Reprint of: Comparative Studies of Rabbit Cardiac and Skeletal Myosins.

Rabbit cardiac myosin contains fewer cysteine residues than the skeletal myosin (7 and 8.8 moles/105 gm. of myosin, respectively). A similar difference is found between the cysteine content of rabbit cardiac and skeletal heavy meromyosins; the cardiac heavy meromyosin contains 8.9 moles/105 gm. of protein as compared to 11 moles in the skeletal heavy meromyosin. In skeletal myosin, actomyosin, and myofibrils the Ca++-ATPase, Ca++-ITPase, and EDTA-ATPase activities are about three times higher than those of cardiac myosin, actomyosin, and myofibrils; whereas the skeletal to cardiac actomyosin-ATPase activity ratio is higher. The ATPase activities of both cardiac and skeletal myosins, actomyosins, and myofibrils, however, are close to each other when determined in the presence of Mg++ at high ionic strength. The abilities of cardiac and skeletal myosins to combine with actin at either high or low ionic strength are also essentially the same. The Ca++-ATPase, Ca++-ITPase, EDTA-ATPase, and actomyosin-ATPase activities of cardiac myosin, heavy meromyosin, and myofibrils, unlike those of skeletal myosin, heavy meromyosin, and myofibrils, do not increase over pH 8.0. The ATPase activities of cardiac and skeletal myosins in the presence of Mg++ at high ionic strength, on the other hand, are affected similarly by changes of pH. In cardiac myosin, heavy meromyosin, and myofibrils, the Ca++activated ATPase is less sensitive to high KC1 concentrations than is that of skeletal myosin, heavy meromyosin, and myofibrils.

Exchange of the actin-bound nucleotide in intact arterial smooth muscle.

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In (32)P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [gamma,beta-(32)P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the beta-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the gamma-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K(+), as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.

Muscular dystrophy in adults: a five-year follow-up.

The aim was to describe the natural history of adults with hereditary muscular dystrophies, including myotonic dystrophy, with respect to muscular function, ventilation and electrocardiogram. In a prospective study, 46 subjects were followed over a period of five years. In 1991 and 1996, their muscle function was assessed according to an observation scheme and their lung vital capacity was measured by spirometer. Electrocardiograms were obtained in 1991, 1993 and 1996. Deterioration of muscular function was seen with regard to both the functional muscle tests and the vital capacity. The proportion of pathological electrocardiograms increased from 38% in 1991 to 54% in 1996 in the 26 patients with myotonic dystrophy without an increase in clinically detected cardiac abnormalities. Timely examinations using standard methods can reveal medically important information on deterioration, which often passes clinically unnoticed because of the insidious progress of the diseases.

Metabolic fate of glucose in vascular smooth muscle during contraction induced by norepinephrine.

The metabolism of glucose in porcine carotid artery was tracked by isotopic methods during sustained isometric contraction induced by 100 microM norepinephrine (NE). In resting muscles, 74 and 18% of glucose taken up was accounted for by glycolysis and glycogen synthesis, respectively. Lactate production accounted for 69%, pyruvate production for 12%, and glucose oxidation accounted for 14% of glycolytic flux. The oxidation of glucose accounted for 57% of the consumption of O2 and thus constituted the primary oxidative substrate. During contraction by NE, glucose-uptake declined modestly below the resting basal rate. Glycolysis of external glucose and lactate production decreased and then increased with sustained contraction. Norepinephrine stimulated simultaneous glycogenolysis and glycogen synthesis, with net glycogen synthesis prevailing over 90 min of isometric contraction. Furthermore, NE modified the distribution of glucosyl units throughout the glycogen pool. The steady state rate of oxidation of glucose did not increase during NE contraction, even though O2-consumption increased. In contrast, increased glucose oxidation was demonstrable during contraction induced by 80 mm KCl. Furthermore, oxidation of exogenous fatty acid could be demonstrated during NE-induced contraction. Thus, NE exerts multiple effects on glucose and glycogen metabolism in smooth muscle, but it does not stimulate glucose oxidation.

Bottle-blowing in hospital-treated patients with community-acquired pneumonia.

A study was carried out to determine whether bottle-blowing has any positive effects in patients with pneumonia. In a prospective open study 145 adults with untreated community-acquired pneumonia requiring hospitalization were randomized to early mobilization (group A), to sit up and take 20 deep breaths on 10 occasions daily (group B), or to sit up and to blow bubbles in a bottle containing 10 cm water through a plastic tube 20 times on 10 occasions daily (group C). Peak expiratory flow (PEF), vital capacity (VC), forced expiratory volume in 1 sec (FEV1) and serum concentration of C-reactive protein (CRP) were determined on admission, and on days 4 and 42. Fever duration and hospital stay were recorded. In a subset of 16 patients, single breath diffusion capacity of carbon monoxide was measured on 3 occasions. The patients in group A were hospitalized for a mean of 5.3 days, group B for 4.6 days and group C for 3.9 days. Treatment was a significant factor (p = 0.037) in a Cox regression model, with group C significantly better than group A (p = 0.01). The number of days with fever was 2.3, 1.7 and 1.6 in groups A, B and C respectively. These differences were not significant (p = 0.28). No significant differences were found between the groups regarding CRP, PEF, VC, FEV1, or diffusion capacity. Intensive bottle-blowing shortens the hospital stay in patients with pneumonia. The underlying mechanism is not clear.

Oxidation of acetate and octanoate and its relation to glucose metabolism in contracting porcine carotid artery.

The oxidation of octanoate and acetate was measured in segments of porcine carotid arteries to ascertain whether the oxidation of exogenous fatty acid substrates (acetate and octanoate) is augmented during contraction induced by K(+)-depolarization. The oxidation of acetate increased from 7 +/- 1 to 14 +/- 2 nmol/min/g (P < 0.01) during sustained isometric contraction. Octanoate oxidation increased from 11 +/- 1 to 14 +/- 1 nmol/min/g (P < 0.05). The rate of oxidation of neither acetate nor octanoate was affected by the presence or absence of glucose either in resting or contracting arteries Acetate or octanoate oxidation could account for the majority of O2 consumption during contraction. Octanoate but not acetate inhibited glucose uptake and glycolysis in resting muscles. In contrast to augmented acetate and octanoate metabolism during contraction, there was a "down-regulation" of glucose metabolism in contracting muscles as evidenced by a decrease in the rate of glucose uptake, glycolysis and lactic acid production during sustained isometric contraction. Thus, contractile activation of vascular smooth muscle is associated with a shifting pattern of substrate utilization. Exogenous acetate or octanoate can serve as the primary oxidative substrate during sustained isometric contraction.

Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.

Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

Calponin phosphorylation does not accompany contraction of various smooth muscles.

Calponin phosphorylation was quantitated in 32P-labeled porcine arterial, uterine, tracheal, stomach and bladder smooth muscles. The negligible amount of 0.003-0.008 mol [32P]phosphate/mol calponin in resting muscles did not increase upon contraction induced by various agents, under conditions when myosin light-chain phosphorylation increased several-fold. The results indicate no involvement of calponin phosphorylation is smooth-muscle contraction.

Proton magnetic resonance spectroscopy of the brain in schizophrenic and affective patients.

Water-suppressed 1H magnetic resonance spectra were recorded from two brain regions of psychiatric patients and normal volunteers. The two regions studied were (a) the basal ganglia structures surrounding the anterior horn of the lateral ventricle and (b) the occipital cortex. N-Acetylaspartate (NAA), phosphocreatine-creatine (PCr-Cr), choline and inositol resonances were seen in both regions. Ratios of metabolite peak integrals to PCr-Cr peak integral were calculated for each spectrum. To control for partial volume effects, comparisons between patients and controls were made only from identical regions i.e. basal ganglia vs basal ganglia, and likewise for occipital cortex. Metabolite ratios from the occipital region of patients were similar to those from the occipital region of normal subjects. Bipolar patients being treated with lithium had elevated NAA/PCr-Cr in the basal ganglia region when compared to normals. These patients also demonstrated elevated choline/PCr-Cr and inositol/PCr-Cr ratios in the basal ganglia region.

Involvement of calponin and caldesmon in sustained contraction of arterial smooth muscle.

The molecular mechanism of smooth muscle contraction was approached by a novel method, covalent 14C-labeling. Intra- and intermolecular protein interactions during contractile activity are reflected by changed reactivity of protein side chains; these can be detected by reagents which readily permeate through the muscle membrane without affecting the contractility and form covalent bonds with proteins in the muscle. The incorporation of 14CH2ICONH2 into proteins of 1-hour histamine contracted versus resting porcine carotid arterial muscles was determined. Out of fourteen 14C-labeled proteins analyzed, only two showed a change in reactivity during sustained contraction. The incorporation of 14CH2ICONH2 into calponin and caldesmon in contracted muscles was about 66% of that into these same proteins in resting muscles. A transformation of calponin and caldesmon molecules from an extended to a more compact conformation explains the decreased reactivity.

Protein phosphorylation during the contraction-relaxation-contraction cycle of arterial smooth muscle.

Porcine carotid arterial muscles were labeled with 32P and then subjected to a resting-contraction-relaxation-contraction cycle. Four different agents were used for contraction: KCl, histamine, norepinephrine, and phorbol dibutyrate. To relax the contracted muscles, they were washed with physiological salt solution. Changes in the [32P]phosphate content of four different proteins--myosin light chain, a 28-kDa cytosolic protein, desmin, and caldesmon--were followed. In a short contraction-relaxation-contraction cycle lasting minutes, induced by K+, histamine, or norepinephrine, only the light chain underwent a phosphorylation-dephosphorylation-rephosphorylation without concomitant cyclic phosphorylation of the 28-kDa protein, desmin, or caldesmon. In a contraction-relaxation-contraction cycle of long duration, 60-min contractions with K+, histamine, or norepinephrine, cyclic phosphorylation of both the light chain and desmin was observed. With 60-min phorbol dibutyrate stimulation, in the long contraction-relaxation-contraction cycle, the phosphorylations of the light chain, desmin, and caldesmon were cycling. It is concluded that under physiological conditions, light-chain phosphorylation initiates both short and sustained arterial contraction. Desmin phosphorylation is likely to be involved in force maintenance during sustained contraction.

Protein phosphorylation in arterial muscle contracted by high concentration of phorbol dibutyrate in the presence and absence of Ca2+.

Porcine carotid arterial muscles were 32P-labeled then contracted with 8 microM phorbol dibutyrate (PDBu) in normal physiological salt solution (PSS) and in Ca(2+)-free PSS containing 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetate. Significant incorporation of [32P]phosphate into the 20-kDa myosin light chain, a 28-kDa protein, desmin and caldesmon was measured with no apparent difference between normal and Ca(2+)-depleted muscles. Ca-determination showed that the Ca(2+)-depleted muscle contained 15% of the total Ca of the normal muscle. However, determination of the actin-bound Ca revealed that all the Ca in the Ca(2+)-depleted muscle could be accounted for by its actin-bound Ca. Accordingly, protein phosphorylation during the slow PDBu-induced contraction may proceed in the virtual absence of free Ca2+. Phosphopeptide mapping of the myosin light chain isolated from muscles contracted with PDBu either in the presence or absence of Ca2+ showed that two-thirds of the incorporated [32P]phosphate was attributable to myosin light chain kinase catalyzed phosphorylation and one-third was due to phosphorylation by protein kinase C. PDBu increased the phosphorylation of the 28-kDa protein, desmin and caldesmon two- to threefold, as compared with that in muscles contracted by KCl depolarization or by the receptor mediated agonists norepinephrine and histamine. Muscles contracted by high concentration of PDBu in the presence or absence of Ca2+ could be fully relaxed and recontracted.

Exchange of 20-kDa myosin light chain-bound phosphate during sustained contraction of arterial smooth muscle.

K(+)-contracted porcine carotid arterial muscles containing phosphorylated 20-kDa myosin light chains (LC) were exposed to carrier-free [32P]orthophosphate in K(+)-stimulating solution during sustained contraction. The covalently bound LC phosphate was completely replaced by [32P]phosphate, indicating that myosin light chain phosphatase and kinase have ready access to the bound phosphate during the sustained contraction. On average, 0.38 mol [32P]phosphate was incorporated per mole LC during the sustained K+ contraction. This value was about half of the maximal value for [32P]phosphate incorporation into LC, 0.74 mol/mol, in muscles contracted with K+ for 1 min. Assuming that sustained contraction involves the maximal number of cross-bridges attached to actin, the data suggest that half of the attached cross-bridges contain phosphorylated LC.

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate.

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.

Absence of calponin phosphorylation in contracting or resting arterial smooth muscle.

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 265, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 32P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [32P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.

Stretch activates myosin light chain kinase in arterial smooth muscle.

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery.

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.

Human leg neuromuscular diseases: P-31 MR spectroscopy.

Phosphorus-31 magnetic resonance (MR) spectra of leg muscles in patients with the neuromuscular diseases Duchenne dystrophy, myotonic dystrophy, postpoliomyelitis, Werdnig-Hoffmann disease, and pedal dystonia were recorded. Ratios of beta-adenosine triphosphate (ATP), inorganic phosphate (Pi), alpha-glycerophosphorylcholine (GPC), and phosphomonoesters to phosphocreatine (PCr) were calculated from peak integrals and compared with normal muscle ratios. In all diseases studied, beta-ATP/PCr and Pi/PCr values showed an increase from normal values. The extent of increase in beta-ATP/PCr was related to the clinical severity of the disease, suggesting that this could be a useful noninvasive means of monitoring effectiveness of therapy for neuromuscular disorders. In myotonic dystrophy and Werdnig-Hoffmann disease, GPC/PCr values increased greatly. The intracellular pH in Duchenne and postpoliomyelitis muscles was slightly elevated compared with that in normal muscles. Hydrogen-1 MR images of muscles showed fat infiltration in all patients, more in weaker muscles and less in stronger muscles.

Volume-selective water-suppressed proton spectra of human brain and muscle in vivo.

Volume-selective water-suppressed proton spectra were recorded from live human brain and muscle at 1.5T by combining a stimulated echo acquisition mode pulse sequence for localization and two saturation pulses for water suppression (Frahm et al., SMRM Abstracts, 1987). Metabolite signals were observed in voxels of size 4-64 cm3. Signals from -CH3 and beta-CH2 of N-acetylaspartate, =N-CH3 and =N-CH2 of phosphocreatine/creatine, -N(CH3)3 of choline and inositol protons were visible in the brain spectra from normal subjects. Differences in metabolite levels were observed between gray and white matters of brain from their water-suppressed spectra. Peaks from =N-CH3 of phosphocreatine/creatine and -N(CH3)3 of choline and carnitine were present in normal muscle spectra along with several resonances from fatty acyl chains.