Prostaglandin release from perfused, isolated hind legs in normal and arteriosclerotic rats

Intraarterial injection of 10 microgram noradrenaline (NA) produced a stronger increase of prostaglandin E-like material (PGE) in the outflow of isolated perfused hind legs of normal rats than of those of arteriosclerotic rats. The vasopressor effect of NA in normal rats was stronger than in arteriosclerotic rats, which could be explained on the basis of PGE release.     

Electrical and mechanical activity of isolated canine stomach perfused with fluorocarbon emulsion

Electrical and mechanical activities were recorded from the isolated canine stomach perfused with fluorocarbon emulsion and oxygenated in vitro. This preliminary experiment was undertaken to determine whether or not these activities can be preserved during extracorporeal bloodless perfusion using a perfusate containing only an oxygen carrier, fluorocarbon and a simulated physiologic salt solution of some electrolytes. We found that electrical control activity of stomachs so perfused was identical with that found under in vivo conditions. The electrical and mechanical response of these stomachs to intra-arterially injected methacholine and pentagastrin and to electrical stimulation of the vagus nerve suggested that the function of the muscular and intramural plexus layers of the gastric wall remained normal. It was also observed that the frequency of the cycles of electrical control activity and the amplitude of antral contractions were significantly increased, when pO2 of the circulating perfusate rose. Results of the biochemical studies of the perfusate suggested utilization of some of its components for the metabolic needs of the perfused organ. The gastric secretion was alkaline and contained particles of fluorocarbon emulsion.     

Acute pancreatitis with hyperlipemia: studies with an isolated perfused canine pancreas

Clinical evidence suggests that in many settings hypertriglyceridemia can initiate an episode of acute pancreatitis. Hydrolysis of triglycerides by pancreatic lipase with the local release of large quantities of free fatty acids (FFAs) has been proposed as the pathogenetic mechanism. To gather information to evaluate this mechanism an isolated, ex vivo, perfused pancreatic preparation was used. Control preparations remained normal in gross appearance, gained little weight (18 gm), extracted oxygen and glucose and released carbon dioxide, and continued to secrete during a 4 hour perfusion period. Serum amylase remained normal (972 CU/100 ml) as did FFAs (1.11 mEq/liter). When triglycerides were added to the perfusate to increase the serum triglycerides to 1,600 mg%, the glands became edematous, hemorrhagic, and gained considerable weight (52 gm) during the 4 hour perfusion period. Serum amylase became markedly elevated (2,624 CU/100 ml), as did the serum FFA (29.19 mEq/liter). When FFAs were added directly to the perfusate, the glands became edematous, hemorrhagic, and gained weight (90 gm), but did so much more rapidly than when triglycerides were added. These studies add support to the concept that hypertriglyceridemia can initiate pancreatic injury. Furthermore, they suggest that the mechanism may be through the release of FFAs.     

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...     

Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented.     

Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4

Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).     

The type-1 repeats of thyroglobulin regulate thyroglobulin degradation and T3, T4 release in thyrocytes

Thyroglobulin (Tg) proteolytic steps are central phenomena in Tg processing and thyroid hormone release in thyrocytes. Based on recent literature data, we propose that the type-1 repetitive units present in the Tg sequence could act as binders and reversible inhibitors of the proteases implicated in Tg processing. The pH-dependent interactions of proteases with the repeats could permit (i) protection from degradation of low iodinated Tg to be recycled; (ii) restriction of early proteolytic attacks to N- and C-terminal hormone formation sites; (iii) increase of the half-time of acidic proteases necessary for the final, extensive degradation steps of Tg.     

UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors

1. Vasoconstrictor responses of the isolated and perfused canine epicardial coronary artery to uridine 5'-triphosphate (UTP) were analysed pharmacologically. 2. At basal perfusion pressure, UTP induced vasoconstriction in a dose-related manner and the vasoconstriction was sometimes followed by a slight vasodilatation at large doses (more than 10 nmol). The rank order of potency for vasoconstriction was UTP = UDP > ATP > TTP > or = ITP > UMP. At raised perfusion pressure by 20 mM KCl, the vasoconstriction was not changed and a small vasodilatation was induced at large doses. The rank order of potency for vasodilatation was induced at large doses. The rank order of potency for vasodilatation was ATP > ITP > or = UDP > UTP > or = TTP. The maximal vasodilator response to UTP was much less than that to ATP. UMP did not induce vasodilatation. 3. The P2X receptor agonist and desensitizing agent alpha, beta-methylene ATP (1 microM) and the P2 receptor antagonist suramin (100 microM) inhibited the vasoconstrictor responses to ATP but not those to UTP and UDP. The P2 receptor antagonist reactive blue 2 (30 microM) did not inhibit the vascular responses to UTP. 4. UTP (200 microM) desensitized the vasoconstrictor responses to UTP, but not either the vasodilator responses to UTP or the vasoconstrictor responses to ATP and UDP. UDP (200 microM) did not desensitize the vascular responses to UTP. 5. Preincubating the UDP stock solution and arterial preparation with hexokinase (10 and 1 uml-1, respectively) did not change the vasoconstrictor responses to UDP. 6. The Ca channel blocker diltiazem (1 microM) inhibited the vasoconstrictor responses to UTP but not those to ATP and UDP. Incubation in a Ca(2+)-free solution containing 1 mM EGTA inhibited the vascular responses to ATP, UTP and UDP. 7. Removal of the endothelium by an intraluminal injection of saponin (1 mg) inhibited the vasodilator responses to UTP. Indomethacin, a cyclo-oxygenase inhibitor (1 microM), inhibited the vasodilator responses to UTP, but NG-nitro-L-arginine, a nitric oxide synthase inhibitor (300 microM), did not have an inhibitory effect. 8. The results suggest that (1) UTP induces vasoconstriction via UTP-preferring P2Y receptors on the smooth muscle and vasodilatation via receptors different from those mediating the vasoconstriction induced by UTP and mediating the vasodilatation by ATP on the endothelium, through mainly the release of prostacyclin in the canine epicardial coronary artery; (2) UDP induces vasoconstriction via UDP-preferring P2Y receptors; and (3) L-type Ca ion channels are involved in the vasoconstriction induced by UTP, but not in that induced by UDP.     

Effects of oxyhemoglobin on vasoconstriction in response to 5-hydroxytryptamine in isolated, perfused canine basilar arteries

OBJECTIVE: Oxyhemoglobin (OxyHb) is thought to be a critical trigger in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage. We investigated whether extraluminally applied OxyHb influenced vascular responses to intraluminally applied vasoactive agents in isolated, perfused, canine basilar arteries. METHODS: The steel cannula insertion method was used to examine vascular responses to intraluminally applied 5-hydroxytryptamine (5-HT) receptor agonists, i.e., 5-HT, 5-carboxamidotryptamine (selective for 5-HT1 receptors), and alpha-methyl-5-hydroxytryptamine (selective for 5-HT2 receptors), potassium chloride, and acetylcholine, before and after extraluminal treatment with OxyHb. RESULTS: Extraluminal application of 2.5 x 10(-5) mol/L OxyHb immediately induced a transient elevation of the basal perfusion pressure, which gradually decreased and then stabilized at a level slightly higher than the control level. Each 5-HT agonist induced dose-dependent vasoconstriction. The potencies of the agonists were not very different, but the efficacies varied, i.e., alpha-methyl-5-hydroxytryptamine > 5-HT > 5-carboxamidotryptamine. Each response was strongly inhibited by ketanserin (a selective 5-HT2 receptor antagonist), indicating that each agonist induces vasoconstriction mediated by 5-HT2 receptors. The vasoconstriction in response to each 5-HT receptor agonist was consistently potentiated by treatment with OxyHb (2.5 x 10(-5) mol/L). 5-HT receptor agonist-induced constrictions after OxyHb treatment were much more markedly inhibited by ketanserin, compared with those before OxyHb treatment. Acetylcholine-induced constrictions were enhanced by OxyHb, but KCl-induced constrictions were significantly decreased by OxyHb. CONCLUSION: It is suggested that OxyHb enhancement of constrictions in response to 5-HT receptor agonists may be mediated by increased sensitivity of 5-HT2 receptors, in addition to actions in the endothelium, in canine basilar arteries. This potentiated vasoconstrictor mechanism may be partially implicated in cerebral vasospasm after subarachnoid hemorrhage.     

The regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the isolated perfused rat liver

The effect of perfusion of an isolated rat liver on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was studied. In liver removed during the basal period of the diurnal cycle of enzyme activity, a 227 +/- 41% increase in enzyme activity occurred after 3 h of a plasma-free perfusion. This could be prevented by the addition of cycloheximide or pure cholesterol (dispersed with lecithin) to the perfusate. In contrast, the continuous addition of taurocholate or taurochenodeoxycholate, alone or in combination, at a variety of rates did not prevent the increase in enzyme activity. The added bile salts were efficiently extracted from the perfusate and excreted in the bile. The addition of these bile salts to a cholesterol-enriched perfusate did not alter the effect obtained with cholesterol alone. If the perfusate contained whole serum, the increase induced by perfusion in the basal period was smaller (88 +/- 27%) than with plasma-free perfusate. Again, the major bile salts of the rat failed to prevent the increase in enzyme activity induced by liver perfusion. If livers were removed and perfused at the height of the diurnal cycle of enzyme activity, the enzyme activity remained high (2 +/- 10% increase) rather than decreasing, as occurs in vivo. If cholesterol was added to these perfusions, a 52 +/- 4% decrease was induced. Bile salt addition induced no decrease. From the results it is concluded that the major bile salts are not direct regulators of hepatic cholesterol synthesis, but pure cholesterol, in the absence of bile salt or lipoprotein, is able to initiate the mechanism that represses hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.     

The effect of aminophylline on pentagastrin-induced secretion and motor activity of totally isolated canine stomach perfused extracorporeally

Totally isolated canine stomachs, perfused extracorporeally with homologous blood of living oxygenerator-dogs, were used for the study of gastric secretion. Aminophylline and pentagastrin were infused into the gastric arterial system separately or combined. Aminophylline, infused alone, did not stimulate gastric secretion. However when aminophylline was administered during the infusion of pentagastrin, it significantly augmented the action of this peptide on gastric acid secretion. Aminophylline did not alter myoelectrical and mechanical response to pentagastrin nor did it affect the myoelectrical activity of the nonstimulated stomach. Gastric arterial perfusion pressure and gastric peripheral resistance were reduced during infusion of aminophylline alone or in combination with pentagastrin.     

Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro

We have investigated the regulatory role of nitric oxide (NO) in corticotropin-releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH-IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy.     

Expression of T3 receptor genes in peripheral lymphocytes from patients with thyroid dysfunction

In order to explore the expression and regulation of T3 receptor genes, the relative amount of peripheral lymphocytes c-erbA alpha and c-erbA beta mRNA from patients with Graves disease and Hashimoto thyroiditis with hypothyroidism (HTH) was determined with hybridization by using human c-erbA alpha and c-erbA beta probes. The results showed that there were two types of c-erbA alpha mRNA (6.0 kb, 3.2 kb) and three types of c-erbA beta mRNA (5.0 kb, 3.0 kb, 2.0 kb). The levels of c-erbA alpha and c-erbA beta mRNA were found to increase in HTH patients, but unaltered in Graves disease patients. This result suggests the presence of up-regulation of nuclear T3 receptor at the gene level in the lymphocytes of hypothyroid patients. The high level of c-erbA alpha and c-erbA beta mRNA may contribute in part to the increase in T3 receptor.     

Absorption of simple nutrients from the in vivo neurally isolated canine jejunum and ileum

BACKGROUND: The effects of intestinal transplantation on enteric absorptive function are not well understood. Our aim was to determine the effects of in situ isolation of the jejunoileum, a large animal model of jejunoileal autotransplantation, on absorption of simple nutrients from the jejunum and ileum separately. METHODS: Four groups of dogs were prepared with modified Thiry-Vella loops: group 1, neurally intact jejunum; group 2, neurally isolated jejunum; group 3, neurally intact ileum; and group 4, neurally isolated ileum. Intestinal loops were perfused with five different isosmolar solutions of NaCl alone, 30 mmol/L glucose, 2.5 mmol/L glycine, 2.5 mmol/L phenylalanine, and 5 mmol/L oleic acid at 1 to 2 weeks and 8 to 9 weeks after operation. RESULTS: Net absorption of water and electrolytes, glucose, glycine, phenylalanine, and oleic acid were not different statistically between neurally intact and neurally isolated intestinal loops at either time point. Ileal loops absorbed more than jejunal loops. CONCLUSIONS: Absorption of simple nutrients from the canine jejunum and ileum is not altered by this model of intestinal autotransplantation. These observations suggest that the extrinsic denervation that accompanies intestinal transplantation does not affect the transport systems for glucose, glycine, phenylalanine, or oleic acid.     

The effects of VIP on intestinal motility: study on ex vivo perfused isolated canine jejunal loops

The effects of VIP on intestinal motility were studied on isolated canine jejunal loops ex vivo perfused at normothermia, under pulsatile flow with heparinized, oxygenated and nonrecirculated canine whole blood, by means of an intraluminal balloon. VIP was administered intraarterially either by 1 min injections or by long-time infusions. The results showed that for arterial concentrations of the polypeptide ranging between 25 pg/ml and 300-500 pg/ml a fast but short-lasting relaxant effect was observed. For higher concentrations VIP usually produced a biphasic response: The relaxant effect is followed by an increase of the basal muscular tone often accompanied, for concentrations higher than about 25 ng/ml, by a marked and transient increase in amplitude of the intestinal rhythmic contractions. During long-time infusions a biphasic response was also observed but both effects were of short duration. A cholingeric origin of the secondary contracting phase was expected but could not be demonstrated because, at blood concentrations at which atropine affected the biphasic response, not only was the contractile effect abolished but also the initial relaxing phase. It is suggested that the secondary contraction may be a "rebound excitation" of myogenic nature or a result of noncholingeric excitatory fiber stimulations. The short-lasting relaxant effect observed under the present experimental conditions, even during long-time infusion of the polypeptide, fails to argue for an important physiological role of VIP as an hormonal inhibitor of intestinal motility. The biphasic response, however, might have a physiological significance in so far as the aboral propulsion of the intestinal content requires a muscular inhibition which rapidly changes to contraction.     

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.     

Factors affecting T3 and T4 proficiency testing

Problem areas within a proficiency testing (PT) program are performance evaluation and sample stability. The different units used in the various T3 uptake methodologies make performance evaluation complex. To facilitate this evaluation, a normalization method for T3 uptake performance evaluation has been developed. Sample stability studies for T3 uptake indicate that, at room temperature, sample values increase after storage for about seven days. Room temperature sample stability studies for T4 using a competitive protein binding (CPB) method indicate that the apparent T4 content of pooled serum increases after about one week. Fatty acids are shown to be an interfering substance in the T4 CPB method as well as the T4 radioimmunoassay (RIA) method. This interference increases with a decrease in carbon chain length from C18 to C12 and with an increase in unsaturation of fatty acids. The B/B0 ration for arachidonic acid at a concentration of 0.48 micronMoles per tube is 17.4 in a CPB method and 87.1 in a radioimmunoassay method indicating that the greater effect is in the CPB method. The increase in T3 uptake values are probably also due to the interfering effect of fatty acids.