Regulation of cross-bridge detachment by Ca ions at high ionic strength in molluscan catch muscle

Changes in the profile of equatorial intensities of X-ray diffraction from an intact, anterior byssal retractor muscle (ABRM) of Mytilus were examined at rest, during contracture brought about by acetylcholine (ACh) and a subsequent rigor-like contraction caused by raising the tonicity of the external solution, and after returning the tonicity to normal. The results suggest that the cross-bridges formed between thick and thin actin filaments during the ACh-contracture were maintained in the hypertonic solution and broken on decreasing the tonicity before the recovery of spacing of the actin filament lattice. A similar rigor-like contraction was induced in glycerinated ABRM by increasing salt concentration during active contraction. The rigor-like force declined rapidly when Ca++ concentration decreased. The results suggest that the detachment of the cross-bridge from the actin filament is regulated by Ca++ at high ionic strength in the ABRM.     

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.     

The effect of nifedipine and verapamil on KCl-induced rhythmic contractions of guinea pig ureter in vitro

Summary Addition of KCl (40 mM) produced rhythmic contractions of guinea-pig ureters in vitro which were unaffected by phentolamine, atropine or tetrodotoxin.KCl failed to elicit rhythmic contractions of ureters incubated in a Krebs solution with no added Ca⁺⁺; in these conditions the addition of CaCl₂ in concentrations of 1.5 mM, or higher, produced rhythmic contractions whose frequency, but not amplitude, was proportional to CaCl₂ concentration in the bathing medium.EDTA reduced the frequency of KCl-induced rhythmic contractions without affecting their amplitude. Nifedipine and verapamil reduced both the frequency and the amplitude of KCl-induced rhythmic contraction; verapamil was more effective than nifedipine in reducing their amplitude.Urethane reduced the amplitude without significantly affecting the frequency of KCl-induced rhythmic contractions. An increase in the extracellular Ca⁺⁺ concentration reverted the suppressive effect of all drugs under study. These results suggest that an influx of Ca⁺⁺ from the extracellular space is responsible for the initiation of KCl-induced rhythmic contractions and is involved in the mechanism(s) which regulates their frequency, but that a separate mechanism regulates their amplitude.     

Influence of ethanol on calcium,inositol phospholipids and intracellular signalling mechanisms

Summary Studies have implicated Ca⁺⁺ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca⁺⁺ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca⁺⁺/Mg⁺⁺-ATPase, the high affinity membrane Ca⁺⁺ pump.Current research suggests that a subtype of the voltage-dependent Ca⁺⁺ channel, the dihydropyridine-sensitive Ca⁺⁺ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases⁴⁵Ca⁺⁺-influx and [³H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca⁺⁺/Mg⁺⁺-ATPase activity in neuronal membranes. Changes in Ca⁺⁺ channel and Ca⁺⁺/Mg⁺⁺-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca⁺⁺ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca⁺⁺/Mg⁺⁺-ATPase activity.The increased sensitivity of three Ca⁺⁺-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Successful cryopreservation of auricle fragments from rat hearts at −196°C

Summary The influence of electrolyte composition and glucose concentration of a cryprotective medium on the survival of auricle fragments from adult rat hearts after storage at –196°C was investigated. Using a K⁺-, Mg⁺⁺-, Ca⁺⁺-rich solution with increased glucose concentration, a high rate of surviving fragments was found after cryopreservation.     

Ambiguous effect of caffeine upon the transmembrane Ca current in mammalian ventricular myocardium

Zusammenfassung Coffein (20 mM) wirkt auf den transmembranären Ca⁺⁺-Strom der Warmblütermyokardfaser biphasisch. Zunächst nimmt der Ca⁺⁺-Strom zu, was auf einer Verbesserung der Ca⁺⁺-Leifähigkeit des langsamen Membrankanals beruht. Nach längerer Einwirkungsdauer von Coffein kommt es dagegen zu einer Abnahme des Ca⁺⁺-Stroms. Als Ursache hierfür wird vor allem eine Verminderung des transmembranären Konzentrationsgradienten für Ca⁺⁺ — infolge eines Anstiegs der freien Ca⁺⁺-Konzentration im Zellinnern — postuliert.     

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.     

Influence of bistramide A on the twitch tension in rat atrial heart muscle

Bistramide A, a new toxin isolated from the UrochordateLissoclinum bistratum Sluiter, was applied to rat auricular heart muscle bundles. At a stimulation frequency of 0.2 Hz, the toxin induces a dose-dependent reduction of the stimulated twitch tension force; it decreases and shortens the duration of the plateau and the slow repolarizing phase of the action potential. In the control solution, switching from a stimulation frequency of 0.2 Hz to 1 Hz decreases the force with which a positive potentiation develops either at a maintained high frequency or after switching from 1 Hz to 0.2 Hz. Bistramide A reduces both the force evoked at 1 Hz and the potentiation. The data suggest that Bistramide A blocks Na⁺ conductance; inhibits Ca⁺⁺ channels in a time-and frequency-dependent manner; reduces Na⁺–Ca⁺⁺ exchange activity; but does not modify the ability of the sarcoplasmic reticulum to be refilled although the rate of Ca⁺⁺ accumulation is decreased.     

Hyposmotic shock-induced discharge in acontia ofCalliactis parasitica is blocked by gadolinium

On acontia ofCalliactis parasitica it was observed that mechanical stimuli applied by a gelatin probe, a method effective in tentacles of Anthozoa, do not induce the discharge of nematocytes. Hyposmotic shock, performed by treatment with NaCl solution 35% hyposmotic with respect to sea water, induces, in the presence of Ca²⁺, the discharge that spreads along the acontial filament, as previously observed following treatment with SCN^–. The hyposmotic shock-induced discharge is blocked by Gd³⁺ at a concentration of 1 M. 10 M Gd³⁺ prevents also the SCN^–-induced discharge. These results suggest the presence of stretch activated cation channels either in nematocytes and/or in supporting cells as well as a possible effect of SCN^– on this class of ion channels.     

The affinity of mitochondria for Ca++

Riassunto E' stata studiata l'affinità dei mitocondri di fegato di ratto e di cuore di coniglio per il Ca⁺⁺, nel sistema dipendente da energia. Si sono usati tamponi EGTA-Ca⁺⁺ per mantenere la concentrazione del Ca⁺⁺ costante durante l'esperimento. Si è dimostrato che laK _m apparente per il Ca⁺⁺ non è lontana da 10^–6 M.

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We are indebted to Dr.B. Chance for many stimulating discussions and for facilitating the progress of the work with the generous hospitality in his Institute.     

Phosphorylation of cockroach antennal polypeptides: effects of second messengers and pheromonal blend

In insect antennal extracts, Schleicher et al.¹ showed that protein kinase C (PKC) inhibitors abolish the transience of pheromone-induced rapid inositol trisphosphate responses, which suggests that pheromonal signals act on phosphorylation of specific proteins. To confirm this hypothesis, we studied the effects of second messengers and a pheromonal blend on phosphorylation of antennal proteins in the cockroachPeriplaneta americana. Proteins from adult male antennae were phosphorylated in vitro in the presence of [³²P] triphosphate, then separated by SDS-polyacrylamide gel electrophoresis. Numerous phosphopolypeptides were visualized. The presence of Ca⁺⁺/calmodulin in the incubation medium resulted in increased phosphorylation of polypeptides with molecular weights of 38, 48, 51, 54 and 58 kDa. Stimulation of PKC by addition of Ca⁺⁺ phosphatidylserine (PS)/phorbol myristate acetate (PMA) resulted in the appearance of three phosphopolypeptides of 36, 70 and 120 kDa. In the presence of cyclic adenosine monophosphate, two new major polypeptides of 46 and 42 kDa appeared; the latter polypeptide also appeared in the presence of cyclic guanosine monophosphate. Comparison with polypeptide composition of tissue from the cerci, leg, brain and fat body showed that the 36 and 48 kDa polypeptides were specific to antennae, whereas the 120 kDa polypeptide was also present in the adult brain. When antennae are subjected to pheromonal stimulation for 16 seconds prior to homogenization, in vitro phosphorylation of the 120, 70, 64 and 38 kDa polypeptides was inhibited, whereas phosphorylation of the 58, 54, 51 and 48 kDa polypeptides was strongly stimulated. It is noteworthy that a 107 kDa polypeptide was observed only after pheromonal stimulation by Ca⁺⁺/PS/PMA. Our findings suggest that Ca⁺⁺-and PKC-dependent protein phosphorylation systems play an important role in the transduction of pheromonal signals in antennae of male cockroachP. americana. We speculate that specific phosphoproteins may modulate sensitivity and signal amplification during the olfactory transduction process.     

Sensitivity of the developing chick myocardium to the positive inotropic effects of calcium and isoproterenol

Summary The present results show that the sensitivity of the chick myocardium to the positive motropic effect of Ca⁺⁺ decreases during development and that the Ca⁺⁺ concentration of the physiological solution used must be lowered below normal to study the effects of positive inotropic agents in preparations from younger embryos. Isoproterenol elicits positive inotropic responses in 7–9-day embryonic ventricle and in newborn chick atria; however, the 4-day embryonic myocardium is unresponsive to isoproterenol.This work was supported by Grant No. HL-15995 from the National Heart Institute (USPHS).The authors would like to thank Dr.F. E. Shideman for the isolated tissue baths used in these experiments.     

Unterschiede zwischen Tyramin und Dimethylphenylpiperazin in der Ca++-Abhängigkeit und im zeitlichen Verlauf der Noradrenalin-Freisetzung am isolierten Kaninchenherzen

Summary On the perfused rabbit heart a constant infusion of tyramine released noradrenaline continuously and independently of the external Ca⁺⁺ concentration. In contrast, noradrenaline release by DMPP was only transient and required the presence of Ca⁺⁺.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Studies of total and protein-bound plasma Mg++ in wild and hatchery-reared coho salmon smolts in freshwater and in seawater

Summary Total plasma Mg⁺⁺ and Ca⁺⁺, Mg⁺⁺ in erythrocytes as well as protein-bound plasma Mg⁺⁺ were investigated in wild and hatchery-reared smolts. The proportion of plasma Mg⁺⁺ which was bound to plasma protein did not change significantly during entry into seawater, even though the in vitro addition of exogenous Mg⁺⁺ to the plasma showed that additional binding was possible.     

Possible role of intestinal alkaline phosphatase activity in thiamine transport

Summary Thiamine deficiency caused a marked decrease of intestinal alkaline phosphatase (al-Pase) activity, but had no effect on the Ca⁺⁺-ATPase activity and Ca⁺⁺-absorption in rats. The al-Pase activity was significantly decreased 1 h after oral administration of ethanol at 0.5 and 2.5 g/kg. In contrast, Mg⁺⁺-, Ca⁺⁺- and (Na⁺+K⁺)-ATPase activities did not change after the administration of ethanol. These findings show that the al-Pase activity, unlike the Ca⁺⁺-ATPase activity, is not related to Ca⁺⁺-absorption. A possible role of al-Pase activity in the active transport of thiamine in the intestine was discussed.     

The effects of the ‘calcium-antagonist’, prenylamine,on the action potential of crayfish muscle (Oronectes virilis)

Zusammenfassung Der organische Ca⁺⁺-Antagonist Prenylamin blockiert die leicht reversiblen Ca⁺⁺-Aktions-potentiale in der Muskelfaser des FlusskrebsesOronectes virilis. Etwas niedrigere Konzentrationen erhöhen die Schwelle und verlängern die Dauer, während schwache Konzentrationen von Prenylamin nur wirksam waren, wenn dieses verwendet wurde, bevor der Muskel dem Aktionspotentialerreger Procain oder Sr⁺⁺ ausgesetzt war.     

Die Abhängigkeit der Ca++-Aufnahme isolierter Mitochondrien des Herzmuskels von der Na+-und K+-Konzentration als mögliche Ursache der inotropen Digitaliswirkung

Summary In isolated mitochondria of heart muscle from rabbits and oxen there is, under suitable conditions, an accumulation of Ca⁺⁺, which is significantly enhanced by elevating the K⁺/Na⁺ quotient of the incubation medium. K-strophanthine (10^–5–10^–7) does not influence the accumulation of Ca⁺⁺ by the mitochondria of heart muscle. Therefore the intracellular increase in exchangeable Ca⁺⁺ observed after digitalis-glycosides could be explained by a decrease of the intracellular K⁺/Na⁺ quotient, which is caused by inhibition of the membrane ATPase and diminishes the capacity for Ca⁺⁺ accumulation in mitochondria.     

Cell-to-cell transmission in the activation of in situ nematocytes in acontia ofCalliactis parasitica

Summary It is reported that Ca²⁺-induced discharge of in situ nematocytes of acontia ofCalliactis parasitica can occur by cell-to-cell transmission along the acontial filament at a speed that averages 9.8·10^–3 cm^–1. The discharge is preceded by protrusion of nematocytes that proceeds along the acontium at a slightly higher speed.     

A specific endogenous inhibitor of two forms of Ca++ activated neutral proteases in platelets

Summary An endogenous, specific inhibitor of high molecular weight has been isolated from bovine blood platelets, which inhibits the activity of the 2 forms of platelet Ca⁺⁺ activated neutral proteases reported previously by us. The inhibition is not due to chelation of Ca⁺⁺ but results from a stoichiometric complex formation.     

Nitroglycerin inhibits the phosphorylation of intermediate filament proteins rather than myosin light chain on porcine coronary artery sustained contraction

The smooth muscle relaxation induced by nitroglycerin is hypothesized to be mediated by an increase in the cytoplasmic concentration of guanosine 3′,5′-monophosphate (cGMP) and subsequent dephosphorylation of the 20-kilodalton myosin light chain (MLC). We investigated this hypothesis in procine coronary arterial smooth muscle stimulated with histamine (3 μM) or K⁺ (30 mM). Stimulation of [³²P]Pi-labeled muscle with histamine or K⁺ for 2 min resulted in a four- or 6.2-fold increase, respectively, in the incorporation of³²P into MLC. After 48 min of exposure to histamine. MLC phosphorylation decreased to the basal level and the phosphorylation of desmin, synemin, and of three unidentified cytosolic proteins was increased. K⁺ stimulation resulted in a sustained increase of MLC phosphorylation but had no effect on the phosphorylation of desmin, synemin, or the three unidentified cytosolic proteins. Application of nitroglycerin (1 μM) 48 min after histamine stimulation inhibited the phosphorylation of desmin, synemin, and the three cytosolic proteins. The sustained phase of histamine-induced contraction was also inhibited to a greater extent then the acute phase of histamine-induced contraction and both the acute and sustained phases of K⁺-induced contraction. These results suggest that MLC phosphorylation is required for both phases of K⁺-induced contraction, whereas phosphorylation of intermediate filament proteins is required for the sustained phase of histamine-induced contraction. Intermediate filament proteins, rather than MLC, may also be the target for the relaxant action of nitroglycerin during histamine-induced sustained contraction.