Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

Phosphoramidon inhibits the intracellular conversion of big endothelin-1 to endothelin-1 in cultured endothelial cells

Effects of various protease inhibitors on the conversion of big endothelin (ET)-1 to ET-1 in cultured endothelial cells were analyzed. A metal protease inhibitor, phosphoramidon, decreases the amount of ET-1 and increase that of big ET-1 released. This effect is dose-dependent and not nonspecific. When the contents of ET-1 and big ET-1 in the cells after culturing in the medium with or without phosphoramidon were measured, the ratio of ET-1: big ET-1 in the cells was 3.3 : 1 and phosphoramidon inverted the ratio in the cells to 1 : 3.5. These data strongly suggest that a phosphoramidon-sensitive protease converts big ET-1 to mature ET-1 intracellularly.     

N-oxidation of imipramine by isolated perfused rat and rabbit lung

Pulmonary uptake and metabolism of imipramine (IMP) was investigated in isolated perfused rat (IPrL) and rabbit (IPRL) lung preparations. Perfusate containing ¹⁴C-IMP (1.2 μmole/g lung) was recirculated through the pulmonary artery in artificially ventilated lungs. The radioactivity in the perfusate declined rapidly and about 80% of the dose was taken up by the lungs within 10 minutes in both IPrL and IPRL preparations. A steady-state was apparently reached thereafter in the IPRL, while a portion of the radiolabel effluxed into the perfusate of the IPrLs, thus reducing the net lung content to 54% of added IMP by 60 minutes. After 60 minutes perfusion, metabolites of IMP accounted for the major radioactivity (80%) in the perfusate, while the lung contained mainly (83%) the unchanged parent compound. The principal metabolite was identified as IMP-N-oxide (IMP-NO) which was found in the perfusate after 5 minutes of perfusion. Only 3% of the added IMP was metabolized by IPRL in 60 minutes. SKF-525A, an inhibitor of cytochrome P-450-mediated monooxygenase system, did not inhibit but enhanced the metabolism of IMP by IPrL to IMP-NO. IMP was principally metabolized to IMP-NO by incubations of 9,000 g supernatant fractions of rat lungs to a significantly higher extent than similar rabbit lung preparations. Including SKF-525A significantly accelerated the metabolism of IMP to IMP-NO in accordance with the perfusion experiments. These results suggest that in contradiction to publishedd reports, IMP is appreciably metabolized by the rat lung $\text{via}$ N-oxidation by non-cytochrome P-450 pathway and the metabolite formed in the lung is released into the circulation indicating its low affinity for the lung tissue.     

Pulmonary disposition of lipophilic amine compounds in the isolated perfused rabbit lung

Audi, S. H., C. A. Dawson, J. H. Linehan, G. S. Krenz, S. B. Ahlf, and D. L. Roerig. Pulmonary disposition of lipophilic aminecompounds in the isolated perfused rabbit lung. J. Appl. Physiol. 84(2): 516-530, 1998.We measured the pulmonaryvenous concentration vs. time curves for [³H]alfentanil,[¹⁴C]lidocaine, and [³H]codeine after thebolus injection of each of these lipophilic amine compounds (LAC) and avascular-reference indicator (fluorescein isothiocyanate-dextran) intothe pulmonary artery of isolated perfused rabbit lungs. A range offlows and perfusate albumin concentrations was studied. To evaluate theinformation content of the data, we developed a kinetic modeldescribing the pulmonary disposition of these LAC that was based onindicator dilution theory, and we sought a robust approach forinterpreting the estimated model parameters. We found that thedistribution of the kinetic model rate constants of the lipophilicamine-tissue interactions can be described by ,, and ,where is a measure of the capacity of the rapidlyequilibrating interactions between the lipophilic amineand the tissue; is a measure of the equilibrium capacity of the slowly equilibrating interactions between the lipophilic amine and the tissue; and isthe mean sojourn time. The values of , , andwere 0.8 ± 0.1 (SE), 0.6 ± 0.1, and 1.6 ± 0.5 s; 1.9 ± 0.1, 5.3 ± 0.4, and 5.6 ± 0.5 s; and 1.1 ± 0.1, 9.8 ± 0.4, and 4.7 ± 0.2 s for alfentanil, lidocaine, and codeine, respectively.These values for , , andreveal the relative dominance of the slowly equilibrating interactions for lidocaine and codeine in comparison with alfentanil. This approachto data analysis may have utility for the potential use of LAC toreveal and to quantify changes in lung tissue composition associatedwith lung disease.

     

Characterization of the contractile and relaxant action of the endothelin-1 precursor, big endothelin-1, in the isolated rat basilar artery

The presence of functional endothelin converting enzyme (ECE) activity in basilar artery ring segments was investigated by measuring the contractile and relaxant effects of big endothelin (ET)-1. Under resting tension conditions cumulative application of big ET1-1 elicited a concentration-related contraction with the concentration-effect curve (CEC) shifted to the right against ET-1 by a factor of 31 and 29 in segments with the endothelium intact or mechanically removed, respectively. Preincubation with the ET(A) receptor antagonist, BQ123, induced an apparently parallel rightwards shift without affecting the maximum contraction. This shift was more pronounced for ET-1 than for big ET-1. With the putative ECE inhibitor phosphoramidon (10(-3) M) in the bath a small rightwards shift of the CEC for big ET-1 was observed in control segments and a more marked one in de-endothelialized segments. In segments precontracted with prostaglandin (PG) F(2alpha) big ET-1 induced a significant although transient relaxation whereas ET-1 did not. However, in the presence of BQ123 both ET-1 and big ET-1 elicited concentration-related relaxation with a significantly higher maximum effect obtained with big ET-1. The potency was 13 fold higher for ET-1, which is markedly less than that found for contraction. The results, therefore, suggest 1) the presence of functional ECE-activity in the rat basilar artery wall, and 2) differences in the functional ECE activity located in the endothelium and media.     

Determination of 5-hydroxytryptamine metabolites in isolated perfused rabbit lung by high-performance liquid chromatography.

An analytical procedure, utilizing high-performance liquid chromatography (HPLC) hasbeen developed for the separation of radiolabeled metabolites of 5-hydroxytryptamine (5-HT) in biological fluids. Four different chromatographic systems are described, which enable the separation of groups of metabolites possessing similar organic functionality to be achieved. As an example of this general analytical method, it is demonstrated that no methylation of 5-HT occurs in perfused rabbit lung, the principal metabolites being 5-hydroxyindoleacetic acid and 5-hydroxytryptophol.     

Comparison of the hemodynamic effects of endothelin-1 and big endothelin-1 in the rat

We compared the hemodynamic effects of continuous i.v. infusion of endothelin-1 and big endothelin-1 in anesthetized rats. Big endothelin-1 was fivefold less potent than endothelin-1 in decreasing cardiac output, heart rate, and stroke volume. However, big endothelin-1 produced a significantly larger increase in mean arterial pressure compared to endothelin-1 at doses that produced identical decreases in cardiac output. These findings support the hypothesis that the hypertensive effects of big endothelin-1 and endothelin-1 are produced by differential effects on systemic vascular resistances.     

Ventilation-perfusion relationships in isolated blood-free perfused rabbit lungs.

The multiple inert gas elimination technique (MIGET) was applied to blood-free perfused isolated rabbit lungs. Commonly accepted criteria for reliability of the method were found to be fulfilled in this model. Ventilation-perfusion (VA/Q) distributions in isolated control lungs corresponded to those repeatedly detected under physiological conditions. In particular, a narrow unimodal dispersion of perfusate flow was observed: perfusion of low-VA/Q areas ranged below 1% and shunt flow approximately 2-3%; perfusion of high-VA/Q regions was not detected. Gas flow was characterized by narrow dispersion in the midrange-VA/Q areas. Application of a low level of PEEP (1 cmH2O) reduced shunt flow to less than 1%, and low-VA/Q areas were no longer noted. By using this PEEP-level, stable gas exchange conditions were maintained for greater than 5 h of extracorporeal perfusion. Graded embolization with small air bubbles caused a typical rightward shift (to higher VA/Q ratios) of mean ventilation, associated with the appearance of high-VA/Q regions and an increase in dead space ventilation. Mean perfusion was shifted leftward, and shunt flow was approximately doubled. Whole lung lavage with saline for washout of surfactant evoked a progressive manifold increase in shunt flow, accompanied by a moderate rise of perfusate flow to low-VA/Q areas. We conclude that the MIGET can be applied to isolated blood-free perfused rabbit lungs for assessment of gas exchange and that typical patterns of VA/Q mismatch are reproduced in this model.     

Production of cortisol from cortisone by the isolated,perfused fetal rabbit lung

Perfusion of the isolated 26 day fetal rabbit lung with ³H-cortisone resulted in its conversion to ³H-cortisol and release into the perfusate. Little conversion of ¹⁴C-cortisol to ¹⁴C-cortisone occurred. Quantitative study of homogenized fetal rabbit lung revealed the development of both the cofactor and the enzyme for 11β-hydroxy steroid dehydrogenase activity between 21 and 29 days gestation. These results suggest increasing production of cortisol from cortisone by the fetal rabbit lung as a function of gestational age. This conversion may be of significance with respect to both lung development and parturition, both events being accelerated by cortisol treatment.     

Critical factors in the radioimmunoassay of endothelin-1, endothelin-3, and big endothelin-1 in human plasma

The possible diagnostic or prognostic significance of changes in circulating level of endothelins in a variety of pathological conditions is currently of interest. Unfortunately, no consensus regarding optimization of sensitivity and extraction procedures for the reliable radioimmunoassay of endothelin-1 (ET-1), big endothelin-1 (BigET-1), and endothelin-3 (ET-3) currently exists. The object of the present study was to evaluate aspects of currently used extraction and assay procedures that limit accurate determination of ET in human plasma and define criteria to reduce variability. Critical parameters include the selectivity of commercial antibodies and the ability to remove interfering material after Sep-Pak absorption by selective washing with 24% ethanol in 4% acetic acid or methylene chloride in 0.1% trifluoroacetic acid. Assay sensitivity and specificity in the physiological range is improved by optimizing total binding parameters for the antibodies to give approximately 15-20% binding of radiolabeled peptide. With these modifications normal plasma values for ET-1, BigET-1, and ET-3 averaged 1.7 +/- 0.06, 2.5 +/- 0.3, and 5.8 +/- 0.2 pg/ml, respectively. These data suggest that such modifications may help to resolve many of the earlier difficulties concerning the role of ET under normal and pathological conditions.     

Phosphoramidon-sensitive endothelin-converting enzyme in vascular endothelial cells converts big endothelin-1 and big endothelin-3 to their mature form.

Incubation of big endothelin-3 (big ET-3(1-41)) with the membrane fraction obtained from cultured endothelial cells (ECs) resulted in an increase in immunoreactive-ET (IR-ET). This increasing activity was markedly suppressed by phosphoramidon, which is known to inhibit the conversion of big ET-1(1-39) to ET-1(1-21). Reverse-phase HPLC of the incubation mixture of the membrane fraction with big ET-3 revealed one major IR-ET component corresponding to the elution position of synthetic ET-3(1-21). When the cultured ECs were incubated with big ET-3, a conversion to the mature ET-3, as well as an endogenous ET-1 generation, was observed. Both responses were markedly suppressed by phosphoramidon. By the gel filtration of 0.5% CHAPS-solubilized fraction of membrane pellets of ECs, the molecular mass of the proteinase which converts big ET-1 and big ET-3 to their mature form was estimated to be 300-350 kDa. Phosphoramidon almost completely abolished both converting activities of the proteinase. We conclude that the above type of phosphoramidon-sensitive metalloproteinase functions as an ET-converting enzyme to generate the mature form from big ET-1 and big ET-3 in ECs.     

Effects of terbutaline on sodium transport in isolated perfused rat lung

We have previously presented evidence that cultured alveolar epithelial cell monolayers actively transport sodium from medium to substratum, and that this process can be stimulated by beta-agonists. In this study the isolated perfused rat lung was utilized to investigate sodium transport across intact mammalian alveolar epithelium. Radioisotopic tracer(s) (22Na and/or [14C]sucrose) were instilled into the airways of isolated Ringer-perfused rat lungs. The appearance of isotope(s) in the recirculated perfusate was measured and a permeability-surface area product was calculated. Pharmacological agent(s) (terbutaline and/or propranolol) were present in the instillate or were added to the perfusate during the experiments. Terbutaline alone, whether in the instillate or perfusate, caused a significant increase in 22Na flux. This increase was prevented by the presence of propranolol. [14C]sucrose fluxes were unaffected by the presence of terbutaline. These data are consistent with the presence of an active component of sodium transport across intact mammalian alveolar epithelium that leads to removal of sodium from the alveolar space.     

ADPase activity of isolated perfused rat lung.

More than 90% of 3H-ADP was metabolized following bolus injection into rat isolated perfused lungs. The major metabolite was inosine, with lesser amounts of adenosine and AMP. The mean pulmonary transit time for the radioactivity associated with ADP and its metabolites was the same as that for the vascular marker 14C-dextran, indicating that ADP is metabolized by enzymes in the pulmonary vessel walls. The metabolism of 3H-ADP was apparently unaffected by the simultaneous injection of prostacyclin or by continuous infusion of indomethacin or aspirin. 3H-ADP was similarly metabolized by the lung following continuous infusion, although relatively higher amounts of adenosine were observed. The metabolism of ADP by the lung represents biological inactivation since over 95% of the platelet-aggregatory activity of ADP was lost on passage through the lung.     

Thromboxane and prostacyclin production in the perfused rabbit lung

We investigated whether prostacyclin formation by the isolated rabbit lung can serve as a measure of pulmonary distress. The basal TXA2 and PGI2 formation was very low, and depended on the preperfusion history of the lung (low or high flow, use of dextran or artificial perfusate). The basal prostanoid production remained unchanged over a time period of 2 h. Neither was it influenced by the serotonin uptake inhibitor chlorimipramine and by small changes in temperature (33 degrees C vs 39 degrees C). The PGI2 formation was almost independent of hemodynamic alterations such as embolism or vasoconstriction. An enhanced production was only seen after a dramatic increase in flow (from 1.7-5 ml/sec), and a transient 3-fold increase was observed after administration of 1 mM H2O2. A substantial (up to 40-fold) but transient increase in TXA2 production was measured after 1 mM of H2O2, and the TXA2 production was positively correlated to the increase in pulmonary arterial pressure. However, thromboxane production was also dramatically augmented by hemodynamic alterations such as embolism, increased flow and--to a lesser extent--vasoconstriction. We conclude that the determination of the prostanoid production (and particularly the TXA2 formation) by the rabbit lung cannot be used as a direct measure of endothelial distress. To this end it is excessively biased by hemodynamic alterations such as recruitment and shear stress.     

1-substituted tetrahydroisoquinoline analogs: beta adrenoceptor blocking agents in the isolated perfused rabbit heart.

The 1-benzyl and 1-methyl congeners of trimetoquinol were tested for antagonism of receptors which mediate inotropy and chronotropy in the isolated perfused rabbit heart. 1-Benzyltrimetoquinol was found to be a blocker of resting, isoproterenol- and dobutamine-stimulated inotropy at concentrations (10^?7?10^?5M) which did not significantly affect chronotropy ( > 10^?5M). 1-Methyltrimetoquinol was found to be a partial agonist in the resting myocardium, weakly blocking inotropy and chronotropy at doses of 10^?7?10^?5M. At a concentration of 10^?4M, 1-methyltrimetoquinol was an agonist of both chronotropy and inotropy. These stimulatory properties appear to be direct (not affected by prior reserpinization) and antagonized by propranolol. In the isoproterenol-stimulated heart, 1-methyltrimetoquinol was a specific negative inotropic agent at doses (10^?7?10^?5M) above which agonist properties were manifest. At 10^?4M, 1-methyltrimetoquinol acted synergistically with isoproterenol to produce positive inotropy and chronotropy significantly greater than that of isoproterenol alone. Currently, it is believed that the receptors which mediate inotropy and chronotropy are $\text{beta}$ adrenergic in nature. Thus, it would appear that 1-benzyltrimetoquinol is a specific antagonist of those $\text{beta}$-receptors which mediate inotropy, while 1-methyltrimetoquinol is a partial agonist of both inotropic and chronotropic $\text{beta}$-receptors. Further, the response to these compounds does not appear to be proportionate in various regions of the myocardium.     

Metabolism of environmental pollutants by the isolated perfused lung.

An isolated perfused rabbit lung system was developed for the study of pulmonary metabolism of foreign compounds. The main features of the system include the use of autologous whole blood, constant pressure perfusion, subatmospheric ventilation, and measurement of a variety of physiological and biochemical parameters. Pulmonary metabolism of benzo(a)pyrene has been investigated with this system. In addition to the 3-hydroxy metabolite, three dihydrodiols and an unidentified polar metabolite were also found. The polar metabolite accounted for approximately 50% of all metabolites found in the five compartments of the perfusion system. Pretreatment with 3-methylcholanthrene increased total metabolism of benzo(a)pyrene and shifted the pattern of metabolites. The perfusion system for the rabbit has been extensively modified for use with rats and guinea pigs. These smaller animals are currently being used to investigate pulmonary metabolism of trichloroethylene.