SYNAPTIC VESICLES ISOLATED FROM RAT HEART:l‐[³H]NOREPINEPHRINE UPTAKE PROPERTIES¹

Abstract

Abstract— A fraction containing synaptic vesicles was isolated from rat heart by differential centrifugation, and the uptake of l‐[3H]norepinephrine was studied in vitro., Uptake was highly dependent upon time and temperature, and was linear for 6 min at 30° or 4 min at 37°C. About 80% of the measured uptake required both ATP and Mg2+ and was inhibited by nanomolar concentrations of reserpine; no inhibition was obtained with cocaine. These properties are characteristic of storage vesicle uptake as opposed to synaptic membrane uptake. Uptake of norepinephrine was saturable and displayed a single Km value of 2 μM. The uptake was completely stereospecific, as unlabeled dl‐norepinephrine was less than half as effective as unlabeled l‐norepinephrine in reducing uptake of l‐[3H]norepinephrine. Norepinephrine uptake could be inhibited by various phenethylamines and indoleamines following the rank order: reserpine > harmaline > 5‐hydroxytryptamine > dopamine > norepinephrine. The vesicle preparation also incorporated [3H]5‐hydroxytryptamine and [3H]dopamine. 5‐Hydroxytryptamine uptake displayed a Km of 0.5 μM and a maximal uptake equivalent to that seen with norepineph‐rine; dopamine uptake followed complex kinetics. Administration of reserpine in vivo or destruction of sympathetic neurons by long‐term guanethidine treatment both eliminated the ability of the preparation to take up norepinephrine. Synaptic vesicles of cardiac sympathetic neurons thus resemble vesicles prepared from other central and peripheral catecholaminergic tissues; this method may be used readily to examine drug effects on rat heart synaptic vesicle function.

Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles

“…Recently, the transport properties of a crude vesicle fraction isolated from rat heart were reported, and the uptake was demonstrated to occur via a high-affinity, ATP2+-dependent carriermediated system (Bareis and Slotkin, 1979). The main difficulties in interpreting the experiments using this crude preparation to determine the molecular characteristics of the transport process are the heterogeneity of vesicle types (e.g., "light" and "heavy" vesicles) (Roth et al, 1968), contamination by other organelles, and correction of the experimental data for two-way fluxes across the membrane.…”

Maturation of Sympathetic Neurotransmission in the Rat Heart

“…It is to be noted that in this study, administra tion of guanethidine was begun prior to devel opment of functional sympathetic nerve con nections to the heart [4,5,7]; the myocardium in these animals therefore has not received neural input in the period in which the transi tion from DNA-dependent to RNA-dependent growth phases occurs [13], Since these results with sympathectomized rats show that basal sympathetic tone is probably not neces sary for normal cardiac growth to occur, it is unlikely that noradrenergic neurons provide the major control mechanism for cardiac cel lular growth and differentiation. Similar re sults were obtained in adult rats lesioned with guanethidine, namely massive sympathec tomy unaccompanied by interference with cardiac growth, RNA or protein synthesis.…”

“…This dose is far above that necessary to cause blockade of transmitter release and actually results in longlasting destruction of sympathetic neurons in both adult and neonatal rats [2,4,8,14,[19][20][21]. Control animals received daily injections of vehicle (0.9% NaCI, 0.1% ascorbic acid, pH 7.4, prepared daily), equal in volume to the guanethidine injections (I ml/ kg).…”

“…A crude fraction containing synaptic vesicles was prepared from the heart homogenate by the method adapted from Seidler el al. [30] by Bareis and Slotkin [4], The homogenate was centrifuged at 1,000# for 15 min and (he pellet discarded. The supernatant was recentrifuged at 20,000 # for 30 min and this supernatant was sedimented at 100,000 g for 30 min in a Beckman Type 40 fixed angle rotor.…”

“…Up take was determined by subtracting the 0°C tissue blank from the 30°C sample and expressed as pmoles of norepinephrine taken up in 4 min per original gram of heart. Although the uptake procedure utilizes a crude microsomal preparation, the incorporation of [3H]-norepinephrine occurs predominantly into the sympathetic storage vesicles [4] and provides a reliable index of development of the sympathetic nerve ter minals in the heart [5,25).…”