High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation.

Nitric oxide synthase (NOS) contributes to estradiol-17beta (E(2)beta)-induced uterine vasodilation, but additional mechanisms are involved, and the cellular pathways remain unclear. We determined if 1) uterine artery myocytes express potassium channels, 2) E(2)beta activates these channels, and 3) channel blockade plus NOS inhibition alters E(2)beta-induced uterine vasodilation. Studies of cell-attached patches identified a 107 +/- 7 pS calcium-dependent potassium channel (BK(Ca)) in uterine artery myocytes that rapidly increased single-channel open probability 70-fold (P < 0.05) after exposure to 100 nM E(2)beta through an apparent cGMP-dependent mechanism. In ovariectomized nonpregnant ewes (n = 11) with uterine artery flow probes and catheters, local BK(Ca) blockade with tetraethylammonium (TEA; 0.05-0.6 mM) dose dependently inhibited E(2)beta-induced uterine vasodilation (n = 37, R = 0.77, P < 0.0001), with maximum inhibition averaging 67 +/- 11%. Mean arterial pressure (MAP) and E(2)beta-induced increases (P

Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure.

Nitric oxide contributes to estrogen-mediated uterine vasodilation; however, the nitric oxide synthases (NOS) involved and their location within uterine arteries are incompletely documented. We investigated the effects of repetitive daily and acute estradiol-17beta (E(2)beta) exposure on uterine hemodynamics and NOS abundance and localization in uterine arteries from nonpregnant ovariectomized ewes receiving daily intravenous E(2)beta (1 microg/kg, n = 5) or no E(2)beta (n = 7) for 5 days to determine NOS abundance, cGMP contents, and NOS immunohistochemistry. Daily E(2)beta increased basal and E(2)beta-mediated rises in uterine blood flow (UBF) 36 and 43% (<0.01), respectively, calcium-dependent NOS activity 150% (P < 0.02) in endothelium-intact and -denuded ( approximately 40% of total NOS) arteries, and cGMP contents 39% (P < 0.05). Endothelial (eNOS) was detected in luminal endothelium, whereas neuronal NOS (nNOS) protein was only in the media. A second group of ewes received E(2)beta (1 microg/kg iv) for 4 days and acute intravenous E(2)beta (n = 8) or vehicle (n = 4) on day 5. UBF rose 5.5-fold (P < 0.001) 115 min after E(2)beta, at which time only endothelium-derived calcium-dependent NOS activity increased 30 +/- 13% (P < 0.05). Daily E(2)beta enhances basal and E(2)beta-mediated increases in UBF, which parallel increases in endothelium-derived eNOS and smooth muscle-derived nNOS. Acute E(2)beta, however, selectively increases endothelium-derived eNOS.

Angiotensin II indirectly vasoconstricts the ovine uterine circulation.

The uterine vasculature of women and sheep predominantly expresses type 2 ANG II receptors that do not mediate vasoconstriction. Although systemic ANG II infusions increase uterine vascular resistance (UVR), this could reflect indirect mechanisms. Thus we compared systemic and local intra-arterial ANG II infusions in six near-term pregnant and five ovariectomized nonpregnant ewes to determine how ANG II increases UVR. Systemic ANG II dose-dependently (P > 0.001) increased arterial pressure (MAP) and UVR and decreased uterine blood flow (UBF) in pregnant and nonpregnant ewes; however, nonpregnant responses exceeded pregnant (P < 0.001). In contrast, local ANG II infusions at rates designed to achieve concentrations in the uterine circulation comparable to those seen during systemic infusions did not significantly decrease UBF in either group, and changes in MAP and UVR were absent or markedly attenuated. When MAP rose during local ANG II, which only occurred with doses > or =2 ng/ml, increases in MAP were delayed more than twofold compared with responses during systemic ANG II infusions and always preceded decreases in UBF, resembling that observed during systemic ANG II infusions. These observations demonstrate attenuated uterine vascular responses to systemic ANG II during pregnancy and suggest that systemic ANG II may increase UVR through release of another potent vasoconstrictor(s) into the systemic circulation.

Ontogeny of vascular angiotensin II receptor subtype expression in ovine development.

Angiotensin II (ANG II) increases arterial pressure in fetal sheep and may modulate cardiovascular adaptation before and after birth. The type 1 angiotensin II receptor (AT1R) predominates in adult vascular smooth muscle (VSM) and mediates vasoconstriction. In contrast, AT2R predominate in fetal tissues and are not known to mediate contraction. Although sheep are commonly used to study cardiovascular development, the ontogeny and distribution of VSM ATR subtypes is unknown. We examined ATR binding characteristics and subtype expression across the umbilicoplacental vasculature and in aorta, carotid, and mesenteric arteries from fetal (n = 44; 126-145 d gestation) and postnatal (n = 65; 1-120 d from birth) sheep using plasma membranes from tunica media and tissue autoradiography. Binding density (Bmax) was similar throughout the umbilicoplacental vasculature (p = 0.5), but only external umbilical arteries and veins and primary placental arteries expressed AT1R, whereas subsequent placental branches and fetal placentomes expressed only AT2R. Systemic VSM Bmax and binding affinity did not change significantly during development (p > 0.1). Fetal systemic VSM, however, expressed only AT2R, and binding was insensitive to GTPgammaS. Transition to AT1R in systemic VSM began 2 wk postnatal and was completed by 3 mo. Before birth, umbilical cord vessels are the primary site of AT1R expression in fetal sheep, and AT2R seem to predominate in systemic VSM until 2-4 wk postnatal.

Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells.

Although it is well known that progesterone alters uterine contractility and plays an important role in maintenance of pregnancy, the biochemical mechanisms by which progesterone alters uterine contractility in human gestation are less clear. In this investigation we sought to identify progesterone-induced adaptations in human myometrial smooth muscle cells that may alter Ca2+ signaling in response to contractile agents. Cells were treated with vehicle or the progesterone analog medroxyprogesterone acetate (MPA) for 5 days, and intracellular free Ca2+ concentration ([Ca2+]i) was quantified after treatment with oxytocin (OX) or endothelin (ET)-1. OX- and ET-1-induced increases in [Ca2+]i were significantly attenuated in cells pretreated with MPA in a dose-dependent manner. Progesterone receptor antagonists prevented the attenuated Ca2+ transients induced by MPA. ETA and ETB receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted in significant downregulation of ETA and ETB receptor binding. MPA did not alter ionomycin-stimulated increases in [Ca2+]i and had no effect on inositol trisphosphate-dependent or -independent release of Ca2+ from internal Ca2+ stores. We conclude that adaptations of Ca2+ homeostasis in myometrial cells during pregnancy may include progesterone-induced modification of receptor-mediated increases in [Ca2+]i.

Differential development of umbilical and systemic arteries. II. Contractile proteins.

In fetal sheep, umbilical responsiveness to ANG II exceeds systemic vascular responsiveness. Fetal systemic vascular smooth muscle (VSM) exhibits an immature phenotype with decreased contractile protein contents, low 200-kDa myosin heavy chain (MHC) SM2, and significant nonmuscle MHC-B expression, whereas umbilical VSM phenotype is incompletely described. We tested the hypothesis that differences in vascular responsiveness could reflect dissimilarities in VSM phenotype. Actin, MHC, MHC isoforms, and active stresses were compared in strips of femoral arteries and aorta from near-term fetal (n = 12) and adult (n = 12) sheep to those in external and intra-abdominal umbilical arteries. Actin contents in fetal femoral artery and aorta were less (P

Differential development of umbilical and systemic arteries. I. ANG II receptor subtype expression.

In fetal sheep umbilical responses to angiotensin II (ANG II) exceed those by systemic vasculature. Two ANG II receptors (AT) exist, AT1 and AT2, but only AT1 mediates vasoconstriction in adult tissues. Thus differences in reactivity could reflect differences in subtype expression. Using competitive radioligand binding assays, we demonstrated AT1 predominance in umbilical arteries and AT2 in femoral arteries. Steady-state responses to intravenous ANG II (0.229-1.72 micrograms/min) were studied in 16 fetuses with umbilical and/or femoral artery flow probes without and with local AT1 (L-158,809) or AT2 (PD-123319) blockade. ANG II dose dependently (P < 0.001) increased umbilical resistance more than arterial pressure (MAP) while decreasing umbilical blood flow. Femoral vascular resistance also increased dose dependently (P = 0.02), but responses were less than umbilical (P = 0.0001) and paralleled increases in MAP; blood flow was unaffected. Cumulative local doses of L-158,809 (125 micrograms) inhibited all responses (P < 0.001); however, 1,000 micrograms of the AT2 antagonist had no effect. Plasma renin activity (PRA) was unaltered by local AT1 blockade, whereas PRA doubled (P = 0.001) after systemic infusion of only 50 micrograms of the AT1 antagonist and remained elevated. Differences in umbilical and femoral vascular responses to ANG II are in large part due to differences in AT subtype expression. Furthermore, in fetal sheep the ANG II negative feedback on PRA is mediated by AT1 receptors, and it is substantially more sensitive to receptor blockade than the vasculature.

Nitric oxide contributes to estrogen-induced vasodilation of the ovine uterine circulation.

Estradiol-17beta (E2beta), a potent vasodilator, has its greatest effects on the uterine vasculature, blood flow (UBF) increasing > or = 10-fold. The mechanism(s) responsible for E2beta-induced vasodilation is unclear. We determined if nitric oxide (NO)-induced increases in cGMP modulate estrogen-induced increases in UBF, and if cyclooxygenase inhibition modifies E2beta responses. Nonpregnant (n = 15) and pregnant (n = 8) ewes had flow probes implanted on main uterine arteries and catheters in branches of the uterine vein and artery bilaterally for blood sampling and infusion of the NO synthase inhibitor L-nitro-arginine methyl ester (L-NAME), respectively. In nonpregnant ewes E2beta (1 microg/kg) caused parallel increases (P < 0.001) in UBF (15+/-3 to 130+/-16 ml/min) and uterine cGMP secretion (23+/-10 to 291+/-38 pmol/min); uterine venous cGMP also rose (4.98+/-1.4 to 9.43+/-3.2 pmol/ml; P < 0.001). Intra-arterial L-NAME partially inhibited increases in UBF dose-dependently (r = 0.66, n = 18, P < 0.003) while completely inhibiting cGMP secretion (P = 0.025). Indomethacin, 2 mg/kg intravenously, did not alter E2beta-induced responses. After E2beta-induced increases in UBF, intraarterial L-NAME partially decreased UBF dose dependently (r = 0.73, n = 46, P < 0.001) while inhibiting cGMP secretion (178+/-48 to 50+/-24 pmol/min; n = 5, P = 0.006); both were reversed by L-arginine. In pregnant ewes, E2beta increased UBF and venous cGMP (9.1+/-0.96 to 13.2+/-0.96 pmol/ml, P < 0.01); however, intraarterial L-NAME decreased basal cGMP secretion 66% (P = 0.02), but not UBF. Acute estrogen-induced increases in UBF are associated with NO-dependent increases in cGMP synthesis, but other mechanisms may also be involved. However, vasodilating prostanoids do not appear to be important. In ovine pregnancy NO is not essential for maintaining uteroplacental vasodilation.

Tissue specific expression of vascular smooth muscle angiotensin II receptor subtypes during ovine pregnancy.

Uteroplacentral responses to infused angiotensin II (ANG II) are less than those elicited by systemic vasculature. This does not reflect ANG II receptor (AT) downregulation but may reflect differences in AT-receptor subtypes expressed. We examined AT-receptor subtypes in smooth muscle (SM) from uterine (UA), mesenteric, renal, and mammary arteries and aorta from nulliparous (n = 12), pregnant (n = 18; 105-140 days, term = 145 days), postpartum (n = 5; 6-9 days after delivery), and nonpregnant parous (n = 14) ewes by assessing displacement of 125I-labeled ANG II binding by [Sar1, Ile8]ANG II (AT1 and AT2), losartan (AT1) PD-123319 (AT2), and CGP-42112A (AT2). AT2 receptors accounted for 75-90% of total binding in UA. Except for mammary arteries, other arteries expressed only AT1 receptors. Receptor subtype expression was not altered by reproductive state in any artery studied. With the use of autoradiography, AT2 receptors appear to predominate in media of small intramyometrial arteries, whereas AT1 receptors predominate in the luminal portion. We therefore determined which subtype mediates endothelium-derived ANG II-induced increases in UA PGI2 synthesis during pregnancy. ANG II (0.05 microM) increased PGI2 synthesis 62%, from 214 +/- 13 to 346 +/- 23 pg.mg-1.h-1 (P < 0.05). Losartan (1.0 microM) inhibited the rise in PGI2 (257 +/- 24 vs. 238 +/- 25 pg.mg-1.h-1), whereas 1.0 microM PD-123319 had no effect (231 +/- 23 vs. 337 +/- 31 pg.mg-1.h-1; P < 0.05). AT2 receptors do not mediate ANG II-induced vasoconstriction, thus differences in uteroplacental and systemic sensitivity to ANG II may reflect predominance of AT2 receptors in UASM and ANG II-induced increases in UA prostacyclin synthesis by endothelial AT1 receptors.

Angiotensin II receptor characteristics and subtype expression in uterine arteries and myometrium during pregnancy.

The uteroplacental vasculature is less sensitive to angiotensin II (ANG II)-induced vasoconstriction than the systemic vasculature. Although the mechanism(s) responsible is unclear, uterine arteries (Ua) may demonstrate ANG II receptor (AT receptors) down-regulation or expression of AT2 receptors, which do not mediate vasoconstriction. We determined AT receptor binding characteristics and subtype expression in Ua from normotensive pregnant (n = 14; 38 +/- 0.5 weeks gestation) and nonpregnant (n = 28) women. Comparative studies were performed with myometrium, a nonvascular smooth muscle. We measured binding density (Bmax) and affinity (Kd) in plasma membrane preparations employing radioligand binding. Receptor subtypes were assessed by inhibiting [125I]ANG II binding with specific antagonists. During pregnancy, the Ua Bmax and Kd were unchanged (P > 0.1; 221 +/- 36 vs. 159 +/- 27 fmol/mg protein and 0.8 +/- 0.1 vs. 0.9 +/- 0.1 nmol/L, respectively). However, myometrial Bmax decreased 92% (580 +/- 129 vs. 44 +/- 7 fmol/mg protein; P < 0.001), and Kd rose 4-fold (1.5 +/- 0.4 to 6.0 +/- 0.6 nmol/L; P < 0.001). AT1/AT2 expression averaged 15%/85% in Ua from nonpregnant and pregnant women, whereas in myometrium, values were 10%/90% and 60%/40%, respectively. In myometrium from laboring women (n = 8), force (1.38 +/- 0.14 to 1.59 +/- 0.12 x 10(4) N/m2; P < 0.04) and contractile frequency (0.038 +/- 0.05 to 0.116 +/- 0.014 contractions/min; P < 0.001) increased with 10(-5) mol/L ANG II and were abolished by AT1 receptor inhibition. Myometrium from nonpregnant women (n = 3) was unresponsive, and AT2 inhibition did not alter responses. In nonpregnant women, AT2 receptors predominate in Ua and myometrium. Although Ua AT receptors are unaltered during pregnancy, myometrial Bmax decreases, reflecting decreases in the expression of AT2 >> AT1 receptors and differential receptor regulation.

Myometrial angiotensin II receptor subtypes change during ovine pregnancy.

Although regulation of angiotensin II receptor (AT) binding in vascular and uterine smooth muscle is similar in nonpregnant animals, studies suggest it may differ during pregnancy. We, therefore, examined binding characteristics of myometrial AT receptors in nulliparous (n = 7), pregnant (n = 24, 110-139 d of gestation), and postpartum (n = 21, 5 to > or = 130 d) sheep and compared this to vascular receptor binding. We also determined if changes in myometrial binding reflect alterations in receptor subtype. By using plasma membrane preparations from myometrium and medial layer of abdominal aorta, we determined receptor density and affinity employing radioligand binding; myometrial AT receptor subtypes were assessed by inhibiting [125I]-ANG II binding with subtype-specific antagonists. Compared to nulliparous ewes, myometrial AT receptor density fell approximately 90% during pregnancy (1,486 +/- 167 vs. 130 +/- 16 fmol/mg protein) and returned to nulliparous values > or = 4 wk postpartum; vascular binding was unchanged. Nulliparous myometrium expressed predominantly AT2 receptors (AT1/AT2 congruent to 15%/85%), whereas AT1 receptors predominated during pregnancy (AT1/AT2 congruent to 80%/20%). By 5 d postpartum AT1/AT2 congruent to 40%/60%, and > 4 wk postpartum AT2 receptors again predominated (AT1/AT2 congruent to 15%/85%). In studies of ANG II-induced force generation, myometrium from pregnant ewes (n = 10) demonstrated dose-dependent increases in force (P < 0.001), which were inhibited with an AT1 receptor antagonist. Postpartum myometrial responses were less at doses > or = 10(-9) M (P < 0.05) and unaffected by AT2 receptor antagonists. Vascular and myometrial AT receptor binding are differentially regulated during ovine pregnancy, the latter primarily reflecting decreases in AT2 receptor expression. This is the first description of reversible changes in AT receptor subtype in adult mammals.

Ontogeny of angiotensin II vascular smooth muscle receptors in ovine fetal aorta and placental and uterine arteries.

OBJECTIVE: Our purpose was to determine if differences in angiotensin II vascular smooth muscle receptor binding account for the attenuated fetal placental and systemic responses to infused angiotensin II relative to maternal responses and for the differences between umbilical and fetal systemic responses. STUDY DESIGN: Using plasma membranes prepared from the medial layer of fetal aorta, fetal placental artery, and maternal uterine artery obtained between 107 and 134 days of ovine gestation (n = 17), we measured and compared angiotensin II receptor binding density (in femtomoles per milligram of protein) and affinity (in nanomoles per liter) in radioligand binding studies with iodine 125-angiotensin II. Maternal and fetal plasma angiotensin II were also compared. RESULTS: A single class of high-affinity receptor was identified in all arteries. Although the binding density was similar at < 100 and > 130 days for fetal aorta (388 +/- 87 [SE] vs 262 +/- 56), placental artery (319 +/- 95 vs 235 +/- 54), and uterine artery (46 +/- 6 vs 50 +/- 13), values for fetal arteries exceeded those for uterine arteries (p < or = 0.018) in spite of higher fetal plasma angiotensin II (74 +/- 18 vs 30 +/- 8 pg/ml, p < 0.01). Affinity did not differ between arteries and, except for fetal aorta (1.4 +/- 0.2 vs 2.1 +/- 0.4, r = 0.49, p = 0.045), was unchanged during late gestation. CONCLUSION: Differences between fetal and maternal responses and fetal placental and systemic responses to infused angiotensin II in sheep do not reflect alterations in total angiotensin II vascular smooth muscle receptor binding density or affinity.

Stability of cephalexin monohydrate suspension in polypropylene oral syringes.

The stability of cephalexin monohydrate suspension in plastic oral syringes was studied. Commercially available cephalexin monohydrate powder for oral administration was reconstituted according to the manufacturer's instructions and stored in the original containers or drawn into 5-mL clear polypropylene oral syringes. The original containers and syringes were divided into groups and stored at -20, 4, 25, 40, 60, or 80 degrees C. Powder from two additional lots was similarly reconstituted and packaged; these original containers and syringes were stored at 80 degrees C only to assess interlot variability. Immediately after reconstitution and at specified times during storage, three syringes and the corresponding three original containers stored at each temperature were removed, and their contents were analyzed for cephalexin concentration using the standard USP iodometric assay for antibiotics. The stability-indicating nature of the assay was documented. Cephalexin monohydrate followed a first-order rate of degradation at temperatures of 40, 60, and 80 degrees C. At temperatures of -20, 4, and 25 degrees C, cephalexin monohydrate exhibited no appreciable degradation during the 90-day study period. Cephalexin monohydrate suspension reconstituted from powder as a suspension and repackaged in clear polypropylene oral syringes was stable for 90 days when stored under ambient, refrigerated, and frozen conditions.