Effect of timing of treatment of the glyburide-reversible cardioprotective activity of BMS-180448.

The effect of the timing of treatment with the ATP-regulated potassium channel agonist BMS-180448 was evaluated in isolated rat heart and ferret models of ischemia and reperfusion. In rat hearts, 10 microM BMS-180448, given before and after global ischemia as well as only during reflow, improved reperfusion contractile function and attenuated lactic dehydrogenase release, although reperfusion-only treatment was less effective. Cromakalim (10 microM) and bimakalim (10 microM) treatment before and after global ischemia afforded a degree of protection similar to that of BMS-180448, although they were not cardioprotective when given only during reperfusion. Pre- and post-treatment cardioprotection were abolished by glyburide. Ischemia/reperfusion significantly increased cytosolic calcium concentration ([Ca++]i) and BMS-180448 given only during reperfusion attenuated this change. In anesthetized ferrets, BMS-180448 (2 mg/kg) or vehicle was infused i.v. during a 40-min interval beginning 1) 10 min before coronary occlusion, 2) at the 45th min of ischemia or 3) at the 5th min of reperfusion. Preocclusion administration of BMS-180448 was associated with a 35% reduction in infarct damage from that recorded in vehicle-treated control ferrets. Drug administered at the midpoint of ischemia reduced infarct size approximately 44%, whereas delaying BMS-180448 infusion until the 5th min of reperfusion reduced, but still provided a significant (17%) level of salvage. The favorable effects of BMS-180448 in the ferret were not associated with changes in either collateral blood flow or peripheral hemodynamics. Thus BMS-180448 shows some protective effects when given only during reperfusion. Cromakalim and bimakalim did not exert similar actions and the difference may be secondary to the faster penetration of BMS-180448.

Glyburide-reversible cardioprotective effects of BMS-180448: functional and energetic considerations.

Adenosine triphosphate (ATP)-sensitive potassium channel openers as a class exert cardioprotective effects, and we can separate vasodilator from glyburide-reversible cardioprotective activity in cromakalim analogs (e.g., BMS-180448). The purpose of this study was to determine the relation between cardiac function, energy status, and cardioprotective effects for BMS-180448 in isolated rat hearts compared with diltiazem. BMS-180448 (1-30 microM) or 0.1-1 microM diltiazem were given 10 min before 25-min global ischemia in rat hearts followed by 30 min of reperfusion. Both compounds significantly increased time to the onset of contracture during ischemia and improved postischemic recovery of contractile function in a concentration-dependent manner. At equivalent cardioprotective concentrations, BMS-180448 depressed preischemic cardiac function significantly less than did diltiazem. During ischemia, diltiazem significantly accelerated the functional decline observed in vehicle-treated hearts, whereas BMS-180448 attenuated the net rate of decline of function. Despite these different effects on preischemic and ischemic cardiac function, diltiazem and BMS-180448 conserved cardiac ATP during ischemia to a similar degree. BMS-180448 enhanced the recovery of ATP (also seen for diltiazem, but not to the same magnitude) and creatine phosphate during reperfusion compared with vehicle-treated hearts. For BMS-180448, this enhanced ATP recovery was accompanied by a significant improvement in the efficiency of oxygen use, which was profoundly reduced in reperfused vehicle-treated hearts. BMS-180448 also significantly enhanced the functional reserve after the 25-min period of global ischemia. Thus BMS-180448 protects ischemic myocardium and conserves ATP with less reduction in cardiac function compared with diltiazem.

KATP-channel openers protect against increased cytosolic calcium during ischaemia and reperfusion.

There is ample evidence that potassium channel openers protect the ischaemic myocardium. Although the protective mechanism is unknown, indirect evidence suggests that potassium channel openers reduce calcium influx during ischaemia which may explain their protective effects. However, recently discovered potassium channel openers such as BMS-180448 are cardioprotective without displaying classical indications of calcium lowering. The current study was designed to provide direct evidence that potassium channel openers delay or prevent increased intracellular free calcium in the myocardium during ischaemia and reperfusion. Cytosolic calcium concentrations were directly measured in perfused rat hearts during global ischaemia by 19F-NMR of the calcium chelator 5F-BAPTA. The cytosolic Ca2+ concentration in vehicle-treated hearts increased from a pre-ischaemia average of 310 +/- 40 nM to 1000 +/- 130 nM during 25 min of ischaemia, followed by partial recovery to 530 +/- 100 nM during 19 min of reperfusion. In contrast, the cytosolic Ca2+ concentration in hearts treated with potassium channel openers BMS-180448 and cromakalim remained low throughout ischaemia, changing from pre-ischaemia averages of 270 +/- 30 nM to 230 +/- 60 nM and from 240 +/- 20 nM to 170 +/- 30 nM during 25 min of ischaemia, respectively. The cytosolic Ca2+ concentrations in these hearts increased to 440 +/- 110 nM in BMS-180448 treated hearts and 290 +/- 60 nM in cromakalim treated hearts during the first 6 min of reperfusion, and were 460 +/- 60 nM for BMS-180448 and 600 +/- 70 nM for after cromakalim 19 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of the membrane in ligand-receptor interactions.

NMR data that underscore the importance of the membrane in ligand-receptor interactions were obtained and analyzed. The following hypothesis for acetylcholine (ACh) binding to the acetylcholine receptor (AChR) is proposed: ACh first binds to the membrane, where it adopts its bioactive conformation, and it then rapidly diffuses along the membrane to bind to the AChR in its already-correct conformation. Data used to support this hypothesis include (a) the NMR-determined binding constant of KM = (2.8 +/- 0.6) x 10(3) M-1 for the binding of ACh to the asolectin membrane, (b) the lipid dependence of AChR activity, (c) the location of the ACh binding site close to the membrane surface, and (d) the conformation of ACh in its membrane-bound state. Additional experiments to test this hypothesis are proposed.

Stroboscopic nuclear magnetic resonance microscopy of arterial blood flow.

NMR microscopy was used to obtain transverse flow profiles of arterial blood flow in the rat carotid artery at 33 microns resolution. The images were gated to the EKG and correspond to identified regions of diastole. The profiles show that flow is laminar during this part of the heart cycle. These results provide the first direct view of blood flow profiles in arteries of submillimeter diameter and suggest that animals as small as juvenile rodents will serve as valuable models for hemodynamic studies. Extensions to flow during systole, stenoses, and flow in the vicinity of the carotid bifurcation are discussed.

Stroboscopic NMR microscopy of the carotid artery.

The non-invasive measurement of vascular dynamics and elasticity is critical in understanding haemodynamic conditions of cardiovascular diseases such as hypertension and atherosclerosis. Although there are numerous invasive and in vitro techniques for such measurements, until now non-invasive methods have been limited. We have now obtained stroboscopic NMR images of the carotid arteries of 80-g rats. The change in the cross-sectional area of arteries of diameter approximately 600-800 microns was correlated with the change in absolute blood pressure. These are the first microimages of a dynamic system and enable the direct visualization of compliance, the non-invasive measurement of Young's modulus, the direct determination of the local effects of vasoconstrictors and vasodilators and the mapping of the entire cardiac cycle.

Conformation of acetylcholine bound to the nicotinic acetylcholine receptor.

We report here the biologically active conformation of acetylcholine when bound to the high-affinity state of the receptor from Torpedo californica. The acetylcholine conformation was determined in the free and bound states by proton NMR two-dimensional nuclear Overhauser effects. In agreement with x-ray crystallographic data, acetylcholine in solution has an extended conformation with an average distance between the acetyl methyl and choline methyl protons of approximately equal to 5 A. When bound to the acetylcholine receptor, acetylcholine adopts a conformation where the acetyl methyl group is close (3.3 A) to the methyl groups of the choline moiety. This bent conformation places the oxygens adjacent to one another and allows the methyl groups to form an uninterrupted hydrophobic surface over the rest of the acetylcholine molecule. The significant difference between the free- and bound-state conformations implies that structure-activity studies based solely on molecular modeling strategies must be approached with caution.

Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments.

A method is presented that uses selective proton Nuclear Magnetic Resonance (NMR) relaxation measurements of nicotine in the presence of the acetylcholine receptor to obtain relative binding constants for acetylcholine, carbamylcholine, and muscarine. For receptors from Torpedo californica the results show that (a) the binding constants are in the order acetylcholine greater than nicotine greater than carbamylcholine greater than muscarine; (b) selective NMR measurements provide a rapid and direct method for monitoring both the specific and nonspecific binding of agonists to these receptors and to the lipid; (c) alpha-bungarotoxin can be used to distinguish between specific and nonspecific binding to the receptor; (d) the receptor--substrate interaction causes a large change in the selective relaxation time of the agonists even at concentrations 100x greater than that of the receptor. This last observation means that these measurements provide a rapid method to monitor drug binding when only small amounts of receptor are available. Furthermore, the binding strategies presented here may be useful for the NMR determination of the conformation of the ligand in its bound state.

Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements.

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies.

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).

1H NMR relaxation studies of the hydrogen-bonded imino protons of poly(dA-dT).

Measurements on the thymine imino proton relaxation rates have been used to study various structural and dynamic properties of 53 +/- 15 base pair long poly(dA-dT). Below 10 degrees C, the relaxation is dominated by dipolar magnetic interactions. At 1 degrees C the relaxation of the transverse magnetization is exponential (R2 = 124 s-1), but the relaxation of longitudinal magnetization is highly nonexponential due to spin-diffusion effects (initial decay rate constant of 28 s-1 and a slower rate of approximately 2.5 s-1 after equilibration of spin polarization). Neither a rigid-rod model nor simple wormlike motions can account for the observed low-temperature relaxation behavior. However, when localized internal motions of the base pairs (three-state jump model) are allowed for, a good fit of the experimental data is obtained by using a correlation time for internal motion of 7 X 10(-10) s and an angular displacement of the bases of +/- 32 degrees relative to the helix axis. The observed R2/R1 ratio for the thymine imino proton yields a value of 1.14 +/- 0.08 A for the imino proton nitrogen distance. Nuclear Overhauser effect (NOE) measurements establish that the base pairing in poly(dA-dT) is Watson-Crick in solution and not Hoogsteen. Exchange of the T-imino protons with H2O dominates the longitudinal relaxation above 28 degrees C (activation energy of 17 +/- 2 kcal and an exchange rate of 5 +/- 2 s-1 at 300 K). Similar values have been reported for the A X T base pairs in DNA restriction fragments and for A X U base pairs in poly(A) X poly(U). These observations can be explained by a model in which exchange of T-imino protons occurs as a result of a single base pair opening, with a rate that is approximately independent of nearest-neighbor sequences and DNA length. Our observations appear to be inconsistent with a soliton model of proton exchange.