The neuromuscular and vascular hypotheses of muscular dystrophy: a possible link via adenine nucleotides and phosphate

A hypothesis is presented that an abnormality of nucleotide synthesis or metabolism might be a primary functional defect underlying Duchenne muscular dystrophy. One aim of this hypothesis is to demonstrate that it is possible to explain a variety of observations in dystrophy, neuromuscular and vascular, on a single hypothesis.     

Roles of prostaglandins suggested by the prostaglandin agonist/antagonist actions of local anaesthetic,anti-arrhythmic,anti-malarial,tricyclic antidepressant and methyl xanthine compounds. Effects on membranes and on nucleic acid function

We have recently found that local anaesthetic, anti-malarial, anti-arrhythmic, tricyclic antidepressant and methyl xanthine compounds behave as prostaglandin antagonists at drug concentrations readily attainable in human body fluids. We have also found that various hormones, including prolactin, vasopressin and angiotensin when present in physiological concentrations trations can be potent stimulators of prostaglandin synthesis. Cortisol has no effect on basal prostaglandin synthesis but in physiological concentrations is able to reverse the effects of other hormones. The drugs are thought to act mainly by interfering with either calcium interactions with membranes, or with cyclic nucleotide synthesis or degradation or with nucleic acid function. We propose that prostaglandins play crucial roles at all three points. The hypothesis leads to new approaches to many aspects of cell regulation, to the functions of many systems, especially the neuromuscular, cardiovascular, renal and respiratory systems. It also has profound implications for drug design and evaluation and for the understanding of many clinical disorders.     

The pathological damage in Duchenne muscular dystrophy may be due to increased intracellular OXY-radical generation caused by the absence of dystrophin and subsequent alterations in Ca2+ metabolism

Recent advances in the genetic and molecular pathogenesis of Duchenne muscular dystrophy and the evidence suggesting a role for oxygen free radicals (oxy-radicals) in the development of this disease are reviewed. In addition, we outline a working of hypothesis as to how disruptions in intracellular Ca2+ homeostasis within the dystrophic cell may initiate cycles of increased oxy-radical fluxes within these cells, leading to intracellular oxidative damage.     

The effect of methylprednisolone on intracellular calcium of normal and dystrophic human skeletal muscle cells.

Clinical trials have shown that a glucocorticoid, the methyiprednisolone (PDN), has a beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients. The aim of this study was to test if the effect of PDN could be mediated via a possible action on intracellular calcium. The intracellular calcium activity, at rest and during calcium mobilizing drug superfusion protocols was recorded in normal and dystrophic human cocultured muscle cells. PDN (10 microM) pretreatment induced an elevation of the resting calcium concentration of 51, 34 and 38% in proliferating normal myoblasts, DMD myoblasts and DMD myotubes, respectively, while normal myotubes resting [Ca2+]i was not altered.     

Stimulation of calcium uptake and cyclic GMP synthesis in rat basophilic leukemia cells by Escherichia coli heat-stable enterotoxin.

The effect of Escherichia coli heat-stable (ST) enterotoxin on calcium and cyclic nucleotide metabolism in rat basophilic leukemia cell cultures was investigated. Addition of ST enterotoxin to rat basophilic leukemia cell cultures resulted in dose- and time-dependent stimulation of calcium uptake and elevation of the intracellular cyclic GMP (cGMP) concentration. The effect of ST enterotoxin on calcium uptake (P less than 0.02) and cGMP synthesis (P less than 0.02) was demonstrated after 5 and 30 min of incubation at 37 degrees C, respectively. In further studies ST enterotoxin did not enhance calcium release or the intracellular concentration of cyclic AMP. The stimulation of calcium uptake and cGMP synthesis by ST enterotoxin was inhibited by pharmacological and chemical agents which block cellular calcium entry and prostaglandin synthesis. These results demonstrate that ST enterotoxin induces calcium uptake and cGMP synthesis in rat basophilic leukemia cell cultures.     

The cellular distribution of calcium in freeze-dried rabbit vas deferens using EM autoradiography

The role of calcium in initiating smooth muscle contraction is widely accepted. The sources of this calcium are thought to be located both intracellularly and extracellularly. We have recently developed a method by which the cellular localization of calcium in smooth muscle can be determined. This method involves exposing the tissue to 45Ca, rapidly freezing and vacuum dehydrating the tissue, and preparing the tissue for electron microscopic autoradiography (EM ARG). In the present study the distribution of calcium in control and potassium-contracted tissue of the rabbit vas deferens was compared. No significant differences in distribution were observed in the two treatments. This finding provides morphological support for the hypothesis that the calcium used in potassium-induced contraction is primarily of extracellular origin. In addition, significant sequestration by intracellular organelles does not occur during a potassium contraction. In other experiments, the effect of rinsing tissue in cold calcium prior to freezing was investigated. From these data is appears that calcium is removed from the cytoplasmic matrix, plasma membrane, and organelles in a nonuniform manner. Further investigation into these findings should enable us to characterize more precisely the intracellular redistribution of calcium that occurs as a result of a variety of physiological manipulations.     

Membrane cross-talk in the early distal tubule segment of frog kidney: role of calcium stores and chloride

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.     

Prostaglandin release from macrophages: An assay system for anti-inflammatory drugs in vitro

It is the intention of this communication to present a simple and reliable in vitro system for the evaluation of anti-inflammatory drugs. The system described appears to combine the advantages of two established procedures, i.e. measurement of the inhibition of prostaglandin (PG) synthesis in vitro using microsomal enzyme preparations and the measurement of the inhibitory effects of drugs on PG synthesis in vivo avoiding major limitations. We propose to measure the effect of drugs on the PG release from mouse peritoneal macrophages in vitro. The influence of culture conditions, phagocytosis and drugs on the PG release from these cells is described. We found that antibody-coated erythrocytes are especially suitable triggers of PG release, and that the release can be inhibited by steroidal and nonsteroidal anti-inflammatory drugs in concentrations resembling their effective doses in vivo.Dedicated to K. Bucher on the occasion of his 65th birthday.     

Distinction between Duchenne and other muscular dystrophies by ribosomal protein synthesis.

Ribosome concentration, ribosome distribution on sucrose density gradients, and in-vitro ribosomal amino-acid incorporation (noncollagen and collagen synthesis) were studied in muscle biopsy samples obtained from 30 patients with Duchenne muscular dystrophy, seven patients with Becker muscular dystrophy, and 10 with facioscapulohumeral muscular dystrophy. Ribosome concentration was normal in Duchenne and facioscapulohumeral and decreased in Becker muscular dystrophy. Distribution of ribosomes in sucrose density gradients showed abnormalities (sharp monosomal peak and fewer polyribosomes) only in Duchenne muscular dystrophy and was normal in the other two types. In-vitro amino-acid incorporation of ribosomes in Duchenne muscular dystrophy revealed high collagen and low noncollagen synthesis of the heavy polyribosomes. This abnormality is controlled by an undetermined enzymatic factor belonging to the soluble enzyme fraction. Supplementation of the dystrophic heavy polyribosomes with normal soluble enzymes restored the synthesis of collagen to that of the controls. Heavy polyribosomes extracted from normals or from carriers produce proportionately more collagen in the presence of soluble enzyme fraction from Duchenne muscular dystrophy than in the presence of their homologous enzymes. In Becker muscular dystrophy, both noncollagen and collagen synthesis of the heavy polyribosomes were increased, under the influence of ribosomal factors. The different protein synthesis in Duchenne and Becker muscular dystrophies suggests that these conditions are non-allelic. In facioscapulohumeral muscular dystrophy the changes in protein synthesis occurred only in the early stage of the disease and consisted of increased noncollagen synthesis of the light polyribosomes, while the heavy polyribosomes had normal activity including collagen synthesis. This reaction was controlled by ribosomal factors.     

The influence of aspirin and indomethacin on the platelet contractile wave.

Recent evidence suggests that a short-lived product of prostaglandin biosynthesis produced on the addition of arachidonic acid to platelet microsomes is involved in physiologic platelet aggregation and mediates the release reaction by producing the platelet contractile wave. An important corollary of this hypothesis is that aspirin and indomethacin, inhibitors of prostaglandin synthesis, should block the platelet contractile process produced by agents which can initiate platelet aggregation. The present study tested this hypothesis and found that aspirin and indomethacin were, indeed, potent inhibitors of the platelet contractile wave stimulated by collagen and epinephrine, but largely failed to inhibit internal transformation induced by thrombin and ADP. The findings confirm the hypothesis that a prostaglandin produced endogenously by platelets initiates platelet contraction and suggests that ADP and thrombin have the ability to stimulate the platelet contractile apparatus by an alternate mechanism not dependent on prostaglandin synthesis.     

Comparative analysis between Duchenne and Becker types muscular dystrophy.

Duchenne and Becker types of muscular dystrophy are usually differentiated according to age of onset and rate of progression criteria which are not sufficient. The aim of this paper was to re-establish the clues for distinguishing Duchenne from Becker types of muscular dystrophy. According to the onset and progression of the disease, one hundred and eleven patients were subdivided into two groups. First group--Becker muscular dystrophy--consisted of 40 patients and second one of 71 patients with Duchenne type of muscular dystrophy. Clinical data confirm some well known differences between Duchenne and Becker muscular dystrophy concerning the age of onset, severity of disease and rate of progression. Electromyographic signs of myopathic changes and spontaneous activity were found in both diseases. Spontaneous activity--bizarre and fibrillation potentials, as well as sharp waves are more common for Duchenne type. The differences between the Becker from Duchenne type of muscular dystrophy can be described on the basis of complex investigations (clinical, electromyographical, histological and biochemical).     

Function and genetics of dystrophin and dystrophin-related proteins in muscle

The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and alpha-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.     

Damage to developing mouse skeletal muscle myotubes in culture: protective effect of heat shock proteins

Damage to skeletal muscle occurs following excessive exercise, upon reperfusion following ischaemia and in disease states, such as muscular dystrophy. Key mechanisms by which damage is thought to occur include a loss of intracellular calcium homeostasis, loss of energy supply to the cell, increased activity of oxidising free radical-mediated reactions and activation of apoptosis pathways. An increased cellular content of heat shock proteins (HSPs) has been shown to protect skeletal muscle against some forms of damage, although the mechanistic basis of this protection is not clearly understood. The aim of this study was to establish a cell culture-based model of damage to C₂C₁₂ skeletal muscle cells using the calcium ionophore, A23187 and the mitochondrial uncoupler, 2,4-dinitrophenol (DNP) as damaging agents. Treatment of cells with 1 m m DNP for 60 min resulted in the release of 63.5 % of intracellular creatine kinase (CK) activity over the 3 h experimental period. Treatment of cells with 10 μ m A23187 for 30 min resulted in the release of 47.9 % of CK activity. Exposure of myotubes to a period of hyperthermia resulted in a significant increase in their content of HSP25, HSP60, HSC70 (heat shock cognate) and HSP70. This increase in HSPs was associated with significant protection against both DNP-induced and A23187-induced damage to the myotubes. These results indicate that an increased content of HSPs may provide protection against the muscle damage that occurs by a pathological increase in intracellular calcium or uncoupling of the mitochondrial respiratory chain.     

New insights in the regulation of calcium transfers by muscle dystrophin-based cytoskeleton: implications in DMD

Calcium mishandling in Duchenne muscular dystrophy (DMD) suggested that dystrophin, a membrane-associated cytoskeleton protein, may regulate calcium-signalling cascades such as calcium entries. Calcium overload in human DMD myotubes is dependent on their contractile activity suggesting the involvement of channels being activated during contraction and/or calcium release. Forced expression of mini-dystrophin in dystrophin-deficient myotubes, reactivates appropriate sarcolemmal expression of dystrophin-associated proteins and restores normal calcium handling in the cytosol. Furthermore, the recombinant mini-dystrophin reduced the store-operated calcium influx across the sarcolemma, and the mitochondrial calcium uptake during this influx. A slow component of calcium release dependent on IP3R, as well as the production of IP3, were also reduced to normal levels by expression of mini-dystrophin. Our studies provide a new model for the convergent regulation of transmembrane calcium influx and IP3-dependent calcium release by the dystrophin-based cytoskeleton (DBC). We also suggest molecular association of such channels with DBC which may provide the scaffold for assembling a multiprotein-signalling complex that modulates the channel activity. This suggests that the loss of this molecular association could participate in the alteration of calcium homeostasis observed in DMD muscle cells.     

Influence of anti-inflammatory drugs on the interaction of lymphocytes and macrophages

The helper function of macrophages in lymphocyte stimulation is well known, but there are indications that macrophages may also exert a suppressor effect on lymphocytes. This effect might be due to prostaglandins secreted by the macrophages. In order to test this hypothesis anti-inflammatory drugs, some of which are known inhibitors of prostaglandin synthesis, were added to a series of phytohaemagglutinin (PHA)-stimulated lymphocyte cultures containing different proportions of macrophages and lymphocytes. The experiments showed that high concentrations of all drugs were inhibitory. Moderate concentrations of some of the PG-synthesis-inhibiting drugs (like indomethacin and mefenamic acid), however, appeared to have a stimulatory effect. The stimulation was more pronounced in cultures containing a high proportion of macrophages. These results support the assumption that macrophages release prostaglandins, which suppress PHA-induced lymphocyte proliferation.     

Clinical and Histopathological Features of Abnormalities of the Dystrophin-Based Membrane Cytoskeleton

The majority (∼70%) of cases of childhood and adult onset muscular dystrophies in males, and ∼10% of dystrophy in girls and women, show underlying primary abnormalities of dystrophin. Approximately 2% of childhood/adult onset muscular dystrophy patients have a primary defect of one of the three sarcoglycan proteins identified to date (α, β, γ). The finding of a sarcoglycan deficiency in muscle generally does not reflect the primary underlying defect, and thus testing of biopsies for sarcoglycans should be used only after normal dystrophin findings, and in conjunction with gene mutation testing. Approximately 30% of neonatal onset congenital muscular dystrophy has been shown to be due α2-laminin (merosin) deficiency. α2-laminin is a component of the myofiber basal lamina, and this protein interacts with the dystrophin-based membrane cytoskeleton. Due to the similar clinical and histopathological features of the different etiologies of muscular dystrophies, molecular testing of peripheral blood DNA and muscle biopsy protein are a critical part of the clinical work-up of dystrophy patients. Many patients carrying a Becker dystrophy or limb-girdle dystrophy diagnosis should be re-evaluated with molecular tests to provide accurate genetic counseling to their families.     

Norepinephrine-induced increase in sympathetic neuron-derived prostaglandins is independent of neuronal release mechanisms.

The contribution of exocytosis to norepinephrine-stimulated prostaglandin release from sympathetic postganglionic neurons was evaluated in homogenates of adult rat superior cervical ganglia. Incubation of ganglion homogenates with norepinephrine (1 mM) for 30 min caused an increased release of prostaglandin E2 and prostaglandin I2 (measured as the stable metabolite, 6-keto-PGF1a). Neither tetrodotoxin (10 mM), K+ (120 mM), nor EDTA in Ca(2+)-free buffer affected prostaglandin generation under basal and norepinephrine-stimulated conditions. These results suggest that the increase in prostaglandin production by sympathetic neurons after norepinephrine administration is not through the release of previously synthesized intracellular stores. Instead, the increase in prostaglandins in response to norepinephrine appears to be explained by de novo synthesis.     

Hypotonic medium increases calcium permeant channels activity in human normal and dystrophic myotubes

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin and an elevated intracellular calcium level. Single-channel recordings were performed with the cell-attached configuration of the patch-clamp technique. The present study shows, on human co-cultured normal and dystrophic muscle cells, the evidence for an increased activity of calcium permeant cationic mechano-sensitive channels under hypotonic medium stimulation. This activity was particularly enhanced in DMD cells. The hypotonic medium induced drastic changes in the single-channel activity characteristics, which are: a large increase of the calcium over potassium permeability ratio; and a great enhancement of the quantity of current crossing through these channels. These channels could contribute to a significant calcium entry, which could participate in the abnormal calcium homeostasis observed in DMD muscle.     

IL-4 primes human endothelial cells for secondary responses to histamine

Interleukin-4 (IL-4) is a multifunctional cytokine, which is involved in numerous disease states, including atopic asthma. IL-4 not only induces direct responses in cells but can also prime for secondary responses to stimuli. Little is known about the priming effects of IL-4 on endothelial cells; therefore, we chose to examine the ability of IL-4 to prime endothelial cells for platelet-activating factor (PAF) synthesis and prostaglandin E(2) (PGE(2)) release. IL-4 alone did not enhance PAF synthesis or PGE(2) release; however, pretreatment with IL-4 primed for PAF synthesis and PGE(2) release in response to subsequent stimulation with histamine. In contrast, tumor necrosis factor alpha (TNF-alpha), oncostatin M (OSM), and IL-1beta did not prime endothelial cells for PAF synthesis in response to histamine. The priming effects of IL-4 occurred without any detectable changes in the requirement for signaling pathways upstream of PGE(2) release. IL-4 treatment increased the expression of mRNA for histamine receptor 1 (HR1) and shifted the inhibition curve for pyrilamine, a specific HR1 antagonist. In addition, the dose-response curve for histamine-induced elevations in intracellular calcium was shifted following IL-4 stimulation. Together, these data indicate that HR1 is up-regulated in IL-4-stimulated human umbilical vein endothelial cells (HUVEC) and suggest that this up-regulation may contribute to the enhanced responsiveness of IL-4-stimulated HUVEC to histamine.