Rabbit kidney function in vitro: the effect of colloids, energy substrate, a vasodilator, perfusion pressure, and bovine serum albumin

An in vitro perfusion system at 37 degrees C for the assessment of rabbit kidney function is described. The purpose of this assay system is to evaluate the effects of cryobiological manipulation on kidney function. The effect of the colloids dextran (MW = 70,000, 80,000, and 180,000) in the perfusate at 110 mm Hg were compared to a reduced perfusion pressure, colloid-free perfusate. Better function was obtained at lower perfusion pressure with the colloid-free perfusate. Less damage was noted histologically on light and electron microscopy. Investigation of energy substrates on rabbit kidney function demonstrated that butyrate, or lactate, in addition to glucose resulted in increased sodium and glucose reabsorption over glucose alone. Substrate-free perfused kidneys exhibited depressed Na transport. Lactate, and to some extent butyrate, decreased net glucose utilization. An alpha-adrenergic blocking agent, isoxsuprine, in the initial flush solution did not appear to be beneficial. An increase of perfusion pressure from 50 to 75 mm Hg resulted in an increase in GFR. Tubular function was enhanced by inclusion of small amounts of BSA in the perfusate.     

Prostaglandin synthesis by lymphoid tissue of mice experiencing a graft-versus-host reaction: relationship to immunosuppression

Studies were carried out on the induction of PGE synthesis during the GVH reaction and its role in GVH-induced immunosuppression. The results demonstrated that spleen, lymph node cells and, to a much lesser degree, thymus cells obtained from adult C57BL/6 × AF₁ mice treated with 50–75 × 10⁶ C57BL/6 lymphoid cells were stimulated to produce PGE during the course of the GVH reaction. The spleen and lymph node PGE production peaked at Day 9 post-GVH induction (30- and 15-fold higher than normal, respectively). Thereafter, it declined to near normal levels by Days 25–30 post-GVH induction. Passage of GVH spleen cells through a rayon column removed macrophages but not mitogen-responsive T and B cells and also removed nearly all of the PGE-producing cells, except during the later course of the GVH reaction. Removal of PGE-producing cells from GVH-immunosuppressed spleen cells significantly reconstituted the mitogen response to PHA and LPS. Treatment of mice experiencing a GVH reaction with indomethacin delayed the onset of suppression of the plaque-forming cell response to sheep erythrocytes. These results suggest that early GVH-induced immunosuppression which may represent an amplified normal regulatory mechanism is mediated by increased macrophage production of PGE which suppresses both B- and T-cell functions, whereas at later stages other immunosuppressive mechanisms become operational.     

Function of rabbit kidneys in vitro at normothermia following equilibration with 3.0 M Me2SO and removal by hypertonic washout at 10 degrees C

In the present study, rabbit kidneys were assayed for function on a 37 °C in vitro perfusion system after perfusion on a 10 °C perfusion system which permits the slow introduction and removal of cryoprotectant. The final concentration of 3.0 M Me₂SO was introduced slowly at two different rates. The washout was achieved by perfusion with Me₂SO-free solutions made hypertonic with mannitol. Two regimens of washout were used: 800, 700, 600, 500, and 400 mOsm/kg; and 600, 500, and 400 mOsm/kg.During perfusion at 37 °C, the glomerular filtration rate was similar in all groups and this increased significantly in all groups with time. Protein leakage was minimal. All three Me₂SO groups showed a depressed Na reabsorption capacity, but the 800 mOsm group was the most severely affected. This was also found with glucose reabsorption. We concluded that rabbit kidneys will function well with the cryoprotectant Me₂SO up to 3 M concentration when introduced slowly and washed out with hypertonic mannitol beginning at 600 mOsm/kg. When 800 mOsm is used at the initial step, the proximal tubular function is severely affected.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Development of the myotomal neuromuscular junction in Xenopus laevis: An electrophysiological and fine-structural study

The normal development of the myotomal neuromuscular junction in Xenopus embryos and tadpoles was investigated electrophysiologically as well as electron microscopically. Spontaneous potentials, considered to be miniature end-plate potentials (MEPPs), were detected by intracellular recording as early as stage 21 and by stage 24 they were observed in every embryo tested. Like MEPPS at later stages they were blocked by curare but not by tetrodotoxin. End-plate potentials (EPPs), subject to block by tetrodotoxin, were evoked by electrical stimulation of the spinal cord in embryos as young as stage 24 and occurred spontaneously as early as stage 22. The durations of MEPPs and EPPs were initially relatively long. Focal external recordings revealed an eightfold decrease in duration during the course of development. Nerve processes emerged from the spinal cord and contacted developing muscle cells as early as stage 21, but junctional specializations were not apparent and vesicles were rare even in stage 24 embryos. During the next 24 hr, between stages 25 and 36, vesicles increased in number and became localized toward the junctional surface of the nerve ending. Basement lamina developed in the cleft and postjunctional ridges and densities were observed. Individual muscle cells also became contacted by several nerve processes. By stages 48–52 there were fewer contacts on individual muscle cells and Schwann cell processes partially covered the nerve endings. Gap junctions were observed between the muscle cells throughout development but occurred less frequently at the later stages. It is concluded that by the time they reach the muscle cells, or very shortly thereafter, at least some of the growing nerve processes can release transmitter, and some of the muscle cells are sufficiently sensitive to acetylcholine in the region of contact to respond with millivolt depolarizations. These earliest functional contacts, however, are morphologically undifferentiated.     

Activation of adenylate cyclase by beta-adrenergic receptors: Investigation of rate limiting steps by simultaneous assay of high affinity agonist binding and GDP release

We report the development and application of a novel assay for high affinity binding of the agonist [³H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [³²P] GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min^?1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 μM and 0.1 μM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [³²P] GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [³H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. ³H-agonist binding and agonist-promoted [³²P] GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.     

Comparative analysis of translation accuracy in an Escherichia coli and a mammalian cell-free system

The effect of environmental stress on the accuracy of protein synthesis in an Escherichia coli and a rat brain cell-free system was investigated. Poly-U was translated in a rat brain and an E. coli cell-free extract under identical ionic conditions. The fidelity of translation, both in the E. coli and the rat brain extracts, was commensurate with what is known about the accuracy of translation in vivo. The incorporation of phenylalanine (code: UUU) and leucine (code: CUU, UUG or A) was measured at various Mg²⁺ concentrations (3 to 22 mm), various pH's (6.6 to 8.6), various temperatures (23 to 42 °C), and in the presence or absence of 2.4% (v/v) ethanol. It was observed that (i) the accuracy of translation was generally higher in extracts from E. coli than from rat brain, and (ii) relative to that in E. coli, the translation fidelity in rat brain extracts was about 2 times more sensitive to ethanol, at least 5 times more sensitive to temperature, and at least 50 times more sensitive to pH. It was found that this differential sensitivity was not due to a differential behavior of the bacterial and the mammalian aminoacyl-tRNA synthetases under stress, but rather to the process of chain elongation itself. It is concluded that the accuracy of protein synthesis is more resistant to environmental stress in E. coli extracts than in extracts from at least one mammalian tissue.