Neprilysin-2 is an important β-amyloid degrading enzyme

Proteases that degrade the amyloid-β peptide (Aβ) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic Aβ accumulation (～30- to 50-fold) and pathological deposition in rodents. The Aβ-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (～1.5- to 2-fold) in Aβ, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/phosphoramidon-sensitive and degrades Aβ. We investigated the role of NEP2 in Aβ degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total Aβ species in the hippocampus and brainstem/diencephalon (～1.5-fold). Increases in Aβ accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, Aβ levels were marginally increased (～1.5- to 2-fold), compared with NEP(-/-)/NEP2(+/+) controls. Treatment of these double-knockout mice with phosphoramidon resulted in elevations of Aβ, suggesting that yet other NEP-like Aβ-degrading endopeptidases are contributing to Aβ catabolism.     

Neutral endopeptidase modulates tachykinin-induced increase in vascular permeability in guinea pig skin

To determine the regulatory role of neutral endopeptidase (NEP) in the tachykinin-induced increase in vascular permeability, we examined the effects of NEP inhibitors and of other protease inhibitors on plasma extravasation induced by intradermal injection of substance P, neurokinin A, and neurokinin B in guinea pig skin. The three tachykinins induced plasma extravasation in concentration-dependent fashions. A significant NEP activity was found to be present in the guinea pig skin. The tachykinin-induced responses were increased by the NEP inhibitors phosphoramidon and thiorphan. However, other protease inhibitors, including a kininase II inhibitor, did not affect the response. We conclude that NEP modulates the tachykinin-induced increase in vascular permeability in the skin.     

Effect of prenatal hypoxia on DNA synthesis in the tracheal epithelium and LPO-AOD system in the lungs of newborn rats

Newborn rats euthanized 24 h after birth were examined. The rats were born to females exposed to chronic hypobaric hypoxia on days 14–19 of gestation. The index of nuclei labeled with³H-thymidine in the tracheal epithelium of newborn rats exposed to prenatal hypoxia was 3 times lower than in the control. The LPO level was higher in posthypoxic animals than in intact rats. Prenatal hypoxia led to the suppression of antioxidant defense in the lungs of newborn rats. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 5, pp. 531–533, May, 1994     

Neuronal activity of somatosensory cortex in a cross-modal (visuo-haptic) memory task

Studies have shown that in the monkey′s associative cerebral cortex, cells undergo sustained activation of discharge while the animal retains information for a subsequent action. Recent work has revealed the presence of such ″memory cells″ in the anterior parietal cortex (Brodmann′s areas 3a, 3b, 1, and 2) – the early stage of the cortical somatosensory system. Here we inferred that, in a cross-modal visuo-haptic short-term memory task, somatosensory cells would react to visual stimuli associated with tactile features. Single-unit discharge was recorded from the anterior parietal cortex – including areas of hand representation – of monkeys performing a visuo-haptic delayed matching-to-sample task. Units changed firing frequency during the presentation of a visual cue that the animal had to remember for making a correct tactile choice between two objects at the end of a delay (retention period). Some units showed sustained activation during the delay. In some of them that activation differed depending on the cue. These findings suggest that units in somatosensory cortex react to visual stimuli behaviorally associated with tactile information. Further, the results suggest that some of these neurons are involved in short-term active memory and may, therefore, be part of cross-modal memory networks. Received: 24 March 1997 / Accepted: 8 May 1997     

Informational capacity of the rat sensorimotor cortex in the postresuscitation period (morphometric analysis of a neuronal population)

The number of neurons was counted in different functional neuronal complexes of the sensorimotor cortex in albino rats in the control and at different times after clinical death caused by asphyxia. A decrease of the number of neurons from 2.5×10⁶ in the control to 1.56×10⁶ toward the 30th day of the postresuscitation period was found in the sensorimotor cortex. The complexes of small neurons in the upper floor (layers II–IV) suffer badly, while the neurons of layer VI are damaged to a somewhat lesser degree and the cells of layer V are minimally altered. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 3, pp. 331–333, March, 1995 Presented by L. V. Poluektov, Member of the Russian Academy of Medical Sciences     

Effect of NMDA on the activity of cortical glutaminergic structures in delayed visual differentiation in monkeys

The effect of intracortical perfusion with the glutamate agonist NMDA on visual recognition and short-term memory, as well as on the responses of visual cortex neurons, were studied in rhesus macaques. A microdialysis technique was used in combination with multichannel microelectrode recording of single cortical cells in the immediate vicinity of the dialysis tube in a behavioral experiment in which the monkey had to solve a task involving delayed visual differentiation of stimuli of different colors. NMDA altered the characteristics of recognition in monkeys. The duration of information storage in short-term memory was increased significantly (2–4-fold), and there was a significant reduction in the motor response time for all delay periods. These changes were accompanied by a significant rearrangement of neuron activity in the visual cortex at all stages of the behavioral task. At different stages of the task, 70–85% of the neurons showed 2–5-fold increases in activity, while 6–20% showed reductions in activity. These results demonstrate an involvement of visual cortex glutaminergic structures in the processes of visual recognition and short-term memory, as well as a nootropic effect obtained by intracortical administration of NMDA. Laboratory for the Regulation of Brain Neuron Function (Director M. O. Samoilov), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 81, No. 5, pp. 23–30, May, 1995.     

Responses to Light of Sensorimotor and Visual Cortex Neurons in Rabbits with a Cryptic Focus of Excitation (a defensive dominant) in the CNS

In the sensorimotor cortex of rabbits with a formed cryptic (subthreshold) focus of excitation in the CNS, the spike frequencies of neurons responding to light stimulation were significantly lower (p = 0.01) than the spike frequencies of neurons not responding to light. Similar findings were obtained in the visual cortex of intact rabbits. In this case too, the spike frequencies of neurons responding to stimulation were significantly lower (p = 0.01) than the spike frequencies of neurons not responding to light stimulation. In both intact rabbits and rabbits with a cryptic focus of excitation formed in the CNS, 36 % of neurons in the sensorimotor cortex responded to light stimuli not specific to this area. In the sensorimotor cortex of rabbits with a cryptic focus of excitation formed in the CNS, as compared with intact rabbits, there were significantly more (p = 0.01) cells responding to light stimuli with latent periods of less than 100 msec and significantly fewer (p = 0.02) responding to light stimuli with latencies of 200–300 msec. In the visual cortex of rabbits with a formed cryptic focus of excitation in the CNS, as compared with intact rabbits, significantly fewer (p = 0.01) neurons responded to light stimuli with latent periods of 50–100 msec.     

Effect of Prenatal Hypobaric Hypoxia on Glutamatergic Signal Transduction in Rat Brain

Ca²⁺-mediated signal transduction of group I metabotropic glutamate receptors (ImGluR) was studied in the brain of young (15 days) and old rats (90 days) exposed to severe hypobaric hypoxia on gestation days 14–16. Changes in the concentration of bound intracellular Ca²⁺ (Ca²⁺ response) were evaluated after repeated application of a selective ImGluR agonist 3,5-dihydroxyphenylglycine (DHPG) to cultured brain slices. Primary application of DHPG for 2 min induced a negative Ca²⁺ response in slices from 15-day-old intact animals, while repeated application caused a positive response. In slices from 90-day-old control animals, both responses were negative. In slices from rats of both age groups subjected to severe prenatal hypobaric hypoxia, both responses were mainly positive, but short-term negative components were present in adult animals. Our results suggest that severe hypobaric hypoxia changes the balance between the two constitutive signal pathways triggered by ImGluR (inosine triphosphate and diacylglycerol pathways). This procedure is followed by the increased influx of extracellular Ca²⁺ (as compared to Ca²⁺ release from the intracellular stores). This imbalance is particularly pronounced at the early stage of ontogeny.     

Differential effects of phosphoramidon and captopril on NK1 receptor-mediated plasma extravasation in the rat trachea

We sought to confirm the identity of the tachykinin receptor subtype that mediates plasma extravasation in the rat trachea, and assess the respective contributions of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) in regulating this tachykinin-induced response. To achieve these aims, we determined the relative potencies of several natural tachykinins and receptor-selective synthetic agonists, both before and after inhibiting NEP with phosphoramidon and ACE with captopril. We also determined the effects of these peptidase inhibitors, and the NK-1 receptor antagonist L-703,606, on the plasma extravasation produced by capsaicin, which releases tachykinins endogenously from sensory nerve endings. We found that the rank order of potency for producing plasma extravasation in the rat trachea was NK-1 receptor agonist ([Sar⁹, Met(O₂)¹¹] SP)>substance P>neurokinin A> neurokinin B. The NK-2 ([Nle¹⁰]NKA (4–10)) and NK-3 ([MePhe⁷]NKB) receptor agonists were without effect. We observed no change in the relative potencies of these peptides after giving rats phosphoramidon or captopril, which suggests that the different peptide potencies are not simply the consequence of different rates of enzymatic degradation. Nevertheless, the responses to substance P and neurokinin A were clearly potentiated in rats given phosphoramidon, indicating that NEP effectively degrades tachykininsin vivo. No significant potentiation was evident for any peptide in rats given captopril. Similarly, the plasma extravasation produced by capsaicin was potentiated in rats given phosphoramidon, but not in those given captopril. Pretreating rats with L-703,606 abolished the response to capsaicin. We conclude from these observations that NK-1 receptors mediate tachykinin-induced plasma extravasation in the rat trachea, and that NEP regulates this response with little or no contribution from ACE.     

Role of Nitric Oxide in Prevention of Cognitive Disorders in Neurodegenerative Brain Injuries in Rats

NO synthesis disturbances play an important role in the development of neurodegenerative damage in Alzheimer disease. We previously showed that adaptation to intermittent hypobaric hypoxia prevents cognitive disturbances in rats with experimental Alzheimer disease [6]. Here we evaluated the role of NO in cognitive disorders and development of adaptive protection during experimental Alzheimer disease. Adaptation to hypoxia in rats was performed in a hypobaric pressure chamber at a simulated altitude of 4000 m (4 h per day for 14 days). Alzheimer disease was simulated by bilateral injections of a toxic fragment of β-amyloid (25–35) into n. basalis magnocellularis. For evaluation of the role of NO in the development and prevention of memory disorders, the rats received intraperitoneally either NO-synthase inhibitor N^ω-nitro-L-arginin (L-NNA, 20 mg/kg, every other day for 14 days) or NO-donor dinitrosyl iron complex (200 μg/kg daily for 14 days). NO-synthase inhibitor potentiated the damaging effect of β-amyloid, abolished the protective effect of adaptation to hypoxia, and produced memory disorders in rats similar to those observed during experimental Alzheimer disease. In contrast, the increase in NO level in the body provided by injections of the NO-donor produced a protective effect against memory disorders caused by β-amyloid similar to that induced by adaptation to hypoxia. We concluded that reduced NO production in the organism plays an important role in the development of cognitive disorders produced by injections of β-amyloid, while prevention of NO deficit by administration of NO-donors or nonpharmacological stimulation of NO synthesis can provide a protective effect in experimental Alzheimer disease. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 10, pp. 371–375, October, 2008     

Distribution of neutral endopeptidase activity in human blood leukocytes

We investigated the distribution of neutral endopeptidase (NEP; EC 3.4.24.11) activity, a possible regulatory enzyme for neuropeptide-induced leukocyte activations, in each cell type of human blood leukocytes. The NEP activity assessed by an NEP inhibitor phosphoramidon-sensitive Met5-enkephalin degrading activity was present in neutrophils and the common acute lymphoblastic leukemia antigen (CALLA)-positive leukemic cells (59 pmol/min/10(6) cells and 62 pmol/min/10(6) cells, respectively); however, the NEP activity was virtually absent in lymphocytes, monocytes, eosinophils, basophils, CALLA-negative leukemic cells, or a promyelocytic cell line HL-60. The enzymatic activity was characterized as NEP on the basis of the values of kinetic parameters (Km = 61 microM, Kcat = 1,692 min-1, and Kcat/Km = 28 min-1 microM-1) and the values of IC50 of two NEP inhibitors phosphoramidon and thiorphan (7.4 nM and 8.4 nM, respectively). The distribution of NEP detected immunocytochemically using anti-NEP monoclonal antibodies was also found to be parallel with the distribution of NEP activity among peripheral blood leukocytes.     

Cytochemically Determined Activity of Glucose-6-Phosphate Dehydrogenase in Morphochemical Characteristics of the Brain of Wistar Rats Differing by Locomotion Parameters

Activity of histochemically determined glucose-6-phosphate dehydrogenase, a key enzyme of the pentose phosphate pathway, was qualitatively determined in layer III and V neurons of the sensorimotor cortex and neurons of the caudate nucleus, nucleus accumbens, and hippocampus (CA3) in mature male Wistar rats with high and low locomotor activity in an open field. A negative correlation was revealed between locomotion of Wistar rats in the open field and activity of glucose-6-phosphate dehydrogenase in the sensorimotor cortex, especially in efferent layer V neurons and neurons of the caudate nucleus and nucleus accumbens, which attested to different capacity of the brain in Wistar rats with high and low open-field locomotion to regeneration of phosphopyridine nucleotides (NADP⁺) and production of pentoses via the pentose phosphate shunt. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 1, pp. 47–49, January, 2009     

Reaction of sensorimotor cortex neurons to stimulation of the lateral hypothalamus in conditions of microiontophoretic application of tetragastrin and bradykinin: The role of food reinforcement

Experimental data were obtained on the nature of rabbit sensorimotor cortex neuron reactions to stimulation of the food motivation center of the lateral hypothalamus in conditions in which food was or was not presented to the animals combined with microiontophoretic application of tetragastrin and bradykinin. These neuropeptides were able to alter the relationship between motivation and reinforcement. This work was supported by the Neurophysiology Program of the Russian-Indian Agreement on Scientific Cooperation. P. K. Anokhin Science Research Institute of Normal Physiology, Russian Academy of Medical Sciences. Science Research Center of Medical Narcology, Ministry of Health of the Russian Federation, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti, Vol. 45, No. 4, pp. 757–764, July–August, 1995.     

Peptidergic correction of behavior in adult progeny of albino rats exposed to acute hypoxia during early organogenesis

Physical development, spontaneous behavior, and training capacity were evaluated in adult progeny of albino rats exposed to acute hypobaric hypoxia on days 9–10 of gestation, corresponding to the early organogenesis period. Prenatal hypoxia caused delayed behavioral disorders, which were more pronounced in females born from mothers with low resistance to hypoxia. Therapeutic intranasal administration of Pro-Gly-Pro peptide in a dose of 1 mg/kg to rat pups on days 13–15 of life completely prevented the negative consequences of acute prenatal hypoxia in adult females and leveled virtually all negative consequences, except delayed physical development, in males. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 38–41, July, 2006     

Thermal Regulation of Spontaneous Spike Frequency and Concomitant Changes in the Amplitude of Spikes from Cortical Neurons

Studies using living slices of guinea pig sensorimotor cortex showed that changes in temperature from 24°C to 37°C produced stepwise increases in neuron spike frequency at two temperature zones: 27–29°C and 34–36°C. Changes in spontaneous activity were accompanied by decreases in spike amplitude at t < 27°C and t > 34°C. After cooling to 24°C, spike amplitude generally recovered completely when the temperature was increased to 32–34°C. The decrease in spike amplitude at t > 35°C could not be restored by decreasing the temperature. It is suggested that these effects are associated with the K⁺ permeability of neuron membranes.     

Neurophysiological correlates of improvements in cognitive characteristics in monkeys during modification of NMDA-ergic structures of the prefrontal cortex

The effects of modification of NMDA-ergic structures by microdialysis perfusion in the prefrontal cortex (field 8) with NMDA glutamate (2 mM) on visual recognition and short-term memory (STM) were studied in rhesus macaques, using methods based on the deferred differentiation of stimuli of different colors. Impulse activity of neurons in the prefrontal and visual cortex was measured during these experiments, both before and after administration of NMDA. NMDA increased (2-fold) the duration of short-term retention of information and significantly reduced the latent period of the motor response at all delay periods, and also produced significant changes in neuron activity in the prefrontal cortex: depending on the stage of behavior, activity decreased in 60–75% of neurons and increased in 8–26%. NMDA produced significant increases in the cross-correlation coefficients between the responses of neurons in the visual and prefrontal cortex. The results showed that glutaminergic structures in the prefrontal cortex are involved in processes of visual recognition and STM in monkeys. The effect of NMDA, which synchronized cortical neuronal processes, improved the short-term understanding of visual information. Laboratory for the Regulation of Brain Neuron Function (Director M. O. Samoilov), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal im. I. M. Sechenova, Vol. 81, No. 1, pp. 32–39, January, 1995.     

Blood flow to the brown adipose tissue of conscious young rabbits during hypoxia in cold and warm conditions

 Brown adipose tissue (BAT) non-shivering thermogenesis is stimulated by cold temperature and depressed by hypoxia. We investigated the extent to which changes in metabolic rate during cold and hypoxia, singly or combined, were accompanied by changes in BAT perfusion. One-month-old rabbits were instrumented for measurements of regional blood flow by the coloured microsphere technique. One group of rabbits was tested in warm (24 °C, n=17), and the other in cold (13 °C, n=9) conditions, first in normoxia (inspired oxygen concentration FIO₂ about 21%, arterial oxygen saturation S _aO₂ approximately 88%) followed by hypoxia (FIO₂ approximately 10%, S _aO₂ approximately 54%). In warm conditions, oxygen consumption (V·O₂, measured by an open-flow method) averaged 22 ml·kg^–1·min^–1 (STPD), and BAT blood flow 98 ml·100g^–1·min^–1. In hypoxia, V·O₂ dropped on average to 87%, whereas BAT flow dropped to 43% of the normoxic values. In the cold during normoxia, V·O₂ averaged 31 ml·kg^–1·min^–1 (STPD), and BAT blood flow was 155 ml·100g^–1·min^–1. In cold and hypoxia V·O₂ dropped to 19 ml·kg^–1·min^–1 (STPD) (i.e. 60% of the normoxic value), whereas BAT blood flow was not altered significantly (148 ml·100g^–1·min^–1). Hence, BAT blood flow decreased in hypoxia in absence of cold stimuli, whereas it remained high when hypoxia occurred during cold, despite the major drop in V·O₂. We conclude that cold is more important than hypoxia in determining BAT perfusion, and that changes in BAT blood flow are not a mechanism for the hypoxic control of V·O₂. Received: 24 June 1998 / Received after revision: 21 September 1998 / Accepted: 29 September 1998     

Single Audiogenic Seizures Induce Increases in Calcineurin and Ca<Superscript>2+</Superscript>-Calmodulin-Dependent Protein Kinase II in the Rat Brain

Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 2B (calcineurin) play critical roles in the formation of nerve cell responses to incoming Ca²⁺ signals. The studies report here addressed how single seizures might affect the functioning of these enzymes. Sounds (80 dB, 12–15 kHz) induced single convulsions in male Krushinskii–Molodkina rats, which have an inherited predisposition to audiogenic seizures. Biochemical investigations were performed two days after seizures. Immunoblotting studies with specific monoclonal antibodies demonstrated increases in the level of the neurospecific α subunit of CaMKII in the sensorimotor cortex and hippocampus of the rats of the experimental group, as compared with controls. The hippocampus of rats which had had seizures also showed increases in the catalytic subunit of calcineurin. CaMKII activity in the hippocampus and cortex of postseizure rats increased, though that of the Ca²⁺-calmodulin-independent functional form of the enzyme did not change. It is suggested that the long-term changes seen here may represent an adaptive mechanism induced by convulsive activity and directed to increasing the threshold of neuron excitability in the brain.     

Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme

A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated S. parasanguis pepO, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni(2+) affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to pepO and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 than to NEP.