We examined the role of cAMP-dependent protein kinase in Ca²⁺ -elicited catecholamine secretion from bovine adrenal chromaffin cells. When the digitonin-treated cells were incubated with the catalytic subunit of cAMP-dependent protein kinase, the secretion of catecholamines from the cells occurred in the absence of Ca²⁺. The effect of the catalytic subunit was dependent on its activity (50-100 units/ml) and the presence of ATP-Mg²⁺ in the incubation medium. However, incubation of the cells with the regulatory subunit of cAMP-dependent protein kinase did not affect the secretion. Ca²⁺ (43 nM -10μM) also increased the secretion, which was ATP-Mg²⁺ -dependent. The catalytic subunit (25-200 units/ml) enhanced the Ca²⁺ -evoked secretion at the suboptimal but not optimal Ca²⁺ concentration, which induced maximal secretion. A potent synthetic peptide inhibitor of cAMP-dependent protein kinase abolished the catalytic subunit-induced secretion, but not the Ca²⁺ -evoked secretion. On the other hand, K- 252a, a potent inhibitor of protein kinases, inhibited both the catalytic subunit-induced and the Ca²⁺ -evoked secretion, but not KT5823, a much less potent inhibitor of protein kinases. These results strongly suggest that the catalytic subunit of cAMP-dependent protein kinase produces the secretion of catecholamines via protein phosphorylation. The results further suggest that the cAMP-dependent protein kinase does not participate in an intrinsic process of Ca²⁺ -elicited secretion but it may act as a modulator.

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To clarify the role of the amygdala in the fulfillment of memory and/or learning, amygdalalesioned mice were tested in passive and active avoidance performances and also in spatial learning tasks. Although the lesioned animals showed deteriorated performances in both passive and active avoidance tests, they executed the spatial learning tasks as well as the control mice. The learning deficit was prominent in the process of memory acquisition of passive and active avoidance tasks, suggesting that the amygdala might be involved in the acquisition processes of these avoidance tests. The locomotor activities of the lesioned animals were slightly increased, but there was no significant difference compared with the control mice. These findings indicate that the amygdala plays a crucial role preferentially in the avoidance learning rather than the spatial learning.

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To clarify the roles of dopamine D₁, and D₂ receptors in behavioral symptoms of Parkinson''s disease, antiparkinsonian effects of various dopamine agonists in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned parkinsonian monkeys were investigated with regard to induction of hyperactivity such as excitability, irritability and aggressiveness. The non-selective dopamine agonist apomorphine ameliorated the parkinsonism, but induced marked hyperactivity dose-dependently. Pretreatment with either the dopamine D₁, antagonist SCH 23390 or the dopamine D₂ antagonist sulpiride markedly suppressed the apomorphine-induced hyperactivity with slight attenuation of the antiparkinsonian effects. Both the dopamine D₂-receptor agonist quinpirole and the dopamine D₁-receptor agonist SKF 82958 ameliorated the parkinsonism in a dose-dependent manner with a slight induction of hyperactivity. Combination treatment of a threshold dose of quinpirole with that of SKF 82958 augmented the antiparkinsonian effects without a marked induction of hyperactivity. However, the combination treatment at higher doses induced marked hyperactivity accompanied by augmented antiparkinsonian effects. These results suggest that stimulation of either central dopamine D₁, or D₂ receptors is requisite for the antiparkinsonian effects and concurrent strong stimulation of both central dopamine D₁, and D₂ receptors causes marked hyperactivity which may be predictive of dopaminergic psychiatric side effects.

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31.5-kD ouabain receptor protein (NORP), which is independent of Na⁺-K⁺ ATPase, was recently isolated selectively from transverse tubule membrane-junctional sarcoplasmic reticulum (TTM-JSR) complexes of cat cardiac muscle. We investigated the role of this NORP in cardiac function with special reference to the positive inotropic effect (PIE) of ouabain, preparing and using a monoclonal antibody (MoAB, immunoglobulin) raised against the receptor protein. Electrically stimulated papillary muscles were immersed in a Tyrode solution containing the anti-NORP MoAB (40 μM), of which the binding potency was high enough for immunological use, for 60 min and then washed out. Thirty minutes after removal of the MoAB, both twitch and K-contracture were still inhibited, but both resting and action potentials and caffeine-induced contracture were unchanged, indicating that NORP plays a key role in excitation (E)-contraction (C) coupling. The intracellular localization of the protein was investigated by immunohistochemical electron microscopy, and the protein was shown to be located on the TTM, the location being probably its external surface and opposite to feet which occupy the TTM-JSR gap. These results indicate that E-C coupling of cardiac muscle cells is mediated through NORP and that ouabain-PIE occurs through the influence of ouabain on NORP in the E-C coupling process.

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Effects of tetramethylpyrazine (TMP), a major constituent of Ligusticum chuanxiong, on spatial cognitive impairment induced by permanent occlusion of bilateral common carotid arteries (2VO) and scopolamine were investigated using 8-arm radial maze performance in rats. Permanent 2VO produced a severe learning deficit in non-pretrained rats. Daily administration of TMP (3 -10 mg/kg, i.p.) from the 3rd day after permanent 2VO significantly improved the learning deficit. TMP did not influence the impairment of the retention task in the pretrained permanent 2VO rats, but it tended to reduce the number of errors elevated by 3-min delay interposition in these rats. In the scopolamine model, scopolamine (0.3 mg/kg, i.p.) significantly decreased the initial correct response and increased the number of errors. Single administration of TMP (1-3 mg/kg, i.p.) dose-dependently reversed the scopolamine-induced impairment of the maze performance. These results suggest that TMP has therapeutic potential for the treatment of dementia caused by cholinergic dysfunction and/or decrease of cerebral blood flow.

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Modulation of learning and memory acquisition, retention and retrieval in the one trial passive avoidance learning task in mice by three inescapable stresses, i.e., footshock (FS), psychological (PSY) and forced swimming (SW) were investigated. Pre-, post-training and pre-test FS-stress (2 mA, 0.2 Hz, 1 sec for 30 min) and pre-training PSY-stress (communication box, 5 min) resulted in enhanced test latencies. On the contrary, SW-stress (20°C, 5 min) immediately or 1 hr after training impaired retention latencies that tended to recover after 2 hr post-training SW-stress, suggesting that at least 2 hr are required to consolidate newly acquired information. In contrast, pre-stress naloxone (Nx), which did not affect FSand PSY-stress induced facilitatory effects, returned to control levels the impaired retention latencies induced by SW-stress. Taken collectively, these results imply the involvement of an opioid-dependent mechanism in the modulation of memory by SW-stress and non-opioid in the case of FS- and PSY-stress. Furthermore, they suggest that different mechanisms are involved in stress-induced memory modifications and the production of stress-induced analgesia (SIA) since in the latter, FS and PSY but not SW stress produce Nx-sensitive antinociception.

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The effects of AH 21-132, a type III and IV phosphodiesterase (PDE) inhibitor, on allergic reactions in the airway were studied by comparing them with the effects of rolipram, a type IV PDE inhibitor, and aminophylline, a non-selective PDE inhibitor. The following results were obtained: 1) AH 21-132, rolipram and aminophylline inhibited the antigen-induced contraction of isolated guinea pig tracheal muscle in vitro. 2) AH 21-132 and aminophylline inhibited antigen-induced histamine release from human lung tissue fragments. 3) Antigen-induced accumulation of inflammatory cells including eosinophils and macrophages in mice bronchoalveolar lavage fluid was clearly inhibited by AH 21-132 and rolipram, but not by aminophylline. 4) AH 21-132, rolipram and aminophylline inhibited immediate phase bronchoconstriction induced by either an intravenous or an aerosol challenge of antigen in guinea pigs. 5) AH 21-132 and rolipram inhibited the aeroantigen challenge-induced late phase increase in the airway resistance in guinea pigs, but aminophylline did not. These results suggest that AH 21-132 has an anti-allergic effect in the airway and that these actions may be beneficial for the treatment of allergic bronchial asthma.

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We determined the changes in gastric functions and systemic blood pressure in response to thyrotropin-releasing hormone (TRH) simultaneously in anesthetized rats and examined the role of the sympathetic nervous system in these changes. TRH injected i.c. increased gastric acid secretion, contraction and mucosal blood flow, and produced hemorrhagic lesions in the glandular stomach. These responses to TRH were almost completely inhibited by bilateral cervical vagotomy or atropine. The increased gastric acid secretion and contraction in response to TRH were significantly augmented by pretreatment with yohimbine but not with prazosin. Bilateral adrenalectomy also potentiated the gastric acid secretory and contractile responses to TRH. Neither prazosin, yohimbine nor adrenalectomy had any appreciable effect on the increased gastric mucosal blood flow induced by TRH. TRH-induced gastric mucosal lesions were significantly aggravated by yohimbine and adrenalectomy. In vagotomized rats, TRH significantly suppressed the gastric functional changes induced by electrical stimulation of the vagus nerves. These data suggest that while gastric functional changes and mucosal lesions induced by TRH mainly occur through stimulation of the vagus nerves, these responses are extensively modified by the sympathetic nervous system including the adrenal glands.

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Xanthine derivatives, pentoxifylline, aminophylline, theophylline and chinoin-170, increased phosphatidylcholine secretion in a primary culture of rat type II pneumocytes. However, these xanthines alone had no effect on intracellular cAMP levels in type II pneumocytes. In contrast, terbutaline-induced secretion of phosphatidylcholine was augmented by these xanthines, and the augmentation depended on the increase of cAMP levels. These results suggest that the xanthines induce phosphatidylcholine secretion possibly through cAMP-dependent and cAMP-independent pathways in the primary culture of rat type II pneumocytes.

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3, 9-Dihydroxy-5H-benzofuro[3, 2-c]quinoline-6-one (KCA-012), the chemical structure of which is closely similar to that of the phytoestrogen coumestrol, inhibited parathyroid hormone-, 1α, 25-dihydroxyvitamin D₃- and prostaglandin E₂-induced bone resorption of cultured fetal rat bones. KCA-012 also increased the calcium content of 9-day chick embryonic femur cultured in vitro. KCA-012 did not show any estrogenic activity as determined by an increase in the uterine weight of ovariectomized rats, whereas coumestrol did. These results indicate that KCA-012 has no estrogenic activity and has unique effects of inhibiting bone resorption and stimulating bone mineralization.

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We investigated the structure-activity relationship for the neurotrophic activity of spermine by comparing the effects of several synthetic spermine analogues on the survival of cultured rat hippocampal neurons. N, N'' -bis(3-aminopropyl)-1, 6-hexanediamine and N, N'' -bis(3-aminopropyl)-1, 3-propanediamine did not promote the neuronal survival, suggesting that the central butanediamine structure is essential for the neurotrophic activity. Furthermore, N¹, N¹²-bis(ethyl)spermine significantly promoted the neuron survival, but its maximum effect was smaller than that of spermine, indicating that two terminal primary amines are required for full agonist activity. The structural requirement for the neurotrophic activity of spermine was different from those for any other reported activities of polyamines.

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We found that substances present in monkey cerebrospinal fluid (CSF) could inhibit [³H]-paroxetine binding in monkey brain preparations. The molecular weight of one of these inhibitory substances was approximately 2000, which is in agreement with earlier studies using human CSF. We also found that the inhibitory effect of the substances present in monkey CSF on [³H]-paroxetine binding decreased after 8 weeks of chronic fluvoxamine (5 mg/kg day, p.o.) treatment. These results suggest that the ability of a drug to decrease the activity of endogenous 5-HT uptake modulators may related to its antidepressive effects.

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We have suggested that cyclopiazonic acid (CPA), a Ca²⁺-ATPase inhibitor, produces nitric oxide (NO) by triggering a Ca²⁺-influx resulting from Ca²⁺-depletion in the endoplasmic reticulum of endothelial cells. The tyrosine kinase inhibitor methyl 2, 5-dihydroxycinnamate, while having no effect on relaxations induced by A23187 or Na nitroprusside, did inhibit the CPA-induced relaxation and cyclic GMP formation in rat aorta. Tyrosine kinase may participate in endothelial NO synthesis through activation of a Ca²⁺ entry mechanism.

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