Measurement of kynurenic acid in mammalian brain extracts and cerebrospinal fluid by high-performance liquid chromatography with fluorometric and coulometric electrode array detection

Kynurenic acid is a broad-spectrum excitatory amino acid (EAA) receptor antagonist which is present in the mammalian central nervous system. We describe a method for the measurement of kynurenic acid using isocratic reverse-phase high-performance liquid chromatography (HPLC) with fluorometric detection enhanced by Zn2+ as a postcolumn reagent. The method requires no prior sample preparation procedures other than extraction with 0.1 M HClO4. The reliability of the primary fluorometric method was verified by comparing measurements of tissue concentrations of kynurenic acid in human cerebral cortex and putamen using three different methods of separation with fluorometric detection, as well as four methods utilizing HPLC with coulometric electrode array system (CEAS) detection. All seven methods produced comparable results. The concentration of kynurenic acid in human cerebral cortex was 2.07 +/- 0.61 pmol/mg protein, and in human putamen, 3.38 +/- 0.81 pmol/mg protein. Kynurenic acid was also found to be present in human cerebrospinal fluid (CSF) at a concentration of 5.09 +/- 1.04 nM. The regional distribution of kynurenic acid in the rat brain was examined. Kynurenic acid concentrations were highest in brainstem (149.6 fmol/mg protein) and olfactory bulb (103.9 fmol/mg protein) and lowest in thalamus (26.0 fmol/mg protein). There were no significant postmortem changes in kynurenic acid concentrations in cerebral cortex, hippocampus, and striatum at intervals ranging from 0 to 24 h. Perfusion of the cerebral vasculature with normal saline prior to sacrifice did not significantly alter kynurenic acid content in rat hippocampus, cerebral cortex, or striatum. The analytical methods described are the most sensitive (10-30 fmol injection-1) and specific (utilizing both excitation and emissions properties and electrochemical reaction potentials, respectively) methods for determining kynurenic acid in brain tissue extracts and CSF. These methods should prove useful in examining whether kynurenic acid modulates EAA-mediated neurotransmission under physiologic conditions, as well as in determining the role of kynurenic acid in excitotoxic neuronal death.     

Morphogenesis of the renal juxtaglomerular apparatus and peripolar cells in the sheep

Summary The morphogenesis of the juxtaglomerular apparatus and peripolar cells was studied in the metanephros of fetal sheep (from 24 to 147 days of gestation) using light and electron microscopy. The first juxtaglomerular apparatus was detected at 45 days of gestation, following constriction of the edges of Bowman's capsule and formation of the vascular pole of the renal corpuscle. Mesenchymal cells gave rise to lacis cells and to smooth muscle and epithelioid cells of the juxtaglomerular arterioles. Epithelioid cells developed only sparse cytoplasmic granulation, first detectable at 92 days. The macula densa developed from tubular cells at the junction of the middle and upper limbs of the S-shaped body of the developing nephron. Peripolar cells arose from epithelial cells in the lower limb of the S-shaped body, at the constricting edges of Bowman's capsule, and formed a cuff around the origin of the glomerular tuft. Cytoplasmic granules were first detected in peripolar cells at 53 days, and remained more prominent than epithelioid cell granulation throughout gestation.     

Spectral mechanisms of the compound eye in the fireflyPhotinus pyralis (Coleoptera: Lampyridae)

Summary Electroretinograms (ERG) were recorded from dark- and chromatic-adapted compound eyes in the dusk-active firefly,Photinus pyralis , at different wavelengths ranging from 320 to 700 run and over 4.5 log units change in stimulus intensity. ERG waveforms differed in the short (near-UV and violet) and long (yellow) wavelengths (Fig. 1). Waveform differences were quantitated by analysis of rise and fall times as a function of the amplitude of the response. Rise times were found to be relatively constant for all stimulus wavelengths. However, variations in the fall times were detected and followed characteristically different functions for short and long wavelengths (Fig. 2).No significant differences in the slopes of the Vlog-I curves at different stimulus wavelengths were observed (Fig. 3).Spectral sensitivity curves obtained from the ventral sector in dark- and chromatic-adapted conditions revealed peaks in the short ( max 400 nm: Fig. 4; max 430 nm: Fig. 5 A; and max 380 nm; Fig. 5B) and long ( max 570 nm: Figs. 4, 5) wavelengths, suggesting the presence of two spectral mechanisms. The long wavelength (yellow) mechanism was in close tune with the species bioluminescence emission spectrum (Fig. 4B).This investigation was supported in part by NIH Research Grant # EY-00490 (to R.M.C.); Research Grant # 01794N from the Research Foundation of the City University of New York (to A.B.L.); NIGMS Training Grant #1 TO 2 GM 05010-01 MARC (to J.A.H.); and NSF Grant # HES-75-09824 (to C.O.T.). We thank Tom Jensen for technical assistance, Barry Schuttler for his courtesy in allowing us to collect fireflies at his farm, Jean Lall for editorial assistance, and the two anonymous referees whose comments added considerably to the quality of this paper.     

Alveolar type II cells: studies on the mode of release of lamellar bodies.

There is increasing evidence that type II alveolar cells are capable of synthesizing surface active material like that obtained from the airways. However a number of problems remain to be solved before it can be stated conclusively that type II cells synthesize the surface active material of the terminal airspace. Among these problems is that of secretion. A number of previous studies have given evidence of the release of lamellar bodies by merocrine secretion. In this study morphologic evidence is presented which supports the view that secretion of lamellar bodies is accomplished by exocytosis. At the apical surface of type II cells, sites can be found where the limiting membrane of the lamellar body is clearly fused with the type II cell plasma membrane and an open channel exists between the contents of the lamellar body and the alveolar space. At these sites the lamellar contents extrude into the airspace with consequent loss of the highly compact organization of intracellular lamellar bodies. The intactness and continuity of the membranes can be traced for the full extent of the exocytosis site. Freeze-etch replicas of the membranes of type II cells show depressions which may represent the sites of discharged lamellae. In addition, tongue-like folds are seen which could be explained as the extensions of cytoplasm which surround the releasing lamellar body and which may flap over the exocytosis pit after discharge. Micrographs of the alveolar space show disorganized lamellar whorls which appear to be unravelling to produce tubular myelin. In view of the unusually large size and lipid composition of lamellar bodies, a mechanism involving hydration of mucopolysaccharide contents as an aid to expulsion of lamellar contents is suggested.