Interleukin-4 inhibits bone resorption and acutely increases cytosolic Ca2+ in murine osteoclasts.

Interleukin-4 (IL-4) is an immune cytokine recently shown to inhibit bone resorption. To determine whether IL-4 directly acts on osteoclasts, we have analyzed its effect on cytosolic calcium concentration [Ca2+]i and bone resorptive function of murine osteoclastic cells generated from bone marrow/stromal cell co-cultures. IL-4 exposure induced an immediate and sustained increase in [Ca2+]i that remained elevated for at least 10 min. This IL-4 effect was dose-dependent, with the maximal effect (209 +/- 15% of baseline, n = 16) at 200 units/ml and an apparent ED0.5 of 60 units/ml. The IL-4-induced [Ca2+]i rise required extracellular Ca2+ influx, since the response was prevented by LaCl3, and voltage-gated Ca2+ channel blockers, although the IL-4 effect was more sensitive to nicardipine and nifedipine than to diltiazem. Depolarization by high extracellular K+ concentration also raised [Ca2+]i, and, under these conditions, osteoclasts failed to respond to IL-4. On the other hand, when intracellular Ca2+ stores were depleted by thapsigargin, IL-4 still induced an increase in [Ca2+]i, although smaller in amplitude and transient. Calcitonin also produced [Ca2+]i increases in osteoclasts, yet it only slightly desensitized these cells to IL-4. Furthermore, IL-4 was much less effective on osteoclasts pretreated (5-10 min) with either forskolin or 8-bromo-cAMP. Both IL-4 and calcitonin were effective even when [Ca2+]i had been increased by exposure to high extracellular Ca2+. Finally, IL-4 dose dependently inhibited the bone-resorptive activity of mature osteoclasts. Therefore, IL-4 signal transduction in osteoclasts involves a rapid and sustained elevation of [Ca2+]i mediated by a voltage-dependent Ca2+ influx, in combination with Ca2+ release from intracellular stores. Modulation of osteoclast [Ca2+]i represents a potential mechanism by which IL-4 inhibits bone resorption.

Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle.

We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.

Characterization of an actin flow artifact that occurs during the presteady state dissociation of actosubfragment-1.

Models for the activation of the myosin subfragment-1 (S-1) ATPase activity by actin describe transitions that occur between kinetic intermediate states during steady state hydrolysis of ATP. These states consist of myosin-nucleotide complexes in rapid equilibrium binding with actin, but steady state measurements of actin binding during hydrolysis lead only to a weighted average of the individual binding constants involved. In the current work, in order to determine the individual binding constants involved in the activation process, we have investigated the presteady state kinetics of the dissociation of actomyosin by ATP. We find that an actin flow artifact appears to dominate the time course of dissociation, and characterization of this artifact reveals that its magnitude rises linearly (approximately) with the concentration of bound S-1. Attempts to subtract the actin flow artifact from the actoS-1 dissociation signal were not entirely successful due at least partially to the transient nature of the bound S-1 concentration in the first few milliseconds. However, further studies reveal that if the order of addition of actin, ATP, and S-1 are varied, the observed light scattering transients are essentially superimposable. One possible explanation of these data is that the binding constants for myosin-ATP and myosin-ADP-Pi to actin are equal. However, it is also possible that the flow artifact is so large that further analysis is precluded. In addition, we show that the actin flow artifact has little effect on the fluorescence measurements of the phosphate burst reported previously. Therefore, the prior interpretation of the fluorescence data remains unchanged.

Effect of limited trypsin digestion on the biochemical kinetics of skeletal myosin subfragment-1.

We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.

Biochemical kinetics of skeletal actosubfragment-1 at high subfragment-1 concentrations.

The actomyosin ATPase activity of skeletal myosin subfragment-1 (S-1) is typically studied by keeping the S-1 concentration low and varying the actin concentration. General agreement exists over the kinetic data observed. Another way of studying the ATPase activity is to keep the actin concentration low and vary the S-1 concentration. The picture that has emerged is that the maximal ATPase rate (per micromolar actin), Vamax, is several fold greater than the Vsmax measured at fixed S-1. Likewise, the apparent activation constant Kam is several fold weaker than KATPase. In addition it is found that Kam, henceforth Kam(At), varies with the total actin concentration At, but controversy continues over the actin dependence of Vamax. Of particular interest is the fact that the Lymn-Taylor and refractory state models could not account for the data. Here we have repeated studies on the ATPase activity at fixed actin concentration in an attempt to determine if the current models for the actin activated myosin ATPase activity can account for both the constant actin and constant S-1 data simultaneously, or if these data imply that new kinetic models need be postulated. We conclude that the current kinetic models can account for the data.