ABSTRACT
OBJECTIVE: The Posturo-Locomotion-Manual (PLM) test, which uses an optoelectronic laboratory system, has here been further developed into an automated, more user-friendly, standardized tool for movement analysis named the QbTestMotus. This paper compares the accuracy of QbTestMotus to the PLM test, in particular the automated data analysis. METHODS: Both QbTestMotus and the PLM recorded data simultaneously from the same 61 patients. The correlation coefficients of movement time (MT), postural time (P), locomotion time (L), and manual time (M) were calculated between the systems. The absolute differences between the result parameters for each patient were also studied. Finally, the differences in MT between the systems were compared with the positive responses in the levodopa (L-dopa) challenges as measured in the PLM test for 11 patients. RESULTS: The comparisons in all the 61 patients showed high correlation coefficients for all four parameters. The absolute differences between the parameters were small and had small standard deviations, and the decreases in MT because of L-dopa in the positive L-dopa responders were much larger than the absolute difference between the systems. CONCLUSION: The PLM test and QbTestMotus are equivalent along all parameters, thus indicating that the test quality is equivalent between the PLM test and the automated QbTestMotus system.
Subject(s)
Image Interpretation, Computer-Assisted/methods , Movement/physiology , Parkinson Disease/physiopathology , User-Computer Interface , Aged , Female , Humans , Male , Middle Aged , Neurologic Examination/methods , Video RecordingABSTRACT
The main characteristics of dysarthria in Parkinson's disease (PD) are monotony of pitch and loudness, reduced stress, variable speech rate, imprecise consonants, and breathy and harsh voice. Earlier treatment studies have shown that dysarthria is less responsive to both pharmacological and surgical treatments than other gross motor symptoms. Recent findings have suggested that repetitive transcranial magnetic stimulation (rTMS) may have a beneficial effect on vocal function in PD. In the present study, 10 individuals with mild PD and no or minimal dysarthria were treated with rTMS as well as placebo stimulation in a blinded experiment. Stimulation was delivered using a frequency of 10 Hz and a stimulation intensity of 90% of the motor threshold. The site of stimulation was the cortical area corresponding to the hand, on the hemisphere contralateral to the patient's most affected side. The participants were audio-recorded before and after both rTMS and sham stimulation. Acoustic analysis was performed on 3 sustained /a:/ for each of the 4 conditions, and analyzed both for the whole group as well as for men and women separately. Results showed that there were no significant differences between any of the conditions regarding duration of sustained fricative or sustained vowel phonation, diadochokinetic rates or intelligibility. Above all, the results of acoustic analyses showed an effect of placebo; there was a significant reduction in fundamental frequency (F(0)) variation, pitch period perturbation, amplitude period perturbation, noise-to-harmonics ratio and coefficient of variation in F(0) between the recordings performed before compared to after sham stimulation.
Subject(s)
Dysarthria/therapy , Parkinson Disease/therapy , Transcranial Magnetic Stimulation , Voice Disorders/therapy , Adult , Aged , Cross-Over Studies , Dysarthria/etiology , Female , Hand/physiopathology , Humans , Male , Middle Aged , Motor Cortex/physiopathology , Parkinson Disease/complications , Parkinson Disease/physiopathology , Phonetics , Psychomotor Performance , Sex Characteristics , Speech Acoustics , Speech Intelligibility , Transcranial Magnetic Stimulation/adverse effects , Voice Disorders/etiology , Voice QualityABSTRACT
This paper describes a system for in vitro cell migration analysis. Adult neural stem/progenitor cells are studied using time-lapse bright-field microscopy and thereafter stained immunohistochemically to find and distinguish undifferentiated glial progenitor cells and cells having differentiated into type-1 or type-2 astrocytes. The cells are automatically segmented and tracked through the time-lapse sequence. An extension to the Chan-Vese Level Set segmentation algorithm, including two new terms for specialized growing and pruning, made it possible to resolve clustered cells, and reduced the tracking error by 65%. We used a custom-built manual correction module to form a ground truth used as a reference for tracked cells that could be identified from the fluorescence staining. On average, the tracks were correct 95% of the time, using our new segmentation. The tracking, or association of segmented cells, was performed using a 2-state Hidden Markov Model describing the random behaviour of the cells. By re-estimating the motion model to conform with the segmented data we managed to reduce the number of tracking parameters to essentially only one. Upon characterization of the cell migration by the HMM state occupation function, it was found that glial progenitor cells were moving randomly 2/3 of the time, while the type-2 astrocytes showed a directed movement 2/3 of the time. This finding indicates possibilities for cell-type specific identification and cell sorting of live cells based on specific movement patterns in individual cell populations, which would have valuable applications in neurobiological research.
Subject(s)
Cell Movement , Microscopy, Video/methods , Stem Cells/physiology , Animals , Cells, Cultured , RatsABSTRACT
Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is also known to induce a wide spectrum of biological responses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not express the glutamate transporter-1 (GLT-1). The V(max) and the K(m) of the glutamate uptake were reduced by 57% and 47%, respectively. Application of the ET(A) and ET(B) receptor antagonists BQ-123 and BQ-788 partly inhibited the ET-1-evoked decrease in the glutamate uptake, whereas the nonspecific ET receptor antagonist bosentan completely inhibited this decrease. Incubation of the cultures with pertussis toxin abolished the effect of ET-1 on the uptake. The ET-1-induced decrease in the glutamate uptake was independent of extracellular free Ca(2+) concentration, whereas the intracellular Ca(2+) antagonists thapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not with the fatty acid-binding protein bovine serum albumin, prevented the ET-1-induced decrease in the glutamate uptake. These results suggest that ET-1 impairs the high-affinity glutamate uptake in cultured astrocytes through a G protein-coupled mechanism, involving PKC and changes in intracellular Ca(2+).
Subject(s)
Astrocytes/metabolism , Endothelin-1/pharmacology , Glutamic Acid/metabolism , Amino Acid Sequence , Animals , Astrocytes/drug effects , Barium Compounds/pharmacology , Blotting, Western , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Chlorides/pharmacology , Depression, Chemical , Immunohistochemistry , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Transduction, GeneticABSTRACT
Recently, knowledge about the role of astrocytes in the brain has increased substantially. As a result we have had to rethink old views regarding how the brain works at the cellular level. Neurons can no longer be regarded as the only cell types of functional significance. The picture instead appears to be far more complex, with an ongoing exchange of information between different cell types, and this interaction is suggested to be particularly important between neurons and astrocytes. Astrocytes express receptors for different classes of neurotransmitters, and have both voltage and receptor operated ion channels. Through active uptake and release of ions, neurotransmitters and water they control the brain interstitium. Intercellular communication via transfer of neuroactive substances through gap junctions makes it possible to coordinate different activities in large areas of the brain. Dysfunction of astrocytic physiology is thought to contribute to the pathogenesis and progress of various neurological disorders such as epilepsy, stroke and cerebral edema.
Subject(s)
Astrocytes/physiology , Brain Diseases/etiology , Brain/cytology , Astrocytes/metabolism , Astrocytes/ultrastructure , Brain/pathology , Brain/physiopathology , Brain Diseases/pathology , Brain Diseases/physiopathology , Cell Communication , Electroencephalography , Gap Junctions/physiology , Humans , Ion Channels , Microscopy, Fluorescence , Receptors, Neurotransmitter/metabolism , Receptors, Neurotransmitter/physiologyABSTRACT
Antibodies against the C-terminus of the beta1-adrenergic receptor were used for staining cultured astrocytes from the rat cerebral cortex. Immunoreactivity was found to be localized exclusively to an intracellular organelle structure similar to the Golgi complex, with no staining of the plasma membrane. The astrocytes stained positive with BODIPY CGP 12177, a FITC-conjugated beta-adrenergic receptor agonist, and this staining was blocked by the beta1-adrenergic antagonist atenolol, indicating that these receptors are expressed on the surface of the astrocytes. The presence of functional plasma membrane beta1-adrenergic receptors was further verified using microspectrofluorometry for measurements of intracellular calcium changes upon beta-adrenergic agonist stimulation. Intracellular immunoreactivity confined to the organelles was also found in astrocytes from mixed astroglial-neuronal cultures. In contrast, the neurons in these cultures showed a strong labeling of the cell bodies by the beta1-adrenergic receptor antibodies. Thus, the beta1-adrenergic receptor antibody, which stains the cell bodies of the neurons, recognizes the astroglial receptors only intracellularly, although functional beta1-adrenergic receptors are present on the astroglial surface. Taken together, these data suggest that the beta1-adrenergic receptors observed intracellularly might be processed on their passage to the surface to a modified form of the final plasma membrane receptor, which is not recognized by the antibodies.
Subject(s)
Adrenergic alpha-Agonists/pharmacology , Astrocytes/metabolism , Calcium/metabolism , Receptors, Adrenergic, beta-1/metabolism , Adrenergic beta-1 Receptor Agonists , Animals , Animals, Newborn , Cells, Cultured , Coculture Techniques , Fluorescent Dyes , Immunohistochemistry , Rats , Rats, Sprague-DawleyABSTRACT
Previous immunohistochemical and radioligand binding studies have shown a cell cycle-dependent regulation of the delta opioid receptor (DOR). The relationship between DOR expression and mitosis in primary astroglial cultures of rat cerebral cortex was investigated in this study. The cultures were arrested during the G(1)/S transition or during mitosis. The DOR protein level increased twofold (P = 0.009) during mitosis and DOR mRNA level increased threefold (P = 0.002) during the G(1)/S transition compared to nonsynchronized cultures. DOR mRNA was also elevated (1.6-fold, P = 0.008) during the G(1)/S transition compared with mitotic cells. A premitotic increase in DOR mRNA suggests that elevated DOR protein levels during mitosis might be regulated during transcription.
Subject(s)
Astrocytes/metabolism , G1 Phase/physiology , RNA, Messenger/metabolism , Receptors, Opioid, delta/metabolism , S Phase/physiology , Animals , Animals, Newborn , Astrocytes/cytology , Cell Cycle/physiology , Rats , Rats, Sprague-DawleyABSTRACT
Astroglia have the capacity to monitor extracellular glutamate (Glu) and maintain it at low levels, metabolize Glu, or release it back into the extracellular space. Glu can induce an increase in astroglial cell volume with a resulting decrease of the extracellular space, and thereby alter the concentration of extracellular substances. Many lines of evidence show that K(+) can be buffered within the astroglial gap-junction-coupled network, and recent results show that gap junctions are permeable for Glu. All these events occur dynamically: the astroglial network has the capacity to interfere actively with neurotransmission, thereby contributing to a high signal-to-noise ratio for the Glu transmission. High-quality neuronal messages during normal physiology can then be maintained. With the same mechanisms, astroglia might exert a neuroprotective function in situations of moderately increased extracellular Glu concentrations, i.e., corresponding to conditions of pathological hyper-excitability, or corresponding to early stages of an acute brain injury. If the astroglial functions are failing, neuronal dysfunction can be reinforced.
Subject(s)
Astrocytes/metabolism , Cell Communication/physiology , Gap Junctions/physiology , Glutamic Acid/physiology , Nervous System Diseases/physiopathology , Animals , Astrocytes/pathology , Astrocytes/physiology , Cell Size , Glutamic Acid/metabolism , Humans , Nervous System Diseases/metabolism , Neurotransmitter Agents/physiologyABSTRACT
The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5-HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Calcium/metabolism , Receptors, Serotonin/physiology , Animals , Astrocytes/drug effects , Blotting, Western , Brain/cytology , Calcium/physiology , Cells, Cultured , Extracellular Space/metabolism , Immunohistochemistry , Rats , Rats, Sprague-Dawley , Receptor, Serotonin, 5-HT2B , Serotonin/analogs & derivatives , Serotonin/pharmacology , Serotonin Receptor Agonists/pharmacologyABSTRACT
The glial cells, especially the astroglia constitute a prominent part of the brain cell volume. Astroglial properties are difficult to study in the intact nervous system. For that reason, different in vitro models have been developed. The development of cell and tissue cultivation conditions has been the prerequisite to our present knowledge of the biochemistry and pharmacology of glial cells and to some extent even neurons. It is, however, an advantage if results from tissue culture can be evaluated in more in vivo like systems. We here describe a method for acute isolation of freshly prepared neurons and glial cells.
Subject(s)
Astrocytes/cytology , Astrocytes/physiology , Brain/cytology , Cell Culture Techniques/methods , Animals , Cell Differentiation/physiology , Cell Division/physiology , Cell Separation/methods , HumansABSTRACT
Endogenous opioid peptides and opioid receptors are expressed by brain cells early during normal development, and exogenous opiate exposure in this period is known to affect brain cell proliferation and maturation. Despite the abundant evidence that opioids affect brain development, little is known about the mechanisms involved. In this study cortical astrocytes in primary culture were examined immunohistochemically by using antibodies against the opioid receptors. The immunoreactivity for delta-opioid receptors was strongly upregulated during mitosis with an increase in immunostaining that started in early prophase and lasted through the M-phase to cytokinesis. Similar effects could not be observed when antibodies against the mu- or kappa-opioid receptor subtypes were used. Cultured neurons and microglia presented a strong and homogenous immunostaining for the delta-opioid receptor and no further upregulation of immunoreactivity could be detected in these cells. The presence of functional delta-opioid receptors on the mitotic astrocytes was verified by using microspectrofluorometry for detection of delta-opioid agonist induced changes in intracellular free calcium concentrations ([Ca2+]i). In these experiments fluo-3/AM incubated cells showed a rapidly induced delta-opioid agonist (DPDPE, 10(-6) M) evoked increase in [Ca2+]i. These results suggest an upregulation of the delta-opioid receptors that could represent a mechanism involved in the response to opioids in the developing brain.
Subject(s)
Astrocytes/metabolism , Mitosis/physiology , Receptors, Opioid, delta/biosynthesis , Animals , Animals, Newborn , Astrocytes/physiology , Cells, Cultured , Enkephalin, D-Penicillamine (2,5)- , Enkephalins/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/drug effects , Spectrometry, Fluorescence , Up-RegulationABSTRACT
A new nonenzymatic method for the acute isolation of astrocytes from rat cerebral cortex is described. A vibratory device was used to dissociate the cells from thin brain slices, and the method yielded fresh and relatively well-preserved astrocytes without previous enzyme incubation. These cells were examined in a microspectrofluorometric system for measurement of changes in intracellular free calcium concentrations ([Ca2+]i), and their expression of various neurotransmitter receptors was determined. Acutely isolated glial fibrillary acidic protein (GFAP)-positive astrocytes (p7-p18) were seen to respond to the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10(-4) M) with increases in [Ca2+]i, and this response was blocked by (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA, 10(-3) M), an antagonist to group 1 metabotropic glutamate receptors. The delta-opioid receptor agonist D-Pen2, D-Pen5-enkephalin (DPDPE, 10(-6) M) evoked [Ca2+]i increases that were blocked by the delta-opioid antagonist ICI 174.388 (10(-5) M). The astrocytes failed to respond to 5-hydroxytryptamine (5-HT, 10(-5) M), although the same cells subsequently were found to respond to other agonists. Furthermore, [Ca2+]i responses evoked by phenylephrine (10(-5) M) were blocked by prazosin (0.2x10(-6) M), suggesting the expression of alpha1-adrenergic receptors on the acutely isolated astrocytes. The cells were also shown to react with [Ca2+]i increases in response to depolarization with high extracellular potassium concentrations (50x10(-3) M). The signals induced by depolarization were not seen in Ca2+-free buffer, indicating the presence of voltage-activated calcium channels in these cells. Thus, the present study confirms some of the results earlier obtained in cell cultures, suggesting that cortical astrocytes in vivo express glutamate, opiate, and adrenergic receptors, coupled to increases in [Ca2+]i, whereas no receptors for 5-HT could be detected.
Subject(s)
Astrocytes/metabolism , Calcium/metabolism , Cell Separation/methods , Receptors, Neurotransmitter/metabolism , Animals , Astrocytes/cytology , Astrocytes/drug effects , Cell Size , Cells, Cultured , Cerebral Cortex , Cycloleucine/analogs & derivatives , Cycloleucine/pharmacology , Enkephalin, D-Penicillamine (2,5)- , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/pharmacology , Enkephalins/pharmacology , Glutamic Acid/pharmacology , Immunohistochemistry , Indans/pharmacology , Norepinephrine/pharmacology , Phenylephrine/pharmacology , Potassium/pharmacology , Prazosin/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Neurotransmitter/agonists , Receptors, Neurotransmitter/antagonists & inhibitors , Serotonin/pharmacologyABSTRACT
Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.
Subject(s)
Astrocytes/metabolism , Calcium/metabolism , Cerebral Cortex/metabolism , GTP-Binding Proteins/metabolism , Neuroglia/metabolism , Receptors, Opioid, delta/physiology , Animals , Astrocytes/drug effects , Calcium Channels/metabolism , Cells, Cultured , Cerebral Cortex/drug effects , Enkephalin, D-Penicillamine (2,5)- , Enkephalins/pharmacology , GTP-Binding Proteins/antagonists & inhibitors , Immunohistochemistry , Neuroglia/drug effects , Neurons/drug effects , Neurons/metabolism , Pertussis Toxin , Rats , Rats, Sprague-Dawley , Receptors, Opioid, delta/agonists , Virulence Factors, Bordetella/pharmacologyABSTRACT
The excitatory effect of presynaptically released glutamate is tightly regulated and terminated by high affinity sodium-dependent glutamate transporters. The regulation of the glial glutamate transporter GLT-1 is potentially important in synaptic modulation. Using astroglial cultures prepared from the rat cerebral cortex, we found that the delta-opioid receptor agonist [D-pen2,D-pen5]-enkephalin decreases and glutamate increases the expression of the GLT-1 transporter mRNA. Corresponding changes in the uptake kinetics were found after incubation for 48 h with the respective agonists when glial glutamate uptake was measured in primary astroglial cultures. The data suggest that long-term receptor activation induces alterations in glial glutamate uptake properties.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Glutamic Acid/pharmacology , Receptors, Opioid, delta/metabolism , Amino Acid Transport System X-AG , Animals , Biological Transport/physiology , Enkephalin, D-Penicillamine (2,5)- , Enkephalins/pharmacology , Gene Expression Regulation/genetics , Glutamic Acid/pharmacokinetics , Kinetics , Microscopy, Fluorescence , Neuroglia , Neurotransmitter Agents/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/antagonists & inhibitors , Synaptic Transmission/physiologyABSTRACT
Recent work from our and other laboratories have shown that glial cells in culture express opioid receptors. In the present study we have analyzed the regulation of delta-opioid receptor mRNA and the regulation of delta-opioid receptor activated astroglial Ca2+ responses in primary cultures from the rat cerebral cortex. Astroglial cultures were incubated with glutamate (Glu) or [D-Pen2,5]enkephalin (DPDPE) for 48 h, and delta-opioid receptor mRNA levels were analyzed using a solution hybridization RNase protection assay. Our results suggest that incubation in Glu or DPDPE upregulates the abundance of delta-opioid receptor mRNA in a dose dependent way. Glu incubated cells showed a maximum upregulation at the highest agonist concentration used (10[-5] M), whereas DPDPE was most effective at low concentrations (l0[-9] M). Furthermore, corresponding Ca2+ imaging experiments showed that incubation in Glu or DPDPE upregulated the responding frequency of delta-opioid receptor activated glial calcium fluxes from a control value of 5% to 14% and 17% responding cells, respectively.
Subject(s)
Astrocytes/metabolism , Cerebral Cortex/metabolism , Enkephalins/pharmacology , Gene Expression Regulation , Glutamic Acid/pharmacology , Receptors, Opioid, delta/metabolism , Aniline Compounds/analysis , Animals , Animals, Newborn , Astrocytes/drug effects , Calcium/metabolism , Cells, Cultured , Cerebral Cortex/drug effects , Dose-Response Relationship, Drug , Enkephalin, D-Penicillamine (2,5)- , Fluorescent Dyes/analysis , Potassium/pharmacology , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Opioid, delta/agonists , Xanthenes/analysisSubject(s)
Emergency Service, Hospital/standards , Internship and Residency , Humans , Sweden , WorkforceABSTRACT
Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B (apoB) 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF (the smooth membrane fraction). The migration of SMF during gradient ultracentrifugation as well as kinetic studies indicated that the fraction was derived from a pre-Golgi compartment, probably the smooth endoplasmic reticulum (ER). Only small amounts of LpB 100 could be detected during these pulse-chase experiments in the subcellular fractions derived from the rough endoplasmatic reticulum (RER). SMF contained the major amount of the diacylglycerol acyltransferase activity present in the ER, while the major amount of membrane bound apoB 100 was present in the RER. Pulse-chase studies of the intracellular transfer of apoB 100 demonstrated the formation of a large membrane-bound preassembly pool in the ER, while no significant amount of apoB 100 radioactivity was present in the membrane of the Golgi apparatus. The maximal radioactivity of LpB 100, recovered from the ER or the Golgi lumen, was small compared with the radioactivity recovered from the ER membrane, indicating that the assembled LpB 100 rapidly leaves the cells. This in turn indicates that the rate-limiting step in the secretion of apoB 100 was the transfer of the protein from the ER membrane to the LpB 100 in the lumen. A portion of the intracellular pool of apoB 100 was not secreted but underwent posttranslational degradation.