Estrogen increases endothelial carbon monoxide, heme oxygenase 2, and carbon monoxide-derived cGMP by a receptor-mediated system.

Carbon monoxide, a gaseous activator of soluble guanylyl cyclase formed by a subtype of the enzyme heme oxygenase designated heme oxygenase-2 in vascular endothelium, has been found to dilate blood vessels independently from nitric oxide. Because of the parallels between nitric oxide and carbon monoxide, we speculated that estrogen might affect carbon monoxide production in vascular endothelium. Endothelial cells of human origin (umbilical vein and uterine artery) were incubated for 4 or 24 h with 10(-12)-10(-6) M 17beta-estradiol. 17beta-Estradiol, at a concentration such as that attained during the ovulatory phase of the menstrual cycle (10(-10) M), administrated for 4 h led to a 2-fold increase in intracellular carbon monoxide production and heme oxygenase-2 protein levels (P < 0.05). A reporter assay, measuring the formation of cGMP as the direct product of carbon monoxide-induced activation of soluble guanylyl cyclase in endothelial cells, also revealed a 56% increase in cellular cGMP after treatment with 10(-10) M E2 17beta-estradiol (P < 0.05). By contrast, higher 17beta-estradiol concentrations had no significant respective effects due to nitric oxide synthase inhibition of carbon monoxide release. This 17beta-estradiol effect appeared to be ER dependent, as preincubation with tamoxifen (10(-6) M) blocked the stimulatory effect of 17beta-estradiol in each instance. Our preliminary data indicate a potential role for carbon monoxide as a biological messenger molecule in estrogen-mediated regulation of vascular tone.

Estrogen does not induce the calcium-dependent nitric oxide synthase in cultured human uterine endothelial and myometrial smooth muscle cells.

In many tissues, estrogen-induced vasodilatation is mediated, at least in part, by the release of nitric oxide (NO). We determined whether human myometrial endothelial and smooth muscle cells express estrogen receptors (ERs) and whether endothelial NO synthase (eNOS) expression in these cells was affected by 17beta-estradiol (10[-13]-10[-6]M). ER was strongly expressed in myometrial smooth muscle cells but was absent from endothelial cells. Expression of eNOS mRNA was strong in endothelial cells, but weak in muscle cells. 17beta-estradiol administration for 24 or 72 h failed to increase eNOS in both cell types. Thus, an increase of human uterine blood flow by estrogens appears not to be mediated by stimulation of myometrial eNOS expression.

High precision measurement of electrical resistance across endothelial cell monolayers.

Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 omega cm2) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric field across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 omega resistance changes of the endothelium on top of a 21 omega series resistance of the support and bathing fluid layers. Histamine (10 microM) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 microM) was less effective, PAF (380 nM) and LTC4 (1 microM) had no effect. TEER was also reduced by the calcium ionophore A-23187 (10 microM). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.

Urinary kallikrein excretion during inhibition of endogenous angiotensin II in the pig.

This study was performed to assess the possible contribution of endogenous angiotensin II (AII) to the regulation of urinary kallikrein excretion. The AII antagonist saralasin or the saline vehicle was infused into the aorta above the renal arteries of pigs under halothane-O2/N2O anaesthesia. Systemic and renal functional parameters were followed for 140 min and during stimulation of the reninangiotensin system by haemorrhage. Urinary kallikrein excretion, determined as kininogenase activity, was increased immediately upon both initiation and termination of the 2 h saralasin infusion into pigs not subjected to haemorrhage. Renal cortical blood flow (RCBF) was maintained, in both saline and saralasin-treated animals at blood pressures as low as 70 mm Hg, while glomerular filtration rate was dissociated during saralasin infusion. As long as RCBF was maintained, urinary kallikrein excretion rate was elevated during the progressive hypotension in both saline and saralasin-treated animals. These findings confirm a close relationship between the maintenance of RCBF and increased activity of the kallikrein-kinin system whether or not AII is antagonized, and indicate that during haemorrhage the kallikrein-kinin system is stimulated by a mechanism not involving AII.

Hemodynamics of the isolated perfused rat kidney in the absence and presence of kallikrein substrate.

Rat kidneys were isolated and perfused for 120 minutes at 80 mm Hg with Krebs-Henseleit buffer containing 7 gm% bovine albumin fraction V. Analysis of the bovine albumin revealed the presence of a contaminating kallikrein substrate which exhibited a MW of 55,000 upon gel-filtration on Sephadex G-200, similar to the reported MW of bovine low molecular weight kininogen. The mean values for renal perfusate flow rate (RPF) and glomerular filtration rate (GFR) in kidneys perfused with this bovine albumin depleted of the kallikrein substrate were significantly lower when compared to standard perfusions. In two other groups of isolated kidneys perfused with kallikrein substrate depleted bovine albumin, purified bovine low molecular weight kininogen (bLMWK, 8.5 micrograms protein in 50 microliters per minute) or synthetic bradykinin (Bk, 1 ng in 10 microliters per minute) were infused after the first 30 minute clearance period into the renal artery. The induced hemodynamic changes were similar in that RPF and GFR significantly increased. These studies show that depletion of bovine albumin from kallikrein substrate results in a concomitant decrease of RPF and GFR, while overall renal function is unaffected. Infusion of both bLMWK or BK to kallikrein substrate depleted bovine albumin has similar effects resulting in a simultaneous increase in RPF and GFR. This suggests that endogenous kallikrein from the perfused kidney releases kinin from infused bLMWK and indicates that kinin primarily affects the afferent arterioles.

Effect of the protease inhibitor aprotinin on renal hemodynamics in the pig.

Aprotinin, the serine protease inhibitor that also inhibits glandular (urinary) kallikrein, or vehicle was infused into the aorta above the renal arteries of anesthetized pigs. Renal hemodynamic and functional parameters were followed over time and during hemorrhagic hypotension. Both renal cortical blood flow and glomerular filtration rate were maintained in vehicle-treated animals at mean arterial pressures as low as 70 mm Hg. As long as renal cortical blood flow and glomerular filtration rate were maintained during the progressive hypotension, urinary excretion rate of kallikrein (as defined by kinin-generating activity) was increased. In contrast, all aprotinin-treated animals had a decreased excretion rate, and the renal cortical blood flow declined with the mean arterial pressure during hemorrhage. The pattern of glomerular filtration rate and plasma renin activity was comparable in both aprotinin-treated and vehicle-treated hemorrhaged animals. Our findings suggest that the endogenous renal kallikrein-kinin system is required for functional renal vasodilatation to maintain renal cortical blood flow during hemorrhage and is therefore directly or indirectly responsible for adjustment of preglomerular resistance.