Use of AORN recommended practices. Applications in ambulatory surgery.
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Ambulatory Surgical Procedures↗
Biomedical subjects
R A Roth
Publications and source records attributed to R A Roth.
Effect of experimental diabetes on insulin binding by renal basolateral membranes.
Removal of insulin from the peritubular vessels involves binding of insulin to specific receptors in the basolateral membranes (BLM); this is followed by phosphorylation of the receptor which may mediate the actions of the hormone. In most tissues receptor number is regulated by plasma insulin levels and is increased in insulinopenic diabetics. To determine whether cortical BLM insulin receptors are similarly regulated, we studied insulin binding to receptors in BLM from normal control rats and rats with streptozotocin diabetes of varying severity. Specific binding of insulin did not differ between control and modestly insulinopenic diabetics but was increased significantly in the severely insulinopenic diabetics. Insulin treatment returned binding to normal. Scatchard analysis suggested an increase in the binding capacity of the severe diabetic BLM rather than an increase in affinity for insulin. This latter was confirmed by competitive experiments in which similar displacement curves were obtained with control and diabetic membranes. Insulin removed by glomerular filtration binds to specific receptors in the luminal membranes but unlike BLM receptors, phosphorylation of these luminal receptors has not been observed. To determine whether luminal and BLM receptors differ structurally, binding sites in both membranes were affinity labelled with 125I-insulin and the cross linking agent, disuccinimidyl suberate, and subjected to SDS-polyacrylamide gel electrophoresis in the presence of a reducing agent. Autoradiograms revealed that the major specifically labelled subunit in both membranes is a 135,000 Mr species which is more abundant in the BLM. We conclude that insulin receptors in cortical BLM respond to severe insulinopenic diabetes as do receptors in most other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease.
Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.
Animals↗
Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity.
The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.
Animals↗
Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase.
Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor beta subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor beta subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.
Animals↗
Linking functional domains of the human insulin receptor with the bacterial aspartate receptor.
A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.
Amino Acid Sequence↗
Mechanisms of receptor-mediated transmembrane communication.
Our experiments with the hIR protein have been designed to address a very general question of transmembrane receptor structure and function: What are the roles and interactions of the various deduced structural domains of such molecules in the initiation of the response of cells to extracellular signals? All of the evidence to date supports the previous hypothesis based on biochemical data that the IR requires ligand-activated TPK functions to initiate the insulin response by cells (for review, see Kahn 1985). Thus, mutations that compromise hIR TPK activity (site-directed point mutations or deletions) result in a concomitant decrease in at least one aspect of insulin action (glucose uptake; Ellis et al. 1986a). Other studies utilizing microinjection of antibodies to inhibit the receptor kinase have extended this conclusion to include a critical role for the receptor kinase in insulin's ability to stimulate ribosomal protein S6 phosphorylation in CHO cells, glycogen synthetase in hepatoma cells, glucose uptake in adipocytes (Morgan and Roth 1987), and frog oocyte maturation (Morgan et al. 1986). Second, analyses of cell lines that express experimentally truncated hIR TPKs demonstrate that, when membrane-anchored, this TPK domain is in fact capable of autonomous hormone-independent IR function: Such cells exhibit a constitutively elevated, insulin-independent uptake of 2-deoxyglucose (Ellis et al. 1987). Finally, by substitution of a homologous TPK for that of hIR, we find that although such a hybrid is capable of insulin-dependent transmembrane signaling (phosphorylation of the hybrid beta-subunit on tyrosine residues), the hybrid IR.ros molecule does not function as an IR in such cells: It mediates neither short-term (uptake of 2-deoxyglucose) nor long-term (incorporation of [3H]thymidine) effects of insulin (L. Ellis et al., in prep.). Together, these results suggest that (1) the hIR TPK domain conveys a substrate specificity for the insulin response and (2) that a functional hIR extracellular domain alone is not sufficient for generation of the insulin response (e.g., ligand-induced aggregation, or simple delivery of insulin into the cell). With the linking of the extracellular and cytoplasmic domains of the hIR molecule has evolved a cellular mechanism for the control of hIR TPK activity; the result is that cells which express the IR are now insulin responsive, and the physiological responses associated with the hormone are ligand-activated. Thus, the uncontrolled state of autonomous TPK activity, with the associated constitutive physiological response (e.g., as exhibited by the spBam hIR mutant), is circumvented.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Upper and midureteral calculi: percutaneous extraction with an occlusion balloon catheter.
In 44 patients with one or more calculi in the upper two-thirds of the ureter, single-stage percutaneous nephrolithotomy was performed through a middle or upper calyceal nephrostomy after cystoscopic placement of an occlusion balloon catheter distal to the calculus; in 42, the procedure was successful. The occlusion balloon catheter permitted retrograde opacification of all systems for enhanced renal puncture. In the last 30 patients an attempt was made either to push the calculus upward mechanically or to flush it upward into the renal pelvis with carbon dioxide or dilute contrast material. This was successful in 24 of these patients. Prior overnight occlusion of the ureter by means of ureteral dilatation further facilitates dislodgment of the calculus, which was successful in 12 of 13 patients.
Catheterization↗
Insulin and insulinlike growth factor receptors and responses in cultured human muscle cells.
Specific receptors for insulinlike growth factors I and II (IGF-I and IGF-II) were found on cultured human myoblasts and myotubes. In contrast, myotubes but not myoblasts specifically bound insulin and were stimulated by nanomolar concentrations of insulin to take up deoxyglucose. In addition, in myoblasts, physiological concentrations of IGF-I and -II and, to a lesser extent, insulin stimulated two- to threefold the uptake of the nonmetabolizable amino acid analogue methylaminoisobutyric acid (MAIB). In myotubes, uptake of MAIB was stimulated preferentially by IGF-I. Monoclonal antibodies that preferentially recognize either the insulin receptor or the IGF-I receptor were utilized to examine which receptors mediated the biological effects of these hormones. The effects of insulin on both myoblasts and myotubes appeared to be mediated in part by the insulin receptor and in part by the IGF-I receptor. In myotubes, the effects of IGF-I and -II both appeared to be mediated through the IGF-I receptor. In myoblasts, the effects of the two IGFs appeared to be in part mediated by the IGF-I receptor and in part mediated by either the IGF-II receptor or another type of IGF-I receptor. The present results suggest that cultured human muscle cells provide a useful model system in which to study the biological actions of insulin and the IGFs.
Aminoisobutyric Acids↗
Purification and characterization of insulin-degrading enzyme from human erythrocytes.
An insulin-degrading enzyme (IDE) was purified from the cytosol of human erythrocytes via the use of ammonium sulfate precipitation and chromatography on columns composed of DEAE-Sephadex, pentylagarose, hydroxylapatite, chromatofocusing resins, and Ultrogel AcA-34. The final preparation was purified greater than 50,000-fold and exhibited a single protein band of Mr = 110,000 on reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Cross-linking of 125I-labeled insulin to the enzyme preparation labeled a protein of the same molecular weight, indicating that this band was in fact the enzyme. Intact insulin, insulin B chain, and glucagon inhibited this cross-linking half-maximally at concentrations of 0.1, 1, and 1.5 microM, respectively. Under nondenaturing conditions, the enzyme had an Mr = 300,000, suggesting that the enzyme may exist under physiological conditions as a dimer or timer. The purified enzyme was inhibited by both sulfhydrylmodifying reagents and chelating agents, indicating that a free thiol and metal were both required for the activity of the enzyme. The purified enzyme was found to degrade physiological concentrations of intact insulin more rapidly than insulin B chain, although at high substrate concentrations (greater than 1 microM) the enzyme degraded B chain to a greater extent. Additional characteristics of the enzyme were a pl of 5.2 and a pH optimum of 7.0. These properties of the red blood cell (RBC) enzyme were very similar to those reported for IDEs from other tissues. Moreover, a polyclonal antiserum to the IDE from skeletal muscle was found to recognize the RBC enzyme.
Chemical Precipitation↗
Monocrotaline pyrrole-induced cardiopulmonary toxicity is not altered by metergoline or ketanserin.
Monocrotaline pyrrole (MCTP) causes endothelial cell damage, pulmonary hypertension and right ventricular hypertrophy in rats by an undetermined mechanism. A role for 5-hydroxytryptamine (5-HT) in the cardiopulmonary response to MCTP has been suggested. To investigate the role of 5-HT, the effects of two 5-HT receptor antagonists were examined in MCTP-treated rats. Cotreatment with metergoline, an antagonist which binds to both 5-HT1 and 5-HT2 receptors, did not alter MCTP-induced elevation of lung weight or right ventricular hypertrophy. 5-HT-induced vascular smooth muscle contractions are mediated by 5-HT2 receptors; therefore, MCTP-treated rats were cotreated with ketanserin (KET), a specific 5-HT2 receptor antagonist. At a dosing regimen of KET that inhibited the 5-HT-induced platelet shape change in platelet-rich plasma and the 5-HT-induced increase in perfusion pressure in isolated lungs, KET did not affect the elevation in lung weight or the increased accumulation of 125I-albumin in the lung tissue of MCTP-treated rats. Moreover, MCTP-induced right ventricular hypertrophy was not attenuated by KET. These results indicate that cotreatment with either of these two 5-HT receptor antagonists does not alter the lung injury or right ventricular hypertrophic response to MCTP and suggest that 5-HT is not necessary for MCTP-induced toxicity.
Angiotensin II↗
Monoclonal antibodies to the insulin receptor as probes of insulin receptor structure and function.
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Antibodies, Monoclonal↗
Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme.
Cytosol extracts high in insulin-degrading activity were cross-linked to 125I-insulin with the bifunctional cross-linker disuccinimidyl suberate. With cytosols from either rat muscle, liver, kidney or brain or human erythrocytes, only a single protein (Mr = 110,000) was specifically labeled. Three different lines of evidence indicated that this labeled protein is insulin-degrading enzyme, a cysteine protease which accounts for most of the insulin-degrading activity in cell extracts. Firstly, the cross-linking of 125I-insulin to this protein is inhibited by unlabeled insulin over the same concentration range of insulin which inhibits degradation. Separated insulin A and B chain were less potent at inhibiting cross-linking, whereas bovine serum albumin and cytochrome c were without effect. Secondly, antibodies to purified insulin-degrading enzyme precipitated the labeled protein in parallel with their ability to precipitate the insulin-degrading activity of the extracts. Thirdly, when the insulin-degrading activity was purified 40,000-fold from erythrocytes, this Mr 110,000 protein co-purified. These results indicate that cross-linking 125I-insulin may be a convenient method for labeling the insulin-degrading enzyme.
Animals↗
The effect of dietary restriction and altered sodium intake on the cardiopulmonary toxicity of monocrotaline pyrrole.
Monocrotaline pyrrole (MCTP) is a reactive metabolite of the plant toxin monocrotaline (MCT), which produces pulmonary vascular injury and right ventricular hypertrophy in rats. In this study, the influence of diet restriction on the cardiopulmonary toxicity of MCTP was examined. In rats fed ad libitum, MCTP treatment resulted in increased lung weight, in elevated lactate dehydrogenase (LDH) activity and protein concentration in cell-free bronchopulmonary lavage fluid, and in right ventricular enlargement. Restriction of feed intake to 40% of normal attenuated the increases in lung weight and lavage protein concentration in MCTP-treated rats and abolished the right ventricular enlargement but did not affect the increased lavage LDH activity. In a study of the effect of diet restriction on the survival of MCTP-treated rats, the percentage of diet-restricted animals surviving was significantly higher than that of surviving animals which ate ad libitum through Day 28, but thereafter there was no significant difference between the two groups. Alterations in dietary sodium intake alone did not affect MCTP-induced toxicity. These results indicate that diet restriction partially protects against the cardiopulmonary toxicity due to MCTP, and that this protective effect cannot be explained by changes in salt intake.
Animals↗
Evaluation of myocardial preservation using 31P NMR.
The purpose of this study was (1) to monitor myocardial high-energy phosphate content and recovery of left ventricular (LV) contractile function following normothermic graded cardiac ischemia and single-dose hypothermic potassium cardioplegia, and (2) to assess the temporal limits of LV functional recovery during single-dose cardioplegia maintained at 17 degrees C. Rabbit hearts (30) were perfused, equipped with an LV balloon, paced at 240 beats/min, and placed in a nuclear magnetic resonance (NMR) magnet. Hearts underwent either graded, global normothermic ischemia or potassium cardioplegia arrest maintained at 17 degrees C for 1 hr. Myocardial high-energy phosphate level, LV contractility, and temperature were monitored continuously. Phosphocreatine (PCr) fell to 10 +/- 2, 2 +/- 1, and 0% of control and ATP to 70 +/- 3, 19 +/- 7, and 0% of control at 10, 40, and 60 min of 37 degrees C ischemia. After 1 hr of reperfusion, regression analysis of final developed pressure (DP) on end ischemic ATP (EIATP) content revealed: DP = 1.02 EIATP + 18 (r = 0.95). Following single-dose cardioplegia, maintained at 17 degrees C, PCr fell to 16 +/- 3% of control at 60 min while ATP fell only to 92 +/- 5% control. With reperfusion, recovery of DP was 100%. It was concluded that (1) PCr serves as an energy buffer for ATP, (2) EIATP predicts recovery of LV function, (3) single-dose cardioplegia maintained at 17 degrees C provides complete myocardial preservation for up to 60 min.
Adenosine Triphosphate↗
Use of retrograde occlusion balloon catheters in percutaneous removal of renal calculi.
The best results in percutaneous renal calculus removal are in patients with solitary pelvic stones and dilated collecting systems. Calyceal and/or infundibular calculi and ureteral calculi constitute more complex problems and are less successfully removed. Placement failure of the nephrostomy tube, and inability to gain access to calyceal calculi, and to engage impacted stones are the usual causes for unsuccessful procedures. To try to improve overall success we inserted occlusion balloon catheters prior to nephrostomy insertion in 60 of 71 patients. In a select group of 23 patients with a small renal pelvis and large calculi or infundibular calculi or patients with ureteral calculi, overnight application of 20 cm of hydrostatic pressure through the ureteric catheter led to marked dilatation in all instances. In all other patients the occlusion balloon catheter was placed immediately prior to the definitive nephrostolithotomy procedure. These ureteral catheters allow for better opacification and for dilatation of a small collecting system thereby facilitating the placement of the nephrostomy tube. They permit manipulation of calyceal and especially ureteral calculi. Placement of ureteral occlusion catheters is a safe adjunct to nephrostolithotomies and, in our experience, resulted in increased overall success.
Catheterization↗
Monoclonal antibodies to the insulin receptor.
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Animals↗
Characterization of an insulin degrading enzyme from cultured human lymphocytes.
An insulin degrading enzyme from cultured human lymphocytes, IM-9 cells, has been purified and characterized. The biochemical, enzymatic and immunological characteristics of this enzyme were all found to be similar to the characteristics of insulin degrading enzymes previously isolated from rat and pig skeletal muscle. Furthermore, this insulin degrading enzyme was found to have no effect on the structure of the insulin receptor nor to be linked to the insulin receptor either on the plasma membrane of cells or when they are shed into the media. The present studies suggest that the IM-9 lymphocytes, which have been extensively used to study the human insulin receptor, may also be a good system for studying human insulin degrading enzymes.
Animals↗