In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).

The kinetics of ATP-induced rigor cross-bridge detachment were studied by initiating relaxation in chemically skinned trabeculae of the guinea pig heart using photolytic release of ATP in the absence of calcium ions (pCa > 8). The time course of the fall in tension exhibited either an initial plateau phase of variable duration with little change in tension or a rise in tension, followed by a decrease to relaxed levels. The in-phase component of tissue stiffness initially decreased. The rate then slowed near the end of the tension plateau, indicating transient cross-bridge rebinding, before falling to relaxed levels. Estimates of the apparent second-order rate constant for ATP-induced detachment of rigor cross-bridges based on the half-time for relaxation or on the half-time to the convergence of tension records to a common time course were similar at 3 x 10(3) M-1 s-1. Because the characteristics of the mechanical transients observed during relaxation from rigor were markedly similar to those reported from studies of rabbit psoas fibers in the presence of MgADP (Dantzig, J. A., M. G. Hibberd, D. R. Trentham, and Y. E. Goldman. 1991. Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J. Physiol. 432:639-680), direct measurements of MgADP using [3H]ATP in cardiac tissue in rigor were made. Results indicated that during rigor, nearly 18% of the cross-bridges in skinned trabeculae had [3H]MgADP bound. Incubation of the tissue during rigor with apyrase, an enzyme with both ADPase and ATPase activity, reduced the level of [3H]MgADP to that measured following a 2-min chase in a solution containing 5 mM unlabeled MgATP. Apyrase incubation also significantly reduced the tension and stiffness transients, so that both time courses became monotonic and could be fit with a simple model for cross-bridge detachment. The apparent second-order rate constant for ATP-induced rigor cross-bridge detachment measured in the apyrase treated tissue at 4 x 10(4) M-1 s-1 was faster than that measured in untreated tissue. Nevertheless, this rate was still over an order of magnitude slower than the analogous rate measured in previous studies of isolated cardiac actomyosin-S1. These results are consistent with the hypothesis that the presence of MgADP bound cross-bridges suppresses the inhibition normally imposed by the thin filament regulatory system in the absence of calcium ions and allows cross-bridge rebinding and force production during relaxation from rigor.

A series of 2-nitrobenzyl derivatives of the alpha 1-selective adrenergic agonist, L-phenylephrine [(R)-N-[2-(3-hydroxyphenyl)-2-hydroxyethyl]-N-methylammonium chloride], have been synthesized and characterized for the purpose of developing biologically inert compounds that can be rapidly converted to L-phenylephrine by near-UV irradiation. The compounds, derivatized on the phenolic oxygen, were O-(1-(2-nitrophenyl)ethyl)phenylephrine (I), O-(2-nitrobenzyl)phenylephrine (II), O-(4,5-dimethoxy-2-nitrobenzyl)phenylephrine (III), and O-(alpha-carboxyl-2-nitrobenzyl)phenylephrine (IV). All four compounds photolyzed to free phenylephrine following a brief exposure to 300-350-nm light or 347-nm laser light with steady-state quantum yields ranging from 0.05 to 0.28. The rates of phenylephrine formation on photolysis were estimated from the decay rates of aci-nitro intermediates detected by absorbance between 380 and 500 nm. Compound IV displayed the highest quantum yield (0.28) and most rapid photolysis rate (1980 s-1) measured under near physiological conditions, pH 7.0, 22 degrees C. Biological properties of the compounds were examined in smooth muscle from rat caudal artery. Laser pulse photolysis of IV at 347 nm initiated a maximal contraction in Krebs buffer, pH 7.1, 25 degrees C, that mimicked the response to 50 microM phenylephrine but was faster in onset. Photoinitiated contractions were characterized by a delay of 0.93 +/- 0.09 s followed by a rising phase with a 10-90% rise time of 3.56 +/- 0.17 s (n = 7). Responses were fully blocked by the alpha 1-selective antagonist prazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of Ca(2+)-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca(2+) released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca(2+)]. The time courses of the rise in stiffness and tension were biexponential at 23 degrees C, pH 7.1, and 200 mM ionic strength. At full activation (pCa < 5.0), the rates of the rapid phase of the stiffness and tension rise were 56 +/- 7 s(-1) (n = 7) and 48 +/- 6 s(-1) (n = 11) while the amplitudes were 21 +/- 2 and 23 +/- 3%, respectively. These rates had similar dependencies on final [Ca(2+)] achieved by photolysis: 43 and 50 s(-1) per pCa unit, respectively, over a range of [Ca(2+)] producing from 15% to 90% of maximal isometric tension. At all [Ca(2+)], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1 +/- 0.8 and 6.2 +/- 1.0 s(-1). The rate of this slower phase exhibited significantly less Ca(2+) sensitivity, 1 and 4 s(-1) per pCa unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca(2+)], suggest a mechanism of regulation in which Ca(2+) controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca(2+)] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases.

We examined the correlation between agonist-stimulated increases in inositol phosphates and force development in vascular smooth muscle. Segments of rat tail artery were preincubated with [3H]inositol and treated with norepinephrine (10(-5) M) for 3-10 s. Tissue levels of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were measured. IP and IP2 increased significantly after 3 s of norepinephrine treatment. IP3 increased significantly after 5 s of norepinephrine treatment. Analysis of tissue extracts by high-pressure liquid chromatography demonstrated that the only isomer of IP3 present in any tissue extract was the 1,4,5-isomer [Ins(1,4,5)P3]. Contractile response to norepinephrine stimulation showed that the increase in inositol phosphates coincides well with the time course of force development. This is the first report demonstrating such an early increase in Ins(1,4,5)P3 in agonist-stimulated vascular smooth muscle. These results are consistent with the hypothetical role of Ins(1,4,5)P3 as a mediator linking agonist-receptor activation to increased intracellular calcium and force development in norepinephrine-stimulated vascular smooth muscle.