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To investigate possible developmental changes in the physiological effect of atrial natriuretic peptide (ANP) after birth, we studied the effect of ANP on the slice cGMP content and the particulate guanylate cyclase activity of aorta, kidney and lung in neonate, 2-week-old and adult rats of both sexes. Incubation with human ANP(99–126) (hANP) increased significantly the slice cGMP content of aorta, kidney and lung in three ages of rats. The hANP-stimulated fraction of cGMP contents of kidney decreased, that of lung increased with development, whereas that of aorta showed no significant change. Consistently, the hANP-responsive particulate guanylate cyclase activity decreased in kidney, increased in lung during development, without significant developmental change in aorta. These results indicate a differential change in the effect of hANP on the slice cGMP content among tissues during development. The developmental change in the effect of hANP on slice cGMP content is probably caused by the ontogenetic change in activation of ANP receptor-linked guanylate cyclase.

Using streptozotocin (STZ)-diabetic mice, we examined the respiratory distress induced by arachidonic acid. Male ddY mice were made diabetic by injecting STZ (170 mg/kg, i.p.) 2 weeks prior to the experiment. Control mice received the vehicle (citrate buffer, pH 4.6). The duration of respiratory distress was observed by a slow i.v.-injection of sodium arachidonic acid (AA) into the caudal vein of mice at the dose of 50 mg/kg. Aspirin was i.p.-administered 30 min before AA. OKY-046 (specific thromboxane A2 (TXA2) synthetase inhibitor), OP-41483 (stable prostacyclin analog) and 9, 11 epithia-11, 12-methano-TXA2 (STA2, stable TXA2 analog) were i.v.-administered 30 min before AA. The duration of respiratory distress induced by AA was significantly reduced in STZ-diabetic mice. Aspirin (10-50 mg/kg) and OKY-046 (25-100 mg/kg) enhanced the AA-induced respiratory distress in STZ-diabetic mice. OP-41483 (1-100 micrograms/kg) reduced the AA-induced effect in both control and STZ-diabetic mice. STA2 (10 micrograms/kg) enhanced the AA-induced effect in both control and STZ-diabetic mice. ONO-1078 (1-10 mg/kg) did not affect the AA-induced effect in both control and STZ-diabetic mice. TMK-688 (0.01-1 mg/kg) reduced the AA-induced effect in the control mice, but not in the STZ-diabetic mice. These results suggest an involvement of leukotriene in the respiratory response to AA in diabetic mice, especially when cyclooxygenase and TXA2 synthetase are inhibited.

Effects of bombesin on brown adipose tissue (BAT) thyroxine (T4) 5'-deiodinase (5'D) activity and rectal temperature were examined in male mice. Immediately following an intracerebroventricular (ICV) or intravenous (IV) injection of bombesin (0.1-100 ng/animal) or vehicle (20 mM bacitracin dissolved in 0.9% saline), the mice were placed in a room at 4 degrees C or 22 degrees C for 30, 60, 120 or 240 min. The ICV injection of bombesin dose-dependently lessened cold-induced increase in BAT 5'D activity and increased hypothermia determined at 120 min of cold exposure, whereas the IV injection of bombesin was without effect. Bombesin (ICV)-induced hypothermia preceded the inhibition of BAT 5'D activity by at least 30 min at 4 degrees C. BAT 5'D activity was not affected by ICV injection of bombesin in mice kept at 22 degrees C, although the rectal temperature was significantly decreased. Bombesin thus appears to prevent cold-induced increase in T4 5'D activity in mouse BAT by its central effect. Bombesin-induced excessive hypothermia itself and/or the decrease in sympathetic tone of BAT by bombesin might decrease cold-induced increase in BAT 5'D activity.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.

Age-related changes of strychnine-insensitive glycine receptors in the rat brain were studied through quantitative in vitro autoradiography with 3H-glycine. 3H-glycine binding sites were most concentrated in the hippocampus, cerebral cortex, and olfactory tubercle, and moderate densities of binding sites were located in the striatum, nucleus accumbens, amygdala, and certain thalamic nuclei. Low densities of 3H-glycine binding sites were observed in the lateral septal nucleus, midbrain nuclei such as the superior colliculus and central gray matter, and granule cell layer of the cerebellum. In aged animals, severe decline of 3H-glycine binding sites was observed in the telencephalic regions including the hippocampus and cerebral cortex. On the other hand, decrease of binding sites in the midbrain nuclei was of lesser degree, and there were no changes in the cerebellum. These results suggest that the decrease of glycine receptors in particular brain regions has some relation with changes of neuronal functions associated with aging process in these areas. The glutamatergic neuronal system, particularly the N-methyl-D-aspartate (NMDA) subtype, has been considered to play an important role in learning and memory. Taking into consideration that strychnine-insensitive glycine receptors are contained in the NMDA receptor complex, the present study implies that the decrease of glycine receptors may be involved in impairments of learning and memory occurring in aged brains.

To evaluate whether the L-arginine-nitric oxide (NO) pathway is involved in the regulation of regional blood flow to brown adipose tissue (BAT), the effects of two specific NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), on the blood flow to interscapular brown adipose tissue (IBAT) were studied in urethane-anesthetized rats. Regional blood flow in IBAT was measured with laser-Doppler flowmetry. An intravenous injection of L-NAME and L-NMMA, but not of either D-enantiomer, caused a transient and dose-dependent increase in IBAT blood flow. Dose-response curves for these NO synthase inhibitors showed that L-NAME was more potent than L-NMMA in increasing IBAT blood flow. We also observed a concomitant pressor effect accompanied by a slight decrease in heart rate following intravenous injection of L-NAME and L-NMMA. An elevation of IBAT blood flow and blood pressure induced by both L-NAME and L-NMMA was reversed by L-arginine in an enantiomerically specific manner. The increase in IBAT blood flow induced by NO synthase inhibitors was of shorter duration and less sensitive to L-arginine than the increase in blood pressure. Our results show that the IBAT blood flow is increased by inhibition of NO synthase and that the response of IBAT vasculature to NO synthase inhibitors is different from that of the resistance vessels which regulate blood pressure. The involvement of L-arginine-NO pathways in modulating microcirculation in IBAT is suggested.

Developmental changes in the response to ganglionic stimulants, nicotine and dimethylphenylpiperazinium, were investigated in rat isolated duodenum by recording isotonic mechanical activity. The duodenal response to nicotine/dimethylphenylpiperazinium (3 x 10(-7) to 10(-3) M) in neonatal rats was contraction, which was blocked by hexamethonium, tetrodotoxin and hyoscine. The response to nicotine/dimethylphenylpiperazinium (10(-6) to 10(-4) M) in the adult duodenum was relaxation, which was blocked by tetrodotoxin and hexamethonium, but by neither guanethidine nor hyoscine. The transition of the response to nicotine/dimethylphenylpiperazinium from contraction to relaxation occurred at around the 3rd postnatal week. Nicotine-induced relaxation of adult duodenum was significantly inhibited by preincubation with alpha-chymotrypsin, a proteolytic enzyme, and a combination of nucleotide pyrophosphatase and 8-phenyltheophylline, a P1 purinoceptor antagonist. Nicotine-induced relaxation was desensitized by alpha, beta-methylene ATP, a stable P2x purinoceptor agonist. These results suggest that the contractile response of isolated duodenum to nicotine is mediated through cholinergic transmission in neonatal rats and the relaxant response is mediated through non-adrenergic, non-cholinergic transmission, which involves both peptidergic and purinergic transmission, in adult rats.

The response of isolated duodenum to neuropeptide Y (NPY) was studied isotonically in neonatal and adult rats. Neuropeptide Y (10 −8 to 10 −6 M ) elicited a biphasic contraction of isolated duodenum from neonatal rats, but monophasic and weak contraction of adult duodenum. The first phase of NPY-induced contraction of neonatal duodenum was concentration dependent and partially inhibited by preincubation with tetrodotoxin, a Na + channel blocker, hyoscine, a muscarinic antagonist, suramin, a P 2 purinoceptor antagonist, and indomethacin, an inhibitor for prostaglandin biosynthesis. Neuropeptide Y(13–36), a specific Y 2 NPY receptor agonist, elicited a concentration-dependent contraction of neonatal rat duodenum. The duodenal response to NPY thus changes during development in rats. Both cholinergic and purinergic transmission and prostaglandin biosynthesis may be involved in the NPY-induced contraction of neonatal duodenum. Neuropeptide Y-induced contraction may be mediated through presynaptic Y 2 receptors.

Receptors for excitatory amino acid, L-glutamate, have been classified into three subtypes named as N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate receptors. In the present study, an effect of age on binding sites of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA), a QA agonist, was studied in the rat brain through quantitative in vitro autoradiography. 3H-AMPA binding sites were most concentrated in the hippocampus and cerebral cortex where glutamate receptors have been demonstrated to play a role in synaptic transmission. In aged rats, 3H-AMPA binding sites in the hippocampus and cerebral cortex were not significantly changed. In our previous studies, it was noticed that strychnine-insensitive glycine receptors, which functionally coupled with NMDA receptors, showed marked age-dependent decreases in telencephalic regions. It has been shown that the glutamatergic neuronal system is involved in learning and memory. Nevertheless, it is considered that AMPA binding sites are not involved in the decline of neuronal functions, especially impairment of learning and memory, accompanying with aging process.

The N-methyl-D-aspartate (NMDA) receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In this study, an effect of age on NMDA antagonist binding sites was investigated through quantitative in vitro autoradiography with 3H-3-((+)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). 3H-CPP binding sites were most concentrated in the hippocampus and cerebral cortex where NMDA receptors have been demonstrated to be involved in synaptic transmission. In aged rats, 3H-CPP binding sites in the hippocampus and cerebral cortex were not significantly changed. As for other brain regions, there was an age-dependent decline of binding sites only in the caudate-putamen and nucleus accumbens. Our previous study revealed that strychnine-insensitive glycine receptors were markedly reduced in telencephalic regions in the aged rat brain. Taking these findings into consideration, it is concluded that glycine receptors but not NMDA antagonist binding sites are severely altered in telencephalic regions of aged animals. It is considered that within the NMDA receptor complex, glycine receptors may be selectively affected in the aging process.

The analgesic effects of μ-and κ-opioid agonists, including morphine, FK33,824, U50,488H, tifluadom and bremazocine, have been determined in C57BL/6J-bgJ (beige) and CXBK mice which are hyporesponsive to μ-opioid receptor-mediated analgesia compared with those of control mice (C57BL/6J (C6J), C57BL/6By (C6By), BALB/cBy (BALB)) using an abdominal constriction assay. The analgesic effect of subcutaneously administered morphine and FK33,824 in both beige and CXBK mice was significantly reduced compared with the controls and the analgesic effect of U50,488H and tifluadom in beige mice was significantly reduced compared with the wild strain (C6J). No reduction of analgesic effect of U50,488H and tifluadom was seen in CXBK compared with its progenitor strains, C6By and BALB, except for a reduction of the effect of tifluadom in CXBK compared with C6By. There was no strain difference in the bremazocine-induced analgesia. These results suggest that the beige mouse has a deficit in analgesia mediated by both μ-and κ-opioid receptors, whereas the CXBK is deficient only in the μ-opioid receptor-mediated analgesia.

Although it has been established that pancreatic cholecystokinin (CCK) receptors are coupled to phosphatidylinositol turnover, the events which follow activation of CCK receptors in the central nervous system have not received much attention. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]1) in response to CCK peptides were measured in single cultured rat striatal neuron by fura-2 fluorometry. CCK peptides dose-dependently increased [Ca2+]i in a monophasic manner. The order of the potencies of CCK peptides to increase [Ca2+]i was as follows: caerulein greater than CCK-8 greater than desulfated CCK-8 greater than CCK-4. The effect of caerulein was completely blocked in a Ca2(+)-depleted medium. In addition, omega-conotoxin GVIA completely inhibited the effect of caerulein, while neither nifedipine nor verapamil affected it. Our results indicate that CCK receptors couple to N-type voltage-sensitive Ca2+ channels in cultured rats striatal neurons.

1. Response to the opioids of isolated small intestine of Suncus murinus, an insectivore, was examined and compared with that of guinea-pig ileum. 2. The mechanical response to morphine and U50,488H, preferential mu- and kappa-agonist respectively, was relaxation which was antagonized by tetrodotoxin and naloxone in the Suncus small intestine. 3. Methionine5-enkephalin and D-alanine2, D-leucine5-enkephalin, both preferential delta-agonists, elicited contraction or relaxation in the Suncus small intestine, while enkephalins elicited sustained contraction in the tetrodotoxin-pretreated intestine. 4. In the guinea-pig ileum, the response to morphine and enkephalins was consistently relaxation which was antagonized by tetrodotoxin and naloxone. 5. Morphine, D-alanine2,D-leucine5-enkephalin and U50,488H inhibited electrically-evoked twitch responses of intestine in a concentration-dependent manner in both animals. 6. The relative potencies calculated from IC50 values of opioids for twitches followed the order, D-alanine2,D-leucine5-enkephalin greater than U50,488H greater than morphine in the Suncus small intestine, whereas U50,488 greater than D-alanine2,D-leucine5-enkephalin greater than morphine in the guinea-pig ileum. 7. Suncus small intestine has unique features such that D-alanine2,D-leucine5-enkephalin was most potent in inhibiting electrically-evoked twitches and that enkephalins induced contraction following tetrodotoxin pretreatment.

The effects of intravenously infused adenosine (ADO) and its analogues, N6-R(-)-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamide adenosine (NECA), on the blood flow to interscapular brown adipose tissue were studied in urethane-anesthetized rats. ADO and NECA, but not R-PIA, caused a dose-dependent increase in interscapular brown adipose tissue blood flow. The relative potency of these compounds to increase this blood flow was NECA greater than ADO greater than R-PIA. Infusing R-PIA and NECA intravenously caused a fall in diastolic blood pressure and heart rate whereas an infusion of ADO up to 380 nmol/rat per min did not elicit any systemic cardiovascular effect. A 10-mg/kg dose of alkylxanthines such as caffeine, 8-phenyltheophylline (8-PT) and 8-sulfophenyltheophylline (8-SPT) inhibited the half-maximum responses of interscapular brown adipose tissue blood flow to ADO by 41, 71 and 65% respectively indicating that caffeine is less potent than 8-PT and 8-SPT. The ADO uptake inhibitor, dipyridamole, significantly potentiated the ADO effect. These results suggest that the increased interscapular brown adipose tissue blood flow caused by ADO might possibly be mediated by A2 receptors.

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor – guanylate cyclase system and opioid receptors.Key words: cGMP, mouse development, carbachol, morphine.

Significance of the intercapsular brown adipose tissue (IBAT) in the thermal response to chlorpromazine (CPZ) during acute and chronic cold exposure was studied in rats. Adult male Wistar-Imamichi rats were exposed to 4 degrees C for 1 (cold-exposed) or 30 days (cold-acclimated). Intraperitoneal injection of 10 mg/kg CPZ caused a more marked and lasted hypothermia in acute cold-exposed rats as compared with that seen in control or in cold-acclimatized rats. Hypothermia induced by CPZ was unaffected by removal of IBAT either in control or acute cold-exposed rats. But in cold-acclimated rats, the removal of IBAT potentiated the hypothermia by CPZ. The relative weight of IBAT in cold-acclimated rats was about 3 times heavier than that in control and acute cold-exposed rats. CPZ had no effect on the relative weight of IBAT in all groups examined. Total lipids in IBAT showed no significant changes following CPZ administration in rats of all groups. In acute cold-exposed rats, serum FFA level progressively decreased after CPZ injection. The noradrenaline concentration in IBAT increased after chronic cold exposure and CPZ suppressed this elevation. The results suggest that the thermogenesis related sympathetic activity in the IBAT plays a mediatory role in thermal response to CPZ during cold acclimation.

1 The effects of adenosine and adenine nucleotides were studied in rat duodenum from postnatal day 1 to day 70. The mechanical activity of duodenal segments was recorded through an isotonic transducer connected to a polygraphic recorder. 2 In rat duodenal segments, adenosine-5′-triphosphate (ATP, 10−4m) and adenosines-5′-diphosphate (ADP, 10−4m) produced a contractile response on postnatal day 1. This response increased with age, peaking on day 7, followed by a gradual decrease and was non-existent by day 21. In contrast, a relaxant response to ATP and ADP was apparent on day 21, and continued to increase up to day 70. 3 The contraction caused by ATP was inhibited by indomethacin or the P2y-purinoceptor antagonist, reactive blue-2 but not by tetrodotoxin or hyoscine. Thus, it may be mediated by production of prostaglandin through the P2y-purinoceptor. The relaxation produced by ATP was inhibited by reactive blue-2 but not by tetrodotoxin, guanethidine or the P1-purinoceptor antagonist, 8-phenyltheophylline indicating that ATP acts on smooth muscle directly through the P2y-purinoceptor. The pD2 for the contractile response to ATP was 5.15 on day 7 and that for the relaxant response, 6.64 on day 70. 4 Adenosine (10−4m) and adenosine-5′-monophosphate (AMP, 10−4m) elicited no response before day 14. On day 14, both adenosine and AMP produced a small relaxant response which increased with age. The relaxation produced by adenosine was inhibited by 8-phenyltheophylline but not by tetrodotoxin or guanethidine, indicating that it is mediated by an action on the P1-purinoceptor of smooth muscle. 5 It is evident from these results that in neonatal rat, a contractile response to ATP and ADP occurs initially in the duodenum, followed by a relaxant response to adenosine and AMP on day 14 and to ADP and ATP on day 21. 6 The smooth muscle of rat duodenum may tentatively be concluded to contain separate purinoceptors for adenosine and AMP (P1) and ADP and ATP (P2) and the responses to P1- and P2-agonists change during the course of development.

The present study deals with the hypnotic effect of pentobarbital (Pento) in relation to its metabolism in hepatic microsomes in streptozotocin (STZ, 170 mg/kg, i.p.) injected mice. Liver weight (mg/10 g body wt.) of STZ-treated mice was larger than that of the controls throughout the experimental period. Although the shortening of sleeping time induced by Pento (60 mg/kg, i.p.) was always observed, Pento-metabolizing enzyme activity (by the method of Kato et al., 1964) increased in mice with diabetes for 2 and 4 weeks but decreased in mice with diabetes for 8 weeks. Induction following phenobarbital (100 mg/kg, s.c.) and inhibition by SKF 525-A (10 mg/kg, i.p.) of hepatic metabolizing enzyme were found in both control and mice with diabetes for 2, 4 and 8 weeks, but these were not definitely correlated to their hepatic Pento-metabolizing enzyme activities. STZ-induced hyperglycemia and shortening of sleeping time by Pento were completely prevented by the pretreatment with nicotinamide (500 mg/kg, i.p.). NPH-insulin injection partially decreased hyperglycemia in STZ-diabetic mice, but sleeping time by Pento was not significantly affected. These results suggest that the hyposensitivity to Pento in STZ-diabetic mice is partially related to an abnormality of metabolism in liver such as the hyperglycemic state.

Met-enkephalin (ENK) induced a dose-dependent transient relaxation in vitro in the rat duodenum while morphine neither induced a response nor affected the response to ENK. The response was not blocked by tetrodotoxin but was abolished by naloxone. The response of guinea-pig duodenum to ENK was either relaxation or pulsatile contractions depending on the dose and was abolished by tetrodotoxin.

The present study on ACTH-administered suckling rats was undertaken to monitor the late behavioral sequelae due to the effects of this drug. The locomotor activity of rats treated with ACTH in the suckling period increased significantly as to the gross size of movements when compared with control rats. The increase in locomotor activity was correlated with the suppression of CNPase activity. These results show that administration of ACTH during the suckling period has suppressive effects on the development of the brain and behavior, and that these effects persist for a longer period after termination of the administration of ACTH than the direct effect of the peptide on endocrine functions.

The effects of thyrotropin-releasing hormone (TRH) administered via the lateral ventricle on the blood flow to the interscapular brown adipose tissue (IBAT) was studied in urethane anaesthetized rats. TRH in i.c.v. doses of 50 or 5 micrograms/kg produced a significant and transient decrease in IBAT blood flow. The effect of 5 micrograms/kg TRH was blocked by bilateral surgical denervation. TRH caused a comparable decrease in blood flow to both the innervated pad of unilaterally denervated rats and the intact pads of non-operated rats whereas no response to TRH was observed in the denervated pad. Adrenal demedullation partly inhibited but did not completely abolish the effect of TRH on IBAT blood flow and lessened the increase in serum adrenaline caused by TRH. The effect of TRH was dose dependently suppressed by pretreatment with prazosin 15 min prior to TRH. Yohimbine did not influence the effect of TRH on IBAT blood flow. Central administration of TRH affects the regional blood flow to IBAT and appears to interfere with neural mechanisms that involve mediation by the alpha 1-adrenoceptors on the vasculature. The effect is in part due to circulating adrenaline from the adrenal medulla.

Electroenteromyographic activity (EMG) of the duodenum was recorded in pentobarbital-anesthetized rats. TRH intraventricularly administered to rats produced changes in EMG such as increased amplitude, decreased frequency of slow waves and the association of bursts of spike potentials with nearly every cycle of the basal electric rhythm (BER). The effect was selectively prompt and marked in the EMG of proximal duodenum. The response was abolished by vagotomy or atropine injection and no response was elicited in the neonatally 6-OHDA-treated rat. Hypophysectomy, cord-transection or acute i.v. injection of 6-OHDA did not block the response. In the brain, TRH seems to stimulate the neuronal system controlling the vagus efferents involved in the regulation of the duodenal enteric nervous system which in turn modulates the myogenic excitability of the duodenum.

A tetrodotoxin- and hexamethonium-sensitive response to thyrotropin-releasing hormone (TRH), an in vitro contraction, first appeared in the duodenum of the 3 day old rat, was increased by day 16, decreased there-after and was extinguished after weaning. A tetrodotoxin-resistant response to TRH, a relaxation, appeared at day 13 and became distinct after weaning. The development of neurogenic receptivity for TRH precedes the appearance of myogenic receptivity in the rat duodenum.