Evidence that corticotropin-releasing factor within the extended amygdala mediates the activation of tryptophan hydroxylase produced by sound stress in the rat

Non-endocrine corticotropin-releasing factor (CRF) is believed to be involved in mediating stress behaviors in rats. The present study investigated the role of CRF in mediating the activation of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, produced in response to sound stress. Bilateral injections of 0.5–3.0 μg of CRF directed towards the central nucleus of the amygdala increased tryptophan hydroxylase activity measured ex vivo when compared to vehicle-injected controls. This increase in enzyme activity, like that due to sound stress, was reversed in vitro by alkaline phosphatase. Intra-amygdala CRF (0.5 μg) also enhanced the in vivo accumulation of 5-hydroxytryptophan (5-HTP) following the administration ofm-hydroxylbenzylamine (NSD-1015, 200 mg/kg). The activation of tryptophan hydroxylase, produced by intra-amygdala CRF, was blocked by the CRF receptor antagonist α-helical CRF_9–41 (10 μg). Additionally, the 5-HT_1A agonist, gepirone, given either systemically (10 mg/kg) or intracerebrally into the region of the dorsal raphe (14 μg), blocked the tryptophan hydroxylase response to CRF. CRF did not increase tissue levels of 5-hydroxyindole acetic acid (5-HIAA) or the ratio of 5-HIAA to serotonin (5-HT) within the striatum of the same animals in which tryptophan hydroxylase activity was quantified, an effect produced by sound stress. Thus, while intra-amygdala CRF failed to mimic the sound stress response in its entirety, these data suggest that CRF is involved in mediating the activation of tryptophan hydroxylase produced by sound stress within the midbrain serotonin neurons.     

Long-term observation of rats after unilateral intra-amygdaloid injection of kainic acid

Electroencephalographic and clinical observations of the rats were done for 3 months after microinjection of kainic acid (KA, 0.2–1.2 μg)into the unilateral amygdala. With high doses of KA (0.6–1.2μg) injection, 64% of rats developed spontaneous limbic siezures 14–25 days after KA injection. Among those which developed spontaneous limbic seizures, 33% of rats developed secondarily generalized seizures 26–71 days after KA injection. This is one of the best chronic models of spontaneous complex partial seizure secondarily generalized in rats, which is economical and easy to prepare.     

Nitroglycerin-induced penile erection and yawning in male rats: Mechanism of action in the brain

The effect of the central administration of nitroglycerin, a potent organic nibate vasodilator, on penile erection and yawning was studied in male rats. When given intracerebroventriculary (ICV), nitroglycerin (33–99 μg) induced the above responses dose-dependently. The minimal effective dose was 33 μg, which was active in 60% of the rats. Nitroglycerin (1.86–6.6 μg) induced penile erection and yawning also when injected in the paraventricular nucleus of the hypothalamus. Nitroglycerin responses were prevented by methylens blue (200–400 μg ICV), by d(CH₂)₅Tyr(Me²-Orn⁸-vaaotocin (0.5–1 μg ICV but not hemoglobin (100–200 μg ICV). In contrast methylene blue (10–20 μg), d(CH₂)₅Tyr(Me)²-Orn⁸-vasotocin (0.05–0.1 μg) and hemogiobin (10–20 μg) were ineffective when injected in the paraventricular nucleus. Systemic haloperidol (0.5–1 mg1kg IP) was also ineffective. The results suggest that nitroglycerin induces penile erection and yawning by activef brain oxytocinergic transmission through the formation of nitric oxide in the paraventricular nucleus of the hypothalamus.     

Tryptophan hydroxylase and 5-HTP-decar☐ylase activity in cockroach brain and the effects of p-chlorophenylalanine and 3-hydroxybenzylhydrazine (NSD-1015)

The activities of two enzymes of the serotonin biosynthetic pathway, namely tryptophan hydroxylase (TPH) and 5-hydroxytryptophan-decar☐ylase (5-HTP-D), were determined in the cockroach, Periplaneta americana, brain using radiometric techniques, significant levels of both enzymes were found, p-Chlorophenylalanine (PCPA) inhibited brain TPH activity strongly both in vitro and in vivo. In vitro, 10 mM PCPA reduced the TPH activity by 76% and in vivo either 15 μg/g or 30 μ/g of PCPA inhibited brain TPH activity by 80–85%. The activity of 5-HTP-D enzyme was inhibited significantly in vivo by 3-hydroxybenzylhydrazine, commonly known as NSD-1015. A recovery in the activity of 5-HTP-D enzyme was observed 12 h after 50 μg/g of NSD-1015 but 100 μ/g caused an inhibition lasting for the 24 h period studied.     

Electroencephalographic spreading depression and concomitant behavioral changes induced by intrahippocampal injections of ACTH1–24 andd-Ala-met-enkephalinamide in the rat

ACTH_1–24 (0.5or10 μg 0.17or3.45nmol) andd-Ala²-Met-enkephalinamide (DAME; 10 μg 17.05nmol) were injected unilaterally into the hippocampus of freely moving rats to examine their effects on EEG activity, DC potentials and behavior. In 85% of the rats DAME elicited spreading depression (SD) with epileptiform discharges preceding and following the wave of SD. The following behavioral changes were recorded. DAME- and KCl-induced SD were accompanied by an increase in locomotor activity and wet-dog shaking behavior, which occurred only during the period of SD. After a wave of SD induced by DAME a biphasic pattern of activity, consisting of an initial depression in locomotion followed by hyperactivity, appeared in 59% of the rats.ACTH_1–24 elicited SD in 13% of the rats tested. Neither the dosage of ACTH_1–24 nor the strain of rats influenced the occurrence of SD and the incidence of ACTH-induced grooming behavior. SD induced by KCl also resulted in excessive grooming comparable to that induced by ACTH_1–24. In the case of KCl-induced SD, grooming began directly after the injection of KCl and was frequently interrupted by short periods of locomotion. ACTH-induced grooming had a later onset and episodes of stretching and yawning were observed. It can be concluded that the behavioral effects of the injection of DAME are unspecific responses to SD and seizure activity. However, ACTH-induced grooming is not solely a byproduct of SD, since it occurred also in the absence of SD.     

Selective FSH-releasing activity of [D-Trp]GAP1–13: Comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs

We had previously shown that fragments of human gonadotropin-releasing hormone associated peptide (GAP) stimulated FSH and LH release in vivo. In particular, GAP₁₋₁₃ had a preferential FSH-releasing activity. To decrease enzymatic degradation, analogs of GAP_1–13 with D-amino acid substitutions were synthesized. The activities were tested in ovariectomized, estrogen-progesterone primed (OEP) rats and compared with those of GAP₁₋₁₃, mammalian (m), chicken II (eII), and lamprey (1) LHRH. The peptides were injected (IV) into conscious, OEP rats and blood samples were obtained via the jugular catheter. [D-Trp⁹]GAP_1–13 selectively stimulated FSH release at a dose of 1 μg. Multiple injections of this analog (10 μg every 30 min for 5 injections) induced a marked elevation of plasma FSH values which peaked (p < 0.001) after the third injection. By contrast, [D-Trp⁹]GAP_1–13 had no effect on LH and prolactin (PRL) release after either single or multiple injections. These doses of [DAla⁴] GAP_1–13 had no effect on the release of FSH, LH or PRL. Both human GAP_1–13 and its [D-Trp⁹] analog exerted a selective FSH-releasing effect at a dose of 10 μg, however, the [D-Trp⁹] analog was more potent than GAP_1–13 on FSH release. The potency of [D-Trp⁹]GAP_1–13 in releasing FSH was approximately that of mLHRH. Chicken II LHRH had slightly selective FSH-releasing activity with a potency that of mLHRH. Lamprey LHRH had a preferential LH-releasing activity and a potency 1000 times less than mLHRH. In conclusion, [D-Trp⁹]GAP_1–13 is a selective FSH-releasing peptide of potential clinical value.     

Adrenal glucocorticoids as a required factor in barbitura-induced changes in functional tolerance and brainstem tryptophan hydroxylase

Male C57BL/6 mice were given phenobarbital via milled food for 9 days; control mice received unadulterated milled food. Blood phenobarbital levels rose to 116 μg/ml during the later days of the experiment. Twenty-fourth after withdrawal of the barbiturate, the animals were given a challenge dose of pentobarbital (PenB, 40 mg/kg, i.p.) and their sleep time was determined as a measure of tolerance. All control animals slept after the injection for an average of 57 min and woke up with brain PenB levels of 22 μg/g. However, none of the phenobarbital-treated animals slept after PenB injection despite brain PenB levels peaking 38% higher than control levels upon awakening (P < 0.001), thus indicating the development of functional tolerance to barbiturate. Adrenalectomized (Adx) animals receiving chronic phenobarbital slept for 13 min and woke up with low brain PenB levels. In the phenobarbital-treated Adx given glucocorticoid replacement (0.5 mg/mouse/day; s.c.), none of the animals slept after the PenB injection, similar to the intact animals.Since a large portion of the tolerance (from 57 min to 0 min) was metabolic in nature, the portion that remained in Adx animals represent lack of brainstem functional tolerance. Since previous studies suggested that functional tolerance to barbiturates may be due to adaptive changes in the serotonergic neurons, brainstem tryptophan hydroxylase activity was examined. The enzyme activity was increased by 64% (P < 0.001) following the chronic barbiturate administration. Interestingly, the barbiturate effect on tryptophan hydroxylase activity was totally abolished in Adx animals, and the effect was restored in Adx animals by hormone replacement with daily injections of corticosterone (0.5 mg/mouse). Thus, there was a correlation between the occurence of barbiturate functional tolerance and the changes in the activity of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin. Our findings support the notion that the development of functional tolerance to barbiturates may involved a serotonergic mechanism, and that adrenal glucocorticoids are a required factor for barbiturate-induced changes in both tolerance and tryptophan hydroxylase.     

Hypothalamic tyrosine hydroxylase activity and plasma gonadotropin and prolactin levels in ovariectomized-steroid treated rats

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase (TH) activity were evaluated in ovariectomized (OVX) estradiol benzoate (EB) or progesterone (P) treated rats. Single injection of 10 μg or daily injection of 5 μg EB/rat for 7 days significantly lowered gonadotropin levels in OVX animals and elevated PRL levels. Single injection of 2 mg or daily injection of 200 μg P/rat for 7 days increased gonadotropin and PRL levels. Hypothalamic TH activity was significantly elevated by estradiol. Single injection of 2 mg P suppressed TH activity in contrast to the elevation in enzyme activity following chronic treatment. These results indicate that hypothalamic noradrenergic as well as dopaminergic neurons participate in the stimulatory or inhibitory feedback effects of ovarian hormones on gonadotropin and PRL secretion.     

Quantitative histochemical measurement of GABA-transminase: Method for evaluation of intracerebral lesions produced by excitotoxic agents

GABA-transminase (GABA-T) activity of fresh frozen coronal sections through rat striatum was evaluated 4–6 weeks after intrastriatal application of kainic or ibotenic acid. 16 μm sections were processed for GABA-T histochemistry and were evaluated quantitatively by computerized densitometry using image analysis. Alternate sections (200 μm) were assayed for GABA-T activity in vitro. Gross examination of sections stained for GABA-T revealed obvious lack of staining in the vicinity of lesions produced by either kainate (0.5–1.0 μg) or ibotenate (10–20 μg); the extent of each lesion was clearly delineated by the stain. Quantitative analysis of stained sections revealed that the lesioned tissue contained 80–90% less GABA-T activity than control tissue. This loss of GABA-T was in agreement with values obtained in adjacent sections assayed in vitro. Similar studies in substantia nigra clearly and quantitatively demonstrated damage induced by ibotenate or kainate in this nucleus as well as in tissue in the overlying reticular formation. Moreover, two compartments of GABA-T were discriminated in substantia nigra: one associated with neural perikarya, which was sensitive to kainic and ibotenic acids (80% of total GABA-T), and a second associated with afferent terminals arising from forebrain projections (20%). Thus, after destruction of neurons with kainic or ibotenic acid, GABA-T activity is largely eliminated. Under these conditions, it appears that glia contribute relatively little to the GABA-T activity measured either histochemically or by direct chemical assay in homogenates.     

Bombesin-induced poikilothermy in rats

The effect of ambient temperature (T_A) on the rectal temperature (T_RE) response to intraventricular injection of bombesin has been evaluated in conscious adult male rats. AtT_A= 4 °C, bombesin (50 ng-1 μg) caused a marked hypothermia which was dose-dependent both in terms of the magnitude and of the duration of the response. The bombesin-induced hypothermia was reduced atT_A= 24 °C, whereas atT_A= 31 or 33 °C, the peptide (1 μg) failed to affect T_RE. AtT_A= 36 °C, bombesin 1–10 μg induced anelevation in T_RE. The hyperthermia observed at high T_A could be reversed to hypothermia by transferring rats to cold. The analogs [D-Trp⁸]bombesin or [D-Leu¹³]-bombesin, tested under the same conditions, failed to produce significant changes in T_RE. These findings demonstrate that bombesin appears to act in the brain as a poikilothermic agent by disrupting thermoregulation at temperatures below or above thermoneutrality.     

Kainate-induced lesion in the optic tectum: dependency upon optic nerve afferents or glutamate

Kainic acid is known to induce characteristic lesions in neurons receiving an intact input with presumed glutamate-mediated neurotransmission. There are indications for glutamate as a transmitter of retinal afferent terminals in the pigeon optic tectum. After tectal injection of kainic acid (0.5–2.0 μg in 0.5 μl) the optic tectum was studied by light and electron microscopy and the following changes were observed: (a) within 1–48 h important neuropil vacuolization predominantly in lower part of layer 5. Such vacuoles were sometimes postsynaptic to identified retinal afferent terminals: (b) within 1 h to 21 days progressive neuronal cell loss throughout the tectal layers. These toxic effects were not observed 2–12 weeks after contralateral retinal ablation but could partially be restored by combined glutamate (0.2 mg) and kainate injection. Thus in the pigeon tectum, kainic acid neurotoxicity is dependent upon an intact retinal input, a finding consistent with a special role for glutamate — possibly as a transmitter — in retinal terminals.     

Corticotropin-releasing factor antagonist blocks microwave-induced decreases in high-affinity choline uptake in the rat brain

Acute (45-min) irradiation with pulsed low-level microwaves (2450-MHz, 2 μsec pulses at 500 pps, average power density of 1 mW/cm², whole-body average specific absorption rate of 0.6 W/kg) decreased sodium-dependent high-affinity choline uptake (HACU) activity in the frontal cortex and hippocampus of the rat. These effects were blocked by pretreating the animals before exposure with intracerebroventricular injection of the specific corticotropin-releasing factor (CRF) receptor antagonist, α-helical-CRF_9–41 (25 μg). Similar injection of the antagonist had no significant effect on HACU in the brain of the sham-exposed rats. These data suggest that low-level microwave irradiation activates CRF in the brain, which in turn causes the changes in central HACU.     

Involvement of the nucleus accumbens–ventral pallidal pathway in postictal behavior induced by a hippocampal afterdischarge in rats

The hypothesis that postictal motor behaviors induced by a hippocampal afterdischarge (AD) are mediated by a pathway through the nucleus accumbens (NAC) and ventral pallidum (VP) was evaluated in freely moving rats. Tetanic stimulation of the hippocampal CA1 evoked an AD of 15–30 s and an increase in number of wet-dog shakes, face washes, rearings and locomotor activity. Bilateral injection of haloperidol (5 μg/side) or the selective dopamine D₂ receptor antagonist, (±)-sulpiride (200 ng/side) before the hippocampal AD, into the NAC selectively reduced rearings and locomotor activity, but not the number of wet-dog shakes and face washes. Injection of R(+)-SCH-23390 (1 μg/side), a D₁ receptor antagonist, or rimcazole (0.4 mg/side), a σ opioid receptor antagonist, into the NAC did not significantly alter postictal behaviors. Bilateral injection of muscimol (1 ng/side), a γ-aminobutyric acid (GABA_A) receptor agonist, into the VP before the AD significantly blocked all postical behaviors. It is concluded that postictal locomotor activity induced by a hippocampal AD is mediated by activation of dopamine D₂ receptors in the NAC and a pathway through the VP.     

Increased binding at 5-HT1A, 5-HT1B, and 5-HT2A receptors and 5-HT transporters in diet-induced obese rats

5-Hydroxytryptamine (5-HT, serotonin), synthesized in midbrain raphe nuclei and released in various hypothalamic sites, decreases food intake but the specific 5-HT receptor subtypes involved are controversial. Here, we have studied changes in the regional density of binding to 5-HT receptors and transporters and the levels of tryptophan hydroxylase, in rats with obesity induced by feeding a palatable high-energy diet for 7 weeks. We mapped binding at 5-HT receptor subtypes and transporters using quantitative autoradiography and determined tryptophan hydroxylase protein levels by Western blotting. In diet-induced obese (DiO) rats, specific binding to 5-HT_1A receptors ([³H]8-OH-DPAT) was significantly increased in the dorsal and median raphe by 90% (P<0.01) and 132% (P<0.05), respectively, compared with chow-fed controls. 5-HT_1B receptor binding sites ([¹²⁵I]cyanopindolol) were significantly increased in the hypothalamic arcuate nucleus (ARC) of DiO rats (58%; P<0.05), as were 5-HT_2A receptor binding sites ([³H]ketanserin) in both the ARC (44%; P<0.05) and lateral hypothalamic area (LHA) (121%; P<0.05). However, binding to 5-HT_2C receptors ([³H]mesulgergine) in DiO rats was not significantly different from that in controls in any hypothalamic region. Binding to 5-HT transporters ([³H]paroxetine) was significantly increased (P<0.05) in both dorsal and median raphe, paraventricular nuclei (PVN), ventromedial nuclei (VMH), anterior hypothalamic area (AHA) and LHA of DiO rats, by 47%–165%. Tryptophan hydroxylase protein levels in the raphe nuclei were not significantly different between controls and DiO rats. In conclusion, we have demonstrated regionally specific changes in binding to certain 5-HT receptor subtypes in obesity induced by voluntary overeating of a palatable diet. Overall, these changes are consistent with reduced 5-HT release and decreased activity of the 5-HT neurons. Reduction in the hypophagic action of 5-HT, possibly acting at 5-HT_1A, 5-HT_1B and 5-HT_2A receptors, may contribute to increased appetite in rats presented with highly palatable diet.     

Bilateral effects of unilateral GDNF administration on dopamine- and GABA-regulating proteins in the rat nigrostriatal system

Dopamine (DA) affects GABA neuronal function in the striatum and together these neurotransmitters play a large role in locomotor function. We recently reported that unilateral striatal administration of GDNF, a growth factor that has neurotrophic effects on DA neurons and enhances DA release, bilaterally increased striatal neuron activity related to locomotion in aged rats. We hypothesized that the GDNF enhancement of DA function and resulting bilateral enhancement of striatal neuronal activity was due to prolonged bilateral changes in DA- and GABA-regulating proteins. Therefore in these studies we assessed dopamine- and GABA-regulating proteins in the striatum and substantia nigra (SN) of 24 month old Fischer 344 rats, 30 days after a single unilateral striatal delivery of GDNF. The nigrostriatal proteins investigated were the DA transporter (DAT), tyrosine hydroxylase (TH), and TH phosphorylation and were examined by blot-immunolabeling. The striatal GABA neuron-related proteins were examined by assay of the DA D₁ receptor, DARPP-32, DARPP-32 Thr³⁴ phosphorylation, and glutamic acid decarboxylase (GAD). Bilateral effects of GDNF on TH and DAT occurred only in the SN, as 30 μg GDNF increased ser19 phosphorylation, and 100 μg GDNF decreased DAT and TH protein levels. GDNF also produced bilateral changes in GAD protein in the striatum. A decrease in DARPP-32 occurred in the ipsilateral striatum, while increased D₁ receptor and DARPP-32 phosphorylation occurred in the contralateral striatum. The 30 μg GDNF infusion into the lateral striatum was confined to the ipsilateral striatum and substantia nigra. Thus, long-lasting bilateral effects of GDNF on proteins regulating DA and GABA neuronal function likely alter physiological properties in neurons, some with bilateral projections, associated with locomotion. Enhanced nigrostriatal excitability and DA release by GDNF may trigger these bilateral effects.     

Differential modulation of angiotensin II and hypertonic saline-induced drinking by opioid receptor subtype antagonists in rats

Opioid modulation of ingestion includes general opioid antagonism of different forms of water intake, μ₂ receptor modulation of deprivation-induced water intake and δ₂ receptor modulation of saccharin intake. Water intake is stimulated by both central administration of angiotensin II (ANG II) and peripheral administration of a hypertonic saline solution; both responses are reduced by general opioid antagonists. The present study examined whether specific opioid receptor subtype antagonists would selectively alter each form of water intake in rats. Whereas systemic naltrexone (0.1–2.5 mg/kg, s.c.) reduced water intake induced by either peripheral ANGII (500 μg/kg, s.c.) or hypeptonic saline (3 ml/kg, 10%), intracerebroventricular (i.c.v.) naltrexone (1–50 μg) only inhibited central ANGII (20 ng)-induced hyperdipsia. Both forms of drinking were significantly and dose-dependently inhibited by the selective κ antagonist, nor-binaltorphamine (Nor-BNI, 1–20 μg). Whereas both forms of drinking were transiently reduced by the μ-selective antagonist, β-funaltrexamine (β-FNA, 1–20 μg), the μ₁ antagonist, naloxonazine (40 μg) stimulated drinking following hypertonic saline. The δ₁ antagonist, [d-Ala², Leu⁵, Cys⁶]-enkephalin (DALCE, 1–40 μg) significantly reduced drinking following ANGII, but not following hypertonic saline; the δ antagonist, naltrindole failed to exert significant effects. These data indicate that whereas κ opioid binding sites modulate hyperdipsia following hypertonic saline, μ₂, δ₁ and κ opioid binding sites modulate hyperdipsia following ANGII. The μ₁ opioid binding site may normally act to inhibit drinking following hypertonic saline.     

Excitatory action of pituitary adenylate cyclase activating polypeptide on rat sympathetic preganglionic neurons in vivo and in vitro

In vivo and in vitro experiments were undertaken to evaluate the effects of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) on rat sympathetic preganglionic neurons (SPNs). Intrathecal injection of PACAP-38 (0.1–1 nmol) via an implanted cannula to the T2-T3 segments of urethane-anesthetized adult rats caused a dose-dependent increase of mean arterial blood pressure from minutes to over 1 h. The pressor response was not antagonized by prior injection of the PACAP type II receptor antagonist PACAP_6-38 (0.5 nmol), but was significantly attenuated by prior intravenous administration of phentolamine (1 mg/kg). As a positive control, intrathecal injection of glutamate (1 μmol) and substance P (SP, 5 nmol) caused a short- and long-lasting pressor response. Vasoactive intestinal polypeptide (VIP, 1 nmol) had no significant pressor effect. In the second series of experiments, whole-cell patch recordings were made from antidromically identified SPNs of immature (12–16-day-old) rat thoracolumbar spinal cord slices. Applied to the spinal cord slices by superfusion, PACAP-38 (10–30 nM) caused intense neuronal discharges with or without a long-lasting membrane depolarization. The depolarization was not prevented by superfusing the slices with tetrodotoxin (0.3 μM) or low Ca²⁺ (0.25 mM) solution, indicating that PACAP-38 directly depolarized the SPNs. The depolarization was insensitive to the type II PACAP receptor antagonist PACAP_6-38. Collectively, these results provide evidence that PACAP-38 exerts a potent and long-lasting excitatory effect on SPNs, leading to an increase of spinal sympathetic outflow and one of the consequences of which is an elevation of blood pressure.     

Inhibitory effect of regucalcin on Ca-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 μg/ml) or dioctanoylglycerol (5 μg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl₂ (1 mM), indicating that Ca²⁺ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10⁻⁷ M). Moreover, regucalcin (10⁻⁷ M) significantly inhibited cytosolic protein kinase activity without Ca²⁺/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10⁻⁷ M) significantly decreased protein kinase activity without Ca²⁺ addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca²⁺-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.     

Effect of intracerebroventricular administration of β-/Tyr/-melanotropin-/9–18/ on rotational behavior induced by substantia nigra lesion in rats

β-/Tyr⁹/melanotropin-/9–18/ administered intracerebroventricularly /icv./ in doses of 1 μ/rat or 10 μg/rat had no influence on the substantia nigra lesion-induced turning activity of rats, however, it was able to potentiate apomorphine-induced contralateral turning. In a dose of 20 μg/rat /icv./ the peptide alone induced clear contralateral turning. The results suggest that at lower doses the peptide facilitates apomorphine-induced dopamine receptor stimulation, while in a higher dose the peptide alone can stimulate dopamine receptor activity.     

N-Monomethyl-l-arginine co-injection attenuates the thermogenic and hyperthermic effects of E2 prostaglandin microinjection into the anterior hypothalamic preoptic area in rats

Prostaglandin E₂ (PGE₂) microinjection (25 ng, 250 nl) into the preoptic area of the anterior hypothalamus (POAH) stimulated heat production in brown adipose tissue (BAT) and increased core temperature in urethane-anesthetized rats. These thermogenic and hyperthermic effects were attenuated by co-injection of N^G-monomethyl-l-arginine (NMMA, 25 μg), a competitive inhibitor of nitric oxide (NO) production froml-arginine. Inclusion ofl-arginine (50 μg), though notd-arginine (50μg) reversed the inhibitory effect of NMMA (25μg) on intra-POAH PGE₂-induced increases in interscapular BAT (IBAT) and core temperatures. Intra-POAH injection of NMMA (25 μg) orl-arginine (50 μg) alone had no effect on IBAT and core temperatures. The results suggest that the effect on thermoregulation induced by action of PGE₂ in the POAH is modulated by a locall-arginine-dependent and NMMA-sensitive NO-generating system.