Dissociation between I2-imidazoline receptors and MAO-B activity in platelets of patients with Alzheimer's type dementia.

The I2-imidazoline receptor is expressed in brain and platelets and could represent a new binding domain on MAO-B enzyme. Brain I2-imidazoline receptors and MAO-B sites have been found to be increased in Alzheimer's disease. The study sought to evaluate I2-imidazoline receptors and MAO-B activity in platelets from patients with Alzheimer's type dementia (ATD) and matched controls. Preliminary saturation experiments of [3H]idazoxan binding to platelet purified mitochondrial membranes were performed to determine the maximal number of binding sites (Bmax) and the apparent dissociation constant (Kd). Afterwards, the I2-imidazoline receptor density ([3H]idazoxan at 8 and 20 nM in the presence of 2 x 10(-6) M efaroxan) was evaluated in 20 patients with ATD and 17 controls. MAO-B activity was quantified by [14C]PEA oxidation. All subjects were screened for cognitive evaluation by the Mini-Mental State Examination. The density of I2-imidazoline receptors was similar in ATD patients (8.4 and 14.3 fmol/mg protein) and controls (8.3 and 14.0 fmol/mg protein). MAO-B activity was 22% higher in ATD subjects. Significant correlations between I2-imidazoline receptors and MAO-B activity were observed. No relationships between I2-imidazoline receptors or MAO-B activity and the cognitive score were observed. In conclusion, platelet I2-imidazoline receptors do not show the increase of I2-imidazoline receptors previously observed in brain of subjects with ATD. The dissociation between I2-imidazoline receptors and MAO-B in platelets suggests that the enzyme contributes to but not exclusively represents the I2-imidazoline receptor.

Guanine nucleotide-specific phosphate transfer by guanine nucleotide-binding regulatory protein beta-subunits. Characterization of the phosphorylated amino acid.

One major substrate protein was phosphorylated with [gamma-32P]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigrated with beta-subunits of heterotrimeric GTP-binding proteins (G proteins) in different gel systems. Upon solubilization of the phosphorylated membranes, the phosphoprotein could be immunoprecipitated by a G protein beta-subunit-specific antiserum. The beta-subunit phosphorylation was transient and was found to be specific for GTP and its analog, guanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was specifically removed by GDP, suggesting that the phosphate can be retransferred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were required for both phosphorylation and dephosphorylation. The phosphorylation was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membranes with the histidine-modifying agent, diethyl pyrocarbonate, resulted in a loss in phosphate incorporation. The data suggest that G protein beta-subunits are involved in a guanine nucleotide-specific enzymatic activity transferring the gamma-phosphate from GTP to GDP, presumably at G protein alpha-subunits, via a phosphohistidine intermediate.

[Nutrition team. Units of nutritional support]. / Equipo de nutrición. Unidades de soporte nutricional.

During the VIII National Congress of the Spanish Society of Parenteral and Enteral Nutrition (SENPE) held in Santander on 5th, 6th and 7th of June last, a subject was raised among several others, which for us was of great current interest and not often found in scientific affairs, related to the organizational affairs of the discipline of Artificial Nutrition, namely the Nutritional Teams or Nutritional Support Units. The aim was to respond to the many problems raised by this discipline: What does it consist of? Is it necessary? What is its purpose? Who is involved in it? What qualifications must these people have? Does it enter into competition with Nutritional, Clinical and Dietetic Services? To reply to these and many other questions, we invited a number of professionals with wide experience in nutritional and other fields, in an attempt to form a group of experts in different specialties with interests in the subject. We were also lucky enough to be able to invite Doctor Rombeau, an internationally-recognized expert, in whose country there exists great experience in the organization of these units. This summary of our Round Table was prepared by the organizer, Doctor Ordóñez, and an attempt was made to respect the spirit of each author's contribution.

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits.

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of recombinant rho A GTP-binding proteins with photoexcited rhodopsin.

The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP[S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.

Interaction of small G proteins with photoexcited rhodopsin.

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.

Effect of ICV pertussis toxin and N-ethylmaleimide on the levels of substance P and serotonin in brain and spinal cord of the rat.

The effects of single intracerebroventricular (icv) injections of either 0.5 microgram pertussis toxin or 5 micrograms N-ethylmaleimide (NEM) on the levels of immunoreactive substance P (ir-SP) and serotonin (5-HT) in the brain and spinal cord of rats have been assessed. At two and six days after pertussis toxin injection, the levels of ir-SP appeared significantly diminished in the spinal cord (about 34%). This reduction was even greater at two days after NEM injection (43%). These two agents did not alter the ir-SP of the midbrain and thalamus, whereas NEM increased the neuropeptide content in the pons-medulla. On the other hand, the thalamic content of serotonin was reduced two days after pertussis toxin (32%) or NEM (20%) injection. The indoleamine levels of the spinal cord were reduced by these treatments (20%), while in the midbrain a slight decrease could be observed. These findings suggest that pertussis toxin and NEM produce these effects by acting upon a common neural substrate.

Intracerebroventricular N-ethylmaleimide differentially reduces supraspinal opioid analgesia in mice.

I.c.v. injection of 1 nmol N-ethylmaleimide (NEM) into mice interfered with opioid-induced supraspinal analgesia, as assessed in the warm water tail-flick test. This effect of NEM was long-lasting (more than 3 days), non-competitive and differentially inhibited by the opioids studied. The analgesia induced by [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2,Met5]enkephalinamide (DAME) and [D-Pen2,D-Pen5]enkephalin (DPDPE) was greatly reduced in NEM-treated mice. The antinociception elicited by [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) was also impaired although to a lesser extent. In contrast, the activity of morphine and etorphine remained unchanged. NEM-sensitive opioids coadministered with morphine antagonized the analgesia elicited by the alkaloid in NEM-treated mice. The administration of naltrexone or DADLE, DAGO, [D-Ala2,N-MePhe4,Met-(O)5-ol]enkephalin (FK-33824) and morphine in doses equivalent to the ED90 doses for inducing analgesia, a few minutes before NEM prevented it from interfering with DADLE-elicited supraspinal analgesia when evaluated 24 h later. In contrast, the selective delta antagonist, ICI 174864, did not protect the DADLE-induced analgesia against the effect of NEM. We suggest that NEM produced its effect by acting upon a site that appears to be distal to the receptor binding site, presumably located on the guanine nucleotide binding regulatory proteins, Gi/Go. Therefore, these transducer proteins might play a key role in the effects displayed by opioids when acting via the mu receptor-Gi/Go complexes.

Chronic naltrexone suppresses platelet aggregation induced by adrenaline and 5-hydroxytryptamine in former heroin addicts.

Naltrexone, an opioid receptor antagonist, is used as an adjunct in the treatment of opiate addiction. In former heroin addicts, long-term treatment with naltrexone (350 mg/week for 5 months) resulted in suppression of adrenaline and 5-hydroxytryptamine (5-HT)-induced platelet aggregation. The results demonstrate that sustained blockade of opioid receptors can impair the functional expression of alpha 2-adrenoceptors and 5-HT2 receptors in human platelets. These findings may have negative clinical implications in the treatment of opiate addiction with naltrexone.

Uncoupling of the platelet alpha 2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity.

Preincubation of platelet-rich plasma with N-ethylmaleimide (NEM) attenuated the inhibitory effect of the alpha 2-adrenoceptor agonist UK 14304 on basal and forskolin-stimulated adenylate cyclase activities. NEM also led to concomitant marked reductions of the specific binding of [3H]UK 14304 to platelet membranes and of the primary aggregation response induced by UK 14304. These results indicate that uncoupling of the receptor adenylate cyclase system by NEM induces down-regulation of platelet alpha 2-adrenoceptor density (3H-agonist binding sites) and of the associated functional response (platelet aggregation).

Modulation of brain alpha 2-adrenoceptor and mu-opioid receptor densities during morphine dependence and spontaneous withdrawal in rats.

The densities of brain alpha 2-adrenoceptors and mu-opioid receptors, quantitated by means of the binding of the agonists [3H]clonidine and [3H]dihydromorphine, respectively, were studied during the development of morphine dependence and spontaneous withdrawal in the rat. The oral administration of morphine (12-130 mg/kg for 3-21 days) led to inconsistent changes in alpha 2-adrenoceptor density while the density of mu-opioid receptors was down-regulated. In contrast, spontaneous opiate withdrawal (3-72 h) significantly increased the density of alpha 2-adrenoceptors while the density of mu-opioid receptors was rapidly up-regulated to control values. In the hypothalamus, but not in other brain regions, the increase in alpha 2-adrenoceptor density after withdrawal followed a time course (3-72 h) related to the severity of the abstinence syndrome. Thus, there was a positive and significant correlation between the severity of withdrawal and the density of alpha 2-adrenoceptors in the hypothalamus. Short-term treatment with clonidine (2 x 0.5 mg/kg, i.p.) prevented the morphine withdrawal-induced increases in alpha 2-adrenoceptor density in various brain regions, but not in the hypothalamus. The main results suggest that modulation of hypothalamic alpha 2-adrenoceptor density during morphine withdrawal is a relevant physiological mechanism by which the opiate abstinence syndrome is counteracted.

Alpha 2-adrenoceptor-mediated inhibition of platelet adenylate cyclase activity in heroin addicts in abstinence.

The inhibition of basal and forskolin-stimulated platelet adenylate cyclase activity by (-)adrenaline was studied in ten heroin addicts during spontaneous withdrawal. Both basal and forskolin-stimulated enzyme activities were increased during heroin withdrawal and the inhibitory effect induced by (-)adrenaline was potentiated with parallel shifts to the left of the concentration-effect curves. The number of binding sites for [3H](-)adrenaline in platelet membranes was also increased during withdrawal. Treatment with clonidine markedly attenuated the inhibitory effect induced by (-)adrenaline on platelet adenylate cyclase activity. The results indicate that the heroin withdrawal syndrome induces supersensitivity of the platelet alpha 2-adrenoceptor-adenylate cyclase system.

Heroin increases the density and sensitivity of platelet alpha 2-adrenoceptors in human addicts.

The density of platelet alpha 2-adrenoceptors, quantified by means of the binding of [3H]clonidine, and the aggregation response induced by adrenaline, was investigated in ten heroin addicts. The number of binding sites for [3H]clonidine was significantly increased during heroin abuse. Concomitantly, there was a potentiation of the adrenaline-induced platelet aggregation, which suggested that continuous heroin use (opiate dependence) is associated with supersensitive platelet alpha 2-adrenoceptors in human addicts. Spontaneous heroin withdrawal further increased in the same addicts the density of platelet alpha 2-adrenoceptors. These results suggest that platelet alpha 2-adrenoceptors may be used as a model to study receptor mechanisms of opiate dependence and withdrawal in humans.

Platelet alpha 2-adrenoceptors in heroin addicts during withdrawal and after treatment with clonidine.

The density of platelet alpha 2-adrenoceptors, quantitated by means of the binding of [3H]clonidine and [3H]yohimbine, and the aggregation response induced by adrenaline were investigated in thirty-two heroin addicts during spontaneous withdrawal. The number of binding sites for [3H]clonidine, but not for [3H]yohimbine, was significantly increased during withdrawal and the increase followed a time course related to the severity of the abstinence syndrome. There was a positive and significant correlation between the severity of withdrawal and the density of platelet alpha 2-adrenoceptors. Concomitantly, the adrenaline-induced platelet aggregation was potentiated. Treatment with clonidine led to significant decreases in receptor densities as well as in functional responses. These results suggest that only alpha 2-adrenoceptors in the agonist state (i.e. number of binding sites for [3H]clonidine) are modulated during the development of the heroin withdrawal syndrome.