Hemorrhage in rats produces reflex decreases in heart rate (HR) and renal sympathetic nerve activity (RSNA). Because serotonergic antagonists attenuate hemorrhage-induced vagal-mediated bradycardia, we determined whether blockade of serotonin synthesis by p-chlorophenylalanine (PCPA) or of serotonin receptors with methysergide would also abolish the renal sympathoinhibition. Mean arterial pressure (MAP), HR, and RSNA were recorded in chloralose-anesthetized rats pretreated with PCPA (300 mg.kg-1.day-1 X 3 days ip, n = 12) or vehicle (0.3 ml saline, n = 9). During hemorrhage, where MAP was maintained at 50 mmHg for 8 min, vehicle-treated rats decreased HR by 27 +/- 13 beats/min and RSNA by -55 +/- 7%. In PCPA-treated rats, HR and RSNA did not change. Cervical vagotomy abolished the bradycardia and sympathoinhibition during hemorrhage. After acute administration of methysergide (400 micrograms/kg iv, n = 8) hemorrhage produced increases of RSNA, whereas vehicle (0.5 ml saline, n = 7) preserved the renal sympathoinhibition to hemorrhage in conscious rats. Finally, volume expansion (0.88 ml blood/100 g body wt) produced comparable decreases in RSNA in sinoaortic-denervated rats pretreated with PCPA (n = 11) or vehicle (n = 10) (-58 +/- 9 vs. 47 +/- 7%, respectively). We conclude that serotonergic mechanisms are critically involved in vagal afferent inhibition of RSNA during severe hemorrhage in rats.

SUMMARY We have studied the dog as a potential model for the human plasma prorenin-renin system. On a regular sodium intake, healthy conscious dogs apparently have a much lower plasma renin activity (PRA) than healthy human volunteers. Cryoactivation of prorenin is virtually absent in dogs, in contrast to that in humans, but becomes more effective after preacidification of the plasma. The concentration of trypsin required for optimal activation of prorenin is 6 to 10 times higher for dog plasma, revealing a prorenin:renin ratio about 10 times greater than in humans. Dialysis of posttryptic plasma decreases the PRA, but it remains 5 times higher than in pretryptic plasma, indicating that activation is not totally dependent on any renin system component that has been rendered dialyzable by trypsin, e.g., substrate converted to tetradecapeptide (TDP). This argues against the view that tryptic activation is attributable to angiotensin production from TDP by the action of cathepsin D, rather than from new renin converted from prorenin. The posttryptic increase in PRA is evident whether plasma incubation is carried out at pH 6.0 or at 7.4, and can be largely blocked by pepstatin, which also implicates a prorenin-renin mechanism rather than TDP-cathepsin. The low PRA in dogs, the negligible cryoactivation and its improvement by preacidification, and the requirement and tolerance of high trypsin concentrations, all point to greater protease inhibition in dog plasma and/or departures from the enzyme(s) responsible for human prorenin activation. Moreover, the tryptic activation of prorenin is not completed quickly as in human plasma, but carries over into the posttryptic stage of angiotensin generation, even in the presence of excess soybean trypsin inhibitor (SBTI), and other potent inhibitors. Such ongoing prorenin activation cannot be attributed only to trypsin itself, nor to kallikrein (both are inhibited by SBTI), but rather to some other enzyme(s) derived by the action of trypsin. This new prorenin convertase activity (possibly renin itself) can be effectively transferred from trypsinized to control dog plasma, in which it greatly accelerates prorenin activation. Thus, contrary to other reports, dog plasma has a high content of activatable prorenin, and with appropriate methodological changes, the dog can be used as an animal model for physiological and biochemical studies of the prorenin-renin system. (Hypertension 5:277-285, 1983) KEY WORDS • trypsin-activation • cryoactivation • pepstatin non-serine proteases • inhibitors * autocatalysls T

To determine the effects of moderate versus severe dietary sodium restriction on the development of 2-kidney, 1-clip (2K,1C) hypertension, young male Wistar rats were placed on diets containing 9, 26, or 101 (control) mumol sodium/g food. Three days later, a solid silver clip (i.d. 0.20 mm) was placed on the left renal artery and diets were continued up to 6 weeks. Adult rats received a 0.25-mm clip. In young clipped rats receiving the 101 mumol/g diet, blood pressure (BP), plasma renin activity (PRA), and BP response to captopril were increased as early as 1 week after clipping and increased further over time. Moderate sodium restriction (26 mumol sodium/g) led to only a slight delay in the development of hypertension; the levels of BP and PRA, the BP response to captopril, and the extent of cardiac hypertrophy achieved by 6 weeks were not different between the 2K, 1C rats receiving 26 or 101 mumol sodium/g. Sodium restriction to 9 mumol/g decreased rate of growth and completely prevented the rise in BP and in left ventricular weight. At 3 and 6 weeks the severely sodium-restricted rats had significantly higher PRA levels than the 2K, 1C control group. However, the BP response to captopril was attenuated relative to the other hypertensive groups. In adult rats, this level of sodium restriction had a small, but significant effect on body weight, but still prevented the increase in BP and in left ventricular weight. In conclusion, dietary sodium restriction can prevent the development of 2K,1C hypertension in both young and adult rats, but only if the restriction is severe. This effect may relate to a marked reduction in the pressor effectiveness of the renin-angiotensin system by low sodium intake per se or by associated metabolic or other changes.

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In human plasma, trypsin activates "prorenin" within 1 min at 23 degrees C. It is quickly neutralized by endogenous inhibitors, and the subsequent hourly expression of old and new renin activity (PRA) is relatively consistent during 15, 30, or 60 min incubation at 37 degrees. In dog plasma, prorenin activation requires much higher trypsin concentrations - 3-5 mg/ml, vs 0.5-1.5 mg in humans - implying a higher content of endogenous protease inhibitors, and/or the lack of some endogenous mediator of the action of trypsin. These could also be partly responsible for the observed lack of cryoactivation in dogs. The effect of trypsin in dog plasma does not end abruptly as in humans. A post-tryptic prorenin "convertase" continues to act at 37 degrees, steadily increasing the hourly rate of angiotensin generation as the incubation is prolonged. Neither lima bean trypsin inhibitor (LBTI) nor endogenous inhibitors fully inhibit this trypsin-induced convertase. It is transferable to normal plasma, where it raises the PRA. Pepstatin severely inhibits this effect, most probably by inhibiting the new renin, possibly also by inhibiting the convertase itself. Rat plasma appears intermediate between human and dog plasmas in some respects. Trypsin activates prorenin well at 4 mg/ml, when exposed for 30 min at 23 degrees, provided the subsequent PRA incubation stage is kept short, e.g. 10 vs 30 min. This implies a low tolerance to effective concentrations of trypsin, presumably attributable to the nature and/or quantity of endogenous protease inhibitors. The amount of prorenin, as judged by activation, equals that of dogs. However, active renin is distinctly higher in rats, possibly due to the stressful influence of anesthesia and blood collection. This greatly reduces the prorenin: renin ratio in rats relative to dogs, and brings them closer to the human ratio. Clamping off the renal blood vessels while blood is collected, lowers the basal PRA, and raises the prorenin:renin ratio. Thus, prorenin is detectable in all 3 species, but the best methods for activating it are quite different, implying marked differences in the mechanisms involved.