Optimized administration regimen of lopinavir for a myocardial ischaemia reperfusion study in Sprague-Dawley rats.

Pharmacokinetics of drugs may differ between small and large mammals (including humans); therefore, drug testing in animal models must be carefully designed. Sprague-Dawley rats were used in cardiac experiments, during which the lopinavir concentration in serum had to match human therapeutic levels (4-10 µg/mL). Lopinavir was administered as a co-formulated drug of lopinavir and ritonavir. It was found that after a single administration of a standard human peroral dose (lopinavir 13.3 mg/kg of body weight), the serum concentration of lopinavir was only one-tenth of the target level. It remained below the minimum target level even after 10-fold the standard dose was administered. After initial pilot tests, a dose escalation study was conducted with oral doses 10- and 15-fold the standard clinical dose of lopinavir (i.e. 133 and 200 mg/kg, respectively). A second administration 2 h later effectively increased and maintained higher concentrations during the experimental ischaemia and reperfusion periods. A dose-dependent increase in serum concentration of the drug was observed. Thus, the target therapeutic serum level of lopinavir in the rats was achieved by administrating 10- to 15-fold the standard human dose twice, separated by a 2 h interval.

[Phalloidin suppresses the force in nebulin-rich lamprey cardiac muscle].

The effect of phalloidin, an agent detaching nebulin from actin in skeletal muscle, on the isometric force in lamprey skinned cardiac muscle, which has nebulin in amounts comparable to that in skeletal muscle, has been studied. We found that, unlike mammalian cardiac muscle expressing nebulin less abundantly and responding to phalloidin by a force increase, lamprey cardiac muscle responds to phalloidin by a force decrease (approximately 50% decrease), thereby resembling the response of skeletal muscle. These results support our hypothesis that nebulin detachment from actin underlies phalloidin-induced force loss and suggest a role of actin-nebulin interaction in contractile function.

[Phallotoxins as an instrument for studying the intramyofibrillar regulation of muscle contraction]. / Fallotoksiny kak instrument dlia issledovaniia vnutrimiofibrilliarnoi reguliatsii myshechnogo sokrashcheniia.

The contraction of myofibrils is regulated by the coordinated action of Ca2+ and myosin cross-bridges. Phallotoxins induce a variety of specific changes in myofibrillar functioning and are therefore a potentially valuable tool for muscle research. There are two greatly differing classes of drugs among phallotoxins: (1) recently discovered secophalloidin and its derivatives. The unique property of secophalloidin is muscle activation without Ca2+, possibly by direct influence on actomyosin interaction. These drugs seem to be especially useful for studying the role of cross-bridges in muscle regulation; (2) the phalloidin group, which includes the majority of phallotoxins. When binding to the phalloidin site on F-actin, they cause muscle-specific changes. In cardiac muscle they work as Ca2+ sensitizers, increasing both the maximal force and Ca2+ sensitivity. An advantage of these drugs is that the target site is known, which allows one to unravel the sequence of molecular events leading to increased contractile function. Presumably, the complex effect of phalloidin in skeletal muscle is related to the disturbance of the actin-nebulin interaction, which may help to clarify the role of nebulin in the regulation contraction.

Different myofilament nearest-neighbor interactions have distinctive effects on contractile behavior.

Cooperativity in contractile behavior of myofilament systems almost assuredly arises because of interactions between neighboring sites. These interactions may be of different kinds. Tropomyosin thin-filament regulatory units may have neighbors in steric blocking positions (off) or steric permissive positions (on). The position of these neighbors influence the tendency for the regulatory unit to assume the on or off state. Likewise, the tendency of a myosin cross-bridge to achieve a force-bearing state may be influenced by whether neighboring cross-bridges are in force-bearing states. Also, a cross-bridge in the force-bearing state may influence the tendency of a regulatory unit to enter the on state. We used a mathematical model to examine the influence of each of these three kinds of neighbor interactions on the steady-state force-pCa relation and on the dynamic force redevelopment process. Each neighbor interaction was unique in its effects on maximal Ca(2+)-activated force, position, and symmetry of the force-pCa curve and on the Hill coefficient. Also, each neighbor interaction had a distinctive effect on the time course of force development as assessed by its rate coefficient, k(dev). These diverse effects suggest that variations in all three kinds of nearest-neighbor interactions may be responsible for a wide variety of currently unexplained observations of myofilament contractile behavior.

Secophalloidin and phalloidin-(S)-sulfoxide as contraction modifiers for comparative study of skeletal and cardiac muscles.

Phalloidin, a toxic product of the mushroom Amanita phalloides, binds specifically to F-actin resulting in strong stabilization of F-actin structure (for review, see; Wieland, 1986). Binding to a specific site on the muscle thin filament F-actin, phalloidin modifies contraction in a tissue specific manner. Phalloidin induced changes depend on functionally important parameters (thin filament activation, cross-bridge kinetics), indicating changes in essential steps of the contractile mechanism. Moreover, there is a different action with different phalloidin derivatives. Such properties make phallotoxins (phalloidin and its derivatives) powerful modifiers for muscle research (for review, see: Bukatina, 1996). Phalloidin-induced changes vary qualitatively with muscle types. In all types of skinned skeletal muscle preparations that have been studied (fast and slow muscles from evolutionarily distant animals), the most general effect of phalloidin is to cause a decrease in tension (Bukatina, Morozov, 1979; Alievskaya et al., 1987; Bukatina et al., 1993). In mammalian skeletal muscles, this decrease in tension may be followed by a slowly developing increase in tension. The resulting tension may considerably exceed the tension before phalloidin administration. In contrast, skinned cardiac muscle responds to phalloidin only by increasing isometric tension from the onset of the response. Moreover, the phalloidin response is completed in approximately one-tenth the time in cardiac muscle that it takes in skeletal muscle. These phalloidin effects in cardiac muscle result in an enhanced Ca2+ responsiveness (Boels, Pfitzer, 1992) with an increase in both the force at maximum Ca2+ activation and the Ca2+ sensitivity (Bukatina et al., 1995).

Stiffness-distortion sarcomere model for muscle simulation.

A relatively simple method is presented for incorporating cross-bridge mechanisms into a muscle model. The method is based on representing force in a half sarcomere as the product of the stiffness of all parallel cross bridges and their average distortion. Differential equations for sarcomeric stiffness are derived from a three-state kinetic scheme for the cross-bridge cycle. Differential equations for average distortion are derived from a distortional balance that accounts for distortion entering and leaving due to cross-bridge cycling and for distortion imposed by shearing motion between thick and thin filaments. The distortion equations are unique and enable sarcomere mechanodynamics to be described by only a few ordinary differential equations. Model predictions of small-amplitude step and sinusoidal responses agreed well with previously described experimental results and allowed unique interpretations to be made of various response components. Similarly good results were obtained for model reproductions of force-velocity and large-amplitude step and ramp responses. The model allowed reasonable predictions of contractile behavior by taking into account what is understood to be basic muscle contractile mechanisms.

Dethiophalloidin increases Ca2+ responsiveness of skinned cardiac muscle.

Phalloidin, an F-actin stabilizing peptide, is known to enhance Ca2+ responsiveness in skinned cardiac muscle. Here we studied the effects of dethiophalloidin (DTPH), a phalloidin derivative which, binding much more weakly to F-actin, on skinned bovine left ventricle muscle. When added to activated skinned muscle, DTPH (15-80 micron), similarly to phalloidin, caused a rapid (within several minutes) enhancement of active force; the relative force enhancement by DTPH became greater as Ca2+ concentration was decreased. Unlike phalloidin, DTPH effects were reversible. Using a value of the force enhancement at 15 micron DTPH (76% of maximum), an apparent equilibrium constant for DTPH binding to myofilaments was estimated at about 5 micron. Force-pCa plots showed that DTPH (80 micron) brought about a 10% increase in the maximal Ca(2+)-activated force and a 0.34 pCa units increase in the Ca2+ sensitivity. Both changes are stronger than those caused by phalloidin in similar conditions (6% and 0.2 pCa units, respectively). As with phalloidin, DTPH did not change the value of the Hill coefficient in the fit tothe force-pCa curve. DTPH and phalloidin interacted as follows: (1) pre-treatment with phalloidin entirely prevented the response to DTPH, indicating the absence of any non-specific DTPH action; and (2) when added after DTPH, phalloidin decreased the force enhancement due to DTPH, reflecting a stronger effect of DTPH to increase force. In conclusion, the stabilization of F-actin structure is not a major factor in the mechanism by which phalloidin modifies contraction.

A study on the mechanism of phalloidin-induced tension changes in skinned rabbit psoas muscle fibres.

The time course of phalloidin induced changes in isometric tension of partially activated skinned rabbit psoas fibres was studied as a function of both phalloidin concentration and time of pre-incubation with phalloidin. Upon addition of phalloidin to non-pretreated (control) fibres there was a fall in tension followed by an increase in tension. The latency of both parts of the response was inversely related to the phalloidin concentration in the range 40-130 microM phalloidin. By preincubating the fibres with phalloidin for varying periods of time it was possible to obtain responses which appeared to represent later portions of the control response. Thus after pre-treatment with 40 microM phalloidin in either rigor or relaxing solution for 5 min (the time corresponding to minimal tension in the control response) the tension response resembled that of the control, beginning from the vicinity of the minimum. The pattern of staining of the fibres by rhodamine-phalloidin was analysed by laser confocal microscopy to relate the mechanical response to phalloidin localization. If fibres were treated with rhodamine-phalloidin for 20-25 min there was a labelling of the I-Z-I segment with intense peaks of fluorescence at the Z-line and the ends of the I filaments. If fibres were pre-incubated for 5 min with phalloidin and then labelled with rhodamine-phalloidin the fluorescence at the Z-line and at the ends of the I filaments was suppressed and the peak of the fluorescence intensity was shifted toward the middle part of the I filament. The data indicate that the decrease in tension caused by phalloidin was associated with binding of phalloidin to the pointed ends of actin filament and the Z-line region, whereas the increase in tension occurred when phalloidin was bound along entire length of the actin filament.

[Phallotoxins as modifiers in the study of muscles]. / Fallotoksiny kak modifikatory v issledovanii myshts.

This review concerns to modification of muscle contractile system functioning by phallotoxins (phalloidin and its derivatives). Binding to actin in thin filament, phallotoxins can change active tension, the spectrum of mechanical relaxation times, ATPase activity, Ca2+ sensitivity. Under some conditions these changes are very pronounced (tens of percents). As a whole phallotoxins action has the following properties: 1) muscle specificity. Qualitatively different changes have been caused by phalloidin in skinned fibers of cardiac, skeletal and smooth muscle. These differences seem to reflect, at least partially, the differences in the muscle specific proteins of the thin filament: 2) dependence on physiological state of the contractile system (thin filament activation, cross-bridge cycle kinetics); 3) specificity in action of different phalloidin derivatives. Due to such properties phallotoxins suppose to be a probe some essential steps in contractile proteins interaction and a potentially power tool for study of mechanism of muscle contraction.

Thin filament activation by phalloidin in skinned cardiac muscle.

Phalloidin binds very tightly and specifically to actin and brings about a marked stabilization of the F-actin filament. In this study the effects of phalloidin on force generation and Ca2+ sensitivity of skinned bovine ventricular muscle were investigated. At all free Ca2+ concentrations addition of phalloidin to activated fibers caused an enhancement of active force. At full Ca2+ activation the force increase was about 6% and the relative force enhancement became greater as the Ca2+ concentration was decreased. Force-pCa plots obtained with fibers pre-treated with phalloidin showed that phalloidin produced an approximately 0.2 pCa unit increase in Ca2+ sensitivity without significant changes in cooperativity of activation. These results suggest that interactions between G-actin subunits may play an important role in cardiac force development.

Effect of phalloidin on the ATPase activity of striated muscle myofibrils.

Phalloidin was shown to increase the ATPase activity and Ca2+ sensitivity of both bovine cardiac and rabbit psoas myofibrils when assayed in a solution containing 50 mM KCl, 100 mM MOPS (pH 7.0), 2 mM MgCl2, 1 mM ATP, 2 mM EGTA, and varying concentrations of Ca2+ (temperature 21-22 degrees C). The phalloidin effect in cardiac myofibrils developed over a time course of several minutes in the presence of 50 microM phalloidin. Relative increase of ATPase activity was maximal at pCa 8 and decreased with decrease in pCa. In cardiac myofibrils the increase was about 70% at pCa 8 and 20% at pCa 4 following 20-30 min pre-incubation with 2 microM or 50 microM phalloidin. The effect persisted after excess phalloidin was washed out. The increase in Ca2+ sensitivity was approximately 0.15 pCa units. For skeletal myofibrils treated with 2 microM phalloidin all changes were considerably less than those seen with cardiac myofibrils and the changes were even less when the myofibrils were exposed to 50 microM phalloidin. These results show that when specifically bound to actin, phalloidin can change the kinetic parameters of the cross-bridge cycle and may also alter the Ca2+ sensitivity of the contractile system. The effects of phalloidin seem to vary with muscle type.

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.

[Study of properties of the actomyosin complex using photo-cleavage of proteins by vanadates]. / Issledovanie svoistv aktomiozinovogo kompleksa s pomoshch'iu obuslovlennogo vanadatami fotorasshchepleniia belkov.

Study of myosin and actomyosin preparations photocleavage conditioned by polyvanadates confirmed the data on V1 and V2 centre cleavage independence of bivalent cations. Actin does not change sufficiently the reaction in V1 centre and considerably slows down the reaction in V2 centre. These actin properties do not depend on bivalent cation (Mg2+), nor on preliminary incubation with vanadate. It was also discovered that preincubation with vanadate in EDTA medium results in myosin molecule cleavage with producing light (M 18 kD) fragments in both cases: with myosin and actomyosin preparations. Besides vanadate-dependent photocleavage of myosin peptide bonds, there were discovered photocrosslinkings of polypeptide chains in myosin and actomyosin preparations also depending on the presence of vanadate. In actomyosin preparations they probably lead to crosslinking of heavy minor proteins to heavy myosin chains.

[Interaction of vanadate with the contractile system of striated muscles in the presence of natural analogs of ATP and ADP]. / Vzaimodeistvie vanadata s sokratitel'noi sistemoi poperechno-polosatykh myshts v prisutstvii estestvennykh analogov ATF i ADF.

Vanadate produced dissociation of rigor-activated (calcium-free) fibres or rabbit m. psoas muscle in the presence of the studied various natural analogs of ATP (NTP) at optimal concentrations. By the degree of sensitivity to vanadate it is possible to establish the order ATP approximately greater than CTP greater than UTP greater than ITP greater than GTP. This series corresponds to the order for actomyosin NTPases qualitatively. Addition of corresponding NDP to fibres produced a decrease of the rigor fibres tension. Vanadate in comparable concentrations does not change the mechanical properties of fibres in the presence of NDP. At high concentration (greater than 10 mM) vanadate produced relaxation of the rigor fibres even in the absence of nucleotides. This effect is irreversible.

[Reduced sensitivity of glycerinated rabbit m.psoas fibers to diprotonated vanadate during decrease in pH]. / Umen'shenie chuvstvitel'nosti glitserinizirovannykh volokon m.psoas krolika k diprotonirovannoi forme vanadata pri ponizhenii pH.

Vanadate (Na3VO4) action of the isometric force of maximal Ca2(+)-activated fibre has been studied at pH from 6.3 to 7.3. Acidification was shown to decrease the force declining vanadate effect: appearance of the dissociation constant estimated from force--H2VO4--dependence increases about two-fold following the pH decreasing from 7.3 to 6.3. Therefore the well-known decline of the force at acidification is accompanied by a decrease of the sensitivity to vanadate. These both phenomena can have a common nature and reflect, for example, a decreasing complex AM.ADP+ responsible for the force and interaction with vanadate. Our findings permit to think that the changes in pH with fatigue increasing the diprotonated form of phosphate simultaneously reduce the sensitivity of the contractile system to this form.

[Mechano-chemical properties of actomyosin-ADP complexes in rabbit psoas muscle fibers]. / Mekhanokhimicheskie svoistva kompleksov aktomiozin-ADF v voloknakh m. psoas krolika.

The relaxing effect of vanadate on active contractile system is found to be completely absent from rigor skinned fibres with ADP even on their stretching up to the forces comparable with the active ones, though vanadate is likely to bind not very firmly with crossbridges not containing inorganic phosphate. Probable reasons of such distinction are considered. The complex actomyosin-ADP in the rigor fibres is supposed to have significantly lower free energy independently of its deformation than the one of the same composition in the active ones. Possible role of different actomyosin-ADP states in the mechanochemical cycle of crossbridge is discussed.

[Changes in the force-pCa characteristics of the striated muscles of the skate after treatment with phalloidin]. / Izmenenie kharakteristiki sila-pCa poperechno-polosatykh myshts skata pod vliianiem falloidina.

The pCa/tension relationship of glycerinated skate thoracic fin muscle was found to fit well in the Hill equation. Phalloidin has been shown to decrease calcium sensitivity (shift of pK to the lower pCa by 0.27 +/- 0.08), tension at saturated pCa by 22 +/- 5%, and Hill coefficient by 0.37 +/- 0.21. As F-actin structural motility is restricted by phalloidin, these results are in agreement with the idea of participation of F-actin conformational changes in its switching by calcium ions and in ensuring cooperativity of this process.

[Different sensitivity of cross-bridges in the muscle fiber sarcomere in Ca2+-activation]. / Razlichnaia chuvstvitel'nost' mostikov v sarkomere myshechnogo volokna pri Ca2+-aktivatsii.

The investigation of transient kinetics of force development by glycerinated rabbit m. psoas fibres at consecutive pCa stepwise drops in the presence of 0.1 mM Na3VO4 was carried out. Stationary content of trapped vanadate in the fibres at low levels of Ca2+-activation was deduced to be attained slowly as compared to the initial rate of force development and vanadate trapping. This result is considered an indication of the fact, that not all cross-bridges can be activated by turns at low levels of Ca2+-activation.

The effect of phallotoxins on the structure of F-actin in myosin-free ghost muscle fibres of rabbit.

Using polarized UV fluorescent microscopy it has been shown that phallotoxins (phalloidin-sulfone, phalloidin-sulfoxide-B, phalloidin-sulfoxide-A and dithio-phalloidin) cause an increase in tryptophan fluorescence anisotropy of F-actin myofilaments in myosin-free ghost muscle fibres of rabbit. The results obtained are considered to be evidence of conformational changes in F-actin, induced by phallotoxins. These changes are irreversible to a significant extent, which points to a high degree of actin binding to both toxic and nontoxic phallotoxins.