Christer Larsson

Age-related reduction of human growth hormone-binding sites in the human brain

Previous studies have shown that alterations in various neuroendocrine functions occur with increasing age. We here report a study of growth hormone (GH)-binding sites in different areas of post-mortem human brains collected from individual males and females of different age. The results indicate that there exists a significant negative correlation between the density of GH-binding sites and increasing age. This phenomenon was observed in both sexes in brain areas such as choroid plexus, hippocampus, hypothalamus, pituitary and putamen but not in e.g. thalamus. In all tissues (except for choroid plexus), the GH binding was significantly higher in those originating from females than those from males. This discrepancy was found likely to be associated with the affinity of GH to lactogenic rather than to somatogenic sites as no pronounced sex difference in binding was observed in the presence of excessive amounts of human prolactin. Data also indicate that the putative GH receptors in the various brain regions differ with regard to binding constants and to the estimated molecular size of the hormone-binding units. The loss of GH receptors in brain of elderly people may have consequences in several physiological courses. The decrease in GH binding at hypothalamic and pituitary levels may be of importance for the mechanisms behind the release or secretion of the hormone.

Comparative Analysis of Nicotine-Like Receptor-Ligand Interactions in Rodent Brain Homogenate

Abstract: The effects of different variables such as incubation time, temperature, tissue protein content, and pH on the interactions of various labelled nicotinic ligands with nicotine‐like binding sites in vitro were studied in rodent brain preparations. The ligands tested were α‐[3H]bungarotoxin (α‐[3H]BTX), [3H]tubocurarine ([3H]TC), and [3H]nicotine ([3H]NIC). The regional distribution of the labelled nicotinic ligand binding was also studied and affinity constants and maximal binding (Bmax) values for the equilibrium [3H]NIC binding are given. Association kinetics for [3H]NIC and [3H]TC binding to brain homogenate were similar, with maximal binding within 5–10 min of incubation, followed by a continuous decrease. In contrast, the binding of α‐[3H]BTX to brain homogenate was much slower, reaching equilibrium after 30–60 min of incubation. Scatchard analysis of equilibrium binding data for [3H]NIC in the hippocampus indicated two binding sites: a high‐affinity site (Bmax 60 pmol/ g protein; KD, 6 nM) and a low‐affinity site (Bmax, 230 pmol/g protein; KD, 125 nM). The data for the high‐affinity [3H]NIC binding site are very similar to previously found data for the high‐affinity binding site of [3H]TC and the binding site of α‐[3H]BTX. Each ligand showed regional differences in binding, and the binding pattern also differed between the ligands.

A comparison of the regional ontogenesis of nicotine- and muscarine-like binding sites in mouse brain.

The postnatal development of the cholinergic neurotransmitter system was studied in the cortex, hippocampus, midbrain and cerebellum of 3‐, 7‐, 12‐, 17‐ and 30‐day‐old NMRI mice. The concentration of muscarine‐like binding sites determined with [3H]quinuclidinyl benzilate as a ligand increased progressively with age. A similar developmental pattern was found for the activity of a presynaptic marker, choline acetyltransferase (ChAT). When expressed as a percentage of the values for 30‐day‐old mice, however, the muscarine‐like receptors were parallel but preceding the development of ChAT in all the brain regions studied. The concentration of nicotine‐like binding sites studied with [3H]α‐bungaro‐toxin as a ligand gradually increased with age in the cortex, hippocampus and midbrain, with a peak between days 7 and 12, followed by a decrease towards day 30. With [3H]tubocurarine as a ligand, on the other hand, the concentration of nicotine‐like binding sites was in general high at 3 days and gradually decreased with age, suggesting that different subpopulations of nicotine‐like receptors might be determined when the two ligands are used.

Effect of long-term nicotine treatment on [3H] nicotine binding sites in the rats brain

Male rats were receiving nicotine (base), 50 mg/kg in their drinking fluid for 9 and 41 weeks, respectively. A markedly reduced intake of fluid was observed during both regimes of nicotine treatment. At withdrawal of nicotine after 9 weeks of treatment the intake of water was immediately increased up to control level while in rats with 41 weeks of exposure the intake of drinking fluid increased to 65% over the control level still 14 days after withdrawal of nicotine. Tolerance to nicotine was measured after 7 days of abstinence in rats treated for 9 weeks. No significant change in cholineacetyltransferase (ChAT) activity or number of muscarinic binding sites in brain was observed after 9 or 41 weeks of nicotine treatment. Twenty-four hours after withdrawal from the 41 weeks nicotine treatment there was a significant increase of 46% in the number of [3H] nicotine binding sites in the hippocampus while the number of binding sites was decreased by 44% in the cortex and unchanged in the midbrain. On day 14th of abstinence these changes had disappeared. Following 9 weeks of nicotine treatment no change in [3H] nicotine binding was observed after 24 h or 7 days of abstinence.

CHRONIC NICOTINE EXPOSURE IN RAT: A BEHAVIOURAL AND BIOCHEMICAL STUDY OF TOLERANCE

Rats received nicotine (50 mg nicotine base/1) in their drinking fluid for 28 days. The daily consumption of nicotine was about 4 mg/kg per day. Controls received plain water. Body weight, food consumption and fluid intake were registered during the whole treatment period and up to 31 days after withdrawal of nicotine. A significant lower body weight was noted in rats, treated with nicotine, in comparison with control rats during the whole treatment period. After withdrawal of nicotine the body weight returned to normal. Only slight effects were seen on the food intake (at the beginning of the nicotine treatment). The fluid intake, on the other hand, was significantly lower in the nicotine group during the whole period of treatment and a reduced intake persisted 28 days after withdrawal of nicotine. Tolerance to nicotine was measured in rats 24 hours after withdrawal of nicotine, using two behavioural tests. No tolerance to nicotine was found after 31 days of withdrawal of nicotine. The number of nicotine-like binding sites was found to be reduced in the midbrain 24 hours after withdrawal of nicotine, while no significant change was found in the cortex and hippocampus.

Regional [3H]acetylcholine and [3H]nicotine binding in developing mouse brain

The postnatal development of nicotine‐like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3‐, 7‐, 12‐, 17‐ and 30‐day‐old mice was studied. Two different nicotinic cholinergic ligands, namely [3H]acetylcholine ([3H]ACh) and [3H]nicotine ([3H]NIC) were used to detect the nicotine‐like binding sites in in vitro binding assays. The postnatal development of the binding sites of [3H]NIC increased gradually with age in all brain regions studied. The [3H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time‐course for the appearance of nicotinic binding sites as observed with [3H]ACh was found to be rather similar to that earlier described for [3H]alpha‐bungarotoxin binding sites, whereas that for [3H]NIC differed from that described for other nicotinic ligands.

Increased number of muscarinic binding sites in brain following chronic barbiturate treatment to rat

Abstract Rats received as their only drinking fluid a solution of sodium barbital (3.33 mg/ml) for more than 40 weeks. In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice. Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C). Synaptosomes from different parts of the brain were incubated with radioactive quinuclidinyl benzilate ( 3 H-QNB) (0.2 nM) for 60 min. A significantly increased number of 3 H-QNB binding sites was found in the striatum and midbrain + medulla oblongata + cerebellum of rats abstinent for 3 days (A3) in comparison with controls (C). Saturation studies indicated that group A3 had significantly more receptors in the midbrain + medulla oblongata + cerebellum than group C, while there was no differences in receptor affinity.

Subchronic treatment of rats with nicotine: Effects on tolerance and on [3H]acetylcholine and [3H]nicotine binding in the brain

The effects of subchronic nicotine treatment on the development of tolerance and on two nicotinic ligand binding sites were investigated. After subcutaneous injection of nicotine (0.45 mg nicotine base/kg) or saline twice a day for 14 days the body weight of rats was significantly lower than that of control animals. A significant tolerance to the acute effects of nicotine on locomotor activity and body temperature was observed after the treatment period. Nicotine treatment also resulted in a significant increase in [3H]acetylcholine (3H-ACh) binding in the midbrain (48.3% increase in comparison with controls) and hippocampus (38.3% increase), whereas the binding of [3H]nicotine (3H-NIC) was unaffected in all brain areas investigated. These results indicate that subchronic s.c. injections of nicotine can differentially affect the binding of two different nicotinic ligands in the brain. It is also concluded that the development of tolerance to the acute effects of nicotine on locomotor activity and temperature is not directly dependent upon changes in binding of [3H]nicotine to the brain.

Characterization of Prolactin Receptors in Human Choroid Plexus

The specific binding of 125I-human prolactin (hPRL) was studied in different areas of the human brain. Particularly high binding affinity of the hormone was found in the choroid plexus and this tissue was therefore selected for further studies. The hippocampus, the hypothalamus and the pituitary were among other regions containing prolactin-binding sites. In the choroid plexus the amount of PRL receptors was significantly higher in females than in males and was also found in both sexes to decrease with age. The binding affinity of 125I-hPRL to choroid plexus was 3.0 x 10(9) M-1 and the binding capacity was 10.3 pmol per mg protein. Following solubilization with Triton X-100 the PRL receptor fraction retained its hormone-binding properties and upon molecular sieve chromatography it behaved as a protein with a molecular mass of approximately 250,000. Cross-linking of 125I-hPRL to receptors from choroid plexus and subsequent sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis indicated a major hormone-binding unit of M(r) 44,000. This value is about 7,000 smaller than that reported earlier by us for the growth hormone receptors from the same tissue, following cross-linking to 125I-human growth hormone (hGH). By affinity column chromatography a complete separation of the hPRL and hGH binding units was achieved. It was thus shown that in choroid plexus the binding sites for GH and PRL occur as discrete entities.

Effects of morphine on prolactin receptors in the rat brain

The effect of chronically given morphine on the binding of ovine prolactin (oPRL) to specific areas in the male rat brain was studied. The drug was delivered through subcutaneously implanted miniosmotic pumps. The results indicated that the density of prolactin binding sites in the hypothalamus and the choroid plexus was significantly decreased in the acute phase of morphine administration but restored to control levels when tolerance to morphine was developed. The decrease in prolactin binding was contrasted by elevated plasma levels of the hormone. A negative correlation was found between the hormone concentration in plasma and the density of its binding sites in the hypothalamus and choroid plexus. The hormone‐binding sites in these two regions were further characterized with regard to binding constants and molecular sizes. The relevance of the present results with respect to the hypothalamic control of prolactin secretion is discussed.