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The formation of A beta and A beta-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of A beta and its C-terminally extended sequences is liberated from the beta-amyloid precursor protein (beta APP) by beta-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM-->NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the "normal' (N peptide) and mutated (delta NL peptide) sequences targeted by beta-secretase(s), we have detected a human brain protease displaying preferred efficiency for the delta NL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative beta-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the delta NL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block A beta secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral delta NL beta APP751 at a 6-fold higher rate than beta APP751 and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of A beta. Altogether, our study indicates that cathepsin D displays several in vitro beta-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease.

In all but one brain region sampled, substance P-like immunoreactivity (SPLI) was unchanged 4 h after a dose of cycloheximide that produced a near-total inhibition of rat brain protein synthesis. This suggests that brain substance P (SP) utilization is slow under basal conditions. The administration of the dopamine antagonist haloperidol to cycloheximide-pretreated rats apparently accelerated striatonigral SP utilization, as evidenced by large depletions in SPLI in the substantia nigra and striatum. Measuring the decline of SPLI a short time after protein synthesis inhibition may provide an index of brain SP utilization.

The origin of the substance P-like immunoreactivity in the rat submandibular gland and its major duct was investigated. Lesions severing the sympathetic, pre- and postganglionic parasympathetic and sensory innervation of the submandibular gland, or treatment with the neurotoxin capsaicin produced only a small non-significant decrease in the substance P-like immunoreactivity content of the gland. In contrast both sensory denervation and capsaicin treatment produced a substantial decrease in the content of substance P-like immunoreactivity in the major duct of the submandibular gland. These observations indicate that the duct innervation is clearly of sensory origin, whilst the substance P-like immunoreactivity of the gland itself appears to be intrinsic.

Neurotensin-like immunoreactivity was detected by radioimmunoassay in the anterior lobe of the pituitary gland of several mammalian species, including man. In the rat, pituitary stalk transection did not change the content of neurotensin-like immunoreactivity. Surgical and chemical thyroidectomy produced a drastic reduction of neurotensin-like immunoreactivity in the anterior pituitary gland, whereas adrenalectomy and gonadectomy were without an effect and the decrease consequent to thyroidectomy was restricted to the pituitary gland. These results suggest an interaction between neurotensin and the hypothalamus/pituitary/thyroid axis at the level of the anterior pituitary gland.

In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland.

The presence of [Lys8-Asn9]-neurotensin8-13-like immunoreactivity was studied by radioimmunoassay in chicken and rat tissues. In the chicken, [Lys8-Asn9]-neurotensin8-13-like immunoreactivity showed a wide distribution throughout the central nervous system and the gastrointestinal tract, and the immunoreactive material co-eluted with the synthetic peptide on reverse-phase high performance liquid chromatography. In the rat, [Lys8-Asn9]-neurotensin8-13-like immunoreactivity was widely distributed when 0.1 M HCl was used as the extraction procedure. However, the immunoreactive material did not co-elute with the synthetic peptide on reverse-phase high performance liquid chromatography; moreover, the addition of the aspartic proteinase inhibitor pepstatin to the extraction medium resulted in a large reduction in the levels of [Lys8-Asn9]-neurotensin8-13-like immunoreactivity and no immunoreactive material could be detected when the tissues were extracted using acetone/HCl. The present results therefore indicate that [Lys8-Asn9]-neurotensin8-13-like immunoreactivity is not present in rat tissues. That which was detected resulted from an extraction artefact.

Neurotensin stimulated inositol phospholipid hydrolysis in matched coronal vibratome sections through the rat brain. This effect was tetrodotoxin-resistant and a good correlation was noticed between the magnitude of neurotensin-stimulated inositol phospholipid hydrolysis and the number of specific [3H]neurotensin binding sites in various brain regions. Neurotensin produced no significant effect on either basal or stimulated cAMP levels.

The regional distribution of xenopsin-like immunoreactivity (XPLI) and of neurotensin-like immunoreactivity (NTLI) was studied by radioimmunoassay in tissues of Xenopus laevis and rat. In Xenopus laevis, XPLI showed a wide distribution throughout central and peripheral tissues with the highest concentration in the skin; NTLI was found in both central and peripheral tissues with the exception of the skin. Gel chromatography on Sephadex G-25 was used in order to characterize the immunoreactive material; both XPLI and NTLI eluted in a single peak at the position of synthetic xenopsin and neurotensin. No XPLI could be detected in central and peripheral tissues from mouse, rat, guinea-pig, rabbit or cat. In contrast, NTLI was found in a variety of peripheral tissues of the rat and it coeluted with synthetic neurotensin on reverse phase high-performance liquid chromatography. It is probably present in preganglionic parasympathetic nerve fibres.

To determine whether the destruction of peripheral sympathetic neurons by anti-NGF-antibodies results from a complement-mediated cytotoxic action or from the deprivation of endogenous NGF or immunologically NGF-like cross-reacting molecules we investigated the time-course of the reversibility of the effect of NGF-antibodies by neutralizing doses of NGF, together with the effect of NGF-antibodies in complement-deficient mice. After administration of a single dose of 50 mg/kg of purified antibodies to newborn rats the TH level was reduced to 75% of controls after 12 h, to 48% after 24 h, 39% after 36, and 28% after 48 h. After this time no further reduction occurred and levels remained constant up to 14 days. The effect of the NGF-antibodies was reversible on addition of NGF up to 48 h after antibody administration. Although the reversibility was not complete (85% of controls) the extent of the reversibility was the same whether NGF was given 12 or 48 h after the antibodies. The incompleteness of the reversibility is reflected by the small number of degenerating neurons apparent as early as 12 h after antibody administration. Since these early degenerative effects were also seen in complement-deficient mice it is concluded that they involve a small population of neurons, sensitive to short-term NGF deprivation whereas the majority of the neurons can withstand deprivation for up to 48 h without sustaining irreversible damage.

At birth, striatal neurotensin-like immunoreactivity amounted to 10% of the adult values which were reached at the age of 5 weeks. In the caudate nucleus neurotensin-like immunoreactivity presented a patchy distribution throughout development that was in register with Met-enkephalin staining, whereas [3H]neurotensin binding sites were most heavily concentrated in the background matrix. Thus, the adult distribution pattern of neurotensin-like immunoreactivity and [3H]neurotensin binding sites is already established at birth.

The sciatic nerve was sectioned unilaterally in rats and nerve growth factor (NGF) applied locally to the nerve stump for the following 10-14 days using an indwelling osmotic pump. The aim of the experiment was to test whether NGF had any effect on the previously reported neurophysiological and neurochemical events that occur central to a peripheral nerve lesion. The method of application allowed the sciatic nerve on the other side to be used as a control. Primary afferent depolarization fell, as expected, to 13% of its control value after chronic nerve section but if NGF was administered it fell to only 43.5% of control. Chronic nerve section is also known to result in expansion of the receptive fields of deafferented dorsal horn cells. NGF treatment reduced the number of such large receptive fields by 50%. The normal depletion of fluoride resistant acid phosphatase from the cut nerve terminals in the dorsal horn did not occur following NGF treatment. Radioimmunoassay of substance P revealed that the 30% reduction in dorsal horn levels that follows chronic sciatic nerve section did not occur when NGF was applied and that the accompanying 60% decrease in dorsal root ganglion levels was changed to a 64% increase by NGF. The results show that chronic NGF treatment of a cut sciatic nerve does partially reverse the central changes that normally follow deafferentation.

The regional distribution of neurotensin-like immunoreactivity (NTLI) was studied by radioimmunoassay in central and peripheral tissues of the cat. In the brain, NTLI showed a wide distribution with highest concentrations in the hypothalamus, the caudate/putamen and the nucleus accumbens. Only low levels of NTLI were measured in the spinal cord and there was no difference between dorsal and ventral horn. In the periphery, NTLI was present in high concentrations in the adrenal medulla and in lower amounts in the superior cervical and the ciliary ganglion. NTLI was present in both lobes of the pituitary gland and throughout the gastrointestinal tract with high concentrations in the ileum. All other peripheral tissues tested contained low but detectable amounts of NTLI. Gel chromatography on Sephadex G-25 was used in order to characterize the immunoreactive material; NTLI in tissue extracts from 3 central and 3 peripheral tissues co-eluted in a single peak at the position of synthetic neurotensin. NTLI is widely distributed throughout cat tissues and there are important differences from the distribution pattern in the rat, the only other species examined in detail to date.

Neurotensin-like immunoreactivity was found in nerve fibers and terminals throughout the corpus striatum of the adult cat. The densest staining was observed in the globus pallidus followed by the caudate nucleus and the putamen. In the caudate nucleus neurotensin-like immunoreactivity had a patchy distribution which was in register with a similar pattern of enkephalin-like immunoreactivity.

The specific binding of [3H]neurotensin binding to the rat striatum was characterized and its localization investigated by using frontal cortex ablations and the neurotoxins kainic acid and 6-hydroxydopamine. Scatchard analysis indicated the existence of a single population of binding sites and the potencies of various neurotensin fragments in competing with [3H]neurotensin for its binding site were in good agreement with the potency values obtained in biological assays. The lesion studies indicated that more than 50% of striatal neurotensin receptors are localized on intrinsic neurones, approximately 30% on dopaminergic nerve terminals and 20% on corticostriatal nerve fibres.

The characteristics of [3H]neurotensin binding were studied using membranes prepared from the rat brain. Binding of [3H]neurotensin was found to be specific, saturable and reversible. Under the conditions of the assay non-specific binding represented less than 20% of the total binding at a radioligand concentration of 2 nM. The specific [3H]neurotensin binding increased linearly with protein concentration and was dependent on the pH and the temperature of the incubation medium. At 25 degrees C equilibrium was reached rapidly and the association kinetics appeared to be monophasic. The dissociation was not monophasic and it could be resolved into two distinct components. Scatchard analysis of the saturation data indicated a single population of binding sites with a density of 432 fmol/mg protein and an equilibrium dissociation constant of 2.85 nM. A Hill transformation of the competitive inhibition of specific [3H]neurotensin binding by increasing concentrations of unlabelled peptide yielded a slope not significantly different from unity. Neurotensin 1-13 and various neurotensin analogues were tested for their ability to compete with [3H]neurotensin for its binding site. Neurotensin 1-13, neurotensin 8-13 and the amphibian skin peptide xenopsin were equipotent and strongly inhibited the specific binding of [3H]neurotensin. Neurotensin 9-13 and the chicken intestinal peptide Lys8,Asn9-neurotensin 8-13 were weakly active, whereas neurotensin 10-13 and the amino-terminal fragments neurotensin 1-6, neurotensin 1-8 and neurotensin 1-11 were inactive. Physiological concentrations of sodium chloride inhibited specific [3H]neurotensin binding, whereas divalent cations and guanyl nucleotides did not produce a significant change in either the equilibrium dissociation constant or the total number of binding sites. There was no apparent correlation between the content of neurotensin-like immunoreactivity in the rat brain and the density of [3H]neurotensin binding sites in the various brain regions. The highest density of binding sites was found in the hypothalamus and the frontal cortex, intermediate levels in striatum, thalamus, midbrain, hippocampus and olfactory bulb, and low levels in cerebellum and pons-medulla oblongata. In general, there was less variation between different brain regions in the number of [3H]neurotensin binding sites than in the content of neurotensin-like immunoreactivity. The characteristics of this binding assay are consistent with [3H]neurotensin binding to a physiological receptor.

A biologically active 2.5S NGF preparation free of renin-activity was prepared. Stimulation of the hypothalamo-pituitary-adrenocortical axis as well as induction of tyrosine hydroxylase in sympathetic ganglia are characteristic NGF effects and are not mediated by renin. These findings are confirmed by observations showing that captopril, an angiotensin converting enzyme inhibitor, failed to block these responses.

Nerve growth factor (NGF) mRNA is widely distributed throughout peripheral and central rat tissues and throughout the human central nervous system. In the rat, high levels were found in cerebral cortex, hippocampus and thalamus/hypothalamus, medium levels in striatum and brainstem and low levels in cerebellum and spinal cord. The hippocampal levels did not change following the surgical transection of the septohippocampal pathway; similarly, the ibotenic acid-induced lesion of the nucleus basalis magnocellularis did not affect the amounts of NGF mRNA in the cerebral cortex. NGF mRNA was also present in high amounts in human cortex and hippocampus, with only low levels in septum/nucleus basalis magnocellularis, suggesting that NGF may also function as a retrograde trophic messenger in the human central nervous system. No evidence was obtained for an insufficient production of NGF mRNA in senile dementia of the Alzheimer type. In peripheral rat tissues, the highest concentrations of NGF mRNA were found in vas deferens, heart, sciatic nerve, submandibular gland and skin, with low levels in tissues such as trigeminal ganglion and pituitary gland.

Specific tritiated neurotensin binding sites were localized in the rat adrenal gland by receptor autoradiography and characterized using a tissue homogenate receptor binding assay. High levels of specific neurotensin binding sites were found in the inner layer of the adrenal cortex and lower amounts in the adrenal medulla with only background labeling in the outer cortical layers. The structure-activity profile of the specific neurotensin binding was consistent with binding to a physiological neurotensin receptor.

The biochemical and morphological changes effected by immunization of adult rats with 2.5 S mouse nerve growth factor (NGF) were studied in sympathetic ganglia and in representative target organs. This immunization procedure maintains high levels of circulating anti NGF-antibody for periods of months. Morphological analysis revealed a general reduction in the size of the adrenergic neurons in the superior cervical ganglion (SCG) which was also reflected at the biochemical level by a 30% decrease in total protein content and a 50--60% reduction in the total activities of all norepinephrine-synthesizing enzymes. However, there was no change in total choline acetyltransferase activity. The biochemical and morphological changes observed in the SCG seem to be confined to the neuronal cell body, since at any stage of immunization target organs (the submandibular and the pineal gland) remained unaffected. All sympathetic ganglia investigated--except the superior mesenteric ganglion--responded in a similar way to the immunization against 2.5 S NGF. These changes in the adrenergic cell bodies were largely reversible. The recovery of normal enzyme activities followed closely the decrease of the antibody titer after cessation of immunization boosting. This indicates that cell death is not caused by anti NGF-antibodies in ganglia of adult animals. Thus, in contrast to adrenergic neurons from newborn animals, which depend on NGF or a crossreacting NGF-like material for survival, differentiated adrenergic neurons need this factor for the maintenance of their normal function but not for survival.

The dopamine receptor antagonist fluphenazine decanoate, when administered for a total period of 10 months, produced a large increase in neurotensin-like immunoreactivity in dopamine-rich brain areas, such as the nucleus accumbens, the striatum and the frontal cortex. A smaller, non-significant increase was observed in the substantia nigra with no change in either the hypothalamus or the spinal cord. The present results provide further evidence in favour of a functional interaction between neurotensin and dopamine in the central nervous system.

The regional distribution of neurotensin-like immunoreactivity was investigated in normal human brain and in brains of patients who had died with neurological illness. In Huntington's disease, neurotensin was increased in the pallidum, whilst in Parkinson's disease no significant changes in neurotensin content were observed. Similarly no changes were found in the telencephalic neurotensin content in senile dementia of the Alzheimer type. High levels of neurotensin-like immunoreactivity were detected in lumbar cerebrospinal fluid from patients and the characterization of the immunoreactive material by high-performance liquid chromatography showed it to be indistinguishable from synthetic neurotensin.