Vesicular Mechanism Research Articles

Translocation of cytosolic acetylcholine into synaptic vesicles and demonstration of vesicular release.

The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.

Does unidirectional vesicular transport occur in retinal vessels?

This paper challenges the hypothesis that the smooth 80 nm plasmalemmal caveolae found in abundance at the abluminal aspect of the endothelium in retinal blood vessels participate in a unidirectional vesicular transport mechanism. Evidence is presented which indicates that horseradish peroxidase, when introduced to the extracellular space of the retina via the vitreous body, may enter the intravascular compartment through junctional incompetence which occurs at or after enucleation of the eye. It is proposed that the plasmalemmal caveolae at the abluminal plasma membrane of endothelial cells in retinal blood vessels are static structures which facilitate the transport of small solutes and ions across the blood retinal barrier.

Biochemical evidence that acetylcholine release from cholinergic nerve terminals is mostly vesicular

The nature of the intraterminal compartments from which acetylcholine (ACh) is released following presynaptic stimulation was investigated. This was pursued by examining the effects of the anticholinergic drug 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of newly synthesized [ 3H]ACh and of endogenous ACh from purified cholinergic nerve terminals (synaptosomes) which were isolated from the electric organs of Torpedo. Preincubation of the synaptosomes, with AH5183 (1–10 μM), does not affect either the intraterminal synthesis of [ 3H]ACh or the uptake of its precursors, but results in a marked inhibition (85%) of the release of the newly synthesized [ 3H]ACh. However, when AH5183 is added following the accumulation of [ 3H]ACh in the nerve terminals, it does not affect [ 3H]ACh release. AH5183 also has no effect on the release of preformed endogenous ACh. These findings, together with the previous in vitro demonstrations that AH5183 is a potent inhibitor of ACh uptake into isolated cholinergic vesicles, suggest that most of the synaptosomal ACh is secreted by a vesicular mechanism.

Secretory component: The polymeric immunoglobulin receptor: What's in it for the gastroenterologist and hepatologist?

The primary function of the SC-pIg system is to secrete pIgs into various external secretions. The cellular mechanism responsible for this transport is schematically depicted in Figure 5. Polymeric immunoglobulin A, which is synthesized by plasma cells that are part of the mucosa-associated lymphoid tissue, gains access to the SC on the albuminal surface of epithelial cells by diffusion from sites of synthesis in mucosae or enters the blood circulation and is cleared, largely by hepatic transport, into bile. The pIgA binds to SC on the abluminal surface of the epithelial cells (and probably hepatocytes) initially by noncovalent interactions that are saturable, reversible, and specific for pIgA and IgM. Subsequently, covalent interaction between SC and its ligand occurs to a variable degree in different species. The SC-IgA complex is endocytosed by the epithelial cell or hepatocyte and is transported across the cell into the external secretions by a microtubule-dependent vesicular transport mechanism. At some point during the transport, the complex is rendered soluble by proteolytic cleavage of the membrane-associated SC molecule to release the soluble sIgA into the gland lumen or the canaliculus. In the intestinal lumen, SC helps protect the sIgA molecule from proteolytic degradation. The sIgA may play a major role in the mucosal defense against pathogenic organisms or harmful antigens.The SC-pIg system differs from many of the other known receptor-ligand interactions in several important ways. First, the synthesis or expression of the receptor (SC), or both, are not regulated by the concentration of the ligand. Second, SC probably is not dissociated from its ligand or recycled to the cell surface as it is secreted in complex with its ligand (pIg) into the external secretions. Third, the interaction of pIgs with their receptor does not function to regulate an intracellular process, but results in transcellular transport of the ligand, which acts in the external environment. Fourth, after initial noncovalent, reversible binding between the receptor and its ligand, the interaction becomes covalent by the formation of disulfide linkages between SC and the pIg. Finally, SC is initially inserted into the abluminal domain of epithelial cells as an integral membrane protein and subsequently is proteolytically cleaved to a soluble molecule which is secreted by the cell. Thus, in contrast to many cell-surface receptor-ligand interactions in which the ligand is ultimately degraded and the receptor is conserved, the SC-pIgA interaction results in partial proteolytic degradation of the receptor and conservation of the ligand.Despite the wealth of information that has accumulated about this receptor-ligand system, a number of very important questions remain to be answered: How is the synthesis and expression of SC in tissues such as the intestine and the liver regulated? What determines how SC is expressed during normal differentiation? What are the cellular mechanisms responsible for the sorting of membrane SC to the abluminal domain of the plasma membrane and then rerouting of SC to and through the luminal plasma membrane? Where and how does the SC-pIg transcellular transport pathway diverge from other receptor-mediated endocytic pathways? What are the specific functions of the sIgA or secretory IgM after secretion into the extracellular environment?From the standpoint of pathophysiology of the liver and digestive organs, it is likely that full understanding of SC and SC-pIg interactions will greatly contribute to understanding of many fundamental aspects of the cellular biology of hepatocytes and intestinal epithelial cells. Moreover, important clues concerning the pathogenesis of major digestive tract diseases, e.g., malignancies and chronic inflammatory diseases, may lie in a better appreciation of SC and the secretory immune system. Thus, study of the SC-pIg system will likely continue to be a fruitful and exciting area of investigation for years to come.

Permeability of blood-brain barrier to various sized molecules.

We studied disruption of the blood-brain barrier (BBB) by acute hypertension and a hyperosmolar solution. The goals were to determine whether 1) disruption of the BBB occurs primarily in arteries, capillaries, or veins, and 2) transport of different-sized molecules is homogeneous or size dependent. Sprague-Dawley rats were studied using intravital fluorescent microscopy of pial vessels and fluorescein-labeled dextrans (FITC-dextran, mol wt = 70,000, 20,000, and 4,000 daltons). The site of disruption was determined by the appearance of microvascular leaky sites. Transport of different-sized molecules was calculated from clearance of FITC-dextran. During gradual hypertension and osmotic disruption, all leaky sites were venular. Rapid hypertension produced venular leaky sites and, in some experiments, diffuse arteriolar extravasation of FITC-dextran. Clearance of different-sized molecules was homogeneous during acute hypertension. In contrast, clearance of molecules during osmotic disruption was size dependent. The findings suggest that 1) venules and veins are the primary sites of disruption following acute hypertension and a hyperosmolar solution; 2) transport of different-sized molecules is homogeneous following acute hypertension, which suggests a vesicular mechanism; and 3) transport following hyperosmolar disruption is size dependent, which suggests that hyperosmolar disruption may involve formation of pores as well as vesicular transport.

Morphology and permeability properties of blood capillaries in extraocular muscles of macaque monkeys.

Extrinsic eye muscles are very different from other skeletal muscles with regard to the morphology of their fibers and response to drugs. In addition, they are provided with an unusually rich blood supply. Since it had been previously reported that the capillaries of extraocular muscles are, at least in part, of the fenestrated type, a feature unusual for skeletal muscles, we have analyzed the morphology of these vessels in macaque monkeys and tested their permeability properties with horseradish peroxidase (HRP). Our study of the blood capillaries in the superior rectus muscle and levator palpebrae superioris in Macaca mulatta and M. fascicularis has demonstrated that these vessels are morphologically similar to the capillaries of other skeletal muscles. Furthermore, when HRP is introduced intravenam, it is transported out of the vessels by a vesicular transcellular mechanism. When HRP is injected in the interstitial spaces of the muscles, it is returned to the lumen by an identical vesicular transcellular transport. Thus, a bidirectional movement of macromolecules can take place across the walls of these vessels, and such a movement is not different from that previously reported for other skeletal muscles.

The release of gamma-aminobutyric acid from horizontal cells of the goldfish (Carassius auratus) retina.

Isolated horizontal cells from goldfish retinas were prepared by enzymatic dissociation using papain and separated from other cells by velocity sedimentation. In the intact retina, H1 horizontal cells possess a high-affinity mechanism for accumulating gamma-aminobutyric acid (GABA). This property is retained in isolated cells, which also release the accumulated GABA in response to depolarization by elevated external K+. L-Glutamic acid and its analogues are highly effective at micromolar concentrations in eliciting the release of preloaded GABA from isolated cells. At saturating concentrations, L-aspartic acid stimulates about one-third as much release as L-glutamic acid. In contrast, the D-isomers of glutamate and aspartate are ineffective. In the intact retina, micromolar concentrations of L-glutamic acid analogues are also capable of eliciting GABA release from H1 horizontal cells. Release of the accumulated GABA from isolated H1 cells is largely independent of external Ca2+ concentrations. In the intact retina, H1 horizontal cells also possess a K+-stimulated GABA release mechanism that is independent of the Ca2+ concentrations in the medium. In addition, there appears to be a small but significant amount of [3H]GABA release that may be Ca2+ dependent. Under our conditions, [3H]GABA release from isolated cells is unaffected by external Na+ concentrations between 20 and 120 mM. However, concentrations of 10 mM or less significantly diminishes this release, with 70% curtailed in Na+-free solutions. Our results, together with morphological observations by a number of other investigators, suggest that there may be two distinct mechanisms for GABA release from goldfish H1 horizontal cells: one being a conventional vesicular mechanism which is Ca2+ dependent, while the other is Na+ driven and Ca2+ independent. H1 horizontal cells in the intact goldfish retina release the accumulated GABA in response to brief incubations in darkness, which is known to be the natural stimulus that depolarizes these neurones.

Ornithodorus tartakovskyi: Quantitation and ultrastructure of cutaneous basophil responses in the guinea pig

Cutaneous lesions elicited in guinea pigs by primary and secondary feeding populations of the argasid tick, Ornithodorus tartakovskyi, were analyzed by light and electron microscopy. Small clusters of basophils appeared at primary bite sites within 24 hr of tick attachment, and by 72 hr constituted approximately 11% of the total leukocytes. Secondary feeding sites exhibited an augmented cellular infiltrate that was dominated by basophils at all times (48–56% of total cells). Eosinophil proliferation was minimal, however, and the remaining cells were of the mononuclear type. Despite mounting a strong cutaneous basophil response of the kind that mediates immune rejection of prolongedfeeding ixodid ticks, the guinea pigs showed no resistance to the fast-feeding Argasidae. It is suggested that argasid ticks probably complete their blood meal prior to basophil arrival at the bite site. Electron microscopy indicated that the number of epidermal Langerhans cells increased with time in both primary and secondary lesions; these cells were more numerous in challenge infections however, and seemed also to occur in the dermis. Basophils at secondary bite sites exhibited three kinds of structural alterations classified as: (1) piecemeal alterations—involving a vesicular degranulation mechanism; (2) an anaphylactic-type of alteration—involving single or compound exocytosis of whole granules; and (3) cytotoxic alterations culminating in complete disintegration. The majority of basophils in 72 hr secondary lesions exhibited cytotoxic alterations. It is suggested that such changes result from contact with tick-derived toxins or enzymes.

Morphometric analysis of effects of spinal cord transection

After spinal cord transection in the rat, endothelial reactivity to horseradish peroxidase (HRP) was quantified at the ultrastructural level. The ratio of the area of labeled endothelial vesicles to area of endothelium (pinocytotic index) was established using morphometric techniques. From 3 h through 7 days the pinocytotic index was significantly elevated as far as 10 mm distal to the transection site. Correlating these findings with a previously established blood vessel classification model, the tracer uptake was identified as both a pinocytotic and vesicular transport mechanism. The latter was associated with an acute response lasting as long as 1 day after transection. Thereafter, the barrier appeared to reestablish its selectivity and only pinocytotic uptake of HRP was apparent.

Nucleotide uptake by isolated cholinergic synaptic vesicles: Evidence for a carrier of adenosine 5'-triphosphate

The in vitro uptake of [ 3H]nucleotides was studied using cholinergic syaptic vesicles isolated from Torpedo electric organ, with a resting membrane potential of 50–60 mV. The osmotically sensitive uptake of [ 3H]adenosine 5'-triphosphate (ATP) was markedly influenced by temperature and external pH, and was maximal after 40–50 min; longer incubation resulted in loss of accumulated radiolabel. Similar characteristics were also observed for adenosine 5'-mono- and diphosphate and guanosine and uridine triphosphates, all of which acted as competitive substrates for the saturable system which transported ATP (K T 1.15 mM). Breakdown of [ 3H]nucleotides in the medium was not a significant factor, and adenosine, guanosine and adenine were very poorly incorporated. Under conditions of V max, vesicle to medium ratios of [ 3H]ATP of 20–25 were observed; the amount of radiolabel was equivalent to 20–50% of the initial endogenous amount of ATP in the vesicles. Atractyloside specifically inhibited nucleotide transport with no modification of hemicholinium-3 sensitive acetylcholine uptake. Antisera raised (a), to whole Torpedo vesicle extract, and (b), to a single purified vesicle polypeptide, greatly stimulated ATP uptake without effect on simultaneous influx of either acetylcholine or glucose. It is concluded that isolated vesicles contain a nucleotide carrier of wide pharmacological specificity (possibly the 34,000 molecular weight protein of Stadler & tashiro [1979]), which is likely to be of physiological relevance. Implications for vesicular refilling mechanisms are discussed.

Evidence for a vesicular transport mechanism in hepatocytes for biliary secretion of immunoglobulin A.

Quantitative electron microscopic autoradiography and diaminobenzidine cytochemistry provide evidence for an uptake and vesicular transport mechanism for iodine-125-labeled immunoglobulin A from plasma to bile by hepatocytes in vivo. The data confirm the existence of a hepatobiliary pathway for secretion of immunoglobulin A into the intestine and are consistent with a vesicular transport mechanism for biliary proteins within liver parenchymal cells.

Bile Secretory Apparatus: Evidence for a Vesicular Transport Mechanism for Proteins in the Rat, Using Horseradish Peroxidase and [125I]Insulin

The morphologic mechanisms involved in the uptake, transport, and secretion of proteins into bile were studied in rat liver in vivo. When both horseradish peroxidase (HRP) and insulin were injected into the portal veins of anesthetized rats, these proteins were subsequently detected in bile. Utilizing the technique of combined cytochemistry and quantitative autoradiography, both HRP and [125I]insulin were coincidentally localized within endocytic vesicles within the interior of hepatocytes at various time points after simultaneous intraportal injection. The data suggest that both proteins followed two pathways involving endocytic vesicles of approximately 1000 A in diameter. In the first pathway these protein-containing vesicles were transported through the hepatocyte and subsequently fused with the bile canalicular membrane, resulting in secretion of contained proteins into the biliary space. The second pathway also involved endocytosis into 1000 A vesicles, but these vesicles were transported to the Golgi region and its associated system of lysosomes and endoplasmic reticulum (GERL). Whether the proteins in these vesicles were later secreted into bile was unclear. Measurement of HRP and [125I]insulin or its metabolites, in bile, provided direct evidence that exogenously administered proteins (or their fragments) gain entrance into the biliary space. Studies in which metabolites of [125I]insulin, [125I]monoiodotyrosine (MIT), and 125I, were injected intraportally, demonstrated that less than 10% of [125I]MIT and less than 1.5% of Na125I were retained in perfusion-fixed and processed liver tissue. This study suggests that proteins in blood plasma are taken up by hepatocytes and secreted into bile via a vesicular transport mechanism.

The mechanism of neuronal noradrenaline and dopamine β-hydroxylase release by a sodium-deficient solution substituted with urea

Isolated rabbit hearts were perfused with a ‘low Na-urea’ solution (the substitution of 132 mM NaCl of Tyrode's solution by 242 mM urea) for 1–30 min. The effect on noradrenaline overflow was compared with that evoked by other NaCl substitutes. ‘Low Na’ solutions released noradrenaline at greatly differing rates and time courses. ‘Low Na-urea’ medium caused a large noradrenaline overflow which in time course and magnitude was similar to that evoked by ‘high K-low Na’. However, the effect of ‘high K-low Na’ was largely dependent on calcium and little affected by lowering the temperature from 36 to 16°C (Q 10 = 1.1) while the noradrenaline output evoked by ‘low Na-urea’ medium was only transiently calcium-dependent and greatly reduced by a drop in temperature (Q 10 = 6.7). Both ‘low Na-urea’ and calcium-free ‘high K-low Na’ solutions evoked the release of α-methyladrenaline which had been incorporated into adrenergic nerves of the hearts of rabbits previously treated with reserpine. Since reserpine blocks the vesicular storage mechanism the amine must have been released from the axoplasm through the neuronal membrane. The re-introduction of Tyrode's solution did not reverse the α-methyladrenaline output evoked by ‘low Na-urea’ although it abolished that after ‘low Na-sucrose’ or calcium-free ‘high K-low Na’ solutions. Similarly, the noradrenaline loss from the normal heart evoked by ‘low Na-urea’ medium was also unaltered by returning to Tyrode's solution. The irreversibility of the action of ‘low Na-urea’ solution on amine release, an initial loss of potassium into the perfusate and previous morphological evidence of myocardial cell damage suggested that the medium produced a hyposmotic shock. As a result, there was a brief calcium-dependent release of noradrenaline and of dopamine β-hydroxylase that was superimposed upon a much larger and persisting non-exocytotic amine overflow which was unaccompanied by release of dopamine β-hydroxylase. The present findings corroborate our previous observation that electron-dense cores in the small vesicles of adrenergic neurons of the rabbit heart are preserved while most of the cardiac noradrenaline is depleted by perfusion with ‘low Na-urea’ solution, and the suggestion by other authors that proteins, such as dopamine β-hydroxylase, rather than amine constitute the electron-dense material.

Perinatal methadone addiction affects brain synaptic development of biogenic amine systems in the rat

Administration of methodone to pregnant and nursing rats or direct treatment of developing pups with methadone resulted in deficits of development of body weight, brain weight and synaptosomal uptake of 5-hydroxytryptamine, dopamine and norepinephrine. Defective synaptic development was most apparent immediately after birth in pups whose mothers received methadone; while some recovery occurred by 3 weeks of age, there was a subsequent deficit in synaptosomal uptake post-weaning. A similar pattern also was seen in development of synaptic vesicle amine uptake. Direct treatment of neonates with methadone also caused reductions in development of synaptosomal and vesicular uptake mechanisms, but the patterns of alteration were different from those in the maternal treatment group. These studies show that the adverse effect of opiates on general brain growth are accompanied by a slowing of synaptic development of biogenic amine systems.

Ontogeny of (−)-[ 3H]norepinephrine uptake properties of synaptic storage vesicles of rat brain

The ontogeny of[3H]norepinephrine uptake mechanisms has been examined in synaptosomes and storage vesicles isolated from rat whole brain. The [3H]norepinephrine accumulated by synaptosomes was low in neonates, but reached adult levels by 15 days of age. In contrast, development of[3H]norepinephrine uptake into isolated rat brain storage vesicles was not complete until 38 days of age. Kinetic analysis of the developing vesicular uptake mechanism revealed no change in Km, while maximal uptake increased progressively from birth to maturity. Storage vesicles from immature and adult rats exhibited similar energy requirements for uptake as determined by their dependence on ATP-Mg2+ concentration; furthermore, the degree of inhibition of [3H]norepinephrine uptake by other amines was the same in both vesicle preparations. Thus, storage vesicles isolated from adult and developing rats display an in vitro[3H]norepinephrine uptake mechanism with properties that are kinetically and pharmacologically similar. The results suggest that, while the number of storage vesicles in the central nervous system increases during development, those vesicles that are present possess a fully functional amine uptake system.

Effects of Vincristine on Endothelial Microtubules in the Spinal Cord

Microtubules (MT) are versatile organelles participating in a wide variety of biological activity. MT involvement in the movement and transport of cytoplasmic components has been well documented. In the course of our study on trauma-induced vasogenic edema in the spinal cord we have concluded that endothelial vesicles contribute to the edema process. Using horseradish peroxidase as a vascular tracer, labeled endothelial vesicles were present in all situations expected if a vesicular transport mechanism was in operation. Frequently,labeled vesicles coalesced to form channels that appeared to traverse the endothelium. The presence of MT in close proximity to labeled vesicles sugg ested that MT may play a role in vesicular activity.

Degranulation mechanisms in human leukemic basophils

The peripheral blood basophils of a patient with chronic myelogenous leukemia, basophilic phase (up to 85% of circulating leukocytes were mature basophils), and substantially elevated levels of urine histamine were studied in the electron microscope in an attempt to relate morphologic changes to possible mechanisms of mediator secretion. By use of criteria developed in earlier studies of allergic contact dermatitis, it was found that more than half of the mature circulating basophils were degranulated and less than 11% of them were fully granulated. Two patterns of basophil degranulation were recognized: (i) anaphylactic-type degranulation, involving fusion of adjacent granule membranes with each other and with the plasma membrane, permitting continuity between granules and the cell exterior, and associated with a concomitant loss of granule contents; (ii) a vesicular transport mechanism involving pinocytotic cytoplasmic vesicles. The anaphylactic mechanism probably accounted for the bulk of histamine release occurring in this patient, in contrast to volunteers with allergic contact dermatitis in whom the vesicular mechanism is more prominent.

The fine structural demonstration of monoaminergic synapses in immature rat neocortex

The radial distribution of monoaminergic (MA) synapses has been studied in lateral neocortex (SI) of infant rats using an electron microscopic histochemical technique. Systemically administered 5-hydroxydopamine crosses the blood-brain barrier and produces small granular vesicles (SGV) in 30% of all synaptic terminals in SI. In the primordium of layer IV, at six days postnatal, 70% of the synaptic terminals contain SGVs. Immature cortex thus contains a high proportion of synapses with a vesicular uptake-storage mechanism for catecholamines. It is proposed that in infant rat there is a major projection from brain stem MA nuclei to the neocortex; this projection may exert a potent influence on the activity of neurons in layer IV.

Vasogenic Edema in the Injured Spinal Cord: Possible Role of Microtubules in Endothelial Vesicular Transport of HRP

The genesis of edema in the central nervous system (CNS) parallels changes in the blood-brain barrier (BBB). The anatomical seat of the BBB is located in the endothelium of the CNS. The presence of interendothelial tight junctions and the low rate of pinocytotic (vesicular) activity constitute the main features of the BBB. Based on the fate of intravascular horseradish peroxidase (HRP), mechanisms of edema genesis have been elucidated. A vesicular mechanism appears to play a role in the formation of edema in the injured (compression) spinal cord.Following spinal cord compression injury (200 gm/cm2-5 min), circulating HRP traversed the endothelium and entered extracellular spaces of the cord parenchyma (Fig. 1). Most microvessels in the injured and adjacent areas exhibited an intensified endothelial vesicular uptake of HRP (Figs. 1, 3). In areas of intense endothelial vesicular activity, the frequency of microtubules (MT) appeared to be increased (Figs. 2, 3).

Maturation of the adrenal medulla—III: Practical and theoretical considerations of age-dependent alterations in kinetics of incorporation of catecholamines and non-catecholamines

Rats were sacrificed at 10-day intervals from birth to 50 days of age and the kinetics of uptake of epinephrine (E) and metaraminol (MA) into isolated adrenal medullary storage vesicles were measured. The K m for epinephrine remained at about 30 μM throughout development, but the maximal uptake ( U max) at birth was 30 nmoles/30 min/100 μg of endogenous catecholamines compared to 2 nmoles at subsequent ages. The K m for MA was about 4 mM at birth, 0·7 mM at 10–20 days of age and 1·2 mM thereafter, while U max was approximately 170 nmoles initially, 50 nmoles at 10–20 days and 70 nmoles thereafter. The preference of the vesicles for uptake of E vs MA (both at 0·1 mM) was approximately 6 to 1 at birth, 1 to 1 at 10 days, 3·5 to 1 at 20 days and 5 to 1 thereafter, while the preference for E vs tyramine was 2 to 1, except at 10 days, when the ratio was 1 to 1. These data suggest that the two vesicular uptake mechanisms, typified by incorporations of E and of MA, develop independently of each other and that the age-dependent alterations are present both at the level of the vesicle membrane and intravesicular storage. The development of a kinetic model aids in the identification of probable sites of specific age-dependent alterations. Maturational changes in uptake and specificity can produce parallel changes in synthesis of catecholamines as determined by conversion of newly incorporated tyramine to octopamine.