Postsynaptic actions of acetylcholine: the coupling of muscarinic receptor subtypes to neuronal ion channels.
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Acetylcholine↗
Biomedical subjects
J Robbins
Publications and source records attributed to J Robbins.
Extracorporeal shock wave lithotripsy for calcified ureteral stent.
Calcification of a ureteral stent has been an unusual complication of long-term stent use for bypassing ureteral obstruction or after surgical repair. Open or percutaneous procedures have usually been required to remove the calcification before the stent can be extracted. We report six cases of calcified ureteral stents successfully managed by extracorporeal shock wave lithotripsy (SWL) followed by cystoscopic extraction, thus avoiding open or percutaneous procedures.
Adult↗
Segregated assembly of muscle myosin expressed in nonmuscle cells.
Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.
Actin Cytoskeleton↗
On the mechanism of M-current inhibition by muscarinic m1 receptors in DNA-transfected rodent neuroblastoma x glioma cells.
1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108-15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)-dependent K+ conductance, producing an outward current, and it inhibits a voltage-dependent K+ conductance (the M conductance), thus diminishing the M-type voltage-dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo-1 using whole-cell patch-clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/- 3.2% (n = 23). Following pressure-application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G-protein was tested by adding 0.5 mM GTP-gamma-S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half-times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml-1 pertussis toxin or on adding up to 10 mM GDP-beta-S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh-induced outward current and/or intracellular Ca2+ transient but did not block ACh-induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach-induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholine↗
Comparison of normal, hypodynamic, and hyperdynamic mouse hearts using isolated work-performing heart preparations.
It is now possible to manipulate the murine genome and produce transgenic mice in which genes encoding myocardial proteins have been ablated, resulting in an altered myocardial performance. In this study, we quantitate myocardial performance in work-performing mouse heart preparations from euthyroid, hypothyroid, and hyperthyroid mice. Our results show that time to peak pressure (TPP) and time to half-relaxation (RT1/2), together with first derivatives of intraventricular pressure (+/- dP/dt), are significant indicators of the quality and quantity of systolic contraction and relaxation. We compared the normal control indicators of contraction and relaxation of three different mouse strains at minimum afterloads (approximately 50 mmHg) and preloads (approximately 5 ml/min) and found them identical in range. All indicators of myocardial performance were significantly higher in the hyperthyroid and lower in the hypothyroid compared with normal mice. The cardiac myosin heavy chain isoform transcript shift (alpha-->beta) associated with hypothyroidism was observed. Because sympathetic activity is greatly enhanced with hyperthyroidism, we studied the effects of isoproterenol and the beta-blocker sotalol on cardiac contractility. Only approximately 50% of the myocardial hyperactivity displayed by hyperthyroid mice could be attributed to beta-adrenergic activity.
Animals↗
Gene targeting. The precise manipulation of the mammalian genome.
Genetics is a powerful tool for studying the function of different gene products as well as the physiological consequences of a normal or aberrant polypeptide. Until recently, the most powerful genetic applications have been restricted to relatively simple organisms whose genomes are more easily manipulated. For a number of years, in lower organisms, it has been possible to create defined genetic changes that are targeted to a specific locus or even to a defined codon or transcriptional regulatory sequence. With the development of gene targeting using embryonic stem cells derived from the preimplantation blastocyst of a mouse, it has become possible to extend these experiments to a mammalian system. Via homologous recombination, one can ablate, or "knock out," a defined genetic locus or mutate a particular set of nucleotides that encodes a peptide domain of interest. These techniques, when applied to genes that underlie normal cardiovascular function, promise to define the exact role(s) different proteins play during the development, growth, and maintenance of the heart. The ability to generate defined animal models of human disease in which the primary genetic defect is known should lead to fundamental advances in the study of the normal and failing heart.
Animals↗
Interleukin-6 inhibits corticosteroid-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells.
Corticosteroid-binding globulin (CBG) belongs to the superfamily of serine proteinase inhibitors which include alpha 1-antitrypsin, alpha 1-antichymotrypsin, and T4-binding globulin. Interleukin-6 (IL-6), the main mediator of the acute phase phenomenon, increases alpha 1-antitrypsin and alpha 1-antichymotrypsin synthesis and decreases T4-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells. This effect is predominantly at a transcriptional level. When Hep G2 cells were exposed to different concentrations of IL-6 for variable time intervals, IL-6 caused a dose- and time-dependent decrease in the amount of [35S]methionine-labeled CBG immunoprecipitated in the culture medium. This effect could be greatly reduced by preincubation of IL-6 with its neutralizing antibody and reversed by removing the cytokine from the culture medium. The secretion rate of CBG was not affected by cell exposure to IL-6. CBG mRNA steady state levels were reduced; changes in mRNA were quantitatively similar to changes in secreted protein. Nuclear run-off assays failed to show a change in the rate of transcription of the CBG gene. These data indicate that IL-6 diminishes CBG synthesis by Hep G2 cells acting at a posttranscriptional level, presumably through a reduced stability of mRNA. In view of the role of IL-6 in the inflammatory process and other acute phase phenomena, these data suggest that its effects on CBG synthesis might influence the bioavailability of cortisol indirectly and play a role in regulating the homeostatic process during these conditions.
Carcinoma, Hepatocellular↗
Characterization of thyroid hormone binding to apolipoprotein-E: localization of the binding site in the exon 3-coded domain.
Apolipoprotein-E (apoE) has been shown by noncovalent binding and photoaffinity labeling with [125I]T4 to possess a single L-T4 binding site with a K5 of about 3 x 10(7) M-1 and a relative affinity for analogs of L-T4 = D-T4 = rT3 = triiodothyroacetic acid > L-T3. T4 binding was not affected by the flavonoid EMD 21388 or heparin, but was inhibited by diclofenac = mefenamic acid > furosemide. Localization of the T4 site to the N-terminal 62-amino acid region of the mature peptide coded by exon 3 was deduced from the following evidence. 1) The N-terminal 15- to 26-kilodalton (kDa) fragments (within residues 1-160 to 210), but not the approximately 10- to 11-kDa fragments (within residues approximately 220-299), were labeled by [125I]T4. 2) Variants apoE2 and apoE4, with nonconservative mutations at positions 112 and 158 (the latter unable to interact with the apoB/E receptor), maintained the ability to bind T4. 3) Monoclonal antibodies MAb 1D7 and 3H1 (epitopes at positions 139-169 and 243-272, respectively) failed to inhibit T4 binding, but MAb 6H7 (epitope at 1-125) decreased labeling by about 24%. 4) Polymers of apoE were specifically labeled despite the interaction between amphipathic alpha-helices of the exon 4-encoded region (63-299). We conclude that apoE, as previously observed with apoA-I and apoB, possesses a T4-binding domain separate from the lipid-binding domain and distinct from both the heparin- and the cell receptor-binding sites. Thyroid hormone binding by apoE may facilitate uptake of the hormone by cells through apoB/E receptors, which are widely distributed in tissues.
Affinity Labels↗
Swallowing after lateral medullary syndrome plus.
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Behavior Therapy↗
Thyroid hormone transport proteins.
TBG, TTR, and albumin are quantitatively the most important thyroid hormone binding proteins in humans. Only a minute fraction of T3 and T4 circulates unbound, but it is this free hormone that is metabolically active at the tissue level, and, therefore, responsible for thyroid status. Inherited or acquired variations in the concentration or affinity of these proteins may produce substantial changes in serum total thyroid hormone levels but do not affect serum free thyroid hormone concentrations and, therefore, do not influence actual thyroid status. Thus, thyroid hormone-binding protein abnormalities must be suspected when abnormally elevated or diminished total thyroid hormone concentrations are encountered in clinically euthyroid subjects. This is crucial to avoid erroneous and potentially detrimental treatments for hyperthyroidism or hypothyroidism.
Biological Transport↗
Collaborative practice: a shared success.
A community hospital reflects on changes made when implementing a collaborative practice theory. Using a philosophy that called both professional groups to "share and work together to provide thorough and consistent patient care," a successful joint practice was operational within a year.
Colorado↗
Neurotransmitter modulation of calcium channels is dependent on the charge carrier used in the recording of currents.
Currents through calcium channels were recorded using calcium, barium and strontium as charge carriers in NG108-15 cells. The mean normalised peak current amplitude at 0 mV was not significantly different between the charge carriers; however, the sustained component (measured at the end of the 500 ms command step) was ca. 3 times larger in barium and strontium. Further, the inhibition by acetylcholine or noradrenaline, although the same at the peak of the current envelope, was significantly greater on the sustained portion of the current for barium and strontium. Increasing internal calcium-buffering (to reduce calcium-dependent inactivation with calcium as the charge carrier) did not increase the amount of inhibition of the sustained portion of current. These results suggest a cautious approach to analysis of neurotransmitter modulation of calcium currents using other charge carriers than calcium.
Acetylcholine↗
Isolated systolic hypertension and subclinical cardiovascular disease in the elderly. Initial findings from the Cardiovascular Health Study.
OBJECTIVE: To assess the association between isolated systolic hypertension (ISH) and subclinical disease in adults aged 65 years and above. DESIGN: Medicare eligibility lists were used to obtain a representative sample of 5201 community-dwelling elderly persons for the Cardiovascular Health Study, a National Heart, Lung, and Blood Institute--sponsored cohort study of risk factors for coronary heart disease and stroke. In this cross-sectional analysis of baseline data, we excluded 3012 participants who were receiving antihypertensive medications, had clinical cardiovascular disease, or had a diastolic blood pressure of at least 90 mm Hg. MAIN OUTCOME MEASURES: For electrocardiogram: myocardial infarction, left ventricular hypertrophy, and left ventricular mass as measures of myocardial damage and strain; for echocardiography: left ventricular mass, fractional shortening, and Doppler flow velocities as measures of cardiac systolic and diastolic function; and for carotid sonography: carotid arterial intima-media thickness as a measure of atherosclerosis. RESULTS: Among the 2189 men and women in this analysis, 195 (9%) had ISH (systolic blood pressure, greater than or equal to 160 mm Hg) and 596 (23%) had borderline ISH (systolic blood pressure, 140 to 159 mm Hg). Systolic blood pressure was associated with myocardial infarction by electrocardiogram (P = .02). Borderline and definite ISH were strongly associated with left ventricular mass (P less than .001). While there was little association with cardiac systolic function, borderline and definite ISH were associated with cardiac diastolic function (P less than .001). Isolated systolic hypertension was also strongly associated with increased intima-media thickness of the carotid artery (P less than .001). CONCLUSIONS: While cohort analyses of future repeated measures will provide a better assessment of risk, both borderline and definite ISH were strongly related to a variety of measures of subclinical disease in elderly men and women.
Aged↗
Characterization of myelin basic protein thyroid hormone response element and its function in the context of native and heterologous promoter.
In this report we have characterized further the myelin basic protein (MBP) gene thyroid hormone response element (TRE) by functional and binding analysis. Mutation and deletion experiments revealed that this TRE, confined to the sequences -184 to -167 of the MBP promoter, is able to function as a classical regulatory element in the context of the native and a heterologous promoter. It is comprised of two regions, containing a motif that is highly conserved among other TREs: AGGACA, arranged as an inverted palindrome. Any mutation within the footprinted region impaired receptor binding and function. Moreover, the deletion of sequences outside of the receptor footprinted region (MBP-TRE-18) resulted in a higher triiodothyronine responsiveness and a concomitant increase in receptor-dependent, hormone-independent repression. Results of transfection assays showed that both receptors alpha and beta elicit indistinguishable triiodothyronine responses when the MBP-TRE functions as a regulator of a heterologous promoter activity. However, a preferential beta receptor transactivation was observed when the MBP-TRE was placed in the context of its native promoter.
3T3 Cells↗
Differential effect of a new thyromimetic on triiodothyronine transport into myoblasts and hepatoma and neuroblastoma cells.
3,5-Dibromo-3'-pyridazinone-L-thyronine (L-94901), a member of a novel class of thyromimetics, reduces cholesterol plasma levels with little effect on cardiac function in rats. Because receptor binding of L-94901 in isolated heart and liver nuclei is similar but binding in liver nuclei in vivo was 50-fold higher than in cardiac nuclei, we studied its effect on triiodothyronine (T3) transport across the plasma membrane of myoblasts, hepatoma cells and neuroblasts. Previously, we had demonstrated saturable, stereospecific and energy dependent transport of T3 into the three cell lines. After equilibrium of intact cells with hormone, nuclear binding of T3 was decreased by L-94901 in all three cell lines. While whole cell uptake and whole cell binding of T3 was only slightly affected by L-94901, kinetic analysis of the initial rate of uptake showed uncompetitive or noncompetitive inhibition and a differential decrease in Vmax. Furthermore, the Ki for the liver and brain derived cells was 10-fold lower than for the muscle derived cells. This effect on the plasma membrane transport of T3 may explain the differential effect reported in the intact animal.
Animals↗
Intracellular Mg2+ inhibits the IP3-activated IK(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording IK(Ca)].
Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP3) can induce an outward Ca(2+)-activated K+ current [IK(Ca)] in some neural cells. We have investigated IK(Ca) activated by intracellular injections of IP3 in whole-cell patch-clamped neuroblastoma x glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg2+ by citrate. Theoretical calculations suggested free [Mg2+] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP3-activated IK(Ca) could be recorded when the free Mg2+ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg2+] blocks IK(Ca). This appeared to be due to inhibition of the response to released Ca2+, since high [Mg2+] also blocked the response to intracellular injections of Ca2+ ions. Mean Mg2+ levels in intact neuroblastoma x glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP3-induced IK(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg2+.
Anions↗
Neurotransmitters inhibit the omega-conotoxin-sensitive component of Ca current in neuroblastoma x glioma hybrid (NG 108-15) cells, not the nifedipine-sensitive component.
Voltage-dependent calcium currents (ICa) in NG 108-15 cells consisted of three pharmacologically distinct components: a transient low-voltage-activated (LVA) current, sensitive to Ni2+; a high-voltage-activated (HVA) current sensitive to the dihydropyridine antagonist, nifedipine and a HVA current sensitive to omega-conotoxin GVIA (CgTx). The voltage sensitivities and decay kinetics of the two HVA currents were indistinguishable. The neurotransmitters acetylcholine (ACh) and noradrenaline inhibited ICa. This inhibition was not occluded by Ni2+ or nifedipine, but was abolished by CgTx. It is therefore concluded that the neurotransmitter-sensitive component of ICa is restricted to that component of HVA current inhibitable by omega-conotoxin.
Acetylcholine↗
Structural and developmental analysis of two linked myosin heavy chain genes.
We have identified at the molecular level two murine myosin heavy chain (MHC) genes which encode adult skeletal isoforms. As is the case for the murine cardiac MHC genes, the genes are closely linked, head to tail. To identify the isoform encoded by each gene, we located and sequenced the 3' termini and assessed the genes' temporal and tissue-specific expression patterns using probes specific for the 3' untranslated regions. These analyses indicate that the myosin encoded by the gene located 5' in the linked pair is the murine 2A isoform. The isoform encoded by the 3'-most gene of the linked pair appears to encode an additional isoform that is expressed in a number of adult skeletal muscles. The pattern of expression of the 3'-most gene indicates that it is tightly controlled developmentally. Transcripts from this gene, when compared to those from the MHC2A and beta-MHC genes, are the predominant MHC transcripts found in the diaphragm, tongue, soleus, and masseter, indicating that it encodes a major skeletal MHC isoform.
Animals↗