Heavy Vesicles Research Articles

Subcellular distribution of neuropeptide Y-like immunoreactivity in guinea pig neocortex

Neuropeptide Y-like immunoreactivity (NPY-LI) was enriched in synaptosomal fractions of neocortex, which on lysis yielded vesicle-rich fractions. The distribution on NPY-LI on a sucrose density gradient was similar to that of somatostatin, with a concentration in heavy vesicles. The peptides were not found in light vesicles in contrast to the distribution of noradrenaline. Both homogenate and vesicular NPY-LI coeluted with synthetic NPY on reverse-phase HPLC.

Effects of Mg 2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg 2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg 2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg 2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg 2+). Calcium uptake velocity in light vesicles correlated with Ca 2+-dependent ATPase activity, suggesting that Mg 2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca 2+-dependent ATPase activity, although a Mg 2+-dependent increase in calcium influx was observed. Thus, Mg 2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg 2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca 2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg 2+ suggested that ruthenium red (0.5 μ M) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.

Synthesis and subcellular transport of sulfogalactosyl glycerolipids in the myelinating mouse brain

In the 17-day-old myelinating mouse brain the site of sulfogalactosyl glycerolipid synthesis and the kinetics of its subcellular distribution were studied by a 2 h pulse-labeling with [ 35S]sulfate followed by a 4 h chase of [ 35S]sulfogalactosyl glycerolipid. At several time intervals after the intraperitoneal [ 35S]sulfate injection, subcellular fractions of brain were obtained by differential and discontinuous sucrose gradient centrifugation. The crude microsomal membrane fraction (17 500 × g supernatant) was further subfractionated into light myelin, plasma membranes, Golgi vesicles, endoplasmic reticulum membranes and heavy vesicles associated with acid hydrolase activities. The results of the [ 35S]sulfogalactosyl glycerolipid labeling kinetics indicate that these lipids are synthesized in the Golgi-endoplasmic reticulum complex and transferred in vesicles associated with lysosomes to the myelin membranes. During this transfer part of the sulfogalactosyl glycerolipids appears to be degraded, similarly as described for brain sulfatides. This double function of lysosomes may be part of a general regulation mechanism of brain myelin glycolipid content.

Vesicular transport of sulfatide in the myelinating mouse brain. Functional association with lysosomes?

Sulfatide synthesis and its subcellular distribution kinetics was followed in the myelinating brain of 17-day-old mice. Pulse-labeling-chasing conditions were achieved by an intraperitoneal injection of (35S)sulfate followed 2 h later by a second injection of a high dose of unlabeled sulfate. At 1, 2, 3, 4, and 6 h after the (35S)sulfate injection, the brains were removed, homogenized, and subcellular fractions were obtained by differential and discontinuous sucrose gradient centrifugation (Eichberg, J., Whittaker, V. P., and Dawson, R. M. (1964) Biochem. J. 92, 91-100). The microsomal membranes were further subfractionated (Siegrist, H. P., Burkart, T., Wiesmann, U. N., Herschkowitz, N. N., and Spycher, M. A. (1979) J. Neurochem. 33, 497-504) into light myelin, plasma membranes, Golgi vesicles, endoplasmic reticulum membranes, and heavy vesicles associated with acid hydrolase activities. The [35S]sulfatide-labeling kinetics was measured in all subcellular fractions. The results indicate that sulfatides are synthesized in the Golgi-endoplasmic reticulum complex and transferred in vesicles at least partially associated with lysosomes to the myelin membranes. The association of sulfatides with lysosomes could explain the existence of the previously described labile pool of newly synthesized sulfatides (Burkart, T., Hofmann, K., Siegrist, H. P., Herschkowitz, N. N., and Wiesmann, U. N. (1981) Dev. Biol. 83, 42-48) and also could be a form of vesicular transport to the myelin.

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle. Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca 2+/μl of water space. After KCl washing both contained less than 4 nmol Ca 2+/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca 2+ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca 2+ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca 2+-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca 2+-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.

Phosphorylation of heavy sarcoplasmic reticulum vesicles: identification and characterization of three phosphorylated proteins.

Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl2, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 μm γ-32P-ATP.32P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl2 (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 μm sodium dantrolene, an inhibitor of Ca2+ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (≈56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.

Dopamine β‐hydroxylase distribution in density gradients: Physiological and artefactual implications

Knowledge of the vesicular origin of circulating dopamine beta-hydroxylase (DbetaH) is indispensable for any attempts to explain the parallelism or lack of it between circulating enzyme and catecholamines as they may relate to physiological stress, forms of hypertension, neurological disorders, and the response to pharmacological agents. The present study represents an effort to evaluate and to place in proper perspective data based on the DbetaH activity found in the region of the light vesicle peak of noradrenaline (NA), which is used as a quantitative measure of a population of small terminal vesicles. Distributions of vesicles and subvesicular components are compared with DbetaH and NA in sucrose-D2O density gradients used to prepare relatively pure fractions of large dense cored vesicles (LDV) from bovine splenic nerve. Although NA in sedimentable particles of the light vesicle peak is likely to be a valid measure of a small vesicle population, the following is demonstrated: (1) A substantial fraction (25%-37%) of the total sedimentable DbetaH activity can be proven to distribute in the region of the light vesicle peak from a tissue with an insignificant small vesicle population. Based on studies of vesicles from sequential nerve segments, this enzyme activity probably corresponds to a population of "immature" LDV which are undergoing axoplasmic transport and have not synthesized their full complement of transmitter. (2) Physical lysis which depletes the matrix of LDV causes redistribution of DbetaH activity from the heavy vesicle peak into the region of the light vesicle peak. Analogously, DbetaH associated with exocytosed LDV and retrograde transport particles is also likely to contaminate the region of the light vesicle peak. (3) Based on available data, it can be calculated that each small dense cored vesicle could contain only 0.1-0.5 molecules of DbetaH and that a contamination of only 0.016% LDV can account for all of the DbetaH reported to occur in the light vesicle peak of normal rat vas deferens preparations.

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid.

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.

Localization of noradrenaline and dopamine-β-hydroxylase in C 1300 mouse neuroblastoma a biochemical and electronmicroscopic study

Investigations were carried out in order to obtain biochemical and electronmicroscopic information on the storage of noradrenaline (NA) and dopamine-β-hydroxylase (DβH) in C 1300 mouse neuroblastoma. By application of two different gradient centrifugation methods only one population of NA vesicles, i. e. heavy NA containing vesicles could be demonstrated. Electronmicroscopic investigation of the tumor revealed the presence of large dense cored vesicles. Electronmicroscopic investigation of gradient fractions further showed that large dense cored vesicles were only present in those fractions of the gradient containing heavy vesicles. The distribution of DβH in relation to the distribution of NA after gradient centrifugation differs from what has been observed in peripheral noradrenergic neurons. In C 1300 mouse neuroblastoma the larger part of the enzyme is associated with the plasma membrane.

Evidence that most of the dopamine β-hydroxylase is not membrane bound in purified large dense cored noradrenergic vesicles

The compartmentalization of dopamine β-hydroxylase was studied in a purified fraction of large, dense-cored noradrenergic vesicles from bovine splenic nerve. A correlative biochemical and morphological approach was used to evaluate physical methods designed to produce more efficient vesicle lysis than has been accomplished previously with freeze-thawing, hypo-osmotic shocks or incubation at 37°C, all of which are relatively ineffective. Of the seven purely physical techniques tested, only those which subjected the vesicles to high pressures followed by rapid decompression produced effective lysis, unmasking of latent enzyme activity, and release of enzyme into the soluble phase. Based on results with the French Press, the best estimate for the percentage of dopamine β-hydroxylase which can be released into the soluble phase is 55–70% of the total enzyme activity produced by the addition of 0.05% v/v Triton X-100. This includes two-thirds of the enzyme which was originally in a latent form. The enzyme is considered to occur primarily, if not entirely, in a granular complex in the vesicle matrix. About one-third is thought to be oriented specifically at the inner surface of the vesicle membrane and is active enzymatically. Physical rupture of the vesicles causes redistribution of partially depleted vesicles and free matrix granules containing dopamine β-hydroxylase activity into less dense zones of a sucrose-D 2O density gradient. After French Press treatment, 30% of the enzyme lost from the heavy vesicle peak is found in the region of the light vesicle peak which is thought to be the equilibrium density of the smaller vesicles characteristic of nerve terminals. The data indicate that much more dopamine β-hydroxylase is potentially available for release from the large, dense-cored vesicles upon nerve stimulation than previously has been believed. This will affect interpretations of exocytotic release from the two vesicle populations in noradrenergic varicosities and will have a bearing on the origin of circulating dopamine β-hydroxylase in response to physiological stress and pharmacological intervention, as well as in neurological diseases.

Vesicles isolated from ATP-depleted erythrocytes and out of thrombocyte-rich plasma.

When human erythrocytes are depleted of endogenous ATP they release spectrin-free vesicles as a light vesicle fraction [Lutz et al: J Cell Biol 73: 548, 1977] and chains of rounded vesicles as well as flattened myelin forms in a heavy vesicle fraction. The heavy fraction retains some spectrin, and glycophorin is partially degraded. The release of both types of fragments is not dependent upon added Ca+2, and 50 micrometer EGTA does not prevent the vesicle release. Concomitant with vesicle release, a large fraction of the major protein components of the cell is found in disulfide-bonded aggregates. A protocol is outlined to recover erythrocyte-specific fragments from thrombocyte-rich plasma. It allows detection of spectrin-free vesicles in whole blood stored under blood bank conditions for 12 days. In freshly drawn blood no such vesicles are observed, but particles are obtained that are different from thrombocyte fragments and that show a prominent glycoprotein running slightly faster than glycophorin.

Characterization of membranes obtained from electric organ of the electric eel by sucrose gradient fractionation and by microdissection.

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a‐bungarotoxin‐binding capacity and Na+‐K+ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS‐polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar.A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [14C]sucrose and 22Na+, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine.The AChE in the light fraction does not appear to be membrane‐bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates.Separated innervated and non‐innervated surfaces of isolated electroplax were obtained by microdissection. α‐Bungarotoxin‐binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na+‐K+ ATPase in the non‐innervated membrane.The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non‐innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.

Adenosine triphosphate-induced rapid calcium release from fragmented sarcoplasmic reticulum

Calcium efflux from skeletal muscle fragmented sarcoplasmic reticulum was studied using a dilution technique and Millipore filtration. In the absence of Mg ++ and external Ca ++, addition of lmM adenosine triphosphate to the suspension resulted in an immediate loss of 26–55% of total vesicular calcium. The amount of calcium released was calculated to be sufficient to effect muscle contraction. After separation of the sarcoplasmic reticulum into light, intermediate and heavy vesicles, the light and heavy fractions were found to be only weakly responsive to adenosine triphosphate, whereas the intermediate fraction lost nearly half of its calcium. The significance of these results with respect to excitation-contraction coupling in muscle is discussed.

Latency of dopamine β-hydroxylase in purified noradrenergic vesicles

Purified large dense core noradrenergic vesicles isolated from bovine splenic nerve were investigated to determine the percentage of total dopamine β-hydroxylase which existed in a latent or masked form. The effects of the nonionic detergent Triton X-100 in concentrations between 0.0025 and 0.1% were studied. At 0.01%, Triton produced a maximum three-fold increase in accessible dopamine β-hydroxylase activity to reach 0.64μmol octopamine per mg protein per min at 37°C, but released only a third of the enzyme into the soluble phase. About 90% of the noradrenaline, 42% of the phospholipids and 17% of the vesicle protein were released. At 0.05%, Triton produced the same maximum enzyme activity with complete solubilization of the enzyme. All the noradrenaline, 90% of the phospholipids, and 48% of the vesicle protein were released into the soluble phase. Ultrastructural examination of the treated vesicles revealed only partial lysis with 0.01% Triton, while 0.05% produced essentially complete disruption of vesicles into membrane fragments and fine granular matrix contents. Equilibrium centrifugation of the control and treated vesicles on a second sucrose-D 2O density gradient showed that emptied and partially depleted vesicles and released matrix granules redistributed from the heavy vesicle peak to less dense regions of the gradient, including the zone of the light vesicle peak. It is proposed that the latency of dopamine β-hydroxylase activity in this noradrenergic vesicle fraction is best explained by the occurrence of the enzyme as an inaccessible complex in the vesicle matrix which may include phospholipid.

Isolation and characterization of two types of sarcoplasmic reticulum vesicles

A purified preparation of sarcoplasmic reticulum from rabbit skeletal muscle has been found to consist of a heterogeneous population of vesicles. Isopycnic centrifugation was used to obtain “light” and “heavy” vesicles from the upper and lower ends of a 25 to 45% (w/w) linear sucrose gradient. Each fraction accounted for about 10 to 15% of the total vesicles. The remainder of the vesicles were of intermediate density and banded between the light and heavy fraction. Light vesicles were composed of about equal amounts of phospholipid and Ca 2+ pump protein which contained approx. 90% of the protein. Heavy vesicles contained in addition to the Ca 2+ pump protein (55–65% of the protein) two other major protein components, the Ca 2+ binding and M 55 proteins which accounted for 20–25 and 5–7% of the protein of these vesicles, respectively. The sarcoplasmic reticulum subfractions had 32P-labelled phosphoenzyme levels proportional to their Ca 2+ pump protein content and contained similar Ca 2+ -stimulated ATPase activities. They were capable of accumulating Ca 2+ in the presence of ATP and of releasing the accumulated Ca 2+ when placed into a medium with a low Ca 2+ concentration. The vesicles differed significantly in that heavy vesicles had a greater number of non-specific Ca 2+ binding sites than light vesicles (approx. 220 vs 75 nmol of bound Ca 2+ per mg protein), in accordance with their high content of Ca 2+ binding protein. Electron dense material could be seen within the compartment of heavy but not light vesicles. Removal of Ca 2+ binding and M 55 proteins from heavy vesicles resulted in empty membranous structures consisting mainly of Ca 2+ pump protein and phospholipid. Electron micrographs of sections of muscle showed dense material in terminal cisternae but not in longitudinal sections of sarcoplasmic reticulum. These experiments are consistent with the interpretation that (1) the electron dense material inside heavy vesicles may be referable to Ca 2+ binding and/or M 55 proteins, and that (2) light and heavy vesicles may be derived from the longitudinal sections and terminal cisternae of sarcoplasmic reticulum, respectively.

Maturation of the adrenal medulla—I: Uptake and storage of amines in isolated storage vesicles of the rat

Albino Wistar rats were sacrificed at 10-day intervals from birth to 50 days of age. Adrenal storage vesicles were analyzed for catecholamines (CA) and for uptake of 14C-epinephrine (E) and 3H-metaraminol (MA). At birth, CA and E and MA uptakes per gland were 3–8 per cent of 50-day levels, about 20 per cent at 10 days, 25 per cent at 20 days, 40 per cent at 30 days, and 65 per cent at 40 days. Although all three parameters increased approximately in parallel, MA uptake slightly exceeded E uptake and CA from birth to 30 days. As a result, MA uptake per 100 μg of CA in the vesicles was 20 per cent above normal at the early time periods, while E uptake per 100 μg of CA was normal. There was no change in the endogenous norepinephrine/epinephrine ratio during development. The rates of efflux of 14C-E and CA from isolated vesicles was normal except at 10 days, when a more rapid efflux was observed, suggesting that storage stability was impaired at that time. Changes in the density of labeled vesicles were evaluated by centrifugation on continuous sucrose density gradients. At birth, both E-and MA-labeled vesicles were more dense than vesicles from 50-day-old rats. By 10 days, however, E- and MA-labeled vesicles were lighter than normal. E-labeled vesicles returned to normal by 20 days, but MA-labeled vesicles did not approach normal densities until 30–40 days. These data suggest that the rate of vesicle synthesis is low at birth but increases rapidly thereafter. The heavy vesicles at birth may represent “overloaded” vesicles, while the lighter ones at later times may be “underloaded” due to the rapid synthesis of new vesicles.