Serotonin uptake and configurational change of bovine pulmonary artery smooth muscle cells in culture

Although it is well known that endothelial cells transport serotonin (5-HT) from extracellular to intracellular locations, it has been generally assumed that smooth muscle cells do not accumulate 5-HT but, rather, respond to 5-HT through a receptor activity unrelated to uptake of this amine or via stimulation of endothelial-derived relaxing factor. In the present study smooth muscle cells (PASMC), isolated and cultured from bovine pulmonary artery, were evaluated for 5-HT uptake under a variety of conditions. 5-HT uptake was linear up to 15 min and the rate was seven- to eightfold higher than that by bovine pulmonary artery endothelial cells. There was intracellular metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA). The uptake was inhibited by exposure to 4 degrees C, absence of Na+ from the medium, and agents such as imipramine, verapamil, ketanserin, and methiothepin. Like that of endothelial cells, 5-HT uptake by PASMC was stimulated by exposure of cells to anoxia for 24 hr. Unlike endothelial cells that showed no morphological changes, PASMC at early passage showed dendritic formation after 30-60 min exposure to 5-HT at a concentration as low as 10(-8) M. Although this configurational change in response to 5-HT was lost with passage of cells, transport of 5-HT by these cells was retained. The configurational change was blocked by agents that inhibited 5-HT uptake, such as imipramine, verapamil, ketanserin, and methiothepin; it was unaffected by inhibitors of protein kinase C, phospholipase C, and calmodulin or absence of Ca2+ from the medium. We conclude that PASMC, as well as endothelial cells, accumulate 5-HT; there appears to be a close relationship between 5-HT uptake and configurational change of early passaged PASMC in culture. The factor(s) required for the configurational change are absent in endothelial cells and lost during passage of PASMC.     

Age determination in the adult screwworm (Diptera: Calliphoridae) by pteridine levels

Fluorescence spectrophotometry was used to assess the possible use of pteridines in the compound eyes to estimate the age of adult screwworms, Cochliomyia hominivorax (Coquerel). Factors affecting the quantities of pteridines include temperature and head size. No difference in pteridine levels was found among flies fed protein or carbohydrate. A regression model for estimating the age of female screwworms was constructed. The model uses head capsule size and relative pteridine quantities and assumes a constant body temperature of 30 degrees C. This regression formula has an r2 of 0.74. Our study extends the use of pteridine accumulation for age determination from obligate sanguinivorous Diptera to an autogenous species that feeds facultatively on nectar and wound exudates. The technique appears to provide a valid means to determine age of these flies.     

Production of a retroviral P15E-related chemotaxis inhibitor by IL-1-treated endothelial cells. A possible negative feedback in the regulation of the vascular response to monokines

The effects of IL-1 on vascular endothelium result in a complex set of alterations which are potentially disruptive of vessel wall and underlying tissue integrity. The present study was aimed at investigating possible regulation of such potentially destructive responses elicited by IL-1 on endothelial cells. Culture supernatants of IL-1-treated human umbilical vein endothelial cells (HEC) were depleted of retroviral p15E-related Ag with immobilized anti-p15E mAb. The monocyte chemotactic and polarizing activity of supernatants of IL-1-treated HEC (presumably related to colony-stimulating factors being released by HEC) was markedly augmented by absorption on immobilized anti-p15E antibodies. Irrelevant IgG had no effect and anti-p15E antibodies did not affect the chemotactic activity of supernatants from unstimulated HEC. The material eluted from Sepharose-bound anti-p15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. The alpha and beta molecular species of IL-1 were equally effective in inducing the production of p15E-related inhibitor. The production of a p15E-related inhibitor of chemotaxis induced by IL-1 in HEC may represent a negative signal in the regulation of the potentially destructive responses to pro-inflammatory cytokines.     

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.     

Sodium and calcium currents in action potentials of rat somatotrophs: their possible functions in growth hormone secretion

We report that both Na+ and Ca2+ currents are involved in the action potentials and in the hormone release from rat somatotrophs in primary culture. Single somatotrophs were identified by reverse hemolytic plaque assay (RHPA) and transmembrane voltage and currents were recorded using the whole-cell mode of the patch-clamp technique. Somatotrophs displayed a mean resting potential of -80mV and an average input resistance of 5.7G omega. Most of the cells showed spontaneous or evoked action potentials. Single action potentials or the initial spike in a burst were characterized by their high amplitude and short duration. Tetrodotoxin (TTX, 1 microM) blocked single action potentials and the initial spikes in a burst, whereas action potentials of long duration and low amplitude persisted. Cobalt (2 mM) plus TTX (1 microM) blocked all the action potentials. Voltage-clamp experiments confirmed the presence of both a TTX-sensitive Na+ current and Co2(+)-sensitive Ca2+ currents. TTX or Na(+)-free medium slightly decreased the basal release of GH but did not markedly modify hGRF-stimulated GH release. However, Co2+ (2 mM), which partially decreased the basal release, totally blocked hGRF-stimulated release. We conclude that (1) Na+ currents which initiate rapid action potentials may participate in spontaneous GH release; (2) Ca2+ currents, which give rise to long duration action potentials and membrane voltage fluctuation, are probably involved in both basal and hGRF-stimulated GH releases.     

The effect of etorphine on nicotine- and muscarine-induced catecholamine secretion from perfused rat adrenal glands

The effect of etorphine on nicotine and muscarine-mediated catecholamine (CA) release from isolated perfused rat adrenal glands was investigated. Nicotine increased CA secretion at the low concentration of 0.5 micrograms while higher concentrations of muscarine (5 micrograms) were required. Moreover, muscarine released primarily epinephrine (EP) from rat adrenal glands while nicotine released norepinephrine (NE) and Ep. Etorphine inhibited NE and EP release evoked by nicotine to the same extent, whereas, muscarine-mediated release of NE and EP was not affected. Mecamylamine and verapamil inhibited nicotine but not muscarine-induced CA secretion. Our results suggest that etorphine preferentially interacts with nicotinic receptors on rat adrenal chromaffin cell membranes.     

Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N₂ fixation, and production of NADP⁺ to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N₂ is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N₂-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N₂ fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.     

Circadian Rhythm of the Prokaryote Synechococcus sp. RF-1

The prokaryotic Synechococcus sp. RF-1 exhibited a nitrogen fixation circadian rhythm with characteristics remarkably similar to the circadian rhythm of eukaryotes. The rhythm had a free-running period of about 24 hours when the length of the preen-trained cycle did not differ too much from 24 hours, and it was insensitive to changes in temperature from 22°C to 33°C. Because the endogenous rhythm of nitrogen fixation was not affected by a phase-shift of its previous cycles, the circadian rhythm in Synechococcus sp. RF-1 was not considered to be controlled simply by a feedback mechanism.     

Water Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells

Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 × 10⁻⁵ molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (23°C) for 7 days acclimated to −28°C (LT₅₀) when treated with ABA, or to −5°C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H₂O) or as grams water per gram dry weight (gram H₂O/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H₂O/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H₂O/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H₂O/10⁶ cells), did not differ in ABA-treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/10⁶ cells increased to 3.3 milligram/10⁶ cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/10⁶ cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur.