Öie Nordström

Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence.

The role of the neuropeptide galanin (GAL) in rat hypothalamus has been studied in different experimental models. Thus, the effect of GAL on potassium-induced dopamine release was analyzed in vitro, and the localization of GAL and GAL binding sites was studied with immunohistochemistry and receptor autoradiography, respectively. In the median eminence GAL and presumably dopamine were found to coexist in nerve endings and this area contained a high density of 125I-GAL binding sites. In vitro experiments revealed that GAL inhibited the release of [3H]dopamine in a dose-dependent manner (IC50 = 7-10 nM), possibly via a presynaptic receptor.

VIP-sensitive adenylate cyclase, guanylate cyclase, muscarinic receptors, choline acetyltransferase and acetylcholinesterase, in brain tissue afflicted by alzheimer's disease/senile dementia of the Alzheimer type

Biochemical parameters were determined in autopsy material from several brain regions of thirteen patients with Alzheimers disease/senile dementia of Alzheimer type (AD/SDAT) (mean age 75 years) and from brains of ten age-matched controls (mean age 76 years). Choline acetyltransferase specific activity was significantly lower in the nucleus caudatus, putamen, left thalamus, hippocampus and the cortex from gyrus hippocampus and the temporal lobe in AD/SDAT, acetylcholinesterase specific activity was significantly lower in the hippocampus, parietal and left frontal lobe in AD/SDAT samples than in corresponding samples from aged-matched controls. A compensatory increase of muscarinic receptors was found in the nucleus caudatus and left frontal lobe samples in AD/SDAT. Guanylate cyclase activity was not significantly altered in AD/SDAT in the brain regions examined. The basal, non-stimulated activity of adenylate cyclase was significantly (p less than 0.05) elevated in hippocampus samples from AD/SDAT patients and the enzyme activity stimulated by the vasoactive intestinal polypeptide VIP (2 microM) or forskolin (10 microM) was also elevated in AD/SDAT although not significantly.

In vivo andin vitro studies on the potentiation of muscarinic receptor stimulation by alaproclate, a selective 5-HT uptake blocker

Alaproclate (10–60 mg/kg) injected i.p. into male mice potentiated and prolonged the oxotremorine and physostigmine-induced tremor in a dosedependent manner. Atropine completely blocked the tremor caused by oxotremorine or physostigmine both in the presence and absence of alaproclate. Pretreatment with the 5-HT receptor antagonist metitepine completely blocked the enhancement of oxotremorine-induced tremor caused by alaproclate. Biochemical studies indicated that the above effects cannot be explained by assuming that alaproclate a) acts as a cholinergic agonist, b) inhibits the acetylcholine esterase, c) interferes with choline uptake or acetylcholine synthesis, or d) directly potentiates the release of acetylcholine. In ligand binding studies alaproclate was found to be a weak competitive inhibitor of muscarinic antagonist binding to membranes from the rat cerebral cortex, rat striatum, human cerebral cortex and human striatum. (Ki∼28–40μM in all four tissues. The present results suggest that alaproclate may potentiate muscarinic responses by a mechanism involving serotonergic receptor mechanisms rather than by a direct interaction with the muscarinic cholinergic receptors.

Muscarinic receptors and receptor-mediated actions on rat thymocytes

Rat thymocytes possess a single class of saturable, high affinity binding sites for muscarinic antagonists of the benzilate type such as [3H]3-quinuclidinyl benzilate ([3H]3-QNB). The average number of receptors per cell is 3000 and the equilibrium dissociation constant of [3H]3-QNB on intact cells is 7.5 nM. In the work reported here we found that perturbation of the thymocyte membrane by addition of phytohemagglutinin (4 micrograms/ml) caused a transient increase in muscarinic antagonist binding, and hydrocortisone (100 mg/kg s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of muscarinic receptor sites on thymocytes by 100%. Atropine treatment, which in other tissues causes increased muscarinic receptor concentration, did not alter the receptor number on thymocytes. Binding of carbachol to the receptor on intact cells resulted in inhibition of cAMP synthesis and stimulation of cGMP synthesis. These muscarinic agonist effects were each inhibited by the simultaneous addition of the muscarinic antagonist atropine (5 X 10(-5) M). No stimulation of phosphatidylinositol turnover by muscarinic agonists was observed.

On the Ionic Mechanism of Presynaptic Muscarinic Receptor Action in Rat Hippocampus

Occupancy of the muscarinic acetylcholine receptor (mAChR) with an agonist may trigger a number of different responses dependent on the localization of the receptor. The mAChR mediates depolarizing actions in Renshaw cells (10–12), in sympathetic ganglion (13) and hyperpolarizing actions in the heart (21). It activates synthesis of cGMP in a number of tissues (1) and inhibits the β-receptor activated adenylate cyclase in heart (25). Some of these actions take place on the presynaptic side and some on the postsynaptic side of the synapse.

MUSCARINIC ACETYLCHOLINE RECEPTOR

ABSTRACT Muscarinic acetylcholine receptors from rat cerebral cortex and small intestine were studied by the high affinity and high specific activity antagonists 3 H-3-quinuclidinyl benzilate (3-QNB) (29.4 Ci/mmol) and 3 H-N-methyl-4-piperidinyl benzilate (4-NMPB) (55.4 Ci/mmol). Equilibrium binding data combined with studies on association and dissociation rates of the receptor antagonist complex are compatible with the reaction sequence: A + R ⇆ AR ⇆ AR * where R and A stand for receptor and antagonist, respectively. For both antagonists of benzilate class the equilibrium constant of the isomerization step was 0.15 in phosphate buffer (Jarv and co-workers, 1979). The results of agonist binding studies carried out under equilibrium conditions can be described as if there were two classes of agonist binding sites with affinities 100 μM and 0.05 μM for carbamylcholine (Hedlund and Bartfai, 1979). It appears that it is the low affinity binding site that is associated with muscarinic activation of cyclic GMP synthesis in cortical slices and in N1E 115 neuroblastoma cells. In the latter the low affinity muscarinic receptor controls a non-voltage dependent Ca 2+ channel (Study and co-workers, 1978) . Calcium channel blocking agents such as D600 and Co 2+ inhibit the receptor mediated rise in cyclic GMP levels. Experiments with the cardiotoxin V II 2 from N. m. mossambica (supplied by Dr. L. Visser) indicate that the cardiotoxin and D600 may share some common site on the receptor-coupled Ca 2+ channel, or on the muscarinic receptor itself.

Increased affinity of choline acetyltransferase for choline in Alzheimer's disease: a steady-state kinetic study

The steady-state kinetics of choline acetyltransferase (CAT) from autopsy samples of human caudate nucleus of aged controls and of patients with Alzheimers disease was studied. In 10 samples from Alzheimers disease-afflicted brains the affinity for the limiting substrate choline (Ch) was significantly higher: Michaelis constant KmCh was for these samples 1.93 +/- 0.72 mM while in the samples from 9 age-matched controls KmCh was 2.53 +/- 0.78 mM. The difference is statistically significant (P less than 0.05). Endogenous choline concentrations in the samples were 124 +/- 39 (n = 10) nmol/g wet wt. in the Alzheimers disease-afflicted samples and 180 +/- 57 (n = 9) nmol/g wet weight (n = 9) in the control samples (P less than 0.05). The initial velocity at 70 microM acetyl co-enzyme (AcCoA) in Alzheimers samples was 171.5 +/- 131.0 pmol [14C]acetyl choline [14C]ACh/mg protein/min as compared to the controls 422.1 +/- 231.0 pmol [14C]ACh/mg protein/min replicating many previous findings about decline of CAT activity in Alzheimers disease. However, in the same samples the affinity for the other substrate acetyl-CoA (AcCoA) was significantly lower for the Alzheimer patients, KmAcCoA = 61 +/- 40 microM, than for the age-matched control patients, KmAcCoA = 28 +/- 8 microns (P less than 0.01). The data suggest some compensation of the loss of enzyme molecules via changed affinity for the limiting substrate, Ch.