Changes in active and inactive renin throughout pregnancy.

In the first trimester of pregnancy, inactive renin in plasma rapidly increases (to 5 times the average concentration in plasma of nonpregnant controls), then declines slowly until midpregnancy, and falls quickly to the normal range after delivery. Inactive renin has the same large apparent molecular weight in pregnancy as in control plasma. Amniotic fluid contains very high levels of inactive renin; its mobility on Sephadex G-100 is the same as that of inactive plasma renin, but a lower molecular weight is indicated by the delayed elution of inactive renin of amniotic fluid from Sephacryl S-200. This anomalous behavior is probably responsible for the different estimates of molecular is probably responsible for the different estimates of molecular weight previously reported. The plasma concentration of active renin in pregnancy is modestly increased in the first trimester, declining gradually until term, and falling quickly after delivery. Although the increased PRA in early pregnancy involves an increase in active renin, increased angiotensinogen appears to play a more important part in sustaining the increased PRA of late pregnancy. The apparent molecular weight of te active renin in pregnancy plasma is larger than that in normal plasma. Gross changes in sodium intake during pregnancy result in changes in active and inactive renin concentrations parallel to those observed in nonpregnant controls. These responses suggest that the kidneys are an important source of the altered plasma renin in pregnancy, but do not exclude a contribution from other sources.

Human kidney cathepsins B and H activate and lower the molecular weight of human inactive renin.

Cathepsins B, H, and D, extracted from human kidneys, activate human inactive renin. Inactive renin, obtained from human kidneys, contains two components of Mr 53,000 and 50,000. Upon incubation with 0.5 microM cathepsin B, the Mr of the larger component decreased progressively to 45,000 (similar to the Mr of active renin) without appreciable loss of renin activity. Cathepsin H also activated and decreased the Mr of kidney inactive renin. Plasma inactive renin was activated by the thiol proteases, cathepsins B and H, with less reduction in Mr than that observed in kidney renin.

Inactive renin of high molecular weight (big renin) in normal human plasma. Activation by pepsin, trypsin, or dialysis to pH 3.3 and 7.5.

Normal plasma contains inactive renin, which becomes active when plasma is dialyzed to pH 3.3 and to pH 7.5, or treated with pepsin or trypsin. Under optimal conditions, each of these procedures activated the same quantity of renin, which was not further increased by repeating or combining two procedures, thus suggesting that the same pool of inactive renin was activated by each procedure. When plasma was fractionated by gel filtration, dialysis activated very little renin in eluates. Trypsin activated renin, but under some conditions also destroyed renin. Pepsin fully activated the inactive renin in eluates without evidence of destruction of renin. The pepsin-activated renin of normal plasma eluted from Sephadex G-100 in a peak of apparent molecular weight (MW) 58,000 and from Sephacryl S-200 with apparent MW 53,000, like big renin in plasma of patients with diabetic nephropathy. Inactive renin was usually increased in amount in plasma of sodium-depleted normal men, but the elution volume did not change with sodium intake. When renin was fully activated in plasma incubated with pepsin or trypsin, the apparent MW of the main peak of big renin did not change appreciably. Inactive renin in plasma was usually increased after sodium depletion, but the elution volume did not change. Active renin of normal plasma had an apparent MW near 41,000 on both gels. Thus, we conclude that big renin is present in normal plasma in amounts at least equal to and usually greater than active renin (the ratio depending on sodium intake) and that pepsin activation readily demonstrates big renin in eluates from gel filtration.

A comparison of cold and acid activation of big renin and of inactive renin in normal plasma.

Normal human plasma contains "inactive renin," whose ability to generate angiotensin I increases after exposure to pH 3.3. Big renin is a partially inactive enzyme of larger molecular weight, which is also activated at pH 3.3, and is found of pregnant women, and in amniotic fluid, but not in normal plasma. We have compared the effects of acid exposure and storage at 4 and -4 C on normal plasma and plasma containing big renin. The concentration of inactive renin in normal plasma was approximately equal to that of normal active renin, and its activity increased slowly on prolonged standing at -4 but not 4 C. In contrast, the activity of big renin increased by 50% as early as 1-3 days at 4 C and increased even more quickly at -4 C. Acid treatment of plasma containing big renin caused 4-10 times greater increase in active renin than similar treatment of normal plasma. During gel filtration, both cold-activated and previously acidified big renin coeluted with unactivated big renin. These data indicate that big renin is highly susceptible to cold or acid activation and that such activation of big renin does not result in a detectable decrease in its molecular weight of 60,000 daltons. Furthermore, acid and cold seem to activate the same pool of inactive renin in normal plasma. Although both normal and big renin are stable for long periods below -20 C, a serious overestimate of plasma renin activity can occur if plasma is stored just above its freezing point before assay.

Big renin in plasma of healthy subjects on high sodium intake.

Normal human plasma contains not only active renin but also an inactive form of renin which, after exposure to low pH, can generate angiotensin I from renin substrate. When healthy volunteers were given first a diet containing 400 mmol sodium and then a diet containing 10 mmol sodium for 4 days the changes in salt intake stimulated large changes in active plasma-renin and smaller changes in inactive renin. Inactive renin comprises a larger fraction of total renin in plasma of salt-loaded healthy subjects than salt depleted subjects. When plasma of healthy men on a high-salt diet was applied to a column of 'Sephadex G-100', renin eluted in two peaks, corresponding to big renin (60 000 daltons) and normal renin of lower molecular weight (40 000 daltons). Active and inactive forms of renin were present in both peaks. Plasma from salt depleted healthy subjects showed a large single peak of renin activity with a maximum at 40 000 daltons. These studies demonstrate that both big and small renin can exist as inactive or active enzyme. Big renin, previously found in certain diseases and in pregnancy, is also present in normal human plasma. These observations suggest a possible physiological role for big renin.

Evidence for the involvement of microtubules in wound contraction.

Many theories have been proposed for the mechanism of wound contraction, that phenomenon of wound closure in which the skin surrounding the tissue defect is drawn into the open wound. When agents that inhibit microtubular function, such as vinblastine and colchicine, were topically applied to actively contracting wounds, contraction stopped. Cytochalasin B, an agent that reportedly disrupts microfilaments, did not alter contraction. These results suggest that wound contraction is related to the functioning of microtubules in fibroblasts within the wound and is proceeding at its maximal rate. The results tend not to support the theory that the microfilament components of cells are involved in wound contraction.

Tissue gas tensions and oxygen consumption in healing bone defects.

A technique for measuring the gaseous environment during the reconstitution of a large bone defect is described. Extensive testing of this system over the past 10 years has demonstrated its validity in measuring the average extracellular tissue pO-2 and pCO-2. Histology of the tissue surrounding the tonometer was obtained and correlated with the gaseous measurements. New bone formation in the healing segmental bone defects has been shown to take place under hypoxic conditions. The oxygen consumption of the surrounding tissue was determined and correlated with the histology and tissue gas measurements. It was demonstrated that the oxygen consumption was not elevated during the period of new bone formation, reflecting a state of anaerobic metabolism. The hypoxic conditions persist until the vascularity increases to match the cellularity. Thephysiological role of oxygen in osseous repair is presented. The present technique as well as the previously published microelectrode technique have demonstrated bone formation in vivo can take place under hypoxic conditions but the mechanism is not known and requires further investigation.