Localization of two glycolytic enzymes in guinea-pig taenia coli.

The bound fractions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of fructose 1,6-diphosphate aldolase (ALD) were measured in intact Taenia coli. ALD was approximately 60% bound and GAPDH was approximately 41% bound. Bound ALD activity remaining in chemically demembranated Taenia coli was similar to that in intact tissue indicating a localization to the contractile apparatus. ALD was found to be specifically bound in the demembranated preparation. Chemical demembranation resulted in almost complete loss of all GAPDH activity indicating a localization of bound GAPDH to cellular membranes.     

Creatine kinase reaction in skinned rat psoas muscle fibers and their myofibrils.

The aim of this study was to evaluate myofibrillar creatine kinase (EC 2.7.3.2) activity on the background of the effect of substrate channeling by myosin ATPase and to compare it with creatine kinase (CK) activity of whole skinned fibers. In order to assess CK activity, skinned fibers were prepared from the rat psoas major muscles defined by light microscopy. The activity in permeabilized fibers after treatment with saponin, Triton X-100 and Ca(2+)-free medium reached 2.80, 6.97 and 3.32 micromol ATP min(-1) mg(-1) protein, respectively, when a coupled enzyme assay system with external hexokinase and glucose-6-phosphate dehydrogenase was used. Transmission electron microscopy (TEM) revealed a possible interference among activities of sarcolemmal, sarcoplasmic, myofibrillar and mitochondrial CK from persisting structures. For evaluation of the myofibrillar CK itself, a pure myofibrillar fraction was prepared. Fraction purity was confirmed by TEM and by enzymatic assays for marker enzymes. Two procedures, i.e. the coupled enzyme assay and the evaluation of phosphocreatine (PCr) concentration before and after the CK reaction, were used for measurement of CK activity in this fraction. The procedures resulted in 3.2 nmol ATP min(-1) mg(-1) protein and 7.6 nmol PCr min(-1) mg(-1) protein, respectively. These alternative approaches revealed a discrepancy between the reacting portions of PCr by more than 50 %, which provides information about the size of the effect, generally described as substrate channeling.     

Creatine kinase: a role for arginine-95 in creatine binding and active site organization

Sequence homology analysis reveals that arginine-95 is fully conserved in 29 creatine kinases sequenced to date, but fully conserved as a tyrosine residue in 16 arginine kinases. Site-directed mutants of rabbit muscle creatine kinase (rmCK) were prepared in which R95 was replaced by a tyrosine (R95Y), alanine (R95A), or lysine (R95K). Kinetic analysis of phosphocreatine formation for each purified mutant showed that recombinant native rmCK and all R95 mutants follow a random-order, rapid-equilibrium mechanism. However, we observed no evidence for synergism of substrate binding by the recombinant native enzyme, as reported previously [Maggio et al., (1977) J. Biol. Chem. 252, 1202-1207] for creatine kinase isolated directly from rabbit muscle. The catalytic efficiencies of R95Y and R95A are reduced approximately 3000- and 2000-fold, respectively, compared to native enzyme, but that of R95K is reduced only 30-fold. The major contribution to the reduction of the catalytic efficiency of R95K is a 5-fold reduction in the affinity for creatine. This suggests that while a basic residue is required at position 95 for optimal activity, R95 is not absolutely essential for binding or catalysis in CK. R95Y has a significantly lower affinity for creatine than the native enzyme, but it also displays a somewhat lower affinity for MgATP and 100-fold reduction in k(cat). Interestingly, R95A appears to bind either creatine or MgATP first with affinities similar to those for the native enzyme, but it has a 10-fold lower affinity for the second substrate, suggesting that replacement of R95 by an alanine disrupts the active site organization and reduces the efficiency of formation of the catalytically competent ternary complex.     

Effects of palytoxin on contractile response and calcium movement in guinea-pig taenia coli

PTX (10(-8)M) induced a rapid increase followed by a gradual decrease in muscle tension in normal physiological salt solution (PSS), while it induced a slow increase in muscle tension in low-Na+ solution. These contractions were inhibited by Ca2+ channel blockers, verapamil and nicardipine. PTX rapidly increased tissue Na+ and decreased tissue K+ contents in normal PSS. In low-Na+ solution, PTX decreased tissue K+ content with a slower rate than that in normal PSS. PTX increased uptake of 45Ca2+ in normal as well as low-Na+ solutions with similar time course as the increase in muscle tension. However, 45Ca2+ uptake still remained high when the PTX-induced transient contraction ceased. These results suggest that PTX increases Ca2+ influx through voltage-dependent Ca2+ channels to cause contraction. After a prolonged exposure to PTX, however, muscle tension is uncoupled from Ca2+ influx.     

Zero extracellular K+ and prostaglandin E release in the guinea-pig taenia coli

Prostaglandin E release rates from isolated strips of guinea-pig taenia coli increased during exposure to zero K+ bathing fluid, from control values of 0.78 +/- 0.11 ng/g per min to levels as high as 29.2 ng/per min. Release rates increased for 40-50 min and then remained constant or fell despite progressive increases in intracellular sodium [Nai+] or fall in intracellular potassium [Ki+]. Readmittance of K+ to the bathing solution resulted in rapid reversal of elevated prostaglandin E release rates. [Nai+] and [Ki+] were markedly more abnormal in strips exposed to zero K+ for 70-201 min compared to 30-min exposures. Upon the readdition of K+ after long zero K+ exposure, the rate of prostaglandin E release fell long before [Nai+] and [Ki+] returned to control levels. After K+ was readded to the bathing solution, the ion concentration of tissues exposed to zero K+ for 30 min returned to normal much more quickly than did those of tissues exposed for the longer time periods, yet the exponential rate constants for fall of prostaglandin E release rate after K+ was added were not significantly different after short or long zero K+ exposure. Thus there was a dissociation between the return of [Nai+] and [Ki+] and the fall of prostaglandin E release rate to control levels. Ouabain augmented prostaglandin E release under conditions where [Ki+] could not fall. Addition of known neurotransmitters present in this tissue to the bathing fluid did not augment prostaglandin E release. Guinea-pig taenia coli strips that had been incubated with [3H]arachidonic acid, constantly released [3H]arachidonic acid and [3H]prostaglandin E and a prostaglandin which cochromatographed with prostaglandin E but could not be converted to prostaglandin B by alkali and was shown to be 6-ketoprostaglandin F1 alpha. Release of [3H]arachidonic acid and [3H]prostaglandin E plus 6-[3H]ketoprostaglandin F1 alpha was increased when strips were exposed to zero K+. Data obtained in this study suggest the augmented prostaglandin E release seen during zero K+ or ouabain is related to increased availability of unbound arachidonic acid at the site of cyclooxygenase in the cell. Augmented prostaglandin E release is apparently not related to alterations in intracellular electrolyte concentrations or release of known neurotransmitters.     

Phosphorus-31 NMR studies of smooth muscle from guinea-pig taenia coli

Phosphorus-31 NMR spectra of superfused isometrically mounted guinea-pig taenia coli were obtained using a horizontal probe at 103.2 MHz. The spectra showed resonances for ATP, phosphocreatine (PCr), and a sugar phosphate resonance. The PCr/ATP ratio was between 1.5 and 2.0 consistent with chemical analysis of tissue extracts. The level of PCr, but not of ATP, decreases reversibly during contraction or inhibition of respiration. These conditions did not cause substantial changes in the intracellular pH, which was 7.0 ± 0.1.     

Development and ageing of intestinal musculature and nerves: the guinea-pig taenia coli

The fine structure of taenia coli was studied by electron microscopy in guinea-pigs from birth to old age (over 2 years old). Smooth muscle cells are ～1,000 μm³ in volume at birth, 2,200 μm³ in young adults and 4,500 μm³ in old age. Muscle growth and muscle cell enlargement continue throughout life, an increase in muscle volume of about 240 times. Differentiated muscle cells divide during development and in adults. Because mitoses are found in any part of the muscle, the tissue grows from within, rather than by addition at the ends or borders. There is progressive increase in nucleus volume, and decrease in surface-to-volume ratio and in nucleus-cell volume ratio in muscle cells. At all ages the taenia consists of a uniform population of muscle cells (apart from dividing cells); there are no undifferentiated cells, no precursor cells or myoblasts, and no degenerating cells. Interstitial cells and fibroblasts are observed at all ages with only small variations in relative number. The amount of intramuscular collagen increases in old age. There is roughly one capillary for every 170 muscle cell profiles at birth, and one for every 200 in adults and in old age. The innervation is dense and reaches all parts of the muscle. In adults there are ～1,300 axons per 10,000 μm² of sectional area, or between 8,000 and 38,000 axons in a full cross section of taenia; this amounts to ～2% of the muscle volume. An answer to the question of why there are so many nerves in the taenia was not found. Expanded axon profiles are part of typical varicose fibres. Varicosities are packed with small clear vesicles and lie at the surface of nerve bundles. Absence of strong, constant patterns indicating specialized contacts of the nerve terminals is a feature of these nerves at all ages. Some varicosities are closest to interstitial cells; more commonly they are close to muscle cells at sites that strongly suggest a neuro-muscular junction. The additional possibility that some varicosities are part of afferent fibres is discussed. The innervation is well developed at birth and the highest density of innervation is found around day 4 when 4% of the taenia consists of nervous tissue. The innervation of immature taenia is characterized by close juxtaposition of axons and muscle cells. Axon profiles packed with vesicles, varicosities and presumptive neuro-muscular junctions are present at birth. The extent of Schwann cells in intramuscular nerves is markedly less than in adults, and virtually all the axons have maximal membrane-to-membrane contact with other axons. In taenia of aged guinea-pigs, the density of innervation is reduced. There is no actual loss of nerve tissue; the total amount of nerve tissue is greater than in young adults, and the apparent reduction reflects a more intense growth of muscle cells. The Schwann cell component becomes more conspicuous than in young adults and there is a greater number of axons fully wrapped by a Schwann cell. Presumptive neuro-muscular junctions are common and probably commoner than in young adults. Growth of muscle cells, changes in their cytological features and in the stroma occur throughout life, including old age. Nerves too continue to grow and undergo structural changes in pattern of distribution, relation with Schwann cells and effector cells.     

Role of creatine kinase in force development in chemically skinned rat cardiac muscle

The influence of phosphocreatine in the presence or absence of MgATP and MgADP was studied in Triton X-100-treated thin papillary muscles and ventricular strips of the rat heart. The pCa/tension relationships, the pMgATP/tension relationships, and the tension responses to quick length changes were analyzed. The results show three major consequences of the reduction of the phosphocreatine concentration in the presence of millimolar concentrations of the MgATP. (a) The resting tension and the maximal Ca2+-activated tension were increased, and the pCa/tension relationship was shifted toward higher pCa values and its steepness was decreased; these effects were enhanced by the inclusion of MgADP. (b) The time constant of tension recoveries after quick stretches applied during maximal activation was increased, while the extent of these recoveries was decreased. (c) The study of pMgATP/tension relationships in low Ca concentrations showed that the decrease in phosphocreatine induced a shift toward higher MgATP values with no changes in maximal rigor tension or the slope coefficient; these effects were increased by the increase in MgADP and were independent of the preparation diameter. Thus, modifications of the apparent Ca sensitivity and resting and maximal tension when phosphocreatine is decreased seem to be due to an increasing participation of rigor-like or slowly cycling cross-bridges spending more time in the attached state. These results suggest that endogenous creatine kinase is able to ensure maximal efficiency of myosin ATPase by producing a local high MgATP/MgADP ratio.     

Quantitative morphological study of smooth muscle cells of the guinea-pig taenia coli

A quantitative study of muscle cells of the guinea-pig taenia coli is reported. Stereological methods were used on electron micrographs and phase contrast micrographs. Smooth muscle cells of taeniae fixed under 1 gram load were about 515 m long. Muscle cell volume was about 3,500 m³ and cell surface 5,300 m². About 168,000 caveolae were found at the surface of each muscle cell, covering about 29 percent of its surface. They produced a 73 percent increase of the cell membrane compared to a smooth-surfaced cell. The ratio surface-to-volume is about 10.67 if the geometrical surface is considered, or 10.39 if the total surface of the cell membrane (including the caveolae) is considered. Mitochondria constituted 3.5–4 percent of the cell volume. A few nexuses were observed, both between two muscle cells and between a muscle cell and an interstitial cell. In serial sections septa of connective tissue and groups of muscle cells were found to disappear within few tens of microns or to merge with other septa, and the taenia did not appear to be divided into clear-cut muscle cell bundles. Bundles of smooth muscle cells were seen passing from the taenia to the underlying circular muscle. The transverse sectional area of the taenia ranged between 0.14 and 0.39 mm²; it showed about 526 blood vessels · mm^-2.     

Ca2+-induced Ca2+ release in skinned single smooth muscle cells isolated from guinea-pig taenia caeci

The Ca2+ release from intracellular Ca2+ storage sites of skinned single smooth muscle cells isolated from guinea-pig taenia caeci was studied. The Ca2+ release from intracellular Ca2+ storage sites of the skinned single cells was enhanced by the presence of submicromolar concentrations of Ca2+ in the solution. The Ca2+ release was enhanced by caffeine and adenine, and suppressed by Mg2+ and procaine. These results suggest that the Ca2+-induced Ca2+ release mechanism may play an important role in the release of Ca2+ from intracellular storage sites of guinea-pig taenia caeci smooth muscle cells.