Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

• Text
• PDF

Abstract

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Authors

J R Balser, H B Nuss, D W Orias, D C Johns, E Marban, G F Tomaselli, J H Lawrence

Increased expression of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of noninsulin-dependent diabetes mellitus subjects.

• Text
• PDF

Abstract

Insulin receptors (IR) and IGF-I receptors (IGF-IR) have been shown to form hybrid receptors in tissues coexpressing both molecules. To date there is no information about the distribution of hybrids in tissues of normal or diabetic subjects. We developed a microwell-based immunoassay to quantitate hybrids in small human tissues samples. Microwells were coated with MA-20 anti-IR antibody or alpha-IGF-IR-PA antibody directed against the IGF-IR alpha-subunit, and incubated with skeletal muscle extracts of patients with noninsulin-dependent diabetes mellitus (NIDDM) and normal controls. Immobilized receptors were incubated with 125I-insulin or 125I-IGF-I in the presence or absence of the two unlabeled ligands. Hybrids were quantified as the fraction of 125I-IGF-I binding immunoadsorbed with MA-20 and expressed as percentage of total IGF-IR (type I+hybrids) immobilized with alpha-IGF-IR-PA. The immunoassay was validated using Western blotting analysis. Relative abundance of hybrids detected in NIDDM patients was higher than in controls. The percentage of hybrids was negatively correlated with IR number and in vivo insulin sensitivity measured by an insulin tolerance test, whereas the percentage was positively correlated with insulinemia. Insulin binding affinity was lower in NIDDM patients than in controls, and was correlated with the percentage of hybrids. Maximal IGF-I binding was significantly higher in muscle from NIDDM patients compared to controls and was positively correlated with the percentage of hybrid receptors whereas IGF-I binding affinity did not differ between the two groups. These results raise the possibility that alterations in expression of hybrid receptors may contribute to decreased insulin sensitivity, and to increased sensitivity to IGF-I. Because IGF-I has been proposed as a hypoglycemic agent in NIDDM, these results are relevant to the development of new approaches to the treatment of insulin resistance of NIDDM.

Authors

M Federici, L Zucaro, O Porzio, R Massoud, P Borboni, D Lauro, G Sesti

Particle-mediated gene transfer with transforming growth factor-beta1 cDNAs enhances wound repair in rat skin.

• Text
• PDF

Abstract

Based on preliminary but variable results with direct DNA transfer into wounds, we evaluated in vivo gene transfer by particle-mediated DNA delivery to rat skin to determine whether overexpression of TGF-beta1 at the site of skin incisions would result in a significant improvement in repair. Optimization of the method with viral promoter-luciferase reporter constructs indicated that expression of luciferase activity persisted up to 5 d and was promoter, pressure, and site dependent (ventral > dorsal). Using cytomegalovirus (CMV)-driven human alpha1-antitrypsin, transgene expression was immunolocalized within keratinocytes of the stratum granulosum at 24 h. We measured tensile strength of skin incisions at 11-21 d in both normal and diabetic rats transfected with TGF-beta1 expression vectors at surgery. Native murine TGF-beta1 under an SV40 promoter produced positive effects, while wound strengthening was more pronounced in diabetic animals using a CMV-driven construct. Transfection of rat skin with constitutively active, mutant porcine TGF-beta1 under the control of the CMV and Moloney murine leukemia virus promoters significantly increased tensile strength up to 80% for 14-21 d after surgery. Transfection 24 h before surgery was more effective. Particle-mediated gene delivery can be used to deliver viral promoter-cytokine expression constructs into rat skin in a safe, efficient, and reproducible fashion. The extent of wound repair, as evidenced by enhanced tensile strength, can be markedly improved in tissues transfected with TGF-beta1 expression constructs.

Authors

S I Benn, J S Whitsitt, K N Broadley, L B Nanney, D Perkins, L He, M Patel, J R Morgan, W F Swain, J M Davidson

Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat.

• Text
• PDF

Abstract

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide produced by K cells of the mammalian proximal small intestine and is a potent stimulant of insulin release in the presence of hyperglycemia. However, its relative physiological importance as a postprandial insulinotropic agent is unknown. Using LGIPR2 cells stably transfected with rat GIP receptor cDNA, GIP (1-42) stimulation of cyclic adenosine monophosphate (cAMP) production was inhibited in a concentration-dependent manner by GIP (7-30)-NH2. Competition binding assays using stably transfected L293 cells demonstrated an IC50 for GIP receptor binding of 7 nmol/liter for GIP (1-42) and 200 nmol/liter for GIP (7-30)-NH2, whereas glucagonlike peptide-1 (GLP-1) binding to its receptor on ++betaTC3 cells was minimally displaced by GIP (7-30)-NH2. In fasted anesthetized rats, GIP (1-42) stimulated insulin release in a concentration-dependent manner, an effect abolished by the concomitant intraperitoneal administration of GIP (7-30)-NH2 (100 nmol/ kg). In contrast, glucose-, GLP-1-, and arginine-stimulated insulin release were not affected by GIP (7-30)-NH2. In separate experiments, GIP (7-30)-NH2 (100 nmol/kg) reduced postprandial insulin release in conscious rats by 72%. It is concluded that GIP (7-30)-NH2 is a GIP-specific receptor antagonist and that GIP plays a dominant role in mediating postprandial insulin release.

Authors

C C Tseng, T J Kieffer, L A Jarboe, T B Usdin, M M Wolfe

In situ microdialysis in bone tissue. Stimulation of prostaglandin E2 release by weight-bearing mechanical loading.

• Text
• PDF

Abstract

In this study we describe, to our knowledge, the first experiment using the microdialysis technique for studying the release of prostaglandin E2 (PGE2) in the proximal tibia metaphysis secondary to mechanical loading. Nine healthy females, six in the loading group and three controls, mean age 34+/-2 (years+/-SEM), participated. A standard microdialysis catheter was inserted into the tibia metaphyseal bone under local anesthesia. Samplings were done every 15 min under a 2-h equilibration period. Thereafter, heel-drops (one impact per second) with as hard impact of the heels into the floor as possible, were done for 5 min by the subjects in the loading group. The control group performed no exercise. Sampling continued after this for another 2-h period. Basal levels of PGE2 in the proximal tibial metaphysis were determined to a mean of 45+/-10 pg/ml (mean+/-SEM, n = 6). The major finding was a 2.5-3.5-fold increase in the release of PGE2 after mechanical loading. The increase was statistically significant (P < 0.05 compared with basal levels) 1 h after the mechanical loading and persisted for the rest of the experimental period. No major alterations were observed in the control group. In conclusion, our data demonstrate that in situ microdialysis is a useful method for studying the PGE2 production in human bone. Furthermore, a rapid and significant increase in PGE2 levels was noticed in response to dynamic mechanical loading.

Authors

K Thorsen, A O Kristoffersson, U H Lerner, R P Lorentzon

Magnetic resonance evidence of hypoxia in a homozygous alpha-knockout of a transgenic mouse model for sickle cell disease.

• Text
• PDF

Abstract

All transgenic mouse models for sickle cell disease express residual levels of mouse globins which complicate the interpretation of experimental results. We now report on a mouse expressing high levels of human betaS and 100% human alpha-globin. These mice were created by breeding the alpha-knockout and the mouse beta(major)-deletion to homozygosity in mice expressing human alpha- and betaS-transgenes. These betaS-alpha-knockout mice have accelerated red cell destruction, altered hematological indices, ongoing organ damage, and pathology under ambient conditions which are comparable with those found in alphaH betaS-Ant[betaMDD] mice without introduction of additional mutations which convert betaS into a "super-betaS" such as the doubly mutated betaS-Antilles. This is of particular importance for testing strategies for gene therapy of sickle cell disease. Spin echo magnetic resonance imaging at room air and 100% oxygen demonstrated the presence of blood hypoxia (high levels of deoxygenated hemoglobin) in the liver and kidneys that was absent in control mice. We demonstrate here that transgenic mice can be useful to test new noninvasive diagnostic procedures, since the magnetic resonance imaging technique described here potentially can be applied to patients with sickle cell disease.

Authors

M E Fabry, R P Kennan, C Paszty, F Costantini, E M Rubin, J C Gore, R L Nagel

Expression and functional assessment of a truncated cardiac troponin T that causes hypertrophic cardiomyopathy. Evidence for a dominant negative action.

• Text
• PDF

Abstract

Mutations in the beta-myosin heavy chain gene are believed to cause hypertrophic cardiomyopathy (HCM) by acting as dominant negative alleles. In contrast, a truncated cardiac troponin T (TnT) that causes HCM implies that altered stoichiometry of contractile proteins may also cause cardiac hypertrophy. Wild-type and HCM-mutant (truncated) TnT were studied in a novel quail myotube expression system. Unexpectedly, antibody staining demonstrated incorporation of both forms of human cardiac TnT into the sarcomeres of quail myotubes. Functional studies of wild type and mutant transfected myotubes of normal appearance revealed that calcium-activated force of contraction was normal upon incorporation of wild type TnT, but greatly diminished for the mutant TnT. These findings indicate that HCM-causing mutations in TnT and beta-myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. This diminished force output is the likely stimulus for hypertrophy in the human heart.

Authors

H Watkins, C E Seidman, J G Seidman, H S Feng, H L Sweeney

Interaction between the insulin-like growth factor family and the integrin receptor family in tissue repair processes. Evidence in a rabbit ear dermal ulcer model.

• Text
• PDF

Abstract

We have determined previously that IGF-I is dependent on the presence of IGF binding protein-1 (IGFBP-1) to act as a wound healing agent. We sought to determine the mechanism whereby IGFBP-1 is able to enhance IGF-I bioactivity. As IGFBP-1 binds both the alpha5beta1 integrin as well as IGF-I in vitro, we asked which of the following interactions were important: (a) the ability of IGFBP-1 to interact with an integrin receptor, and/or (b) the binding of IGF-I by IGFBP-1. We used an IGF-1 analogue (des(1-3)IGF-I) with a > 100-fold reduction in affinity for IGFBP-1 as well as an IGFBP-1 mutant (WGD-IGFBP-1) which does not associate with the alpha5beta1 integrin to selectively abrogate each of these interactions. We also tested the ability of IGFBP-2, a related binding protein which has an arginine-glycine-aspartate sequence but does not associate with integrin family members, to enhance IGF-I bioactivity. Full-thickness dermal wounds were created on rabbit ears; various combinations of native IGF-I, native IGFBP-1, native IGFBP-2, and their respective analogues/mutants were applied to each wound. Wounds were harvested 7 d later for analysis. Only native IGF-I in combination with native IGFBP-1 was effective as a wound healing agent, enhancing reepithelialization and granulation tissue deposition by 64+/-5 and 83+/-12% over controls (P = 0.008 and 0.016, respectively). The same doses of IGF-I/WGD-IGFBP-1, des(1-3)IGF-I/IGFBP-1, and IGF-I/IGFBP-2 were ineffective. We propose that IGF-I physically interacts with IGFBP-1 and that IGFBP-1 also binds to an integrin receptor, most likely the alpha5beta1 integrin. This interaction is unique to IGFBP-1 as the closely related IGFBP-2 had no effect, a finding consistent with its inability to bind to integrin receptors. Our results suggest that activation of both the IGF-I receptor and the alpha5beta1 integrin is required for IGF-I to stimulate wound healing.

Authors

R D Galiano, L L Zhao, D R Clemmons, S I Roth, X Lin, T A Mustoe

Thioredoxin: a redox-regulating cellular cofactor for glucocorticoid hormone action. Cross talk between endocrine control of stress response and cellular antioxidant defense system.

• Text
• PDF

Abstract

Adaptation to stress evokes a variety of biological responses, including activation of the hypothalamic-pituitary-adrenal (HPA) axis and synthesis of a panel of stress-response proteins at cellular levels: for example, expression of thioredoxin (TRX) is significantly induced under oxidative conditions. Glucocorticoids, as a peripheral effector of the HPA axis, exert their actions via interaction with a ligand-inducible transcription factor glucocorticoid receptor (GR). However, how these stress responses coordinately regulate cellular metabolism is still unknown. In this study, we demonstrated that either antisense TRX expression or cellular treatment with H2O2 negatively modulates GR function and decreases glucocorticoid-inducible gene expression. Impaired cellular response to glucocorticoids is rescued by overexpression of TRX, most possibly through the functional replenishment of the GR. Moreover, not only the ligand binding domain but the DNA binding domain of the GR is also suggested to be a direct target of TRX. Together, we here present evidence showing that cellular glucocorticoid responsiveness is coordinately modulated by redox state and TRX level and propose that cross talk between neuroendocrine control of stress responses and cellular antioxidant systems may be essential for mammalian adaptation processes.

Authors

Y Makino, K Okamoto, N Yoshikawa, M Aoshima, K Hirota, J Yodoi, K Umesono, I Makino, H Tanaka

D-glucose-induced dysmorphogenesis of embryonic kidney.

• Text
• PDF

Abstract

An organ culture system was used to study the effect of D-glucose on embryonic kidneys, and to delineate the mechanism(s) relevant to their dysmorphogenesis. Metanephroi were cultured in the presence of 30 mM D-glucose. A notable reduction in the size and population of nephrons was observed. Ureteric bud branches were rudimentary and the acuteness of their tips, the site of nascent nephron formation, was lost. Metanephric mesenchyme was atrophic, had reduced cell replication, and contained numerous apoptotic cells. Competitive reverse transcriptase-PCR analyses and immunoprecipitation studies indicated a decrease in expression of heparan sulfate proteoglycan (perlecan). Status of activated protein-2 was evaluated since its binding motifs are present in the promoter region of the perlecan gene. Decreased binding activity of activated protein-2, related to its phosphorylation, was observed. D-glucose-treated explants also had reduced levels of cellular ATP. Exogenous administration of ATP restored the altered metanephric morphology and reduced [35S]sulfate-incorporated radioactivity associated with perlecan. The data suggest that D-glucose adversely affects the metanephrogenesis by perturbing various cellular phosphorylation events involved in the transcriptional and translational regulation of perlecan. Since perlecan modulates epithelial/mesenchymal interactions, its deficiency may have led to the metanephric dysmorphogenesis and consequential atrophy of the mesenchyme exhibiting accelerated apoptosis.

Authors

Y S Kanwar, Z Z Liu, A Kumar, M I Usman, J Wada, E I Wallner