Differential localisation and synthesis of porcine factor VIII related antigen (VIIIR:AG) in vascular endothelium and in endothelial cells in culture

Porcine and human umbilical vein and adult blood vessels were studied for the presence and synthesis of factor VIII related antigen (VIIIR:Ag) and fibronectin (Fn) by immunofluorescence histology and immunoautoradiography. Investigation of human tissue confirmed the widespread distribution of VIIIR:Ag on the endothelium of all blood vessels examined but observations on porcine tissue gave different results. Porcine umbilical vein and porcine adult veins were positively stained for VIIIR:Ag whilst porcine aorta and other pig arteries appeared to be negative or only weakly positive. Some blood vessels (?venous) in the adventitia of porcine aorta were positively stained whilst adjacent ones (?arterial) were negative. Radiolabelled methionine was added to culture medium and proteins synthesised by cultured EC were examined by two dimensional crossed immunoelectrophoresis and autoradiography. Identification of radiolabelled precipitin arcs provided a highly sensitive method for confirming the specificity of antisera and for detecting VIIIR:Ag and Fn. Examination of cultured human umbilical vein EC confirmed the synthesis of VIIIR:Ag and distinguished between VIIIR:Ag and Fn. Studies of porcine umbilical vein EC in culture gave similar results to those observed with corresponding human EC. However, cultures derived from porcine aorta did not demonstrate synthesis of VIIIR:Ag and microscopy failed to locate VII IR:Ag in these cells with certainty. The results confirmed the synthesis of VIIIR:Ag by human and porcine umbilical vein EC but differences in staining reactions and the apparent inability to synthesise VIIIR:Ag by cells derived from porcine aorta suggested that porcine EC at different anatomical sites may subserve different functions.     

D-600 blocks spontaneous discharge,excitability and contraction of cultured embryonic chick heart cells

Standard microelectrode techniques and a new microphotometric system were used to study the effects of D-600 on spontaneously beating or electrically driven cultured embryonic chick heart cells. The effects of the addition of 10^?6m D-600 to the superfusing solution were always first cessation of automaticity or triggered activity and then inhibition of excitability as measured by anodal-break excitation. Triggered and spontaneous activity faded during D-600 influence in a way similar to the damping of an inharmonic oscillation. Finally a stable membrane potential of about ?40 mV was reached. Simultaneous measurement of cell pulsation by photometric means showed that each excitation elicited a contraction, as long as the amplitude of spontaneous action potentials or of anodal-break excitations displayed overshoot of the zero line. Excitations of lower amplitude were not sufficient to trigger a measurable contraction. The inhibitory effects of D-600 could not be overcome by resting periods but were antagonized by membrane hyperpolarization. These findings indicate that reduction of the slow inward current by D-600 inhibits excitation and contraction in cultured embryonic chick heart cells.     

Disobutamide-induced cytoplasmic vacuoles in cultured dog coronary artery muscle cells

Smooth muscle cells released by protease from the coronary artery of dogs were cultured in M199 supplemented with 10% fetal calf serum. Cells were grown on glass coverslips to semiconfluence and exposed to disobutamide at 0,1, 2, 3, 6, 8, and 10 × 10^–4 M for 24, 48, and 72 h, examined in situ by light microscopy, then fixed in 100% methanol, stained with May-Grünwald Giemsa and examined by light microscopy. Cells were also exposed to drug for 24 h, pelleted, fixed and prepared routinely for electron microscopy. Control cells and cells exposed to 6 × 10^–4 M disobutamide were examined. Vacuole formation was dose and time dependent between 2 x 10^–4 M and 10^–3 M. By light microscopy, morphologic alterations induced by the drug were clear cytoplasmic vacuoles in live cells, vacuoles in dead cells and dead cells without vacuoles. Small round vacuoles were an early change. Dark granules were dispersed among the vacuoles. The vacuoles increased in size at higher doses or longer times. Eventually all the cytoplasm was occupied by vacuoles and the cells were enlarged. By electron microscopy, the vacuoles were round, primarily membrane-bound, contained mostly electron-lucent material and occasionally small flocculent bodies. There was vacuolar coalescence. The dose response of vacuole induction in confluent and semiconfluent cells was similar. Cytoplasmic vacuoles without cell death can be induced by disobutamide at 2 or 4 × 10^–4 M during a 3-day exposure. Cell culture is a suitable biological system for studying cytoplasmic vacuoles of the type induced by disobutamide.Presented in part as an Abstract (# 2924) in the 1983 Annual Meeting of the Fed. Am. Soc. Exp. Biol., April 10–14, 1983, Chicago, Illinois, and published in Fed. Proc. 42(4): 972.     

Function of Culturing Monolayer Hepatocytes by Collagen Gel Coating and Coculture with Nonparenchymal Cells

Abstract: Since 1987, we have been developing a bioartificial liver (BAL) using multiplated cultured hepatocyte monolayers. With the goal of promoting hepatic functions of cultured hepatocyte monolayers, we combined the use of a collagen gel layer over the monolayers of hepatocytes and/or cocultured hepatocytes with nonparenchymal cells (NPCs). The study was divided into four groups according to culture configurations: Group 1: hepatocyte monolayer culture (control); Group 2: coculture of hepatocytes and NPCs; Group 3: hepatocyte monolayer with a overlaid collagen gel layer; and Group 4: coculture with a overlaid collagen gel layer. The culture continued for 14 days. Morphological changes and hepatic functions were evaluated by urea and albumin syntheses. The morphological status of the hepatocytes remained for 2 weeks in Groups 3 and 4. Deterioration and detachment of hepatocytes and/or NPCs started in Group 1 and 2 on the third day in culture. Significantly high urea synthesis was noted in Group 4 (p < 0.001 compared with Group 1 and 2: p = 0.0014 compared with Group 3). Although there was no significant difference in albumin synthesis among the four groups, those hepatocytes covered by the collagen gel (Groups 3 and 4) tended to secrete albumin throughout the observation period. These results indicted that the environment, although artificial (but close to the in vivo state), supplied with collagen gel and the coculture, enhanced the activities of the cultured hepatocyte monolayers. We suggest that use of cocultured hepatocytes under a collagen gel is a promising candidate for a bioreactor of multiplated BAL.     

Computer analysis in the electron microscopic assessment of cellular injury.

Computer programs have been developed to characterize data obtained from the electron microscopic assessment of diverse types of cellular injury using cultured cell preparations.     

Naloxone interferes with granulocytopoiesis in long-term cultures of mouse bone marrow; buffering by the stromal layer

Long-term cultures of mouse bone marrow cells were treated with naloxone, starting at the time of culture initiation or in the 2nd or 4th week of culture. Cell proliferation was suppressed and the ratio of immature and mature granulocytes to macrophages diminished by naloxone treatment. The effect depended on the timing of naloxone addition to the cultures and on its concentration, with a bell-shaped dose-response curve. High and low concentrations of naloxone (10⁻⁴, 10⁻⁶, 10⁻¹⁴ M) interfered with hematopoiesis more strongly than the intermediate concentrations (10⁻⁸ to 10⁻¹² M). Early cultures lacking the stromal layer were more sensitive to naloxone than the cultures with established stroma. The bell-shaped dose-response curve has been attributed to an interplay of specific (opioid-receptor-mediated) and nonspecific mechanisms. Opioidergic mechanisms apparently participate in the regulation of hematopoiesis.     

Subtypes of adrenergic receptors and intracellular mechanisms involved in modulatory effects of noradrenaline on glutamate

We have previously reported that the response of cultured chick cerebellar neurons to glutamate is enhanced by noradrenaline (NA) or isoproterenol and suppressed by clonidine. The present study was carried out to further specify the adrenergic receptor subtypes involved in the facilitatory effect of NA or isoproterenol and the suppressive effect of clonidine, and to examine the intracellular mechanisms underlying these modulatory effects of NA. The clonidine effect, which was mimicked by NA iontophoresed with large ejecting currents, was blocked by yohimbine and tolazoline (alpha 2 antagonists) and also by dibutyryl cyclic AMP or forskolin which augmented the glutamate response by itself. Prazosin, an alpha 1 receptor antagonist did not block the clonidine effect. NA- or isoproterenol-induced facilitation, which was mimicked by denopamine (beta 1 agonist), was antagonized by acebutolol (beta 1 antagonist) and not by ICI 118,551 (beta 2 antagonist). Pretreatment of neurons with pertussis toxin for more than 24 h blocked the suppressive action of clonidine without affecting the facilitatory action of isoproterenol. Furthermore, intracellular injection of GDP beta S inhibited the modulatory effects of either clonidine or isoproterenol. These results indicate that the facilitatory and inhibitory modulatory effects of NA may be mediated by beta 1 and alpha 2 receptors linked to cAMP systems, respectively, and the former is coupled with the stimulatory G protein (Gs) and the latter is with the inhibitory G protein (Gi).     

Light and electron microscopic analysis of insulin binding sites on neurons in dissociated brain cell cultures

The distribution of insulin binding sites on primary cultured neurons and glia from the fetal rat was examined by the immunoperoxidase method using a specific insulin receptor antiserum. Light and electron microscopic analysis revealed a homogenous distribution of insulin binding sites on selective neuron-like cells of the dissociated cell culture system. To determine the influence of medium insulin on the distribution of insulin binding sites, dissociated cell cultures were maintained in the presence or absence of porcine insulin for varying time periods. We observed a significant increase in the number of insulin stained neuron-like cells maintained in insulin free defined medium compared to neuron-like cells maintained in insulin supplemented defined medium. Further, we examined the distribution of insulin binding sites after incubation with the antibody, which has agonistic properties in peripheral tissues, for varying time periods prior to fixation. Under these conditions, the light microscopic analysis revealed a heterogeneous (patchy) distribution of immunoreactive insulin binding sites, suggesting that the ligand receptor complex migrates. These results demonstrate the presence and distribution of insulin binding sites on neurons maintained in vitro, and provide morphological evidence to support a functional role for insulin in CNS tissues.     

The subcellular distribution of [14C]dichloromethylenebisphosphonate and [14C]1-hydroxyethylidene-1,1-bisphosphonate in cultured calvaria cells

Summary Rat calvaria cells were cultured for 6 days in the presence or absence of [¹⁴C]dichloromethylenebisphosphonate ([¹⁴C]Cl₂MBP) or [¹⁴C]1-hydroxyethylidene-1, 1-bisphosphonate ([¹⁴C]HEBP), after which cell organelles were separated by differential centrifugation. The distribution of protein, glutamate dehydrogenase, acid phosphatase, and 5'-nucleotidase was similar for cells treated or not treated with Cl₂MBP. About 70–80% of the [¹⁴C]Cl₂MBP and [¹⁴C]HEBP was found to be present in the supernatant. This was the only fraction that showed a ratio higher than 1 for the relative specific radioactivity, indicating that the bisphosphonates accumulated mainly in the cytosol. Rapid separation of particulate components and soluble cytoplasm of cells treated with [¹⁴C]Cl₂MBP confirmed this finding, showing that it is unlikely that the result was due to leakage from the organelles. The uptake of [¹⁴C]Cl₂MBP into cells was similar in different cell types. The binding of both bisphosphonates to macromolecules in the medium was 0.1–0.2% and 1–4% in the cells. This binding is not due to metabolic activity of the cells. About 15–20% of [¹⁴C]HEBP and [¹⁴C]Cl₂MBP was modified by the living cells.     

Vitamin D metabolism and phosphate transport in developing kidney: effect of diet and mutation

In order to obtain a better understanding of the molecular mechanisms involved in phosphate reabsorption and vitamin D hormone production by mammalian kidney, we have devoted our efforts to the study of a mutant mouse model (Hyp). Studies from our laboratory have demonstrated that Na⁺-dependent phosphate transport is significantly reduced in renal brush border membrane vesicles derived fromHyp mice and that the regulation of the renal mitochondrial enzymes which metabolize 25-hydroxyvitamin D₃ (25-OH-D₃) is impaired in the mutant strain. The demonstration of abnormal phosphate transport and 25-OH-D₃ metabolism in proximal tubule cells derived fromHyp kidney after 6–8 days in culture indicates that the mutant renal phenotype is independent of circulating factors and, therefore, intrinsic to the kidney. However, the precise relationship between these two proximal tubular abnormalities is poorly understood. Because theHyp mutation segregates as a Mendelian trait, it is very likely that one mutant gene is responsible for the biochemical and clinical phenotype. Several hypotheses are put forth to explain the nature of the primary mutation in theHyp mouse.