Fitzpatrick, Lorraine A.

Smoking is a major risk factor for failure of coronary artery bypass grafts (CABG). Experiments were designed to determine effects of transdermal nicotine, independent of smoking, on structure and function of CABG. Saphenous veins were placed as CABG in untreated dogs (control) or in dogs treated with transdermal nicotine (one 11-mg or two 22-mg patches/day) for 5 wk. Serum nicotine and plasma nitric oxide were measured. Grafts were removed and prepared for organ chamber studies and histology. Serum nicotine averaged 12.1 and 118.7 ng/ml in the 11 mg/day and 44 mg/day groups, respectively. Plasma nitric oxide was higher in dogs treated with 11 mg/day doses compared with controls. In organ chamber studies, endothelium-dependent relaxations to thrombin and A-23187 and endothelium-independent relaxations to nitric oxide were greatest in grafts from dogs treated with 11 mg/day doses. Intimal thickness of the grafts were similar among groups. However, staining for bone sialoprotein was increased in the media of grafts from the 11 mg/day treatment group. These data suggest that transdermal nicotine in doses comparable and double to those used for conventional smoking cessation treatment in humans does not adversely affect early patency of canine CABG up to 4 wk postoperatively. Transdermal nicotine, however, may increase production of and response to nitric oxide in bypass grafts.

It currently remains controversial as to the intracellular mechanisms coupled to the inhibition of parathyroid hormone (PTH) secretion that are modulated by extracellular divalent cations. To study mechanisms responsible for regulation of PTH release by cations, we investigated the effect of Mg2+ on cytosolic Ca2+ levels ([Ca2+]i) and PTH secretion in single isolated bovine parathyroid cells. Addition of 9.0 mM Mg2+ evoked a spike of [Ca2+]i in approximately 80% of cells in the presence of extracellular Ca2+. Mg2+ decreased steady-state [Ca2+]i, which represented inhibition of influx of extracellular Ca2+ in 13-78% of cells. The percentage of cells that had a decline in steady-state [Ca2+]i after exposure to Mg2+ was dependent on the extracellular Ca2+ concentration. The effect of Mg2+ on intracellular Ca2+ response was dose dependent. Extracellular Mg2+ inhibited PTH secretion in cells that showed decline in steady-state [Ca2+]i, although cells that showed a spike after addition of Mg2+ secreted more PTH than cells that did not show a spike. The spike of [Ca2+]i and decline in steady-state [Ca2+]i that occur in response to extracellular Mg2+ may be caused independently by two distinct mechanisms that differentially regulate secretion of PTH.

Experiments were designed to determine whether endogenous physiological fluctuations in sex steroid hormones affect expression or functional responses of endothelin receptors. Coronary arteries from sexually mature male, female, and ovariectomized pigs were prepared either for receptor binding or measurement of isometric force in organ chambers. Competitive binding of 125I-labeled endothelin-1 was significant for a one-site model with unlabeled endothelin-1 and a two-site model with unlabeled endothelin-3 and sarafotoxin S6c in all pigs. The total number of binding sites for all endothelin ligands was not different between male and female pigs. Binding affinities for the high-affinity binding site for both endothelin-3 and sarafotoxin S6c were significantly greater (lower inhibition constant) in membranes prepared from female pigs with high endogenous estrogen. In organ chamber experiments, contractions to endothelin-1 but not endothelin-3 or sarafotoxin S6c were significantly greater in coronary arterial rings from female compared with male pigs and were not affected significantly by removal of the endothelium or by treatment of the rings with either indomethacin (10(-5) mol/l) or the combination of indomethacin and NG-monomethyl-L-arginine (10(-4) mol/l). These results suggest endogenous fluctuations in estrogen are associated with an increase in affinity of a high-affinity endothelin receptor in coronary arterial smooth muscle of female pigs. In addition, independent of endogenous estrogen status, coronary arteries from female pigs generate significantly greater contractions to endothelin-1 compared with male pigs. This phenomenon occurs at the level of smooth muscle and is not dependent on the endothelium or synthesis of nitric oxide or prostaglandins.

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

Background Coronary calcium identified by electron-beam computed tomography (EBCT) correlates poorly with luminal atherosclerotic narrowing, but calcium, an intimate part of coronary plaque, may be more directly related to atheromatous plaque area. Methods and Results Thirty-eight coronary arteries from 13 autopsy hearts were dissected, straightened, and scanned with EBCT in 3-mm contiguous increments. Coronary calcium area was defined as one or more pixels with a density >130 Hounsfield units (0.18 mm 2 /pixel). Each artery was divided into corresponding 3-mm segments, representative histological sections were stained, and atherosclerotic plaque area per segment (mm 2 ) was quantified. Coronary artery calcium and coronary artery plaque areas were correlated for the hearts as a whole, for individual coronary arteries, and for individual coronary artery segments. The sums of histological plaque areas versus the sums of calcium areas were highly correlated for each heart and for each coronary artery. However, coronary plaque area was on the order of five times greater than calcium area. Furthermore, minimal diffuse segmental coronary plaque could be present despite the absence of coronary calcium detectable by EBCT. Conclusions This histopathologic study confirms an intimate relation between whole heart, coronary artery, and segmental coronary atherosclerotic plaque area and EBCT coronary calcium area but suggests that there is a threshold value for plaque area below which coronary calcium is either absent or not detectable by this methodology.

AbstractSmooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and β-galactosidase (AdβGal). In contrast to AdβGal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]l-arginine to [3H]l-citrulline), had increasing cyclic 3′,5′ cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.