Multifunctional nanocatalyst-based ultrasensitive detection of human tissue transglutaminase 2.

Tissue transglutaminase 2 (TGM2) is a novel tumor marker, which can be used as the complementary marker for alpha-fetoprotein to improve overall diagnostic performance of hepatocellular carcinoma at the clinical level. In this work, we have developed a multifunctional nanocatalyst-based electrochemical method for TGM2 assay with an ultrahigh sensitivity. Here we firstly functionalize carboxyl-modified graphene oxide with poly-lysine and copper ion to form an electrochemical nanocatalyst. On the one hand, the nanocatalyst can function as the substrate of TGM2. On the other, the nanocatalyst can be applied for signal amplification to enable high sensitivity of the detection. With the specific glutamine-donor-peptide of TGM2 modified on the electrode, the exertion of the transamidation activity by TGM2 can lead to the tethering of the nanocatalyst with the peptide on the electrode, inducing obvious changes of the electrochemical signals. Therefore, simple and sensitive detection of TGM2 can be achieved. Moreover, TGM2 is also detected effectively in complex serum samples, suggesting potential diagnostic applications of the new method proposed in this work.

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system.

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.

Total substitution of fish oil by vegetable oils in gilthead sea bream (Sparus aurata) diets: effects on hepatic Mx expression and some immune parameters.

The use of vegetable oils in fish nutrition has been extensively studied; and recent work has focused attention on replacing fish oil with alternative fatty acid sources and their effect on the immune system. However, little is known about the effect of these oils on immune parameters such as the fish interferon system. In this study we evaluate the effect of two vegetable oils (linseed and soybean) on gilthead sea bream Mx expression and other innate immune parameters. Experimental diets were formulated where fish oil was totally replaced by vegetable oils or for a mixture of them (50% linseed and 50% soybean). Another diet prepared with pure fish oil was used as a control. Two experiments were carried out in order to evaluate growth, feed utilization, serum alternative complement pathway activity, serum lysozyme and phagocytic activity of head kidney leucocytes as well as Mx expression in the liver. In the first experiment fish were fed with experimental diets for 6 months and then, growth and feed utilization as well as immune parameters were analyzed. In the second experiment, fish from the previous feeding trial were injected with either a sub-lethal dose of Photobacterium damselae subsp. piscicida (94/99) or a synthetic dsRNA (Poly I:C) in order to stimulate an Mx response. The results show that total substitution of fish oil by vegetable oils decreased the growth of gilthead sea bream juveniles. Furthermore, both phagocytic activity and serum alternative complement pathway activity were significantly reduced by the inclusion of either vegetable oil individually in the sea bream diets, but the diet with mixed vegetable oils had no significant effect. There was no effect on serum lysozyme levels but the basal constitutive levels of Mx transcript expression in the liver were elevated in the fish fed the vegetable oil diets. The time-course of the Mx response to injection of Poly I:C was shorter in the fish fed the fish oil diet and the fish fed the diet based on a mixture of both vegetable oils showed a faster Mx response to bacterial injection. Following stimulation with Poly I:C or PDP the fish fed the vegetable oil based diets still maintained higher basal levels of hepatic Mx expression than the fish fed the fish oil diet which returned to undetectable levels.

Consumption of virgin olive oil influences membrane lipid composition and regulates intracellular signaling in elderly adults with type 2 diabetes mellitus.

We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCalpha) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Galphao, Gbeta, and PKCalpha were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Galphas, Gbeta, and PKCalpha in both groups, reduction in Galphai was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCalpha with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.

Tempol reduces oxidative stress and restores renal dopamine D1-like receptor- G protein coupling and function in hyperglycemic rats.

Dopamine via activation of renal D1-like receptors inhibits the activities of Na-K-ATPase and Na/H exchanger and subsequently increases sodium excretion. Decreased renal dopamine production and sodium excretion are associated with hyperglycemic conditions. We have earlier reported D1-like receptor-G protein uncoupling and reduced response to D1-like receptor activation in streptozotocin (STZ)-treated hyperglycemic rats (Marwaha A, Banday AA, and Lokhandwala MF. Am J Physiol Renal Physiol 286: F451-F457, 2004). The present study was designed to test the hypothesis that oxidative stress associated with hyperglycemia increases basal D1-like receptor serine phosphorylation via activation of the PKC-G protein receptor kinase (GRK) pathway, resulting in loss of D1-like receptor-G protein coupling and function. We observed that STZ-treated rats exhibited oxidative stress as evidenced by increased lipid peroxidation. Furthermore, PKC activity and expression of PKC-betaI- and -delta-isoforms were increased in STZ-treated rats. In addition, in STZ-treated rats there was increased GRK2 translocation to proximal tubular membrane and increased basal serine D1-like receptor phosphorylation. Supplementation with the antioxidant tempol lowered oxidative stress in STZ-treated rats, led to normalization of PKC activity, and prevented GRK2 translocation. Furthermore, tempol supplementation in STZ-treated rats restored D1-like receptor-G protein coupling and inhibition of Na-K-ATPase activity on D1-like receptor agonist stimulation. The functional consequence was the restoration of the natriuretic response to D1-like receptor activation. We conclude that oxidative stress associated with hyperglycemia causes an increase in activity and expression of PKC. This leads to translocation of GRK2, subsequent phosphorylation of the D1-like receptor, its uncoupling from G proteins and loss of responsiveness to agonist stimulation.

Differences in regional and subcellular localization of G(q/11) and RGS4 protein levels in Alzheimer's disease: correlation with muscarinic M1 receptor binding parameters.

Deficits in M1 muscarinic receptor system signaling in Alzheimer's disease (AD) prompted an analysis of components of these systems, namely, the G(q/11) protein and the regulator of G-protein signaling (RGS) 4 protein. In AD parietal cortex, total levels of G(q/11) and RGS4 proteins were significantly lower than age-matched control cases by 40% and 53%, respectively. However, the levels of membrane-bound G(q/11) and RGS4 protein in AD parietal cortex were maintained at levels comparable to controls. Furthermore, in the frontal cortex and cerebellum both the total and membrane levels of G(q/11) and RGS4 protein were not altered in AD cases compared to control cases. To our knowledge, this is the first report to examine RGS proteins in AD. Using receptor binding assays on the parietal cortex membrane fractions from AD cases, we found the muscarinic agonist carbachol still bound to high- and low-affinity sites (two-site fit) and the potency of 5-guanylylimidodiphosphate (GppNHp) to shift receptors from the high- to low-affinity state (based on the ternary complex model) was greater in AD cases compared to controls. In contrast, we previously reported a lack of high-affinity agonist binding sites in the frontal cortex in AD cases even in the absence of GppNHp. The data suggest that the equilibrium dynamics between the cytosolic and membrane levels of G(q/11) and RGS4 may contribute to the regional differences in the coupling of muscarinic M1 receptors in AD and have implications for the variability in effects of cholingeric treatment strategies currently in place.

Expression of the galanin receptor subtype Gal-R2 mRNA in the rat hypothalamus.

The distribution of galanin receptor subtype 2 (Gal-R2) mRNA-expressing cells was examined by in situ hybridization in the rat hypothalamus using a full-length rat 35S-riboprobe. Gal-R2 receptor mRNA-expressing cells were found at moderate to high levels of expression in most nuclei and regions of hypothalamus. The labeling was observed within well-defined anatomical nuclei: preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, dorsomedial, mammillary nuclei. The supraoptic and ventromedial nuclei were almost devoid of labeling. Some scattered labeled cells were also observed in the pituitary. This distribution of Gal-R2 mRNA-expressing cells corresponds well with that of galanin binding sites studies. As compared to the distribution of the galanin receptor subtype 1 (Gal-R1), our results indicate that the Gal-R2 type is differentially distributed, although a significant overlap exists in some regions such the preoptic area, arcuate and dorsomedial nuclei. The functional implications of these results are discussed in light of the role of galanin receptors plays in neuroendocrine regulation and feeding behavior.

delta Opioid receptor subtypes activate inositol-signaling pathways in the production of antinociception.

To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists.

Galanin receptors in the hippocampus and entorhinal cortex of aged Fischer 344 male rats.

Galanin (GAL) has been proposed to be an inhibitory modulator of cholinergic memory pathways because it acts within the hippocampus to inhibit the release and antagonize the postsynaptic actions of acetylcholine. Here we have used: 1) slice binding and quantitative autoradiography to assess the density and occupancy of GAL receptors; and 2) in situ hybridization histochemistry to assess expression of the GALR1 receptor subtype in the ventral hippocampus of 3-month-old and 21-month-old Fischer 344 male rats. We detected a small but significant (p < or = 0.0003) age-related reduction in 125I-GAL binding-site density in the ventral hippocampus and entorhinal cortex under standard binding conditions. Post-hoc analysis indicated that this reduction with age persisted in the CA1 radiatum and entorhinal cortex following GTP-induced desaturation to unmask pre-existent GAL receptors occupied by endogenous ligand. It was not associated with a significant change in peak GALR1 gene expression in the hippocampus. Because a portion of GAL receptors in this region have been postulated to function as presynaptic auto-receptors on cholinergic fiber terminals, the reduction in GAL binding sites with age may be a consequence of age-related alterations in GAL receptor expression by basal forebrain cholinergic neurons which project to the ventral hippocampus.

A desensitization of hypothalamic 5-HT1A receptors by repeated injections of paroxetine: reduction in the levels of G(i) and G(o) proteins and neuroendocrine responses, but not in the density of 5-HT1A receptors.

The aim of the present study was to determine whether the previously observed desensitization of hypothalamic 5-hydroxytryptamine1A (5-HT1A) receptors, during daily injections of fluoxetine, is mediated by sustained blockade of 5-HT reuptake. In the present study, we examined the time course effects of another 5-HT uptake inhibitor, paroxetine. Paroxetine reduced the oxytocin, adrenal corticotropic hormone and corticosterone responses to a challenge with the 5-HT1A agonist 8-hydroxy-2-(dipropylamino)tetralin. These reductions in hormone responses were significant after 3 daily injections and reached a maximum after 7 daily paroxetine injections. These hormone responses remained maximally suppressed after 14 daily injections of paroxetine. A single day of paroxetine treatment did not alter the hormone responses to 8-hydroxy-2-(dipropylamino)tetralin. Repeated injections of paroxetine did not reduce the density of 5-HT1A receptors in any brain region but did produce a gradual reduction in the levels of G(i) and G(o) proteins in a region-specific manner. The time course of the paroxetine-induced reduction in the level of G(i1) and G(i3) proteins in the hypothalamus was similar to the effect previously observed with fluoxetine and was also similar to the time course of paroxetine-induced reductions in oxytocin and adrenal corticotropic hormone responses to 8-hydroxy-2-(dipropylamino)tetralin. In conclusion, these results suggest that blockade of 5-HT uptake sites produces a delayed and gradual desensitization of 5-HT1A receptors in the hypothalamus. This desensitization is not due to changes in the density of hypothalamic 5-HT1A receptors. Reduction in the hypothalamic level of G(i3) proteins may play a role in the desensitization of 5-HT1A receptor systems. However, reductions in G(i1) or G(o) proteins cannot be excluded as potential mediators of the desensitization of 5-HT1A receptor systems.

Molecular cloning of a lobster G alpha(q) protein expressed in neurons of olfactory organ and brain.

We have isolated from an American lobster (Homarus americanus) olfactory organ cDNA library a clone, hG alpha(q), with >80% identity to mammalian and arthropod G alpha(q) sequences. In brain and olfactory organ, hG alpha(q) mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. G alpha(q) protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hG alpha(q) plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hG alpha(q) mRNA species (6 kb) in the olfactory organ, and the localization of hG alpha(q) mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hG alpha(q) is to mediate olfactory transduction.

Chronic fluoxetine induces a gradual desensitization of 5-HT1A receptors: reductions in hypothalamic and midbrain Gi and G(o) proteins and in neuroendocrine responses to a 5-HT1A agonist.

The time course of fluoxetine-induced desensitization of hypothalamic 5-hydroxytryptamine1A receptors was examined in rats. Daily injections of fluoxetine (10 mg/kg/day) for 0, 3, 7, 14 or 22 days gradually produced a shift to the right in the dose-response curve effects of 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) on plasma adrenal corticotropic hormone, corticosterone and oxytocin. A partial reduction was observed for the adrenal corticotropic hormone and oxytocin responses to 8-OH-DPAT (50 micrograms/kg s.c.) after 3 days, and a maximum reduction of all hormone responses was observed after 14 days of fluoxetine injections, when the adrenal corticotropic hormone and oxytocin responses to the 50-micrograms/kg dose of 8-OH-DPAT were virtually blocked. To begin to examine the mechanism of 5-hydroxytryptamine1A receptor desensitization, we determined levels of Gi and G(o) proteins in the hypothalamus, midbrain and frontal cortex by using immunoblots. The hypothalamic levels of Gi1 and Gi3 proteins were significantly reduced after 7 and 14 days of fluoxetine injections. The levels of G(o) and Gi2 proteins in the midbrain were significantly decreased after 3 days and remained reduced for the duration of fluoxetine injections. Fluoxetine did not reduce the concentrations of Gi and G(o) proteins in the frontal cortex at any time. The similarity in time course between fluoxetine-induced reductions in hormone responses to 8-OH-DPAT and the reduction in hypothalamic levels of Gi1 and Gi3 proteins suggests that a reduction in hypothalamic levels of Gi3 and/or Gi1 proteins plays a role in the gradual desensitization of 5-hydroxytryptamine1A receptors induced by fluoxetine.

Effects of verapamil on acute murine Chagas' disease.

Continuous administration of verapamil significantly reduced the mortality rate of acute murine Trypanosoma cruzi infection (P < 0.05). The mechanistic basis for these observations was investigated. Verapamil and other calcium-channel blockers did not inhibit the growth of epimastigotes in culture. Furthermore, verapamil did not inhibit the intracellular growth of amastigotes in endothelial cells as determined by the uptake of 3H-uracil. There were no significant differences in parasitemia between infected mice that were untreated and those treated with verapamil. Twenty days postinfection infected, untreated mice had a parasitemia of 5.8 x 10(6) trypomastigotes/ml (SD +/- 2 x 10(6)), whereas infected, verapamil-treated mice had a parasitemia of 2.2 x 10(6) trypomastigotes/ml (SD +/- 0.5 x 10(6)). There was no significant difference in mortality between mice administered verapamil only for the initial 10 days of murine infection compared to those treated continuously. A 3-day delay in the initiation of verapamil administration reduced the mortality rate, but a 10-day delay did not. Propranolol (beta-adrenergic blocker), prazosin (alpha 1-adrenergic blocker), and diltiazem (another calcium-channel blocker) reduced the mortality but not significantly (P = 0.07). In biochemical studies of the beta- adrenergic signal transduction complex, we determined that verapamil and propranolol reversed the infection-associated decrease in myocardial beta- adrenergic adenylyl cyclase activity. In contrast, complementary western blot analysis revealed no significant changes in the G-proteins of the beta- adrenergic receptor complex 45 days postinfection. Therefore, these results suggest that the basis of verapamil's influence on the early critical period of infection is multifactorial and independent of a direct trypanocidal effect.

Treatment in hypertensive cardiac hypertrophy, II. Postreceptor events.

We investigated the effect of pharmacological treatment with captopril, nitrendipine, and captopril plus nitrendipine on myocardial heterologous adenylyl cyclase desensitization and the underlying postreceptor defects in spontaneously hypertensive rats (SHR). In myocardial membranes from SHR, stimulation of adenylyl cyclase with guanylylimido-diphosphate (P < .001) and forskolin (P < .05) was significantly reduced, whereas no difference with forskolin was obtained in the presence of manganese chloride. Reconstitution of Gs alpha into Gs alpha-deficient S49 cyc- mouse lymphoma cells revealed no difference between SHR and control rats. In contrast, pertussis toxin labeling of Gi alpha was significantly increased in SHR. The reduction of adenylyl cyclase in SHR was abolished after pertussis toxin treatment of membranes. Treatment with captopril, nitrendipine, or both reduced Gi alpha and increased guanylylimidodiphosphate-stimulated adenylyl cyclase activity in SHR. In summary, heterologous adenylyl cyclase desensitization due to an increase of Gi alpha but in the presence of an unchanged activity of Gs alpha or the catalyst occurs in SHR. This alteration, which could contribute to the progression of contractile dysfunction by producing adrenergic subsensitivity, is sensitive to pharmacological treatment most likely because of a reduction of sympathetic activity.

Attenuation of muscarinic receptor-G-protein interaction in Alzheimer disease.

Cortical M1 muscarinic receptor-G-protein coupling, high-affinity, guanine nucleotide-sensitive agonist binding (Flynn et al., 1991; Warpman et al., 1993) and muscarinic receptor-stimulated [3H]PIP2 hydrolysis (Ferrari-DiLeo and Flynn, 1993) are known to be defective in Alzheimer disease. Whether this defect reflects an alteration in the M1 muscarinic receptor, its respective guanine nucleotide binding (G) protein or both is not known. This study compares the number and both basal and muscarinic receptor-mediated function of G-proteins in synaptosomal membranes from cerebral cortical samples of age-matched control subjects and Alzheimer disease patients. Immunoblotting with anti-G alpha q/11 and anti-G beta antibodies demonstrated no alteration in the number of these G-protein subunits in Alzheimer disease. Basal [35S]GTP gamma S binding and hydrolysis of [gamma-32P]GTP by high-affinity GTPase also were not significantly altered in Alzheimer disease compared to control membrane samples. However, muscarinic agonist-stimulated GTP gamma S binding and GTP hydrolysis were significantly reduced (80-100%) in Alzheimer disease cortical samples. Diminished agonist-stimulated GTP gamma S binding and GTP hydrolysis correlated with the loss of guanine nucleotide-sensitive, high-affinity agonist binding (KL/KH) ratio) to the M1 receptor subtype. These data provide further evidence for the loss of muscarinic receptor-G protein coupling in Alzheimer disease and support the hypothesis that muscarinic receptor-mediated cortical activation may be compromised in Alzheimer disease.

Selective patterns of expression of G protein alpha subunits during in vitro development of hypothalamic neurons.

Expression of different alpha subunits of G proteins was studied in hypothalamic primary cultures grown in defined medium and enriched in either neurons or glial (astrocyte) cells. In parallel, the cellular distribution of Gi, Gs, and GoA subunits was visualized by in situ hybridization. Immunoblots using specific antisera and hybridization of mRNAs with specific oligonucleotide probes allowed us to characterize Gs, Gi2, and GoA as major neuronal G proteins in the hypothalamus, whereas the glial cells expressed mostly Gs, Gi2, and GoB forms. Gi was found to be expressed very early and transiently in the culture, whereas expression of Gs and GoA increased regularly with time.

Cloning and pharmacologic characterization of a thromboxane A2 receptor from K562 (human chronic myelogenous leukemia) cells.

Pharmacologic and molecular evidence conflicts in regard to the existence of tissue-specific subtypes of thromboxane A2 receptors (TXR). The full length TXR complementary DNA (cDNA) was cloned from a platelet-like cell line. It was expressed and its pharmacology was characterized. Northern analysis of TXR transcripts in multiple tissues showed strong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDNA (K562TXR) was isolated. K562TXR encodes a protein identical to the previously characterized placenta TXR cDNA, except for a single amino acid substitution (Glu21-->Lys). Similar to thromboxane receptors on K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/293) bound the thromboxane agonist 125I-labeled [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5- heptenoic acid ([125I]BOP) with a Kd of 5.5 +/- 1.1 nM, a Bmax of 289,056 +/- 60,220 sites/cell and a Hill coefficient of -0.94 +/- 0.01 (n = 6). K562TXR/293 cells also demonstrated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid. In contrast to the single [125I]BOP binding site observed in K562TXR/293, [125I]BOP binding to placental membranes resulted in a Hill coefficient significantly less than unity with a statistically superior two-site model for binding [KdH 0.63 +/- 0.18 nM and KdL of 12.5 +/- 5.0 nM, with Bmaxs of 29 +/- 9 and 212 +/- 41 fmol/mg of protein, respectively (n = 7)].(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of phosphoinositide-specific phospholipase C-beta 1 and GTP-binding protein, Gq alpha, in bovine iris sphincter membranes: characteristics of the phospholipase and its coupling to cholinergic muscarinic receptors.

Previously, we have established that treatment of iris sphincter smooth muscle with carbachol (CCh) results in increased phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) and in muscle contraction. To throw more light on the mechanism of muscarinic stimulation of PLC in this tissue we have investigated the properties of this enzyme and its regulation by GTP analogs and protein phosphorylation in bovine iris sphincter membranes. The data obtained can be summarized as follows: (1) the presence of PLC-beta 1 and a GTP-binding protein, Gq alpha, was detected in the microsomal (membrane) fraction by anti-PLC beta 1 and anti-Gq alpha antibodies, respectively. The membrane PLC hydrolysed exogenously added PIP2 and this hydrolysis was increased dose-dependently by Ca2+ (1-10 microM) but the enzyme activity was inhibited by Mg2+. (2) Addition of guanosine 5'-O-thiotriphosphate (GTP gamma S, 0.1 microM) to the membrane fraction increased PIP2 hydrolysis by 30%, whereas addition of CCh (10 microM) was without effect. However, when added together, CCh and GTP gamma S increased PIP2 hydrolysis by 46%. This effect was significantly inhibited by atropine and by the anti-PLC-beta 1 and anti-Gq alpha antibodies. (3) Removal of PLC-beta 1 from the membranes with 2 M KCl resulted in a significant reduction of the CCh-induced PIP2 hydrolysis, and this effect of the muscarinic agonist was restored when the membrane fraction was supplemented with PLC-beta 1 purified from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of muscarinic receptors to GTP proteins in postmortem human brain--alterations in Alzheimer's disease.

The coupling of muscarinic agonist receptors to guanine nucleotide-binding (G) proteins was investigated in the frontal, temporal cortices and thalamus of control and Alzheimer brains by using carbachol in competition experiments with [3H]QNB. In the presence of GppNHp, the carbachol/[3H]QNB competition binding data showed a 6-fold increase in the high-affinity muscarinic agonist coefficient (Ki high) in the thalami of control brains and a significantly increased proportion of low-affinity agonist binding sites (Bmax low) in the temporal cortices of control brains, while no significant effect of GppNHp was observed in Alzheimer brains. The results suggest a disturbance of the muscarinic receptor-G protein coupling in Alzheimer's disease.

Detection of small GTP-binding proteins in the outer envelope membrane of pea chloroplasts.

We found small GTP-binding proteins in the outer envelope membrane of pea chloroplasts. The proteins in this membrane were separated by SDS-PAGE, transferred to a nitrocellulose filter, and incubated with [alpha-32P]GTP. Three GTP-binding proteins with the molecular weight of 24,000 were found. Binding was prevented by 10(-8)-10(-7) M GTP or by 10(-7) M guanosine 5'-[gamma-thio]triphosphate or GDP; binding was unaffected by 10(-8)-10(-6) M ATP. Thermolysin treatment of intact chloroplasts resulted in the loss of GTP-binding activity, suggesting that these proteins were in the cytosolic side of the outer envelope membrane.