Thrombin attenuates the stimulatory effect of histamine on Ca2+ entry in confluent human umbilical vein endothelial cell cultures

Human umbilical vein endothelial cells were grown to confluence, as first passage cells, on coverslips. Treatment with ionomycin or histamine caused a sustained rise in intracellular Ca2+ (measured by Fura-2 fluorescence), but after treatment with thrombin, only a transient rise in Ca2+ was observed. Furthermore, the addition of thrombin after ionomycin or histamine lowered the raised Ca2+ back to near control levels. This effect of thrombin was concentration dependent, with increasing concentrations producing increases in both the rate and extent of the lowering of Ca2+. A similar effect of thrombin was seen on video imaging of Fura-2-loaded cell monolayers. The Ca2(+)-lowering effect of thrombin was not mimicked by phorbol 12-myristate 13-acetate nor blocked by staurosporine, indicating a lack of involvement of protein kinase C; intracellular pH also does not appear to be involved. The mechanism by which thrombin lowers cytoplasmic Ca2+ is due mainly to inhibition of Ca2+ entry since thrombin prevented the stimulated influx of Mn2+ caused by histamine or ionomycin. It may therefore be that in vivo under certain physiological or pathological conditions, thrombin's effects on intracellular Ca2+ are more transient than those of histamine, and thrombin also may induce transience in histamine's actions.     

Metoclopramide increases plasma but not cerebrospinal fluid vasopressin levels in man: study in hydrocephalic patients.

Arginine vasopressin (AVP) concentrations were determined in plasma and in cerebrospinal fluid (CSF) in 8 adult male patients suffering from hydrocephalus of various etiologies, before and after intravenous administration of 10 mg metoclopramide. Metoclopramide was able to increase the plasma (2.6 +/- 0.2 ng/l in basal conditions and 6.1 +/- 0.6 ng/l at 30 min) but not the CSF AVP levels. The results suggest that the neurons which secrete AVP into the CSF may be functionally different from those secreting into the peripheral circulation.     

Bacterial conversion of glycerol to beta-hydroxypropionaldehyde

beta-hydroxypropionaldehyde (3-HPA) can be oxidized to acrylic acid, an industrially important chemical used in the manufacture of synthetic plastics and other polymers. Of 19 genera and 55 strains tested, 3 Klebsiella and 2 Enterobacter strains produced 3-HPA. The most efficient strain was Klebsiella pneumoniae NRRL B-4011. Under optimum conditions (28 degrees C; 40 g of semicarbazide hydrochloride per liter, 70 g of glycerol per liter; and pH 6.0), 3.1 g of B-4011 cells per liter accumulated 22 g of 3-HPA per liter at a specific rate of 0.83 g/g per h; however, 14.5 g of cells per liter accumulated 46 g of 3-HPA per liter at a specific rate of 0.41 g/g per h.     

Vasculature of the head and respiratory organs in an obligate air-breathing fish,the swamp eel Monopterus (=Amphipnous) cuchia

Methyl methacrylate vascular corrosion replicas were used to examine the macrocirculation in the head region and the microcirculation of respiratory vessels in the air-breathing swamp eel Monopterus cuchia. Fixed respiratory tissue was also examined by SEM to verify capillary orientation. The respiratory and systemic circulations are only partially separated, presumably resulting in supply of mixed oxygenated and venous blood to the tissues. A long ventral aorta gives rise directly to the coronary and hypobranchial arteries. Two large shunt vessels connect the ventral aorta to the dorsal aorta, whereas the remaining ventral aortic flow goes to the respiratory islets and gills. Only two pairs of vestigial gill arches remain, equivalent to the second and third arches, yet five pairs of aortic arches were identified. Most aortic arches supply the respiratory islets. Respiratory islet capillaries are tightly coiled spirals with only a fraction of their total length in contact with the respiratory epithelium. Valve-like endothelial cells delimit the capillary spirals and are unlike endothelial cells in other vertebrates. The gills are highly modified in that the lamellae are reduced to a single-channel capillary with a characteristic three-dimensional zig-zag pathway. There are no arterio-arterial lamellar shunts, although the afferent branchial artery supplying the gill arches also supplies respiratory islets distally. A modified interlamellar filamental vasculature is present in gill tissue but absent or greatly reduced in the respiratory islets. The macro- and micro-circulatory systems of M. cuchia have been considerably modified presumably to accommodate aerial respiration. Some of these modifications involve retention of primitive vessel types, whereas others, especially in the microcirculation, incorporate new architectural designs some of whose functions are not readily apparent.     

A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.