Colonic mucin discharge by a cholinergic agonist, prostaglandins, and peptide YY in the isolated vascularly perfused rat colon

Efforts to understand the function of dystrophin, the protein product for the Duchenne muscular dystrophy gene, resulted in the purification of the dystrophin-glycoprotein complex. Over the past year several novel components of this complex have been identified. Recent studies have extended the number of muscular dystrophies associated with the oligomeric complex to six genetically distinct diseases, including three new forms of limb-girdle muscular dystrophy and one form of congenital muscular dystrophy.     

Evaluation of biologic gastrin activity of compound CI-988 in the isolated, vascularly perfused rat stomach

BACKGROUND: The peptoid CI-988 has previously been shown to have high affinity for the cholecystokinin (CCK)-B/gastrin receptor and has been reported to be a powerful CCK antagonist in many systems, although it has agonist activity on histidine decarboxylase in the rat. METHODS: In the present study the effect of CI-988 on acid secretion and histamine release in the totally isolated, vascularly perfused rat stomach was assessed. RESULTS: CI-988 was found to be a gastrin agonist with regard to the stimulation of both histamine release and acid secretion. CONCLUSION: Thus, in this stomach model CI-988 behaved as a CCKB/gastrin agonist. The present study underlines the importance of testing the biologic activity of ligands in models with sufficient sensitivity.     

Peptide YY, glucagon-like peptide-1, and neurotensin responses to luminal factors in the isolated vascularly perfused rat ileum

Exposure of the ileum to nutrients markedly inhibits several upper gastrointestinal functions. Hormonal peptides of the ileal wall, i.e. peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and neurotensin (NT), are thought to play a role in this negative feedback mechanism. The present study was conducted to comparatively assess the secretion of PYY, GLP-1, and NT upon luminal infusion of a variety of individual luminal factors in the isolated vascularly perfused rat ileum preparation. PYY, GLP-1, and NT were measured in the portal effluent with specific RIAs. Glucose (250 mM) induced a pronounced release of the three peptides, whereas a physiological concentration of 5 mM did not induce peptide secretion. Peptone (5%, wt/vol) evoked a sustained release of PYY, GLP-1, and NT. Only NT secretion was increased upon luminal administration of 100 mM sodium oleate. Short chain fatty acids (20 mM) evoked an early and transient release of the three peptides. In contrast, taurocholate (20 mM) induced a sustained release of PYY, GLP-1, and NT, but the threshold concentration for peptide release was lower for NT than for PYY or GLP-1. Cellulose or pectin (0.5%, wt/vol) did not modify peptide secretion. In conclusion, glucose and peptone are potent stimulants of PYY, GLP-1, and NT release. Only NT is released upon oleic acid stimulation. Finally, taurocholate is a potent stimulant of the release of the three peptides. Overall, PYY, GLP-1, and NT may participate cooperatively in the ileal brake. As relatively high concentrations of the various stimulants were required to elicit peptide release, it seems likely that this mechanism operates in cases of maldigestion or malabsorption.     

The isolated air perfused rat heart

A simple method has been developed for continuous monitoring of metabolic activity of an isolated, perfused rat heart by O2/CO2 respirometer. Since respirometer provides vital data on oxygen consumption and carbon dioxide production of a preserved organ on a continuous basis over a long period of time, it will be possible to use this method to monitor viability of not only isolated heart but also any given donor organ under preservation.     

Regulation of renin release and intrarenal formation of angiotensin. Studies in the isolated perfused rat kidney

1. Isolated rat kidneys were perfused at a constant pressure of 90 mmHg in a single-pass system with either a cell-free medium or a suspension of washed bovine red blood cells, free of the components of the renin-angiotensin system. In red blood cell perfused kidneys renal haemodynamics and sodium reabsorption corresponded closer to values observed in the intact rat than in cell-free perfused kidneys. 2. In red blood cell-perfused kidneys in the absence of plasma renin substrate autoregulation of renal blood flow was almost complete at pressures above 90 mmHg, provided that perfusion pressure was changed rapidly. 3. Renin release varied inversely with perfusion pressure within a pressure range from 50 to 150 mmHg; the greatest changes of renin release occurred, when perfusion pressure was reduced from 90 to 70 mmHg; maximal stimulation of renin release was observed at 50 mmHg. After reduction of perfusion pressure, renin release immediately started to rise and reached a new level within 5 min. Local reduction of perfusion pressure in small arteries and arterioles by the injection of microspheres induced a short-lasting decrease in renal plasma flow and a transient stimulation of renin release. 4. High concentrations of furosemide stimulated renin release by a direct intrarenal mechanism. 5. Isoproterenol stimulated renin release in low concentrations without a concomitant vasodilation, whereas high concentrations induced an increase in both renal plasma flow and renin release. The effects of isoproterenol were completely blocked by propranolol. 6. Sodium nitroprusside induced similar increases in renal plasma flow, as did high concentrations of isoproterenol, but only a small and slow increase in renin release was observed. 7. Angiotensin II (AII) suppressed renin release in concentrations corresponding to plasma levels measured in the intact rat independently of its vasoconstrictor effects, whereas vasopressin in antidiuretic concentrations did not affect renin release. 8. AII, AI, synthetic tetradecapeptide renin substrate (TDP), crude and purified rat plasma renin substrate induced a dose-dependent reduction in renal plasma flow. SQ 20 881, a competitive inhibitor of converting enzyme, and low doses of 1-Sar-8-Ala-AII (saralasin), a competitive antagonist of AII, did not change renal plasma flow, whereas high concentrations of saralasin had a vasoconstrictor effect on their own. 9. Saralasin inhibited the vasoconstrictor effects of AII and TDP to a similar degree. SQ 20 881 inhibited the vasoconstrictor effects of AI and purified renin substrate, but did not influence the actions of TDP and the crude renin substrate preparation. 10. From these data it is concluded, that AI is converted into AII within the kidney at a rate of 1-2%. The vasoconstriction induced by the crude renin substrate probably does not involve the AII receptors. TDP may act by itself on the AII receptors or via the direct intrarenal formation of AII...     

Effects of thyroxine pretreatment and calcium on the isolated perfused rat heart

Phosphorylase a activity was the same in isolated perfused hearts from euthyroid and thyroxine-pretreated rats. Perfusion with 3.6 mM Ca2+ caused an increase in phosphorylase a in hearts from euthyroid as well as those from thyroxine-pretreated animals, but the Ca2+-induced stimulation of phosphorylase activity was similar in both groups over the time course studied. Greater conversion of phosphorylase b to a occurred with 7.2 mM than with 3.6 mM Ca2+ in both groups, but once again thyroxine pretreatment did not significantly influence the conversion of phosphorylase b to a. Isometric systolic tension increased in response to 3.6 mM and 7.2 mM Ca2+ in hearts from normal and thyrotoxic rats, but thyroxine pretreatment did not appreciably alter the nature of this response. While spontaneous heart rate was higher in hearts from thyroxine-pretreated rats, perfusion with 3.6 mM or 7.2 mM Ca2+ had no significant effect on heart rate in hearts from euthyroid or thyrotoxic rats.     

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.     

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.     

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.     

The effect of hepatocyte enlargement on the hemodynamic characteristics of the isolated perfused rat liver preparation

The influence of hepatocyte enlargement on intrahepatic hemodynamics was assessed in the isolated perfused rat liver preparation (IPRL) using two experimental models: hypotonic liver cell swelling and phenobarbitone-induced hepatocyte hypertrophy. The analysis of pressure-flow data obtained from the portal vascular bed over a flow range of 0 to 70 mL/min in the presence of a maximally-effective concentration of the vasodilator agent papaverine hydrochloride (6 x 10(-4) mol/L) enabled the calculation of P0, an estimate of the pressure required to passively distend the intrahepatic vasculature, and Gmax, the maximal portal vascular conductance. By comparison with an isotonic perfusion medium (Krebs-Henseleit buffer [KH] containing 2.5% bovine serum albumin [BSA]), perfusion with a hypotonic medium induced a significant increase in mean hepatocyte cross-sectional area (H(A)) (590 +/- 21 vs. 324 +/- 23 microm(-2), p < .05), a fall in Gmax (0.39 +/- 0.08 vs. 2.02 +/- 0.18 mL/min/g/mm hg, P < .001), and an increase in P0 (2.96 +/- 0.38 vs. 1.58 +/- 0.07 mm hg, P < .001). Phenobarbitone administered in drinking water (0.5 g/L) over a period of 60 days also induced a significant degree of hepatocyte enlargement (HA, 510 +/- 29 microm2, P < .05). On day 7, portal pressure measured in vivo in this group was significantly elevated compared with untreated controls (10.5 +/- 0.3 vs. 8.4 +/- 0.2 mm hg, P < .001), while in the IPRL Gmax was reduced (0.48 +/- 0.01 mL/min/g/mm hg, P < .001), and P0 was increased (2.23 +/- 0.17 mm hg, P < .05). However, with continued phenobarbitone treatment portal pressure, Gmax and P0 returned toward control values. The results confirm that hepatocyte enlargement is associated with a significant disturbance of intrahepatic hemodynamics but also that some adaptation occurs if hepatocyte enlargement is sustained over a prolonged period of time.     

Effect of unconjugated bilirubin on the uptake of p-aminohippuric acid by the isolated perfused rat kidney

The effect produced by unconjugated bilirubin (UB) on p-aminohippuric acid (PAH) translocation from plasma to the urine, was analyzed by using an isolated rat kidney preparation applying the multiple indicator dilution technique. Arterial -to- urine mean transit times and fractional recoveries of creatinine and PAH in the venous effluent, were calculated before and after the incorporation of UB into the system. PAH influx into cells, its efflux from the cells to the capillaries, and its movement between lumen and tubular cells were also calculated. The results indicated that PAH influx into cells was impaired in the presence of UB. The phenomenom was shown to be dose-dependent. On the contrary, the efflux of PAH to the capillaries that was also impaired by UB, seemed to be unrelated to the pigment concentration within the system. Movement of PAH between lumen and tubular cells also appeared to be affected by UB. The results obtained suggested that UB interferes with the renal handling of PAH but the mechanism involved could not be clarified with the model used.     

Effect of metabolic inhibitors on Na+ transport in isolated perfused rat lungs

A quality assurance system for generic screening of patient care has been developed in a university hospital in The Netherlands. This system is based on the Medical Management Analysis System, developed in the United States. Patient care is systematically screened for adverse patient occurrences (APOs). Screening criteria have been developed and some adjustments have been made to the original system, because of structural differences in health care between the United States and The Netherlands. The results of a study comparing two screening strategies are presented: screening by a medically trained, outside reviewer and screening by the Ear, Nose and Throat specialists themselves. The reviewer screened almost all admissions to the department as opposed to the specialist, who filled in screening forms of only 77.5% of the admissions. Specialists are found to register more APOs in their own domain (42%) as compared to the screener (28%). Issues concerning the nursing process receive less attention by the specialists (14%) than by the screener (22%). Because the personnel delivering direct patient care are more familiar with and informed about this process of care, the importance is stressed of getting specialists as well as nurses involved in the evaluation of the care they deliver to patients.     

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.