Therapeutic targets in liver transplantation: angiotensin II in nonsteatotic grafts and angiotensin-(1-7) in steatotic grafts.

Numerous steatotic livers are discarded as unsuitable for transplantation because of their poor tolerance of ischemia-reperfusion(I/R). The injurious effects of angiotensin (Ang)-II and the benefits of Ang-(1-7) in various pathologies are well documented. We examined the generation of Ang II and Ang-(1-7) in steatotic and nonsteatotic liver grafts from Zucker rats following transplantation. We also studied in both liver grafts the effects of Ang-II receptors antagonists and Ang-(1-7) receptor antagonists on hepatic I/R damage associated with transplantation. Nonsteatotic grafts showed higher Ang II levels than steatotic grafts, whereas steatotic grafts showed higher Ang-(1-7) levels than nonsteatotic grafts. Ang II receptor antagonists protected only nonsteatotic grafts against damage, whereas Ang-(1-7) receptor antagonists were effective only in steatotic grafts. The protection conferred by Ang II receptor antagonists in nonsteatotic grafts was associated with ERK 1/2 overexpression, whereas the beneficial effects of Ang-(1-7) receptor antagonists in steatotic grafts may be mediated by NO inhibition. Our results show that Ang II receptor antagonists are effective only in nonsteatotic liver transplantation and point to a novel therapeutic target in liver transplantation based on Ang-(1-7), which is specific for steatotic liver grafts.

Characterization of vascular reactivity in dorsal hand veins after oral rosiglitazone treatment in healthy subjects.

OBJECTIVE: In clinical studies with diabetic patients thiazolidinediones have been shown to restore abnormal vascular function which might be attributed to improved blood sugar control or to restoration of vascular endothelium and smooth muscle responsiveness. The present study was undertaken to investigate whether rosiglitazone modulates vascular responsiveness to different vasoactive agents and exerts renin-angiotensin-system (RAS)-inhibiting properties in healthy subjects in vivo. METHODS: 24 healthy male subjects were randomized to receive either rosiglitazone or placebo. Venoconstrictor responses to angiotensin II (Ang II) and phenylephrine, and endothelium-dependent response to histamine and insulin, and endothelium-independent response to glyceroltrinitrate were compared using the dorsal hand vein compliance method. Effects on the RAS were investigated by plasma level determinations of Ang II and angiotensin-(1-7). Treatment effects on the systemic arterial system were investigated by standardized pulse-wave-analysis. RESULTS: Rosiglitazone significantly inhibited venoconstrictor responses to Ang II by 19% (-70% vs. -51% constriction, p = 0.034) and in the presence of rosiglitazone the ED80 for phenylephrine was increased (ED80: 317 A+/- 86 ng vs. 531 A+/- 102 ng; p = 0.010). Rosiglitazone treatment was without effect on endothelium-dependent dilation, blood pressure, pulse-wave-velocity and plasma angiotensin peptide levels. CONCLUSIONS: The data of the present study in veins of healthy subjects are consistent with data from in vitro and animal studies supporting a direct effect of rosiglitazone on venous tone by modulation of the vascular smooth muscle response via AT1-receptor-downregulation.

Effects of hormone therapy on blood pressure and the renin-angiotensin system in postmenopausal women.

Observational studies have documented an association between lower rates of cardiovascular disease and hormone therapy (HT). Meanwhile, randomized clinical trials have documented increased rates of myocardial infarction and stroke in women receiving hormone therapy. These seemingly discordant findings have stimulated new research to examine estrogen's effects on the cardiovascular system, including its effects on blood pressure, regulation of the renin-angiotensin system (RAS), and the clinical consequences of hypertension. In the last 6 years several studies have better defined the mechanisms by which HT affect the RAS, blood pressure, and the clinical effects of hypertension. Recent studies documented increases in angiotensinogen synthesis and the suppression of active renin with estrogen replacement. Genotype may be a factor in determining the degree of suppression of angiotensin converting enzyme levels that occurs with estrogen therapy. Estrogen supplementation in postmenopausal women increases systemic angiotensin II, a potent vasoconstrictor. This vasopressor effect is attenuated by an estrogen-induced reduction of angiotensin II type 1 receptor expression. Renal blood flow reduction, in the absence of blood pressure changes, have been reported after estrogen replacement, and an increased risk of total stroke has been demonstrated in hypertensive women on HT compared to normotensive women on this therapy. Estrogen replacement affects many components of the RAS, but its effect on this system has little effect on blood pressure. Further studies are needed to describe the effects of estrogen replacement on abnormal vasculature and how these effects relate to myocardial infarction and stroke.

Distribution of angiotensin-(1-7) and ACE2 in human placentas of normal and pathological pregnancies.

This work was designed to study the expression of the vasodilator peptide angiotensin-(1-7) [Ang-(1-7)] and its generating enzyme (ACE2) in the uteroplacental interface. Placentas were obtained from 11 early pregnancy failures (5 miscarriages and 6 ectopic pregnancies), 15 normotensive, and 10 preeclamptic gestations. In placental villi, the main sites of immunocytochemical expression of Ang-(1-7) and ACE2 were the syncytiotrophoblast, cytotrophoblast, endothelium and vascular smooth muscle of primary and secondary villi. Syncitial Ang-(1-7) expression in samples obtained from miscarriages and ectopic pregnancies was increased compared to normal term pregnancy [2.0 (2.0-2.25 for the 25 and 75% interquartile range) vs 1.3 (1.0-1.9), p<0.01]. In the maternal stroma, Ang-(1-7) and ACE2 were expressed in the invading and intravascular trophoblast and in decidual cells in all 3 groups. Ang-(1-7) and ACE2 staining was also found in arterial and venous endothelium and smooth muscle of the umbilical cord. The expression of Ang-(1-7) and ACE2 was similar in samples obtained from normal term or preeclamptic pregnancies, except for increased expression of ACE2 in umbilical arterial endothelium in preeclampsia [0.5 (0.5-0.8) vs 0.0 (0.0-0.0), p<0.01]. The uteroplacental location of Ang-(1-7) and ACE2 in pregnancy suggests an autocrine function of Ang-(1-7) in the vasoactive regulation that characterizes placentation and established pregnancy.

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague-Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats.

The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague-Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17beta-estradiol (E2). We evaluated estrogen's influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1-7) [Ang-(1-7)] and on aminopeptidase (AMP), which degrades Ang-(1-7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31+/-0.03 nmol/min/mg versus 0.18+/-0.04 nmol/min/mg of protein, p<0.05) and Tg(+) (0.26+/-0.04 nmol/min/mg versus 0.13+/-0.02 nmol/min/mg of protein, p<0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p<0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1-7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.

Episodic secretion of renin.

Renin is secreted episodically in the absence of overt stimulation in the pentobarital-anesthetized, saline-loaded cat. Ninety min postlaparotomy, arterial and renal venous plasma renin concentrations (PRC) were determined at 15-min intervals for a 2 hr period. The pattern of renin secretion consisted of significant secretory peaks and periods in which little or no renin was secreted. In only a few instances could renin secretion be attributed to a preceding arterial blood pressure drop. The secretory episodes were also dissociated from flow.

Angiotensin-(1-7) dilates canine coronary arteries through kinins and nitric oxide.

Angiotensin-(1-7) [Ang-(1-7)] was recently recognized to have novel biological functions that are distinct from those of Ang II. In these studies, we determined the vasoactive effects of Ang-(1-7) together with the endothelium-dependent mediator(s) of these responses in canine coronary arteries. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers perfused with 95% O2/5% CO2 at 37 degrees C. Ang-(1-7) caused significant concentration-dependent vascular relaxation (2.73 +/- 0.58 micromol/L, EC50) of rings precontracted with the thromboxane A2 analogue U46,619. Pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (1 mol/L) abolished the vasodilator response to Ang-(1-7), whereas treatment with the cyclooxygenase inhibitor indomethacin (10 micromol/L) was without effect. The vasodilator response produced by Ang-(1-7) was blocked by 75% with the bradykinin B2 receptor antagonist Hoe 140 (1 micromol/L) or by 80% with the nonselective Ang II antagonist [Sar1,Thr8]-Ang II (1 micromol/L). In contrast, the selective AT1 or AT2 Ang II antagonists CV 11974 (1 micromol/L), and PD 123319 (1 micromol/L), respectively, were ineffective in inhibiting the Ang-(1-7)-elicited vasodilation. Furthermore, pretreatment of the coronary rings with 2 micromol/L Ang-(1-7) markedly potentiated the bradykinin response. These results suggest that Ang-(1-7) elicits coronary vasodilation that is specifically mediated by the endothelium-dependent release of nitric oxide. These responses involve a B2 bradykinin receptor and a non-AT1, non-AT2, angiotensin receptor. These data suggest that increases in circulating levels of Ang-(1-7) accompanying long-term administration of converting enzyme inhibitors or Ang II receptor blockers may contribute to the cardioprotective actions of these drugs.

Characteristics of hormonal and neurogenic mechanisms of deoxycorticosterone-induced hypertension.

We characterized the hemodynamic and endocrine changes associated with the evolution of steroid-induced hypertension in conscious, trained, instrumented dogs given intramuscular injections of deoxycorticosterone (DOC) pivalate on Days 1 (20 mg/kg) and 14 (10 mg/kg) of the study. Because hypertension could be produced in these dogs without salt loading and unilateral nephrectomy, the research afforded a novel opportunity to determine the primary effects of DOC excess on the renin-angiotensin and sympathetic nervous systems, and on vasopressin levels. Both before and during 28 days of DOC treatment, regular measurements of mean arterial pressure, heart rate, cardiac output, and total peripheral resistance were coupled with serial determinations of plasma and cerebrospinal fluid levels of angiotensin II, vasopressin, norepinephrine, and electrolytes. DOC induced a progressive rise in mean arterial pressure associated with increased cardiac output and no change in heart rate. These hemodynamic changes were accompanied by sustained decreases in plasma renin activity, and in plasma, but not cerebrospinal fluid, angiotensin II. In contrast, plasma and cerebrospinal fluid vasopressin rose transiently on the 7th and 14th days of the study, respectively. After anesthesia with morphine and chloralose, the hemodynamic response to occlusion of a sole innervated carotid artery was evaluated on the 5th week before and after cervical vagotomy. Compared to normal animals, dogs with DOC-induced hypertension showed a reduced pressor response to carotid occlusion associated with suppression of reflex tachycardia; vagotomy partially restored the pressor response to normal levels. The data suggest that DOC-induced hypertension changes central hormonal influences of cardiovascular function, and also alters cardiopulmonary baroreceptor reflex control of peripheral sympathetic nerve activity.

In vivo metabolism of angiotensin I by neutral endopeptidase (EC 3.4.24.11) in spontaneously hypertensive rats.

We investigated the processing enzymes involved in the formation of circulating angiotensin-(1-7) after intravenous administration of angiotensin I to conscious spontaneously hypertensive and Wistar-Kyoto rats. Immunoreactive products, including angiotensin I, angiotensin II, and angiotensin-(1-7), were measured in arterial blood by three specific radioimmunoassays. Angiotensin I infusion (2 nmol) induced a rapid increase in immunoreactive angiotensin II and angiotensin-(1-7). Pretreatment with the angiotensin converting enzyme inhibitor enalaprilat (2 mg/kg) eliminated angiotensin II formation and augmented circulating levels of angiotensin I and angiotensin-(1-7) in spontaneously hypertensive and Wistar-Kyoto rats. The elevated levels of angiotensin-(1-7) in enalaprilat-treated rats were blocked by concurrent treatment with the neutral endopeptidase (EC 3.4.24.11) inhibitor SCH 39,370 (15 mg/kg) in both strains. Administration of SCH 39,370 alone decreased angiotensin-(1-7) levels in spontaneously hypertensive rats, whereas angiotensin II levels increased in both strains (p less than 0.01). Comparisons of the metabolism of angiotensin I in the two rat strains showed increased formation of angiotensin-(1-7) in spontaneously hypertensive rats not given any of the enzyme inhibitors. In addition, levels of angiotensin I were higher after administration of SCH 39,370 in hypertensive rats. These novel findings reveal that neutral endopeptidase EC 3.4.24.11 participates in the conversion of angiotensin I to angiotensin-(1-7) and in the metabolism of angiotensin II in the circulation of both spontaneously hypertensive and Wistar-Kyoto rats. Our results suggest that neutral endopeptidase EC 3.4.24.11 is a major enzymatic constituent of the circulating renin-angiotensin system.

Angiotensin-(1-7) augments bradykinin-induced vasodilation by competing with ACE and releasing nitric oxide.

Recent studies have shown that angiotensin-(1-7) [Ang-(1-7)] interacts with kinins and augments bradykinin (BK)-induced vasodilator responses by an unknown mechanism. In this study, we evaluated whether the potentiation of the BK-induced vasodilation by Ang-(1-7) may be attributable to inhibition of BK metabolism, release of nitric oxide, or both. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers. 125I-[Tyr0]-BK metabolism was determined in vascular rings by assessing the degradation of the peptide by high-performance liquid chromatography. Ang-(1-7) augmented the vasodilation induced by BK in a concentration-dependent manner in rings preconstricted with the thromboxane analog U46619. The EC50 of BK (2.45 +/- 0.51 nmol/L versus 0.37 +/- 0.08 nmol/L) was shifted leftward by 6.6-fold in the presence of 2 mumol/L concentration of Ang-(1-7). The response was specific for BK. since Ang-(1-7) did not augment the vasodilation induced by either acetylcholine (0.05 mumol/L) or sodium nitroprusside (0.1 mumol/L). Moreover, neither angiotensin I nor angiotensin II (Ang II) duplicated the augmented BK response of Ang-(1-7). Pretreatment of vascular rings with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NA; 100 mumol/L) completely abolished the effects of Ang-(1-7) on BK-induced vasodilation whereas pretreatment with indomethacin (10 mumol/L) was without effect. The potent specific BK B2 receptor antagonist, Hoe 140. nearly abolished the BK and the Ang-(1-7) potentiated responses at 2 mumol/L, whereas at a lower concentration (20 nmol/L) Hoe 140 shifted the response curve to the right for both Ang-(1-7) and vehicle; however, the augmented response to Ang-(1-7) persisted. Preincubation of vascular rings with 20 mumol/L of the AT1 (CV11974), AT2 (PD123319), or nonselective (Sar1 Thr8-Ang II) receptor antagonists had no significant effect on the Ang-(1-7)-enhanced vasodilator response to BK. Lisinopril (2 mumol/L) significantly enhanced the BK-induced vasodilator response while at the same time it abolished the synergistic action of Ang-(1-7) on BK. In addition, pretreatment with 2 mumol/L Ang-(1-7) significantly inhibited the degradation of 125I-[Tyr0]-BK and the appearance of the BK-(1-7) and BK-(1-5) metabolites in coronary vascular rings. Ang-(1-7) inhibited purified canine angiotensin converting enzyme activity with an IC50 of 0.65 mumol/L. In conclusion. Ang-(1-7) acts as a local synergistic modulator of kinin-induced vasodilation by inhibiting angiotensin converting enzyme and releasing nitric oxide.

Differential regulation of angiotensinogen transcripts after renin infusion.

To investigate angiotensinogen regulation in high-renin hypertension, we infused porcine renin intravenously at either a low (4 mU/kg per hour, n = 6) or high (20 mU/kg per hour, n = 9) dose into male Sprague-Dawley rats (225 to 250 g) for 5 days using osmotic minipumps. Control rats received 0.9% NaCl. In renin-infused rats, mean arterial pressure and plasma renin activity were significantly elevated. Both low- and high-renin infusions lowered plasma angiotensinogen levels. Plasma angiotension II was elevated in rats given renin but reached statistical significance only at the higher dose. Angiotensinogen mRNA isolated from the liver, adrenal gland, kidney, and brain was measured by slot blot analysis. Both renin doses were associated with significant decreases in the levels of liver and hypothalamic angiotensinogen mRNA. In the medulla oblongata, angiotensinogen mRNA was reduced only by the higher renin dose. The lower dose increased angiotensinogen mRNA in the adrenal gland, and in kidney, angiotensinogen mRNA level was unchanged by renin infusion. Angiotensinogen mRNA visualized on Northern blots showed that the number of mRNA species in liver decreased from three in control rats to a single mRNA species after renin infusion. Tissue differences in the size of the major angiotensinogen mRNA species were also apparent. This, together with changes in the total hybridization signal of angiotensinogen mRNA in tissues, suggests that renin differentially affects the different angiotensinogen mRNA transcripts. Results of this study indicate that angiotensinogen gene expression is regulated not only by alterations in levels of circulating angiotensin II but also by other mechanisms, presently unidentified, that are activated by renin infusions.

Effects of chronic sodium depletion on canine brain renin and cathepsin D activities.

The activities of brain renin and cathepsin D were measured in brain regions of 10 dogs on a normal sodium intake (65 mEq Na+/day) and 10 other dogs placed on a low sodium diet (less than 4 mEq Na+/day) for 21 days and given a diuretic. The purpose of this study was twofold: to assess the effect of sodium depletion on brain renin activity; and to assess in the same regions alterations in brain renin and cathepsin D activities. Sodium depletion caused a ninefold increase in plasma renin activity, hemoconcentration, and hyponatremia. In the presence of marked hyperreninemia, the average cerebral renin activity was reduced significantly; the most pronounced changes occurred in the upper and lower brain-stem regions. Cerebrospinal fluid renin was increased by 30%, but this change was not significant in sodium-depleted dogs. There were no significant alterations in cathepsin D activity whether assessed in total or regional brain areas. These observations support the view that there is an inverse relationship between plasma and brain renin activity in chronically sodium-depleted dogs. Additionally, evidence is provided that brain renin activity is modified independently from cathepsin D activity.

Effect of chronic sodium depletion on cerebrospinal fluid and plasma catecholamines.

To test the role of central neurogenic factors in sodium-depleted states, cerebrospinal fluid (CSF) norepinephrine, epinephrine, and dopamine were measured in mongrel dogs first on a normal sodium intake (65 mEq sodium/day) and then on a 21-day regime of low sodium diet (4 mEq/day combined with diuretics). Plasma catecholamines were measured in the same group of dogs. Three weeks of sodium depletion supplemented with diuretics caused a 24-fold increase in plasma renin activity, hemoconcentration, and elevated serum protein concentration. Both plasma and CSF sodium decreased significantly. After sodium depletion, plasma norepinephrine rose 76% but epinephrine and dopamine did not change. The same pattern was observed whether samples were obtained in conscious or anesthetized animals. In CSF, norepinephrine rose 44% during sodium depletion, while epinephrine and dopamine remained unchanged. The CSF norepinephrine was related inversely to the CSF sodium concentration and directly to plasma renin activity. These observations support the view that the combined procedure of restricted dietary sodium intake and diuretic therapy causes alterations in CSF norepinephrine in a direction compatible with possible overactivity of central noradrenergic neurons.

Angiotensin-(1-7). A member of circulating angiotensin peptides.

We measured the concentrations of three principal products of the renin-angiotensin system and seven of their metabolites in the plasma of anesthetized normal dogs and in dogs 24 hours after bilateral nephrectomy. The levels of the angiotensin peptides were measured by high-performance liquid chromatography combined with radioimmunoassay using three specific antibodies that recognized different epitotes in the sequences of angiotensin I, angiotensin II, and angiotensin-(1-7). The analysis revealed that angiotensin-(1-7) is present in the plasma of intact (4.9 +/- 2.2 fmol/ml) and nephrectomized (0.5 +/- 0.5 fmol/ml) dogs. An intravenous injection of purified hog renin (0.01 Goldblatt unit/kg) increased plasma levels of angiotensin I, angiotensin II, and angiotensin-(1-7) both before and after nephrectomy. These changes were associated with parallel increases in the concentrations of fragments of the three parent peptides. Administration of MK-422 led to the disappearance of circulating angiotensin II and its fragments both before and after a second injection of the same dose of renin. In contrast, MK-422 augmented the plasma levels of both angiotensin I and angiotensin-(1-7). The concentrations of these two peptides, but not the blood pressure, were again augmented by a second injection of renin given after blockade of converting enzyme. These effects were observed both before and after bilateral nephrectomy. These findings show that angiotensin-(1-7) circulates in the blood of normal and nephrectomized dogs. In addition, we found that angiotensin-(1-7) is generated in the blood from the cleavage of angiotensin I through a pathway independent of converting enzyme (EC 3.4.15.1).

Production of angiotensin-(1-7) by human vascular endothelium.

The heptapeptide angiotensin-(1-7) is a circulating biologically active product of the renin-angiotensin system. In this study, we evaluated the role of the vascular endothelium in the formation of angiotensin-(1-7). Metabolism of 125I-angiotensin I was investigated using confluent cultured bovine and human aortic and umbilical vein endothelial cells. The fetal calf serum-supplemented medium was replaced by serum-free medium containing 0.2% bovine serum albumin. One hour later, this medium was replaced by serum-free medium containing 125I-angiotensin I. After incubation of 125I-angiotensin I for various intervals at 37 degrees C, the medium was collected and analyzed for formed products by high-performance liquid chromatography. Products of angiotensin I metabolism were identified by comparison of their retention times with those of radiolabeled standards. The contribution of proteases released into the medium was evaluated by incubation of 125I-angiotensin I with medium previously incubated for 1 hour with endothelial cells. Incubation of 125I-angiotensin I with bovine and human endothelial cells produced a time-dependent generation of 125I-angiotensin-(1-7) greater than 125I-angiotensin II greater than 125I-angiotensin-(1-4). Generation of angiotensin peptides was not due to the presence of proteases in the medium. When human umbilical endothelial cells were incubated in the presence of the angiotensin converting enzyme inhibitor enalaprilat (1 microM), generation of angiotensin II was undetectable. In contrast, angiotensin-(1-7) production increased by an average of 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue renin-angiotensin systems in renal hypertension.

Angiotensinogen messenger RNA (mRNA) levels were measured in the brain (hypothalamus, lower brain stem, cerebellum), liver, kidneys, and adrenal glands of rats made hypertensive by ligation of the aorta between the renal arteries. We also measured renin mRNA in the kidneys of these renal hypertensive rats. The early phase of hypertension (day 6) was associated with significant increases in plasma renin activity and levels of circulating angiotensin II. The circulating renin-angiotensin system was not activated in the later phase of hypertension (day 24). Angiotensinogen mRNA levels were elevated in the lower brain stem of hypertensive rats at both stages of hypertension. In contrast, angiotensinogen mRNA levels in the hypothalamus were increased only at day 6 after aortic ligation. Decreased levels of angiotensinogen mRNA were observed in the cerebellum in both the early and later phases of the hypertension. Angiotensinogen mRNA levels in the adrenal gland below the ligature fell in the early phases but rose in the later phases of hypertension. Renin mRNA levels of the ischemic kidney remained elevated at both the early and later phases, whereas in both ischemic and nonischemic kidneys, levels of angiotensinogen mRNA remained below sham values throughout the period of study. These results indicate differential expression of renin-angiotensin system mRNAs in tissues of renal hypertensive rats. The differential changes in the expression of angiotensinogen mRNA over the course of development and maintenance of renal hypertension suggest that factors in addition to angiotensin II are important in modulating the expression of renin-angiotensin system genes.

Basal levels of plasma epinephrine and norepinephrine in the dog.

Conscious (n = 62) and anesthetized (n = 34) dogs were studied to establish basal levels and ranges for plasma epinephrine (E) and norepinephrine (NE) in this species. Trained conscious dogs were familiarized to recording conditions and personnel for 2 to 3 weeks and acclimatized to the laboratory for at least 15 minutes prior to blood sampling from a chronically implanted catheter. Their basal values were 65 +/- 47 pg/ml for E and 145 +/- 58 pg/ml for NE, which were significantly lower (p less than 0.05) than values in a second group of conscious dogs trained in the same manner but sampled soon after arrival to the laboratory (E = 144 +/- 93 pg/ml; NE = 193 +/- 86 pg/ml). Catecholamine levels in dogs anesthetized with one of three different regimes commonly used in cardiovascular studies were shown to be similar to the basal values found in conscious dogs acclimatized to the laboratory. The weak correlations found between basal plasma catecholamines and hemodynamic variables in all groups of conscious dogs reflect the complexity of factors interacting with the sympathetic nervous system in the maintenance of arterial pressure. These results document the variability that can be expected when using catecholamine levels as an index of sympathetic nervous system activity and the necessity of standardizing conditions for sample collection.

Counterregulatory actions of angiotensin-(1-7).

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.

Estrogen regulation of angiotensin-converting enzyme mRNA.

Estrogen replacement therapy is cardioprotective in postmenopausal women; however, the precise molecular mechanisms for this modulation are not fully elucidated. We previously showed that chronic estrogen replacement therapy reduced angiotensin-converting enzyme (ACE) activity in tissue extracts and serum with an associated reduction in plasma angiotensin II. A reverse transcriptase-polymerase chain reaction assay was developed to determine whether estrogen treatment regulates tissue ACE mRNA concentration. Total RNA was isolated from kidney cortex, kidney medulla, lung, and aorta of ovariectomized Sprague-Dawley rats after 21 days of chronic 17beta-estradiol replacement therapy (5 mg pellet per rat SC) or placebo. A marked decrease in densitometric intensity ratios of amplified ACE cDNA to elongation factor-1alpha control cDNA was observed in all tissues from placebo-treated rats compared with the estradiol-treated rats (renal cortex: 0.29+/-0.04 versus 0.14+/-0.02; renal medulla: 0. 37+/-0.04 versus 0.24+/-0.03; lung: 4.49+/-0.37 versus 2.49+/-0.59; and aorta: 0.41+/-0.04 versus 0.29+/-0.02; all P<0.05). A comparable reduction in ACE activity was detected in tissue extracts from kidney cortex, kidney medulla, and lung of hormone-treated animals. Incubation of purified rat lung ACE with 1 or 10 micromol/L 17beta-estradiol had no effect on enzyme activity. These results suggest that estrogen treatment regulates tissue ACE activity by reducing ACE mRNA concentrations. Thus, the beneficial cardiovascular effects of estrogen may be mediated in part by downregulation of ACE with a consequent reduction in the circulating levels of the vasoconstrictor angiotensin II, a decrease in the metabolism of the vasodilator bradykinin, and an increase in the production of the vasorelaxant angiotensin-(1-7).

Catecholamines and serotonin in the area postrema of normal and sodium-depleted dogs.

Sodium depletion, a maneuver that is accompanied by a 14-fold elevation of plasma renin activity (PRA), alters the norepinephrine concentration of the canine area postrema (AP), a circumventricular organ of the 4th ventricle known to be sensitive to circulating angiotensin II. The norepinephrine concentration of the AP after 3 weeks of sodium depletion decreased by 43%, whereas the concentration of epinephrine and dopamine and the activity of phenylethanolamine-N-methyltransferase (PNMT) did not change. In the pyramidal tract (PT) and choroid plexus (CP) catecholamines were present in significantly lower amounts than in the AP; their concentrations were unaffected by sodium depletion in the PT, but in the CP the norepinephrine concentration was reduced. Serotonin was present in the AP but its concentration was unaltered by sodium depletion. These findings provide evidence that sodium depletion produced an alteration in the concentration of norepinephrine of the area postrema without any change in the concentration of epinephrine, dopamine or serotonin.