Biomedical subjects

P F Dillon

Publications and source records attributed to P F Dillon.

28 records · Page 2Linked to original sources

PubMed1988

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.

Adenosine Diphosphate↗

PubMed1987

Phosphorylation potential and adenosine release during norepinephrine infusion in guinea pig heart.

This study tested the hypothesis that adenosine released from isolated guinea pig hearts (n = 5) in response to norepinephrine is related to the cellular phosphorylation potential (PP; [ATP]/[ADP][Pi]), where Pi is inorganic phosphate. 31P-nuclear magnetic resonance (NMR) was used to measure the relative concentrations of Pi, phosphocreatine (PCr), and ATP. Hearts were Langendorff perfused with a physiological salt solution containing 0.1 mM Pi. The venous effluent was collected for measurement of adenosine and partial pressure of oxygen (PO2). After a control period, norepinephrine (6 X 10(-8) M) was infused for 20 min during which 31P-NMR spectra and samples of venous effluent were collected every minute. With norepinephrine infusion, PCr decreased rapidly to 72% of control (P less than 0.05) by 8 min and then recovered to 80% of control for the remaining 12 min. ATP fell slowly to 70% of control (P less than 0.01) over 20 min. Pi increased to a peak at 2 min (P less than 0.01), then declined slowly to a steady state (60% of the peak and 3.5 X control) from 8 to 20 min. Adenosine release increased from 11 +/- 6 to a peak of 250 +/- 68 pmol.min-1.g-1 (P less than 0.01) at 7 min and then slowly fell (P less than 0.05) to a steady state of approximately 110 pmol.min-1.g-1 (P less than 0.01 vs. control) from 10 to 20 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1987

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)

Animals↗

PubMed1986

31P NMR spectroscopy, chemical analysis, and free Mg2+ of rabbit bladder and uterine smooth muscle.

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.

Adenosine Triphosphate↗

PubMed1983

The role of myosin light chain phosphorylation in regulation of the cross-bridge cycle.

Ca2+ binding to myofibrillar regulatory sites can produce conformational changes allowing cross-bridge attachment and cycling. Measurements of smooth muscle actomyosin ATPase activity suggested that Ca2+ might act indirectly to mediate cross-bridge attachment by stimulating myosin light chain phosphorylation. However, the predicted obligatory relationship between developed force and myosin phosphorylation was not always observed in living smooth muscle. The observation that myosin phosphorylation was always tightly correlated with average cross-bridge cycling rates estimated from isotonic shortening velocities suggested that Ca2+ has two regulatory roles. One action is exerted via a Ca2+-binding protein whose identity is unknown in smooth muscle. This regulatory site acts like other Ca2+-binding regulatory proteins in muscle to permit cross-bridge interaction and to determine active stress. The second regulatory role involves stimulation of myosin light chain kinase and light chain phosphorylation. Increasing the level of phosphorylated cross-bridges increases shortening velocities or rate of force development. We suggest that the dephosphorylated cross-bridges are noncycling or slowly cycling in activated smooth muscle. Smooth muscle may be a particularly favorable experimental preparation for demonstrating a general regulatory role of myosin phosphorylation in modulating the kinetics and energetics of muscle contraction.

Actins↗

PubMed1982

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.

Animals↗

PubMed1982

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.

Animals↗

PubMed1981

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.

Actins↗

PubMedDate not supplied

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.

Adenosine Diphosphate↗

PubMedDate not supplied

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.

Animals↗