Calcium binding in chemically skinned fibers of rat myocardium during force development

Summary The amout of bound calcium and force were measured in chemically skinned rat myocardial fibers during isometric contraction at different concentrations of free Ca²⁺. The data obtained suggest that calcium binding by cardiac myofibrils is cooperative and probably depends on mechanical tension.The author expresses his gratitude to Dr V. I. Kapelko for valuable discussion of the experimental results.     

Effect of high doses of somatostatin on adenylate cyclase activity in peripheral mononuclear leukocytes from normal subjects and from acute leukemia patients

Summary In normal lymphocytes somatostatin non-competitively inhibited basal (ID₅₀ 5×10^–4 M) and isoproterenol- and forskolin-stimulated adenylate cyclase activity (Ac). In acute leukemia blasts, non-responsive to isoproterenol, forskolin, which activates the catalytic subunit, stimulated and somatostatin inhibited Ac, thus indicating the leukemic enzyme, though defective, retains the inhibitory pathway and catalyst function.     

Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity

The adenylate cyclase activator forskolin and its pharmacologically inactive derivative 1,9-dideoxyforskolin were found to inhibit in a dose-dependent fashion the ecdysone 20-monooxygenase activity associated with wandering stage larvae ofDrosophila melanogaster and fat body and midgut from last instar larvae of the tobacco hornworm,Manduca sexta. The concentrations of these labdane diterpenes required to elicit a 50% inhibition of the cytochrome P-450 dependent steroid hydroxylase activity in the insect tissues ranged from approximately 5×10^–6 to 5×10^–4 M.     

Die Wirkung von Theophyllin auf den cellulären Ca-Stoffwechsel des Warmblüterherzens

Summary In isolated, electrically driven, left guineapig atria, theophylline (5×10^–4 g/ml) increased the rate of⁴⁵Ca uptake and release without affecting the total myocardial Ca content and the amount of exchangeable cellular Ca. In sheep and calf heart preparations, theophylline (10^–4–10^–3 g/ml) increased Ca inward current during excitation (as examined indirectly by Ca dependent changes of membrane potential in TTX-containing solutions) as well as tension development. It is concluded that the positive inotropic effect of theophylline in mammalian hearts is due to an increase in Ca influx during the excitation process.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

Summary The contraction induced by a Ca²⁺-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (F_o), immediate elastic recoil (SE), unloaded shortening velocity (V_us), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca²⁺-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. F_o developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. V_us in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than V_us induced by Ca²⁺ (1.6x10^–6M) in the control fibers. Addition of Ca²⁺ (1.6x10^–6M) to a contraction induced by MLCK-resulted in small increases in both F_o and V_us. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca²⁺. We conclude that with respect to actin-myosin interaction, MLCK- and Ca²⁺-induced contractions are similar.     

Low temperature-induced contracture of depolarized smooth muscle and the effects of calcium and multivalent cations

Summary Lowering of temperature caused tension development in the smooth muscle of the guinea-pig vas deferens, which was dependent on extracellular Ca. Mn²⁺ and La³⁺ reversed the effect and induced phasic contraction on rewarming.Part of the results were presented at the 14th Int. Congress of Physiological Science, Budapest 1980.     

Decrease of mRNA levels and biosynthesis of sucrase-isomaltase but not dipeptidylpeptidase IV in forskolin or monensin-treated Caco-2 cells

Treatment for 48 h of differentiated, confluent Caco-2 cells with 2.5 10^?5 M forskolin or 10^?6 M monensin, which produces a significant decrease of the de novo biosynthesis of sucrase-isomaltase, does not change quantitatively the de novo biosynthesis of dipeptidylpeptidase IV. Western blot analysis and silver nitrate staining indicate that neither drug induces any modification in the steady state expression of these two brush border hydrolases. Northern blot analysis shows that the level of dipeptidylpeptidase IV mRNA does not change in treated as compared to control Caco-2 cells. In contrast, forskolin and monensin dramatically decrease the level of sucrase-isomaltase mRNA. These observations suggest a separate regulation of biosynthesis for sucrase-isomaltase and dipeptidylpeptidase IV in intestinal cells. The mechanisms responsible for such a difference are discussed. Among them, the role of glucose metabolism, which is perturbed by both drugs, appears to be of crucial importance.     

Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.     

Differential sensitivity of amphibian nodal and paranodal K+ channels to 4-aminopyridine and TEA

Summary Voltage-dependent K⁺ channels are blocked by several drugs, including 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP is most widely used to localize K⁺ channels in mammalian and non-mammalian nerve fibers, but 4-AP and TEA alter various K⁺ channels and/or preparations in specific ways. The reason is not known, in part because dissociation constants for 4-AP and TEA have not been measured for nodal and internodal K⁺ channels in the same fibers. Smith and Schauf showed that the density of nodal versus paranodal K⁺ channels in frog nerves depends on fiber diameter. This size dependence was used to determine the relative sensitivity of nodal and internodal K⁺ channels to 4-AP and TEA, and to compare voltage- and time-dependent activation. The results show nodal and internodal K⁺ channels activate similarly. However, internodal channels are selectivity blocked by 4-AP while TEA is more effective on nodal channels. A high sensitivity of internodal K⁺ channels may explain why 4-AP improves symptoms in diseases such as multiple sclerosis.     

Zur Wirkung des Calciums auf die Myofibrillen-ATPase des Herzmuskels

Summary The inhibition of cardiac myofibrillar ATPase activity by EDTA can be completely reversed by Ca⁺⁺ under certain experimental conditions. This shows that there is no fundamental difference between the reaction of cardiac myofibrillar ATPase and the tension development of glycerinated cardiac fibres, as was supposed byBriggs andHannah on account of the results ofParker andBerger.     

Evidence for cross bridge slippage in a stretched muscle fibre

Summary Bundles of glycerol-extracted psoas fibres which were contracted by immersion in a saline containing 15 mM MgATP and 12 M free Ca⁺⁺ were subjected to up to 3 stretches (rise time 0.8 msec) each of amplitude 1% L_i at intervals of 10 msec. The elastic tension responses to these stretches were all of comparable size and the peak tensions reached during the stretches were in each case followed by a rapid tension decline almost to the tension values before the stretches. This indicates that stretch-induced detachment and reattachment of cross bridges to the actin filament occurred within 10 msec (slippage).Supported by the Deutsche Forschungsgemeinschaft, grant Ru 154/12. The editorial assistance of Mr D.J. Williams is gratefully acknowledged.     

The conduction velocity,number and diameter of unmyelinated fibers in Remak's nerve

Summary Remak's nerve in the chicken was examined ultrastructurally and electrophysiologically to determine the characteristics of fibers in the nerve trunk. The ratio of unmyelinated fibers to myelinated ones was 1111. The mean number of unmyelinated fibers was 3555±232 (SEM, n=5) and they had a mean diameter of 0.502±0.034 (SEM) m. The compound action potential consisted almost entirely of a large diphasic waveform which had a mean peak conduction velocity of 0.62±0.031 (SEM, n=5) m·s^–1 at 37°C.     

Differential vascular effects of neuropeptide Y(NPY) selective receptor agonists

Neuropeptide Y (NPY) increases blood pressure either directly or indirectly by potentiating the effect of various vasoconstrictors. Only one (the Y₁-receptor) of two subtypes of receptors (Y₁ and Y₂) is thought to mediate the vascular smooth muscle contraction. To test this hypothesis we challenged isolated rat mesenteric arteries that had a functional endothelium with (1–36) NPY and with specific Y₁-receptor ([Leu³¹, Pro³⁴] NPY) and Y₂-receptor ([Ahx^5–24, -Glu²--Lys³⁰] NPY) agonists. The Y₁-receptor agonist elicited a contractile response similar to that of NPY, whereas the Y₂-receptor agonist had no effect on wall tension. We also found that the presence of a functional endothelium has no influence on the contractile response to NPY. From these data we conclude that the direct contractile effect of NPY in the mesenteric artery is mediated by stimulation of Y₁-receptors and is not endothelium-dependent.     

Effect of norepinephrine and vasopressin on carotid sinus baroreceptor activity in the anesthetized rabbit

Summary Norepinephrine (NE) 10^–6 M or vasopressin (VP) 12.5 U/ml were injected into the isolated carotid sinus of anesthetized rabbits. The sinus was exposed either to the arterial pressure or to a pressure controlled reservoir. Multifiber and single fiber recordings were made. Both NE and VP increased baroreceptor activity at all sinus pressures but decreased activity in a few fibers. The results are consistent with the drugs having their effects on smooth muscle fibers in the adventitia.Acknowledgments. This work was supported by grants from the Medical Research Council of Canada and the B.C. Heart Foundation. The authors are grateful to J. Sharp and D. Morton for their excellent assistance.     

Blockade by zinc ions of K+-induced contraction and calcium in guinea pigTaenia coli

Preincubation with 0.3 mM Zn²⁺ markedly inhibited both the tonic response and Ca²⁺ binding at low affinity sites induced by K⁺ (60 mM), with smaller effects on the phasic response and the high affinity Ca²⁺ sites, inTaenia coli. However, when the muscle was kept in Zn²⁺-containing medium following the first stimulation with the K⁺, the phasic response and the high affinity Ca²⁺ sites were more severely inhibited during the second stimulation with K⁺. This probably indicates that Zn²⁺ reduced the tonic tension response to K⁺ mainly by inhibiting Ca²⁺ influx at the cell membranes ofTaenia coli. However, when Zn²⁺ is continuously present, Ca²⁺ is not supplied at the storage sites and is not available for the phasic response to a second stimulation with K⁺.     

Frequency-dependent effect of verapamil on rat soleus muscle

Summary In the presence of verapamil (0.1 mM) rat soleus muscle fibers failed to generate action potentials with overshoots. In fibers with their V_m set to a local level of –90 mV, verapamil produces a gradual reduction in the amplitude of the repetitive action potentials; this effect is more pronounced at high rates of stimulation (100 Hz). Our results suggest a local anesthetic action of this drug that could contribute with its calcium channel blocking effect to the diminished mechanical tension observed in the presence of the drug.Acknowledgments. We thank A. Losavio and M. Stefanolo for technical assistance. This work was supported by grants from CONICET and SUBCYT, Buenos Aires, Argentina and Muscular Dystrophy Association, USA.     

Effect of phosphorylation of myosin light chains on interaction of heavy meromyosin with regulated F-actin in ghost fibers

Summary The binding of phosphorylated heavy meromyosin to regulated F-actin in ghost fibers at high Ca²⁺ concentration increases, and at low Ca²⁺ concentration decreases, the anisotropy of intrinsic tryptophan fluorescence of F-actin. The effect is opposite to the effect of the binding of dephosphorylated heavy meromyosin.     

Zur Wirkung von Oxytocin auf einen autorhythmischen glatten Gefässmuskel

Summary The influence of oxytocine on the spontaneously contractile portal vein of the rat has been investigated. This octapeptide decreases the active tension and abolished it completely at a concentration of 5×10^–7g/ml. The effect is not caused by an adrenergic mechanism but by a direct action on the smooth muscle cell.     

Effect of metabolic versus respiratory acid-base changes on isolated coronary artery and saphenous vein

Summary Experiments were performed on helically cut strips from coronary artery and saphenous vein to determine the relative influence of metabolic versus respiratory acid-base changes. Tensions were measured over a range of various HCO₃ ^– concentrations and pCO₂'s. The results suggest that tension is influenced by extracellular pH and is independent of pCO₂.Supported by USPHS grant No. HL24232.     

Skinned smooth muscle: Time course of force and ATPase activity during contraction cycle

Summary The time course of ATPase activity and force has been determined during contraction and relaxation in skinned (hyperpermeable) anterior byssus retractor muscle, ABRM, ofMytilus edulis and compared with corresponding measurements on skinned taenia coli of guinea-pigs. Following a calcium-induced contraction, lowering the [Ca⁺⁺] to 10^–8 M rapidly reduces ATPase activity within 2 min to resting levels while force declines only to about 30–50% of maximal tension within the same time. Thus slow relaxation is due to a catch-like-state which is common to different kinds of smooth muscles and can be reduced with cAMP in ABRM and by P_i in taenia coli.The support by the Deutsche Forschungsgemeinschaft and the excellent technical assistance of Mrs K. Winnikes are gratefully acknowledged.