Beta-adrenergic receptors in rat myocardium: Direct detection by a new fluorescent beta-blocker

Summary A new fluorescent -blocker, 9-amino-acridin propranolol (9-AAP), was administered i.v. to rats. Multiple fluorescent 9-AAP binding sites were observed on cardiac muscle cells in frozen sections. Intensity and density of cardiac 9-AAP fluorescence were markedly reduced following pretreatment with (±)- and (–)-propranolol but not with (+)-propranolol. Our findings suggest that 9-AAP may label -adrenergic receptor sites in rat myocardium.D. A. is supported by a fellowship from the Lady Davis Fellowship Trust and by a Research Grant from the Bat-Sheva de Rothschild Fund for The Advancement of Science and Technology. The technical assistance ofA. Itzhak is gratefully acknowledged.     

High affinity binding of 5-hydroxytryptamine to some membrane preparations from cattle brain. Relationship to lysergic acid diethylamide (LSD)]

5 hydroxytryptamine binds to crude brain membrane preparations with two different affinities (KD = 1 to 2 X 10(-9) M for the highest, 1 to 2 X 10(-8) M for the lowest). LSD also binds with two affinities (KD = 3 to 4 X 10(-9) M and KD = 2 to 3 X 10(-8) M). Subcellular distribution of these sites shows that binding involves the two binding affinities in microsomal membranes but solely the high affinity binding sites are present in purified synaptosomal membranes. High affinity sites for 5 HT and for LSD are different as no direct competitive inhibition is observed in that case. On microsomal membranes, direct relationship occurs between low affinity binding for 5 HT and high affinity binding for LSD.     

Modification by levo-propranolol of tremors induced by harmine in mice

Summary It has been recently reported that the levo isomer of propranolol possesses anti-serotonin properties in animals. Since harmine-induced behavioural changes in mice are reported to be mediated through central serotonergic receptors, an attempt was made to test whether 1-propranolol would also modify harmine-induced responses by virtue of its antiserotonergic or anti-adrenergic property. The results indicated that 1- and dl-propranolol inhibited central serotonin receptor mediated responses to harmine in mice, a finding that is analogous to other recent observations.     

An analysis of the effects of urethane on cardiovascular responsiveness to catecholamines in terms of its interference with Ca++ mobilization from both intra and extracellular pools

Urethane (1 X 10(-2) - 1 X 10(-1) M) reduced, in a concentration-dependent manner, both intra and extracellular Ca++ dependent noradrenaline-induced contractions of perfused rabbit ear artery as well as the tonic contractions produced by perfusion with high K+ solution. However, a quantitative analysis of the data indicated that for urethane concentrations similar to those found in plasma during anesthesia urethane antagonism is confined to noradrenaline-induced contractions which depend upon the mobilization of Ca++ from intracellular storage sites. In KCl-contracted arteries, urethane enhanced the relaxant effects of isoprenaline. - Urethane reduced the amplitude of contractions of spontaneously beating guinea-pig right atrium at concentrations which have only a limited effect on frequency. In addition, it decreased in a concentration-dependent manner the amplitude of isoprenaline-activated electrically driven, and K+ depolarized guinea-pig right ventricular strips. Urethane had no effect on the chrono and inotropic actions of isoprenaline on cardiac preparations. In in vivo experiments the chronotropic response to low doses of isoprenaline was significantly higher in urethane-treated as compared to unanesthetized rats. The higher dose of isoprenaline tested produced a significant fall in systolic blood pressure in urethane-anesthetized rats. A significant correlation exists between the chronotropic response to isoprenaline and resting heart rate values in urethane-anesthetized rats. These results indicate that urethane, at concentrations similar to those found in plasma during anesthesia selectively interferes with mobilization of Ca++ from intracellular storage sites. In addition, the interference of urethane anesthesia with the isoprenaline chronotropic effect 'in vivo' cannot be explained by a direct interference of urethane with beta-adrenoceptors at cardiac level.     

The anticonvulsant effects of propranolol and β-adrenergic blockade

Summary The anticonvulsant activity of racemic and (+)-propranolol was studied in rats. Neither drug changed the current to produce a minimal seizure in 50% of animals. Both drugs were effective in the maximal electroshock seizure test, the (+) isomer being more potent than the racemic form. Since the (+) isomer is practically devoid of -adrenergic blocking activity, the anticonvulsant effects of propranolol do not result from -adrenergic blockade.We wish to thank Hana Boucek for skilful technical assistance. Propranolol and (+)-propranolol were obtained through the courtesy of Ayerst Laboratories Ltd, in Montreal. This research was supported by the Medical Research Council of Canada, grant MA-4754.     

Effects of pCai and pHi on cell-to-cell coupling

Internal longitudinal resistance (ri), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H+ and Ca2+ may modulate ri is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure ri during various interventions. In some experiments, intracellular pH (pHi) was recorded simultaneously to study the pHi-ri relation. Both intracellular Ca2+ and H+ independently modified ri. However, internal resistance of cardiac fibers was insensitive to pHi changes compared to other tissues. A latent period preceded the pHi-related changes in ri and the amount of change depended upon methodology. The results suggest that direct action of protons or ri may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H+- or Ca2+-dependent phosphorylation of junctional proteins.     

Chemical and photooxidation of thiothixene (Navane®): Structure of the thiothixene fluorophor

Summary The spontaneous photooxidation of thiothixene (Navane^®) to 2-(N,N-dimethyl-sulfonamido)-9-thioxanthone is described. The corresponding sulfoxide is demonstrated to be the fluorescent species obtained upon permanganate oxidation of thiothixene in the fluorometric assay procedure of Mjörndal and Oreland.Acknowledgments. The authors wish to acknowledge the facilities and helpful suggestions provided by Drs R. N. Adams and G. L. Grunewald of the University of Kansas. This work was supported by UPHS grant GM 1341.     

The anticonvulsant effects of propranolol and beta-adrenergic blockade.

The anticonvulsant activity of racemic and (+)-propranolol was studied in rats. Neither drug changed the current to produce a minimal seizure in 50% of animals. Both drugs were effective in the maximal electroshock seizure test, the (+) isomer being more potent than the racemic form. Since the (+) isomer is practically devoid of beta-adrenergic blocking activity, the anticonvulsant effects of propranolol do not result from beta-adrenergic blockade.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts.

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilatation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Isolierung fluoreszierender Stoffe ausAstacus fluviatilis

Summary We succeeded in isolating three fluorescent substances fromAstacus fluviatilis (crayfish) which were recently found also inDrosophila melanogaster. These compounds are Isoxanthopterin (2-amino-6, 9-dioxypterin), 2-amino-6-oxypterin and HB₂.     

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50?% of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72?h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.     

Intercellular junctions of hyperplastic retinal pigment epithelium

In rats with retinopathies induced by excess fluorescent light or injections of urethane, the retinal pigment epithelium (RPE) undergoes focal hyperplasia. Neither intravascularly injected horseradish peroxidase or lanthanum nitrate penetrated the sensory retina at these hyperplastic sites. Electron microscopy revealed that this was due to the persistence of intact tight junctions among a single layer of hyperplastic cells facing the sensory retina. These junctions prevented intraocularly injected microperoxidase from passing as well. Cells within the hyperplastic foci were connected only by adherent junctions that presented no permeability barrier.     

Action,in vitro et in vivo,de l'angiotensine II sur le captage cardiaque de la noradrénaline

Summary The action of angiotensin II on cardiac uptake of norepinephrine was investigated in the rat in vivo and in vitro. In contrast to desipramine, neither infusion of subpressive (10 ng/kg/min) or pressive (50–150 ng/kg/min) amounts of angiotensin on intact and/or binephrectomized rats, nor incubation of cardiac slices with angiotensin II (10^–5; 10^–9 M) impair the accumulation of tritiated norepinephrine and the level of metabolites. It is thus concluded that there is no inhibiting action of angiotensin II on the cardiac uptake of norepinephrine.     

Modification by levo-propranolol of tremors induced by harmine in mice

It has been recently reported that the levo isomer of propranolol possesses anti-serotonin properties in animals. Since harmine-induced behavioural changes in mice are reported to be mediated through central serotonergic receptors, an attempt was made to test whether 1-propranolol would also modify harmine-induced responses by virtue of its anti-serotonergic or anti-adrenergic property. The results indicated that l- and dl-propranolol inhibited central serotonin receptor mediated responses to harmine in mice, a finding that is analogous to other recent observations.     

Regulation of the cardiac sodium pump

In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

3[H]-Sulpiride labels mesolimbic non-dopaminergic sites that bind antidepressant drugs

3[H]-(-)-Sulpiride and 3[H]-spiperone binding was compared in rat amygdala, nucleus accumbens and striatum, using (+/-)-sulpiride to define specific binding. 3[H]-(-)-Sulpiride bound to twice as many sites in amygdala and nucleus accumbens as 3[H]-spiperone. 3[H]-(-)-Sulpiride binding was directed to these additional sites by using 1 microM spiperone to mask dopaminergic binding. The binding of 3[H]-(-)-sulpiride to these sites was high affinity, reversible, Na+-dependent, but not stereospecific. Metoclopramide, tiapride and antidepressant medications, but not other neuroleptics, ADTN, or serotonin displaced 3[H]-(-)-sulpiride binding to these sites. These data suggest that 3[H]-(-)-sulpiride labels mesolimbic sites other than dopamine receptors which may mediate antidepressant effects.     

Quantitative differences in the pharmacological effects of (+)- and (-)-cathinone

The optically pure isomers of cathinone were prepared by separating synthetic cathinone racemate and used to study central and peripheral effects of these indirect sympathomimetics in rats and guinea pigs. The (-)-isomer was significantly more potent than the (+)-isomer in stimulating locomotor activity whereas no difference was observed with respect to their cardiac effects. In analogy to observations with (+)- and (-)-amphetamine such variable isomer discrimination may be due to different stereoselectivities of amine uptake mechanisms in the target tissues.     

Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.