Rabbit Myocardial Membranes Research Articles

Structure-activity relationships of inotropic bipyridines: crystallographic analysis of four milrinone analogues

1,6-Dihydro-2'-methyl-6-oxo-[3,4'-bipyridine]-5-carbonitrile (I), C 12 H F N 3 O, M r = 211.23, monoclinic, C2/c, a = 13.152(2), b = 8.987(2), c = 16.538(3) A, β = 95.60 (1)°, V = 1945.4(5) A 3 , Z = 8, D x = 1.442 Mg m -3 , λ(Mo Kα) = 0.71073 A, μ = 0.090 mm -1 , F(000) = 880, T = 90K, R = 0.066 for 1997 observed reflections. 1,6-Dihydro-2,2'-dimethyl-6-oxo-[3,4'-bipyridine]-5-carbonitrile (II), C 13 H 11 N 3 O, M r = 225.25, triclinic, P1, a = 6.704 (1), b = 6.846 (1), c = 13.273 (3) A, α = 81.31 (2), β = 85.28 (2), γ = 66.02 (2)°, V= 550.0 (2) A 3 , Z = 2, D x = 1.360 Mg m -3 , 1(Mo Kα) = 0.71073 A, μ = 0.084 mm -1 , F(000) = 236, T = 90K, R = 0.056 for 2301 observed reflections. 5-Amino-2-methyl-[3,4'-bipyridin]-6(1H)-one (III), C 11 H 11 N 3 O.HCl.2H 2 O, M r = 273.72, triclinic, P1, a = 7.200(2), b = 10.695(2), c = 17.556(4) A, α = 77.20(2), β = 87.94(2), γ = 83.58 (2)°, V = 1310.0 (7) A 3 , Z = 4, D x = 1.388 Mg m -3 , λ(Mo Kα) = 0.71073 A, μ = 0.293 mm -1 , F(000) = 576, T = 291 K, R = 0.067 for 4921 reflections. 5-Amino-2'-methyl-[3,4'-bipyridin]-6(1H)-one (IV), C 11 H 11 N 3 O, M r = 201.23, monoclinic, P2 1 /c, a = 9.5350(9), b = 14.310(2), c= 7.4961(8) A, β = 105.280(1)°, V = 986.7(4) A 3 , Z = 4, D x = 1.355 Mg m -3 , λ (Mo Kα) = 0.71073 A, μ = 0.085 mm -1 , F(000) = 424, T = 160K, R = 0.047 for 1732 reflections. There is a broad range of molecular conformations for these bipyridines, as reflected by the bipyridine torsion angle C(2)-C(1)-C(1')-C(2'). The largest angles are observed for the two 2'-methyl milrinone analogues (I) and (II), which have values of 144.2(2) and 74.3 (2)°, respectively, compared with that of 42.3° observed for milrinone. The addition of either a 2- or 2'-methyl to amrinone causes the almost coplanar parent structure to adopt twist angles of 38.5 (2) and -36.8 (2)° for molecules 1 and 2 of (III), respectively, and 59.4 (3)° for (IV). A common feature of these structures is a hypercentric distribution of their normalized structure factors. Ring-ring stacking interactions observed in these structures contribute to the hyperparallelism observed in some crystal lattices or the creation of superlattices by the addition of noncrystallographic symmetry in other structures. These bipyridine molecules form a network of hydrogen bonds with the keto O or pyridine N atoms acting as acceptors and the pyridone 3-N or 5-amino group acting as donors. Each of the two independent bipyridinium cations of (III) form alternate layers in the lattice and are involved in a two-dimensional network of hydrogen bonds. Biochemical activity, as measured by the stimulatory effect on rabbit myocardial membrane Ca 2+ -ATPase activity, reveals that only (II) and (III) have appreciable activity with 49 and 57% of enzyme stimulation by milrinone, respectively.

P1-173 - Agonist-independent inhibition by Gi of isoproterenol-stimulated adenylate cyclase activity in rabbit myocardial membranes

P1-173 - Agonist-independent inhibition by Gi of isoproterenol-stimulated adenylate cyclase activity in rabbit myocardial membranes

Antiarrhythmic agent amiodarone possesses calcium channel blocker properties.

Amiodarone possesses multiple pharmacologic properties, including peripheral and coronary vasodilatation, negative inotropy, and negative chronotropic and dromotropic effects. These properties are shared by the group of drugs termed calcium channel blockers. We examined the interaction of amiodarone with receptors for the 1,4-dihydropyridine (DHP) calcium blockers in rat and rabbit myocardial membrane particulates. Amiodarone displaced specifically bound [3H]nitrendipine in both rat and rabbit preparations in a competitive, concentration-dependent manner at a single class of binding sites (Ki approximately 0.27 micxroM). Calcium channel activity was determined pharmacologically in a tissue bath with electrically stimulated rabbit right ventricular strips, KCl-induced aortic ring contraction, and 45Ca2+ uptake in K(+)-depolarized cultured rat cardiomyocytes. Amiodarone completely inhibited myocardial contraction (EC50 = 1.7 microM), completely antagonized depolarization-induced aortic ring contraction (EC50 = 24 nM), and significantly reduced (29% vs. control) 45Ca2+ uptake into cultured cells. The calcium channel blocking effects of amiodarone may contribute significantly to its pharmacologic profile.

O-79 - Influence of pertussis toxin treatment on binding properties of muscarinic and β-adrenergic receptors on rabbit myocardial membranes.

O-79 - Influence of pertussis toxin treatment on binding properties of muscarinic and β-adrenergic receptors on rabbit myocardial membranes.

Histamine receptors mediating a positive inotropic effect in guinea pig and rabbit ventricular myocardium: distribution of the receptors and their possible intracellular coupling processes.

The difference in histamine receptor subtypes that are involved in the positive inotropic effect of histamine in guinea pig and rabbit ventricular myocardium was analytically characterized. In guinea pig papillary muscles, the positive inotropic effect of histamine was antagonized by cimetidine but not by mepyramine. The converse was true in rabbit papillary muscles. However, histamine evoked a positive inotropic effect through H1- and H2-receptors after blockade of H2- and H1-receptors in guinea pig and rabbit papillary muscles, respectively. Adenylate cyclase was significantly activated by histamine via H2-receptors in guinea pig but not in rabbit myocardial ventricular membranes. Accumulation of [3H]inositol monophosphate in ventricular strips prelabeled with myo-[3H]inositol was increased by histamine via H1-receptors to a similar extent in rabbits and guinea pigs. Radioligand binding experiments with [3H]mepyramine and [3H]tiotidine showed an increased number of H1-receptors and a decreased number of H2-receptors in guinea pig compared with rabbit ventricular myocardium. These results suggest that the positive inotropic effects of histamine are dominated by an H1-receptor-mediated effect in rabbits and by an H2-receptor-mediated one in guinea pig ventricular myocardium, and the positive inotropic effect manifested by one subtype apparently restricts the expression of the positive inotropic effect mediated by the other subtype. This species difference is not due to a difference in densities of the receptor subtypes, but may be partly related to a difference in the extents of coupling of H2-receptors to adenylate cyclase.

The Role of L-Type Voltage-Dependent Calcium Channels in Anesthetic Depression of Contractility

In this chapter data are presented to show that not only halothane but also enflurane and isoflurane decrease the density of binding sites for the calcium antagonist [ 3 H]isradipine in adult and newborn rabbit myocardial membranes. The chapter provides an observation that halothane can interfere with the binding of calcium channel antagonist (CCA) to cardiac L-type voltage-dependent Ca 2+ channel (VDCC). It has been convincingly demonstrated that the other volatile anesthetics, enflurane and isoflurane, are both able to interfere with the binding of a radiolabeled dihydropyridine, [ 3 H]isradipine, to both cardiac and skeletal muscle membranes. This phenomenon is relevant to anesthetic depression of contractility in an intact organ. Halothane can actually protect VDCC from blockade by isradipine. However, the sites have been protected by halothane appear to have a markedly lower affinity for [ 3 H]isradipine than the sites that has been untouched by halothane. The decreased affinity that has been observed in the VDCC, “protected” by halothane suggests that halothane is either modifying the VDCC or selecting an independent group of VDCC. Isradipine is known to bind to inactive channels with high affinity, and it is possible that halothane “protects” channels that are in the “unavailable” state or that have been modified by phosphorylation or other mechanisms resulting in a change of the K d for isradipine. This is a distinctly different result from previous in vitro data and from the K d values obtained from control hearts and hearts perfused with isradipine alone.

Rabbit skeletal muscle sarcoplasmic reticulum Ca 2+-ATPase activity: stimulation in vitro by thyroid hormone analogues and bipyridines

Sarcoplasmic reticulum-enriched membranes from rabbit skeletal muscle contained Ca 2+-ATPase activity which was significantly enhanced (26% increase, P < 0.001) in vitro by physiological concentrations (10 −10 M) of L-thyroxine (T 4) and 3,3′,5-triiodo- L-thyronine (T 3). In contrast, the biologically inactive iodothyronine analogues D-T 4 and 3,3′,5,5′-tetraiodothyroacetic acid (Tetrac) (10 −10 M) were without effect on enzyme activity. 3,5-Dimethyl-3′-isopropyl- L-thyronine (Dimit), a bioactive analogue, was highly effective as a Ca 2+-ATPase stimulator, increasing enzyme activity by 43% ( P < 0.02 vs. T 4 effect). A bipyridine cardiac inotropic agent, milrinone, has been reported to be thyromimetic in a myocardial membrane Ca 2+-ATPase system, and in concentrations from 10 −10 to 10 −5 M enhanced skeletal muscle SR membrane Ca 2+-ATPase activity in vitro ( P < 0.001). Milrinone analogues which have been previously shown to enhance rabbit myocardial membrane Ca 2+-ATPase activity, and which have a twist relationship of the pyridine rings, were also striated muscle Ca 2+-ATPase stimulators. We conclude that (1) striated muscle is a mammalian tissue in which physiological levels of biologically relevant thyroid hormone analogues, particularly Dimit, stimulate Ca 2+-ATPase activity in vitro by a non-genomic mechanism; (2) cardiac bipyridine analogues which are thyromimetic in vitro in rabbit heart, and which have structural homologies with thyroid hormone, are stimulators of rabbit striated muscle sarcoplasmic reticulum Ca 2+-ATPase activity.

Affinity labeling of forskolin-binding proteins comparison between glucose carrier and adenylate cyclase

An [125I]iodoazidosalicylic acid derivative of forskolin was synthesized for identification of the diterpene's binding sites on the catalytic subunit of adenylate cyclase and on glucose transport proteins. The affinity label was selectively incorporated into proteins of Mr 40 000–60 000 in membranes from human erythrocytes and from various other tissues. The iodoazidosalicylic acid derivative also specifically labeled the catalytic moiety of adenylate cyclase from rabbit myocardial membranes. However, the structural requirements of the two forskolin-binding sites must be different, since the affinity of the photolabel for the glucose carriers is much higher than that for the cyclase catalyst. Furthermore, the label is readily competed with by D-glucose and cytochalasin B for its binding site on the glucose carrier but not on adenylate cyclase.

Species Differences in the Negative Inotropic Response of 1,4-Dihydropyridine Calcium Channel Blockers in Myocardium

To determine if species differences exist in myocardial response to 1,4-dihydropyridine (DHP) calcium channel blockers, the binding and pharmacologic responses of a series of DHP compounds were examined in both rat and rabbit myocardium. [3H]Nitrendipine was used to label specific binding sites in myocardial membrane particulates. The results of saturation binding experiments (n = 3) indicated no statistically significant difference in either Kd or Bmax between rat and rabbit myocardial membranes (0.19 +/- 0.02 nM and 157 +/- 29 fmol/mg protein in rat and 0.14 +/- 0.06 nM and 227 +/- 125 fmol/mg protein in rabbit). Furthermore, [3H]nitrendipine binding inhibition experiments using 12 unlabeled DHP analogues yielded Ki values for each compound that were almost identical in myocardium from rat and rabbit, resulting in an excellent 1:1 correlation when data for all of the compounds were compared (r = 0.997, p less than 0.001). The negative inotropic effect of five of these DHP compounds was studied in vitro in isolated right papillary muscles from rabbit and right ventricular strips from rat, and concentration required to displace 50% of ligand binding (IC50) values for inhibition of contraction were determined. The IC50 values were significantly greater in rat myocardium than in rabbit myocardium (p less than 0.003). Therefore, a significantly lower potency of DHP calcium channel blockers has been demonstrated in rat compared with rabbit myocardium, and this species difference cannot be explained by a difference in the DHP binding site. Rat myocardium differs from rabbit myocardium in a number of ways that may explain this lower potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium channel blocker inhibition of the calmodulin-dependent effects of thyroid hormone and milrinone on rabbit myocardial membrane Ca 2+-ATPase activity

The Ca 2+-ATPase activity of rabbit myocardial membranes is stimulated in vitro by l-thyroxine and by milrinone, a bipyridine. These effects are concentration dependent and calmodulin requiring. The calcium channel blockers nifedipine and verapamil have been reported to have anticalmodulin effects in other assay systems. In this study we have examined the effects of nifedipine and verapamil on rabbit myocardial membrane Ca 2+-ATPase activity, in the absence (basal activity) and presence of exogenous l-thyroxine (T4), 10 −10M, and milrinone, 10 −7 M. Basal enzyme activity was inhibited by a minimum of 10 −6 M nifedipine ( ic 50 of 3.4 × 10 −5 M) and 10 −5 M verapamil ( ic 50 of 1.5 × 10 −4 M). Both calcium antagonists inhibited enzyme stimulation by T 4 and milrinone, with halfmaximal inhibition of T 4 and milrinone effects, respectively, at 2.9 × 10 −5 M and 9.0 × 10 −6 M nifedipine and 3.0 × 10 −5 M and 5.2 × 10 −5 M verapamil. The addition of exogenous purified calmodulin, 40 ng/μg, membrane protein, in the presence of 10 −5 M nifedipine or verapamil restored T 4-stimulated enzyme activity. Nifedipine and verapamil, each at a concentration of 10 −6 M, significantly inhibited binding of radioiodinated calmodulin to rabbit heart membranes in vitro. These studies provide evidence that nifedipine and verapamil have an anti-calmodulin effect in this myocardial enzyme system. Through interaction with calmodulin, the channel blockers inhibit thyroid hormone and milrinone stimulation of myocardial membrane Ca 2+-ATPase.

Competition of milrinone, a non-iodinated cardiac inotropic agent, with thyroid hormone for binding sites on human serum prealbumin (TBPA)

Milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1 H)-one] is a positive cardiac inotropic agent recently shown to have thyromimetic activity in vitro in a rabbit myocardial membrane Ca 2+-ATPase system [K. M. Mylotte et al., Proc. natn. Acad. Sci. U.S.A. 82, 7974 (1985)]. In the present studies, milrinone was examined for activity as an inhibitor of iodothyronine binding by human serum thyroid hormone transport proteins, thyroxine-binding globulin (TBG), prealbumin (TBPA) and albumin. Polyacrylamide gel electrophoresis at pH9.0 of sera equilibrated with [ 125I]thyroxine showed that milrinone competed with l-thyroxine (T 4) for binding sites on TBPA (10 and 100 μM milrinone caused 61 and 73% reductions, respectively, in T 4 binding to TBPA, P < 0.01); T 4 displaced from TBPA was bound by TBG and albumin. Comparable reductions in T 4 binding to TBPA were observed in electrophoretic studies conducted at pH 7.4. Binding of triiodo- l-thyronine (T 3) to TBPA was electrophoretically confirmed and shown to be decreased in the presence of milrinone. Electrophoresis of purified TBPA also demonstrated that [ 14C]milrinone co-migrated with this transport protein and that milrinone displaced tracer T 4 from TBPA. Amrinone, the 2-H-5-NH 2 analog of milrinone, had less than 5% of the activity of milrinone as an inhibitor of T 4 binding in electrophoretic studies. Scatchard analysis of T 4 and milrinone binding to purified TBPA, measured by equilibrium dialysis, showed two classes of binding sites, with association constants, respectively, of 6.1 × 10 7 M −1 and 1.6 × 10 6 M −1 for T 4, and 1.7 × 10 6 M −1 and 8.9 × 10 2 M −1 for milrinone. Computer graphic modeling of the binding of milrinone to the T 4 site in the crystal structure of TBPA showed that milrinone best occupied this site when the substituted bipyridine ring overlapped the phenolic ring of T 4. In this orientation the 5-cyano group, which has an electronegativity similar to that of iodine, occupied the same volume as the 5'-iodine of T 4. The 5-amino group of amrinone lacks these characteristics. In this orientation, the keto function of milrinone overlapped the T 4 4'-hydroxyl and could participate in similar intermolecular interactions. Thus, milrinone, a non-iodinated bipyridine, and thyroid hormone share structural and biochemical homologies and compete for the same binding site on TBPA.

Forskolin-stimulated adenylate cyclase activity in fetal and adult rabbit myocardial membranes

It has previously been reported that there is an increase in the concentration of myocardial β-adrenergic receptors as well as an increase in the sensitivity of adenylate cyclase stimulation by l-isoproterenol in fetal rabbit myocardial membranes from 21 to 31 days' gestation. To investigate whether a change in the catalytic component of the β-adrenergic receptor-adenylate cyclase complex is responsible for the observed increase in adenylate cyclase activity, I have measured the latter in the presence of forskolin and manganese chloride, compounds that can directly stimulate the catalytic component. Myocardial membranes were obtained from fetal rabbits at 21, 28, and 31 days' gestation and from pregnant adult animals. There was an increase in basal adenylate cyclase activity from the fetal to the adult stage. The percent of maximal stimulation by l-isoproterenol in fetuses from 21 through 28 to 31 days' gestation and in the adult animal. However, although forskolin was the most potent stimulator of adenylate cyclase, the degree of stimulation was equivalent in all groups tested. Similar results were obtained with manganese chloride. We conclude that the catalytic component of the β-adrenergic receptor-adenylate cyclase complex in myocardial membranes is stimulated to an equivalent degree at all gestational ages in fetal and aduit rabbits and shows no maturational changes.

Differential activities of tolbutamide, tolazamide, and glyburide in vitro on rabbit myocardial membrane Ca2+-transporting ATPase activity.

At clinically achievable concentrations (10(-9) to 5 X 10(-6) M), tolbutamide and tolazamide are in vitro inhibitors of Ca2+-transporting ATPase activity in sarcolemma-enriched rabbit myocardial membranes (sulfonylurea IC50, 10(-7) M). Thyroid hormone stimulation of this calcium pump-associated enzyme in vitro has been previously reported; in our study, this hormonal action was shown to be inhibited by tolbutamide and tolazamide. In contrast to these two sulfonylureas, glyburide (up to 5 X 10(-6) M) had no effect on basal or thyroid hormone-stimulable Ca2+-ATPase activity in vitro. Studies of binding of radiolabeled purified calmodulin to heart membranes showed that tolbutamide and tolazamide inhibited this interaction, whereas glyburide had no effect on calmodulin binding. Addition of purified calmodulin (5-40 ng/micrograms membrane protein) to myocardial membranes incubated with 10(-7) M tolbutamide or tolazamide restored Ca2+-ATPase activity and thyroid hormone responsiveness of the enzyme. Inhibition by tolbutamide and tolazamide of myocardial sarcolemmal Ca2+-ATPase is a mechanism by which these two sulfonylureas may at least transiently raise resting sarcoplasmic Ca2+ concentration. This effect of sulfonylureas on Ca2+-ATPase is not expressed in the presence of the benzamide side chain of glyburide. The inhibitory action of certain sulfonylureas on Ca2+-ATPase is mediated by interference of the agents with the binding of calmodulin to cardiac membranes.

Differential activities of tolbutamide, tolazamide, and glyburide in vitro on rabbit myocardial membrane Ca2+-transporting ATPase activity

Differential activities of tolbutamide, tolazamide, and glyburide in vitro on rabbit myocardial membrane Ca2+-transporting ATPase activity

Catalytic unit of adenlyate cyclase: purification and identification by affinity crosslinking.

The guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocardial membranes was purified approximately equal to 60,000-fold to a specific activity of 15 mumol X mg-1 X min-1 by Lubrol PX extraction, affinity chromatography, and gel permeation HPLC. The major purification (greater than 2000-fold) was achieved by affinity chromatography on forskolin-Sepharose, a method previously developed in this laboratory. The final product appeared as two major peptides of Mr 150,000 and 42,000 and one minor peptide of Mr 45,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. It is suggested that the Mr 42,000 and 150,000 components represent the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein (GS) and the catalytic unit, respectively, because upon crosslinking of a reconstituted adenylate cyclase containing the [32P]ADP-ribosylated alpha-subunit of GS (Mr, 42,000), a single radiolabeled product of Mr 190,000 appeared on NaDodSO4/polyacrylamide gels. Further identification is based on the correlation of the Mr 150,000/42,000 bands with enzymatic activity when the purified enzyme was analyzed by various chromatographic procedures.

Adenylate Cyclase Activity in Fetal Rabbit Myocardium

Adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was determined in vitro in fetal rabbit myocardial membranes from individual fetal pups at 21 to 31 days gestation (term, 31 days). Basal and NaF-stimulated adenylate cyclase activities did not change during 21-31 days gestation. Significant stimulation of the enzyme by l-isoproterenol was observed only in the presence of guanosine triphosphate (100 microM). Under these conditions, maximal adenylate cyclase stimulation by l-isoproterenol (100 microM) was significantly higher at 25-31 than at 21 days gestation. Moreover, EC50 (Kact) for l-isoproterenol at 25-31 days was significantly lower than at 21 days gestation. We conclude that, in fetal rabbit myocardial membranes, there is an increase in the sensitivity of adenylate cyclase stimulation by l-isoproterenol from 21 to 25-31 days gestation.

Rabbit myocardial membrane Ca 2+-adenosine triphosphatase activity: Stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca 2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca 2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5′-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 ± 3.3 μmol P i mg membrane protein −1 90 min −1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to l-thyroxine (T 4) (10 −10 m) increased Ca 2+-ATPase activity to 29.2 ± 3.8 μmol P i ( P < 0.001). Dose-response studies conducted with T 4 showed that maximal stimulatory response was obtained at 10 −10 m. Hormonal stimulation was comparable for l-T 4 and triiodo- l-thyronine (T 3) (10 −10 m). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and d-T 4, each at 10 −10 m, significantly decreased enzyme activity compared to control (basal) levels. The action of l-T 4 on myocardial membrane Ca 2+-ATPase activity was inhibited by trifluoperazine (100 μ m) and the naphthalenesulfonamide W-7 (50–100 μ m), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca 2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 μg mg membrane protein −1) in the myocardial membrane fraction and 0.35 μg mg −1 in cytosol. Myocardial Ca 2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.

Isolation of homologous and heterologous complexes between catalytic and regulatory components of adenylate cyclase by forskolin—Speharose

Homologous and heterologous complexes between catalytic and GTP-binding components can be isolated by means of immobilized succinyldeacetylforskolin (forskolin—Sepharose). A heterologous complex is formed by reconstitution of forskolin—Sepharose bound catalytic function from rabbit myocardial membranes with the homogeneous [ 3H]methyl-GTP-binding protein from duck erythrocyte membranes. Analysis of the reconstituted complex by sodium dodecyl sulfate polyacrylamide gelelectrophoresis reveals that only the M r 42 000 component of the GTP-binding protein's M r 42 000/ M r 35 000 heterodimer contributes to the formation of active adenylate cyclase.

Ethanolamine plasmalogen methylation by rabbit myocardial membranes

Enzymatic methylation of alkenylacylglycerophosphoethanolmine to form alkenylacylglycerophosphocholine was observed in rabbit myocardial membranes, and was compared to the corresponding methylation sequence for diacyl substrates. Membranes were incubated with S- adenosyl- l -[ methyl- 3 H] methionine and assayed for incorporation of radioactivity into selected lipids. The rate of incorporation of methyl groups into diacylglycerophosphocholine exceeded that for alkenylacylglycerophosphocholine, 12.0 ± 3.6 vs. 3.9 ± 0.7 pmol product fonned/ mg per h ( mean ± S. D.), even when normalized for ethanolamine substrate concentration (5.7 ±1.6 vs. 1.8 ± 0.4 pmol CH 3 incorporated/μmol diradylglycerophosphoethanolamine). Rabbit myocardial phospholipid methyltransferase activity is optimal at basic pH for each substrate, is moderately stimulated by added Ca 2+ or Mg 2+ and is completely inhibited by S-adenosylhomocysteine. An apparent K m of 0.2 mM for S-adenosylmethionine applies to diacyl- and alkenylacylglycerophosphocholine formation; at low concentrations of methyl donor (0.003 mM), the monomethylated products accumulate.

Role of Ca 2+ in maintenance of rabbit myocardial cell membrane structural and functional integrity

The structure and function of the sarcolemma's superficial layers and the sarcolemma's dependence upon [Ca] 0 was studied in the isolated arterially perfused rabbit interventricular septum. Perfusion of tissue for 20 min with 0 Ca (< 5 μ m) solution produced a distinctive separation of membrane superficial laminae. These separations were found to be irreversible and associated with an increased cellular 45Ca content and irreversible contracture when normal [Ca] 0 was reperfused. Perfusion with 50 μ m Ca produced no alteration of membrane structure, contractile function or 45Ca uptake. 0 Ca perfusion resulted in a 14% mean cellular 42K loss and this appeared to be independent of superficial membrane separations. Mg supplements in 0 Ca perfusate resulted in membrane separations and contracture intermediate to those of 0 Ca without supplements and 50 μ m Ca; Mg supplements completely protected against cellular K loss. Zero [Ca]-induced separation of superficial laminae correlates with increased Ca permeability and suggests that this membrane region is involved in the control of contractile Ca. Ca ion is also essential for control of K permeability, probably in the lipid bilayer. The importance of the presence of [Ca] 0 to the normal structure and function of these membrane sites is emphasized.