Skeletal tissues in Mozambique tilapia (Oreochromis mossambicus) express the extracellular calcium-sensing receptor.

Molecular phylogenetic analysis suggests that the extracellular calcium-sensing receptor (CaSR) emerged evolutionarily in association with the chordate-vertebrate lineage. Our studies overall explore the evolution of CaSRs, and the possible historical linkage of CaSRs to vertebrate skeleton as functional components of calcium homeostasis through regulated storage and/or release. We applied both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) to evaluate Casr gene and CaSR protein expression, respectively, in skeletal tissues of a cichlid teleost, the Mozambique tilapia (Oreochromis mossambicus).

Extracellular calcium-sensing receptor distribution in osmoregulatory and endocrine tissues of the tilapia.

The extracellular calcium-sensing receptor (CaSR) serves an important detector function in vertebrate Ca(2+) homeostasis. In this study, we surveyed using immunohistochemistry the tissue and cellular distribution of the CaSR protein in the Mozambique tilapia (Oreochromis mossambicus) and the Japanese eel (Anguilla japonica). Specifically, we examined receptor expression in ion-transporting barrier tissues that may be directly responsive to extracellular Ca(2+) levels, and in tissues that are implicated in endocrine signaling to homeostatic effectors such as Ca(2+)-transporting epithelia.

cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus).

The complete cDNA sequence of the tilapia extracellular Ca(2+)-sensing receptor (CaR) was determined. The transcript length of tilapia CaR (tCaR) is 3.4 kbp and encodes a 940-amino acid, 7-transmembrane domain protein that is consistent in its structural features with known mammalian and piscine CaRs.

Impact of prednisone on TGF-beta1 and collagen in diaphragm muscle from mdx mice.

The purpose of this study was to assess the impact of prednisone treatment for 8 weeks on the level of transforming growth factor-beta 1 (TGF-beta1), hydroxyproline (HYP) concentrations, and level of the mature, nonreducible collagen cross-link hydroxylysylpyridinoline (HP) in diaphragm muscle from 12-week-old mdx mice. Diaphragm muscle from untreated mdx mice had a significantly higher level of TGF-beta1, HYP, and HP cross-link compared with normal C57BL/10J (control) mice. Prednisone treatment significantly reduced the level of TGF-beta1 and HYP in diaphragm from mdx mice to values similar to control mice, but resulted in a higher level of the HP cross-link compared with untreated mdx mice.

Pre-clinical screening of drugs using the mdx mouse.

The genetically dystrophin-deficient mdx mouse, with its characteristic and regular exercise-induced loss of strength, is a useful experimental platform on which to screen potential drug therapies in the treatment of some dystrophic diseases. Pharmacological agents of several chemical and functional classes were examined in their ability to reduce the loss of muscular strength in young exercised mdx mice. Therapeutic intervention over the period 4-10 weeks of age was evaluated in weekly tests of whole-body strength.

Natriuretic peptides in fish physiology.

Natriuretic peptides exist in the fishes as a family of structurally-related isohormones including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and ventricular natriuretic peptide (VNP); to date, brain natriuretic peptide (or B-type natriuretic peptide, BNP) has not been definitively identified in the fishes. Based on nucleotide and amino acid sequence similarity, the natriuretic peptide family of isohormones may have evolved from a neuromodulatory, CNP-like brain peptide. The primary sites of synthesis for the circulating hormones are the heart and brain; additional extracardiac and extracranial sites, including the intestine, synthesize and release natriuretic peptides locally for paracrine regulation of various physiological functions.

Local synthesis of natriuretic peptides in the eel intestine.

The intestine is a major osmoregulatory organ in euryhaline fishes which allows them to survive in the sea, and natriuretic peptides have been implicated in regulation of transmural transport. Atrial (ANP) and ventricular natriuretic peptide (VNP) were identified in eel intestine. Elution profiles of ANP and VNP from high-performance liquid chromatography (HPLC) were determined by radioimmunoassay using highly specific antisera.

Cromolyn increases strength in exercised mdx mice.

Exercised mdx mice were used to evaluate the efficacy of two pharmacologic entities, cromolyn and compound 48/80. Beginning at 2 weeks of age, mdx mice were treated with either cromolyn (50 mg/kg/day), prednisone (2mg/kg/day), compound 48/80 (1mg/kg/day), or diluent vehicle. At 4 weeks of age, treated mice were subjected to twice weekly, forced treadmill running which has previously been shown to cause expressed weakness in mdx mice (Hudecki, Pollina et al.

Duchenne-like myopathy in double-mutant mdx mice expressing exaggerated mast cell activity.

Dystrophin-deficient female mdx mice were bred with male Tsk+/+ pa mice to examine the role played by mast cells in the pathophysiology of dystrophin deficiency. Resultant mdx/Tsk double-mutant mice were then examined functionally, biochemically, and histologically. While mdx mice remained as strong as their normal counterparts, mdx/Tsk double-mutant mice became progressively weak with age.

Efficacy of drug regimen exceeds electrostimulation in treatment of avian muscular dystrophy.

Autosomal-recessive dystrophic chickens were treated in three experimental groups with an intraperitoneal multicomponent drug mixture (50 mg/kg Ep475, 20 mg/kg Cinanserin, 10 mg/kg stanazolol, 100 mg/kg leucine, 0.1 mg/kg insulin, 100 mg/kg glucose, and 50 mg/kg carnitine), percutaneous high-frequency electrostimulation of the pectoralis muscle, or a combination of both drug and electrostimulation treatments. Therapeutic efficacy was determined in each group by measurements of strength, righting ability, and histomorphometric analyses of the pectoralis musculature.

Enhanced sensitivity of mdx mice to intramuscular injection of compound 48/80.

Species-specific differences in the inflammatory response, specifically with regard to mast cells, have been proposed to explain the phenotypic variation among dystrophin-deficient humans, and mdx mice (Gorospe et al., 1994). To test this hypothesis we have intramuscularly injected a mast cell secretogogue into both dystrophin-negative mdx and dystrophin-positive normal mice.

Strength and endurance in the therapeutic evaluation of prednisolone-treated MDX mice.

The dystrophin-deficient, X-linked dystrophic mouse (mdx) was used to evaluate the efficacy of prednisolone treatment. A test protocol was used to take advantage of the quantifiable weakness and disability as well as molecular genetic defect shared with the X-linked Duchenne muscular dystrophy (DMD). Whole-body weakness and fatigue were determined by non-invasive force-transducer physiographic and variable-speed treadmill techniques, respectively.

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity.

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin.

Cell and fiber-type distribution of dystrophin.

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue.

Effects of denervation on spectrin concentration in avian skeletal muscle.

The effect of denervation on avian muscle alpha-spectrin was examined in fast and slow muscles. Using immunofluorescence, the surgically denervated fast-twitch posterior latissimus dorsi (PLD) exhibited a significant increase in spectrin antigen associated with the sarcolemma and within the sarcoplasm compared with the contralateral innervated control muscle. Using gel electrophoresis followed by immunoblotting, we found a two- to three-fold increase in the levels of spectrin in the denervated PLD over that found in the innervated PLD.

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle.

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res.

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken.

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal.

Increased concentration of spectrin is observed in avian dystrophic muscle.

A significant increase in the concentration of spectrin has been observed in dystrophic chicken pectoralis major muscle when compared to normal fast-twitch muscle. In normal muscle, alpha-spectrin-specific immunofluorescence delineates each myofiber with a network pattern of staining at the sarcolemma with little staining within the cytoplasm. In dystrophic fibers, numerous intensely stained areas occur within the cytoplasm and staining at the sarcolemma is increased, thereby obscuring or eliminating the highly regular network arrangement of spectrin usually seen in this region.

Effects of percutaneous electrical stimulation on functional ability, plasma creatine kinase, and pectoralis musculature of normal and genetically dystrophic chickens.

The breast musculature of genetically dystrophic Line 413 and genetically related normal Line 412 chickens were treated in three separate trials with high-frequency electrical stimulation (ES). Beginning on days 7 or 14 ex ovo, each bird received three ES treatments per week. Each stimulation cycle repeated five times per day consisted of 15 s "on" followed by 50 s "off".

Characterization of lymphocyte interferons with different species specificities from normal and genetically dystrophic chickens.

Lymphocytes from thymus and spleen of normal (Line 412) and genetically dystrophic (Line 413) chickens produce two types of interferons (IFNs) with different host cell specificities. The first type, referred to as ChIFN-alpha, demonstrates antiviral activity on primary normal chicken embryo (CE) cells. This activity is stable at 60 degrees C for 1 h and, in this respect, ChIFN-alpha is similar to the standard ChIFN-beta.

In vivo effects of three calcium blockers on chickens with inherited muscular dystrophy.

Genetically homozygous Line 413 dystrophic chickens were given in separate trials daily i.p. injections of aqueous solutions of the calcium blocker drugs, diltiazem, verapamil, or nifedipine.

Limited benefit to genetically dystrophic chickens from a synthetic proteinase inhibitor: Ep475.

Chickens with inherited muscular dystrophy (Line 413) were treated in two separate trials with daily intraperitoneal injections of 10% DMSO-water solutions containing the proteinase inhibitors, Ep475 and E64. Drug therapy in each case significantly prolonged the functional ability of the treated chickens. Diluent control chickens around day 35 ex ovo characteristically reached a maximum ability to right from the supine position in a standardized functional test for muscle weakness.

Parenteral branched-chain amino acid treatment and avian dystrophy.

Genetically homozygous line 413 dystrophic chickens were given twice-daily intraperitoneal injections of solutions containing branched-chain amino acids (BCCA-leucine, valine, isoleucine) either alone or in combination; and their alpha-ketoacid analogs (alpha-ketoisocaproic and alpha-ketoisovaleric acids). Another trial consisted of an amino acid mixture containing BCAA. Amino acid supplementation in each case significantly prolonged righting ability measured regularly by a standardized flip-test procedure.