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1. Isolated hearts of guinea-pigs were perfused in vitro with a physiological salt solution via a retrograde aortic cannulation (Langendorff preparation) at constant perfusion pressure. Bolus intra-arterial injections of various vasodilator drugs were made and the coronary flow responses were measured with an electromagnetic flow probe placed in the arterial inflow circuit. Inhibitory drugs were infused intra-arterially. 2. Nitro-L-arginine (NLA; 500 microM), an NO synthesis inhibitor, decreased coronary baseline flow by 16 +/- 0.8%, converted acetylcholine-induced coronary vasodilatation to vasoconstriction and had no effect on coronary flow responses to adenosine or papaverine. Sodium nitroprusside-induced responses were enhanced during NLA infusion by 46 +/- 11%. 3. Adenosine 5'-triphosphate (ATP) increased coronary flow but coronary flow responses to ATP were not altered by infusion of NLA. 4. ATP-induced coronary dilatation was not significantly attenuated by infusion of the adenosine receptor antagonist XAC, (xanthine amine congener; 2 microM), whereas XAC decreased coronary flow responses to adenosine by 75% +/- 5%. 5. ATP-induced coronary flow responses were reduced by only 31 +/- 4% during indomethacin infusion (2.8 microM) whereas indomethacin completely eliminated the initial vasoconstriction phase and greatly attenuated the peak flow and duration of the later vasodilatation phase seen in response to arachidonic acid (0.75 nmol). Indomethacin had no effect on vasodilatations produced by adenosine or prostaglandin I2. 6. These results indicate that ATP-induced coronary dilatation in the isolated, perfused heart of the guinea-pig is not dependent upon NO production or upon degradation of ATP to adenosine. The coronary dilator action of ATP may be partially dependent (approximately 30%) upon the production of vasodilator prostaglandins.
Isolation-perfusion of the liver was performed in ten pigs using 5-fluorouracil administered in the perfusion circuit at doses of 100, 250, 500, and 1000 mg/kg body weight. Perfusion was performed for 60 minutes at normothermic (37 C) or hyperthermic (41 C) temperatures. One animal died shortly after perfusion. Incomplete isolation of the hepatic vasculature in two animals resulted in significant drug leakage into the systemic circulation with resulting hematologic toxicity. Perfusion with 5-fluorouracil at 1000 mg/kg produced hepatic necrosis. Perfusion with 5-fluorouracil at doses of 100, 250, or 500 mg/kg produced no hepatic toxicity except for transient elevations of hepatic enzymes and resulted in no systemic drug toxicity. Levels of 5-fluorouracil tolerated by the liver in the isolation-perfusion system were more than 1000-fold greater than the maximum drug levels achievable by routine systemic, intra-arterial, or intraperitoneal administration.
The isolated perfused rat lung was used as a model to study the possible hormonal regulation of lipid metabolism in the mammalian adult lung. Experimental diabetes, whether induced by alloxan or streptozotocin, decreased the incorporation of [U-14C]glucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60-80%. Glucose incorporation into phosphatidylcholine and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the incorporation of glucose into the phosphatidylcholine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidylcholine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mammalian pulmonary surfactant complex.
The isolated Krebs-perfused rat kidney was used for the quantitative and qualitative evaluation of the family of peptides derived from rat atrial extracts. Renal resistance changes were measured in perfused rat kidneys continuously infused with norepinephrine. The low molecular weight peptide fraction from rat atrial extracts was purified to obtain six peptides. The 21 amino acid peptide, designated atriopeptin I, was previously demonstrated to be natriuretic and to relax intestinal but not vascular smooth muscle strips (in vitro) and to be an extremely weak renal spasmolytic in the isolated perfused rat kidney. On the other hand, the 23 amino acid peptide (which has a Phe-Arg carboxy-terminal extension on atriopeptin I), designated atriopeptin II, and atriopeptin III (the Phe-Arg-Tyr carboxy-terminal extension) were natriuretic and spasmolytic (in vitro) on both intestinal and vascular smooth muscle. Both atriopeptin II and III produced a profound concentration-dependent decrease in renal resistance in the norepinephrine-constricted rat kidney preparation. A comparative study of the six peptides isolated from atrial extracts indicates that the Phe-Arg or Phe-Arg-Tyr carboxy-terminal extension of the basic 21 amino acid sequence is essential for the renal vasorelaxant activity. A purified high molecular weight peptide (designated atriopeptigen) is impotent relative to the low molecular weight atriopeptin II and III as a renal spasmolytic in isolated perfused rat kidneys and reduces renal resistance only after in vitro proteolytic cleavage. Thus, the low molecular weight peptides atriopeptin II and III appear to be the active species that mediate the renal vasodilation produced by atrial extracts.
In isolated nonworking hearts perfused with substrate-free medium for 90 min, 2000 microU insulin.mL-1 reduced the rates of glycogenolysis and lipolysis, whereas 50 microU insulin.mL-1 reduced the rate of lipolysis with no significant effect on glycogenolysis. Glycogen and triglycerides were the chief energy sources during the course of depletion in the presence and absence of insulin. In hearts perfused with medium containing 5 mM glucose for 3 h, glycogen and triglycerides contributed to the heart energy requirement, but in the presence of insulin (50 or 2000 microU.mL-1) endogenous triglycerides contributed to the heart energy requirement with net glycogen synthesis. In hearts preperfused with substrate-free medium for 90 min and perfused for another 90 min with medium containing 5 mM glucose, the depletion of endogenous glycogen and triglycerides stimulated the utilization of glucose and the synthesis of glycogen and triglycerides. In hearts preperfused with substrate-free medium for 90 min and perfused for another 90 min with medium containing glucose (5 mM) and insulin (50 or 2000 microU.mL-1), insulin enhanced the utilization of glucose and the synthesis of glycogen and triglycerides. Glucose utilization, glycogenolysis, lipolysis, and synthesis of glycogen and triglycerides were sensitive to the physiological insulin concentration and were also affected by the heart content of glycogen and triglycerides, in the isolated nonworking perfused rat heart.
An isolated perfused lung [IPL) preparation was used to investigate the effects of SO2 (1-2 ppm) on the metabolism of benzo[a]pyrene (BaP), a ubiquitous potent carcinogen that has been associated with the increased incidence of a human bronchiogenic carcinoma in occupational and urban populations. [14C]BaP, with and without crude air particulate (CAP), was administered intratracheally to the IPL in conjunction with SO2 or after pretreatment of the whole animal with SO2. Metabolites were isolated from serial blood samples up to 3 h after the administration of [14C]BaP to the IPL. Metabolites were also isolated from lung tissue, washout fluid, macrophage, and trachea and bronchi at the end of the perfusion at 180 min. Patterns of BaP metabolites were determined by thin-layer and high-performance liquid chromatography and scintillation counting. SO2 given in conjunction with BaP on the IPL or given to the whole animal followed by BaP on the IPL, in comparison with BaP only on the IPL, resulted in a twofold increase in the total rate of appearance of metabolites of BaP in the blood with changes in the metabolic pattern. SO2 given in conjunction with BaP and CAP on the IPL, in comparison with BaP plus SO2 on the IPL, resulted in a threefold decrease in the total rate of appearance of metabolites of BaP in the blood with changes in the metabolic pattern.
The isolated perfused lung (IPL) preparation is a well-established model for the study of alveolar epithelial sodium transport. We noted that preparations of normal fluid-filled rat lungs with recirculated perfusate reproducibly lost weight, whereas preparations in which the perfusate was discarded after a single pass through the lungs had a variable and lesser weight change. To confirm this, we performed IPL experiments by using male Sprague-Dawley specific-pathogen-free rats (175-225 g). In 10 IPLs, perfusate initially was discarded after passing through the lungs and then was recirculated continuously. During the single-pass period, the rate of weight change was +0.7 +/- 2.0 mg/min compared with -9.0 +/- 1.3 mg/min for the recirculating period. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulated during recirculation. The weight loss induced by recirculation was reproduced by perfusion with 8-bromoadenosine 3',5'-cyclic monophosphate or terbutaline in single-pass fashion and blocked when the kinase inhibitor H-8 or phosphodiesterase was present in the recirculating perfusate. In summary, perfusate recirculation in the IPL stimulates fluid resorption at least partially via cAMP. This should be factored into the design and interpretation of IPL experiments.
The isolated perfused rat heart model can be used to evaluate cardiotoxicity, and is especially useful in distinguishing direct vs indirect cardiac injury. Various perfusion systems can be used to characterize the pathophysiologic as well as morphologic changes induced by drugs or chemicals of interest. The isolated perfused heart was used in the studies described herein to characterize the mechanism of allylamine cardiotoxicity. Rat hearts were perfused with Krebs-Henseleit buffer containing 10 mM allylamine and a latex balloon was inserted into the left ventricle to monitor pressure. Coronary flow in hearts perfused with 10 mM allylamine was similar to control hearts at 5, 10, and 30 min, but was reduced by 1 hr (11.5 +/- 0.6 ml/min/g wet heart weight vs 16.0 +/- 0.7, p less than 0.01). Peak left ventricular systolic pressure increased in hearts perfused with allylamine for 5 min (156 +/- 8 mm Hg vs 103 +/- 9, p less than 0.01), but by 2 hr was decreased compared to controls (89 +/- 6 vs 105 +/- 5, p less than 0.05). End diastolic pressure was markedly increased at 2 hr (58 +/- 3 vs 4 +/- 0.8, p less than 0.01). Morphologically, allylamine perfused hearts exhibited significant contraction band changes as well as numerous cells with marked swelling of the sarcoplasmic reticulum. The findings in this study suggest that allylamine produces direct myocardial damage that appears to be independent of coronary flow. These studies demonstrate that the isolated perfused rat heart model can be used to evaluate mechanisms of acute cardiotoxicity.
The isolated perfused rat liver was used for the study of the characteristics of bilirubin transfer from perfusate to bile in comparison to those of sulfobromophthalein (BSP). The parameters estimated included mean extraction ratio, removal half life, hepatic clearance, intrinsic or metabolic clearance rate of biliary excretion, hepatic concentration and biliary concentration for both compounds. The results obtained favoured the conclusion that bilirubin was less efficiently handled than BSP by the isolated rat liver.
Isolated perfused kidneys from prehypertensive Dahl salt-sensitive (DS) rats demonstrated marginally increased glomerular responsiveness when nitrendipine or verapamil was superimposed upon norepinephrine vasoconstriction. This was manifested by a greater increase in the glomerular filtration rate of DS rat kidneys, as compared to kidneys from Dahl salt-resistant (DR) rats. This glomerular response to nitrendipine by isolated kidneys was not affected by the development of salt-induced hypertension in DS rats, but was eliminated by antecedent salt loading in DR rats. In further experiments designed to assess more directly the reactivity of renal vascular calcium channels in DS and DR rats, the calcium channel agonist BAY-K-8644 and its pure agonist isomer elicited greater increases in renal vascular resistance in kidneys from prehypertensive DS rats than in kidneys from similarly prepared DR rats. Renal vascular reactivity to both BAY-K-8644 and its agonist isomer were greatly magnified following salt-induced DS rat hypertension. These results suggest that a genetically conferred abnormality of calcium channel function may contribute to the renal functional characteristics of the DS rat kidney.
The isolated perfused human pancreas was employed as a model in which electrical stimulation of the celiac mixed neural bundle was performed in the presence or absence of selective neural blockers. The insulin and glucagon responses to hyperglycemia alone or in the presence of splanchnic nerve stimulation were similar in magnitude to the results obtained in a preliminary report on isolated human pancreatic function and in studies using animal models. Stimulation of the celiac neural bundle in the presence of hyperglycemia resulted in an inhibition of insulin release and in an augmentation of glucagon release. alpha-adrenergic stimulation resulted in a strong suppression of insulin secretion and a mild suppression of glucagon secretion. beta-adrenergic fiber stimulation caused a mild augmentation of both insulin and glucagon release, whereas the cholinergic fibers strongly stimulated both alpha- and beta-cell secretion. The predominant effects of celiac neural bundle stimulation are insulin inhibition by was of an alpha-adrenergic effect and glucagon stimulation by way of a cholinergic effect. Thus, in this in vitro human model, our data confirm that the splanchnic innervation of the pancreas has a potent regulatory role on pancreatic hormone release in human subjects.
Using the isolated perfused neonatal sheep liver model, we examined the disposition of propranolol (n = 8, age 0.25-10 days) and compared our findings with our previous study from the perfused near-term fetal sheep liver (Ring JA, et al. 1995. Drug Metab Dispos 23:190-196). Within 45 min of dosage, perfusate propranolol levels had fallen by three orders of magnitude to be less than the limit of detection. Perfusate disappearance curves were monoexponential in six experiments and biexponential in two experiments. The mean shunt-corrected hepatic extraction ratio was 0.92 +/- 0.09, much greater than that seen in the fetal sheep liver (0.26 +/- 0.13, P < 0.0001) but still less than values in the adult sheep (0.97). At the conclusion of the perfusion, 4-hydroxypropranolol was the major metabolite present and 5-hydroxypropranolol and N-desisopropylpropranolol were minor metabolites. We conclude that the isolated perfused neonatal sheep liver is a useful model with which to study the maturation of neonatal hepatic drug oxidation. Our study shows that propranolol is rapidly eliminated by the neonatal liver to form several metabolites at rates far greater than in the fetal liver, but rates of elimination have not yet reached that reported in the adult sheep liver.
The isolated perfused porcine skin flap (IPPSF) is a novel alternative, humane in vitro model consisting of a viable epidermis and dermis with a functional microvasculature. For this study, 200 microliters of either 10.0, 5.0, 2.5, 1.25, 0.50, or 0.20 mg/ml of bis (2-chloroethyl) sulfide (HD) in ethanol or ethanol control was topically applied to a 5.0 cm2 dosing area of the IPPSF and perfused for 8 h with recirculating media. HD dermatotoxicity was assessed in the flap by cumulative glucose utilization (CGU), vascular resistance (VR), light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). HD produced a statistically significant dose relationship for gross blisters and microvesicles. The HD-treated IPPSFs were also characterized by a decrease in CGU and an increase in VR. Light microscopic changes included mild intracellular and slight intracellular epidermal edema, multifocal epidermal-dermal separation, and dark basal cells. Ultrastructural alterations consisted of cytoplasmic vacuoles, pyknotic basal cells, nucleolar segregation, and epidermal-dermal separation occurring between the lamina lucida and lamina densa of the basement membrane. The severity of these changes increased in a dose-dependent manner. Morphologically, the IPPSF appeared similar to human skin exposed to HD with the formation of macroscopic blisters and microscopic vesicles. In conclusion, the IPPSF appears to be an appropriate in vitro model with which to study the pathogenesis of vesicant-induced toxicity.