Inhibition of a voltage-dependent Ca current by concanavalin A

Summary Incubation of the hypotrichous ciliateStylonychia mytilus in fluorescein-labeled concanavalin A (Con A, 0.1–0.5 g/ml) produced a strong fluorescence of its membranelles, but comparatively weak fluorescence of the other compound cilia and of the somatic membrane. Compared to untreated cells, the frequency of spontaneous backward movements was reduced in the presence of 0.5 g/ml ConA. In electrophysiological experiments Con A altered the excitability of the cell membrane. The two-peak action potential lost its second component which is associated with voltage-dependent Ca channels in the membranelles. The corresponding Ca current (Ca current I) was inhibited by low concentrations of Con A (0.2–0.5 g/ml). A second voltage-dependent Ca current (Ca current II) was not affected. Reducing the K outward current by intracellular Cs and/or extracellular tetraethylammonium, or changing the holding potential, did not restore the Con A-sensitive Ca current I. Con A also inhibited this current when Ca was replaced by Ba. The inhibitory effect of Con A on the voltagedependent Ca current I was prevented by 10–30 mM -methyl-d-mannoside, and the lectin wheat germ agglutinin (20 g/ml) did not affect the Ca currents, indicating that the Con A effect was mediated by binding to specific sugar residues on the excitable membrane. The succinylated dimeric derivative of Con A did not inhibit Ca current I up to concentrations of 5 g/ml. It is concluded that the two voltage-dependent Ca currents inStylonychia can be chemically isolated due to their different sensitivity to Con A, which appears to bind preferentially to sites near or at the Ca channel in the membranellar membrane.     

Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents

Summary A tightly-sealed cardiac sarcolemmal patch preparation of large diameter (10–16 m with 5–25 G seals) has been developed to study regulation of selected electrogenic mechanisms. Formation of a large readily accessible membrane surface area is achieved by incubating freshly isolated myocytes in a high KCl/zero calcium solution, which promotes separation of sarcolemma from myofilaments. The formation of large-diameter, high resistance seals is facilitated by depositing a neutral hydrocarbon film on electrode tips. Sodium currents in excised patches are stable for patch life-times of 15–40 min with peak current densities of 120 to 350 A/cm². Outward sodium-calcium exchange current is identified by its specific dependence on external calcium and internal sodium, inhibition by cobalt and dichlorobenzamil, and activation by <0.1M internal free, calcium. Maximum exchange current density is >20 A/cm².Supported by a Grant-in-Aid and an Established Investigatorship of the American Heart Association.     

Phenotypic differences between induced and spontaneous 2μ-plasmid-free segregants of Saccharomyces cerevisiae

Summary The maximum specific growth rates (_max) of 2 -plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the _max of their 2 -plasmid-containing ([cir ⁺]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 -based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The _max of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir ⁺] parents. In all cases, however, the induced [cir°] strains had a _max which was significantly less than that of their [cir ⁺] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in _max was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.     

The use of microelectrodes for measurement of local H+ activity in the cortical subarachnoidal space of cats

Summary pH microelectrodes with pointed tip (Hinketype) were constructed for the continuous measurement of the local pH in the perivascular space of pial arteries in the feline cerebral cortex. The sensitive tip had a length of 20–60 and a base diameter of 10–25 . As reference electrode, a micropipette (tip diameter 2 ), filled with 150 mM KCl was used. Calibration curves were linear and showed a sensitivity of 54.5–57.5 mV/pH unit at 38°C. Advantages of such electrodes are the easy penetration of the subarachnoid membrane, the long life span, the quick response, and a minimal drift.—The electrodes were tested in vivo during hyper- and hypoventilation and during local perivascular injection of mock spinal fluid at varying pH. A close correlation was observed between the change in perivascular pH and the corresponding change in pial arterial diameter.A preliminary report of these investigations was presented at the autumn meeting of the German and Austrian Physiological Society, Vienna, Sept. 23–26, 1975, Pflügers Arch., Suppl. R148 (1975)Supported by the Deutsche Forschungsgemeinschaft     

Lectin-ferritin binding on dystrophic and normal retinal pigment epithelial membranes

Summary Phagocytosis in the rat retina is a process which involves uptake of shed photoreceptor outer segments by the overlying retinal pigment epithelium (RPE). In rats with inherited retinal degeneration, there is a defect in phagocytosis. One aspect of this phagocytic defect may be an alteration in glycoconjugate-containing membrane components on RPE membranes which mediate this phagocytic interaction. Lectin binding sites have been studied in order to localize the distribution of glycoconjugates on pigment epithelial microvilli in normal and dystrophic retinas and to determine if there are differences in the dystrophic retinas which would provide a clue about the defect.The following ferritin-conjugated lectins were used in this study: Concanavalin A (Con A-fe) or lens culinaris haemagglutinin (LcH-fe) for mannosyl-containing glycoconjugates; and wheat germ agglutinin (WGA-fe) forn-acetylglucosamine and sialic acid-containing glycoconjugates. Control tissue was incubated in the lectin in the presence of its competitor sugar. The number of ferritin particles or lectin-ferritin binding sites per micrometre of microvillous membrane was quantified from electron micrographs using a computer and a digitizing tablet. The number of WGA-fe binding sites on normal RPE microvillous membranes (56.0/m) was statistically equivalent to the dystrophic membranes (48.8/m). The number of Con A-fe binding sites on normal (27.3/m) and dystrophic RPE (26.7/m) was also the same. A dramatic difference in LcH-fe binding sites was demonstrated on normal (1.5/m) as compared to dystrophic RPE (19.1/m). Our results indicate that more mannosyl residues are accessible on dystrophic microvillous membranes and, based on what is currently known about LcH binding, that these residues belong to glycoconjugates having fucosyl-containing carbohydrate cores. The data also suggest that in normal animals without a phagocytic defect such fucosyl-containing glycoconjugates are not as accessible and may be masked by other sugar residues in the oligosaccharide chain.     

Experimental studies of membrane tethers formed from human neutrophils

Membrane tethers (thin, cylindrical pieces of membrane) have been implicated in the rolling of neutrophils along the endothelium. In our studies, these tethers were formed from passive, stimulated (0.1 M fMLP), and osmotically swollen (170–180 mOsm) human neutrophils; as well as neutrophils treated with 0.3 M latrunculin A to disrupt the cytoskeleton. This tether formation was accomplished by micropipette suction of latex beads coated with antibodies to proteins on the neutrophil membrane surface. From plots of force versus velocity for the tether formation process, we calculated adhesion energies per unit area of the lipid membrane to the cytoskeleton and the viscous resistance (effective viscosity) that occurs during the formation of these tethers at finite velocity. Most of the properties of the neutrophil were altered once it had been treated as described above. We were also able to show mechanical reversibility of membrane tethers, as well as an unexpected formation rate at high tether forces. Since membrane tethers have been implicated in the rolling of neutrophils, then the changes in tether formation may ultimately alter how these cells roll. © 2002 Biomedical Engineering Society. PAC2002: 8716Dg     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

Modulation of bradykinin responses in airway smooth muscle by epithelial enzymes

We have studied the effect of epithelium removal on responses of guinea pig trachea to bradykinin (BK). BK (1 nM–10 M) gave a concentration-dependent relaxation when epithelium was present (E+: EC₅₀=10±3 nM). Epithelium removal resulted in a biphasic response to BK with relaxation at low concentrations (E–: EC₅₀=3.0±1.0 nM) and a recontraction to baseline at higher concentrations (EC₅₀=2.0±1 M). Phosphoramidon (10 M), an inhibitor of neutral endopeptidase (NEP), which cleaves BK into inactive peptides, potentiated relaxation (EC₅₀=1.0±0.9 nM and 0.1±0.1 nM in E+ and E respectively) and contraction in trachea with intact epithelium (EC₅₀=0.08±0.03 M). Inhibition of cyclooxygenase by indomethacin (5 M), inhibited relaxation to BK in E+ tracheal segments, resulting in a slight contraction (EC₅₀=1.0 M), whereas a potent contractile response was observed in E–segments (EC₅₀ 1.6 M, maximal contraction >1 g). In the presence of both indomethacin and phosphoramidon BK caused contraction, even in the presence of epithelium (EC₅₀=0.2±0.11 M), and the response in the absence of epithelium was similar to the response observed in trachea with intact epithelium (EC₅₀=0.25±0.1 M). The contractile effect of BK on airway smooth muscle may be inhibited by a protective role of epithelium, due to release of relaxant prostanoids and by degradation by epithelial NEP. In asthma, bronchoconstrictor responses to BK may be partly explained by loss of airway epithelium.     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1

Summary The circular plasmid pKD1 (or 1.6 m DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology. pKD1 can be transferred to Kluyveromyces lactis where it is replicated stably. Using recombinant molecules derived from pKDl, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2 -based Saccharomyces cerevisiae transformation system.     

Stimulation of protein kinase C activity may increase microvascular permeability to colloidal carbon viaα-isoenzyme

The vasculature of the isolated mesentery and small intestine was perfused with a gelatin-containing physiological salt solution in vitro. Various phorbol-related compounds that are known to have different affinities for the protein kinase C (PKC) isoenzymes, and bradykinin (BK), were tested for their ability to cause the microvascular endothelium to become permeable to injected colloidal carbon (CC). Phorbol 12,13-dibutyrate (PDB), 12-deoxyphorbol 13-phenylacetate (DOPPA), thymeleatoxin (TMX), and resiniferatoxin (RFX), each at a concentration of 1M, were found to increase permeability. Pretreatment with the PKC inhibitor Ro 31–8220 (1M) significantly reduced the response to all of these compounds. Indomethacin (1M), on the other hand, reduced only the effect of RFX. 12-Deoxyphorbol 13-phenylacetate 20-acetate (DOPPAA) (1M) and BK (10M) did not increase CC leakage. These results suggest that the Ca²⁺-dependent PKC-isoenzyme was involved in the increase in endothelial permeability. BK does not appear to stimulate PKC activity in this experimental situation.     

The effect of EMD 57033, a novel cardiotonic agent,on the relaxation of skinned cardiac and skeletal muscle produced by photolysis of diazo-2, a caged calcium chelator

Summary EMD 57033 is thought to produce its potentiating effect by increasing the apparent calcium sensitivity of myofibrils. We have investigated the effect of 10M EMD 57033 on relaxation speed, induced by flash photolysis of 2mM diazo-2 (a caged Ca²⁺ chelator), in skinned semitendinosus frog muscle fibres and guineapig trabeculae. 10M EMD 57033 has no effect on the relaxation speed of semitendinosus fibres. In trabeculae, EMD 57033 slightly increases the relaxation speed slightly, in contrast to ADP which produces a slowing. 1mM ADP combined with 10M EMD 57033 slows relaxation but not to the degree seen with ADP alone. Like ADP, EMD 57033 increases the number of cross-bridges in the force producing state, but unlike ADP does not affect the transition rates involved in relaxation.     

The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at -molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10–100 ms.In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1–3 m internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had to be found in which channels open at a relatively low frequency.     

The analgesic drug buprenorphine inhibits osteoclastic bone resorption in vitro,but is proinflammatory in rat adjuvant arthritis

We have examined the effect of the -opioid analgesic buprenorphine on osteoclastic bone resorption in vitro and in the rat adjuvant arthritis model. In the bone slice assay buprenorphine inhibited osteoclastic bone resorption with an IC₅₀ of 1 M. This effect was not mimicked by the -opioid agonist ([D-Ala,N-Me-Phe, Gly-ol]-enkephalin and was not prevented by the -opioid antagonist naloxone. Since other agents that inhibit osteoclastic bone resorption, such as bisphosphonates and calcitonin prevent bone erosion in the rat adjuvant arthritis model, we also examined the effect of buprenorphine in this model. Surprisingly, buprenorphine exacerbated inflammation measured by paw volume and increased joint destruction assessed by X-ray scores, in the injected paws and particularly in the non-injected paws. These studies also show that attempts to ameliorate animal suffering in this chronic model by using centrally acting analgesics such as buprenorphine may lead to complications in interpreting screening results obtained with novel, potential anti-arthritic compounds.accepted by I. Ahnfelt-Rønne     

Dose-response curve of glutamate applied by superfusion to crayfish muscle synapses

Summary Single muscle fibers were space clamped to a membrane potential of –75 to –80 mV, and the synaptic currents elicited byl-glutamate (gEPSCs) were recorded. The bathing solutions flowing across the fibers at high speed could be switched rapidly and repeatedly through valves actuated by solenoids. Glutamate solutions were applied for periods of 7 s or 1 s, and the responses to repeated applications were averaged. For glutamate concentrations of 10–50 mol/l, applied for 7 s, the gEPSCs reached a steady state. In this concentration range the amplitude of the gEPSC rose steeply proportional to the powern=2.5 ton=6 (average of 12 experimentsn=4.0) of the glutamate concentration. At higher concentrations, after rising for a few seconds the gEPSC was reduced by desensitization. At 500 mol/l glutamate complete desensitization was reached with an approximate time constant of less than 1 s. The glutamate concentration that elicited a half maximum gEPSC wasK=70 mol/l. If glutamate was superfused only for 1 s, similar dose-response curves were observed. In these experimentsn was between 4 and 6. The results obtained by superfusion agree quantitatively with those published for electrophoretic applications.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Pharmacological modulation of plasminogen activator secretion by P388D1 cell line

P388D1 is a murine macrophage cell line which spontaneously secretes plasminogen activator (PA; activated function) and lysozyme (LYS; constitutive function). Compounds which decrease PA secretion without affecting LYS secretion have potential as down-regulators of macrophage function and, hence, of the immune system. Glucocorticoids (e.g., dexamethasone, IC₅₀<0.01 M) and auranofin (IC₅₀=1 M) are positive in this model. In contrast, cyclooxygenase inhibitors (indomethacin, ibuprofen and piroxicam, all at 1 M) boost PA secretion; lipoxygenase inhibitors (REV-5901, NDGA and piriprost, all at 10 M) have little or no effect.Dexamethasone, but not auranofin, induces a urokinase-inhibitory activity which elutes between 0.13 and 0.19M NaCl upon anion exchange HPLC (TSK-DEAE-5-PW). Fibrin overlay following SDS-PAGE of the HPLC peak reveals a urokinase-inhibitory band at 90 Kd.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

A force transducer and a length-ramp generator for mechanical investigations of frog-heart myocytes

An apparatus for studying the mechanics of isolated frog heart myocytes is described. The cells are held horizontal in a through of Ringer solution by means of two suction micropipettes. Myocyte force is measured with an opto-electronic system recording the deflection of the tip of one micropipette, which acts as a cantilever force probe. The force probes are selected for compliance according to the force a myocyte is expected to develop in a given condition, so as to limit myocyte shortening during force development to no more than 1% of the slack cellular length (l ₀). The other micropipette, which is stiff relative to the forces measured, is mounted on an electromagnetic-loudspeaker motor by which controlled-velocity length changes, of preset size and in either direction, are imposed on myocytes. The force transducer has a sensitivity of 5–10 mV/nN, with a frequency response of 700–900 Hz in Ringer solution and a resolution of 0.5–1 nN. The motor with a suction micropipette can complete controlled-velocity length ramps within 1.5–2.0 ms, across a range of ±100 m at a resolution of 8.0 nm. These values correspond, for frog-heart myocytes 200 m and 400 m long, to 25%–50% l ₀ and 0.002%–0.004% l ₀ respectively.     

Contractile properties and ultrastructure of three types of muscle fibre in the dogfish myotome

Summary Three main types of fibre can be differentiated in the adult dogfish myotome at the immediate post-anal level. An outer band of muscle consists of 80–90 pale multiply innervated fibres (superficial fibres). These fibres are 80–90 m in diameter, lack M-lines and have a low Ca²⁺-activated myosin ATPase activity. Volume densities of myofibrils (V_v(my,f)) and mitochondria (V_v(mt,f)) are respectively 76 and 9.5%. Beneath this layer are around 8000 red multiply innervated fibres. These have an average diameter of 25–40 m. V_v(my,f) and V_v(mt,f) are 62 and 21% respectively, and M-lines are present. Around 11000 white focally innervated twitch fibres lie beneath the red fibre zone. White fibres with an average diameter of 80–120 m have a high Ca²⁺-activated myosin ATPase activity and V_v(my,f) and V_v(mt,f) are 78 and 5% respectively.Contractile properties of single skinned fibres were determined at 12° C. Maximum Ca²⁺ activated tensions (kN m^–2) and unloaded contraction speeds (muscle lengths s^–1) were 49 and 0.5 for superficial, 70 and 1.4 for red and 180 and 4.4 for white muscle fibres.Superficial fibres have not been reported in other elasmobranchs with the exception of the closely related nursehound (Scyliorhinus stellaris L.) It is suggested that they are specialized for sustained force generation, having a tonic (postural) rather than a locomotor role.