Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Light and electron microscopic studies onMyxobolus cotti El-Matbouli and Hoffmann, 1987 infecting the central nervous system of the bullhead (Cottus gobio)

Myxobolus cotti (Myxozoa: Myxosporea) is described as found in the central nervous system of the bullhead (Cottus gobio) caught in the Alpine lake Königssee and in a brook in the Bavarian Forest, Federal Republic of Germany (El-Matbouli and Hoffmann 1987). Aggregations of spores and polysporoblastic trophozoites compressed and replaced large areas of the white and grey matter of the brain and spinal cord. These aggregations may be surrounded by a thin, connective tissue capsule; in a few cases they were associated with loose infiltrates of glial cells. Neither conspicuous tissue reactions nor inflammatory responses were evident. No other organs were seen to be infected withM. cotti. Mature spores are oval, with a tapering anterior end, and the pyriform polar capsules are nearly equal in size. Fresh spores measured 8.9–15.1 m in length (mean, 12.4 m) and 8–12.4 m in width (mean, 9.6 m); polar capsules were 4.3–9 m long (mean, 6.4 m); and 2–3.8 m wide (mean, 2.9 m). Light microscopy, the ultrastructure of pansporoblasts, sporogenesis and mature spores are described.     

Bradykinin Antagonizes the Effects of α-Thrombin

-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1M AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 × 10^–6 cm/sec to 4.92 × 10^–6 cm/sec. BK (1M) produced a similar increase to 4.46 × 10^–6 cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1M), prior to addition of AT, reduced the AT permeability coefficient to 2.69 × 10^–6 cm/sec. Addition of NA-BK (1 M) for 5 minutes, then BK (1 M) for 5 minutes, inhibited the effect of BK and of AT (1 M) on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 × 10^–6 cm/sec). AT (1 M) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 M) for 2 minutes also inhibited the effects of -thrombin (1 M) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an -thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 M) with -thrombin (1 M) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators -thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion,secretion and aggregation

Adrenaline (1 to 10M) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U-46619 and the Ca²⁺ ionophore A23187. Adrenaline (0.5 M) potentiates the aggregation and secretion induced by all the previous agonists in citrated plateletrich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: i) nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC₅₀ 0.22 M and 0.28 M respectively); ii) nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC₅₀ ranging from 0.1 to 2.5 M) or in washed platelets (IC₅₀ ranging from 0.1 to 0.8 M); iii) nicergoline inhibits the biding of³H-yohimbine to washed human platelets (IC₅₀ 0.26 M); iv) the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly theex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC₅₀ range 108 to 670 M) and in washed platelets (IC₅₀ range 27 to 140 M) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activationin vivo could justify the use of nicergoline (Sermion^®), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

β-Adrenoceptor stimulation activates large-conductance Ca2+-activated K+ channels in smooth muscle cells from basilar artery of guinea pig

We studied the effect of isoproterenol on the Ca²⁺-activated K⁺(BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca²⁺ concentration, [Ca²⁺]_i. Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V _H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from aV _H of 0 mV was highly sensitive to block by external tetraethylammonium·Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. With [Ca²⁺]_i between 0.1 and 1.0 M, 0.4 M isoproterenol increased this current by 58.6±17.1%, whereas with [Ca²⁺]_i at 0.01 M a sixfold smaller increase was observed. With [Ca²⁺]_i0.1 M, 100 M dibutyryl-adenosine 3:5: cyclic monophosphate (cAMP) and 1 M forskolin increased this current by 58.5±24.1% and 59.7±10.3%, respectively. The increase with isoproterenol was blocked by 4.0 M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from aV _H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-outwhole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260-pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca²⁺]_i from 0.01 to 1.0 M. Outside-out-whole-cell recordings with [Ca²⁺]_i0.1 M indicated that 100 M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152±115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca²⁺]_i, with a 35-fold larger effect observed with 0.1–0.5 M Ca²⁺ compared to 0.01 M Ca²⁺. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by-adrenoceptor stimulation, that the effect depends strongly on [Ca²⁺]_i, and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.     

Effect of steroids on γ-aminobutyrate-induced currents in cultured rat astrocytes

Cultured astrocytes from rat cortex respond to the inhibitory neurotransmitter -aminobutyric acid (GABA) by the activation of Cl^– channels [Bormann J, Kettenmann H (1988) Proc Natl Acad Sci USA 85:9336–9340]. The glial response shares many pharmacological properties with those mediated by neuronal GABA_A receptors, but differs in its sensitivity to inverse benzodiazepine agonists [Backus KH, Kettenmann H, Schachner M (1988) Glia 1:132–140]. To compare glial GABA receptors further with their neuronal counterparts, we analysed the effect of steroids, which have recently been shown to modulate neuronal GABA_A-receptor-mediated responses, on GABA-induced currents in astrocytes. The agonist allotetrahydrodeoxycorticosterone (THDOC) at concentrations of 100 nM and 1 M enhanced GABA-evoked (with 10 M GABA) currents up to 115% and 162.4% of controls respectively. The antagonist dehydroisoandrosterone 3-sulphate (DHEAS) at concentrations of 1 M, 10 M and 100 M depressed GABA-evoked (10 M) currents to 72%, 42.8% and 21.4% of controls respectively. The steroids were less effective at higher GABA concentrations. 100 M DHEAS directly elicited a membrane current, while THDOC (1 M) did not exert any direct response. This study demonstrates that steroids modulate GABA-evoked currents and thus may interfere with any of the functions of glial GABA receptors that are at present under discussion.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

A diffractometer using a lateral effect photodiode for the rapid determination of sarcomere length changes in cross-striated muscle

A simple method is devised to record rapid sarcomere length changes of muscle fibres using a lateral effect diode. In the standard position the diffractometer records length changes between 1.65 and 3.8 m, the output being linear 1 V/m with a frequency response of –3 dB at 1.2 kHz. The absolute error is<0.05 m between 1.65 and 2.80 m and <0.1 m between 2.81 and 3.30 m. The resolution of length changes is<0.005 m over the whole range. By varying the detector position the length range can be extended to either side, and spatial resolution can be improved at the expense of length range.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Effects of dipyridamole on adenosine concentration,insulin sensitivity and glucose utilisation in soleus muscle of the rat

Adenosine has been shown to modulate the sensitivity of skeletal muscle to insulin (Budohoski et al. 1984). In an attempt to further characterize the modulatory action of adenosine on insulin sensitivity inskeletal muscle we have investigated the effect of the nucleoside transport inhibitor dipyridamole in isolated incubated soleus muscle strips. At a concentration of 50 M, dipyridamole increased the concentration of adenosine in the soleus muscle by 36% and in the incubation medium by 32%. At this concentration of dipyridamole, the basal rates (in the presence of 1 unit of insulin/ml) of lactate formation, 2-deoxy [2,6-³H]glucose phosphorylation and glucose oxidation were decreased by 48%, 43% and 47% respectively, whilst the rate of glycogen synthesis was unaffected. Insulin-stimulated rates (in the presence of 10000 unit of insulin/ml) of lactate formation, 2-deoxy [2,6-³H] glucose phosphorylation, glycogen synthesis and glucose oxidation were decreased by 70%, 30%, 26% and 20% respectively in the presence of 50 M dipyridamole. Although 50 M dipyridamole was required to exert a significant effect on medium and soleus muscle adenosine concentrations, statistically significant effects on glycolytic rate were observed at concentrations as low as 2 M dipyridamole.It is concluded that the results are not consistent with dipyridamole exerting an effect on skeletal muscle carbohydrate metabolism solely through elevation of the intracellular or interstial adenosine concentration, but strongly suggest that dipyridamole inhibits glucose transport and/or phosphorylation in skeletal muscle.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Effects of ketamine on GABA-evoked excitability of peripheral nerve

Summary The effects of the dissociative anaesthetic, ketamine on GABA-evoked changes in the excitability of myelinated fibers of dorsal and ventral roots of isolated bullfrog sciatic nerves were examined. Ketamine alone (0.01–1000 M) evoked small increases (<5%) in dorsal root fiber excitability, as reflected in the half-maximal A-fiber compound action potential when concentrations were >10 M; with 0.1 M even larger increases (10%) were elicited in the ventral root fibers. As well, the increases evoked by 10 M ketamine were followed by graded decreases. 0.1 and 10 M ketamine, concentrations which by themselves had small or no effect, produced a dose-dependent depression of the large increases in excitability which are induced by activation of GABA receptors. In the presence of ketamine GABA concentration-response curves of the dorsal root fibers showed depression of the maximal response, while those of the ventral root fibers were shifted to the right. This apparent antagonism of GABA responses by ketamine may arise from blockade of receptor-mediated effects (e.g. K⁺/Cl^- currents and/or secondary depolarization from K⁺ accumulation), but could also be caused by a selective potentiation of hyperpolarizing receptors.     

Fibre types inLimulus telson muscles: morphology and histochemistry

Summary Using a variety of techniques, we have demonstrated the presence of at least two fibre types inLimulus median telson levator muscle. By light and electron microscopy, large (21 56 m² mean cross-sectional area) fibres have A-bands of 4.1 m, one-half I bands of 2.15 m and Z lines 0.5 m in width. Few mitochondria are found in these fibres, which comprise 54% of those present in a given microscope field and which occupy 82% of the total cross-sectional area. Small fibres (484 m² mean cross-sectional area) have A bands of 6.3 m, one-half I bands of 3.1 m and Z lines between 0.5 and 1.0 m in width and are rich in mitochondria. Although small fibres comprise nearly one-half (46%) of the fibres in a field, they occupy only 18% of the total cross-sectional area.Histochemical staining for alkaline-stable myofibrillar ATPase activity and mitochondrial reduced -nicotinamide adenine nucleotide (-NADH) tetrazolium reductase activity confirms the presence of two fibre types. The large fibres react positively for the myofibrillar ATPase activity and negatively for the mitochondrial enzyme activity. The reverse is seen with the small fibres. Some fibres of intermediate size, having intermediate staining characteristics, were also observed. Native gel electrophoresis of both myofibrillar and purified myosin preparations supports the observed differences in myofibrillar ATPase activity in that two myosin isozymes are resolved on pyrophosphate gels. Although the thick filaments isolated from unstimulated small fibres are longer (>6.0 m) than those isolated from unstimulated large fibres (4.26 m), all have a similar appearance with respect to the arrangement of myosin heads on their surfaces, and similar diameters. The implications of the observed heterogeneity of fibre types is discussed with reference to previously reported phenomena inLimulus telson muscle, including changes in length of thick filaments on fibre stimulation and the shape of the length-tension curve obtained from fibre bundles.     

Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-μm DNA

Summary YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-m DNA, or lager brewing yeast 2-m DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-m DNA under these conditions. By construction of laboratory-lager 2-m DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-m DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.     

Anterograde transport of opioid receptors in rat vagus nerves and dorsal roots of spinal nerves: pharmacology and sensitivity to sodium and guanine nucleotides

Summary We have utilized the technique of in vitro autoradiography to ascertain that opioid receptors are transported in the rat vagus nerve and in the rat dorsal spinal root fibers. In the dorsal roots, opioid receptors accumulated on both sides of the ligatures. In the vagus nerve, a distal accumulation of binding sites was difficult to detect, however, proximal to the ligatures, vagal receptors accumulated in a linear fashion during the first 12 h of ligation. At longer periods after ligation, accumulation was less than expected and the receptors appeared to migrate retrogradely. The receptor transport could be blocked by intravagal colchicine injection and the receptor translocation could be elicited in isolated vagal nerve segments suggesting that the receptors move by fast transport. Sodium chloride, present in the incubation medium, inhibited [³H]dihydromorphine ([³H]DHM) binding to receptors adjacent to and far from the proximal aspect of the ligature with IC₅₀'s of 42 mM and 51 mM, respectively. The addition of GTP in the incubation medium also inhibited [³H]DHM binding to proximal and far proximal receptors with IC₅₀'s of 0.27 M and 1.0 M, respectively. The presence of GTP also inhibited [³H]naloxone ([³H]Nal) binding to proximal and far proximal receptors with IC⁵⁰'s of 0.34 M and 0.66 M, respectively. The transported vagal opioid receptors bound the ligands in a stereospecific manner. Using [³H]DHM, [³H]D-ala²-D-leu⁵-enkephalin ([³H]DADL), and [³H]ethylketocyclazocine ([³H]EKC), we found that most of the transported vagal receptors have mupharmacology although kappa and delta receptors are present.