Elements Of Endoplasmic Reticulum Research Articles

Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.

An assay for endoplasmic reticulum (ER)-through-Golgi transport has been developed in streptolysin O-permeabilized murine erythroleukemia (MEL) cells. The reporter proteins are metabolically labeled native murine glycophorins, which display a distinctive shift in electrophoretic mobility after acquisition of O-linked oligosaccharides. The O-linked sugars are acquired at a site distal to a brefeldin A block, presumably in a cis Golgi compartment, and sialylation occurs in middle and/or trans Golgi compartments. In permeabilized cells supplemented with cytosolic proteins and an ATP-generating system, 20-50% of the radiolabeled precursor glycophorins can be converted to the mature, sialylated form. This maturation process is ATP- and cytosol-dependent and is blocked by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). Electron microscopy of permeabilized MEL cells shows retention of ER elements, stacked Golgi cisternae, free polysomes, and other subcellular components. In the presence of GTP[gamma S], dilated vesicles accumulate around the Golgi stacks. Antisera to the carboxyl terminus of the Golgi resident alpha subunit of Gi3 inhibit maturation of glycophorin. To our knowledge, a transport assay utilizing O-glycosylation of an endogenous protein as a monitor of ER-through-Golgi traffic in permeabilized cells has not been reported previously. Furthermore, the data provide evidence for heterotrimeric GTP-binding protein involvement in Golgi function.

Properties of Johnsongrass Chlorotic Stripe Mosaic Virus

AbstractPhysicochemical, biological, and cytopathological properties of Johnsongrass chlorotic stripe mosaic virus (JCSMV) found in Iran were investigated. Virus particles were polyhedral, showed a knobbed surface structure, were c. 30 nm in diameter and had a buoyant density of 1. 359 g/ml in cesium chloride. Virions contained one major protein with a molecular weight of 41 kd and a single species of ssRNA with a molecular weight of 1. 43 × 106 d. Acid hydrolysis of the virus followed by thin‐layer electrophoresis gave the following molar percentages of the bases: A: 23. 5, G: 27. 5, C: 26 and U: 23. Separation of nucleotides of the viral RNA using alkaline hydrolysis was not successful. Mechanical inoculation of freshly purified virus or isolated RNA failed to infect Johnsongrass or maize plants. The virus was readily detected by ELISA in seeds from infected plants and young seedlings raised from such seeds, but not in later stages of growth. Ultrathin sections of infected cells showed high concentrations of virus particles in the cytoplasm and vacuoles. Virus‐like particles also occurred in the stroma of chloroplasts and mitochondria. Cisternae of endoplasmic reticulum (ER) were often extremely inflated and filled by a fine fibrillar material. Small membrane‐associated vesicles were frequently found in ER elements and occurred also in the permuclear space. Based on particle structure, properties of the nucleic acid, molecular weight of the coat protein and cytopathology, the virus resembles carmoviruses. However, lack of mechanical transmissibility is not known for any virus classified with this group. No serological reaction was detected with a total of 30 antisera to carmoviruses and other isometric viruses.

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization.

The K-pyroantimonate/OsO4 (PA) cytochemical method coupled with EGTA and X-ray microanalytical controls has been used to localize Ca2+ at egg activation in Discoglossus pictus eggs. The results show that: 1) the PA method is able to selectively localize Ca2+ pools mobilized by activating stimuli; 2) the smooth endoplasmic reticulum (SER) elements located in the animal dimple region, i.e. in the predetermined site of fertilization, are the first egg components labeled by precipitates; 3) a decreasing gradient of precipitates is present from the center beyond the boundaries of the dimple region; 4) precipitates are lacking in the remainder of the egg even at late times after activation. The possibilities are discussed that a) SER is the major Ca2+ -releasing store at activation in Discoglossus, and b) the observed gradient of pyroantimonate-detected Ca2+ reflects an ionic Ca2+ gradient.

The Origin of Cortical Vesicles and their Role in Egg Envelope Formation in the "Spiny" Eggs of a Calanoid Copepod, Centropages velificatus

The mature oocytes of the marine calanoid copepod, Centropages velificatus, contain two morphologically distinct populations of cortical vesicles that undergo sequential exocytoses at the time of spawning. The contents of the primary cortical vesicles are released first and form the primary egg envelope. This is followed by the exocytosis of the secondary cortical vesicles. These contain numerous intracisternal granules that, upon release into the perivitelline space, transform into a mass of fine fibers. The continual accumulation of fibers constitutes an extracellular matrix between the primary envelope and the egg's plasmalemma. Further amassment of the fibers beneath the primary egg envelope results in the formation of long, spiny projections. The evolution of the cortical vesicles was traced to the early vitellogenic oocytes and appears to be unique. The two populations of cortical vesicles are synthesized together within the same cisternal elements of rough endoplasmic reticulum (RER). The RER originates from membranous blebs off both the nuclear membrane and stacks of annulate lamellae in the early vitellogenic oocytes. Numerous intracisternal granules are present within the RER. Some of these granules fuse, forming a dense, ring-like structure in the extremities of the cisternae. These bud off from the RER to become the primary cortical vesicles. The unfused intracisternal granules remain as discrete bodies within irregular profiles of vesicular ER and comprise the secondary cortical vesicles.

Colloidal gold ultraimmunocytochemical localization of DNA and RNA adducts in rat hepatocytes.

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-methylguanine (O6-metG) and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2 h to 5 mM N-nitrosodimethylamine (NDMA). In NDMA-treated cells, the O6-metG-induced immunoreactive sites do not appear at random but seem to be concentrated in the nucleus, and in the cytoplasm, in areas rich in rough endoplasmic reticulum (RER) elements. Mitochondria were not significantly labelled. Untreated control preparations showed no specific immunogold labelling. After RNase digestion of ultrathin sections obtained from cells exposed to NDMA and subsequent immunogold labelling, most of the immunolabelling in the cytoplasm had disappeared, and that over the nucleus had only been slightly reduced, as compared to undigested specimens from NDMA-treated cultures. After similar digestion with DNase, a strong reduction of the labelling of the nucleus was observed, but labelling of the cytoplasm was practically unaffected by this enzymatic treatment, as compared to what was observed in undigested preparations of NDMA-treated hepatocytes. The results provide evidence of preferential formation of O6-metG at the DNA and RNA levels, in the nucleus and cytoplasm RER, respectively. Furthermore, this study demonstrates the applicability of the high-resolution protein A-gold technique for ultrastructural detection of nucleic acid adducts in NDMA-treated hepatocytes using affinity-purified anti-O6-metG polyclonal antibodies.

Freeze-fracture organization of chromatin and cytoplasm in neurons and astroglia of rat cerebellar cortex

The cytology of the cell nucleus and cytoplasm of neurons and astroglia of the rat cerebellar cortex has been investigated by freeze-fracture electron microscopy. The main differential characteristics in the cytoplasm of the several cell types of the cerebellar cortex were: (1) the organization of endoplasmic reticulum elements, including special configurations of lamellar bodies and hypolemmal complexes, (2) the polarity, extension and arrangement of Golgi cisterns and associated tubulovesicular elements; (3) the connection pattern among different membrane-bounded cellular compartments; and (4) the architecture of endomembranes (i.e. presence of pits and fenestrations). In the nucleus, the main differential features were the the three-dimensional view of the nuclear envelope, the distribution of nuclear pores and the aggregation pattern of chromatin, visualized as clusters of nuclear particles in cross-fractures. The quantitative analysis of chromatin revealed four peaks of nuclear particle sizes (8, 12, 17 and 21 nm) that may correspond to variable degrees of coiling of the polynucleosomal chain in the chromatin fibre. Significant differences were observed in the proportion, numerical density and size distribution of aggregated nuclear particles in heterochromatin domains among the different cell types of the cerebellar cortex. The percentage of nuclear particles in aggregates varied from 10% in Purkinje cells to 64% in granule cells. Astrocytes and Bergmann glia showed intermediate values (about 40%). The percentage of nuclear particles in aggregates showed a significant (P less than 0.05) negative linear correlation with the nuclear volume, the number of pores per unit nuclear volume and the total number of pores per nucleus. In granule cells and astroglia, heterochromatin domains had a greater percentage of large nuclear particles (greater than 10 nm) than did euchromatin domains, whereas in interneurons, Purkinje and Golgi cells heterochromatin and euchromatin showed a similar proportion of large particles. Nuclear particles in euchromatin exhibited a similar pattern of distribution in all cerebellar cells.

Cortical endoplasmic reticulum in plants

ABSTRACT Structural observations provide persuasive evidence for the existence of a cortical network of endoplasmic reticulum (ER) in a large number of plant and animal cells. The network in plants generally possesses a polygonal pattern in which smooth, tubular elements are joined by intervening lamellar segments. The individual elements of ER are often positioned extremely close to the plasma membrane (PM), and may form appositional contacts, but fusion does not occur. The network arises at cytokinesis and establishes continuity between the cortical ER of daughter cells in the form of tightly furled membranetubules that traverse the plasmodesmata. The specific function of the cortical ER complex is unknown but different possibilities seem attractive. It may serve key roles in anchoring the cytoskeleton and in facilitating secretion. The cortical ER might also participate in the communication of signals between the exterior of the cell and cytoplasm. As a consequence of its ability to release and/or sequester Ca, the ER could control the cytoplasmic activity of this ion and thus a host of physiologically and developmentally important reactions.

Intracellular sterol trafficking

Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.

Reversible neuronal damage in hippocampal pyramidal cells with triethyllead: the role of astrocytes.

A single dose (19 mg kg-1) of triethyllead given to weanling rats produces necrosis in a small number of hippocampal pyramidal (CA3) and hilar neurons with reversible changes in the remaining neurons of this region. The sequence of events has been studied by light and electron microscopy over a period from 12 h to 14 days after dosing. Early changes resemble those previously described for trimethyltin, with the formation of characteristic tubulo-vesicular dense bodies by 12 h accompanied by vacuolation of Golgi and smooth surfaced endoplasmic reticulum (SER) elements which became generalized by 24 h. Large numbers of secondary dense bodies, formed from tubulo-vesicular dense bodies as well as from autophagosomes, were present by 48 h, whilst very little rough surfaced endoplasmic reticulum (RER) and few polyribosomes remained and vacuolation was much reduced. In those animals which did not die from seizures, the majority of hippocampal pyramidal cells were able to recover from these changes with astrocytes playing a significant role in the elimination of the dense bodies. This involved astrocytes inserting processes into the neuronal perikaryon from where the secondary dense bodies were selectively transferred into the astrocyte cytoplasm. This activity was first seen at 48 h, reached a peak at 4 days, when most CA3 neurons contained one or more astroglial intrusions and subsided soon after. The surviving neurons returned to apparent normality over the period from 3 to 7 days with a gradual return of polyribosomes. Golgi elements and RER.

Transitional endoplasmic reticulum membranes and vesicles isolated from animals and plants

The process of formation from endoplasmic reticulum and transfer to Golgi apparatus of small 50–70 nm transition vesicles has been reconstituted in a cell-free system. Fractions enriched in transition elements derived from part-rough, part-smooth transitional regions of the endoplasmic reticulum were prepared from elongation zones of hypocotyls of etiolated seedlings of soybean and coleoptiles of maize and were compared with those from rat liver. When activated with nucleoside triphosphate, cytosol and an ATP regenerating system, time- and temperature-dependent transfer of membranes to Golgi apparatus acceptor was demonstrated. The fractions enriched in transition elements were radioiodinated with125I by the Bolton-Hunter procedure. Acceptor Golgi apparatus stacks were immobilized to nitrocellulose strips to facilitate analysis. In heterologous transfer experiments, the plant and animal acceptors and donors could be interchanged. The transfer was limited primarily by the donor (rat liver > soybean hypocotyl > maize coleoptiles) and determined secondarily by the source of the acceptor. The acceptor fractions were most efficacious when prepared from the same source as the donor. Thus, 50–70 nm vesicles bud from transitional endoplasmic reticulum elements of plants function in a manner similar to those of animal cells to transfer membrane materials to the Golgi apparatus. The recognition signals that determine vesicle fusion appear to be conserved both among species and between the plant and animal kingdoms to the extent that donor and acceptor sources may be interchanged with only small reductions in overall efficiency of transfer.

Dynamic behavior of endoplasmic reticulum in living cells

Endoplasmic reticulum (ER) was studied by fluorescence microscopy of living CV-1 cells treated with the fluorescent carbocyanine dye DiOC 6(3). Using video recording and image processing techniques, several distinct forms of highly localized movements of ER were documented, categorized, and analyzed in terms of mechanism and structural implications. These include tubule branching, ring closure, and sliding. These localized movements have been observed to generate the basic elements of ER: linear tubules, polygonal reticulum, and triple junctions. We propose that as such they act as the mechanism for constructing the polygonal lattice of interconnected membrane tubules that constitutes ER. The nature of these movements suggests possible involvement of the cytoskeleton, and, in view of the close correlations in the distributions of ER and microtubules, and the accompanying paper (Dabora and Sheetz), it is possible that microtubules may play a role in generating ER motility and in constructing and maintaining the ER network in living cells.

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin

The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin. Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.

Proteinaceous inclusions in specialized thallus cells of Chylocladia verticillata (Rhodymeniales, Rhodophyta)

Abstract The marine red alga Chylocladia verticillata (Rhodymeniales, Rhodophyta) possesses numerous scattered small cells on the inner face of the hollow thallus which are characterized by the presence ofa great number of cytoplasmic membrane-bound fibrillar inclusions. Application of the Coomassie Brilliant Blue R histochemical technique and treatment of tissue with enzyme solutions indicated that these inclusions are constituted of protein. The frequent, close association of the proteinaceous inclusions with endoplasmic reticulum elements and the occasional coalescence of the endoplasmic reticulum membranes to the inclusion membrane suggest that these inclusions may originate from the endoplasmic reticulum. Fibrillar elements of cytoplasmic inclusions appear to be organized into successive arrays of arcs arranged in nearly parallel stacked levels, as interpreted by examining different planes of inclusion sections. Analysis of the diffraction pattern of such sections has revealed that the proteinaceous ...

Ultrastructural Study of Spermiogenesis in the ChitonAcanthopleura haddonifrom the Jordan Gulf of Aqaba (Red Sea)

Summary Ultrastructural study of spermiogenesis in the chiton Acanthopleura haddoni (Mollusca, Polyplacophora) reveals that early spermatids have round to oval nuclei with granular chromatin. A few mitochondria, a Golgi body, two centrioles, and elements of endoplasmic reticulum are present. Middle spermatids have nuclei with filamentous chromatin material, and the nuclear envelope exhibits a posterior implantation fossa to which a basal plate is aligned. The proximal and distal centrioles are at right angles, and pericentriolar bodies are observed lateral to the proximal centriole. A primordium of the tail appears early in spermiogenesis. No proacrosomal vesicles were observed, and the Golgi body was always located posterior to the nucleus. In late spermatids, the nucleus is bullet-shaped, and chromatin is sheet-like. In fully developed spermatids, the nucleus is extremely dense, and has an anterior extension. No acrosome is present. Three mitochondria constitute a very short middle piece, and an eccentr...

Effects of caffeine and cyclic adenosine 3′,5′-monophosphate on adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes

The adenosine triphosphate-dependent calcium uptake by endoplasmic reticulum elements of lysed synaptosomes from rat brain cortex was studied. Caffeine exhibited a biphasic effect on this calcium uptake activity: concentrations of 1, 2, 5, 10 or 30 mM caffeine stimulated calcium uptake by 62, 111, 73, 88 and 60% respectively, whereas calcium uptake was inhibited by 55% at a 60-mM concentration of caffeine. Calcium release from endoplasmic reticulum elements of lysed brain synaptosomes was stimulated by 10 mM caffeine. Cyclic adenosine 3',5'-monophosphate stimulated calcium uptake in the lysed synaptosome preparation: exogenous concentrations of 0.05, 0.5, 5, 50, or 500 microM stimulated uptake by 67, 67, 95, 38 or 67% respectively. To explore the possibility that caffeine stimulated calcium uptake through inhibition of phosphodiesterase and consequent preservation of cyclic adenosine 3',5'-monophosphate, we have tested whether caffeine retained its ability to stimulate calcium uptake under conditions of maximal stimulation by cyclic adenosine 3',5'-monophosphate. The combined presence of 10 mM caffeine and 5 microM cyclic adenosine 3',5'-monophosphate resulted in an approximate doubling of the calcium uptake as compared to the uptake in the presence of the cyclic nucleotide alone, indicating that the stimulation due to caffeine does not occur via cyclic adenosine 3',5'-monophosphate.

Transition vesicle-enriched fractions isolated from rat liver exhibit a unique peptide pattern

Isolated fractions enriched in transition vesicles isolated by preparative free-flow electrophoresis from transition elements of endoplasmic reticulum incubated with ATP and a cytosol fraction in a cell free system, exhibited a polypeptide composition distinct from that of the original transitional endoplasmic reticulum fractions. The transition vesicles were deficient in peptides of apparent molecular mass between 200 and 270 kD, in the vicinity of 65 kD and less than 40 kD. Immunoblot analysis suggested a 140 kD protein to be concentrated in transition vesicles and low or absent from the endoplasmic reticulum fractions from which the transition vesicles were derived.

Ultrastructure of the Pineal Gland of the Tropical Bat <i>Rousettus leschenaulti</i>

The ultrastructure of the pineal organ was studied in the tropical megachiropteran Rousettus leschenaulti. The pineal lies deep beneath the hemispheres adjacent to the third ventricle and is traversed by the habenular commissure anteriorly. Its parenchyma consists of a uniform population of light and occasional dark pinealocytes which appear to differ only in the degree of cytoplasmic staining. Pinealocytes are characterized by well developed Golgi bodies associated with numerous small vesicles, many mitochondria and polyribosomes, and frequent subsurface cisternae. Lipid droplets and elements of smooth endoplasmic reticulum are scant. Cisternae of granular endoplasmic reticulum are occasionally dilated. A distinct feature is the abundance of clear vesicles in the pinealocyte pericapillary terminals, which also frequently contain granular vesicles and a very large vacuole. The pineal is further characterized by the presence of a small number of glial cells and myelinated nerve fibers. A broad perivascular space investing numerous capillaries contains glial-cell and pinealocyte processes, collagen fibrils and abundant unmyelinated nerve fibers. Tortuous extensions of the perivascular space enter the pineal parenchyma where they come in close proximity to branched intercellular channels or canaliculi characterized by specialized junctions and microvilli. Differences between the pineal of the non-hibernating megachiropteran Rousettus and that of the hibernating microchiropteran bats, and structural similarities to the pineal of tropical rodents are discussed.

Cytological studies of the susceptible reaction of sugarbeet roots to Heterodera schachtii

The cellular alterations induced in sugarbeet by Heterodera schachtii were studied at 3 and 12 days after nematode inoculation. Three days after inoculation the procambial cells involved in the nematode feeding were already transformed into a syncytium with ultrastructural features which indicated high metabolic activity. Feeding tubes produced by interaction of nematode saliva with the cell contents were observed; in their vicinity the cytoplasm was devoid of cell organelles except for smooth endoplasmic reticulum. Spiny structures occurred in the parenchyma syncytial cells and they appeared to be related to endoplasmic reticulum and dictyosomes; the relation could be functional and it is possible that the spiny component is involved in protein synthesis. Nuclei were enlarged and amoeboid in form and sometimes they migrated through a space in the cell wall. Conspicuous elements of rough endoplasmic reticulum, arranged in concentric layers and actively synthesizing, and plastids with dense inclusions indicating bulk protein synthesis, were detected in parenchyma cells, adjacent to syncytia. Twelve days after nematode inoculation the ground cytoplasm of the syncytia was densely packed, but without vacuoles and the membrane system resembled endoplasmic reticulum. At this time the modification of the cytoplasm and cell wall appeared to be involved in the rapid solute uptake which would be essential for nematode development.

Ultrastructural and immunohistochemical studies on steroid-secreting cells of testis and ovary of normal and 3-methylcholanthrene-treated mice.

The testis and ovary of normal and 3-methylcholanthrene-treated mice were studied ultrastructurally and immunohistochemically in order to learn whether steroid-secreting cells of the gonads are involved in drug metabolism. The steroid-secreting cells, i.e., Leydig cells of the testis, and theca interna cells, interstitial gland cells, and corpus luteum cells of the ovary of 3-methylcholanthrene-treated mice show a strong positive reaction to the antiserum against, hepatic microsomal cytochrome P-450, of liver which is the terminal oxidase of the drug-metabolizing enzyme complex. In addition, it was found that elements of smooth endoplasmic reticulum (SER) in drug-treated mice become well developed as compared with those in control animals. These findings indicate that the steroid secreting cells in testis as well as ovary are involved in the metabolism of both endogenous and exogenous chemical compounds.

Epidermal growth factor receptors and human placenta

s: European Placenta Group/Rochester Trophoblast Joint Conference EPIDERMAL GROWTH FACTOR RECEPTORS AND HUMAN PLACENTA C. V. Rao & N. Chegini (University of Louisville School of Medicine, KY, USA) 47I Term human placenta is one of the richest sources of receptors for epidermal growth factor (EGF). EGF is present in maternal and fetal circulation and in amniotic fluid. However, the source of EGF in these compartments is not known. The EGF receptors are present primarily in syncytiotrophoblasts. By light microscope autoradiography, we have found that these receptors were high at mid pregnancy and decreased towards term. This is the opposite of biochemical binding measurements. The latter observations can be explained by the presence of more connective tissue in placental cotyledons at mid term compared with term pregnancy. The receptors are enriched in microvillous plasma membranes, which are exposed to maternal circulation. The receptors are also found in basolateral plasma membranes, which are in close proximity to fetal blood capillaries. Since EGF may not cross the placenta, maternal EGF may influence syncytiotrophoblast function by binding to receptors in microviilous plasma membranes, whereas fetal EGF may also influence syncytiotrophoblast function via receptors in basolateral plasma membranes. The EGF in amniotic fluid presumably interacts with receptors in fetal membranes and decidua. In addition to the two outer cell surface membranes of syncytiotrophoblasts, EGF binding and EGF responsive kinase activities were also found in lysosomes, rough and smooth endoplasmic reticulum and Golgi elements. Quantitative electron microscope autoradiographic studies revealed that 12SI-EGF internalizes and associates with all of the above intracellular organelles in addition to nuclei. While the placental 125I-EGF binding differed among some pregnancy states, fetal membrane 12SI-EGF binding was similar in all the pregnancy states. This suggested that placental and fetal membrane EGF receptors are regulated differently. PERMEABILITY OF THE IN SITU PERFUSED RAT PLACENTA N. Robinson, R. Jenkins, P. Thomas & C. Sibley (St Mary's Hospital, Manchester, UK) A method for the in situ perfusion of the rat placenta has recently been developed. This preparation has allowed simple measurement of the permeability of the rat placenta to a range of hydrophilic molecules as follows: At 2I + i days of gestation, animals were anaesthetized with sodium thiobutabarbital and, after laparotomy, the uterus was exposed and the fetal circulation of one of the placentae was perfused, via an umbilical artery cannula with a modified Krebs' Ringer, at o.5 ml/min. A range of radiolabelled hydrophilic molecules, of increasing molecular size, were injected as a bolus into the mother and three samples of maternal plasma were taken at to--min intervals for measurement of radioactivity whilst fetal side perfusate was collected at 4min intervals from a cannula in the umbilical vein. The permeability surface area product (PS) was calculated as: PS (/d/rain/g)= (venous perfusate radiolabel concentration • flow rate) (mean maternal plasma radiolabel concentration x placental weight). The ratio of PS/D, where D is the diffusion coefficient in water of the particular molecule, was independent of molecular size for 14C-mannitol, 51CrEDTA and 14C/3H-inulin, whilst the PS/D for ~25Ialbumin suggested steric hindrance of this molecule. By contrast, the PS/D of 22Na+, 15.06 + i.oocm/g suggested additional non-diffusional transport. These results are close to those obtained in the intact rat placenta, and therefore, unlike the guinea pig, perfusion in the rat does not appear to be associated with increased placental porosity.