Nociceptin and dynorphin A(1-17) produce antianalgesia through independent systems in mice

The administration of dynorphin A(1-17), Dyn, intrathecally (i.t.) or of nociceptin, intracerebroventricularly (i.c.v.) produces antianalgesic actions against i.t. morphine in the tail flick test in mice. The antianalgesic action of nociceptin is mediated by spinal PGE2 and attenuated by i.t. PGD2 or indomethacin. The Dyn response is mediated by release of IL1beta in the spinal cord to activate an ascending pathway to the brain and in turn releases IL1beta in the brain which activates a descending pathway to the spinal cord. The present work investigated the possibility that the action of IL1beta in the Dyn system might release prostaglandins so that the Dyn and nociceptin antianalgesic systems would overlap at these points. The results indicated that in the Dyn system neither the IL1beta in the spinal cord or brain implicated prostaglandin release because i.t. and i.c.v. PGD2 and indomethacin did not affect Dyn-induced antianalgesia. In addition, nociceptin-induced antianalgesia did not involve components in the Dyn system. Thus, the Dyn and nociceptin antianalgesic systems did not overlap and each were independent systems.     

Prostaglandin E2 stimulates adrenocorticotrophin and cortisol secretion via a hypothalamic site of action in fetal sheep.

The hypothesis that prostaglandins stimulate fetal adrenocortical activity via a central site of action within the fetal brain was tested in chronically catheterized fetal sheep. At day 120 gestation (term = 145 days) fetal sheep were surgically prepared with catheters in the lateral cerebral ventricle, jugular vein and carotid artery and experiments began five days later. Intravenous (i.v.) infusion of prostaglandin E2 (30 or 120 micrograms.h-1) caused a significant dose-related increase in fetal plasma concentrations of ACTH. Despite this increase in ACTH, cortisol was only stimulated after the highest dose of prostaglandin E2. Intracerebroventricular (i.c.v.) infusion of PGE2 (30 micrograms.h-1) also stimulated ACTH secretion although the peak response was delayed and considerably less compared with the same dose administered intravenously. Prostaglandin F2 alpha administered i.v. or i.c.v. had no effect on circulating concentrations of either ACTH or cortisol. These data provide evidence that prostaglandin E2 can stimulate fetal ACTH secretion by acting in the fetal brain. Furthermore, the greater release of ACTH after i.v. compared with i.c.v. prostaglandin E2 suggests that a site of action other than the brain, such as the pituitary gland, may also be important. These results provide further evidence that during late gestation circulating prostaglandins can act to stimulate fetal pituitary-adrenal maturation.     

Non-opioid nociceptive activity of human dynorphin mutants that cause neurodegenerative disorder spinocerebellar ataxia type 23

We previously identified four missense mutations in the prodynorphin gene that cause human neurodegenerative disorder spinocerebellar ataxia type 23 (SCA23). Three mutations substitute Leu(5), Arg(6), and Arg(9) to Ser (L5S), Trp (R6W) and Cys (R9C) in dynorphin A(1-17) (Dyn A), a peptide with both opioid activities and non-opioid neurodegenerative actions. It has been reported that Dyn A administered intrathecally (i.t.) in femtomolar doses into mice produces nociceptive behaviors consisting of hindlimb scratching along with biting and licking of the hindpaw and tail (SBL responses) through a non-opioid mechanism. We here evaluated the potential of the three mutant peptides to produce similar behaviors. Compared to the wild type (WT)-peptide, the relative potency of Dyn A R6W, L5S and R9C peptides for SBL responses was 50-, 33- and 2-fold higher, and Dyn A R6W and L5S induced the SBL responses at a 10-30-fold lower doses. Dyn A R6W was the most potent peptide. The SBL responses induced by Dyn A R6W were dose dependently inhibited by morphine (i.p.; 0.1-1 mg/kg) or MK-801, an NMDA ion channel blocker (i.t. co-administration; 5-7.5 nmol). CP-99,994, a tachykinin NK1 receptor antagonist (i.t. co-administration; 2 nmol) and naloxone (i.p.; 5 mg/kg) failed to block effects of Dyn A R6W. Thus, similarly to Dyn A WT, the SBL responses induced by Dyn A R6W may involve the NMDA receptor but are not mediated through the opioid and tachykinin NK1 receptors. Enhanced non-opioid excitatory activities of Dyn A mutants may underlie in part development of SCA23.     

Effect of neuropeptide Y on the hypothalamic-pituitary-adrenal axis in the dog

There is increasing evidence that neuropeptide Y (NPY) affects the release of pituitary hormones, including adrenocorticotropic hormone (ACTH). The present study was designed to clarify the mechanism by which NPY activates the hypothalamic-pituitary-adrenal (HPA) axis in the dog. Mongrel dogs were equipped with a chronic cannula allowing intra-third (i.t.v.) or intra-lateral (i.l.v.) cerebroventricular administration. A 1.19 nmol, i.t.v. dose of NPY produced as great an ACTH and cortisol response as did equimolar ovine corticotropin releasing factor (CRF). This action of NPY was dose-dependent and shared by peptide YY (PYY) and pancreatic polypeptide (PP), other members of the PP family peptide. Intravenously (i.v.) administered NPY (1.19-11.9 nmol) was much less potent than i.v. CRF in stimulating ACTH and cortisol secretion. However, i.v. NPY significantly increased plasma ACTH and cortisol concentrations, raising the possibility that NPY may modulate the activity of corticotrophs. We have next investigated the possible relationship between NPY and CRF on the HPA axis. Pretreatment with a novel CRF antagonist, alpha-helical CRF9-41 (130.9 nmol i.t.v. or 261.8 nmol i.v.), partly but significantly attenuated the ACTH and cortisol responses to i.t.v. NPY (1.19 nmol). Furthermore, adding a subthreshold dose of i.t.v. NPY (0.119 nmol) to i.t.v. CRF (1.19 nmol) or i.v. NPY (2.38 nmol) to i.v. CRF (0.595 nmol) resulted in the potentiation of CRF-induced ACTH secretion. These results indicate that NPY may activate the HPA axis in concert with CRF probably at hypothalamic and/or pituitary levels. The present findings that NPY evokes ACTH secretion and potentiates the effectiveness of CRF as a secretagogue, together with high concentrations of NPY in the hypothalamus and pituitary portal blood, suggest that NPY is involved in the multihormonal control of ACTH release.     

Excitation of nigral dopamine neurons by the GABA(A) receptor agonist muscimol is mediated via release of glutamate

Previous electrophysiological studies have shown that the GABA(A)-receptor agonist muscimol is able to markedly increase the firing rate of rat nigral dopamine (DA) neurons. This action of the drug is paradoxical since local microiontophoretic application of the drug is associated with a clearcut inhibition of this neurons. In the present electrophysiological study, an attempt was made to analyze the mechanism of this action of the drug. Administration of muscimol (0.25-4.0 mg/kg, i.v.) was associated with a dose-dependent increase in firing rate as well as an increased bursting activity of the nigral DA neurons. Both these effects of muscimol were clearly antagonised by intravenous administration of the NMDA receptor antagonist MK 801(1 mg/kg) or by intracerebroventricular administration of the broad-spectrum excitatory amino acid receptor antagonist kynurenic acid. Furthermore, pretreatment with PNU 156561A (40 mg/kg, i.v., 5-8h), a compound that raised endogenous kynurenic acid levels about 9 times, also clearly antagonised the actions of muscimol. Indeed, this treatment reversed the excitatory action of muscimol into an inhibitory effect on the nigral DA neurons. Here, we report that the excitatory action of muscimol is mediated indirectly by release of glutamate.     

The influence of NMDA receptor agonist and antagonist on morphine state-dependent memory of passive avoidance in mice

The measurement of step-down latency in passive avoidance has been used to study memory in laboratory animals. The pre-training injection of 5 mg/kg morphine impaired memory, which was restored when 24 h later the same dose of the drug was administered. To explore the possible involvement of NMDA modulators on morphine-induced memory impairment, we have investigated the effects of intracerebroventricular (i.c.v.) administration of NMDA and the competitive NMDA antagonist, DL-AP5, on morphine-induced memory impairment or recall, on the test day. Morphine (5 mg/kg, s.c.) was administered 30 min before training to induce impairment of memory and 24 h later, 30 min before test to improve it. Pre-test administration of NMDA (0.00001, 0.0001 and 0.001 microg/mouse, i.c.v.) did not alter the retention latency compared to the saline-treated animals. But restored the memory impairment induced by pre-training morphine (5 mg/kg, s.c.). Pre-test administration of DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) by itself decreased the retention latencies. The same doses of DL-AP5 increased pre-training morphine-induced memory impairment. Co-administration of NMDA (0.0001 and 0.001 microg/mouse, i.c.v.) and morphine (5 mg/kg, s.c.) on the test day increased morphine memory improvement. Conversely, DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) inhibited morphine-induced memory recall. It is concluded that NMDA receptors may be involved, at least in part, in morphine state-dependent learning in mice.     

Insulin sensitivity is mediated by the activation of the ACh/NO/cGMP pathway in rat liver

The hepatic parasympathetic nerves and hepatic nitric oxide synthase (NOS) are involved in the secretion of a hepatic insulin sensitizing substance (HISS), which mediates peripheral insulin sensitivity. We tested whether binding of ACh to hepatic muscarinic receptors is an upstream event to the synthesis of nitric oxide (NO), which, along with the activation of hepatic guanylate cyclase (GC), permits HISS release. Male Wistar rats (8-9 wk) were anesthetized with pentobarbital sodium (65 mg/kg). Insulin sensitivity was assessed using a euglycemic clamp [the rapid insulin sensitivity test (RIST)]. HISS inhibition was induced by antagonism of muscarinic receptors (atropine, 3 mg/kg i.v.) or by blockade of NOS [NG-nitro-L-arginine methyl ester (L-NAME), 1 mg/kg intraportally (i.p.v.)]. After the blockade, HISS action was tentatively restored using a NOdonor [3-morpholynosydnonimine (SIN-1), 5-10 mg/kg i.p.v.] or ACh (2.5-5 microg.kg(-1).min(-1) .i.p.v.). SIN-1 (10 mg/kg) reversed the inhibition caused by atropine (RIST postatropine 137.7 +/- 8.3 mg glucose/kg; reversed to 288.3 +/- 15.5 mg glucose/kg, n = 6) and by L-NAME (RIST post-L-NAME 152.2 +/- 21.3 mg glucose/kg; reversed to 321.7 +/- 44.7 mg glucose/kg, n = 5). ACh did not reverse HISS inhibition induced by L-NAME. The role of GC in HISS release was assessed using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 nmol/kg i.p.v.), a GC inhibitor that decreased HISS action (control RIST 237.6 +/- 18.6 mg glucose/kg; RIST post-ODQ 111.7 +/- 6.2 mg glucose/kg, n = 5). We propose that hepatic parasympathetic nerves release ACh, leading to hepatic NO synthesis, which activates GC, triggering HISS action.     

CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

The effect of the new oral hypoglycemic agent A-4166 on glucose turnover in the high fat diet-induced and/or in the hereditary insulin resistance of rats.

A-4166, a phenylalanine derivative, is a hypoglycemic agent, which has been shown to improve blood glucose levels mainly due to the rapid and short term stimulation of insulin release. Nevertheless, a possible extrapancreatic action of A-4166 has not yet been investigated. Therefore, insulin action (euglycemic hyperinsulinemic 6.4 mU.kg-1.min-1 clamp plus 3H-2-deoxyglucose tracer administration) was studied after 3 weeks on either standard (BD) or high fat (HF) diet in normal control (C) or in hereditary insulin resistant (hHTg) rats which were given a single dose of A-4166 (10 mg per kg BW, i.v.) 60 min after clamp commencement. HF feeding reduced the glucose infusion rate (GIR) required to maintain euglycemia to about 50% of C (p < 0.001). In hHTg rats, HF did not further pronounce the pre-existing decrease of GIR of hHTg animals fed BD. A-4166 changed GIR neither in C, nor in the hHTg group. The estimated glucose disposal (Rd) (C-BD: 32.3 +/- 1.9 vs C-HF: 25.5 +/- 1.9 mg.kg-1.min-1, p < 0.001) and glucose metabolic index (Rg') in skeletal muscles (Q. femoris: C-BD: 25.6 +/- 1.5 vs C-HF: 12.3 +/- 1.1 mmol.100 g-1.min-1, p < 0.001) were reduced by HF in control rats but were not restored by a concomitant bolus of A-4166. Nevertheless, in hHTg rats fed the HF diet a single dose of A-4166 brought back their Rd (hHTg-HF: 23.5 +/- 1.3 vs hHTg-HF plus A-4166: 31.0 +/- 3.5 p < 0.03) and Rg' (Soleus muscle: hHTg-HF: 29.2 +/- 3.2 vs hHTg-HF plus A-4166: 41.3 +/- 4.0) to values of the control group on BD. In summary, a) a single bolus administration of A-4166 to the control or to the insulin resistant hHTg rats, fed either the BD or HF diets, did not abolish the reduction of GIR required to maintain euglycemia during hyperinsulinemic clamps; b) nevertheless, A-4166 caused a significant increase of the estimated plasma glucose disposal (Rd) and skeletal muscle glucose metabolic index (Rg') of hHTG rats fed the HF diet; c) we suggest that A-4166 may have an extrapancreatic action but this needs to be proven using a long-term administration plan of A-4166.     

Effect of subdiaphragmatic vagotomy and cholinergic agents in the hypothalamic-pituitary-adrenal axis activity.

In the present study, we examined whether the vagus nerve is involved in mediating the stimulation of hypothalamic-pituitary-adrenal (HPA) axis by cholinergic muscarinic and nicotinic agonists, carbachol and nicotine. The site of HPA axis muscarinic stimulation was determined using peripheral (i.p.) and intracerebroventricular (i.c.v.) administration of carbachol, atropine sulphate (AtrS) and atropine hydrobromide (AtrBr). The i.p. carbachol-(0.5 mg/kg)-induced corticosterone response was significantly reduced by i.p. pretreatment with AtrBr (0.1 mg/kg), but was not diminished by i.c.v. AtrS (0.1 mug). The increase in corticosterone secretion induced by i.c.v. carbachol (2 microg) was totally abolished by i.c.v. pretreatment with AtrS (0.1 microg) but was not altered by i.p. AtrBr. Subdiaphragmatic vagotomy performed 2 weeks earlier substantially decreased the i.p. carbachol (0.2 mg/kg)-induced ACTH response and markedly augmented ACTH and corticosterone response to a higher dose of carbachol (0.5 mg/kg) in comparison with the responses in sham operated rats. Vagotomy abolished the stimulatory effect of i.p. nicotine in a low dose (1 mg/kg) on ACTH and corticosterone secretion; the ACTH response to higher dose (2.5 mg/kg) was considerably reduced, while corticosterone response remained unaffected. These results suggest that carbachol given i.c.v. evokes considerable corticosterone response by stimulation of central cholinergic muscarinic receptors. A major part of the i.p. carbachol-induced corticosterone secretion results from peripheral cholinergic muscarinic receptor stimulation. Subdiaphragmatic vagotomy moderately intensified the carbachol-induced ACTH and corticosterone secretion. Vagotomy significantly reduced the nicotine-induced ACTH secretion, possibly by the involvement of vagal afferents. The nicotine-induced corticosterone secretion is not exclusively regulated by circulating ACTH but by various intra-adrenal regulatory components.     

Stimulation of gastric acid secretion by progesterone metabolites as neuroactive steroids in anesthetized rats.

The effect of neuroactive progesterone metabolites, 5alpha- and 5beta-pregnan-3alpha-ol-20-one, and their stereoisomers at the 3 C site, 5alpha- and 5beta-pregnan-3beta-ol-20-one, on gastric acid secretion was investigated in urethane-anesthetized rats. Both 5alpha- and 5beta-pregnan-3alpha-ol-20-one dose-dependently (0.3-3 mg x kg(-1), i.v.) stimulated gastric acid secretion with an early onset of action. Their potency and efficacy were almost the equivalent of one another. In contrast, their stereoisomers did not have a significant effect even at 10 mg x kg(-1) (i.v.). The 5beta-pregnan-3alpha-ol-20-one (3 mg x kg(-1), i.v.)-stimulated gastric acid secretion was remarkably inhibited by bilateral vagotomy or pretreatment with atropine (1 mg x kg(-1), i.v.). An antagonist of the GABA(A) receptor, picrotoxin, at 3 and 6 mg x kg(-1) (i.v.), significantly inhibited the 5beta-pregnan-3alpha-ol-20-one (3 mg x kg(-1), i.v.)-stimulated gastric acid secretion. These results indicate that naturally occurring neuroactive steroids, 5alpha- and 5beta-pregnan-3alpha-ol-20-one, stimulate gastric acid secretion in a stereoselective and dose-dependent manner in urethane-anesthetized rats. It is likely that the action of these neuroactive steroids is of central origin and that interaction with GABA(A) receptors and stimulation of vagal pathway are involved in its mechanism of action.     

Effect of constitutive- and inducible-cyclooxygenase in the carbachol-induced pituitary-adrenocortical response during social stress.

Acetylcholine potently stimulates the hypothalamic-pituitary-adrenal (HPA) axis. Cholinergic receptor agonist carbachol, given intraperitoneally (i.p.) or into the lateral cerebral ventricle (i.c.v.) to non-anesthetized rats acts via multiple pathways to stimulate the HPA axis. The present study sought to determine 1) the functional selectivity of carbachol for cholinergic muscarinic and/or nicotinic receptors involved in the stimulation of HPA axis; 2) the involvement of prostaglandins (PGs) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the carbachol-induced ACTH and corticosterone secretion in non-stressed rats and animals exposed to social crowding stress for 7 days (24 per a cage for 6). Carbachol was given i.c.v. or i.p. and cholinergic receptor antagonists or cyclooxygenase isoenzyme antagonists were given by the same routes 15 min earlier. One hour after the last injection trunk blood was taken for ACTH and corticosterone determinations. Atropine (0.1 microg i.c.v.), a cholinergic receptor antagonist, totally abolished the carbachol (2 microg i.c.v.)-induced ACTH and corticosterone secretion and mecamylamine (20 microg i.c.v.), a selective nicotinic receptor antagonist, did not affect this secretion. This finding indicates that carbachol functions as a selective central cholinergic muscarinic receptor agonist for the HPA axis stimulation. Crowding stress significantly diminished the carbachol (0.2 mg/kg i.p.)-induced plasma ACTH and corticosterone levels measured 1 hr after administration. Pretreatment with indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase inhibitor, significantly diminished the ACTH and corticosterone responses to carbachol (0.2 mg/kg i.p.) in control rats and moderately decreased these responses in stressed rats. Piroxicam (0.2 and 2.0 mg/kg i.p.), a COX-1 inhibitor, considerably impaired the carbachol-induced ACTH and corticosterone responses in control rats and markedly diminished these responses in stressed rats. A selective COX-2 blocker, compound NS-398 (0.2 and 2.0 mg/kg i.p.), substantially decreased the carbachol-induced hormones secretion in control rats but did not markedly alter this secretion in stressed rats. These results indicate that in the carbachol-induced HPA axis activation PGs generated by COX-1 are considerably and to a much greater extent involved than PGs generated by COX-2. Social stress markedly diminishes the mediation of PGs generated by COX-1 but PGs synthesized by COX-2 do not substantially participate in the carbachol-induced HPA response.     

Effects of some putative amino acid neurotransmitters on the stimulated TSH secretion in male rats

The importance of several amino acids (glycine, L-glutamic acid, L-serine, taurine and beta-alanine) in the regulation of the stimulated secretion of TSH was studied in male rats using both peripheral and central administration of the amino acids. Glycine (10-200 mg/kg i.p.), L-glutamic acid (10-500 mg/kg i.p.) and L-serine (500 mg/kg i.p.) decreased significantly the cold-induced TSH secretion whereas beta-alanine (1-500 mg/kg i.p.) and taurine (10-100 mg/kg i.p.) were not effective. The effect of L-glutamic acid (100 mg i.p.) was partially antagonized by bicuculline (1 mg/kg i.p.) but not by picrotoxin (1 or 2 mg/kg i.p.). Only glycine (50 and 100 mg/kg i.p.) inhibited the TRH-stimulated TSH secretion. When the intracerebroventricular route was used, L-serine (50 micrograms/rat) decreased the TSH could response whereas glycine and L-glutamic acid (1-50 micrograms/rat) had no clear effect. We conclude that glycine, glutamate and serine inhibit the cold-induced TSH secretion in the male rat. The action of serine and glycine is possibly mediated through the periventricular hypothalamus and the anterior pituitary, respectively. The inhibition caused by glutamate seems to be partially mediated through the bicuculline-sensitive GABA receptors in the hypothalamus. Taurine and beta-alanine play no role in the control of rat TSH secretion.     

Complex role of 5-HT in the regulation of TSH secretion in the male rat

The role of 5-hydroxytryptamine (5-HT) in the regulations of TSH secretion was studied in male rats using both peripheral and central administration of the drugs. Basal TSH levels were not modified by moderate doses of 5-HT (subcutaneously) or its precursors or antagonists (intraperitoneally) given 1 h before decapitation. The cold-stimulated TSH secretion was decreased by L-tryptophan (L-TRP, 400 mg/kg i.p.), quipazine (10 mg/kg i.p.) and 5-HT (1 or 5 mg/kg s.c. or i.v.) as well as by p-chlorophenylalanine (pCPA, 20 or more mg/kg i.p.) when the drugs were given 1 h before sampling. pCPA (100-400 mg/kg i.p.) was active 24-48 h after the injection but repetitive administration did not affect TSH levels. 5-HT (5 mg/kg s.c.) was effective also in pinealectomized animals. L-TRP and 5-hydroxytryptophan potentiated the TRH-stimulated TSH secretion when given 1 h before killing. 5-HT (10 microgram/rat) infused into the third ventricle enhanced the cold-stimulated TSH secretion when given 30-45 min before sampling. When injected into the medial basal hypothalamus, 50-HT (1-10 microgram/rat) had no effect on basal or stimulated TSH levels. The results suggest: (1) 5-HT does not play any role in the regulation of basal TSH secretion; (2) in the cold-stimulated TSH secretion 5-HT has a stimulatory action evidently inside the blood-brain barrier and also an inhibitory effect obviously outside this barrier.     

Effect of adrenergic antagonists and cyclooxygenase inhibitors on the nicotine-induced hypothalamic-pituitary-adrenocortical activity.

Nicotine is a potent stimulus for the hypothalamic-pituitary-adrenal (HPA) axis. Systemic nicotine acts via central mechanisms to stimulate by multiple pathways the release of ACTH from the anterior pituitary corticotrops and corticosterone from the adrenal cortex. Nicotine may stimulate indirectly the hypothalamic paraventricular nucleus, the site of the corticotropin-releasing hormone (CRH) neurons which activates ACTH release. In the present studies an involvement of adrenergic system and prostaglandins synthesized by constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) in the nicotine-induced HPA response in rats was investigated. Nicotine (2.5-5 mg/kg i.p.) significantly increased plasma ACTH and corticosterone levels measured 1 hr after administration. Adrenergic receptor antagonists or COX inhibitors were injected i.p. 15 min prior to nicotine and the rats were decapitated 1 hr after the last injection. Prazosin (0.01-0.1 mg/kg), an alpha1-adrenergic antagonist, significantly decreased the nicotine-evoked ACTH and corticosterone secretion. Yohimbine (0.1-1.0 mg/kg), an alpha2-adrenergic antagonist, moderately diminished ACTH response, and propranolol (0.1-10 mg/kg), a beta-adrenergic antagonist, did not significantly alter the nicotine-induced hormones secretion. Pretreatment with piroxicam (0.2-2.0 mg/kg), a COX-1 inhibitor, considerably impaired the nicotine-induced ACTH and corticosterone secretion. Compound NS-398 (0.2-5.0 mg/kg), a selective COX-2 blocker did not markedly alter these hormones secretion, and indomethacin (2 mg/kg), a non-selective COX inhibitor significantly diminished ACTH response. These results indicate that systemic nicotine stimulates the HPA axis indirectly, and both adrenergic system and prostaglandins are significantly involved in this stimulation. Noradrenaline, stimulating postsynaptic alpha1-adrenergic receptors, and prostaglandins, synthesized by COX-1 isoenzyme, are of crucial significance in the nicotine-induced ACTH and corticosterone secretion.     

Effects of (+/-)-sotalol, (+/-)-propranolol, and (-)-propranolol on norepinephrine release upon hepatic nerve stimulation in the dog liver in vivo

The study was carried out to determine whether the diminished release of norepinephrine (NE) upon sympathetic activation in the presence of sotalol can be attributed to the blockade of beta-adrenoceptors in the liver. NE release from the liver was measured in hepatic venous blood collected during direct hepatic nerve stimulation in anesthetized dogs. The mean basal NE concentration in hepatic venous and aortic blood was 0.046 +/- 0.003 and 0.244 +/- 0.041 ng/mL, respectively. NE release increased significantly as stimulation frequency increased, while aortic NE concentration remained unchanged. The increasing response of NE release upon stimulation in the vehicle control group remained stable during the whole experimental period. In dogs treated with sotalol (5 mg/kg, i.v.), NE release was reduced approximately by 30-43%, and the difference was statistically significant (P less than 0.01) at 8 Hz. (+/-)-Propranolol (2.5 mg/kg, i.v.) tended to diminish it, but the difference was not significant. (-)-Propranolol (0.1 mg/kg, i.v.) did not alter NE release at any frequency tested. The beta-blocking action of these drugs in the liver, as determined by the antagonism against the hepatic arterial vasodilating response to isoproterenol, was most effective with (+/-)-propranolol (100%), followed by (-)-propranolol (90%) and sotalol (70%). The results suggest that the inhibitory effect of sotalol on NE release may be related to a mechanism other than its beta-blocking action in the dog liver.     

The synaptic and nonsynaptic glycine transporter type-1 inhibitors Org-24461 and NFPS alter single neuron firing rate in the rat dorsal raphe nucleus. Further evidence for a glutamatergic-serotonergic interaction and its role in antipsychotic action

Single neuron firing rate was recorded from dorsal raphe nucleus of anesthetized rats. The firing rate of raphe neurons varied from 4 to 8 discharge per second before drug administration and this neuronal activity was decreased by L-701,324 (2 mg/kg i.v. injection), a competitive antagonist of glycineB binding site of N-methyl-D-aspartate (NMDA) receptors. The glycine transporter type-1 (GlyT1) antagonists Org-24461 (10 mg/kg i.v.) and NFPS (3 mg/kg i.v.) reversed the inhibitory effect of L-701,324 on single neuron activity recorded from dorsal raphe nucleus of the rat. Org-24461 and NFPS both tended to increase the raphe neuronal firing rate also when given alone but their effect was not significant. This finding serves further evidence that glutamate released from axon terminals of the cortico-striatal projection neurons stimulates serotonergic neurons in the raphe nuclei and this effect is mediated at least in part by postsynaptic NMDA receptors. Thus, GlyT1 inhibitors are able to reverse the hypofunctional state of NMDA receptors, suggesting that these drugs may have beneficial therapeutic effects in neurological and psychiatric disorders characterized with impaired NMDA receptor-mediated transmission.     

Dynorphin and the hypothalamo-pituitary-adrenal axis during fetal development

Although dynorphin has long been considered an endogenous opioid peptide with high affinity for the kappa-opioid receptor, its biological function remains uncertain. The high concentration of dynorphin peptides and kappa-opioid receptors in the hypothalamus suggest a possible role for dynorphin in neuroendocrine regulation. This review will summarize evidence that support a role for dynorphin in regulation of the developing hypothalamo-pituitary-adrenal (HPA) axis. Dynorphin can exert dual actions on adrenocorticotropin (ACTH) release: (i) via activation of hypothalamic kappa-opioid receptors leading to release of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and (ii) via a non-opioid mechanism that involves N-methyl-D-aspartate (NMDA) receptors and prostaglandins, and which is not dependent on CRH or AVP. The primary site of action of dynorphin and NMDA appears to be the fetal hypothalamus or a supra-hypothalamic site. The non-opioid mechanism does not mature until a few days prior to parturition and is active for only the brief perinatal period. In contrast, the opioid mechanism behaves as a constitutive system with sustained activity from prenatal to postnatal life. It is likely that the two mechanisms may respond to different stress stimuli and play a different role during development.     

Effects of i.c.v. losartan on the angiotensin II-mediated vasopressin release and hypothalamic fos expression in near-term ovine fetuses

Our previous studies have shown that central administration of angiotensin (ANG II) causes arginine vasopressin (AVP) release in the fetus at 70-90% gestation. This is evidence that the hypothalamic-neurohypophysial system is relatively mature before birth. However, few data exist regarding central ANG receptor mechanisms-mediated AVP response during fetal life. To determine roles of brain ANG receptor subtypes in this response, AT1 and AT2 receptor antagonists, losartan and PD123319, were investigated in the brain in chronically prepared ovine fetuses at the last third of gestation. Application of losartan intracerebroventricularly (i.c.v.) at 0.5 mg/kg suppressed central ANG II-stimulated plasma AVP release. Losartan at 5 mg/kg (i.c.v.) demonstrated a significant enhancement of AVP increase to i.c.v. ANG II. Associated with the increase of plasma vasopressin levels, c-fos expression in the hypothalamic neurons was significantly different between the low and high doses of losartan. The low dose losartan markedly reduced the dual immunoreactivity for FOS and AVP in the supraoptic nuclei and paraventricular nuclei after i.c.v. ANG II, whereas the high dose losartan together with ANG II, significantly increased the co-localization of positive FOS in the AVP-containing neurons than that induced by i.c.v. ANG II alone. Central ANG II induced fetal plasma vasopressin increase was not altered by PD123319. The data suggest that losartan in the fetal brain has remarkably different effects based on the doses administrated on central ANG II-related neuroendocrine effects at the late gestation, and that the AT1 mechanism is critical in the regulation of fetal body fluid homeostasis related to plasma AVP levels.     

Stimulatory effects of endogenous and exogenous nitric oxide on gastric acid secretion in anesthetized rats

We previously reported the stimulatory effect of endogenous nitric oxide (NO) on gastric acid secretion in the isolated mouse whole stomach and histamine release from gastric histamine-containing cells. In the present study, we investigated the effects of endogenous and exogenous NO on gastric acid secretion in urethane-anesthetized rats. Acid secretion was studied in gastric-cannulated rats stimulated with several secretagogues under urethane anesthesia. The acid secretory response to the muscarinic receptor agonist bethanechol (2 mg/kg, s.c.), the cholecystokinin(2) receptor agonist pentagastrin (20 microg/kg, s.c.) or the centrally acting secretagogue 2-deoxy-D-glucose (200 mg/kg, i.v.) was dose-dependently inhibited by the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 or 50 mg/kg, i.v.). This inhibitory effect of L-NNA was reversed by a substrate of NO synthase, L-arginine (200 mg/kg, i.v.), but not by D-arginine. The histamine H(2) receptor antagonist famotidine (1 mg/kg, i.v.) completely inhibited the acid secretory response to bethanechol, pentagastrin or 2-deoxy-D-glucose, showing that all of these secretagogues induced gastric acid secretion mainly through histamine release from gastric enterochromaffin-like cells (ECL cells). On the other hand, histamine (10 mg/kg, s.c.)-induced gastric acid secretion was not inhibited by pretreatment with L-NNA. The NO donor sodium nitroprusside (0.3-3 mg/kg, i.v.) also dose-dependently induced an increase in acid secretion. The sodium nitroprusside-induced gastric acid secretion was significantly inhibited by famotidine or by the soluble guanylate cyclase inhibitor methylene blue (50 mg/kg, i.v.). These results suggest that NO is involved in the gastric acid secretion mediated by histamine release from gastric ECL cells.