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Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z′ factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC50values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.

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Length-tension and force-velocity relationships and responsiveness to calcium were measured for isthmus of rabbit oviducts removed 24 h after injection of human chorionic gonadotropin (hCG), 72 h after hCG, 24 h after hCG with progesterone treatment, and 72 h after hCG with estrogen treatment. The resting tension at which maximum active tension was obtained was reduced in progesterone-treated tissues. The maximum active tension and maximum rate of tension development of contractions induced by field stimulation were greatest in tissues removed 72 h after hCG and lowest 72 h after hCG with estrogen treatment. Time to peak tension was reduced in tissues removed 24 h after hCG. Force-velocity curves were hyperbolic and were fitted to the Hill equation. The maximum load and maximum velocity of shortening were greatest in tissues removed 72 h after hCG and/or 24 h after hCG with progesterone treatment. ED50for calcium-induced contraction in depolarizing solution was lowest for tissues removed 24 h after hCG, but maximum tension was reduced in tissues from this group. These data suggest that the properties of the smooth muscle of the oviduct, and hence the conditions of ovum transport, are at least partially regulated by changes in contractility; entry of ova into the uterus is associated with increased force and velocity of contractions.

The acute effects of cocaine and/or Δ9-tetrahydrocannabinol (THC) were studied in ovariectomized female rats. Intermediate doses of cocaine (10 or 20 mg/kg) caused an increase in serum luteinizing hormone (LH) and a decrease in serum prolactin levels, whereas a higher dose (40 mg/kg) caused a decrease in serum LH. THC (10 mg/kg) attenuated serum LH and prolactin levels. The THC effect on LH was blocked by cocaine. Neither drug alone or in combination affected serum follicle-stimulating hormone levels. The cocaine-induced changes in LH levels were closely paralleled by changes in hypothalamic norepinephrine content, suggesting a neurochemical basis for cocaine’s action on LH release.

A porphyrinic sensor was developed and used to monitor the release of nitric oxide (NO) by a fetal bovine aorta endothelial (FBAE) cell culture and a primary bovine aorta endothelial (BAE) cell culture, with populations of several thousand cells. The sensor consisted of either a platinum mesh or reticulated vitreous carbon support covered with several layers of a p-type semiconducting metalloporphyrin and a cation exchanger, Nafion. Tissue and cell cultures can be grown directly on the surface of the sensor and NO release can be measured by amperometric or differential pulse voltammetry methods. The detection limit of the sensor deposited on reticulated vitrous carbon and platinum mesh support is 10 nM and 0.1 μM respectively. The response time is about one millisecond and precision 5-6%. The peak NO concentration released from FBAE cells was 190 ± 20 nM and 70 ± 20 nM when agonized by calcium ionophore (A23187) and bradykinin respectively. The maximum NO concentration released from the primary culture BAE cells was 770 ± 50 nM and 400 ± 50 nM when agonized by angiotensin II and bradykinin respectively.

The endothelium of the rabbit aorta (in contrast to the endothelium of the rabbit mesenteric artery) did not possess an ETB receptor mediating vascular relaxation. The smooth muscle of the rabbit aorta had both ETA and ETB receptors both of which mediated a contractile response. The characteristics of the contractions of rabbit aorta to endothelin-1 (mixed ETA and ETB receptor agonists) and to Sarafotoxin 6c (selective ETB receptor agonist) were different. Endothelin-1 was a full agonist and the contractile response and the responses to this agonist showed little tachyphylaxis. Sarafotoxin 6c was a partial agonist and the responses of the rabbit aorta to this agonist exhibited substantial tachyphylaxis. The specific ETA receptor antagonist, BQ 123 inhibited responses of the rabbit aorta to endothelin-1 without affecting responses to sarafotoxin 6c. The non-specific endothelin receptor antagonist, PD 142893 inhibited responses of the rabbit aorta to both endothelin, and sarafotoxin 6c. It is concluded that the rabbit aorta has no ETB receptors on the endothelium mediating relaxation. It is also concluded that the smooth muscle of the rabbit aorta has both ETA and ETB receptors both of which mediate contraction.

The contraction and electrical conduction velocity of the longitudinal and circular muscle of the oviduct from rabbits in estrus, 24 or 72 h following administration of human chorionic gonadotropin (hCG) or 14 days following castration have been compared. Two populations of conduction and contraction velocities were identified, with the faster velocity being associated with the longitudinal muscle. There was a large overlap between longitudinal and circular contraction, suggesting complex relationships between longitudinal and circular muscle. From the results it appears that during ovum transport the circular velocities increased, with the slowest rates at estrus. The values obtained 72 h following hCG injection most closely resembled those in the castrate group of animals. In the longitudinal orientation, however, the velocities were greater 24 h following hCG injection than at estrus or 72 h following hCG injection, suggesting a complex relationship between both longitudinal and circular muscle of the oviduct following ovulation.