Selective localization of calcium-binding protein in human brainstem, cerebellum and spinal cord.

The 28,000-Da vitamin D-dependent calcium-binding protein, CaBP, which is induced by one hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, was localized by immunocytochemistry in the human brainstem, cerebellum and cervical segment of the spinal cord. Positive structures (neurons and their processes) were restricted to some well-defined motor and sensory pathways. In motor regions, the highest density of immunoreactive sites was found in the Purkinje cell layer of the cerebellar cortex, and CaBP-positive neurons were also found in the reticular formation and the inferior olivary nucleus.

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation.

Regional heterogeneity of glycoconjugate distribution in the glomerulus revealed by lectin-gold cytochemistry and SDS-PAGE.

The authors have used SDS-PAGE and lectin overlay analysis in parallel with lectin-gold cytochemistry to identify Helix pomatia lectin (HPL) binding glycoconjugates in rat kidney glomeruli. Previous work revealed HPL binding sites only beneath podocyte foot process bases, where they contact the glomerular basement membrane. It is shown here that after neuraminidase digestion of thin sections of glomeruli before incubation with HPL-gold complexes, the number of HPL binding sites is markedly increased.

Internalization and recycling of 125I-photoreactive insulin-receptor complexes in hepatocytes in primary culture.

When the insulin receptor is tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to it by UV irradiation, the fate of this labeled receptor can be followed both morphologically and biochemically. In the present study we have applied this tool to trace the pathway followed by 125I-photoreactive insulin-receptor complex in hepatocytes in primary culture. As determined by quantitative electron microscopic autoradiography, the internalized labeled material first associates with clear vesicles, second is found in multivesicular bodies, third associates with dense bodies and fourth returns to the cell surface via clear vesicles.

Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells.

Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.

Basic fibroblast growth factor induces angiogenesis in vitro.

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries.

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing.

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons.

Transport of horseradish peroxidase from the cell surface to the Golgi in insulin-secreting cells: preferential labelling of cisternae located in an intermediate position in the stack.

We have used serial sectioning to study the topology of Golgi cisternae in insulin-secreting cells during secretion-stimulated endocytotic uptake of exogenous horseradish peroxidase (HRP). HRP-labelled cisternae were followed on several series of consecutive sections. This revealed that labelled cisternae could always be traced to a position in the Golgi stack intermediate between the cis and the trans poles.

Direct cell-to-cell transmission of vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) infection of kidney-derived, LLC-PK1 epithelial cells resulted in the budding of new viral particles into the basolateral space of the cultures. In lateral regions where cells were in close apposition, the majority of assembling viral particles in the process of budding from the producing cell had their apex already engaged in clathrin-coated pits of the neighbouring cell surface. These observations suggest that the viral envelope-plasma membrane interaction triggers the focal formation of clathrin-coated pits; they also show how VSV infection could spread throughout a tissue with only minimal exposure to a host's extracellular environment.

Role of intracellular calcium and protein kinase C in the endocytosis of transferrin and insulin by HL60 cells.

The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin.

Interactions of lectins with specific cell types in toad urinary bladder. Surface distribution revealed by colloidal gold probes and label fracture.

Colloidal gold probes were used in conjunction with pre-embedding labeling and label-fracture to show the plasma membrane distribution of Helix pomatia lectin (HPL) and wheat germ lectin (WGL) binding sites on different epithelial cell types of toad urinary bladder. Mitochondria-rich cells were virtually unlabeled with HPL, but showed a strong affinity for WGL. Granular cells were weakly labeled with WGL but had a variable affinity for HPL.

Loss of polarization of plasma membrane domains in transformed pancreatic endocrine cell lines.

Using enveloped RNA viruses that bud selectively from either the apical or basolateral surface in polarized epithelial cells, we have recently provided evidence for polarization of plasma membrane domains in cultured pancreatic islet cells. In this study, we have followed the same experimental strategy to establish whether these polarized properties are maintained in transformed pancreatic endocrine cells. We find that influenza virus and vesicular stomatitis virus emerge from both the attached and free surfaces of cultured insulinoma cells (RIN cells) and SV40-transformed beta-cells (HIT cells).

Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.

To assess whether junctional coupling is involved in the secretory activity of pancreatic acinar cells, dispersed rat acini were incubated for 30 min in the presence of either heptanol (3.5 mM) or octanol (1.0 mM).

A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack.

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack.

Receptor-mediated endocytosis of polypeptide hormones is a regulated process: inhibition of [125I]iodoinsulin internalization in hypoinsulinemic diabetes of rat and man.

Much data suggest that receptor-mediated endocytosis is regulated in states of hormone excess. Thus, in hyperinsulinemic states there is an accelerated loss of cell surface insulin receptors. In the present experiments we addressed this question in hypoinsulinemic states, in which insulin binding to cell surface receptors is generally increased.

Calcitonin and vasopressin affect epithelial properties in a renal cell line.

We have used a stable clonal variant (D + Sc), isolated from the LLC-PK1 pig kidney-derived cell line and selected for its extensive capacity to form domes, in order to study the hormonal modulation of epithelial permeability in culture. Calcitonin, vasopressin, and other agents that raise intracellular adenosine 3',5'-cyclic monophosphate levels caused a rapid and dramatic decrease in the size and number of domes. This effect was independent of RNA and protein synthesis, and thus appeared unrelated to the production of urokinase, a proteinase synthesized by the cells in response to these agents.

LLC-PK1 cells: cloning of phenotypically stable subpopulations.

The LLC-PK1 pig kidney-derived cell line is morphologically and functionally heterogeneous. We have clonally derived three sublines that differ in their response to calcitonin and in their ability to form domes. The three clones were analyzed for their basal and hormonally induced plasminogen activator production.

Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate.

Cultured microvascular endothelial cells isolated from fenestrated capillaries have been shown to express many properties of their in vivo differentiated phenotype, yet they contain very few diaphragmed fenestrae. We show here that treatment of capillary endothelial cells with the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, induces more than a fivefold increase in the frequency of fenestrae per micron 2 of cell surface, as determined from a quantitative evaluation on freeze-fracture replicas. In quick-frozen, deep-etched preparations, the endothelial fenestrae appeared to be bridged by a diaphragm composed of radial fibers interweaving in a central mesh, as previously observed in vivo.

Horseradish peroxidase uptake and crinophagy in insulin-secreting cells.

Upon exposure of pancreatic B cells to exogenous horseradish peroxidase (HRP), a population of secretory granules becomes HRP-labelled. In isolated islets of Langerhans, we studied the fate of HRP-labelled secretory granules during a pulse-chase experiment with HRP in order to assess their relationship with lysosomes containing secretory granule cores. These structures (crinophagic or multigranular bodies) were previously shown to be a site of insulin degradation (Orci et al.

The "coat" of kidney intercalated cell tubulovesicles does not contain clathrin.

Intercalated cells of kidney collecting ducts contain a population of tubulovesicles in their apical cytoplasm, whose limiting membranes are decorated by arrays of dense, club-shaped projections oriented toward the cytoplasm. These tubulovesicles have been implicated in endo-exocytotic events in these cells. To determine a possible relationship between this "coating" material and clathrin, the coat protein associated with endocytotic coated pits and coated vesicles in other cell types, we applied a monospecific, affinity-purified anti-clathrin antibody to thin sections of rat kidney embedded at low temperature in Lowicryl K4M.

Tris(hydroxymethyl)aminomethane inhibits the synthesis and processing of proinsulin in isolated rat pancreatic islets without affecting release of insulin stores.

Isolated rat islets of Langerhans were pulse-labeled (5 min, [3H]leucine) and then exposed to 10 or 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 during an 85-min chase period. There was a dose-related inhibition of the conversion of labeled proinsulin to insulin by Tris.

Intracellular pathway followed by the insulin receptor covalently coupled to 125I-photoreactive insulin during internalization and recycling.

After it interacts with a specific receptor on the cell surface, insulin is internalized in its target cell by an adsorptive endocytotic process and eventually degraded in lysosomes. It was also recently shown that the initial surface interaction between the hormone and its receptor is followed by an internalization of the receptor, which later is recycled back to the cell surface. In the present study the insulin receptor was tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to the insulin receptor by ultraviolet irradiation.

A Simple Method for Quick-Freezing.

In conventional freeze-fracture replicas produced from tissue cryoprotected with glycerol, the hydrophobic inner surfaces of membranes are revealed, but hydrophillic structures are obscured in the surrounding ice. Quick-freezing of tissue obviates the need for glycerol, which prevents the removal of this ice by etching or freeze-drying, but the major problem in freezing without glycerol cryoprotection is ice crystal formation. We describe here a simple method for quick-freezing tissue, in the absence of glycerol, on a nitrogen-cooled copper block with a hand-held specimen holder.