Disposition of the bromosulfophthalein-glutathione conjugate in the isolated perfused rat kidney

Renal elimination of the bromosulfophthalein-glutathione conjugate (BSP-GSH) after its i.v. administration in the rat in vivo is negligible. In our study we wanted to establish whether the high albumin-binding of BSP-GSH constitutes the major restrictive factor toward the urinary excretion of the compound. The renal disposition of BSP-GSH was studied in the isolated rat kidney during perfusions with or without albumin in the perfusate. The urinary clearance of BSP-GSH in the absence of albumin was very low (< 60 microliters/min) as compared to the inulin clearance (approximately 300 microliters/min). This indicates that albumin-binding is not the major reason for the low urinary clearance of BSP-GSH. Addition of albumin to the perfusate further decreased the urinary excretion by 60%. BSP-GSH is metabolized by the kidney into two major metabolites: the cysteinylglycine conjugate and the di-glutathione conjugate. Both metabolites appear in perfusate, which suggests that BSP-GSH undergoes tubular (re-)uptake. The di-glutathione conjugate is further metabolized to the di-cysteinylglycine conjugate. The di-glutathione conjugate and the di-cysteinylglycine conjugate are the major urinary components and the urinary elimination of BSP-GSH may depend on their formation. Inhibition of gamma-glutamyl transpeptidase activity with acivicin largely prevented the degradation to the cysteinylglycine and dicysteinylglycine conjugates of BSP. The total rate of urinary excretion, however, was only slightly lowered by acivicin. Apparently, cleavage of the gamma-glutamyl moiety is not relevant for the total urinary elimination of BSP-GSH.     

Vitamin B2 metabolism in the isolated perfused rat liver

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage lambda: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.     

Steroid biosynthesis in the whelk, Buccinum undatum

Cell-mediated immunity (CMI) to homologous spermatozoal antigens was studied in sixty-two males following vasectomy of a duration of 1-10 years. A group of twenty-two normal, fertile non-vasectomized males was also included in the study. The inhibition in the leucocyte migration test (LMT), in the presence of spermatozoal antigen, was taken as an index of CMI. Twenty of the sixty-two vasectomized males (32.2 percent) showed a positive LMT reaction. When the results were analysed with reference to the duration of vasectomy, it was noted that four cases each (22.2 percent), showed a positive LMT reaction in the groups 0-2 years, and 3.5 years. On the other hand twelve cases gave a positive reaction in the group 6-10 years (46.1 percent). It appears that the incidence of CMI to spermatozoa increases with the duration in vasectomy.     

Studies on the viability of the isolated vascularly perfused rat colon

The colon contains large numbers of endocrine cells. Insight into their physiological function is limited. This is due to the fact that no sufficient model of isolated endocrine colon cells is available. In the present study we introduce an isolated vascularly perfused colon model for in vitro studies. This model offers the advantage that it keeps the endocrine cells in their physiological orientation and environment. The gut mucosa is highly sensitive to ischemia. Therefore, a careful validation of its viability is crucial in gut organ preparations. This study demonstrates that, by utilizing an oxygenated vascular medium supplemented with 25% washed bovine erythrocytes, a perfusion of the colon is achieved for at least 1 h without obvious tissue injuries. During this time parameters such as perfusion pressure, venous lactate dehydrogenase release, glucose consumption, lactate output, oxygen consumption, perfusate loss by the preparation and morphology were analyzed. Dependent on stimulation, the endocrine L cells of the colon released glucagon-like peptide-I upon arterial perfusion of methacholine or gastrin-releasing peptide. In conclusion, a model for the isolated perfusion of the colon is introduced which is suitable for studies of endocrine colon cells.     

The interaction of endothelin receptor responses in the isolated perfused rat lung

To gain more insight into the complex pulmonary interactions of endothelins (ET), we studied airway and vascular responses to endothelins in isolated perfused rat lungs in the presence of the novel ET(B)-receptor antagonist BQ788. In particular we focused on airway responses and on prostacyclin release. The effectiveness of BQ788 in our system was shown by its ability to concentration-dependently prevent vasoconstriction (IC50 0.1 microM), bronchoconstriction (IC50 0.1 microM) and prostacyclin production (IC50 < 0.1 microM) induced by the ET(B)-receptor agonist IRL1620 (1 nmol). Airway responses to ET-1: ET-1-induced bronchoconstriction was aggravated by BQ123 (1 or 8 microM), while BQ788 pretreatment (1 or 8 microM) showed no significant effect. Simultaneous treatment with 8 microM BQ123 and BQ788 attenuated the ET-1-induced bronchoconstriction. Vascular responses to ET-1: ET-1 (1 nmol)-induced vasoconstriction was potentiated by BQ788 (1 or 8 microM), but attenuated by the ET(A)-receptor antagonist BQ123 (1 microM). In the presence of BQ788 diminished amounts of the stable prostacyclin metabolite 6-keto-PGF1alpha were detected in the perfusate. Simultaneous treatment with 8 microM BQ123 and BQ788 completely prevented the ET-1-induced vasoconstriction. Conclusions: Both ET(A)- and ET(B)-receptors contribute to ET-1-induced vasoconstriction and bronchoconstriction. The ET-1-induced vasoconstriction is attenuated by stimulation of ET(B)-receptors, a response that is partly mediated by prostacyclin. Due to the mutual interactions between ET(A)- and ET(B)-receptors, simultaneous inhibition of both receptors is required to prevent the deleterious effects of ET-1 on lung functions.     

Glucose transport in isolated perfused proximal tubules of snake kidney

Glucose transport was studied in isolated, perfused snake (Thamnophis spp.) renal tubules. When 14C-labeled and unlabeled glucose concentrations for bath and perfusate were identical, net transepithelial glucose transport occurred from lumen to bath. Maximum rates of transport were 1.24 X 10-12 and 2.17 X 10-12 mol min-1 mm-1 in proximal-proximal and distal-proximal segments, respecitvely. Glucose concentration in cells of perfused tubules of both segments was less than that of bath and lumen when tubules spontaneously stopped transporting glucose. Transepithelial glucose permeability ath leads to lumen) was about 0.25 X 10-5 cm sec-1 for both segments. Peritubular membrane permeability (bath leads to cell) was about 0.50 X 10-5 cm sec-1 for both segments. Luminal membrane permeabilities (cell leads to lumen) were 0.29 X 10-5 and 0.65 X 10-5 cm sec-1 for proximal-proximal and distal-proximal segments, respectively. Luminal membrane permeability in opposite direction (lumen leads to cell) was about 10.0 X 10-5 cm sec-1 for both segments. These results indicate that, during maximum glucose absorption, glucose enters cells down concentration gradient across luminal membrane by a mediated process and is transported out of the cells against concentration gradient at peritubular membrane.     

Uptake and metabolism of nicotine by the isolated perfused rabbit lung

In order to investigate the possibility of pulmonary first-pass metabolism of nicotine inhaled in tobacco smoke, the absorption and disposition of 14C-nicotine were studied in an isolated perfused rabbit lung preparation after nicotine administration directly into the perfusing blood and tobacco smoke administration via in the inspired tracheal air. After administration into the perfusing medium, the rate of nicotine metabolism was first-order and dose-independent at the two doses studies (0.1 and 1.0 mg) but lung metabolic clearance was quite low (3 ml/min) relative to whole body clearance (140 ml/min) measured by administering 14C-nicotine to intact rabbits. Accumulation of nicotine by lung was not extensive (13-23% of the dose administered). After administration of tobacco smoke from 14C-nicotine-spiked cigarettes, absorption of nicotine was rapid but the rate of metabolism was markedly reduced compared to the studies in which drug was administered in the perfusing medium. This reduction in the rate of metabolism was apparently caused by some component of tobacco smoke but was shown to be unrelated to the level of carbon monoxide in the perfusate. The slow clearance of nicotine by rabbit lung (which is further reduced after smoke administration) compared to a high pulmonary blood flow rate makes unlikely the possibility of significant first-pass lung metabolism in smokers.     

Disposition of morphine in the rat isolated perfused kidney: concentration ranging studies

The rat isolated perfused kidney was used to investigate the linearity of the renal disposition of morphine and its potential oxidative and glucuronidative metabolism by the kidney. In a set of single-dose experiments, morphine was administered to recirculating perfusion medium to achieve initial concentrations of 0.2, 2 and 20 microM (n = 4 at each concentration). In a set of multiple-dose experiments, morphine was administered to perfusate as sequential bolus doses to achieve concentrations of 0.2, 2, 20 and 200 microM (n = 6). HPLC was used to determine the concentration of morphine in perfusate and urine. Normorphine, morphine-3-glucuronide and morphine-6-glucuronide could not be detected in perfusate or urine, a result that suggests an absence of oxidative and glucuronidative metabolism of morphine by the rat kidney. The volume of distribution of morphine within the kidney was high (31 +/- 3 ml/g at 0.2 microM), which indicates extensive accumulation, and remained constant with increasing perfusate concentration. The ratio of unbound renal excretory clearance to glomerular filtration rate was always greater than unity for all kidneys, which indicates that the renal excretion of morphine involves net tubular secretion. This ratio was constant (P > .05) over the 100-fold concentration range of the single-dose study. In the multiple-dose study, the ratio was marginally but significantly (P < .05) higher at concentrations of 2, 20 and 200 microM than at 0.2 microM, a difference that cannot be explained by saturation of tubular secretion. The results suggest that the tubular secretion of morphine is not saturated over a wide range of concentrations (0.2-200 microM).     

The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas

Gastric inhibitory polypeptide (GIP) produced a dose-related increase in immunoreactive insulin (IRI) from the perfused isolated rat pancreas. The doses employed were within physiological limits. This effect was glucose-concentration-dependent in that there existed a threshold concentration of glucose above which GIP exerted the insulinotropic action, and that, at a fixed concentration of GIP, increased glucose concentrations stimulated IRI release in more than an additive manner. A biologically active fragment of the GIP molecule was isolated and purified. All criteria have been satisfied that GIP is an insulinotropic hormone.     

Effect of ventricular dilatation on defibrillation threshold in the isolated perfused rabbit heart

INTRODUCTION: Ventricular dilatation has important electrophysiologic effects, but its effect on ventricular defibrillation threshold (DFT) is unknown. METHODS AND RESULTS: A fluid-filled, latex balloon was placed in the left ventricular cavity of 19 isolated rabbit hearts. In each experiment, an undilated volume (equivalent to a left ventricular end-diastolic pressure of approximately 0 mmHg) was compared to a dilated volume achieved by adding 1.0 mL of saline (n = 10) or 5% dextrose (n = 9) to the intracavitary balloon. Left ventricular effective refractory period (ERP) and DFT were determined at each volume. Defibrillation was attempted with a monophasic shock delivered between a patch electrode positioned over the posterior left ventricle (cathode) and a metallic aortic cannula (anode). DFT was determined using a modified "down/up" protocol with 10 V steps. Ventricular dilatation increased the left ventricular end-diastolic pressure from 0 +/- 0.5 mmHg to 35 +/- 3 mmHg (P < 0.001), decreased the average left ventricular ERP 15% (from 116 +/- 3 msec to 99 +/- 3 msec; P < 0.001), and increased the average DFT 30% (from 96 +/- 4 V to 125 +/- 7 V; P < 0.001). In one third of experiments, the dilated DFT was > or = 150% of the DFT at zero volume. The mechanism of the observed increase in DFT is unknown but may be related to the decrease in refractoriness observed with ventricular dilatation. CONCLUSION: Acute ventricular dilatation in this model increased DFT an average of 30%, an effect not previously described. This observation may have implications for patients with implantable cardioverter defibrillators.     

The influence of glycerol on the Ca flux in the isolated perfused rat heart

Perfusion of the isolated rat heart with a fluid containing 800mM glycerol in addition to its normal constituents causes a contraction height decrease of about 50%. A change to normal perfusion fluid causes a period of contracture to occur. After this period the heart is perfectly viable. Mechanical and osmotic effects could be ruled out as possible causes. No gross disturbances were seen in the electron micrographs. An effect on intermediary metabolism is equally unlikely. Presumably, we must seek the explanation in a direct effect of the glycerol on the Ca++ fluxes across the cellular and intracellular membranes.     

Protective effect of vanadate on oxyradical-induced changes in isolated perfused heart

In order to examine the mechanisms of the beneficial effects of vanadate on cardiac dysfunction in chronic diabetes, rat hearts were perfused with xanthine plus xanthine oxidase, an oxyradical generating system in the absence or presence of vanadate. The heart failed to generate contractile force and increased the resting tension markedly within 5 min of perfusion with xanthine plus xanthine oxidase. These changes were prevented by the addition of 4 microM vanadate in the perfusion medium. The protective effects of vanadate on the loss of developed tension and increased resting tension due to xanthine plus xanthine oxidase were dose-dependent (0.1-5 microM). Perfusion of the hearts with glucose-free medium did not abolish the protective actions of vanadate. The sarcolemmal Ca(2+)-pump (ATP-dependent Ca2+ uptake and Ca(2+)-stimulated ATPase) and Na(+)-dependent Ca2+ uptake activities were decreased upon perfusing the hearts with a medium containing xanthine plus xanthine oxidase for 5 min; these effects were prevented by the addition of 2-4 microM vanadate in the perfusion medium. The signals for superoxide radicals produced by xanthine plus xanthine oxidase, as detected by electron paramagnetic resonance spectroscopic technique, were inhibited by 5-100 microM vanadate. These results suggest that vanadate is an oxyradical scavenger and thus may prevent heart dysfunction under some pathological conditions by its antioxidant action.     

Intravascular and interstitial degradation of bradykinin in isolated perfused rat heart

Porphyromonas gingivalis, a Gram-negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine-specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg-specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real-time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease-degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme-linked biotin-avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of L-Arg, or oligopeptides containing the Arg residue at the C-terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C-terminus. Taken together, these studies indicate that P. gingivalis arginine-specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C-terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae-Arg interaction (a novel host-parasite relationship).