Nicotine patches are available as an over-the-counter medication for aid in smoking cessation. This study was designed to determine how nicotine patch therapy over time and dose ranges used in smoking cessation programs in humans affects endothelium-dependent relaxations. Dogs were treated with nicotine patches (11, 22, or 44 mg/day) for 2 and 5 wk. Circulating nicotine and oxidized products of nitric oxide (NOx) were measured. Coronary arteries were prepared for measurement of isometric force and aortic endothelial cells were prepared for measurement of mRNA or NO synthase (NOS) activity. Circulating nicotine increased with increasing concentrations of nicotine patches. After 5 wk of treatment with 22 mg/day patches, circulating NOx was reduced but NOS activity was increased. NOS mRNA was similar among groups. Only after 5 wk of treatment with 22 mg/day patches were endothelium-dependent relaxations reduced to alpha(2)-adrenergic agonists, ADP, and the calcium ionophore A-23187. These results suggest a time and biphasic dose effect of nicotine treatment on endothelium-dependent responses that may be related to bioavailability of NO. This complex relationship of duration and dose of nicotine treatment may explain, in part, discrepancies in effects of nicotine on endothelium-dependent responses.

Experiments were designed to study effects of raloxifene, a selective estrogen receptor modulator, on venous endothelium and smooth muscle. Rings of femoral veins with and without endothelium from adult gonadally intact, and ovariectomized female pigs were suspended for measurement of isometric force in organ chambers. Concentration-response curves to raloxifene (10-9-10-5 M) were obtained in rings at baseline tension or following contraction with prostaglandin (2 x 10-6 M) in the absence or presence of NG-monomethyl-l-arginine (l-NMMA) (nitric oxide synthase inhibitor), 1H-(1.2.4) oxadiazolo (4,3-A) quinoxalin-1-one (ODQ, soluble guanylate cyclase inhibitor), tetraethylammonium acetate (TEA; potassium channel blocker), or indomethacin (cyclooxygenase inhibitor). Raloxifene caused acute, concentration-dependent relaxations that were greater in rings with than in rings without endothelium from both groups. The l-NMMA significantly inhibited relaxations to raloxifene in rings with endothelium from ovariectomized females whereas TEA only inhibited relaxations in rings with endothelium from intact female pigs. ODQ and indomethacin significantly inhibited relaxations in rings with endothelium from both groups. These results suggest that raloxifene acutely relaxes femoral veins through release of endothelium-derived factors and by direct stimulation of vascular smooth muscle cells. Whether nitric oxide or potassium channel activation contributes to relaxations by raloxifene may depend on ovarian hormonal status of the animal.

endothelium; hyperpolarizing factor; nitric oxide; potassium channels OBSERVATIONAL AND EPIDEMIOLOGICAL studies suggest that oral hormone replacement therapy provides primary prevention of coronary artery disease in postmenopausal women while it also increases the risk of venous thrombosis (5,20, 23,40,49,54,55). The reasons for these apparent opposite effects on the arterial and venous systems are not known. Effects of estrogen (17␤-estradiol) on the arterial circulation are well characterized (35). For example, 17␤-estradiol has both genomic and nongenomic effects on arteries. Some of these effects include rapid and sustained production and release of endothelium-derived nitric oxide (NO), changes in activation of K ϩ and Ca 2ϩ channels, and regulation of intracellular Ca 2ϩ (12,13,36,37,42,45,59). However, it is unknown whether 17␤-estradiol could cause effects in veins as endothelial and vascular smooth muscle cells from veins have estrogen receptors (ER)-␣ (ER␣) and -␤ (ER␤) (22). Information is needed to begin to understand mechanisms of how estrogen treatment increases the risk of venous thrombosis. Therefore, the experiments were designed to determine the acute affects of 17␤-estradiol on femoral veins and whether or not these effects were modulated by the ovarian status of the animal with the use of ovariectomy (by laparoscopy) to mimic menopause. This study fills an important gap in the literature because the acute effects of 17␤-estradiol in arteries are well documented (35), but the effects of 17␤-estradiol on veins are unknown. METHODS Animals.Gonadally intact and ovariectomized (Ovx) female Yorkshire pigs (6 mo old, 110-120 kg) were used for these experiments. The external genitalia from gonadally intact females showed changes associated with an estrus cycle. Serum levels of estrogen from gonadally intact females range from 10 to 30 pg/ml and estrogen levels in Ovx females are below the sensitivity level of assay (4, 57). Uterine weight was used as a bioassay for the efficacy of surgery and was significantly less in Ovx females (51.3 Ϯ 7.8 g) compared with gonadally intact females (86.6 Ϯ 12.3 g). All pigs were fed twice a day with Lean Grow (Land O'Lakes Farmland Feed), given free access to water, and were housed at 22°C on a 12:12-h light-dark cycle.Protocol. Four weeks after ovariectomy, Ovx and agematched gonadally intact female pigs were anesthetized (ketamine and xylazine, 12 and 8 mg/kg, respectively), and the femoral veins were removed. The veins were placed immediately into cold modified Krebs-Ringer bicarbonate solution of the following composition (in mmol/l): 118.3 NaCl, 4.7 KCl, 2.5 CaCl 2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 0.026 Ca 2ϩ disodium EDTA, and 11.1 dextrose. After the adventitia was trimmed, the veins were cut into rings ϳ4-5 mm long. Some rings were stored at Ϫ80°C for subsequent Western blotting to evaluate expression of ERs. Other rings were prepared for organ chamber experiments or immunohistochemistry.Organ chamber experiments. Rings with and without endothelium were ...

-Experiments were designed to determine how ovariectomy modulates mitogenic factors in platelets and how these factors affect proliferation of coronary arterial smooth muscle. Platelet-derived growth factors (PDGFAB and PDGFBB), transforming growth factors (TGF-␤1 and TGF-␤2), and vascular endothelial growth factor (VEGF165) were quantified in platelet lysates and platelet-poor plasma from adult gonadally intact and ovariectomized female pigs by ELISA. Proliferation of cultured coronary arterial smooth muscle cells (SMCs) from both groups of pigs was determined in response to autologous or heterologous platelet lysates. Platelet concentrations of PDGFBB, but not PDGFAB, TGF-␤1, and TGF-␤2, increased with ovariectomy. VEGF165 was not detected in platelets from either group. Proliferation of SMCs from ovariectomized females was significantly greater on exposure to autologous or heterologous platelet lysates than proliferation of SMCs from intact females. These results indicate that ovariectomy increases concentrations of PDGFBB in platelets. Higher levels of PDGFBB in platelets in synergy with other plateletderived products could contribute to increased proliferative arterial response to injury after ovariectomy.

Estrogen replacement increases risk of venous thrombosis. In this study, we determined responses in vitro to platelets and platelet products in veins from adult male and intact and ovariectomized female pigs. When contracted with prostaglandin F(2alpha), platelets (25,000 platelets/microl) caused relaxation in veins with endothelium. Higher numbers of platelets caused contraction in veins with and without endothelium. In veins without endothelium, contractions were greater in veins from male than in veins from female pigs, and contractions in intact female pig veins were greater than in ovariectomized females pig veins. Platelet products 5-hydroxytryptamine and thromboxane (analog U-46619) caused comparable contractions in all veins; contractions to prostacyclin were less in veins from intact female pigs. ADP caused comparable endothelium-dependent relaxations in all groups. These relaxations were increased by indomethacin in veins from intact males and females; with inhibition of nitric oxide, relaxations were comparable in all groups. These results suggest that venous responses to platelets vary with sex and presence of ovaries in female pigs. These variations reflect differences in type and quantity of substances released from platelets as well as the sensitivity of the smooth muscle to some vasoactive substances. In addition, products of cyclooxygenase may reduce endothelium-dependent relaxations in veins.