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Phosphocreatine (PCr) and intracellular pH changes were monitored by 31P-NMR spectroscopy in isolated, arterially perfused cat biceps and soleus muscles, while the pH of the CO2-bicarbonate buffered perfusate was decreased from 7.1-7.4 to 6.4-6.7 by increasing the CO2 in the equilibrating gas from 5 to up to 70%. In biceps (fast twitch) muscles, intracellular pH decreased from 7.0 to 6.6 (30% CO2, 30 degrees C), peak tetanic force decreased by 8%, but the rise and relaxation times of tetanic were not significantly changed. In soleus muscles, intracellular pH decreased from 7.0 to 6.6 (30% CO2, 30 degrees C), peak tetanic force was unchanged, but the rise and relaxation times of tetani were increased by 27 and 112%, respectively. In both muscles greater decreases in tetanic force were observed during repetitive or ischemic stimulation, which resulted in intracellular pH similar to that produced by hypercapnia. Contrary to previous reports, there was no significant decrease in PCr level in either muscle type with decreased intracellular pH. In the soleus at 30 degrees C there was a significant increase in PCr level with decreased pH.

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The relationship between extracellular calcium concentration ([Ca2+]o), isometric force (Fo), unloaded shortening velocity (Vus), and the rate of ATP utilization (JATP) were studied in vascular smooth muscle (VSM) during the steady state of an isometric contraction at 37 degrees C. Experiments were conducted on porcine carotid artery media strip and ring preparations that were stimulated with 109 mM KCl substituted for NaCl. Unloaded shortening velocity was estimated by the "slack" test method. Both Fo and Vus were dependent on [Ca2+]o. Vus at 7.5 mM [Ca2+]o was 1.7 times greater than Vus at 1.6 mM [Ca2+]o. The difference in force at these two Ca2+ concentrations was more variable than Vus, but in general was less than the change in Vus. The rate of ATP utilization was assessed from steady-state measurements of tissue O2 consumption. Increasing [Ca2+]o in the range of 0.15-7.5 mM resulted in an increase in both JATP and Fo. The relation between JATP and Fo was nonlinear with JATP increasing proportionately more than Fo between 1.6 and 7.5 mM Ca2+. The calcium-dependent increase in JATP appears to be primarily related to contractile protein interaction, since the effect of [Ca2+]o on JATP was substantially reduced during stimulation at short muscle lengths (Lmin), where tension development is absent. The economy of tension maintenance was 3.3. times greater when measured in the [Ca2+]o range of 0.15 and 1.6 mM than when measured at 1.6 to 7.5 mM [Ca2+]o. These data indicate that [Ca2+]o may regulate both the number and the rate of cycling of cross bridges in porcine carotid artery.

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In smooth muscle isometric force, shortening velocity, the ATP utilization under isometric conditions, and tension cost are all functions of calcium. This suggests that both cross bridge number and cycle rate are dependent on calcium. On the other hand, the series elastic component does not exhibit a significant dependence on calcium under conditions in which Vus can be increased with little change in Fo. And although the data is not as extensive, the efficiency in working producing contractions is not a strong function of calcium. Our challenge is to fit these mechanical and energetic data into a mechanism which fits the ever growing and controversial body of evidence on the biochemical basis for the action of calcium on the contractile apparatus. At present the most striking feature is the observation that calcium can control the velocity in a manner which is independent of isometric force and efficiency. This would appear to favor a mechanism in which calcium directly affected cross bridge cycle rate in the absence of an internal load.

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31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.

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The rate of ATP utilization ( JATP ) and unloaded shortening velocity ( Vus ) of porcine carotid artery were studied during tension development and maintenance to assess the importance of mechanisms proposed to account for its high economy of tension maintenance. Previous work from our laboratory established that the rate of O2 consumption ( JO2 ) can be used to measure rates of tissue phosphagen utilization as early as 30 s after initiation of contraction. Studies at 37 degrees C of tissue JO2 indicated the following. 1) The basal JO2 was 0.069 +/- 0.017 mumol O2 X min-1 X g-1) (n = 10). During steady-state tension maintenance suprabasal JO2 was 0.079 +/- 0.012 mumol O2 X min-1 X g-1 for tissues stimulated with KCl (n = 11) and 0.213 +/- 0.022 (n = 3) for tissues stimulated with KCl + histamine. 2) For both stimulation conditions an initial elevated (peak) suprabasal JO2 was observed that correlated with the rate of tension development. The peak suprabasal JO2 was approximately twice the steady-state suprabasal JO2 measured 10 min after stimulation. 3) There was no significant change in JO2 during tension redevelopment after a rapid length step at 15-20 min after stimulation (n = 4). Studies of Vus indicated the following. 1) During steady-state tension maintenance Vus was 0.008 +/- 0.001 tissue lengths/s for tissues stimulated with KCl (n = 13) tissue lengths/s for tissues stimulated with KCl + histamine (n = 6). 2) An initial twofold peak in Vus was observed at 0.25-0.5 min in that decreased to steady-state levels by 4 min after KCl stimulation, whereas minor decreases in Vus still occurred until 10-20 min after KCl + histamine stimulation. 3) The measured transient in Vus , although biphasic, does not temporally correlate with the transient in JO2 even after the JO2 time course was corrected for delays resulting from O2 diffusion and the electrode response time. The mechanism underlying the observed maximum change of two- to fourfold in both JATP and Vus is not sufficient to account for the high economy of tension maintenance.

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ATP utilization (delta approximately P) during an isometric contraction has been studied in terms of both measurements of oxygen consumption and lactate production as well as of the tissue nucleotide and metabolite levels. The contribution of breakdown of preformed ATP and phosphocreatine (PCr) pools to delta approximately P during contraction is minor compared to that made by metabolic synthesis of ATP. For tonic vascular smooth muscle (VSM), in fact, no change in ATP or PCr from resting levels can be measured. In contrast to amphibian skeletal muscle, a P:O of 3 can be demonstrated in VSM. In both tonic and phasic VASM, delta approximately P is biphasic with contraction duration, attaining a maximal value before that of isometric force and declining to a steady-state value approximately 60% of the maximal suprabasal rate during the maintenance of constant isometric force. The steady-state rate of ATP utilization per unit force maintained increases with extracellular Ca2+. Both the pre-steady-state temporal dependence and the steady-state dependence on Ca2+ are consistent with the hypothesis that myosin phosphorylation modulates the cross-bridge cycle rates. VSM metabolism, when viewed in terms of ATP synthesis, is primarily oxidative. However, even under fully oxygenated conditions, lactate is the major end product of glucose catabolism. Recent work has shown that aerobic lactate production is specifically coupled to Na-K transport in many, but not all, vascular tissues. Oxidative metabolism, on the other hand, is strongly related to active isometric force. The biochemical basis of this functional compartmentation was investigated at the level of substrate specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

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Previous studies (Paul, R. J. Chemical energetics of vascular smooth muscle. In: Handbook of Physiology: The Cardiovascular System. Bethesda, MD: Am. Physiol. Soc., 1980, p. 201-235) have shown that vascular oxygen consumption reaches a steady state at approximately twice the basal rate during maintenance of isometric contraction. The time course of the attainment of a metabolic steady state, the metabolic signal for the observed increase in respiration, and the contribution of endogenous phosphagens to the energetics of isometric contraction are not known with certainty. To this end, the time course of the tissue content of ATP, ADP, AMP, phosphocreatine (PCr), inorganic phosphate (Pi), and lactate were measured during a KCl-induced isometric contraction in porcine carotid artery and compared with values in the basal state. Oxygenated unpoisoned strips were frozen at 0, 0.5, 1, and 15 min of contraction, and tissue extracts were analyzed using analytical isotachophoresis. No statistically significant changes from the basal levels of ATP and PCr were measured. A small but significant increase in ADP was seen at all times. An increase in Pi of 1.25 mumol/g was observed at 0.5 min, which decreased in time. Tissue lactate content increased by 1.79 mumol/g after 1 min of contraction. The calculated range of cellular free ADP (ADPfree), 44-123 microM, may be sufficient to saturate oxidative phosphorylation. This and the apparent lack of change of ADPfree from basal during contraction suggest that it does not play a role in the coordination of metabolism and contractility. From as early as 0.5 min, when less than 40% of peak isometric force is attained, intermediary metabolism provides the total ATP required for contraction.

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Myosins isolated from mixed rabbit skeletal muscle and several smooth muscles differ with respect to the rate of ATP hydrolysis and the degree of inhibition of the potassium-activated ATPase activity at saturating actin concentrations. Kinetic studies indicate that smooth muscle myosins are tightly bound to skeletal F-actin in the presence of ATp. This observation led to the hypothesis that ATP is a poor dissociating agent for smooth muscle myosin and skeletal F-actin. The kinetic interpretation was verified by electron microscopic observations of myosin binding to F-actin filaments in the presence of ATP.

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A study of the K+-activated myosin ATPase activity, which was measured at high ionic strength in the absence of divalent cations, permitted estimates of the actin-myosin interaction under conditions where (i) myosin-myosin interactions were prevented, (ii) the actin-myosin interaction could be studied in the presence of ATP, and (iii) variation in myosin light chain phosphorylation did not alter smooth muscle myosin ATPase activity. A comparison of myosins isolated from swine carotid arteries and mixed (leg and back) rabbit skeletal muscle was conducted in the presence and absence of rabbit skeletal actin. It was found that (i) arterial myosin, like skeletal myosin, exhibited hyperbolic kinetics for ATP hydrolysis, (ii) specific ATPase activities were significantly higher for skeletal myosin, (iii) saturating concentrations of actin appear to totally inhibit the arterial myosin ATPase activity, but only partially inhibit skeletal myosin activity, (iv) the free actin concentration required for half-maximal inhibition was significantly lower for the arterial myosin ATPase activity than for the skeletal myosin activity; (v) unlike skeletal actomyosin, arterial actomyosin exhibits tight binding characteristics in the presence of ATP, (vi) the binding stoichiometry for arterial myosin to skeletal F-actin was 2 mol of actin monomer/mol of myosin. These observations reveal differences in the interaction of arterial and skeletal myosin with actin, and may in part, explain the high force-generating characteristics of arterial smooth muscle.

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