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We describe a technique which allows perfusion of an isolated, ventilated rat lung with an extracorporeal veno-venous circuit from a support animal. This model provides stable and reliable function of the isolated lung for up to 3 h, thus overcoming some of the disadvantages of previous models. It may prove a potentially useful model for assessing ischaemic lung injury.
In this study, the effects of indomethacin (prostaglandin synthase inhibitor), propranolol (beta adrenergic receptors blocker), tetraethylammonium (TEA) (calcium-dependent potassium channel blocker) and glibenclamide (ATP-sensitive potassium channel blocker), NG nitro-L-arginine (NO synthetase inhibitor) and naloxane (nonselective opioid receptor antagonists) on the responses induced by sufentanil and remifentanil were investigated in the isolated perfused rat kidney. Renal arter was cannulated. Then the kidney was perfused continuously with warmed (37 degrees C) and aerated (95% O2 and 5% CO2). Krebs Henselieit solution by using a peristaltic pump delivering a constant flow (8-10 ml/min). Vascular responses were detected as changes in perfussion pressure, which was monitored continuously with a pressure transuder and recorded on polygraph. After phenilephrine (PE)-induced vasoconstriction had reached a platoe, sufentanil or remifentanil were given. Vasodilatation was recorded. Antagonists or inhibitors were added and responses were recorded. At the end of each experiment; papaverine was used to obtain the maximum dilatation. None of the used antagonists or inhibitors were not effected the submaximum PE construction. The used opioids were not alter in basal perfusion pressure. Antagonists or inhibitors had no effect on papaverine-induced dilatation. Bolus addition of sufentanil and remifentanil produced concentration dependent vasodilation. Indomethacine L-NAME, propranolol, naloxone and glibenclamide did not significantly alter responses of both of the opioids (p > 0.05). But, sufentanil and remifentanil induced dilatation were significantly affected by TEA (p < 0.05). The present results demonstrated that sufentanil and remifentanil decrease perfusion pressure in the isolated rat kidney and such mechanism may involve the calcium active K+ channels activation.
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.
The kinetics of nicotine elimination was investigated in isolated perfused lung and liver of phenobarbital (PB)- and 5,6-benzoflavone (BF)-pretreated rats. The estimated kinetic parameters demonstrated a high nicotine elimination rate in rat lung approaching the capacity of liver when both organs were in an uninduced state. The concentration-time profiles of cotinine as the main metabolite were almost identical for isolated lung and liver. In both organs the cotinine plasma concentrations reached a plateau level after 60 min of perfusion. Pretreatment of rats with 5,6-benzoflavone did not affect the rate of nicotine elimination and cotinine formation either in the lung or in the liver. Phenobarbital treatment, however, induced nicotine clearance in lung approximately 2-fold. This effect is quantitatively lower than the PB-related 8-fold induction of hepatic nicotine elimination observed in a previous study. The present results also indicate that the turnover of cotinine is markedly enhanced after PB induction. The elimination half-lives and clearance values for cotinine as the substrate were approximately 10-fold increased in rat liver after PB pretreatment. Thus, an important contribution of extrahepatic tissues to nicotine metabolism in rats has to be assumed. Moreover, since cotinine elimination is significantly increased after PB induction it is questionable whether cotinine plasma concentrations can further be used as suitable parameter for nicotine consumption.
Simultaneous recordings of pressure and flow were made from the renal artery in perfused isolate canine kidneys. The system was perturbated by the administration of angiotensin and prostaglandin A2. The data were recorded on frequency-modulated analog tape and converted to digital form, and a Fast Fourier Transform program was used to calculate input impedance. Changes in total renal volume and renal arterial pressure data were used to determine compliance values. in compliance were found, along with changes in both magnitude of impedance and phase angle, as a result of the perturbations introduced. Seemingly, renal vascular compliance has an important role in the autoregulatory mechanisms of the kidney.
Homologous and autologous rat erythrocytes, human erythrocytes as well as glutaraldehyde-treated and hemolysed homologous red blood cells suspended in an isotonic, buffered saline, free of immunoglobulins were perfused through the isolated rat liver. The mononuclear phagocyte system of the liver (mainly Kupffer cells) was able to recognize damaged rat and heterologous human erythrocytes and to sequester them by a direct membrane-erythrocyte mechanism. Hemoglobin and homologous and autologous rat erythrocytes were not sequestered or only to a minor extent. High sequestration rates are reflected by an increased energy demand with subsequent increase of the lactate-pyruvate and beta-hydroxybutyrate-acetoacetate-quotient.
Using the model of in vitro perfusion of the isolated rabbit ovary, in which follicular ruptures can be observed, in this paper we report on the photographic and cinematographic recording of the gross anatomy of ovulation. The observed sequence of events and their timing were very similar to that which has been reported for ovulation observed in vivo. In addition, the use of infrared film made it possible to visualize intrafollicular events prior to rupture.
The secretion and molecular nature of immunoreactive neurotensin (NT) was studied following stimulation of an isolated perfused porcine ileal segment with glucose, triglyceride and intra-arterial infusion of gastrin-releasing peptide (GRP). Secreted peptides were separated using gel chromatography and analyzed with 3 sequence-specific radioimmunoassays towards NT. Glucose (5%) and GRP both stimulated NT secretion from the ileal segment whereas pure triglyceride did not. Maximal secretion of NT during glucose perfusion was 0.448 nmol/min and 6.9 nmol/min during GRP infusion (medians, n = 5). GRP infused in doses from 10(-10) to 10(-8) M stimulated NT release in a dose-related manner. Following gel chromatography only the intact peptide and no smaller or larger molecular size immunoreactive components were observed. The study showed that both luminal and humoral stimuli release NT from the isolated pig ileum. Apparently no fragments or other NT-related immunoreactive components were cosecreted with the peptide.
Parameters of the cell cycle of lymphoid cells and the rate of production of small lymphocytes and mature plasma cells were estimated in human spleens. 12 normal and 1 enlarged spleen with lymphatic hyperplasia were labelled either by a pulse or continuously with 3H-TdR-thymidine during normothermic perfusion of the isolated spleens. Biopsies of splenic tissue were taken at different intervals and evaluated autoradiographically. The mean initial labelling index of immunoblasts was 73% and for plasmoblasts 52%. The labelling index for small lymphocytes and mature plasma cells increased initially from 0% to 1%, and up to 25% respectively after 14 h. The duration of the S-phase, as determined by double labelling, was 8.26 h for lymphoid blasts. Data from the percentage labelled mitosis curve resulted in a minimal tG2 of about 1 h and tG2 + tM of about 4 h. The estimated generation time was about 11.5 h. The results found on the hyperplastic spleen were comparable to those on the normal spleen.
Renal metabolism of salicylic acid (SA) to salicyluric acid (SU), as well as the metabolism of SU to SA, was demonstrated in the isolated perfused rat kidney. SU formation was dependent upon the inclusion of a glycine pool and glycine concentration influenced the rate of SU excretion. The total conversion of SA to SU in 1 hr was 7.7% after the administration of 1 mg SA. Administration of increasing amounts of SA diminished this extent of SU formation. The addition of a competitive substrate, benzoic acid, produced a rapid formation and excretion of hippuric acid with a corresponding inhibition of SU formation and excretion. It is important to note that when isolated kidneys were perfused with 2.5 mg SU, 20--30% of the dose was metabolized to SA. Increasing the dose of SU to 10 mg decreased the amount of SA formed. In view of observed reversible SA/SU metabolism, a larger renal contribution to overall salicylate disposition is suggested.
Antioxidant properties of thioctic and dihydrolipoic acid have been demonstrated in membranes and low density lipoproteins (LDL) in vitro. In vivo studies with dietary supplementation of thioctic acid to rats showed that it can also protect tissues against oxidative damage. Presumably, this action is due to a thioctic acid dihydrolipoic acid (TA/DHLA) coupled antioxidant mechanism, which enhances the activity of other antioxidants (i.e. ascorbate, alpha-tocopherol) by regenerating them from their radical form. In the present study, thioctic acid proved to protect against ischemia/reperfusion injury to Langendorff perfused hearts. Hearts isolated from rats fed thioctic acid and subjected to ischemia exhibited better mechanical recovery (left ventricular developed pressure) after reperfusion and lower lactate dehydrogenase leakage. Thioctic acid supplementation also decreased the appearance of fluorescent lipid peroxidation products after ischemia/reperfusion, lowered the rate of 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN) induced lipid peroxidation in heart homogenates, and prevented the loss of alpha-tocopherol. The total sulfhydryl group content in thioctic acid fed animals was higher and the decrease due to ischemia-reperfusion was not as marked in this group as observed in the control. These results show that dietary supplementation with thioctic acid in vivo provides protection against ischemia/reperfusion injury in the Langendorff heart model.
Some of the metabolites of arachidonic acid formed in the lipoxygenase and cyclooxygenase pathways stimulate insulin release. We studied the relative importance of each of these pathways in the modulation of glucose-induced insulin release by using inhibitors of arachidonate metabolism. Perfusion of the isolated rat pancreas with two chemically different inhibitors of cyclooxygenase, flurbiprofen and sodium salicylate, markedly inhibited prostaglandin E2 release, but had little effect on glucose-induced insulin release or on potentiation of insulin release caused by prior exposure to glucose. On the other hand, nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, not only inhibited both phases of glucose-induced insulin release but also abolished the potentiation effect. These effects of NDGA prevailed, when it was administered together with flurbiprofen, which caused profound inhibition of prostaglandin E2 release. We conclude that 1) lipoxygenase pathways play a dominant role in glucose-stimulated insulin release, and 2) endogenous lipoxygenase metabolites influence the potentiating effect of glucose on the release of insulin in response to a subsequent stimulation.
A newly developed model for perfusion of the isolated rat ovary was employed to study the interactions of Prl with PGF2 alpha in respect to the effects of LH on cAMP formation and progesterone production in the 5 day old corpus luteum of the PMSG-treated rat. An inhibitory effect of PGF2 alpha on both basal and LH stimulated progesterone secretion was found. This block also involved inhibition of the ovarian cAMP release which was not associated with a reduction of the flow of the medium to the ovary. When Prl was present in the medium the PGF2 alpha block of LH-induced cAMP release was reversed. However, Prl failed to restore block of LH stimulation of progesterone secretion in 4 out of 9 experiments, indicating an additional site of action of PGF2 alpha distal to the cAMP in these experiments.
Perfused non working isolated rat heart preparations have been used to study whether alanine (0,89 gl-1) could represent an energy source for the myocardium. Its influence on glucose and lactate metabolism in the heart was also studied. The use of 14C labelled compounds permitted the estimation of the oxidation rate of these three metabolites. The results show that: 1) Alanine was not utilized by the heart. 2) Alanine did not alter myocardial glucose metabolism or lactate oxidation. 3) Alanine lowered lactate uptake by the myocardium which may indicate that alanine influences lactate transport through the cell membranes. One may speculate that in vivo, alanine will not interfere with glucose or lactate metabolism by the heart as, even in acute physiological conditions (exercice, hypoxie, fasting) lactate is always present in blood at higher concentration than alanine.
The routes by which secretory proteins leave the pancreas have been studied during single-pass perfusion of the isolated cat pancreas with a physiological salt solution. The amounts of amylase and total protein appearing in pancreatic juice, venous effluent, and the exudate escaping from the surface of the gland (probably representing lymph) were measured during stimulation with secretin alone and together with bolus injections of acetylcholine. During stimulation with secretin alone, the amylase output in venous effluent was four times greater than in pancreatic juice or exudate. In contrast, most protein appeared in exudate, some in the venous effluent, and a very small amount in pancreatic juice. Acetylcholine transiently increased the output of both amylase and total protein in pancreatic juice to values that greatly exceeded those in venous effluent and exudate. After a delay of 10 min, it also increased the output of amylase and total protein in exudate, but had no effect on venous effluent. In conclusion, secretory proteins enter the venous circulation at a constant rate (i.e., not influenced by acetylcholine stimulation). Some also appear in exudate (lymph), the proportion of which is determined by stimulation. Whether the proteins in these two fluids and in pancreatic juice are derived from the same intracellular pool remains to be determined.
Aminomethylphosphonate (NMePo) and 2-aminoethylphosphonate (NEthPo) were evaluated as alternative pH indicators in the isolated perfused rat liver using (31)P-nuclear magnetic resonance (NMR). NMePo did not distribute within cells and remained in the extracellular space. It exhibited pH titration with a low pK(a) value (5.35). This behaviour makes NMePo useful for extracellular volume or acidic pH determination. In contrast, NEthPo accumulated within cells without altering liver energetic steady state, evaluated from nucleosides triphosphates resonances, even for prolonged (100 min) experiments. Withdrawal of NEthPo from perfusate revealed a residual resonance corresponding to the internalized amount of this phosphonate. This fraction was almost stable vs time and allowed determination of spin-lattice relaxation time constant T(1) within the liver (2.2 +/- 0.3 s; n = 6). Comparison of the titration curves for NEthPo and inorganic phosphate revealed that the accuracy of pH determination within physiologic or acidic range in both cases was comparable. Finally, when extracellular pH was decreased, the NEthPo resonance frequency was found to undergo the same chemical shift variations as observed for cytosolic P(i) signal, which was in good agreement with a cytosolic accumulation of this phosphonate. Therefore, NEthPo could be considered as an interesting cytosolic pH probe suitable for (31)P-NMR measurements, especially when experimental conditions prevent reliable observation of cytosolic Pi resonance.
We studied the effects of a series of vasodilators on intrahepatic vascular resistance of isolated perfused cirrhotic rat livers in basal conditions and during norepinephrine-induced vasoconstriction. Cirrhosis was induced by repeated intraperitoneal injections of carbon tetrachloride. The vasodilators were a nonselective beta-adrenergic antagonist (propranolol), an alpha 1-adrenergic antagonist (prazosin), a nonselective beta-adrenergic agonist (isoproterenol), an alpha 2-agonist (clonidine), nitrovasodilators (nitroglycerin and nitroprusside), calcium channel blockers (verapamil, diltiazem, nifedipine), papaverine, diazoxide and pentoxifylline. In basal conditions, isoproterenol, nitroglycerin, papaverine, pentoxifylline and nitroprusside demonstrated significant vasodilatory activity. However, the response was weak and isoproterenol was the only drug active in the therapeutic range of concentrations. Propranolol, prazosin, verapamil, diltiazem, nifedipine and diazoxide were ineffective. Prazosin, papaverine and pentoxifylline reduced norepinephrine-induced vasoconstriction, whereas isoproterenol, clonidine and propranolol were ineffective. We conclude that several vasodilators can reduce resistance in the cirrhotic rat liver, but their potency is low and few are effective at therapeutic concentrations. Furthermore, their activity may be blunted when resistance is increased by norepinephrine.
The role of microcirculation in the pathogenesis of alcoholic liver injury was investigated in isolated perfused livers from fed rats. Infusion of ethanol into the portal vein at concentrations ranging from 25 to 200 mmol/L increased portal pressure, which is an indicator of hepatic vasoconstriction, in a concentration-dependent fashion. Portal pressure started to rise immediately on initiation of ethanol load and remained at higher than basal levels throughout the period of ethanol infusion. Release of lactate dehydrogenase, an indicator of cell injury, into the effluent perfusate began to increase after 20 to 30 min of ethanol infusion and continued to increase until the end of the experiment (60 min after the initiation of ethanol infusion). The lactate dehydrogenase level in the effluent perfusate at 60 min was dependent on the ethanol concentration (0 mmol/L, 8 +/- 3 IU/L; 25 mmol/L, 22 +/- 3 IU/L; 50 mmol/L, 51 +/- 11 IU/L; 100 mmol/L, 60 +/- 7 IU/L; 200 mmol/L, 120 +/- 7 IU/L). Simultaneous infusion of sodium nitroprusside (100 mumol/L), a known vasodilator, inhibited significantly the ethanol-induced increases in portal pressure and lactate dehydrogenase release by abolishing hepatic vasoconstriction. In histological examinations focal hepatocellular necrosis, evidenced by trypan blue staining of cell nuclei, was detected predominantly in midzonal and pericentral areas of the liver lobule after 60 min of ethanol infusion. Change in portal pressure during 60 min of ethanol infusion correlated significantly with levels of lactate dehydrogenase after ethanol infusion (r = 0.82; p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)