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We assessed the amount of protein kinase C (PKC) in samples from postmortem normal human and Alzheimer's disease (AD) brains by a two-site enzyme immunoassay that quantitatively identified types alpha, beta, and gamma isozymes. In the normal human brain matter, type beta was the main type present, the majority of each isozyme of PKC being present in the membranous fraction of the brain tissues. In AD brains, the amount of type beta PKC was significantly reduced in the membranous fraction of the temporal cortical tissues. The amounts of types alpha and gamma in the membranous fraction and types alpha, beta, and gamma in the cytosolic fraction in AD brains were lower than in the control brains, but the difference was not significant. There was also a significant decrease in the levels of PKC in the membranous fraction of AD brains, as measured by radioactive phorbol ester binding. These results suggest that the type beta PKC isozyme is mainly present in the human temporal cortex and that reduced levels of type beta PKC in the membranous fraction may reflect a biochemical deficit related specifically to the pathogenesis of AD.

The magnitude of the noradrenaline-induced contractions of dog middle cerebral arteries was less than that seen in the vertebral, common carotid, femoral and renal arteries. Noradrenaline and clonidine produced a similar magnitude of maximum contractions in the middle cerebral arteries, whereas methoxamine produced no significant contractions in the same arteries. In the extracranial arteries, noradrenaline and methoxamine produced significantly larger contractions than clonidine. Binding studies revealed no specific 3H-prazosin binding sites in the cerebral arteries, though such binding sites were evident in the case of extracranial arteries. 3H-Yohimbine binding studies revealed the presence of two classes of binding sites with high and low affinities in both cerebral and extracranial arteries. After superior cervical ganglionectomy, noradrenaline- and clonidine-induced contractions of the denervated middle cerebral arteries were not altered, compared with the control arteries. A 3H-yohimbine binding study was also performed using the denervated cerebral arteries. This study revealed that there was a low affinity 3H-yohimbine binding site, whereas high affinity 3H-yohimbine binding site was not detectable. These results suggest the presence of two different binding sites with high and low affinity for alpha 2 adrenoceptors, which we are classifying into alpha 2H and alpha 2L subtypes. The high affinity sites, alpha 2H adrenoceptors, are presynaptically located while the low affinity sites, alpha 2L adrenoceptors, located postsynaptically. The noradrenaline-induced contractions are probably mediated by postsynaptic low affinity sites of alpha 2 adrenoceptors (alpha 2L adrenoceptors) in the cerebral arteries and mainly by alpha 1 adrenoceptors in the extracranial arteries.

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a K m value of 0.14 μM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET B receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.

We studied benzodiazepine receptors in brains obtained at autopsy from 7 normal controls and 7 patients with Alzheimer-type dementia, using 3H-flunitrazepam as a ligand. Compared with controls, 3H-flunitrazepam binding sites were significantly reduced in number in frontal and temporal cortex and hippocampus in the Alzheimer-type dementia brains. These results suggest that there are significant changes in the number of benzodiazepine receptors in selected brain regions of patients with Alzheimer-type dementia.

Using ligand binding techniques, we studied beta-adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer-type dementia (ATD). Inhibition of [3H]dihydroalprenolol [( 3H]DHA) binding by the selective beta 1 antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of beta 1/beta 2-adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of beta-adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. beta 1-Adrenergic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas beta 2-adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that beta 1- and beta 2-adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.

Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer-type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (-)-[3H]nicotine and [3H]quinuclidinyl benzilate [( 3H]QNB), respectively. Compared with the controls, (-)-[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.

The nature of alpha-adrenergic receptors in human cerebral arteries was characterized, and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. Norepinephrine content of control arteries was also analyzed and compared with that of arteries after subarachnoid hemorrhage. Norepinephrine content in human cerebral arteries in cases of subarachnoid hemorrhage was about 5% of the control group. Specific binding of [3H]yohimbine, a selective alpha 2-antagonist, to cerebral arteries of the control group indicated two classes of binding sites: high-affinity sites with KD of 0.5 nM and Bmax of 18 fmol/mg protein and low-affinity sites with KD of 29 nM and Bmax of 248 fmol/mg protein. In cerebral arteries obtained from the subarachnoid hemorrhage group, [3H]yohimbine binding sites were of a single class with KD of 53 nM and Bmax of 456 fmol/mg protein. These results suggest that sympathetic denervation and subsequent alterations in alpha 2-adrenergic receptors occurred after subarachnoid hemorrhage in human cerebral arteries. These changes in sympathetic innervation to cerebral arteries were considered to be one of the antecedents of delayed vasospasm after subarachnoid hemorrhage.

The nature of beta adrenergic receptors in human cerebral arteries was characterized and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. The specific 3H-dihydroalprenolol, a beta adrenergic antagonist, binding to human cerebral arteries was saturable and of high affinity (KD = 12.3 nM) with a Bmax of 790 fmol/mg protein. Ki values and Hill coefficients of adrenergic agents for 3H-dihydroalprenolol were as follows; propranolol, 4.1 X 10(-8)M, 1.01; isoproterenol, 1.7 X 10(-6)M, 0.80; epinephrine, 8.3 X 10(-6)M, 0.48; norepinephrine, 2.3 X 10(-5)M, 0.45; metoprolol, 6.8 X 10(-8)M and 7.9 X 10(-6)M, 0.62; butoxamine, 2.2 X 10(-8)M and 2.1 X 10(-6)M, 0.43. The analysis of inhibition of specific 3H-dihydroalprenolol binding by these adrenergic agents suggests that human cerebral arteries contain a high density of beta adrenergic receptors and that the receptors are classified into two types, namely beta 1 and beta 2 adrenergic receptors. The calculated beta 1/beta 2 ratio from Hofstee plots was approximately 4/6. KD and Bmax of 3H-dihydroalprenolol binding to the cerebral arteries after subarachnoid hemorrhage were compared with those of control group. KD and Bmax of 3H-dihydroalprenolol binding of subarachnoid hemorrhage group were 13.9 nM and 1140 fmol/mg protein, respectively. The calculated beta 1/beta 2 ratio was approximately 6/4. These data suggest that the density of total beta adrenergic receptors increased without any significant change in the affinity after subarachnoid hemorrhage and that the increase of beta 1 adrenergic receptors was dominant.

(-)-[3H]Nicotine was found to bind specifically to membranes of human brains obtained at autopsy. The binding was stereospecific, (-)-nicotine being 40 times more potent than (+)-nicotine in displacing labeled (-)-nicotine. Saturation binding studies revealed the presence of two binding sites with dissociation constant (KD) values of 8.1 and 86 nM, and maximum binding capacity (Bmax) values of 36 and 90 fmol/mg protein, respectively. In competition studies, nicotinic agonists were 1,000 times more potent than ganglionic, neuromuscular, and muscarinic blocking drugs in displacing labeled (-)-nicotine. IC50 values for cholinergic drugs of (-)-[3H]nicotine binding were as follows: (-)-nicotine, 0.51 nM; acetylcholine, 12.6 nM; (+)-nicotine, 19.9 nM; cytisine, 27.3 nM; and carbachol, 527 nM. IC50 values of alpha-bungarotoxin, hexamethonium, d-tubocurarine, and atropine were larger than 50 microM. (-)-[3H]Nicotine binding was highest in the nucleus basalis of Meynert and thalamus and lowest in the cerebral cortex and caudate in the brain regions tested. These results suggest that nicotinic cholinergic receptors are present in human brain and that there are regional differences in the density of these receptors.

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.

We examined the effects of a transient exposure of rats to severe (5% O2, 95% N2 for 30 min) or mild (10% O2, 90% N2 for 30 min) hypoxia and a chronic exposure to mild hypoxia (10% O2, 90% N2 up to 48 h) on carbachol-stimulated phosphoinositide (PI) turnover and [3H]quinuclidinyl benzilate (QNB) binding in 4 regions of the brain (cerebral cortex, striatum, hippocampus and cerebellum). When the rats were exposed to hypoxia transiently, significant changes were caused only by severe hypoxia. In all 4 regions examined, basal incorporation of [3H]inositol into inositol phosphates (IPs) in the absence of carbachol was significantly increased following transient severe hypoxia and remained high for 48 h. Carbachol stimulation of PI turnover (% of basal) was significantly enhanced only in the hippocampus. The increase in carbachol stimulation in the hippocampus was observed only after a 6-h exposure to room air following the severe hypoxia and lasted for at least 48 h. In Ca2+-free incubation medium, the increase in basal incorporation of [3H]inositol into IPs elicited by severe hypoxia was not observed but carbachol still had an enhanced stimulatory effect. Binding studies showed that maximum binding capacity (Bmax values) of [3H]QNB binding was significantly increased only in the hippocampus and only after a 6-h exposure to room air following the severe hypoxic exposure and remained increased for 48 h. Kd values showed no significant change. While a transient exposure of rats to mild hypoxia caused no significant change either in carbachol-stimulated PI turnover or in [3H]QNB binding parameters, a chronic exposure to mild hypoxia for more than 6 h caused a significant increase in [3H]QNB binding capacity localized in the hippocampus, which was accompanied by an enhanced stimulatory effect of carbachol on PI turnover. These changes observed with mild hypoxia, however, were transient and tended to return to control levels following a 48-h exposure. These results suggest an up-regulation of muscarinic cholinergic receptor-coupled PI turnover in the rat brain caused either by transient severe hypoxia or by chronic mild hypoxia.

In this study, we analyzed the mechanism of selective motor neuronal death, a characteristic of amyotrophic lateral sclerosis, using embryonic rat spinal cord culture. When dissociated cultures were exposed to low-level glutamate (Glu) coadministered with the Glu transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC) for 24 hours, motor neurons were selectively injured through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. Nitric oxide synthase (NOS) inhibitors attenuated this toxicity, and long-acting nitric oxide (NO) donors damaged motor neurons selectively. Nonmotor neurons survived after exposure to low-dose Glu/PDC, but Glu-induced toxicity was potentiated by coadministration of an NO-dependent guanylyl cyclase inhibitor. In addition, 8-bromo-cyclic GMP, a soluble cyclic GMP analogue, rescued nonmotor neurons, but not motor neurons, exposed to high-dose Glu/PDC. Twenty-four hours' incubation with PDC elevated the number of neuronal NOS-immunoreactive neurons by about twofold compared with controls, and a double-staining study, using the motor neuron marker SMI32, revealed that most of them were nonmotor neurons. These findings suggest that selective motor neuronal death caused by chronic low-level exposure to Glu is mediated by the formation of NO in nonmotor neurons, which inversely protects nonmotor neurons through the guanylyl cyclase-cyclic GMP cascade. Induction of neuronal NOS in nonmotor neurons might enhance both the toxicity of motor neurons and the protection of nonmotor neurons, which could explain the pathology of amyotrophic lateral sclerosis.

Parkinson's disease is characterized by the mesencephalic dopaminergic neuronal loss, possibly by apoptosis, and the prevalence is higher in males than in females. The estrogen receptor (ER) subtype in the mesencephalon is exclusively ER beta, a recently cloned novel subtype. Bound with estradiol, it enhances gene transcription through the estrogen response element (ERE) or inhibits it through the activator protein-1 (AP-1) site. We demonstrated that 17beta-estradiol provided protection against nigral neuronal apoptosis caused by exposure to either bleomycin sulfate (BLM) or buthionine sulfoximine (BSO). BLM and BSO-induced nigral apoptosis was blocked by inhibitors for caspase-3 or c-Jun/AP-1. The antiapoptotic effect by estradiol was blocked by ICI 182,780, an antagonist for ER, but not by a synthesized peptide that inhibits binding of the ER to the ERE. Estradiol had no effects on caspase-3 activation and c-Jun NH(2)-terminal kinase (JNK), which were activated by BLM. It also suppressed apoptosis by serum deprivation, which was independent of caspase-3 activation. Therefore, the antiapoptotic neuroprotection by estradiol is mediated by transcription through AP-1 site downstream from JNK and caspase-3 activation. Furthermore, 17alpha-estradiol, a stereoisomer without female hormone activity, also provided an antiapoptotic effect. Therefore, the antiapoptotic effect is independent of female hormone activity.

Estrogens have been reported to exert neuroprotection in the brain, but there have been no reports of such neuroprotection in spinal motor neurons, the neurons selectively involved in amyotrophic lateral sclerosis (ALS). In this study, we demonstrated that 17beta-estradiol and its biologically inactive stereoisomer, 17alpha-estradiol, prevented glutamate- and nitric oxide (NO)-induced selective motor neuronal death observed in primary cultures of the rat spinal cord. The dose of estradiols required for motor neuron protection was greatly reduced by co-administration with glutathione. The results of this study shows that estradiol protects spinal motor neurons from excitotoxic insults in vitro, and may have application as a treatment for ALS.

Parkinson’s disease is characterized by dopaminergic neuronal death and the presence of Lewy bodies in the substantia nigra pars compacta (SNpc). α-Synuclein and ubiquitin are components of Lewy bodies, but the process of Lewy body formation and the relationship between inclusion formation and dopaminergic neuronal death have not been resolved. In this study, unilateral intranigral microinjection of 6-hydroxydopamine caused a significant loss of tyrosine hydroxylase-immunopositive neurons in both the substantia nigra and striatum and apomorphine-induced contralateral rotation. The co-administration of proteasome inhibitors, such as lactacystin or carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), significantly prevented both dopaminergic neurodegeneration and apomorphine-induced rotational asymmetry. Proteasome inhibitors markedly formed intracellular protein inclusions labeled by thioflavin-S in the SNpc. Inclusion-like immunoreactivities for α-synuclein and ubiquitin were detected after 4 weeks. These results suggest that proteasome plays an important role in both the early phase of dopaminergic neuronal death and inclusion body formation. Keywords:: proteasome inhibitor, neuroprotection, dopaminergic neuron, substantia nigra, Parkinson’s disease

It has been known that rotenone and 1-methyl-4-phenylpyridinium ion (MPP(+), a metabolite of MPTP), which inhibit mitochondrial complex I, are useful tools for parkinsonian models in vertebrates such as primates and rodents. Planarian, an invertebrate flatworm, has a high potential for regeneration, and dopamine plays a key role in its behavior. In the present study, we examined a cloned planarian, the GI strain from Dugesia japonica. Planarians that were treated with rotenone or MPTP underwent autolysis and individual death in a concentration- and time-dependent manner. In addition, these effects induced by rotenone or MPTP were inhibited by several antiparkinsonian drugs and caspase inhibitors. These results suggest that the degeneration of planarian dopaminergic system induced by rotenone or MPTP may be mediated through caspase-like activation.

Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathogenesis of Alzheimer's disease (AD). Here we show the signal transduction mechanism involved in nicotinic receptor-mediated protection against beta-amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an alpha7 nicotinic receptor antagonist (alpha-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. The alpha7 nicotinic receptor was physically associated with the PI3K p85 subunit and Fyn. These findings indicate that the alpha7 nicotinic receptor transduces signals to PI3K in a cascade, which ultimately contributes to a neuroprotective effect. This might form the basis of a new treatment for AD.