Biotransformation of glyceryl trinitrate and elevation of cyclic GMP precede glyceryl trinitrate-induced vasodilation.

In this study, we examined glyceryl trinitrate (GTN) biotransformation and cyclic GMP elevation in vascular smooth muscle before onset of GTN-induced relaxation. Isolated rabbit aortic strips (RAS) and strips of bovine pulmonary artery (BPA) and bovine pulmonary vein (BPV) were contracted submaximally and incubated with [3H]GTN. Before onset of GTN-induced vasodilation, the tissues were freeze-clamped and then analyzed for GTN, glyceryl-1,2-dinitrate (1,2-GDN), and glyceryl-1,3-dinitrate (1,3-GDN) and for cyclic GMP. Before onset of relaxation of RAS, BPA, and BPV, there was significant biotransformation of GTN to GDN and significant elevation of cyclic GMP. There was significantly greater biotransformation of GTN and elevation of cyclic GMP by BPV than by BPA incubated with the same concentration of GTN, which was temporally related with the more rapid onset of relaxation induced in BPV than in BPA. These results are consistent with the hypothesis that the magnitude of GTN biotransformation before vasodilation is the important determinant of subsequent tissue relaxation. In GTN biotransformation before vasodilation, there was preferential formation of 1,2-GDN. These data indicate that the mechanism of GTN biotransformation to 1,2-GDN is related to elevation of cyclic GMP and subsequent vasodilation.

Relationship between biotransformation of glyceryl trinitrate and cyclic GMP accumulation in various cultured cell lines.

We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured rat lung fibroblasts (RLF), porcine kidney epithelial (PK1), bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Biotransformation of 0.1 microM GTN was linear over 30 min and the percentage of glyceryl dinitrate (GDN)/10(6) cells for BAE, BASM, RLF and PK1 at 30 min was 3.1, 2.3, 5.8 and 21.7%, respectively. At low GTN concentration (0.01-0.1 microM) there was a highly selective formation of 1,2-GDN, whereas at higher GTN concentration (greater than 1 microM) this selectivity was lost. Cyclic GMP accumulation did not occur in BAE or BASM at any GTN concentration, whereas for RLF and PK1 it was highly correlated to the rate of GDN formation. Upon re-exposure to GTN after treatment of RLF or PK1 cells for 3 hr with 0.1 mM GTN, there was an almost complete loss of the cyclic GMP response, GTN biotransformation was attenuated markedly and the selective formation of 1,2-GDN at low GTN concentration was absent. However, when GTN-treated cells were incubated for 18 hr in GTN-free media, there was a recovery of the cyclic GMP response, GTN biotransformation and selective 1,2-GDN formation toward control values.(ABSTRACT TRUNCATED AT 250 WORDS)

A facile, reliable method for staining blood vessel endothelium.

A modification of the silver-based stain for outlining endothelial cell borders of rabbit aorta is described. The present method uses ammonium sulfide in place of exposure to light to develop the stain. Rabbit aortic rings were sequentially immersed in 280 mM dextrose, 15 mM silver nitrate, 280 mM dextrose, dilute ammonium sulfide, and 280 mM dextrose solutions. They were then cut open and mounted on microscope slides with glycerin jelly. The procedure is rapid, easily mastered, and suited for use in most pharmacology laboratories. The resulting preparations can be viewed under reflected or transmitted light.

Mechanism of glyceryl trinitrate-induced vasodilation. II. Lack of evidence for specific binding of GTN to bovine pulmonary vein.

Radioligand binding studies were conducted to test the hypothesis that specific receptor sites for glyceryl trinitrate (GTN) exist in vascular smooth muscle. The radioligand, [3H]GTN (9.98 Ci/mmol), was incubated with whole homogenate or subcellular fractions (10,000 X g pellet, 100,000 X g pellet and 100,000 X g supernatant) of bovine pulmonary vein (BPV) at 4 degrees C, 37 degrees C or room temperature. After incubation for 5, 15, 30 or 60 min, unbound and bound ligand were estimated by physical separation and liquid scintillation spectrometry. Separation of bound from free ligand was accomplished by vacuum filtration, centrifugation, equilibrium dialysis, gel filtration or precipitation. No evidence of specific binding of [3H]GTN to BPV whole homogenate or any subcellular fraction was observed. The viability of the BPV preparations was verified by demonstrating specific binding of [3H]nitrendipine to the 100,000 X g pellet. To ensure that the ligand was present throughout the incubation procedure in sufficient concentration to detect specific binding, the ability of BPV whole homogenate and subcellular fractions to biotransform GTN to glyceryl dinitrate was measured. After 60-min incubation at 37 degrees C, greater than 95% of the GTN added was unaltered; at 90 min, more than 83% of the radioactivity was associated with GTN. Our data are interpreted as not supporting the concept of a GTN receptor in BPV.

Mechanism of glyceryl trinitrate-induced vasodilation. I. Relationship between drug biotransformation, tissue cyclic GMP elevation and relaxation of rabbit aorta.

This study was conducted to test the hypothesis that biotransformation of glyceryl trinitrate (GTN) is involved in GTN-induced relaxation of vascular smooth muscle. The temporal relationship between GTN biotransformation, elevation of cyclic GMP content and vasodilation in rabbit aortic strips (RAS) was determined. Isolated RAS were contracted submaximally with phenylephrine, and then were incubated with 0.62 microM [3H]GTN in a 30-sec time course study. GTN-induced relaxation (inhibition of phenylephrine-induced tone) of RAS was monitored; tissue cyclic GMP content was measured by radioimmunoassay; and GTN, glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN) concentrations in RAS were measured by thin-layer chromatography and liquid scintillation spectrometry. There was time-dependent biotransformation of GTN to GDN by the RAS and a time-dependent increase in cyclic GMP content in the RAS. Statistically significant (P less than .05) biotransformation of GTN and elevation of cyclic GMP content in the tissue occurred at 10 sec, whereas the onset of GTN-induced relaxation of RAS occurred at 12 sec. During the tissue biotransformation of GTN, there was preferential formation of 1,2-GDN compared with 1,3-GDN, with 1,2-GDN/1,3-GDN ratio of 3.0/1 at 10 sec, 5.3/1 at 20 sec and 5.8/1 at 30 sec. The results of this study are consistent with the hypothesis that GTN is a prodrug, such that biotransformation to an active metabolite is involved in GTN-induced relaxation of vascular smooth muscle. The data also indicate that the mechanisms of GTN biotransformation and GTN-induced activation of guanylate cyclase may be related intimately.

A comparative study of glyceryl trinitrate biotransformation and glyceryl trinitrate induced relaxation in bovine pulmonary artery and vein.

Recent evidence supports the hypothesis that the mechanism by which glyceryl trinitrate induces relaxation of vascular smooth muscle involves the biotransformation of glyceryl trinitrate. This study was conducted to determine if there was a direct correlation between the capacity of vascular smooth muscle preparations to biotransform glyceryl trinitrate and their sensitivity to the relaxant effect of this organic nitrate. Isolated bovine pulmonary arteries and veins were contracted submaximally and cumulative dose-response relationships to glyceryl trinitrate were obtained; the vein was approximately 10 times more sensitive than the artery to glyceryl trinitrate induced relaxation. In a separate series of experiments, these vascular tissues were contracted submaximally and incubated with 0.5 microM [14C]glyceryl trinitrate for 2 min, during which glyceryl trinitrate induced relaxation was monitored. At 2 min, tissue samples were taken for determination of glyceryl trinitrate and glyceryl-1,2- and 1,3-dinitrate content by thin-layer chromatography and liquid scintillation spectrometry. Biotransformation of glyceryl trinitrate to glyceryl dinitrate occurred concomitantly with relaxation of these blood vessels. The concentration of glyceryl dinitrate in the vein was significantly less than that in the artery (p less than or equal to 0.05), even though significantly greater relaxation of the vein than the artery was observed (p less than or equal to 0.05). From these data, a simple linear relationship between glyceryl trinitrate biotransformation and relaxation is not apparent.

In vivo bupivacaine reduces the in vitro sensitivity of the human vas deferens to phenylephrine.

The isolated human vas deferens (HVD) has been the subject of a limited number of pharmacological studies. Furthermore, it has not been established whether the local or general anesthetics used in obtaining the HVD affect the responses of this isolated tissue. Therefore, the aim of this study was to determine if the local anesthetic, bupivacaine (BPV), alters the sensitivity of the HVD to agonists. It was shown that BPV, injected into subjects undergoing vasectomy, was present in the excised HVD and in sufficient concentration to attenuate the phenylephrine (PE)-induced contraction. Bupivacaine assays were done by gas chromatography - mass spectrometry. Following 3 h of equilibration with repetitive washing, 95.0 +/- 2.1% (SD) of the BPV could be eliminated from the tissue, which correlated well with an observed increase in sensitivity of the HVD to PE. The sensitivity to PE of HVD isolated from subjects under general anesthesia was less than the sensitivity of tissues obtained with bupivacaine and in this case the depressant effect was not reversed by washing. These results indicate that it is important to consider the anesthetic procedure used to acquire the HVD and also the equilibration procedure prior to pharmacological studies.