Particulate Membrane Research Articles

Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions

Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.

Development of biodegradable polyesterurethane membranes with different surface morphologies for the culture of osteoblasts.

To evaluate the biocompatibility of biodegradable polyesterurethane membranes with different surface morphologies for their possible use as orthopedic biomaterials, rat osteoblasts were cultured on smooth, sunken, and particulate polyesterurethane membranes. A close interaction between cells and exposed particles on the particulate membranes was found. Cells on the particulate surfaces were well spread and flattened and had the greatest adhesion while cells on the smooth surfaces were more rounded, less spread, and less adhered. In addition, in order to investigate their in vivo degradation rates, the morphologic changes in retrieved membranes from 2, 4, and 8 weeks after subcutaneous implantation were observed by scanning electron microscopy and their average molecular weight changes were determined by gel permeation chromatography. These analyses showed that smooth membranes, compared with the two other surface membrane types, had the greatest rate and degree of molecular weight change. In contrast, the molecular weight of particulate membranes, which favor the osteoblast culture, had not changed significantly at 8 weeks postimplantation. Thus particulate polyesterurethane membrane surfaces may be of use as an orthopedic biomaterial, and polyesterurethane membranes certainly provide an ideal system for further study of the relative contributions to biocompatibility and degradation derived from surface morphology.

Interesting behavior for filtration of macromolecules through EVAL membranes

The objective of this article is to investigate the permeation characteristics of several polydispersed macromolecules through the asymmetric and the particulate poly (ethylene-co-vinyl alcohol) (EVAL) membranes. The solutes in examination include two nonionic solutes, dextran and polyethylene glycol (PEG), and one anionic solute, sodium polyacrylate (SPA). The ultrafiltration results indicate that the rejection coefficients increase monotonically with the molecular weights of test solutes permeating through the asymmetric EVAL membranes. This is consistent with the behavior of normal commercial ultrafiltration membranes. For the particulate membranes, the rejection curves show different trends. In most cases, there exist two local minima in the rejection curves, having the shape of “W”. This unusual behavior is found to be associated with the dual-pore character of the particulate membrane. To this context, a parallel composite membrane model is proposed to explain this specific rejection in terms of rejection coefficients for the small and the large pores.

Morphology of crystalline Nylon-610 membranes prepared by the immersion-precipitation process: competition between crystallization and liquid–liquid phase separation

Nylon-610 membranes were prepared at 25°C by direct immersion of various dope solutions into either formic acid/water or 1-octanol bath. Depending on the dope and bath conditions, the precipitated membranes demonstrated characteristics derived from crystallization and/or liquid–liquid phase separation during the precipitation process. As a good dope solution was immersed in a harsh bath, e.g., water, precipitation occurred initiated by liquid–liquid phase separation. The formed membrane exhibited a cellular structure similar to that commonly observed in amorphous membranes. Alternatively, when a metastable dope (with respect to crystallization) was immersed in a soft bath containing a substantial amount of formic acid, crystallization dictated the precipitation process to yield bi-continuous, particulate membranes. Membranes with extensive macrovoids were observed, in the event that the dope contained a large amount of solvent. In the latter case, precipitation took place immediately after immersion, consistent with Strathmann and Smolder’s results for several membrane forming systems. In addition, skinless microporous membranes were prepared by precipitation of the dope solutions in a 1-octanol bath, in which precipitation occurred slowly and the formed membrane was composed of “sheaf-like” crystallites that interlocked into a homogeneous bi-continuous network.

G protein G alpha q/11 and G alpha i1,2 are activated by pancreastatin receptors in rat liver: studies with GTP-gamma 35S and azido-GTP-alpha-32P.

In the liver, pancreastatin exerts a glycogenolytic effect through interaction with specific receptors, followed by activation of phospholipase C and guanylate cyclase. Pancreastatin receptor seems to be coupled to two different G protein systems: a pertussis toxin-insensitive G protein that mediates activation of phospholipase C, and a pertussis toxin sensitive G protein that mediates the cyclic GMP production. The aim of this study was to identify the specific G protein subtypes coupling pancreastatin receptors in rat liver membranes. GTP binding was determined by using gamma-35S-GTP; specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Activation of G proteins was demonstrated by the incorporation of the photoreactive GTP analogue 8-azido-alpha-32P-GTP into liver membranes and into specific immunoprecipitates of different Galpha subunits from soluble rat liver membranes. Pancreastatin stimulation of rat liver membranes increases the binding of gamma-35S-GTP in a time- and dose-dependent manner. Activation of the soluble receptors still led to the pancreastatin dose-dependent stimulation of gamma-35S-GTP binding. Besides, WGA semipurified receptors also stimulates GTP binding. The binding was inhibited by treatment with anti-Galphaq/11 (85%) and anti-Galphai1,2 (15%) sera, whereas anti-Galphao,i3 serum failed to affect the binding. Finally, pancreastatin stimulates GTP photolabeling of particulate membranes. Moreover, it specifically increased the incorporation of 8-azido-alpha-32P-GTP into Galphaq/11 and Galpha, but not into Galphao,i3 from soluble rat liver membranes. In conclusion, pancreastatin stimulation of rat liver membranes led to the activation of Galphaq/11 and Galphai1,2 proteins. These results suggest that Galphaq/11 and Galphai1,2 may play a functional role in the signaling of pancreastatin receptor by mediating the production of IP3 and cGMP respectively.

Effect of Solubilization of Porcine Thyrotropin (TSH) Receptor on TSH Binding and on Radio-Receptor Assay for Anti-TSH Receptor Antibodies

The effect of solubilization of porcine thyrotropin receptor (TSHR) on TSH binding and on the radio-receptor assay for anti-TSHR antibodies was examined. After TSHR solubilization with 1% dodecylpolyethyleneglycoether, TSH binding affinity was increased, from Kd = 1.15nM to 0.45nM, and TSH binding capacity was slightly increased, from 0.15nM to 0.19nM. With a particulate membrane suspension from thyroid cells, blocking of TSH binding to the membrane suspension by anti-thyrotropin receptor antibody was observed only for thyroid stimulation blocking antibody (TSBAb), not for thyroid-stimulating antibody (TSAb). After the solubilization of TSHR, both TSBAb and TSAb blocked TSH-binding to the solubilized TSHR. We speculate that TSAb interacts with the TSHR in the native conformation without interfering with TSH binding, and that after the solubilization, any anti-TSHR antibody interferes with TSH-binding due to the conformational change in TSHR. With these particulate thyroid cell membrane preparations, we can detect only TSBAb by the radio-receptor assay.

Formation of crystalline EVAL membranes by controlled mass transfer process in water–DMSO–EVAL copolymer systems

The effect of solvent content in the coagulation bath on the formation of poly(ethylene-co-vinyl alcohol) (EVAL) membranes by isothermal immersion–precipitation in water–dimethylsulfoxide (DMSO)–EVAL system has been investigated. As a homogeneous EVAL dope is immersed in a harsh bath, e.g., water, instant precipitation occurs initiated by the liquid–liquid demixing process. The formed membrane exhibits an asymmetric morphology with extensive finger-like macrovoids, similar to that observed in most amorphous polymeric membranes. On the other hand, if precipitation takes place in a soft bath containing a substantial amount of solvent, crystallization, rather than liquid–liquid demixing, starts to dominate and a skinless, bi-continuous, particulate membrane becomes the precipitated product. The immersion–precipitation process for the present system has been modeled as a ternary mass transfer problem. The calculated diffusion trajectories and concentration profiles illustrate reasonably the membrane morphologies formed in various immersion conditions.

Specific Non-Genomic, Membrane-Localized Binding Sites for Progesterone in the Bovine Corpus Luteum1

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.

Effects of precipitation conditions on the membrane morphology and permeation characteristics

The permeability and permselectivity of asymmetric and particulate membranes towards glucose and proteins of various molecular sizes were studied. It was found that the skin layer of asymmetric membranes was permeable to glucose and insulin but effectively prevent the permeation of immunoglobulins. This result parallels our interest for the development of artificial pancreas. It was also found that skinless particulate membranes exhibited not only high permeation rates with respect to albumin and immunoglobulins but also good selectivity between these components. Thus, particulate membranes has the potential to be used in separating albumin from immunoglobulins for treating disorders related to immunoglobulin abnormalities.

Solute rejection of dextran by EVAL membranes with asymmetric and particulate morphologies

EVAL copolymer was precipitated from water and octanol to form, respectively, asymmetric and particulate membranes in an isothermal immersion precipitation process. Permeability of these membranes was examined with respect to dextran samples of various sizes. The results indicate that asymmetric membranes reject large dextran molecules and let through small molecules for the pressure range of 0.25–0.75 kg/cm 2 . The particulate membranes, however, exhibit an unusual filtration behaviour; i.e. there exists a maximum rejection at intermediate dextran molecular weight. This implies that small molecules tend to be trapped inside the nano-pores within the EVAL particles whereas large molecules travel through the tortuous channels outside the particles during a filtration operation.

Flux enhancement by a continuous tangential gas flow in ultrafiltration hollow fibres for drinking water production: Effects of slug flow on cake structure

A new process is proposed to reduce particulate membrane fouling by injecting air into the feed stream, creating a gas/liquid two-phase flow on the membrane surface. The injected air is supposed to form air slugs inside the hollow fibres; these slugs create high wall shear stresses and flow instabilities. These phenomena may prevent filtered particles from settling on the membrane surface, and so enhance the ultrafiltration flux. Experiments were carried out with clay suspensions in hollow fibre membranes. A range of air velocities and particle concentrations was examined. The air injection process led to an increase in the permeate flux, depending on the liquid velocity and transmembrane pressure, for all the various concentrations studied. For specific conditions, the flux can be increased by 155% using a critical gas velocity. Above this critical value, the flux is no longer enhanced. The air injection clearly modifies the cake structure, and seems to expand the cake. Evolutions of cake thickness, porosity and specific resistance with gas velocity are given and analysed.

3H]Raclopride Binding to Brain Tissue from Subjects with Schizophrenia: Methodological Aspects

The binding of [ 3H]raclopride to particulate membrane and frozen sections (with quantitative autoradiography) from the caudate-putamen, obtained at autopsy from schizophrenic and non-schizophrenic subjects, was measured. The affinity of [ 3H]raclopride to particulate membrane was significantly decreased in the schizophrenic compared to non-schizophrenic subjects. The density of [ 3H]raclopride binding to tissue from subjects with schizophrenia was increased, unchanged or decreased depending on the methodology used. Finally, there was an age-dependent decrease in [ 3H]raclopride binding in the frozen sections from the caudate-putamen of the non-schizophrenic subjects. This age-dependent decrease was not apparent using particulate membrane from schizophrenic or non-schizophrenic subjects or tissue sections from the schizophrenic subjects. We conclude that the binding of [ 3H]raclopride is dependent on methodology and therefore data from in vitro and in vivo studies using this drug should be interpreted with caution. © 1997 Elsevier Science Ltd.

Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes

The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-zeta. In addition to primary monocytes, activation of PKC-zeta in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide epsilon and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-zeta from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase. Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-zeta. Furthermore, activation of PKC-zeta failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-zeta activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-zeta is activated downstream of PI 3-kinase in monocytes in response to LPS.

Aging-related regulation of myo-inositol 1,4,5-trisphosphate signal transduction pathway in the rat striatum.

To determine the effects of the aging process on the regulation of phosphoinositide signal transduction pathway, inositol 1,4,5-trisphosphate and inositol 1,4,5-trisphosphate receptor-associated parameters were examined in the striatum of brains removed from young (3 months), adult (12 months) and senescent (25 months) male Fischer 344 rats. Inositol 1,4,5-trisphosphate content was significantly increased (P < or = 0.01) at 25 months of age compared to 3 and 12 months. No age-related differences in phosphatidylinositol 4,5-bisphosphate hydrolysis were found in striatal slices after stimulation with trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate, a metabotropic glutamatergic receptor agonist. Phosphatidylinositol 4,5-bisphosphate hydrolysis following stimulation with (R,S)-alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid, a glutamatergic/quisqualate agonist, showed a significantly increased accumulation of net [3H]inositol 1,4,5-trisphosphate in senescent striatum whereas the muscarinic cholinergic agonist carbachol induced highest response in the young striatum. In each case, agonist-stimulated response was significantly reduced in the presence of the receptor-associated antagonist. The density of inositol 1,4,5-trisphosphate receptor in the particulate membranes derived from 12- and 25-month-old rats was decreased (P < 0.01) compared to that from young rats. Binding affinity of inositol 1,4,5-trisphosphate receptor for [3H]inositol 1,4,5-trisphosphate was increased (P = 0.05) only at 25 months of age when compared with 3 months of age. Incubation of partially purified inositol 1,4,5-trisphosphate receptor with striatal cytosol in the presence of Ca2+ showed an age-dependent susceptibility to proteolytic degradation of this receptor that was completely inhibited by calpain I inhibitor peptide. Paradoxically, the quantity of inositol 1,4,5-trisphosphate receptor mRNA-encoding transcripts was increased (P < or = 0.01) at 25 months of age, suggesting an age-dependent change in either transcriptional rate, stability or processing of inositol 1,4,5-trisphosphate receptor mRNAs in the striatum. The activity of inositol 1,4,5-trisphosphate3-kinase decreased (P < or = 0.01) with age whereas the activity of soluble inositol 1,4,5-trisphosphate 5-phosphatase was highest at 3 months but significantly decreased at 12 months of age. However, the activity of inositol 1,4,5-trisphosphate 5-phosphatase remained unchanged between 12 and 25 months of age, suggesting possible developmental modulation of the activity of the enzyme. Taken together with the established 'cross-talk' between signal transduction systems, the present data suggest that molecular/cellular changes in striatal inositol 1,4,5-trisphosphate/Ca2+ signal transduction pathway along with neuronal cell loss may contribute to aging-related decrease in striatal functioning.

Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes

BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) improves liver function in patients with chronic cholestatic liver diseases by an unknown mechanism. UDCA is conjugated to taurine in vivo, and tauroursodeoxycholic acid (TUDCA) is a potent hepatocellular Ca2+ agonist and stimulates biliary exocytosis and hepatocellular Ca2+ influx, both of which are defective in experimental cholestasis. Protein kinase C (PKC) mediates stimulation of exocytosis in the liver. The aim of this study was to determine the effects of TUDCA on PKC in isolated hepatocytes. METHODS: The effect of TUDCA on the distribution of PKC isoenzymes within the hepatocyte was studied using immunoblotting and immunofluorescence techniques. In addition, the effect of TUDCA on the accummulation of sn-1,2-diacylglycerol (DAG), the intracellular activator of PKC, and hepatocellular PKC activity was studied using radioenzymatic techniques. RESULTS: Immunoblotting studies showed the presence of four isoenzymes (alpha, delta, epsilon, and zeta). The phorbol ester phorbol 12-myristate 13-acetate (1 mumol/L) induced translocation of alpha-PKC, delta-PKC, and epsilon-PKC from cytosol to a particulate membrane fraction, a key step for activation of PKC. TUDCA, but not taurocholic acid, selectively induced translocation of the alpha-PKC isoenzyme from cytosol to the membranes. In addition, TUDCA induced a significant increase in hepatocellular DAG mass and stimulated membrane-associated PKC activity. CONCLUSIONS: TUDCA might stimulate Ca(2+)-dependent hepatocellular exocytosis into bile in part by activation of alpha-PKC. (Gastroenterology 1996 May;110(5):1553-63)

PGE2 and PGI2 inhibit ET-1 secretion from endothelial cells by stimulating particulate guanylate cyclase.

Prostaglandins (PG)E2 and prostacyclin (PGI2) can cause vasodilation in selective vascular beds and could act in part by inhibiting the production of the vasoconstrictor endothelin-1 (ET-1). We recently reported that these prostanoids inhibit ET-1 production/secretion from cultured endothelial cells via the generation of guanosine 3'-5'-cyclic monophosphate (cGMP). It is unclear whether this results from the stimulation of the particulate (membrane) of soluble (cytosolic) form of guanylate cyclase, and whether these effects are through an intermediate, such as nitric oxide. PGE2 and PGI2 each caused a three- to fourfold increase in both membrane and whole bovine aortic endothelial cell guanylate cyclase activity. The stimulations were significantly reversed (80-90%) by the compound LY-83583, an antagonist to cGMP generation, but were unaffected by methylene blue (MB), an inhibitor of nitric oxide-induced soluble guanylate cyclase. In contrast, the prostaglandins did not generate cGMP in cytosolic fractions. The prostaglandins inhibited ET-1 secretion from the intact cells, which was significantly prevented by LY-83583, but not by MB. Neither prostaglandin stimulated NO synthase activity, an indicator of nitric oxide generation. We conclude that PGE2 and PGI2 are likely to inhibit ET-1 secretion through the activation of the particulate guanylate cyclase, identifying a novel mechanism by which the prostanoids signal in the endothelial cell.

Regulation of phosphatidylinositide transduction system in the rat spinal cord during aging.

Age-related functional alterations in a variety of neurotransmitter systems result in modulation of interneuronal communications which has some relevance in neurological deficits observed in the aging process. The synergistic interactions between protein kinase and inositol 1,4,5-trisphosphate (insP3)/Ca2+ pathways underlie a variety of cellular responses to external stimuli. To determine whether age-dependent changes occur in the regulation of protein kinase C and inositol 1,4,5-trisphosphate/Ca2+ pathways, insP3 contents as a marker for the release of intracellular calcium, saturation binding analysis of Ins P3 receptor using [3H]inositol 1,4,5-trisphosphate, slot/northern blot analysis of Ins P3 receptor-encoding mRNA transcripts, and the activities of Ca2+/phospholipid-dependent protein kinase C isozymes were investigated in the rat spinal cord. Inositol 1,4,5-trisphosphate content and [3H]inositol 1,4,5-trisphosphate binding site density (Bmax) were quantified in the spinal cords of young (three months old), adult (12 months old) and senescent (25 months old) male Fischer 344 rats. Spinal cord content of inositol 1,4,5-trisphosphate was increased (P < 0.01) in the 25-month old compared to the three- and 12-month old animals. The density of Ins P3 receptor in particulate membranes derived from the 25-month old rats was reduced (P < or = 0.01), but the binding affinity (Kd) was increased (P < or = 0.04) by a factor of 2.2 and 3.2 at 25 months of age when compared with three- and 12-month old animals, respectively. Young and middle-aged animals showed no differences in both inositol 1,4,5-trisphosphate contents and [3H]inositol 1,4,5-trisphosphate binding site density. The quantity of Ins P3 receptor mRNA was significantly increased with age in the order 25 >> 12 > 3 months of age. Total functional cytosolic and membrane-associated PKC activities were decreased (P < or = 0.05) in the 25-month compared to the three- and 12-month old rats in which activity remained unchanged. Total membrane/cytosolic activity ratios were unchanged by the aging process. In all cases, the activities of membrane-associated conventional protein kinase C isozymes (alpha, beta and gamma), determined by immunoprecipitation followed by in situ quantification of protein kinase C activities in the immunoprecipitates, showed age-dependent decline. The activities of protein kinase C-alpha and beta were significantly decreased in age-related manner. However, the activity of the gamma-isozyme was not significantly changed at 12- and 25-months of age, although it was higher (P < or = 0.03) in young rats. Western blot analyses using affinity purified polyclonal antibodies specific for each isozyme indicated a single protein with an apparent molecular mass of approximately 80 x 10(3) molec. weight for all isozymes except for the beta isozyme that also had an appreciable immunoreactive band at approximately 36 x 10(3) molec. weight. Overall, the aging process did not affect the electropheretic mobility of each isozyme. With decreased protein kinase C activity, the present data suggest that the aging process would decrease protein kinase C-induced phosphorylation of membrane proteins including Ins P3 receptor. A significant change in Ins P3 receptor affinity combined with increased levels of Ins P3 receptor mRNA-encoding transcripts in senescent rats suggests not only a modification (possibly by phosphorylation) of Ins P3 receptor protein but also the existence of multiple (spliced) variants of Ins P3 receptor in spinal neurons with increasing age. The present data indicate that the spinal contents of inositol 1,4,5-trisphosphate increased with age, but with decreased efficacy and number of inositol 1,4,5-trisphosphate-activatable Ca2+ channels in the spinal cord of senescent rats. These age-related changes may contribute to the attenuated responsiveness of spinal cord neurons by phosphoinositide-coupled receptors during the aging process.

Serotonin 2 receptors and the serotonin transporter in the schizophrenic brain

The binding of [ 3H]paroxetine and [ 3H]ketanserin to particulate membranes from frontal cortex of subjects who had or did not have schizophrenia was measured as was [ 3H]paroxetine binding to particulate membranes from the hippocampus and caudate nucleus. There was no change in either the affinity or density of [ 3H]ketanserin binding to membranes from the frontal cortex of subjects who had schizophrenia. Similarly, there was no difference in the density of [ 3H]paroxetine binding to membranes from subjects who had or did not have schizophrenia. The affinity of [ 3H]paroxetine binding in the frontal cortex and putamen did not differ in subjects who had schizophrenia. By contrast, there was a significant decrease in the affinity of [ 3H]paroxetine binding to the hippocampal membrane from subjects who had schizophrenia (0.40 ± 0.06 nM vs 0.26 ± 0.02 nM; p < 0.05). Furthermore, this difference was more apparent in the subjects who had schizophrenia and committed suicide (0.49 ± 0.09 nM) than it was in those who had schizophrenia but did not commit suicide (0.32 ± 0.09 nM). As [ 3H]ketanserin binds to the serotonin 2 receptor our data suggest that this receptor is not changed in the Brodmann's area 9 of the frontal cortex. By contrast, [ 3H]paroxetine binds to the serotonin transporter and therefore our data suggest that the serotonin transporter is altered in the hippocampus of subjects with schizophrenia.

Protein kinase C and diacylglycerol content in basilar arteries during experimental cerebral vasospasm in the dog

Sixteen dogs were entered into a study of the double subarachnoid hemorrhage (SAH) model of cerebral vasospasm. Six animals were sacrificed 72 hours after the first experimental SAH, and the remaining 10 animals were killed 72 hours after the second experimental SAH; ten additional animals served as controls. Basilar arteries were rapidly excised from the dogs and frozen. Multiple segments of the frozen arteries were analyzed independently for total protein and 1,2-diacylglycerol (DAG) content, which averaged 3.17 (+/- 0.27 standard error of the mean; SEM) pmol DAG/microgram protein for all 25 arteries analyzed. A slight decreasing trend in DAG content relative to that of control vessels was found in vessels chronically constricted in situ by subarachnoid blood clot; however, this trend did not attain statistical significance. Two segments of the same vessels were assayed independently for protein kinase C (PKC) activity, which averaged 1.21 (+/- 0.08 SEM) pmol phosphate incorporation per minute per microgram protein for all 24 arteries analyzed. A small decrease in PKC content was noted in vessels that experienced a single SAH; however, PKC returned to near control value in vessels subjected to double SAH. The ratio of particulate (membrane bound) to soluble PKC activity, an indicator of PKC translocation to the membrane and hence PKC activation, showed a small but statistically significant trend to increase with experimental SAH.

3H]paroxetine binding is altered in the hippocampus but not the frontal cortex or caudate nucleus from subjects with schizophrenia.

[3H]Paroxetine binding to particulate membrane from tissue, obtained at autopsy, from the hippocampus, frontal cortex, and caudate nucleus from subjects who had or had not had schizophrenia was measured. The density of [3H]paroxetine binding to membranes from subjects who had or had not had schizophrenia did not differ. Similarly, the affinity of [3H]paroxetine binding in the frontal cortex and caudate nucleus was not different. By contrast, the affinity of [3H]paroxetine binding to hippocampal membrane from subjects who had schizophrenia was significantly lower than the affinity of binding for the nonschizophrenic subjects (0.40 +/- 0.06 vs. 0.26 +/- 0.02; p < 0.05). As [3H]paroxetine binds to the serotonin transporter, these data suggest that the serotonin transporter is altered in the hippocampus in subjects with schizophrenia.