Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle.

Phosphorylation of the 20,000-dalton light chain of myosin is closely correlated with cross-bridge cycling in arterial smooth muscle. Evidence is presented that dephosphorylation can produce an attached, noncycling cross-bridge (latch-bridge) which is responsible for the high economy of force maintenance in this tissue.

Mechanical and metabolic toxicity of 3-(trimethylsilyl)propanesulfonic acid to porcine carotid arteries.

3-(Trimethylsilyl)propanesulfonic acid (TMSPS) is used as a water-soluble NMR frequency marker. It has its major resonance at 0.00 ppm relative to trimethylsilane, and smaller resonances at 0.62, 1.77 and 2.85 ppm. Its toxicity was tested by exposing contracted porcine carotid strips to increasing concentrations of TMSPS. Up to 3 mM, no statistical change in tension was found. Tension decreased 94 +/- 2% (S.E.) after 30 min in 10 mM TMSPS. An intermediate concentration of TMSPS (6 mM) caused a small fall in phosphocreatine in unstimulated perfused porcine carotid arteries (82 +/- 2% S.E.). A larger decrease (59 +/- 6% S.E.) occurred during K+ contractures in the presence of 6 mM TMSPS. From those experiments it appears the TMSPS is non-toxic in concentrations up to 3 mM, but at greater concentrations inhibits both contraction and phosphorus metabolism.

Augmentation of aortic ring contractions by angiotensin II antisense peptide.

Previous biochemical experiments have revealed two antisense peptide antagonists to human angiotensin II (Ang II), one encoded in the cDNA in the antiparallel reading, the other in the parallel reading. Neither peptide's ability to produce physiological antagonism has been demonstrated previously. Both peptides were tested for their ability to antagonize Ang II-induced contractions on rabbit aorta smooth muscle. Neither peptide had any direct contractile activity. The antiparallel Ang II peptide had physiological antagonism to Ang II contractions at a lower sensitivity than reported in biochemical studies, and its antagonist activity was partially blocked by Ang II antiserum, suggesting that it is not an antipeptide but an Ang II homologue. The parallel Ang II antipeptide also required high concentrations for physiological inhibition. Its contractile inhibition was not affected by Ang II antiserum and diminished the Ang II contraction at high micromolar concentrations, findings consistent with physicochemical data showing that it is an Ang II complement. The concentration of either peptide required to produce an antagonistic physiological effect was too high to predict any pharmacological usefulness. The parallel antipeptide, however, significantly increased the force of muscle contractions at high nanomolar concentrations, thus displaying a unique dual augmentation/antagonist activity. This antipeptide seems to have highly sequence-specific activity because other similar parallel antipeptides had no activity. The parallel antipeptide augmentation mimics the shift in the Ang II dose-response curve produced in hypertension studies of the slow pressor effect of Ang II and may be useful in deducing the currently unknown cause of the slow pressor effect. It may also have some uses in migraine studies.

Influence of cellular energy metabolism on contractions of porcine carotid artery smooth muscle.

A number of cellular metabolites, including inorganic phosphate and ADP, have been proposed to regulate the contractions of smooth muscle. Hypothesizing that one of these would have a greater influence than the others, parallel experiments using tissue mechanics and (31)P-NMR allowed comparison of several metabolic components with the generation of force in porcine carotid artery smooth muscle during long-term contractions. P(i), ADP, ATP, PCr, free energy, pH, and free Mg(2+) were determined from phosphate spectra during a control-hypoxia-postcontrol sequence generated during K(+) stimulation by replacement of oxygen with nitrogen using either pyruvate or glucose as substrate. Both pH and free Mg(2+) were significantly lower in control pyruvate-supplied tissues than in glucose-supplied tissues. Mechanical experiments following the same protocol produced variations in force. The pyruvate series produced the greater range of mechanical and metabolic changes. Linear and logarithmic regression analysis found the order of correlation with force to be highest for P(i), followed by pH, free energy, PCr, ATP, ADP, and free Mg(2+). The results are consistent with models for the regulation of myosin ATPase by free phosphate inhibition. The results are inconsistent with models of ADP as a regulator of smooth muscle force. Perturbations which alter intracellular phosphate, such as creatine loading, may produce side effects on the contractions of vascular smooth muscle.

Phenylphosphonate: a 31P-NMR indicator of extracellular pH and volume in the isolated perfused rabbit bladder.

31P-NMR has been used extensively to estimate intracellular pH. It also can be used to measure extracellular pH and volume when an NMR-detectable extracellular phosphorous probe is used. Phosphonic acids have been suggested as useful 31P-NMR extracellular markers. The present study was designed to assess the utility of phenylphosphonic acid (PPA) as a 31P-NMR extracellular marker in perfused smooth muscle. Rabbit bladder strips were exposed to PPA concentrations of 1-20 mM. Tension development in response to maximal carbachol challenges (10 microM) was independent of PPA concentration. Addition of PPA (6 mM) to the perfusate supplying the isolated resting rabbit bladder had no effect on 31P-NMR-detectable phosphatic compounds. PPA's resonance frequency was distinctly downfield from endogenous phosphates and demonstrated a pH-dependent chemical shift of +/- 1.12 ppm/pH unit over the range of 6.4 to 7.6 with a pK' of 7.09 at 23 degrees C. The time courses for washing PPA in and out of the resting bladder were best described by monotonic exponential growth (r = 0.972; n = 3) and decay (r = 0.972; n = 3) equations, respectively. Rate and time constants for PPA wash-in (0.039 +/- 0.004 min-1 and 25.7 +/- 2.3 minutes) and washout (0.038 +/- 0.000 min-1 and 26.3 +/- 0.0 minutes) were not significantly different. Using steady state PPA and ATP peak intensities and concentrations, an extracellular-to-intracellular ratio was calculated to be 0.31 +/- 0.03 (n = 3). These data indicate that PPA remains distributed exclusively in the extracellular spaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct determination of ADP in hypoxic porcine carotid artery using 31P NMR.

31P-NMR spectroscopy was performed on vascular smooth muscle (VSM; porcine carotid artery) superfused with a substrate-free high K(+)-PSS. Scans were collected before (control), during (hypoxia), and after (post-control) hypoxia, and chemical measurements of ATP (0.070 +/- 0.13 mumoles/g wet wt.) and creatine (2.04 +/- 0.14 mumol/g wet wt.) were made. During hypoxia, well-defined beta-ADP signals were consistently resolved. Their areas indicated that after 30, 60, and 90 min of hypoxia, free ADP was 0.05 +/- 0.01, 0.09 +/- 0.01, and 0.12 +/- 0.01 mumol/g wet wt., respectively. The apparent tissue equilibrium constant (Kck) for creatine kinase (CK) was calculated using 90 min hypoxic data and was 7.6 +/- 0.6 x 10(8) M-1. It was used to compute free ADP levels (mumol/g wet wt.) for control (0.028 +/- 0.002) and post-control (0.23 +/- 0.003) periods, since ADP signals could not be directly detected, and for the 30 and 60 min hypoxic periods (0.05 +/- 0.01 and 0.08 +/- 0.01, respectively). The Kck-dependent ADP values for the 30 and 60 min hypoxic periods periods were the same as the ADP values determined directly from the beta-ADP peak areas, suggesting that the CK reaction is in equilibrium in smooth muscle. These data show that 31P-NMR provides a means of directly measuring free ADP in hypoxic smooth muscle and a more accurate means of computing free ADP levels in normoxic VSM through the use of an in situ tissue Kck vs an assumed or in vitro Kck.

Capillary electrophoretic measurement of tissue metabolites.

A method for the measurement of tissue metabolites from rabbit urinary bladder using capillary electrophoresis (CE) has been developed. The method generates a reproducible electropherogram containing > 20 peaks, including NAD, NADH, lactate, UDP-glucose, phosphocreatine, creatine, ATP, ADP, GTP, and UTP, from < 20 nl of extract solution generated from 1.1 nl (or approximately 1.2 micrograms) of tissue in < 40 min. Multiple samples from the same bladder produce SE comparable with enzymatic or nuclear magnetic resonance (NMR) measurements of metabolites: phosphorus-NMR measurement requires 10(6) more tissue than CE; individual enzymatic measurements using 100 microliters/sample require 2,000 microliters, a 10(5) greater volume than required by CE for the same number of metabolites. CE detects about three times more peaks than phosphorus-NMR on a similar time scale. Comparable measurements using enzymatic analysis would require approximately 10 times longer. The combination of minimal tissue volume requirements, rapid measurement, and reproducibility makes CE a valuable tool in the investigation of simultaneous changes in multiple metabolites from minute tissue samples.

The theory of diazymes and functional coupling of pyruvate kinase and creatine kinase.

The physical-chemical principles governing the interactions of enzymes having common metabolic products are presented. Methods for comparing the dissociation rates of the metabolic product and the rates of enzyme-enzyme interaction are given. Using muscle pyruvate kinase (PK) and creatine kinase (CK) as an example, it is shown that the probability of forming an enzyme-product-enzyme complex is much greater than the rate of ATP dissociation from either enzyme. Experimental evidence using 31P-NMR demonstrates that in the presence of both pyruvate kinase and creatine kinase, there is exchange of phosphate between phosphocreatine and phosphoenolpyruvate without a change in the intermediate, ATP. This confirms the formation of a PK.ATP.CK complex in an aqueous solution without enzyme attachment to a substructure. Enzymes capable of forming these mobile clusters are defined as diazymes, and the criteria for their formation are given. The metabolic implications of diazymes are discussed.

31P NMR study of insulin effects on the isolated perfused rabbit urinary bladder.

Insulin stimulates hexose transport, intermediary metabolism, and cell growth and development. These effects are well-documented in skeletal but not smooth muscle. 31P NMR spectroscopy was performed on rabbit urinary bladders (n = 4) to characterize insulin's actions on smooth muscle. The bladder and its vasculature were surgically isolated from the animal and perfused with a PSS/red blood cell perfusate. After a control steady state was achieved (approx 1-2 h), insulin (0.100 mU/ml) was added to the perfusate. Relative levels of intracellular phosphorylated compounds, pH, and free Mg2+ were measured and compared to control values. Also, extracellular pH and fractional volume were assessed using phenylphosphonate, a 31P NMR extracellular pH and volume indicator. Insulin induced significant increases in PCr (16 +/- 9%) at the expense of Pi, intracellular pH (delta pH 0.24 +/- 0.07), and fractional extracellular volume (49 +/- 1%). Intracellular free Mg2+ and extracellular pH did not change. These results indicate that in situ smooth muscle is sensitive to physiological levels of insulin. In fact, insulin improves the energy state of smooth muscle cells and the overall tissue perfusion.

Glucose dependence of sequential norepinephrine contractions of vascular smooth muscle.

The effects of successive, norepinephrine (NE)-stimulated, contractions of porcine carotid artery intima-media strips as a function of recovery medium were studied. Recovery following NE stimulation required the presence of glucose in the bathing medium for subsequent force production in response to NE stimulation. Potassium stimulation following failed NE contractions produced maximal contractions. Phosphorous nuclear magnetic resonance studies under similar conditions indicated that the energy state of the tissue was not impaired during and after recovery from NE stimulation without glucose. This study shows that the phasic phase of norepinephrine-stimulated contractions is dependent on the availability of extracellular glucose during the poststimulation recovery period for successive NE contractions.

Phosphocreatine and creatine kinase in energetic metabolism of the porcine carotid artery.

OBJECTIVES: Magnetization exchange experiments and force analysis were performed on porcine carotid arteries with varied phosphocreatine (PCr) levels. The aim of these experiments was to determine the creatine kinase (CK) kinetics and the role in hypoxic relaxation. METHODS: The magnetization exchange techniques used were multisite saturation transfer (MST) and conventional saturation transfer (CST). The two techniques were used because CST assumes a two-site exchange while MST allows one to assume a three-site exchange. Mechanical parameters of tension generation and relaxation were measured to determine the energetic effects on contractility of carotid strips. RESULTS: Measurements of molecular exchange between ATP and PCr found the pseudo first-order rate constant (kf) of 0.17 +/- 0.04 S-1 (PCr-->ATP) and kr = 0.12 +/- 0.03 S-1 (ATP-->PCr) in unstimulated porcine carotid artery. In the carotids, despite increased PCr and K+ stimulation, no magnetization exchange is observable with MST. This result indicates that the ATPase was less than 0.04 mumol/g/s (below the NMR resolution) while CK was 0.11 mumol/g/s. Creatine-loaded carotids showed no significant differences in force measurements: maximal force, resting tension, and the rate of hypoxia were all unchanged. CONCLUSIONS: The flux ratio (flux forward over flux reverse) was 0.94 +/- 0.13 which was considered to be indicative of CK being at equilibrium in the resting porcine carotid artery. The rate of the CK reaction is rapid enough to assume a two-site kinetic exchange not limiting energetic supply during hypoxia-induced relaxation.

Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase.

The interaction of pyruvate kinase from skeletal (SKPK) and smooth (SMPK) muscle with MM-creatine kinase (MMCK) and BB-creatine kinase (BBCK) was assessed using temporal absorbance changes, variations in absorbance at different wavelengths, concentration dependence, association in an electric field, and PK kinetic activity. SKPK exhibits a time course of absorbance increase in the presence of MMCK with a time constant of 29.5 min. This increase occurs at all wavelength from 240 to 1000 nm. At 195 nm, the combination of SKPK and MMCK produces a decrease in absorption with electric fields of both 0 and 204 V/cm. The change in SKPK-MMCK is saturable. SKPK activity is significantly increased by the presence of MMCK in solutions of 0-32% ethanol. These results indicate specific SKPK-MMCK interaction. SMPK and BBCK did not exhibit similar coupling when the BBCK concentration dependence of absorbance or SMPK activity in solutions of 0-32% ethanol was determined. Both MMCK and BBCK increased SKPK activity; neither MMCK nor BBCK increased SMPK activity. The ability to form diazymatic complexes with creatine kinase appears to reside in SKPK. This coupling may account for the increased flux through PK without significant substrate changes seen during skeletal muscle activation. This coupling will not occur in smooth muscle.

Tonic force maintenance with reduced shortening velocity in arterial smooth muscle.

The isotonic shortening velocity of swine carotid media tissues contracting in response to high K+, histamine, norepinephrine, or AC electrical stimulation rapidly increased to a maximum value and then declined to a steady-state level while force was still increasing or steady. The maximum shortening velocity calculated for no external load on the tissue (Vo) also decreased during the course of contractions when active stress remained constant. The fall in velocity with time was not Ca2+ dependent, because reductions in the [Ca2+] in high K+ solutions that significantly reduced the maximum stress (Fo) had no effect on Vo in the steady state. On washout of high K+ solutions, the ability of the tissue to shorten on isotonic quick release fell rapidly to low levels before isometric stress exhibited significant declines. The data indicate that cross-bridge cycling rates are modulated in this tissue. We suggest that this reflects the formation of attached, noncycling cross bridges (termed latch bridges), which constitute an internal load on the contractile system during tonic contractions.

High force development and crossbridge attachment in smooth muscle from swine carotid arteries.

In experiments designed to achieve maximal activation, the active force/cell cross-sectional area in tissues prepared from the swine carotid media was 6.7 +/- 0.3 (sd) X 10(5) N/m5. This value exceeds that reported for other vertebrate muscle cells and is striking because of the low smooth muscle myosin content. The hypothesis that high force generation may, in part, reflect an increase in the crossbridge duty cycle, i.e., the fraction of the cycle during which force is generated, was tested by determining the rate of force redevelopment after a step shortening and the ration of the load-bearing capacity of the contractile system to the developed stress during the course of isometric contractions. Maximal crossbridge cycling rates estimated by the rate of force redevelopment occurred 30 seconds after the onset of a high K+-induced contraction, and decreased thereafter, although the load-bearing capacity or maximum active stress was maintained. These results from isometric experiments support the hypothesis and provide further evidence that attached, non-cycling crossbridges contribute to force maintenance in tonically contracting arterial smooth muscle.

Molecular complementarity I: the complementarity theory of the origin and evolution of life.

We assert that molecular complementarity is much more widespread than is commonly acknowledged in biological systems, if not actually ubiquitous. It creates the coupling necessary for non-equilibrium systems to form. It stabilizes aggregates against degradation, thereby increasing concentrations to levels adequate to foster the formation of prebiotic systems and represents the earliest form in which natural selection was manifested. Complementarity confers on all interacting parts of such systems in formation carrying capacity. RNA or DNA are not, therefore, necessary to the emergence of life, but represent specialized forms of complementary molecules adapted specifically to information storage and transmission. Non-genetic information exists in metabolic functions and probably preceded genetic information historically. Complementarity also provides the basis for homeostasis and buffering of such systems not only in a chemical, but also in structural and temporal terms. It provides a mechanism for understanding how new, emergent properties can arise, and a basis for the self-organization of systems. We demonstrate that such aggregates can have properties not predictable from their individual components, thus providing a means for understanding how new functions emerge during evolution. Selection is for modules rather than individual components. The formation of functional sub-systems that can then be integrated as modules greatly increases the probability of the emergence of life. The result of such modular evolution alters the standard view of evolution from a tree or bush-like image to an integrated network composed of alternating periods of integration (as molecules and molecular aggregates merge) and divergence (as molecules and aggregates undergo variations). This provides a mechanism for evolution by punctuated equilibria. Molecular complementarity puts strict limits on variations, however, preventing evolution from being random. The evolutionary, physiological and embryological consequences of this view of life are outlined, and various models and experiments described that further characterize it.

Molecular complementarity II: energetic and vectorial basis of biological homeostasis and its implications for death.

The energetic basis of molecular complementarity is presented. In biological systems requiring both homeostasis and non-equilibrium state maintenance, molecular complementarity provides a framework for the co-existence of these states within an organism. Smoothly changing homeostatic and thermodynamic systems, such as regulation of pH or an ensemble of asynchronous muscle crossbridges, are modeled using Liapunov functions. When biological systems undergo discontinuous state changes, such as the initiation of the heartbeat, life/death transition or the detachment of molecules, alternative analytical systems such as catastrophe theory provide information that continuous analytical methods cannot. Catastrophe theory produces a model of biology in which death can occur by two distinct mechanisms: loss of homeostatic control or loss of sufficient free energy. Molecular complementarity buffers molecules from temporal and physical changes. The usefulness of molecular complementarity is limited to association energies near the ambient energy, kT. Within this range, complementarity will alter molecular functions and will convert scalar biochemical reactions into vectorial physiological processes. Both thermodynamic and catastrophic models can be used to link energetic and homeostatic processes: the former providing quantitative information from continuous systems; the latter providing qualitative information from discontinuous systems involving state changes.

Multisite saturation transfer using DANTE and continuous wave.

The DANTE pulse sequence was modified to produce selective resonance saturation similar to that produced by the continuous wave (CW) technique. A combined DANTE and CW saturation technique can be used to perform multisite saturation transfer experiments because of the similar saturation produced by the two techniques.

The use of 31P-NMR to study smooth muscle.

The use of 31P NMR to study smooth muscle is hindered by low metabolite concentrations and low tissue mass. These disadvantages can be overcome due to low rates of energy utilization and the temporal stability of smooth muscle. Smooth muscle free Mg++, ADP, and the creatine analogue beta-guanidinopropionate can be studied in ways distinctive from 31P NMR of striated muscle. The pharmacological effects of naturally occurring agents such as insulin, glucose, or norepinephrine, and of NMR markers such as phenylphosphonate or 3-(trimethylsilyl)-1-propane-sulfonate on both vascular smooth muscle and perfused smooth muscles can be measured. Given sufficient collection time, 31P NMR is as useful in assessing the behavior of smooth muscle as it is of other muscle types.

Dynamics of Imbibition in Phaseolus vulgaris L. in Relation to Initial Seed Moisture Content.

The seed moisture level marking the onset of imbibitional injury (breakpoint) was determined for two cultivars of Phaseolus vulgaris L. cvs ;Tendercrop' (TC) and ;Kinghorn Wax' (KW). At 20 degrees C the breakpoints were 0.15 gram H(2)O/gram dry weight (gram per gram) for TC and 0.11 gram per gram for KW. When seeds were imbibed at 5 degrees C, the breakpoints were 0.19 gram per gram (TC) and 0.16 gram per gram (KW). Below the breakpoint germination changed 4.6%/0.01 gram per gram for all treatments. Imbibition rates were maximal at 0.07 gram per gram and 0.33 gram per gram after 20 minutes imbibition. Rates of electrolyte leakage were correlated with the imbibition rate maximum at 0.07 gram per gram but were unaffected by the maximum at 0.33 gram per gram. The transition from tightly bound to semibound water occurred at 0.09 gram per gram and 0.11 gram per gram for KW and TC, respectively. T1 values increased exponentially as seed moisture decreased from 0.47 gram per gram to 0.05 gram per gram. (13)C-NMR sugar signals increased at moisture levels above 0.14 gram per gram and plateaued at approximately 0.33 gram per gram seed moisture. These results suggest that the breakpoint moisture level for imbibitional damage is a function of temperature while the injury process is similar at both 5 and 20 degrees C. Imbibition and leakage rate maxima reflect transitions in the states of seed water. NMR data support the application of the Water Replacement Hypothesis to seeds. Thus, imbibitional injury may be related to specific, temperature dependent moisture levels that are determined by water binding characteristics in the seed tissue.

Antioxidant-independent ascorbate enhancement of catecholamine-induced contractions of vascular smooth muscle.

Ascorbate reduces the oxidation rate of catecholamines and, by an independent mechanism, enhances rabbit aortic ring contractions initiated by catecholamines. The largest significantly different fractional increases in force produced by ascorbate enhancement of norepinephrine (NE), epinephrine, phenylpropanolamine (PPA), and ephedrine (Eph) are 5.5, 1.8, 1.6, and 1.3 times, respectively. In physiological salt solutions bubbled with 95% O(2) at 37 degrees C, NE, PPA, and Eph have oxidation rate constants of 1.24, 247, and 643 h, respectively. Ascorbate significantly enhances 100 nM NE contractions by at least twofold at all ascorbate concentrations >15 microM, including the entire physiological range of 40-100 microM. Ascorbate preloading and washout followed by NE exposure produces significantly greater contractions than NE without ascorbate preloading but significantly lower than NE simultaneously with ascorbate. Ascorbate does not enhance K(+)- or angiotensin II-induced contractions. Ascorbate enhancement of catecholamine contractions occurs in addition to the reduction in oxidation rate, because the increases in force occur faster than oxidation can occur, the increases occur with compounds that have negligible oxidation rates, and the increases occur when ascorbate and NE are not physically present together. These results are consistent with ascorbate acting on the adrenergic receptor. Ascorbate may play a role in shock and asthma treatments and potentiate the cardiovascular health consequences of PPA and Eph (Ephedra).