Inhibitory effect of somatostatin analogue, SMS 201-995, on secretin-stimulated exocrine secretion in isolated perfused rat pancreas

In order to clarify the effect of somatostatin of the ductal secretion of the exocrine pancreas, we measured pancreatic juice and protein secretion stimulated with 10 pM secretin and/or 10 pM cholecystokinin (CCK) in the presence or absence of somatostatin analogue, SMS 201-995 (SMS) utilizing the isolated perfused pancreas of rats. SMS significantly inhibited both pancreatic juice flow and protein output elicited by 10 pM secretin without affecting basal secretion. The inhibitory effect of SMS was dose-dependent and maximal inhibition was observed with 1-10 nM. Half-inhibitory dose of SMS for juice secretion was 140 pM. Because CCK is thought to potentiate secretin action on the ductal system, we examined the effect of SMS on pancreatic secretory response to 10 pM secretin in combination with 10 pM CCK. In the experimental system we used, the amounts of pancreatic juice and protein secreted during a 30-min stimulation with secretin and CCK were additive. SMS inhibited both pancreatic juice and protein secretion to the level comparable with that obtained with either stimulus and SMS. SMS had no effect on CCK-stimulated pancreatic juice secretion but significantly inhibited protein output. The present study demonstrated, therefore, that SMS inhibits ductal secretion in response to physiological concentration of secretin.     

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 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.     

Effect of an experimental malaria infection on the metabolism of phenacetin in the rat isolated perfused liver

1. The effect of infection with the rodent malaria parasite Plasmodium berghei on the metabolism of phenacetin has been investigated in a rat isolated perfused liver preparation. 2. A bolus dose of phenacetin (10 mg) was introduced into the perfusate reservoir of both control (n = 4) and malaria-infected (n = 4) liver preparations, and samples of bile and perfusate were collected (0-4 h) for hplc analysis of phenacetin, paracetamol and its phase II metabolites. 3. Whereas malaria had no effect on the hepatic clearance of phenacetin (control: 0.64 +/- 0.15 versus malaria: 0.66 +/- 0.14 ml min-1), there was a significant reduction in the hepatic clearance of generated paracetamol (control: 1.22 +/- 0.15 versus malaria: 0.41 +/- 0.08 ml min-1) and the total recovery in bile and perfusate of paracetamol glucuronide (control: 1.18 +/- 0.44 versus malaria: 0.29 +/- 0.20 mg). There was no significant change during malaria infection in the total recovery of either phenacetin (control: 1.30 +/- 0.73 versus malaria: 0.79 +/- 0.36 mg) or paracetamol sulphate (control: 0.81 +/- 0.25 versus malaria: 0.74 +/- 0.16 mg),     

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.     

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.     

Influence of pentobarbital-Na on stimulation-evoked catecholamine secretion in the perfused rat adrenal gland

The relationship between leukocyte migration and parenchymal cell death in vivo remains poorly documented. Accordingly, cell killing in the rat mesentery, as recorded by propidium iodide staining, was investigated with an intravital approach. Superfusion of platelet-activating factor (PAF, 10(-8) M) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) led to extensive leukocyte extravasation but no significant cell death. In contrast, pretreatment with 10(-8) M PAF or fMLP for 1 h, followed by superfusion of PAF in combination with fMLP (both at 10(-8) M) led to an increase in cell death. Mesenteric parenchymal cells but no endothelial cells were killed. Some of the dead cells were identified as granulocytes/monocytes that were already in the tissue at the start of the experiment. The incidence of cell death was lower but not eliminated when leukocyte migration was blocked with a monoclonal antibody against CD18. A xanthine oxidase inhibitor, BOF-4272, failed to diminish cell death, whereas a hydroxyl radical scavenger, dimethylthiourea, attenuated cell killing without an effect on the number of adhering and migrating leukocytes. These observations demonstrate that leukocytes serve as a factor in the killing of extravascular cells only after the development of a level of stimulation that differs from that required to induce a migratory stimulus into the extravascular space.     

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.     

Effect of a new CCK-A receptor antagonist, dexloxiglumide, on the exocrine pancreas in the rat

The effect of dexloxiglumide, a new potent cholecystokinin (CCK) antagonist, on pancreatic enzyme secretion and growth was studied in the rat. Pancreatic exocrine secretion was studied both in vitro (isolated and perfused pancreatic segments) and in vivo (anaesthetized animals with cannulation of the common bile duct) whereas the trophic effect was investigated after short-term (7 days) administration of the CCK-agonist, caerulein, or camostate (a potent trypsin inhibitor), with or without dexloxiglumide. CCK-8 stimulated amylase release from in vitro pancreatic segments in a concentration-dependent manner. Dexloxiglumide displaced the concentration response curves to CCK-8 to the right without affecting the maximum response, suggesting a competitive antagonism. The Schild plot analysis of data gave a straight line with a slope (0.90 +/- 0.36) not significantly different from unity. The calculated pA2 for dexloxiglumide was 6.41 +/- 0.38. In vivo experiments confirmed results from in vitro studies since intravenous dexloxiglumide reduced pancreatic exocrine secretion induced by submaximal CCK-8 stimulation (0.5 nmol/kg/h) in a dose-dependent manner, the ID50 being 0.64 mg/kg. Both exogenous and endogenous (released by camostate) CCK increased the weight of the pancreas, the total pancreatic protein and DNA, trypsin and amylase content. Dexloxiglumide (25 mg/kg), administered together with caerulein (1 microgram/kg), reduced the peptide-induced increase in pancreatic weight, protein and enzyme content. Similarly, when dexloxiglumide was given together with camostate (200 mg/kg), all the observed changes were reduced by concomitant administration of the antagonist. These results demonstrate the ability of dexloxiglumide to antagonize the effects of CCK on pancreatic secretion and growth, suggesting that this compound is a potent and selective antagonist of CCK-A-receptors in the pancreas.     

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.     

Histamine receptor subtypes mediating hyperpolarization in the isolated, perfused rat mesenteric pre-arteriolar bed

Histamine is a general dilator of rat blood vessels. We investigated the relative contribution of receptor subtypes to the rat mesenteric dilator responses initiated by histamine and related agonists. Histamine initiated dose, and endothelium-dependent, dilation of constricted mesenteric beds with an ED50 of 0.4 +/- 0.1 nmol. The ED50 was increased 10-fold by 0.1 microM chlorpheniramine (a histamine H1-receptor selective antagonist). Histamine H2 receptor blockade with tiotidine (0.1 microM) slightly decreased, while thioperamide (1 microM), a selective histamine H3 receptor antagonist, did not block histamine-induced dilation. Mesenteric bed dilation initiated by histamine H2 receptor selective agonists, amthamine and dimaprit, were antagonized markedly by tiotidine. However, the dilation initiated by the putative histamine H3 receptor selective agonists, R(-)- or S(+)-alpha-methylhistamine and imetit were not affected by thioperamide (1 microM). Histamine H2- and H3-receptor mediated dilator effects were endothelium-independent and were blocked by either excess (80 mM) extracellular K+, or 1 mM tetrabutylammonium (a non-selective K+ channel blocker), as well as by 1 microM dequalinium, a non-peptide blocker of the small conductance Ca2+-activated (SKCa) K+ channels. We conclude that (i) histamine H1 receptor subtype predominantly mediates endothelium-dependent dilator effect of histamine, and (ii) vascular hyperpolarization through opening of K+ channels (SKCa) mediate the dilator responses to histamine H2 receptor (amthamine and dimaprit) and the putative histamine H3 receptor (R(-)-alpha-methylhistamine and imetit) agonists.     

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.     

Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: comparison with calcitonin gene-related peptide effects

Adrenomedullin (ADM), a vasodilatatory peptide contained in adrenal medulla, was found to induce a dose-dependent increase in aldosterone (ALDO) and corticosterone (B) release by the in situ perfused rat adrenal gland, along with a rise in the flow rate of the perfusion medium. The minimal effective dose for ALDO response was three and two orders of magnitude less than those able to evoke B and medium flow rate responses. Calcitonin gene-related peptide (CGRP), another vasodilatatory peptide contained in adrenal medulla and showing a slight homology in its amino acid sequence with ADM, elicited similar effects. CGRP (8-37), a specific antagonist of CGRP1 receptors, annulled all the effects of both ADM and CGRP, whereas l-alprenolol, a beta-adrenoceptor antagonist, partially reversed only ALDO response to the peptides. In light of these findings the following conclusions are drawn: i) ADM and CGRP stimulate rat adrenals in vivo to release B by raising blood flow rate; ii) ADM and CGRP enhance ALDO secretion via an indirect mechanism probably requiring the release of catecholamines by medullary chromaffin cells; and iii) the effects of ADM and CGRP on the rat adrenal gland are mediated by a common receptor of the CGRP1 subtype.     

Effect of S-adenosyl-L-methionine and dilinoleoylphosphatidylcholine on liver lipid composition and ethanol hepatotoxicity in isolated perfused rat liver

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.