The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.

C terminus of presenilin is required for overproduction of amyloidogenic Abeta42 through stabilization and endoproteolysis of presenilin.

Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.

Effects of calcitonin gene-related peptide on membrane currents in mammalian cardiac myocytes.

We examined the effects of calcitonin gene-related peptide (CGRP) on the membrane currents of single atrial and ventricular cells of guinea pig heart. The tight-seal whole-cell voltage-clamp technique was used. In atrial cells, like isoproterenol, CGRP increased the L-type Ca channel current (ICa.L) in a concentration-dependent manner. Human CGRP-(8-37), a putative CGRP receptor antagonist, completely abolished the CGRP-induced increase of ICa.L. Although the effects of CGRP were similar to those of isoproterenol, propranolol, a beta-adrenergic receptor antagonist, did not affect the CGRP-induced increase of ICa.L. After ICa.L had been maximally activated by isoproterenol (2 microM) or intracellular cyclic adenosine 5'-monophosphate (100 microM), CGRP failed to increase ICa.L. Acetylcholine antagonized the effects of CGRP on ICa.L. Unlike the effects on atrial cells, CGRP had no significant effects on the membrane currents of ventricular myocytes. These results indicate that CGRP increases ICa.L via adenylate cyclase activation by binding to specific membrane receptors in cardiac atrial myocytes. Furthermore, CGRP receptors are expressed in atrial cells but probably not in ventricular cells.

Coenzyme Q10 attenuates cyanide-activation of the ATP-sensitive K+ channel current in single cardiac myocytes of the guinea-pig.

The effect of coenzyme Q10 (CoQ10) on the cyanide (CN-)-induced ATP-sensitive K+ channel current (KATP) was examined in single atrial myocytes, using the patch clamp technique. Superfusion of the cells with a CN-/low glucose bathing solution induced an outward current in the whole-cell clamp condition. Glibenclamide (1 microM) abolished this current, indicating that the current was carried through the KATP channel. After steady-state activation by CN-, pinacidil (a KATP channel opener, 300 microM) failed to further increase the current. In cell-attached patches, CN-, when applied to the bath, induced bursting openings of an 80 pS channel (the KATP channel). In cells preincubated for 30 min in a solution containing CoQ10 (100 micrograms/ml), CN(-)-activation of the KATP channel was markedly attenuated both at the whole cell and at the single channel level. At the steady-state effect of CN- in CoQ10-treated cells, pinacidil (300 microM) activated the current to the maximum level achieved by CN- in the control cells. These results suggest that CoQ10 reduces in the CN(-)-induced KATP current not by affecting the channel itself but by preventing depletion of intracellular ATP caused by CN-.

Glibenclamide specifically blocks ATP-sensitive K+ channel current in atrial myocytes of guinea pig heart.

Effects of glibenclamide on the control membrane ionic currents, acetylcholine or adenosine-induced K+ current, and nicorandil-induced K+ current were examined in single atrial myocytes of guinea pig heart. The nystatin-whole cell clamp technique was used. Nicorandil evoked the time-independent K+ current which is probably the current through the ATP-sensitive K+ channel. Glibenclamide inhibited this current in a concentration-dependent fashion, although it had no effect on the other currents. We concluded that glibenclamide specifically inhibits the ATP-sensitive K+ channel current in cardiac myocytes.

Adenosine-5'-triphosphate-induced sinus tachycardia mediated by prostaglandin synthesis via phospholipase C in the rabbit heart.

Effects of adenosine 5'-triphosphate (ATP) and adenosine on cardiac sinus pacemaker activity were examined in the rabbit heart. Electrocardiograms of hearts were recorded while using the Langendorff perfusion method. Both adenosine and ATP, added to the perfusate, slowed the sinus pacemaker activity in a concentration-dependent manner. But in about 40% of the cases. ATP higher than 300 microM initially accelerated and then slowed the heart. The sinus slowing caused by adenosine and ATP was blocked by theophylline (a P1 receptor antagonist) and disappeared in the hearts pre-treated with islet-activating protein (1AP). In contrast, the ATP-induced sinus acceleration was not affected by either theophylline or IAP. In about 75% of the IAP-treated hearts. ATP persistently accelerated the sinus pacemaker. In the remaining 25% of the hearts, ATP caused junctional tachycardia, which may have masked the ATP-induced sinus acceleration. Apamin specifically blocked the ATP-induced sinus acceleration, suggesting that P2 receptors are involved. Among various adenine nucleotide analogues, the order of potency in inducing tachycardia in IAP-treated hearts is adenosine-5'-[gamma-thio]triphosphate greater than adenylyl imidodiphosphate greater than adenosine 5'-[alpha, beta-methylene]triphosphate = ATP greater than adenosine diphosphate = adenosine 5'-[beta, gamma-methylene]triphosphate. ATP-induced acceleration was partially blocked by indomethacin and aspirin (cyclooxygenase inhibitors), but not by nordihydroguaiaretic acid (a lipoxygenase inhibitor). These results suggest that cyclooxygenase and not lipoxygenase metabolites of arachidonic acid, e.g. prostaglandins, may be involved in the generation of tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin uncouples the muscarinic receptors from GK protein in the atrial cell membrane of the guinea-pig heart.

The effects of heparin on activation of the G protein-gated muscarinic K+ channel were examined in atrial cells of guinea-pig heart. The inside-out patch clamp technique was used. The pipette solution contained 1.1 microM acetylcholine (ACh). In the inside-out patches, intracellular GTP activated the muscarinic, K+ channel. When heparin (0.05-5 units/ml) was further added to the intracellular side of the patch membrane, the channel openings were depressed in a concentration-dependent fashion. The effects of heparin were reversible after wash-out. Heparin did not affect GTP-gamma S-induced activation of the K+ channel. Therefore, it is suggested that heparin may uncouple the muscarinic receptors from GK protein in the cardiac atrial cell membrane.

Cardiac Ca current does not run down and is very sensitive to isoprenaline in the nystatin-method of whole cell recording.

The conventional L type Ca2+ channel current (ICa.L) was measured in single atrial and ventricular myocytes, with a new whole-cell recording technique using an ionophore, nystatin. The membrane of a cell-attached patch was gradually permeabilized by nystatin (100-200 micrograms/ml), added to the pipette solution, within 2-5 min after formation of a G omega-seal. The electrical activity of the cells was measured through the pipette. ICa.L, measured with the nystatin-whole cell recording technique, did not exhibit the so-called "run-down" phenomenon for up to 90 min. The response of ICa.L to isoprenaline was also well preserved during the measurement. The half maximal concentration for the isoprenaline-induced increase of ICa.L was 8.2 x 10(-9) M, which is a much smaller value than that reported previously. Thus, the nystatin-whole cell clamp recording is a useful technique to measure membrane currents of cardiac myocytes with preserving the physiological intracellular milieu.

Frequency- and voltage-dependent effects of recainam on the upstroke velocity of action potential in rabbit ventricular muscle.

The effects of recainam (Wy 42,362) on transmembrane action potentials were examined in isolated rabbit right ventricular papillary muscles. Recainam (3 x 10(-5) to 3 x 10(-4) M) caused a concentration-dependent decrease in the Vmax of the action potential. At 3 x 10(-4) M, there was a slight decrease in the amplitude of the action potential. The resting potential and the action potential duration were not affected. Use-dependent block of Vmax was tested over a wide range of pacing frequencies (from 0.1 to 3.0 Hz). At 1.0 Hz, recainam 10(-4) M produced exponential decreases in Vmax with a rate constant of 0.17 per action potential and 39.8% reduction at steady state. This use-dependent block was augmented at the higher stimulation frequencies. The time constant for the recovery of Vmax from use-dependent block (offset) was 17.2 s. In papillary muscles depolarized with 10 mM [K+]0, the use-dependent block was augmented but tonic block and the rates of onset and offset of the use-dependent block were similar to those in normally polarized preparations in 4 mM [K+]0. The curves relating membrane potential and Vmax in preparations stimulated at a low frequency (0.01 Hz) were not shifted by 10(-4) M recainam. These findings suggest that recainam is a specific sodium-channel blocker and has kinetically slow but potent affinity for the channel during action potentials. This selective binding during action potential was further augmented by depolarization and is likely to play a significant role in the control of ventricular arrhythmias by the drug.

Anti-cholinergic effects of quinidine, disopyramide, and procainamide in isolated atrial myocytes: mediation by different molecular mechanisms.

Effects of quinidine, disopyramide, and procainamide on the acetylcholine (ACh)-induced K+ channel current were examined in single atrial cells, using the tight-seal, whole-cell clamp technique. The pipette solution contained guanosine-5'-triphosphate (GTP) or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S, a nonhydrolysable GTP analogue). In GTP-loaded cells, not only ACh but also adenosine induced a specific K+ channel current via GTP-binding proteins (G) by activating muscarinic ACh or adenosine receptors. Quinidine and disopyramide depressed the ACh-induced K+ current quite effectively. Procainamide had a weak inhibitory effect. Quinidine also depressed adenosine-induced K+ current, while the effect of disopyramide on adenosine-induced current was much smaller than that on ACh-induced current. In GTP-gamma S-loaded cells, the K+ channel was uncoupled from the receptors and was activated irreversibly, probably due to direct activation of G proteins by GTP-gamma S. Quinidine depressed the GTP-gamma S-induced K+ current just as in the cases of ACh- and adenosine-induced currents of GTP-loaded cells. Disopyramide had only a weak inhibitory effect and procainamide showed no effect. From these results, it is strongly suggested that the major mechanisms underlying the anti-cholinergic effects of quinidine, disopyramide, and procainamide are different; quinidine may inhibit the muscarinic K+ channel itself and/or G proteins, while disopyramide and high doses of procainamide may mainly block functions of muscarinic ACh receptors in atrial myocytes.

Anti-cholinergic effect of verapamil on the muscarinic acetylcholine receptor-gated K+ channel in isolated guinea-pig atrial myocytes.

Effects of verapamil on the acetylcholine (ACh)-induced K+ current were examined in single atrial cells, using the tight-seal whole-cell clamp technique. The pipette solution contained guanosine-5'-triphosphate (GTP) or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S, a non-hydrolysable GTP analogue). In GTP-loaded cells, ACh induced a specific K+ current, which is known to be mediated by pertussis toxin-sensitive GTP-binding (G) proteins. Verapamil (0.1-100 microM) depressed the ACh-induced K+ current in a concentration-dependent fashion. In GTP-gamma S-loaded cells, the K+ current remained persistently after wash-out of ACh, probably due to irreversible activation of G proteins by GTP-gamma S. Verapamil (0.1-100 microM) also depressed the intracellular GTP-gamma S-induced K+ current. However, the magnitude of verapamil-depression of the K+ current in GTP-gamma S-loaded cells was significantly smaller than that in GTP-loaded cells at concentrations between 1 and 10 microM of the drug. From these results, it is suggested that verapamil may block not only the function of muscarinic ACh receptors but also of G proteins and/or the K+ channel itself and thereby depress the ACh-induced K+ current in isolated atrial myocytes.

Electrophysiologic effects of a new antiarrhythmic agent, recainam, on isolated canine and rabbit myocardial fibers.

Recainam (Wy 42,362) is a new antiarrhythmic agent undergoing clinical evaluation, but its electrophysiologic effects in cardiac muscle are poorly defined. With microelectrode techniques, its profile in isolated preparations of dog and rabbit hearts was determined using drug concentrations of 10 to 300 microM. Recainam induced a concentration- and frequency-dependent decrease in the maximal rate of rise of the phase 0 of the action potential (Vmax), action potential amplitude and overshoot potential, with little or no change in the effective refractory period except in Purkinje fibers, in which it was markedly reduced. At a 300 microM concentration, Vmax was reduced 51% (p less than 0.001) in ventricular muscle and 44% (p less than 0.001) in atrial muscle, with no change in action potential duration or effective refractory period. At the same drug concentration in Purkinje fibers, Vmax was decreased by 41% (p less than 0.01), action potential duration at 90% repolarization by 36% (p less than 0.01) and effective refractory period by 34% (p less than 0.01). Recainam had no significant effect on the sinoatrial node, but it depressed phase 4 depolarization in isoproterenol-induced automaticity in Purkinje fibers. The drug had no effect on slow channel potentials induced by high concentrations of potassium and isoproterenol. The data indicate that the electrophysiologic profile of recainam in isolated cardiac muscle is consistent with the overall effects of class IC agents without having an effect on the slow calcium channel. Its major action is to depress Vmax, with little effect on refractoriness.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of 3,3'5'-triiodothyronine (reverse T3) on cardiac function in the rabbit.

In a recent study, we have shown that the antiarrhythmic drug, amiodarone caused significant elevations of serum rT3 levels and inhibition of the peripheral conversion of T4 to T3 (Kannan et al. Endocrinology 115:1710-1716, 1984). In this study we have examined whether or not rT3 has a direct effect on the monodeiodinase enzyme activity and electropharmacology of the rabbit myocardium. Six male New Zealand White rabbits were administered 50 ug rT3 (a dose about 100 times the daily rT3 production rate) i.p. for 5 days, while the control group received saline. Serum T4, T3 and rT3 concentrations, and myocardial as well as liver and kidney 5'monodeiodinase enzyme activities were measured. Surface ECG and electrophysiological parameters in the atrial, ventricular and sinoatrial node in control and rT3 groups were obtained. Surface ECG did not show any difference in heart rate, RR, PQ, QRS or QT intervals in the two groups. Reverse T3 administration did not produce any significant alterations in APA, MRP, Vmax, APD90 and ERP of atrial and ventricular muscle. Despite administration of a massive dose of rT3, serum levels of T3 and T4 were unchanged as compared to control group. Reverse T3 administration did not produce any alterations in the 5'monodeiodinase activities in the myocardium, liver and kidney. The studies suggest that rT3 does not appreciably affect myocardial function and that the effects of amiodarone on the heart occur via mechanisms other than its ability to cause an increase in serum rT3 levels.

Noninvasive study of the presystolic component of the first heart sound in mitral stenosis.

Echophonocardiography and pulsed Doppler echocardiography were performed in 30 patients with mitral stenosis (19 with atrial fibrillation and 11 with sinus rhythm) to investigate the genesis of the presystolic component or small apical vibrations preceding the first heart sound in mitral stenosis. In 27 patients, mitral valve closure preceded or coincided with tricuspid valve closure regardless of the preceding RR interval. Of three patients whose tricuspid valve closed prematurely, two had a prolonged PR interval. The soft apical vibrations, which were recorded during the final rapid closing motion of the mitral valve echogram (B-C slope), began with the upstroke of the apexcardiogram. During this event the pulsed Doppler echocardiogram revealed a deceleration in the velocity of mitral inflow. In two exceptional patients with a prolonged PR interval, this apical sound was separated from a presystolic rumble that occurred during an accelerated phase of mitral inflow or at the A wave of mitral valve echograms. In conclusion, the tricuspid valve is not a factor contributing to the genesis of the small apical vibrations preceding the first heart sound in mitral stenosis. These vibrations are caused by acceleration of left ventricular contraction and deceleration of mitral inflow in the presence of a stenotic valve.

[Mechanism of systolic anterior motion and left ventricular outflow obstruction in hypertrophic obstructive cardiomyopathy].

Two-dimensional echocardiographic studies were performed for 22 patients with asymmetric septal hypertrophy and systolic anterior motion (SAM) of the mitral valve to clarify the mechanism of SAM and left ventricular outflow obstruction. The long-axis view of the left ventricle showed that a distal portion of the anterior mitral leaflet became surplus when the mitral valve was closed in 15 of 16 patients with SAM-septal contact and in four of six patients without it. In these patients, the mitral leaflet coaptation was displaced anteriorly, and/or the anterior leaflet was elongated, showing an anterior motion. In an unusual patient with SAM-septal contact, a distal portion of the long posterior mitral leaflet was surplus. The remaining two patients without SAM-septal contact had a rather widely coapted portion of the mitral leaflets. The distal surplus portion and the coapted portion of the mitral valve were displaced anteriorly prior to the onset of ejection, and moved forward and protruded into the outflow tract more rapidly and more excessively than the papillary muscles during early to mid systole in all patients. Then, the distal mitral valve and the chordae tendineae adopted the configuration of an inverted V in mid systole. The tip of the surplus leaflet contacted the interventricular septum in patients with SAM-septal contacts. In the short-axis view of the left ventricle, the central portion of the distal mitral valve moved forward excessively and bent like an inverted U during early to mid systole. Therefore, SAM of the distal mitral valve is thought to be caused by the hydrodynamic force generated by the ejection flow. This force is supposed to be not only due to the Venturi effect, but is more properly understood as a "pushing force". Left ventricular outflow obstruction is considered to be formed by the protruding distal mitral valve and the hypertrophied septum.

[Echocardiographic features of small heart].

To clarify the clinical significance of a small heart; i.e., a small cardiac silhouette on chest radiography, an echocardiographic study was performed. Sixty persons with small heart according to cardiothoracic ratios less than or equal to 40%, and 23 age- and sex-matched normal controls (42% less than cardiothoracic ratio less than or equal to 50%) received two-dimensional and M-mode echocardiography. The body weights and body surface areas (BSA) in the small heart group were significantly less than those in the control group. On the lateral chest radiographs, numerous cases with small heart had straight spines and chests with decreased anteroposterior diameters. More than half of the small heart group had a variety of cardiovascular complaints, including chest pain, palpitation, dyspnea, and dizziness. Echocardiographic measurements were performed and hemodynamic indices were calculated. The results were as follows: Left ventricular dimension at end-diastole (LVDd), left ventricular dimension at end-systole (LVDs), left atrial dimension (LAD), and left ventricular mass (LV mass) of the small heart group were significantly less than those of the control group. There were, however, no differences in the values corrected by BSA (LVDd/BSA, LVDs/BSA, LAD/BSA and LV mass/BSA) between the two groups. We found no differences in hemodynamic indices (heart rate, stroke volume, ejection fraction, and cardiac output) at rest between the small heart and control groups. Nineteen cases (32%) had mitral valve prolapse (MVP) on echocardiography in the small heart group. Characteristic phonocardiographic findings were found in 11 cases with MVP (systolic click in four, mitral regurgitant murmur in three, and both in four).(ABSTRACT TRUNCATED AT 250 WORDS)

[Pulsed Doppler echocardiography and pharmacodynamic phonocardiography in the diagnosis of silent aortic regurgitation: a correlative study].

To determine the usefulness of pulsed Doppler echocardiography (PDE) in diagnosing aortic regurgitation (AR), the following two studies were performed. PDE and phonocardiography (PCG) were evaluated for the diagnosis of AR in 36 patients for whom angiography was performed. In 24 patients with grade 2 to 4 by Sellers' classification, all PDE studies were positive for AR (sensitivity of 100%) and PCG studies were positive in 21 patients (sensitivity of 88%). Among 10 patients of grade 1, PDE studies were positive for eight (sensitivity of 80%) and PCG studies were positive for seven (sensitivity of 70%). Angiography was negative for AR in two PCG-proven cases, and PDE also proved AR in one of these. Thus, in all patients, the sensitivity of PDE and PCG were 94 and 82%, respectively. These results indicate that AR may be missed during auscultation, or even when using the latest type PCG. The ability of detecting silent AR was studied in 160 consecutive patients with AR. PDE was positive for AR in 156 of 160 consecutive patients who were diagnosed as having AR by PDE or PCG. In the remaining four patients, three were diagnosed by PCG alone and one by pharmacodynamic phonocardiography using methoxamine (ME-PCG). On the other hand, in 137 of 160, Ar was proven by PCG, and the remaining 23 were so-called "silent AR". ME-PCG was performed for 11 cases of silent AR, but an unequivocal AR murmur was detected in only five. Therefore, the sensitivity of PDE in diagnosing silent AR was 96 percent, while that of ME-PCG was only 45 percent. Thus, compared to PCG and even pharmacodynamic PCG, PDE is a much more sensitive method of diagnosing AR. However, in mild AR cases, angiography, PDE and PCG, all have some limitations in diagnosing AR.

[Generalized lentigo in a case of hypertrophic cardiomyopathy with right ventricular outflow tract obstruction].

A case was reported in which generalized lentigo was associated with hypertrophic cardiomyopathy and right ventricular outflow tract obstruction. A 34-year-old man was admitted to our hospital for cardiac evaluation. He had been noted to have a heart murmur since his childhood and had had a diagnosis of pulmonary stenosis in his high school age. Physical examination revealed precordial murmurs and numerous lentigines over his whole body surface including palms and soles. Two-dimensional and M-mode echocardiography disclosed asymmetric septal hypertrophy, systolic anterior motion of the mitral valve (SAM) and systolic semiclosure of both semilunar valves. A systolic turbulent flow was recorded at the outflow tracts of both ventricles by pulsed Doppler technique. Cardiac catheterization demonstrated a pressure gradient of 13 mmHg across the right ventricular outflow tract. We considered that the present case was an atypical LEOPARD syndrome with predominant right ventricular outflow tract obstruction which was precisely evaluated by noninvasive techniques.