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Angiotensin-converting enzyme 2 (ACE2), a monocarboxypeptidase capable of metabolizing angiotensin II (Ang II) into angiotensin-(1-7) [Ang-(1-7)], has emerged as a potential therapeutic target. We hypothesized that ACE2 is a negative regulator of Ang II-mediated pathological effects in vivo. In Wistar-Kyoto (WKY) rats, Ang II infusion (0.1 μg min(-1) kg(-1)) induced a pressor response, activation of NADPH oxidase and generation of superoxide in the heart, kidney and blood vessels; these effects were significantly blunted by recombinant human ACE2 (rhACE2; 2 mg kg(-1)), in association with a lowering of plasma Ang II and elevation of Ang-(1-7) levels. In the spontaneously hypertensive rat (SHR) model, rhACE2 (2 mg kg(-1) day(-1)) delivered over a 14 day period partly corrected the hypertension, the NADPH oxidase activation and the increased superoxide generation in the heart, kidney and blood vessels. Treatment with rhACE2 inhibited Ang II-mediated phosphorylation of the myocardial extracellular signal-regulated kinase 1/2 pathway in WKY rats, with congruent results seen in SHR hearts. Hence, rhACE2 is an important negative regulator of the Ang II-induced pressor response and NADPH oxidase activation and suppresses pathological myocardial signalling, thereby providing a novel therapeutic agent with which to antagonize an activated renin-angiotesin system.

In portal hypertension, development of a hyperdynamic circulation is preceded by transient mesenteric vasoconstriction. Portal hypertension increases splenic venous outflow pressure. We hypothesized that this causes direct reflex activation of mesenteric vasoconstrictor nerves and splenorenal reflex-mediated activation of the renin–angiotensin system. In anaesthetized male rats, we measured mesenteric efferent nerve activity and mesenteric vascular conductance (MVC) after selectively elevating splenic venous pressure. Partial splenic vein occlusion raised splenic venous pressure (from 4.8 ± 0.4 to 24.1 ± 0.3 mmHg; n= 18) and induced a significant increase in mesenteric efferent nerve activity (from 23.2 ± 3.3 to 31.6 ± 3.5 spikes s−1; n= 11); this response was abolished by prior splenic denervation (from 32.4 ± 2.4 to 31.2 ± 1.6 spikes s−1; n= 7). Mesenteric vascular conductance, the ratio of superior mesenteric artery blood flow to mean arterial pressure, fell upon splenic vein occlusion (ΔMVC =−0.0120 ± 0.0014 ml min−1mmHg−1; P < 0.05, n= 10). This was attenuated by splenic denervation (ΔMVC =−0.0044 ± 0.0018 ml min−1mmHg−1; P < 0.05, n= 8), but unaffected by mesenteric denervation (ΔMVC =−0.0145 ± 0.0020 ml min−1mmHg−1; n= 6) or bilateral renal denervation (ΔMVC =−0.0106 ± 0.0021 ml min−1mmHg−1; n= 5). Localized blockade of mesenteric vascular angiotensin II type 1 (AT1) receptors significantly attenuated the response (ΔMVC =−0.0058 ± 0.0017 ml min−1mmHg−1; P < 0.05, n= 5), whereas blockade of both AT1 and α1-adrenergic receptors caused a significant increase in mesenteric conductance (ΔMVC =+0.0033 ± 0.0010 ml min−1mmHg−1; P < 0.05, n= 6). Our evidence suggests that increased splenic venous outflow pressure reflexly activates adrenergic/angiotensinergic mesenteric nerves, vasodilator mesenteric nerves and the renin–angiotensin system. We propose that obstruction to splenic venous outflow, such as would normally accompany portal hypertension, induces reflex mesenteric vasoconstriction independently of the increase in portal venous pressure.

We developed a novel model using fluorescent intravital microscopy to study the effect of atrial natriuretic peptide (ANP) on the extrasplenic microcirculation. Continuous infusion of ANP into the splenic artery (10 ng min−1 for 60 min) of male Long–Evans rats (220–250 g, n= 24) induced constriction of the splenic arterioles after 15 min (−7.2 ± 6.6% from baseline diameter of 96 ± 18.3 μm, mean ±s.e.m.) and venules (−14.4 ± 4.0% from 249 ± 25.8 μm; P < 0.05). At the same time flow did not change in the arterioles (from 1.58 ± 0.34 to 1.27 ± 0.27 ml min−1), although it decreased in venules (from 1.67 ± 0.23 to 1.15 ± 0.20 ml min−1) and increased in the lymphatics (from 0.007 ± 0.001 to 0.034 ± 0.008 ml min−1; P < 0.05). There was no significant change in mean arterial pressure (from 118 ± 5 to 112 ± 5 mmHg). After continuous ANP infusion for 60 min, the arterioles were dilated (108 ± 16 μm, P < 0.05) but the venules remained constricted (223 ± 24 μm). Blood flow decreased in both arterioles (0.76 ± 0.12 ml min−1) and venules (1.03 ± 0.18 ml min−1; P < 0.05), but was now unchanged from baseline in the lymphatics (0.01 ± 0.001 ml min−1). This was accompanied by a significant decrease in MAP (104 ± 5 mmHg; P < 0.05). At 60 min, there was macromolecular leak from the lymphatics, as indicated by increased interstitial fluorescein isothiocyanate–bovine serum albumin fluorescence (grey level: 0 = black; 255 = white; from 55.8 ± 7.6 to 71.8 ± 5.9, P < 0.05). This study confirms our previous proposition that, in the extrasplenic microcirculation, ANP causes greater increases in post- than precapillary resistance, thus increasing intrasplenic capillary hydrostatic pressure (Pc) and fluid efflux into the lymphatic system. Longer-term infusion of ANP also increases Pc, but this is accompanied by increased ‘permeability’ of the extrasplenic lymphatics, such that fluid is lost to perivascular third spaces.

We have previously shown that the splenorenal reflex controls renin release through splenic afferent and renal sympathetic nerves. We proposed that this reflex would also affect renal blood flow (RBF). RBF was measured in male Long Evans rats using transit-time flow probes. There were no significant differences between any of the experimental groups with respect to baseline values of RBF (8.9 +/- 0.4 ml min(-1), n= 25) or mean arterial pressure (MAP, 98.7 +/- 2.5 mmHg, n= 25). Splenic venous pressure was selectively raised (from 7.9 +/- 0.6 to 21.6 +/- 0.3 mmHg, n= 25) in anaesthetized rats by partial ligation of the splenic vein. This caused an immediate fall in RBF (-2.1 +/- 0.2 ml min(-1), n= 7) and in MAP (-12.4 +/- 2.8 mmHg, n= 7). The fall in RBF, but not the fall in blood pressure, was attenuated by renal denervation (DeltaRBF: - 0.7 +/- 0.1 ml min(-1), n= 6), splenic denervation (DeltaRBF: -0.8 +/- 0.1 ml min(-1), n= 6) and close renal arterial injection of the alpha1-adrenergic blocker phenoxybenzamine (12.5 microg; DeltaRBF: -0.8 +/- 0.1 ml min(-1), n= 6). Renal conductance fell only in the intact control group, i.e. the residual fall in RBF in the denervated and phenoxybenzamine-treated animals could be attributed to the fall in MAP. We also showed that splenic vein occlusion increased both splenic afferent (from 3.0 +/- 0.3 to 6.6 +/- 0.6 spikes s(-1), n= 5) and renal efferent (from 24.8 +/- 2.0 to 50.2 +/- 4.9 spikes s(-1), n= 9) nerve activity. We conclude that obstruction to splenic venous outflow, such as would occur in portal hypertension, initiates increased splenic afferent nerve activity and renal vasoconstriction through the splenorenal reflex, as well as a fall in blood pressure. We propose that this contributes to the renal and cardiovascular dysfunction observed in portal hypertension.

Atrial natriuretic factor (ANF(1-28); ANF) is important in fluid volume homeostasis. It is released in response to atrial distention, as would normally occur during hypervolaemia (Levin et al. 1998). Exogenously administered ANF causes an increase in haematocrit and a decrease in plasma volume, which cannot be accounted for by urinary losses (De Bold et al. 1981). We have shown that these responses are abolished by splenectomy (Kaufman, 1992) and that ANF causes a sustained increase in the haematocrit of blood as it passes through the spleen (Deng & Kaufman, 1996). Since the rat spleen is non-contractile and has virtually no blood storage capacity (Reilly, 1985), the additional red cell mass cannot originate from splenic reservoirs. The increase in haematocrit must therefore derive from a reduction in plasma volume. Indeed, further investigations have revealed that, under normovolaemic conditions, about 25 % of fluid volume flowing into the spleen is removed from the circulating blood and drains into the systemic lymphatic system (Chen & Kaufman, 1996). These findings have led us to believe that the spleen is a major site of ANF-induced translocation of fluid out of the intravascular space. ANF could potentially cause this increase in fluid extravasation by altering capillary permeability within the spleen, as it has been shown to do this at high doses in a variety of tissues (Trippodo & Barbee, 1987). However, splenic capillary beds have been proven to have a discontinuous endothelium (Takubo et al. 1999), which makes it unlikely that fluid extravasation results from increased capillary permeability. Furthermore, we have shown that there is no change in plasma protein concentration of blood flowing through the spleen, and that lymph draining from the spleen is isoncotic to the plasma; this indicates that the capillary beds are indeed freely permeable to plasma proteins (Kaufman & Deng, 1993). Therefore, we have proposed that changes in splenic fluid extravasation in response to physiological levels of ANF occur, not through changes in capillary permeability, but via alterations in splenic haemodynamics to increase intrasplenic filtration pressure. Such a mechanism would be analogous to the role of ANF in the kidney, where it raises glomerular filtration pressure, thereby increasing glomerular filtration rate (Marin Grez et al. 1986). The present study was designed to determine the mechanism by which ANF increases fluid efflux from the spleen. The experiments were done in a blood-perfused rat spleen, which enabled us to measure changes in arterial and venous blood flow, intrasplenic microvascular pressure, and pre-/post-capillary resistance in response to close-arterial infusion of ANF.

1. Atrial natriuretic factor (ANF) causes a reduction in plasma volume that is abolished by splenectomy. Experiments were conceived to investigate whether ANF acts within the spleen to increase efflux of fluid from the intravascular to the extravascular space. 2. ANF, infused into the splenic artery of anaesthetized rats at rates of 1, 5 and 20 ng min -1, caused a dose-dependent increase in the arteriovenous difference in haematocrit as blood flowed through the spleen (basal difference, 0.18 +/- 0.10%; difference after 10 min at 20 ng min -1 ANF, 1.5 +/- 0.18%; n = 6). There was no such change in plasma protein concentration. 3. ANF (20 ng min -1) did not alter splenic arterial blood flow. However, splenic venous blood flow fell so that the arteriovenous difference increased significantly (basal difference, 0.34 +/- 0.19 ml min -1; difference at 60 min, 1.1 +/- 0.20 ml min-1, n = 7). There was no change in mean arterial pressure. 4. These data confirm our hypothesis that ANF acts within the spleen to increase fluid efflux from the intravascular to the extravascular space. Since there is no change in total splenic blood flow, we propose that the effects of ANF are mediated by dilatation of the splenic afferent arterioles and constriction of the efferent venules, thus increasing filtration pressure.

1. Previous evidence has suggested that fluid is extracted from the blood during its passage through the splenic circulation. In order to further investigate this phenomenon, flow probes were placed around the splenic artery and vein of rats. Splenic blood flow and mean arterial blood pressure were measured immediately after surgery, while the rats were still anaesthetized. Five days later, the measurements were repeated first in conscious rats, and then after administering sodium pentobarbitone. Final measurements were made in the conscious animals at day 10 post surgery. 2. Splenic arterial blood flow was lowest at the time of surgery. It had doubled by day 5, and remained stable thereafter at about 8 ml min-1. Splenic venous blood flow also increased after surgery, but less so than the arterial flow, so that the arteriovenous difference had increased to 2.0 +/- 0.4 ml min-1 by day 5. 3. In response to sodium pentobarbitone (20 mg I.V.), there was a transient fall in splenic arterial blood flow from 6.7 +/- 0.9 to 4.9 +/- 0.7 ml min-1. There was no significant change in the arteriovenous difference of blood flow. 4. In conclusion, we have shown that splenic blood flow is considerably higher than previously reported. We have also revealed a significant new path for fluid efflux from the intravascular space; at least 25% of fluid volume flowing into the splen is removed from the circulating blood. 5. Although anaesthesia modulates splenic blood flow, the changes are not as pronounced as those observed in the acute surgically prepared animal.

1. We investigated the control of atrial natriuretic factor (ANF) secretion during pregnancy. 2. Plasma ANF levels were measured in conscious virgin female rats under basal conditions, and after atrial distension with an indwelling balloon catheter. The rats were then mated, and the measurements repeated at 7, 14 and 21 days of pregnancy, and at 1 week postpartum. Plasma ANF levels were also measured in ovariectomized rats injected with progesterone, oestradiol, or oestradiol plus progesterone. 3. Basal plasma ANF levels were elevated at 7 and 14 days of pregnancy, but returned to prepregnant levels by 21 days. At 1 week postpartum, they were again elevated. 4. In response to atrial stretch, plasma ANF increased significantly in virgin rats (from 100 +/- 10 to 148 +/- 13 pg ml-1, P < 0.001, n = 20). In contrast, there was no such secretory response observed in the pregnant and postpartum animals i.e. stretch-induced secretion of ANF was markedly attenuated. 5. Treatment with exogenous oestradiol caused a significant increase in plasma ANF levels in acyclic rats. However, neither progesterone nor a combination of oestradiol plus progesterone had any effect. 6. It is concluded that basal and stretch-induced ANF secretion are differentially influenced by pregnancy; oestradiol is identified as a potential stimulatory factor.

1. We tested the hypothesis that hypervolaemia causes an increase in intrasplenic filtration of cell-free fluid out of the vasculature. To this end we developed a preparation in the anaesthetized rat whereby the splenic vein could be non-occlusively cannulated. 2. Haematocrit and plasma protein concentrations were measured in the splenic afferent and efferent blood supplies. 3. In response to volume loading with saline (1% body weight), there was a sustained increase in the arterial-venous differential of haematocrit, i.e. there was a relative increase in the haematocrit of the blood draining from the spleen. There was no such change in plasma protein concentration. By contrast, this degree of volume loading had no effect on the haematocrit of blood passing through the hindquarters of the animal. 4. Following volume expansion, there was no significant difference in the protein concentration of the plasma and the lymph fluid collected from the splenic lymphatic duct. 5. Distension of the superior vena caval-right atrial junction by means of a small inflatable balloon, caused a similar increase in the splenic venous haematocrit, and again, no change in plasma protein concentration. 6. We interpret these results to mean that, in response to expansion of the intravascular space, there is increased intrasplenic filtration of plasma out of the blood and into the lymphatic system.

1. Extracellular fluid deficits of 33% were produced in male Long-Evans rats by peritoneal dialysis. The conscious, unrestrained animals were then infused I.V. for 30 min with atriopeptin III at doses of 0.01, 0.1, 0.5 and 1.0 micrograms/min. At 5 min into the infusion, the rats were offered water and subsequent intakes were monitored. Since atrial natriuretic peptide (ANP) causes hypotension, one group of control animals was given an injection of diazoxide sufficient to match this fall in blood pressure. 2. A similar group of rats was prepared for measurement of plasma ANP achieved by infusion. 3. Relative to the saline-infused controls, atriopeptin III did not reduce water intake. Indeed, intake was increased at the highest dose of 1.0 micrograms/min. 4. Relative to the diazoxide controls, water intake was influenced by atriopeptin III in a dose-dependent manner, the greatest attenuation being observed at infusions of 0.1 microgram/min. 5. Infusion of atriopeptin III at 0.1 microgram/min caused plasma ANP levels to rise from 252 +/- 21 to 532 +/- 136 pg/ml (n = 9, P less than 0.05) at 15 min. The lowest dose (0.01 microgram/min) caused no detectable increase in plasma levels. 6. It is concluded that, in groups of hypovolaemic rats matched for blood pressure, atriopeptin III caused a dose-related reduction in water intake.