Sara G. Horowitz

Evidence for loss of brain [3H]spiroperidol and [3H]ADTN binding sites in rabbit brain with aging

[3H]Spiroperidol and [3H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [3H]spiroperidol binding sites and a 140% higher number of [3H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [3H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [3H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [3H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.

Striatal met-enkephalin concentration increases following nigrostriatal denervation

Following specific lesion of the nigrostriatal dopaminergic pathways in rat brain, striatal met-enkephalin on the lesioned side increased to 245% of that on the non-lesioned side. This increase was evident only after a lag period of 7 days and the increase was maintained for at least 2 months after lesion. By contrast, there was no change in striatal somatostatin or vasoactive intestinal polypeptide concentration, indicating that the effect was not a generalised one. Levels of all three of these neuropeptides were unchanged in frontal cortex. These findings support the concept of a dopaminergic-enkephalinergic functional interrelationship in the striatum. In addition, the findings provide evidence that, following destruction of nigrostriatal dopaminergic neurons, not only is there a gradually developing postsynaptic dopamine receptor supersensitivity but also a compensatory alteration in the enkephalinergic system.

Properties of dopamine agonist and antagonist binding sites in mammalian retina.

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.

Dopamine stimulates rat cortical somatostatin release

Release of somatostatin from slices of rat frontal cortex was studied. Increasing the potassium ion concentration in the medium from 6 mM to 55 mM resulted in a significantly increased release of somatostatin. Dopamine increased the release of somatostatin from cortex and this effect of dopamine was blocked by haloperidol and other dopaminergic antagonists. Other catecholamines as well as serotonin, histamine and acetylcholine failed to stimulate the release of somatostatin. The stimulatory effect of dopamine on release of somatostatin from cortical slices provides an approach for examination of the receptor properties and function of dopamine in this brain region.

3H-spiroperidol binding and dopamine-stimulated adenylate cyclase: Evidence for multiple classes of receptors in primate brain regions

Abstract Studies of displacement by agonist and antagonist drugs of 3H-spiroperidol binding in brain regions of Cebus and rhesus monkeys revealed one type of receptor in caudate nucleus and a second type of receptor in both frontal and anterior limbic cortex. Compared with caudate, the cortical regions were more sensitive to clozapine and loxapine, equally sensitive to fluphenazine and relatively less sensitive to haloperidol. Also, the cortical regions were insensitive to molindone. Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex. In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of 3H-spiroperidol receptors are coupled to adenylate cyclase.

Retina contains guanine nucleotide sensitive and insensitive classes of dopamine receptors

Calf retina contains high affinity, stereospecific and saturable binding sites, characterized as dopaminergic, for 3H-spiropheridol (Bmax from Scatchard analysis = 38 +/- 4 fmoles/mg protein) and for 3H-ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene). The 3H-ADTN sites include a discrete and saturable sub-set of sites sensitive to guanine nucleotide inhibition (Bmax = 50 +/- 6 fmoles/mg protein) and a separate saturable sub-set of sites insensitive to guanine nucleotides (Bmax = 63 +/- 7 fmoles/mg protein). The binding of agonists to 3H-spiroperidol sites is also inhibited by guanine nucleotides.

Heart conditioned medium promotes central cholinergic regeneration in vivo

The aim of the present study was to evaluate the effect of a proposed cholinergic growth factor, heart conditioned medium (HCM), on the regeneration of the cholinergic septo-hippocampal pathway in rats. At 0, 4, 8 and 16 days after mechanical injury to septo-hippocampal axons, animals received either repeated intraseptal injections of HCM or control vehicles. Recovery of choline acetyltransferase (CAT) activity in iris implants placed in the hippocampus was used as an index of cholinergic regeneration. The results indicate that CAT activity in iris implants from animals receiving HCM was significantly greater than in control implants. Thus, this is the first report of the in vivo activity of heart conditioned medium as a cholinergic growth factor.

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts

Abstract cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.

Biogenic Amine-Stimulated Adenylate Cyclase and Spiroperidol-Binding Sites in Rabbit Brain: Evidence for Selective Loss of Receptors with Aging

Evidence for selective decreases in biogenic amine receptor function with age in the rabbit has been obtained. Dopamine-stimulated adenylate cyclase activity in the striatum (caudate-putamen) of rabbit brain declined by about 50 percent as rabbits aged from less than 1 to 4 to 5 years of age. Similar decreases in transmitter-stimulated adenylate cyclase activity were found for histamine as well as for dopamine and norepinephrine in hypothalamus, frontal cortex and anterior limbic cortex. Isoproterenol-stimulated activity was also decreased with age in frontal cortex. These changes appeared to represent decreases in maximal response and not alteration in affinity for amine. In contrast, dopamine-stimulated adenylate cyclase of retina and transmitter-independent (basal or Gpp(NH)p-stimulated) activity in each of the regions studied were not altered with age. Dopamine receptors in striatum directly assessed by measurement of [3H]-spiroperidol binding revealed a comparable decrease in the number of binding sites without change in ligand affinity. Preliminary data also indicated decreased spiroperidol binding sites in the cortical regions of older animals. These changes in striatum and cortex were evident in the absence of decreases in either dopamine content or choline acetylase activity, an activity presumed to be present in neurons containing dopamine receptors. It is proposed that selective age-dependent decreases in postsynaptic biogenic amine receptor content occur in the absence of, or independent from, neuronal cell loss, possibly by a mechanism involving receptor desensitization. These changes occur in the animal model in those brain regions which in man are thought to be of importance in the loss of cerebral function that is found with senscence.

Heart conditioned medium elicits post-lesion muscarinic receptor recovery in vivo.

Intraseptal administration of heart conditioned medium (HCM) stimulates the growth of injured cholinergic fibers into iris implants placed in the anterodorsal hippocampus. The aims of the present report were to monitor muscarinic cholinergic receptor concentrations during denervation and central innervation of the peripheral tissue targets, and to evaluate the effect of HCM on these changes. For 0, 4, 8, 16 and 28 days after mechanical injury to septohippocampal axons, animals received either intraseptal injections of HCM or control vehicle. Binding of [3H]quinuclidinylbenzilate ([3H]QNB) within iris implants was used as an index of cholinergic muscarinic receptor concentration. The results indicate that within the iris implants: (1) a dramatic drop in the number of muscarinic receptors is observed 4 days after denervation; (2) under control conditions, central cholinergic innervation is not associated with muscarinic receptor recovery; and (3) after administration of HCM, muscarinic receptor levels begin to increase within two weeks and approach the pre-lesion endogenous concentration following 28 days of treatment. These results support the hypothesis that trophic factors may facilitate the restoration of effective, appropriate connections between nerve fibers and their targets.