action
`is blocked completely by indomethacin but only partially by an AT receptor blocker.
` The
`31
`1
`peptide induces relaxation of porcine and canine coronary artery,
` piglet arterioles,
` and
`32 33
`34
`the feline mesenteric bed, possibly via release of NO through a non-AT , non-AT
`1
`2
`angiotensin receptor.
` Unlike Ang II, Ang-(1-7) does not elicit vasoconstriction, aldosterone
`35
`release, or stimulation of thirst and salt appetite, nor does it produce a pressor response
`after intraventricular administration in normotensive rats. Indeed, Ang-(1-7) facilitates the
`baroreflex and displays depressor effects in sites within the dorsal medulla.
` These
`5 36
`effects in the dorsal medulla are blocked by a selective antagonist to Ang-(1-7), [d-Ala ]
`7
`-Ang-(1-7).
` Thus, increasing evidence supports the concept that Ang-(1-7) opposes the
`37
`actions of Ang II and may do so through a novel receptor.
`
`Actions That Oppose the Effects of Ang II Are Enhanced in Models of
`Hypertension
`Ang-(1-7), similar to losartan and ACE inhibitors, counteracts the actions of Ang II.
`7
`Ang-(1-7) may contribute to the antihypertensive effects produced by ACE inhibitors, since
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`Counterregulatory Actions of Angiotensin-(1-7)
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`circulating levels of Ang-(1-7) increase 25-fold to 50-fold during ACE inhibition
` and
`19 21 22
`Ang-(1-7) alone can produce antihypertensive effects in hypertensive animals.
` In the
`38
`spontaneously hypertensive rat (SHR), chronic infusion of Ang-(1-7) produces significant
`increases
`in urinary excretion of prostaglandin E and 6-keto-prostaglandin F
`2
`1α
`accompanied by diuresis, natriuresis, and a decrease in blood pressure.
` Systemic
`38
`administration of Ang-(1-7) attenuates the vasoconstrictor actions of phenylephrine and Ang
`II in hypertensive but not normotensive rats,
` in contrast with the potentiation of
`39
`α-adrenoceptor-mediated pressor responses by Ang II. Moreover, intravenous infusions of
`Ang-(1-7) reverse the inhibitory effects of Ang II on the reflex control of heart rate in both
`SHR and Wistar-Kyoto rats and improve the impaired slope of the reflex control of heart
`39
`rate in SHR after either peripheral or central administration.
`36 39
`A recent study in a genetic model of hypertension that is associated with heightened
`activity of the brain angiotensin system clearly demonstrated the opposing actions of
`Ang-(1-7).
` In this important research, we evaluated the hemodynamic effects of delivering
`40
`either a specific, affinity-purified Ang-(1-7) antibody or an Ang II monoclonal antibody
`(KAA8) into the brain of conscious homozygous mRen2(27) renin transgenic [Tg(+)] rats
`(Fig 2). Cerebroventricular administration of the affinity-purified Ang-(1-7) antibody in
`conscious Tg(+) hypertensive rats caused significant dose-related elevations in blood
`pressure and heart rate.
` The hypertensive response was augmented in transgenic rats
`40
`studied 7 to 10 days after cessation of lisinopril therapy. In contrast, all doses of the Ang II
`antibody produced hypotension and bradycardia. The magnitude of the depressor response
`was significantly augmented in transgenic rats weaned off lisinopril therapy. Central
`administration of either the Ang-(1-7) or Ang II antibodies had no effect on normotensive
`Sprague-Dawley rats. These data demonstrate that Ang-(1-7) opposes the action of Ang II
`on the central mechanisms that contribute to the maintenance of this model of hypertension.
`In addition, these studies showed an important contribution of the brain renin-angiotensin
`system to the maintenance of this form of monogenetic hypertension.
`There is also evidence that Ang-(1-7) can act as an antagonist to the actions of Ang II in
`the vasculature.
` Therefore, mechanisms other than activation of prostaglandins and NO
`41
`may play a role in mediating the depressor effects of Ang-(1-7). It is not currently possible to
`determine the exact contribution of prostaglandins versus other mechanisms to the effects
`produced by Ang-(1-7) in the SHR or Tg(+) hypertensive rats from these initial studies. In
`addition, the mediators stimulated by Ang-(1-7) may differ, depending on the vascular bed
`and species studied. In recent studies we showed that the Ang-(1-7)–induced prostacyclin
`release from aortic VSMCs of Tg(+) rats was greater than that from VSMCs isolated from
`Sprague-Dawley control rats.
` Similarly, in the renovascular hypertensive dog, the
`42
`depressor component of the response to systemic Ang-(1-7) is exaggerated.
` Thus, the
`43
`degree of activation of these depressor systems is influenced by the state of activation of
`the renin-angiotensin system.
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`Counterregulatory Actions of Angiotensin-(1-7)
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`Evidence for Ang-(1-7) Vasodilator Actions in Canine Coronary Vessels
`and Interactions With Kinins
` as well as isolated feline
`Ang-(1-7) relaxes canine or porcine coronary artery rings,
`32 33
`mesenteric beds.
` This effect is blocked in both canine and porcine rings by removal of the
`35
`endothelium or pretreatment with an NO synthase inhibitor. Moreover, the vasorelaxant
`activity of Ang-(1-7) is markedly attenuated by the bradykinin B receptor antagonist Hoe
`2
`140 and does not appear
`to be associated with
`the synthesis and release of
`prostaglandins.
` Assessment of
`the angiotensin receptor subtypes mediating
`the
`33
`responses to Ang-(1-7) revealed that these effects are not inhibited by subtype-selective
`AT or AT receptor antagonists but are markedly attenuated by prior exposure to the
`1
`2
`competitive nonselective Ang II peptide receptor antagonist [Sar , Thr ]-Ang II. These
`1
`8
`results suggest that Ang-(1-7) has a direct effect on the endothelium, through the release of
`NO and kinins, mediated by an angiotensin receptor pharmacologically distinct from AT
`1
`and AT receptor subtypes. Furthermore, Ang II and Ang-(1-7) at equivalent concentration
`2
`ranges produced diametrically opposite changes in the contractile state of coronary artery
`rings (Fig 3).
`Additionally, Ang-(1-7) potentiated synergistically bradykinin-induced vasodilation. These
`actions of Ang-(1-7) may contribute to the cardioprotective effects of chronic ACE inhibition.
`Ang-(1-7)’s potentiating effect on the response to bradykinin was first described by Paula et
`al,
` who showed that low concentrations of Ang-(1-7) given intravenously augmented by
`44
`2-fold to 10-fold the vasodepressor response elicited by bradykinin. In isolated canine
`coronary arteries, Ang-(1-7) has a synergistic, concentration-dependent action on
`bradykinin-induced vasodilation that is dependent on the release of NO but not
`prostaglandins.
` The response is specific for Ang-(1-7), since neither acetylcholine, sodium
`45
`nitroprusside, nor prostaglandins were able to augment the bradykinin-induced relaxation.
`45
`This synergistic effect of Ang-(1-7) is not mediated by a known angiotensin receptor, since
`the effect persists in the presence of AT , AT , and [Sar , Thr ]-Ang II receptor antagonists.
`1
`8
`1
`2
`In fact, in contrast to a receptor-mediated effect of Ang-(1-7),
` the peptide may augment
`33
`vasodilation in coronary arteries by acting as a local modulator of ACE activity. Li et al
`45
`found that Ang-(1-7) significantly inhibits the degradation of
`I-[Tyr ]-bradykinin and the
`125
`0
`appearance of the bradykinin-(1-7) and bradykinin-(1-5) metabolites in coronary vascular
`rings while it also inhibits purified canine ACE activity with an IC of 0.65 μmol/L. These
`50
`findings indicate that Ang-(1-7) may inhibit ACE activity to elevate bradykinin levels as one
`mechanism of promoting vasodepressor actions.
`
`Antiproliferative Actions of Ang-(1-7) in VSMCs
`In previous studies in porcine and rat VSMCs, Ang II activates phospholipase C and D and
`releases prostaglandins, whereas Ang-(1-7) releases only prostaglandins.
` The
`29 30 46 47
`activation of phospholipase C by Ang II in VSMCs is known to stimulate growth. Because
`prostaglandins inhibit vascular growth, we speculated that Ang-(1-7) might also prevent the
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`Counterregulatory Actions of Angiotensin-(1-7)
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`growth of VSMCs. The effect of Ang-(1-7) on cell growth was determined by measuring [ H]
`3
`thymidine incorporation into rat aortic VSMCs in the presence and absence of various
`mitogens.
` The amount of [ H]thymidine incorporation was increased by treatment with
`48
`3
`fetal bovine serum, platelet-derived growth factor, or Ang II. In the presence of Ang-(1-7),
`the incorporation of [ H]thymidine in response to fetal bovine serum, platelet-derived growth
`3
`factor, and Ang II was significantly attenuated in a dose-dependent manner (Fig 4). Thus,
`Ang II and Ang-(1-7) have opposite effects on VSMC growth.
`Attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) is unaffected by
`antagonists selective for AT or AT receptors. However, the sarcosine derivatives of Ang II
`1
`2
`are effective antagonists, indicating that growth inhibition by Ang-(1-7) is a result of
`angiotensin receptor activation. In contrast, Ang II stimulation of [ H]thymidine incorporation
`3
`is attenuated by the AT -selective antagonists. Thus Ang-(1-7) inhibits VSMC growth
`1
`through activation of a non-AT , non-AT receptor.
`48
`1
`2
`Novel Receptor Identified in Bovine Aortic Endothelial Cells
`The inhibition of vascular growth by Ang-(1-7) through a non-AT , non-AT receptor
`1
`2
`suggests that the heptapeptide activates a unique angiotensin peptide receptor. Since
`previous studies strongly suggested that the endothelium also responds to Ang-(1-7)
`through activation of a non-AT , non-AT angiotensin peptide receptor,
` we isolated
`28 33
`1
`2
`endothelial cells from bovine thoracic aorta to determine whether they contain a high-affinity
`I-Ang-(1-7) binding site. Scatchard analysis of saturation isotherms of endothelial cells
`125
`showed that
`I-Ang-(1-7) binds to bovine aortic endothelial cells with an affinity of 19
`125
`nmol/L and a density of 1351 fmol/mg protein.
` In competition studies, the specific binding
`49
`of
`I-Ang-(1-7) is blocked by [Sar , Ile ]-Ang II and by [d-Ala ]-Ang-(1-7), a selective
`125
`1
`8
`7
`blocker of responses to Ang-(1-7).
` In contrast, neither the AT -selective nor the
`49
`1
`AT -selective antagonists significantly competes for
`I-Ang-(1-7) binding. Further proof
`125
`2
`that Ang-(1-7) does not bind to a typical AT or AT receptor is derived from studies showing
`1
`2
`that bovine aortic endothelial cells do not contain the mRNA encoding an AT or AT
`1
`2
`receptor. In preliminary experiments, we also showed that
`I-Ang-(1-7) binds specifically
`125
`and with high affinity to the endothelial layer of canine coronary arteries, using the technique
`of in vitro emulsion autoradiography, as previously described (Fig 5). Binding to canine
`50
`coronary endothelium was effectively competed for by either unlabeled Ang-(1-7) or [d-Ala ]
`7
`-Ang-(1-7). These results are in agreement with endothelial cell binding data as well as
`49
`previous studies in which we showed that Ang-(1-7) causes vasodilation of canine coronary
`arteries by a non-AT , non-AT receptor.
`33
`1
`2
`ANG-(1-7) AND THE KIDNEY
`A critically important target organ for Ang II is the kidney. Ang II causes renal
`vasoconstriction, release of aldosterone, reduced glomerular filtration rate, stimulation of
`sodium and bicarbonate transport in the proximal tubules, and mesangial cell hypertrophy.
`51
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`In contrast, Ang-(1-7) exhibits diuretic and natriuretic properties in hypertensive animals, as
`evidenced by our studies in SHR.
` Similar natriuretic and diuretic actions occur in the in
`38
`vivo isolated and in vivo perfused kidney.
` The natriuretic and diuretic responses to
`52 53 54
`Ang-(1-7) are associated with increases in prostaglandin release,
` and the majority of the
`53
`renal effect of Ang-(1-7) is attenuated with the cyclooxygenase inhibitor indomethacin. In
`isolated proximal tubules, very low doses (10
` to 10 mol/L) of Ang-(1-7) inhibit transport-
`−13
`−9
`dependent oxygen consumption and bicarbonate transport.
` These actions of Ang-(1-7)
`54
`55
`are at least partially blocked by antagonists selective for the AT receptor. In marked
`1
`contrast to the effects of Ang II on the kidney, Ang-(1-7) does not alter renal blood flow or
`stimulate aldosterone release.
`53 54 56
`The renal excretory response to low doses of Ang-(1-7) is markedly different from the
`antinatriuresis and antidiuresis reported
`for
`low doses of Ang
`II. Subthreshold
`vasoconstrictor doses of Ang II activate AT receptors on the proximal tubule, leading to a
`1
`decrease in urinary sodium and water excretion. The natriuretic effect of Ang-(1-7) may be
`due to decreased AT receptor activation resulting from receptor competition with
`1
`endogenous Ang II. However, the renal vascular constrictor response to Ang II (AT
`1
`receptor mediated) is unaltered when coinjected with nonvasoconstrictor doses of
`Ang-(1-7), suggesting that Ang-(1-7) does not functionally antagonize the AT -mediated
`1
`actions of Ang II in the kidney.
` Alternatively, the natriuretic effects of high doses of Ang II
`54
`may be due to conversion of Ang II to Ang-(1-7).
`infusions of Ang-(1-7) in
`In
`the above studies, differences were observed to
`normotensive and hypertensive rats. While transient depressor and diuretic/natriuretic
`effects and suppression of plasma vasopressin levels were seen in the SHR, minimal
`changes occurred
`in
`the Wistar-Kyoto
`rats.
`
`Interestingly, Ang-(1-7) stimulates
`38
`antinatriuresis and a nonsignificant rise in plasma vasopressin in water-loaded Wistar rats.
`57
`Santos et al
` recently reported antidiuretic effects in an isolated collecting duct preparation,
`58
`which were blocked by the [d-Ala ]-Ang-(1-7) antagonist. These antidiuretic effects are in
`7
`direct contrast to the effects observed in the isolated and in situ perfused kidney. One
`obvious difference in the studies with intact animals was that the experiments were
`performed after water loading. Burnier et al
` reported conflicting results concerning AT
`59
`1
`treatment of patients after an acute water load. In addition, in isolated tubules, the route for
`administration of the Ang-(1-7) and the segment of the nephron accessed was different than
`in isolated kidneys or whole-animal experiments. These findings suggest that the overall
`state of sodium and water balance (and perhaps the overall activity of the renin-angiotensin
`system) may influence the effects of Ang-(1-7) in the kidney, as discussed below. In
`addition, these results emphasize that the route of administration and the site of the
`nephron exposed to Ang-(1-7) may determine the direction of observed actions.
`Ang-(1-7) was reported to inhibit transcellular sodium flux in cultured renal tubular
`epithelial cells, an action that may be mediated through the activation of phospholipase
`A .
` Interestingly, the phospholipase A activation in response to Ang I is markedly
`60
`2
`2
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`Counterregulatory Actions of Angiotensin-(1-7)
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`potentiated by captopril. These data indicate that Ang-(1-7) plays an important role in
`modulating sodium handling, most likely at the level of the tubule, and reinforce the concept
`that Ang-(1-7) is a biologically active member of the renin-angiotensin system. The data
`also suggest that the tubular epithelium can convert Ang I to Ang-(1-7) with only minor
`amounts of Ang II formed. Inhibition of neprilysin blocked the majority of Ang-(1-7)
`formation.
` In contrast, Ang II was metabolized preferentially to Ang-(1-4) by neprilysin and
`61
`shorter N-terminal fragments such as Ang-(2-8) and Ang-(3-8) by peptidyl and dipeptidyl
`aminopeptidases. Thus, the kidney contains the necessary substrates and enzymes for the
`intrarenal generation of Ang-(1-7).
`
`Presence of Angiotensin-(1-7) in Urine: A Marker of Renal Function?
`A further insight into the role of Ang-(1-7) in renal function was gained by demonstration of
`large quantities of the heptapeptide in rat urine. Urinary excretion of Ang-(1-7) averages
`4.8±0.4 pmol/24 h compared with 0.73±0.04 pmol/24 h for Ang II.
` Urinary Ang-(1-7) levels
`62
`are increased by 88% after a 5-day exposure of the rats to lisinopril (20 mg/kg, PO).
`Combined treatment with lisinopril and a neprilysin inhibitor returned the concentration and
`excretion of Ang-(1-7) to control levels and augmented Ang II concentration. These data
`suggest that the kidneys are an important source for the production of Ang-(1-7) and
`reinforce the concept that neprilysin participates in the renal processing of Ang I into
`Ang-(1-7).
`The demonstration of high concentrations of Ang-(1-7) in rat urine is an important finding
`that could lead to greater knowledge of the mechanisms that account for the progressive
`decline in renal function associated with hypertension and end-stage renal disease. A
`significant step into this problem has been gained with the characterization of Ang-(1-7) in
`the urine of 31 healthy volunteers and 18 untreated essential hypertensive subjects.
` In
`63
`these studies, the concentration of Ang-(1-7) in the urine of normal subjects averaged
`62.6±22.6 (SD) pmol/L, corresponding to a urinary excretion rate of 98.9±44.7 pmol/24 h.
`Concurrent measurements of plasma Ang-(1-7) showed that the content of Ang-(1-7) in
`urine was 2.5-fold higher than that measured in the plasma. In contrast, untreated essential
`hypertensive subjects had lower concentrations and 24-hour urinary excretion rates of
`Ang-(1-7) averaging 39.4±18.0 pmol/L and 60.2±14.6 pmol/24 h, respectively (P<.001).
`Differences in the excretory rate of Ang-(1-7) between normal volunteers and essential
`hypertensive subjects were not modified by normalization of the data by urinary creatinine
`excretion rates. In addition, urinary concentrations of Ang-(1-7) correlated inversely with
`arterial pressures (r=−.48, P<.001), whereas both urinary Ang-(1-7) (odds ratio of 0.92 [95%
`confidence interval: 0.88-0.97]) and age were independent predictors of systolic blood
`pressure.
`These studies demonstrate the presence of Ang-(1-7) in urine and the existence of
`reduced levels of the heptapeptide in individuals with untreated essential hypertension. The
`relatively higher concentrations of Ang-(1-7) in urine compared with plasma are in
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`Counterregulatory Actions of Angiotensin-(1-7)
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`agreement with data showing that Ang-(1-7) may contribute to the regulation of blood
`pressure. The inverse association between Ang-(1-7) and arterial pressure provides a
`potential marker for the characterization of forms of essential hypertension associated with
`reduced production or activity of vasodilator hormones.
`
`CONCLUDING REMARKS
`Studies of the role of Ang-(1-7) in the control of blood pressure suggest that the renin-
`angiotensin system possesses the ability to limit the pressor and proliferative actions of Ang
`II through a mechanism that relies on the alternative generation of Ang-(1-7). In this context,
`Ang-(1-7) may act as the signal peptide for the activation of the negative feedback limb that
`limits the pressor and proliferative actions of Ang II through stimulation of vasodilator
`prostacyclin, NO, or both. Increased Ang II production is a critical component of the short-
`term cardiovascular response to acute changes in blood pressure and blood volume.
`Therefore, regulatory mechanisms must exist to ensure that increased production of
`Ang-(1-7) will not negate the important compensatory actions of Ang II in both the short- and
`long-term regulation of blood pressure. The specific feedback mechanisms that regulate the
`formation of Ang-(1-7) independently from Ang II have yet to be determined. Our working
`hypothesis is that one aspect of the regulatory feedback mechanism may entail modulation
`of Ang II production by ACE, since Ang-(1-7) is metabolized by ACE and exhibits an affinity
`comparable to that for bradykinin (approximately 0.7 mol/L).
` Our studies also suggest that
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`the actions of Ang-(1-7) are most evident in situations associated with long-term rather than
`short-term increases in Ang II production or activity. Likewise, augmented Ang-(1-7)
`formation follows chronic rather than acute inhibition of ACE.
` While further studies are
`20
`required to assess the mechanisms that determine the factors that control production of
`Ang-(1-7) over Ang II formation from their common Ang I substrate, the data gathered to
`date suggest that the net actions of the renin-angiotensin system in the long-term regulation
`of blood pressure may depend on a balance between the tissue concentrations of Ang II
`and Ang-(1-7; Fig 6).
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`Counterregulatory Actions of Angiotensin-(1-7)
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`Figure 1. Diagram showing the active pathways involved in the production of Ang-(1-7). Ang I is processed
`to biologically active peptides by distinct enzymes. Ang I is hydrolyzed at Phe -His by angiotensin-
`8
`9
`converting enzyme (ACE) or chymase (CHYM) to yield Ang II. Ang-(1-7) is produced by the hydrolysis of
`Ang I at Pro -Phe by neutral endopeptidases 24.11 and 24.15 (NEP) and prolyl endopeptidase (PE). PE
`7
`8
`and prolyl carboxypeptidase (PCP) hydrolyze Ang II to Ang-(1-7).
`
`Figure 2. Contrasting effects of cerebroventricular injection of either a specific polyclonal Ang-(1-7) (▪, solid
`line) or monoclonal angiotensin II (▴, dashed line) antibody on the mean arterial pressure of homozygous
`mRen2(27) transgenic hypertensive rats. Data were redrawn from Reference 40. Values are mean±SE.
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`Counterregulatory Actions of Angiotensin-(1-7)
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`Figure 3. Average cumulative dose response to angiotensin (Ang) II (left) and Ang-(1-7) (right) in canine left
`anterior descending coronary vessels precontracted with 10 nmol/L U46 619, a thromboxane A analogue.
`2
`All concentrations of Ang II and Ang-(1-7) are given as negative logs. The ordinate shows the percent
`contraction or relaxation of the precontracted coronary vessels. Data were redrawn from Reference 33.
`
`Figure 4. Vascular smooth muscle cells made quiescent by 48 hours in serum-free media and then treated
`for an additional 48 hours with either Ang-(1-7) in the presence of 1% fetal bovine serum (serum stimulated)
`or Ang II. Left axis, Inhibition of serum-stimulated [ H]thymidine incorporation by increasing concentrations
`3
`of Ang-(1-7) is presented as the percentage of serum-stimulated activity (29 442±3253 cpm per well). Right
`axis, Stimulation of [ H]thymidine incorporation by increasing concentrations of Ang II is presented as the
`3
`percentage of basal activity (13 217±228 cpm per well). Data were redrawn from Reference 48.
`
`Figure 5. In vitro emulsion autoradiography showing specific binding of
`I-Ang-(1-7) to endothelial cells on
`125
`a canine coronary artery frozen-sectioned at 14 μm and incubated with 0.75 nmol/L
`I-Ang-(1-7) in the
`125
`presence or absence of either 1 μmol/L Ang-(1-7) or d-Ala -Ang-(1-7). Sections were processed for
`7
`emulsion autoradiography as previously described.
` The arrows indicate the endothelium lining the lumen.
`50
`Abundant silver grains, i