Biomedical subjects

W K Engel

Publications and source records attributed to W K Engel.

At least 109 records · Page 6Linked to original sources

PubMed1988

Dihydropyridine-sensitive Ca2+ channel in aneurally cultured human muscles. Relationship between high-affinity binding site and inhibition of calcium uptake.

Dihydropyridine-sensitive Ca2+ channels and the relationship between binding of dihydropyridine derivatives and depolarization-induced Ca2+ uptake have been studied in aneurally cultured human muscle. Analysis of the equilibrium binding of the 1,4-dihydropyridine derivative (+)-PN200-110 revealed a single high-affinity binding site with a Kd of 0.15 +/- 0.05 nM and a Bmax of 87 +/- 12 fmol/mg protein. Inhibition of (+)-[3H]PN200-110 binding by nitrendipine revealed a Ki of 0.8 nM for the nitrendipine-receptor complex. Depolarization of cultured human muscle achieved by elevating the K+ concentration increased the uptake 45Ca2+ which was inhibited by nitrendipine with an IC50 of 1.1 nM. This study demonstrates that aneurally cultured human muscle has dihydropyridine-sensitive voltage-dependent Ca2+ channels which are functional when the fibers are depolarized.

Calcium↗

PubMed1988

Histoenzymatic profile of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord.

We examined histoenzymatic characteristics of human muscle fibers grown in monolayer culture and innervated de novo in culture for 60-90 days by fetal rat spinal cord neurons. Serial cryostat cross sections were obtained using a freshly frozen sandwich of adult rat muscle and cultured human muscle. An advanced degree of morphologic and histoenzymatic maturation of cultured human muscle was reached after innervation. In contrast to aneurally cultured human muscle fibers, the innervated muscle fibers were smaller in diameter and had myonuclei preferentially located at the periphery of the fiber. The innervated fibers contained a well-developed intermyofibrillar network revealed by the NADH-TR and SDH reactions. Phosphorylase activity was strong to moderate in most muscle fibers. Although most of the innervated cultured muscle fibers were still not fully differentiated into two histochemical fiber types because they had strong ATPase activity after both alkaline and acid preincubation, a few of them had an ATPase profile similar to type 2 fibers in human adult muscle and had reciprocal staining with phosphorylase and NADH-TR reactions. This is the first evidence of differentiation into different histochemical fiber types of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord.

Animals↗

PubMed1988

Age-dependent requirements of cultured spinal cord neurons.

Biochemical response of spinal motor neurons (SMNs) to putative trophic factors present in the fetal calf serum (FCS) or released from the native glial cells was evaluated in ventral spinal cord cultures of 12- to 14- and 16- to 18-day embryos (before and after death of many SMNs in vivo) considering choline acetyltransferase and 2',3'-cyclic 3'phosphohydrolase as the biochemical markers of SMNs and oligodendrocytes respectively. Cultured SMNs of older embryos were more dependent than those of younger embryos on glial cells and on FCS putative trophic factors. FCS seemed more important than glial cells for survival of both older and younger SMNs and its withdrawal resulted in more prominent and longer neurite outgrowth. Our study demonstrates that the response of SMNs to trophic factors depends on the age of the rat embryos from which they were cultured.

Animals↗

PubMed1987

Accumulation of CK-MM is impaired in innervated and contracting cultured muscle fibers of Duchenne muscular dystrophy patients.

No specific abnormalities have been reproducibly manifested in aneurally cultured muscle of Duchenne muscular dystrophy (DMD) patients. We now report that the accumulation of the muscle-"specific" isozyme of creatine kinase (CK-MM) was significantly and preferentially impaired in long-term innervated contracting muscle fibers cultured from 4 DMD patients (DMD-InnCMFs) compared to: i) their noninnervated sister-cultured muscle fibers, and ii) innervated contracting control cultured human muscle fibers (Control-InnCHMFs). Accumulation of other muscle-"specific" isozymes (MSIs), viz. glycogen phosphorylase, phosphoglycerate mutase, and lactic dehydrogenase, was not significantly impaired. We have not observed preferentially-impaired CK-MM accumulation in any Control-InnCHMFs from 22 patients (children and adults) with a variety of neuromuscular diseases. There was no apparent difference between DMD-InnCMFs and Control InnCHMFs regarding: acceptance of innervation; neuronally-driven, virtually continuous muscle-fiber contractions; characteristic myofiber organization by phase-contrast microscopy, and increased longevity of the innervated fibers.

Animals↗

PubMed1987

Kinetic analysis of thyrotropin-releasing hormone binding in the central nervous system: evidence for receptor desensitization.

To investigate the mechanism(s) of experimentally and clinically observed refractoriness of spinal lower motor neurons (LMNs) to the excitatory effects of high-dose TRH, we examined the kinetics of dissociation of [3H]TRH from its CNS-receptor. At 23 degrees C, the receptor was rapidly (40 min) and completely converted from a form with fast dissociation kinetics (complex I; t1/2 20-30 min) to one from which the peptide dissociated much more slowly (complex II; t1/2 greater than 120 min). This conversion required the presence of added agonist ([3H]TRH) and was not prevented by the GTP-analog Gpp(NH)p. We suggest that complexes I and II may respectively represent active and inactive (desensitized) forms of the TRH-receptor and that TRH-induced I to II conversion of the receptor is responsible for refractoriness of LMNs to the drug.

Amygdala↗

PubMed1987

De novo neuromuscular junction formation on human muscle fibres cultured in monolayer and innervated by foetal rat spinal cord: ultrastructural and ultrastructural--cytochemical studies.

Ultrastructural features of neuromuscular junction formation and transverse tubule development were studied utilizing a newly developed model in which human muscle fibres cultured in monolayer are innervated by foetal rat spinal cord with dorsal root ganglia attached. At early innervation (7-10 days), when distinct 'boutons' are contacting muscle fibres, the contacts of nerve terminals with the muscle fibres are, ultrastructurally, superficial and unorganized, and there is no basal lamina-like material between nerve terminals and muscle fibres. A bouton consists, ultrastructurally, of a cluster of small nerve terminals contacting the muscle fibre. At 2-3 weeks of innervation, shallow 'beds' are formed on the muscle fibre just beneath nerve terminals, and occasionally there are irregular and miniscule fragments of basal lamina-like material in the cleft. There is no Schwann cell apposing the nerve terminal at this stage of innervation. After 4-5 weeks of innervation there is more definite basal lamina material in the cleft and suggestive postsynaptic plasmalemmal densities and invaginations. However, there is no Schwann cell apposing the nerve terminal at this stage. At 6-8 weeks of innervation, deep postsynaptic folds are present, a Schwann cell apposes the nerve terminal, and basal lamina surrounds the entire muscle fibre. At all four stages of innervation examined, ultrastructural cytochemistry of alpha-bungarotoxin binding reveals that nicotinic ACh receptors are located exclusively at the neuromuscular junctions. After 1-2 weeks of innervation, very few lanthanum-positive transverse tubules are observed and only in close proximity to the surface membrane. After 3 weeks of innervation, more lanthanum-positive tubules are present, and they are located deeper within the muscle fibre. Five weeks after innervation, somewhat more elaborated tubules (but no lateral sacs) appear, and honeycomb structures are often present. After 6-7 weeks of innervation the tubular system is very elaborate and lateral sacs are present. Hence, this study describes consecutive stages of the formation of neuromuscular junctions and transverse tubules in innervated cultured human muscle, and provides an important basis to which similar studies related to the diseased human muscle can be compared.

Animals↗

PubMed1987

Developmental expression of the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer and innervated by fetal rat spinal cord.

The electrophoretic pattern of phosphoglycerate mutase of adult innervated normal human muscle is composed predominantly of the muscle-specific isozyme, whereas the electrophoretic pattern of aneurally cultured human muscle is composed only of the brain-specific isozyme. We studied the transition of the isozymes (phosphogluterate mutase) in human muscle cultured in monolayer and innervated for 20 to 83 days by rat embryo spinal cord explants. In this culture system, regions of innervated muscle fibers in close proximity to the ventral part of the spinal cord explant continuously contracted and the contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated cultured human muscle fibers, the total activity of phosphoglycerate mutase was increased and the muscle-specific isozyme was expressed. The amount of muscle-specific isozyme directly correlated with the duration of innervation. This study demonstrated that expression of the gene for the muscle-specific isozyme of phosphoglycerate mutase in human muscle cultured in monolayer is influenced by de novo innervation.

Animals↗

PubMed1987

Effects of electrical stimulation and tetrodotoxin paralysis on expression of muscle-specific isozymes of four enzymes in aneurally cultured embryonic rat muscle.

We studied the effect of electrical stimulation and a sodium channel blocker (tetrodotoxin) on the expression of muscle-specific isozymes of creatine kinase, glycogen phosphorylase, phosphoglycerate mutase, and lactate dehydrogenase in aneurally cultured embryonic rat muscle. Muscle contractile activity slightly accelerated the accumulation of muscle-specific isozyme of creatine kinase in early cultures (4 days of experiment), but no increase in the expression of muscle-specific isozymes of any enzyme was present in older cultures (11 days of experiment). We conclude that muscle contractile activity is not a main regulator of isozyme maturation in this system.

Animals↗

PubMed1987

Prednisone-responsive limb-girdle syndrome: a special disorder?

An eight-year-old boy had non-familial generalized muscle weakness, greater in the girdle and proximal limb muscles, sparing eye and face. It was insidiously progressive from age 14 months to age 20 months and then static, with major weakness. Two biopsies from very weak muscles, showed only type II muscle fiber atrophy. Muscle carnitine was normal. The EMG was "myopathic". Edrophonium test, antibodies to achR and systemic curare test were negative. There was a dramatic clinical improvement with prednisone, sustained now seven years later. Neither the pathogenesis nor cell mainly affected (neuron or muscle) is known. Not typical of a known disease, this unique patient demonstrates the potential therapeutic importance of steroid trial in patients with a severe chronic "limb-girdle syndrome" whose electro-physiology is non-specific and the muscle morphology shows changes "too minimal" to explain severe muscle weakness.

Biopsy↗

PubMed1987

Lack of usefulness of DN-1417 for characterization of a CNS receptor for thyrotropin-releasing hormone.

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)

Amygdala↗

PubMed1987

Insulin enhances development of functional voltage-dependent Ca2+ channels in aneurally cultured human muscle.

Voltage-dependent Ca2+ channels were studied by the binding of the potent Ca2+ channel antagonist PN200-110 and by the K+-induced 45Ca2+ uptake in human muscle cultured aneurally in the presence of insulin, fibroblast growth factor, and epidermal growth factor, added in combination or individually. Compared to the muscle grown in medium without growth factors, 14-15 days of treatment with insulin (10 micrograms/ml) alone or in combination with two other growth factors caused a 3.4- and 3.8-fold increase per culture dish in the number of PN200-110 binding sites, respectively. There was no change in the affinity of the ligand-receptor complex. Under the same conditions, there was also fourfold increase of the K+-induced 45Ca2+ uptake in cultured human muscle. Neither fibroblast growth factor nor epidermal growth factor alone influenced PN200-110 binding sites. Our study demonstrates that insulin enhances the development of functional voltage-dependent Ca2+ channels in cultured human muscle.

Calcium↗

PubMed1987

Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation.

Adult human muscle cultured in monolayer was cocultured with explants of 13-14-d-old rat embryo using (a) ventral spinal cord (VSC), (b) transverse section of whole spinal cord (WSC), and (c) WSC with dorsal root ganglia (DRG) attached (WSC + DRG). AChR clusters and AChE-positive patches, both at the nerve-muscle contacts, were studied at 5, 12, and 21 d of coculture with each of the 3 spinal cord preparations. In addition, AChE-positive patches were studied after 31-64 d of coculture with WSC + DRG to evaluate further organization of those patches. Compared to VSC and WSC cocultures, WSC + DRG induced significantly more AChR clusters per muscle fiber at the nerve-muscle contacts at 5 d of coculture, and the percentage of muscle fibers containing AChR clusters was higher at all 3 time points quantitated. The number of AChE-positive sites was the same with all 3 spinal cord preparations in early (day 5) cocultures. Between 12 and 21 d of coculture, the number of muscle fibers containing AChE patches increased significantly only with WSC + DRG, correlating with the increased number of contracting muscle fibers in that coculture system. Only in human muscle cocultured with WSC + DRG was successful innervation of the cultured muscle fibers achieved, as manifested by (1) contractions in a continuous rhythm of large groups of muscle fibers that were reversibly blocked by 1 mM d-tubocurarine (aneurally cultured human muscle does not spontaneously contract); (2) well-developed cross-striations throughout the fiber; (3) well-organized AChE-positive sites; and (4) a trend from multifocal toward unifocal innervation of those muscle fibers. Our studies demonstrate that adult human muscle cultured in monolayer can be innervated by fetal rat spinal cord and that, in our system, DRG are essential for achieving functional innervation.

Acetylcholinesterase↗

PubMed1986

Analogs of thyrotropin-releasing hormone: hypotheses relating receptor binding to net excitation of spinal lower motor neurons.

Experimentally and clinically, treatment with high-doses of TRH produces a net excitation of spinal lower motor neurons (LMNs) that is subsequently reduced or completely lost through continuous or repeated exposure to the peptide. This is operationally termed "autorefractoriness" (AR). We have performed biochemical and in vivo pharmacologic experiments to investigate the mechanism(s) of AR. Biochemically, we classified TRH and several analogs into three groups based on their binding by spinal-cord TRH-receptors (TRH-Rs): high-affinity, (low nanomolar range; MeTRH, TRH); intermediate-affinity (mid-nanomolar range; MK-771, RX77368) or low-affinity (micromolar range; DN-1417, PNP). When tested in vivo for LMN excitatory activity in cordotomized (T8) rats, TRH and MK-771 produced rapid-onset excitation followed AR. In contrast, sustained excitation with much less AR was produced by the low affinity analog DN-1417. Based on these results, we have formulated two receptor-based hypotheses to explain AR: a) rapid TRH-R desensitization (conversion to an inactive form) by high- but not low-affinity TRH-analogs; and b) a slower down-regulation (cellular internalization) of the agonist-receptor complex, most evident with high-affinity agonists. Thus, low-rather than high-affinity TRH-analogs may be superior to TRH for providing sustained LMN excitation (increase of strength) in motor neuron degenerative disorders.

Animals↗

PubMed1986

Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle.

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.

Animals↗

PubMed1985

Fibroblast growth factor, epidermal growth factor and insulin exert a neuronal-like influence on acetylcholine receptors in aneurally cultured human muscle.

Fibroblast growth factor (FGF), epidermal growth factor (EGF) and insulin added in combination to the culture medium in which normal human muscle was cultured caused a 4.0-fold (P less than 0.005) increase of the total number of nicotinic acetylcholine receptors (AChRs) and a 4.5-fold (P less than 0.001) increase in AChR aggregation. Individually, only FGF caused a 3.0-fold increase (P less than 0.005) in AChR aggregation, without influencing the total number of AChRs. To the contrary, insulin alone caused a 2.0-fold increase (P less than 0.05) in the total number of AChRs without influencing AChR aggregation. These findings show that these three polypeptide growth factors exert a neuronal-like influence on cultured human muscle in regard to AChRs.

Culture Techniques↗

PubMed1985

Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implications.

TRH has rapid-onset (30 sec), slow-offset (1-12 days) clinical benefit in patients with amyotrophic lateral sclerosis and other motor neuron disorders. This benefit is probably receptor-mediated and may have at least 2 components. To obtain a better understanding of the various responses to TRH of the spinal lower motor neurons (LMNs) in patients, and possibly to help guide selection of additional therapeutic agents, we utilized rat CNS (spinal-cord and brain membranes) to analyze the ability of certain molecules to inhibit specific binding of [3H]methyl TRH [( 3H]MeTRH) to the TRH receptor. We found: a) lack of high-affinity binding of the TRH-analog DN-1417 by spinal-cord and brain TRH receptor, despite its known strong TRH-like action physiologically on LMNs; b) lack of high-affinity binding of the TRH-product cyclo(His-Pro) by spinal-cord and brain TRH receptor despite its having some strong TRH-like physiologic actions on the CNS; and c) lack of any identifiable high-affinity receptor for cyclo(His-Pro) in spinal cord and brain. From these data we hypothesize that the acute transmitter-like action of DN-1417, TRH, and possibly other TRH-analogs and products on LMNs is via a non-TRH receptor, such as an amine or amino acid neurotransmitter receptor, e.g. a 5-hydroxytryptamine receptor. We further postulate that the CNS TRH-receptor may modulate a trophic-like influence of TRH on LMNs.

Amyotrophic Lateral Sclerosis↗

PubMed1985

Thyrotropin-releasing hormone enhances motor neuron-evoked contractions of cultured human muscle.

Thyrotropin-releasing hormone (TRH), 1 to 2.7 mM, introduced to the medium of monolayer-cultured human muscle innervated by cocultured fetal rat spinal cord with dorsal root ganglia, increased muscle fiber contractions 4.9 times (p less than 0.005). Doses higher than 5 mM, after increasing contractions for a few seconds, caused complete inhibition of contraction (autorefractoriness). Neither aneurally cultured human muscle nor innervated cultured human muscle blocked by 1 mM d-tubocurarine was excited by TRH. Thus, in this system TRH appeared to excite muscle contractions through direct or indirect action on motor neurons.

Animals↗

PubMed1985

Cells of neural crest origin as possible models to investigate thyrotropin releasing hormone action in the central nervous system.

Since TRH has been linked to trophic events in the motor neuron and is being used for symptomatic treatment of motor neuron disorders, we have examined seven neural crest cell lines for TRH-receptors (TRH-Rs) and for the enzymes choline acetyltransferase (ChAT) and creatine kinase (CK), with the intent to identify one that can be used for studies of TRH-R mediated cellular events in the CNS. Three of the seven (neuroblastomas) were ChAT-positive but were without detectable TRH-Rs and showed no major alterations in ChAT/CK-activities following acute (24 hr) treatment with 0.01-10 microM TRH. In contrast, high affinity TRH receptor sites were detected in a melanoma (rpmI 3460) cell line. Linking cellular events with these receptors could lead to the use of this established cell line as a tool for the biochemical investigation of the pathways and mechanisms of TRH action in the CNS.

Animals↗