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M Camps

Publications and source records attributed to M Camps.

87 records · Page 5Linked to original sources
PubMed1991

Failure of [32P]ADP-ribosylation by pertussis toxin to determine Gi alpha content in membranes from various human tissues. Improved radioimmunological quantification using the 125I-labelled C-terminal decapeptide of retinal transducin.

The quantitative determination of pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G-proteins) in cell membranes is still a problem. Pertussis-toxin-catalysed [32P]ADP-ribosylation strongly relies on the substrate quality of the alpha-subunits and is influenced by the concentration of nucleotides, beta gamma-subunits, the physicochemical properties of the membranes influencing the availability of Gi alpha for pertussis toxin, and covalent modification of Gi alpha. Quantification of immunoreactive material on Western blots can be only imprecisely performed by two-dimensional densitometry. In order to generate a method for quantification of pertussis-toxin-sensitive G-proteins in membranes we have developed a fast and sensitive radioimmunoassay. The C-terminal decapeptide of retinal transducin alpha (KENLKDCGLF) was 125I-labelled and used as tracer. Polyclonal antiserum (DS 4) was raised against this peptide. Gi alpha proteins were determined by competition of solubilized membranes for 125I-KENLKDCGLF binding to DS 4 using dilutions of retinal transducin alpha as standard. The interassay variation was less than 10%, with a sensitivity of 2.5 micrograms/ml. The density of Gi alpha was highest in human adipose tissue, followed by HL60 cells, lung, mononuclear leucocytes, thrombocytes and left ventricular myocardium. A striking difference was observed between the density of Gi alpha and the amount of incorporation of [32P]ADP-ribose into the 40 kDa membrane proteins by pertussis toxin in the same samples. This is also demonstrated by comparison of the weak [32P]ATP-ribosylation of pertussis toxin substrates with the density of immunoreactive Gi alpha on Western blots in tissues such as lung. This study shows that the Gi alpha content can be exactly determined by a sensitive and fast radioimmunoassay using iodinated synthetic peptide homologues of Gi alpha proteins. Radioimmunological quantification of Gi alpha might be able to detect the 'true' Gi alpha content of membranes without being hampered by influences on the [32P]ADP-ribosylation reaction. It is concluded that this newly developed method may become an important tool for studying expression of Gi alpha proteins in a variety of tissues or cell types, and for precisely quantifying the changes caused by pathological conditions.

Adenosine Diphosphate Ribose↗
PubMed1990

Guanosine 5'-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate in HL-60 granulocytes. Evidence that the guanine nucleotide acts by relieving phospholipase C from an inhibitory constraint.

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-[beta-thio]diphosphate. ATP, adenosine 5'-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

Cytosol↗
PubMed1990

Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level.

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Autoradiographic localization of dopamine D 1 and D 2 receptors in the brain of several mammalian species.

Dopamine D 1 and D 2 receptor distributions were studied in the brain of the mouse, rat, guinea pig, cat and monkey by means of in vitro quantitative autoradiography using [3H]SCH 23390 and [3H]CV 205-502 to label D 1 and D 2 subtypes respectively. The distribution of both subtypes of receptors was similar within the basal ganglia of all species investigated. The highest densities for both subtypes were found in the nucleus caudatus, putamen, nucleus accumbens, olfactory tubercle and substantia nigra. Outside of the basal ganglia, differences in the distribution of both receptors were found among the species examined in regions such as cerebellum, cortex, hippocampus, superior colliculus and olfactory bulb. In all species D 1 receptor densities were higher than those of D 2. The absolute amount of both subtypes, however, varied among species. These results indicate that dopamine receptor distribution is well preserved in the basal ganglia during evolution, although differences among species exist in their distribution outside the basal ganglia and their absolute amount.

Aminoquinolines↗
PubMed1990

Insulin resistance of skeletal muscle during pregnancy is not a consequence of intrinsic modifications of insulin receptor binding or kinase activities.

Insulin action in skeletal muscle is markedly depressed at late pregnancy. The purpose of this study was to investigate whether insulin resistance of skeletal muscle during pregnancy is associated to intrinsic alterations in the biological activities of insulin receptor. To that end, insulin receptors from mixed, red and white skeletal muscle from control and 19-20 days pregnant rats were partially purified and insulin binding and tyrosine kinase activities were evaluated. Muscle insulin receptors from diabetic rats were also studied provided that changes in receptor number and tyrosine kinase activities had been clearly substantiated. Total high affinity insulin binding sites expressed either per gram of tissue or per milligram of protein were similar in muscles from control and pregnant rats, in contrast to diabetic rats in which an increased high affinity receptor number was observed. No differences in affinity were detected for high affinity binding sites in any of the groups investigated. The integrity of the partially purified insulin receptors from control and pregnant groups was identical as determined by affinity cross-linking of [125I-TyrB26]insulin to the receptor and by beta-subunit phosphorylation. Autophosphorylation of the beta-subunit and the pattern of phosphopeptides obtained after digestion of phosphorylated beta-subunit with trypsin, elastase, and staphylococcal V8 protease were indistinguishable in control and pregnant groups. Tyrosine receptor kinase was also similar in receptor preparations from control and pregnant muscle. This is in contrast to diabetes in which a defective tyrosine kinase was confirmed. In order to detect possible differences due to the fiber type, further sets of experiments were performed in receptor preparations from red and white muscle. In keeping with previous data, tyrosine kinase activity of the insulin receptor was 2.5-fold greater in red muscle than white muscle; however, under these conditions, receptor kinase activity was unmodified in preparations from pregnant rats in red and white muscle fibers. Recent evidence has revealed the existence of an insulin binding inhibitor in muscle extracts. We detected the presence of such an inhibitor in the flow-through fraction after WGA chromatography. This inhibitory activity was found to be greater in muscle extracts obtained from pregnant rats as compared to fractions from control rats. We conclude that insulin resistance of skeletal muscle at late pregnancy is not explained by intrinsic modifications of insulin receptor binding or kinase activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Dopamine receptors in human brain: autoradiographic distribution of D1 and D2 sites in Parkinson syndrome of different etiology.

The distribution and density of dopamine D1 and D2 receptors were examined by autoradiography in postmortem brain tissue from patients with pathological diagnosis of Parkinson's disease, status lacunaris, clinical parkinsonism without neuropathological lesions and in age-matched controls. The D1 antagonist [3H]SCH 23390 and the D2 agonist [3H]CV 205-502 were used as ligands. No significant differences in the distribution or density of D1 or D2 receptors were found in Parkinson's disease in the areas examined, including the nucleus caudatus, putamen, globus pallidus and substantia nigra. In contrast, cases presenting lacunar lesions in the striatum showed marked decreases in D1 and D2 receptor densities in this region. Patients clinically diagnosed as parkinsonians but without Parkinson's disease lesions or striatal lacunar softenings showed reduced densities of D2 receptors in the nucleus caudatus and putamen, while in the substantia nigra the densities were comparable to controls. In the basal ganglia of these cases D1 receptors were slightly decreased.

Aged↗
PubMed1989

Acute effects of 1-methyl-1,4-phenylpyridinium ion (MPP+) on purine metabolism in rat striatum studied in vivo using the microdialysis technique.

In order to obtain further insight into the interactions between the purinergic and dopaminergic pathways in the striatum, we studied both metabolisms simultaneously, using a microdialysis technique in 1-methyl-1,4-phenylpyridinium ion (MPP+) unilaterally-denervated conscious rats. In these rats the contralateral side was used as control. The perfusates were collected every 20-25 min using 4 mm dialysis probes, implanted in each striatum, and assayed for dopamine and purine metabolites. After MPP+ administration, all adenosine metabolites - with the exception of uric acid - and dopamine levels were significantly increased in the extracellular medium. However, the time-course change in dopamine level did not correlate with the adenosine and inosine time-courses, suggesting a different mechanism of liberation in response to MPP+ administration.

1-Methyl-4-phenylpyridinium↗
PubMed1989

Dopamine receptors in human brain: autoradiographic distribution of D2 sites.

We have studied the detailed anatomical distribution of D2 receptors in human post mortem brain tissue using quantitative autoradiographic techniques. D2 receptors were labeled using the specific D2 agonist [3H]CV 205-502 and the antagonist [3H]spiroperidol. The pattern of D2 receptor distribution observed with the two ligands was very similar. The highest densities were found in the nucleus caudatus, putamen, nucleus accumbens and olfactory tubercle followed by the substantia nigra, where D2 receptors were mainly concentrated in the pars compacta. Lower but still significant densities were associated with the lateral part of the globus pallidus and CA1 and CA3 fields of the hippocampus. The medial part of the globus pallidus, the dentate gyrus and the amygdala showed low to very low densities of D2 receptors. Almost negligible amounts of binding were observed in the olfactory bulb, diencephalon, brainstem, cerebellum and most parts of the neocortex. Our results are comparable with previously reported localizations of D2 receptors in the human and rat brain. We also report the lack of the so-called spirodecanone binding sites in the human brain. The localization of D2 receptors is compared with the distribution of D1 receptors.

Aging↗
PubMed1989

Dopamine D1 and D2 receptors visualized in MPTP treated C57 mice by in vitro autoradiography: lack of evidence of receptor modifications in parkinsonian mice.

The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on D1 and D2 dopamine receptors was assayed using in vitro quantitative autoradiography. D1 receptor subtype was labeled using 1 nM of 3H-SCH 23390 and D2 receptor subtype was labeled using 0.4 nM of 3H-spiroperidol. The results are compared to the effect of MPTP on the striatal levels of dopamine and its metabolites, in BL C57 mice. While 2 and 5 doses of MPTP 30 mg/kg/day intraperitoneally reduced the content of striatal dopamine and its metabolites, no modifications were detected in D2 receptor subtype in any cerebral area studied. However, D1 receptors were reduced in the substantia nigra 24 hr after the last of 2 doses, but not later. We suggest a compensatory mechanism of the surviving dopaminergic neurones as well as the participation of spare receptors. This would explain the lack of receptor modification after the lesion obtained as seen by the striatal reduction of dopamine and metabolites content, after MPTP administration.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1988

In vivo labeling of brain dopamine D2 receptors using the high-affinity specific D2 agonist [3H]CV 205-502.

The high-affinity selective dopamine D2 agonist [3H]CV 205-502 was utilized to in vivo label brain dopamine D2 receptors in the rat. Intravenous administration of [3H]CV 205-502 resulted in a selective accumulation of radioactivity in the striatum and in the pituitary. Smaller amounts of binding were found in the hypothalamus and cortex and non-significant binding was seen in the cerebellum. The binding of [3H]CV 205-502 was stereospecifically blocked by (+)-butaclamol but not by (-)-butaclamol. In vivo binding of [3H]CV 205-502 was also dose-dependently blocked by other neuroleptics including sulpiride, haloperidol and spiroperidol, dopamine agonists such as bromocriptin, apomorphine and C1 201-678 but not by dopamine D1 antagonists or serotonin-2 antagonists. The regional distribution of the sites labeled in vivo by [3H]CV 205-502 was investigated by autoradiography and compared with the distribution seen after in vitro labeling in the consecutive sections, following washing of the label. The autoradiograms reveal the labeling of the same areas seen when in vitro autoradiography was used. High densities of binding were localized in the nucleus accumbens, striatum and olfactory tubercle as well as in the olfactory bulb. Lower densities were seen in the substantia nigra pars compacta and in the ventral tegmental area as well as in the stratum lacunosum molecular of the hippocampus and in the superior colliculus. The intermediate lobe of the pituitary also presented high densities while in the anterior pituitary only intermediate densities of receptor binding were observed. These results demonstrate that [3H]CV 205-502 is the first high-affinity agonist useful for the autoradiographic visualization of dopamine D2 receptors after in vivo labeling. This compound could be modified for utilization in positron emission tomography imaging of dopamine D2 receptors in the living animal.

Aminoquinolines↗
PubMed1988

Mapping dopamine receptors in the human brain.

We have investigated the anatomic localization of dopamine D1 and D2 receptors in the human brain using selective high affinity ligands for both types of dopamine receptors and the technique of receptor autoradiography. Dopamine D1 receptors were labeled in postmortem human brain tissue sections using the antagonist [3H]SCH 23390. Dopamine D2 receptors were labeled in consecutive tissue sections using the agonist [3H]205-502 and the antagonist [3H]spiroperidol. D1 and D2 dopamine receptors presented a heterogeneous distribution in the human brain. The highest concentrations of both D1 and D2 receptors were found in parts of the basal ganglia, particularly the nucleus caudatus and putamen. Lower concentrations were seen in other areas for example, the lateral globus pallidus was enriched in D2 receptors and the medial globus pallidus in D1 receptors. The substantia nigra contained intermediate densities of both D1 and D2, D1 receptors being present in higher concentrations. Dopamine D1 receptors were also localized in areas outside of the basal ganglia, particularly in the neocortex, amygdala and hippocampal formation. Dopamine D2 receptors were also present in areas outside of the basal ganglia, the most significant densities being found in the hippocampal formation. We observed a marked age-dependent decline in the density of D1 receptors during the first decades of life. In contrast, D2 receptor concentrations appeared to be unaltered with age. The distribution and densities of dopamine receptors were examined in 12 cases of Parkinson's disease and compared to a control adult population. No significant differences in density and distribution were seen for either D1 nor D2 receptors.

Adolescent↗
PubMed1987

Autoradiographic localization of dopamine D2 receptors in the rat brain using the new agonist [3H]N-0437.

The selective dopamine D2 agonist [3H]N-0437 was used to label dopamine receptors in vitro in slide-mounted rat brain microtome sections. The characteristics of the binding of [3H]N-0437 to tissue section were similar to those observed previously in membrane preparations and indicated that this ligand labels sites with the properties of a dopamine D2 receptor. The regional distribution of these receptors was examined by autoradiography and quantified by computer-assisted microdensitometry. The highest densities of [3H]N-0437 sites were observed in the nucleus caudate-putamen, accumbens, olfactory tubercle, island of Calleja and the glomerular layer of the olfactory bulb. Lower densities of binding sites were seen in stratum griseum superficialis of the superior colliculus, substantia nigra pars compacta and area ventral tegmental, entorhinal cortex and in the molecular layer of the 9th and 10th lobules of the cerebellum. Very low densities were seen in the neocortex and hippocampal formation. The density of [3H]N-0437 binding sites in the rat striatum are higher than those observed with other dopamine D2 [3H]agonists and comparable to those seen with [3H]antagonists. [3H]N-0437 is a new useful tool for the anatomical localization of dopamine D2 receptors in brain.

Animals↗
PubMed1983

Embedding iliac bone biopsies at low temperature using glycol and methyl methacrylates.

A mixture of pure and anhydrous glycol methacrylate and methyl methacrylate is used as an embedding medium for iliac bone biopsies. Infiltration is carried out at -20 C with the embedding medium and a cold inactivated catalyst-initiator system. Raising the temperature to 4 C initiates polymerization and limits the peak temperature of polymerization to 25 C. In this way, such thermolabile enzymes as osteoclastic acid phosphatase are preserved. After staining, sections are dehydrated in polyethylene glycol 400 30% in 2-propanol. This gives flat sections and improves staining properties.

Acrylates↗
PubMed1982

A simple and reliable method for purifying glycol methacrylate for histopathological studies.

Pure glycol methacrylate (GMA) is an excellent embedding medium which allows High Performance Optical Microscopic studies. However presence of methacrylic acid (MA) small amounts (usually 1 to 3%) leads to intense background staining of the section. A simple and reliable chemical purification procedure is exposed which leads to an extra-pure GMA free of MA and hydroquinone.

Acrylates↗