Reduced inhibitory effects of clonidine and neuropeptide Y on3H-noradrenaline release from synaptosomes of the medulla oblongata of the spontaneously hypertensive rat

Summary The release of³H-noradrenaline (³H-NA) evoked by high-K⁺ (15 mM) was studied in synaptosomes isolated from the medulla oblongata of the normotensive Wistar-Kyoto and spontaneously hypertensive male rat (14 weeks old) using a superfusion apparatus. Based on concentration-response curves clonidine was shown to have a reduced ability to inhibit³H-NA release in synaptosomes isolated from the spontaneously hypertensive rat (SHR) versus the normotensive rat. Furthermore, only a high concentration of NPY (100 nM) had the ability to enhance the inhibitory effects of clonidine on³H-NA release in synaptosomes isolated from the medulla oblongata of the SH male rat, while 1 nM of NPY was effective in synaptosomes isolated from the medulla oblongata of the normotensive Wistar-Kyoto rat. These results may indicate a reduced presynaptic 2-adrenoceptor and NPY receptor function to inhibit³H-NA release from NA and/or adrenaline (A) nerve terminals in the medulla oblongata of the adult 14 weeks old SHR.     

Effects of verapamil on the release of different neurotransmitters

The effect of verapamil on resting and depolarization-induced monoamine release was investigated in rat hippocampal synaptosomes prelabeled with [³H]-5-hydroxytryptamine (HT) or [³H]-norepinephrine (NE) and rat striatal synaptosomes prelabeled with [³H]-dopamine (DA). Verapamil (50 μM) completely abolishes high K⁺-induced [³H]-NE release, but paradoxically facilitates high K⁺-induced [³H]-5-HT and [³H]-DA release. All these high K⁺-evoked responses were Ca²⁺ dependent. Verapamil does not modify [³H]-NE baseline release, but increases dose dependently [³H]-5-HT and [³H]-DA baseline release. Verapamil (10 μM, for 5 min) increases endogenous DA release (70%) and endogenous 5-HT release (40%) independently on the presence of external Ca²⁺. The total amount of these monoamines (released plus retained by the preparation) and their metabolites (DOPAC and 5-HIAA) was similar in control and verapamil-treated synaptosomes. Verapamil displaces [³H]-spiroperidol specific binding (K_i of 2.4 × 10^?6M) and [³H]-SCH-23390 specific binding (K_i of 9 × 10^?6M) from striatal synaptosomal membranes, and [³H]-5-HT specific binding (K_i of 3 × 10^?5M) from hippocampal synaptosomal membranes. It is concluded that in addition to the Ca²⁺ antagonistic properties of verapamil on the Ca²⁺-dependent, depolarization-induced release of some neurotransmitters [gamma aminobutyric acid (GABA and NE)], another mechanism probably mediated by presynaptic receptors underlies the effects of verapamil on DA and 5-HT release from discrete brain regions. © 1995 Wiley-Liss, Inc.     

Presynaptic modulation of sympathetic neurotransmission in rat left ventricle slices: effect of pressure overload

Summary. The purpose of this study was to establish the rat left ventricle (LV) tissue slice system for examination of norepinephrine (NE) release from sympathetic nerve terminals. Moreover, initial experiments were performed to use the LV tissue slice system to examine differences in NE uptake and release following cardiac pressure overload induced by abdominal aortic constriction (AC). Kinetic parameters (Vmax, Km) for the specific uptake of [³H]-NE demonstrated high affinity (Km, 1.94 ± 0.83 μM) and moderate capacity uptake (Vmax, 182 ± 6 fmol/mg/weight/min). Following 10 days of pressure overload, the Vmax for [³H]-NE uptake was significantly reduced (by 46%) in LV slices from AC rats compared to sham-operated (SO) controls. In control rat LV slices preloaded with [³H]-NE, electrically evoked [³H]-overflow was calcium- and stimulus pulse number-dependent. The neuronal uptake inhibitor, desipramine (DMI), increased (by 60%) evoked [³H]-overflow from LV slices. The α₂-agonist, UK14304, decreased evoked [³H]-overflow from LV slices in a concentration-dependent manner (maximal reduction of 75%). The β₂-agonist, salbutamol, increased evoked [³H]-overflow from LV slices in a concentration-dependent manner (maximal increase of 200%). In separate experiments, the LV tissue slice system was used to examine the effect of pressure overload on evoked [³H]-overflow. Following 10 days of pressure overload, evoked [³H]-overflow from LV slices of AC rats was increased (by 50%) compared to SO control. Increases in evoked [³H]-overflow from LV slices of AC rats compared to SO controls remained evident in the presence of DMI. These results demonstrate the relative importance of NE release and uptake using an in vitro LV tissue slice system. Sympathetic nerve terminals innervating rat LV were demonstrated to possess functional presynaptic α₂- and β₂-adrenergic receptors. Finally, using this LV tissue slice system, reductions in the uptake velocity and increases in evoked NE release were demonstrated in response to acute cardiac pressure overload. Received August 12, 1999; accepted January 12, 2000     

Neurotransmitter synthesis, storage and release by aggregating cell cultures of rat brain

Rotation-mediated aggregating cell cultures of mechanically dissociated fetal (15–16 days gestation) rat brains between 25 and 35 days in vitro were examined for their ability to synthesize neurotransmitters and putative neurotransmitters from radioactively labeled precursors added to the culture medium. Cultures derived from whole brain synthesized [³H]acetylcholine from [³H]choline, [³H]γ-aminobutyric acid from l-[³H]glutamic acid, [³H]dopamine from l-[³H]tyrosine, [³H]dopamine and [³H]norepinephrine from l-[³H]dihydroxyphenylalanine, and [³H]serotonin from l-[³H]tryptophan. Veratridine increased and tetrodotoxin decreased the rate of [³H]-dopamine synthesized by aggregates derived from midbrain plus hindbrain. In chase experiments in which aggregates were incubated for 4 h with radioactively labeled precursors and then for 4 h with non-radioactively labeled precursors, addition of veratridine (50 μM) during the second 4 h incubation significantly decreased the amounts of radioactively labeled acetylcholine, l-glutamic acid, dopamine and serotonin recovered from aggregates. Tetrodotoxin (5 μM) present during the chase significantly increased the amounts of [³H]acetylcholine and [³H]dopamine recovered from the aggregates. In addition, reserpine (4 μM) markedly depleted [³H]dopamine from aggregates in these experiments. These results indicate that these cultured cells synthesized neurotransmitters and in addition suggest that some of these compounds are stored by and released from electrically active cells within the aggregates.     

The effect of ovarian steroids on the accumulation of 3H-labelled monoamines by hypothalamic tissue in vitro

In vitro accumulation of³H-labelled monoamines into hypothalamic slices taken from rats in different endocrine conditions was investigated. Accumulation of³H-labelled noradrenaline into hypothalami from ovariectomised rats was increased by pretreatment for 3 days with 1 μg oestradiol. Accumulation of³H-labelled dopamine (DA) was decreased by 4 mg progesterone given together with 0.05 μg oestradiol for 3 days.³H-labelled 5-hydroxytryptamine (5HT) accumulation was not affected by any of the hormonal treatments. When oestradiol (3.6 × 10^?12?3.6 × 10^?6M) or progesterone (1.6 × 10^?6?1.6 × 10^?4M) was added to the incubation flask both hormones inhibited accumulation of all 3 amines.In hypothalami taken from immature rats induced to ovulate, accumulation of [³H]NA and [³H]5HT on the day before ovulation was not different from untreated controls; in both groups there was a diurnal rhythm. Accumulation of [³H]DA in these hypothalami significantly increased at the beginning of the critical period and decreased at the end, indicating a dopaminergic control of the timing of the critical period.     

Opposing effects of thapsigargin on the survival of developing cerebellar granule neurons in culture

Elevated levels of potassium (K⁺) promote maturation and survival of cerebellar granule neurons in culture. When switched from a culture medium containing high K⁺ (25 mM) to one with low K⁺ (5 mM) mature granule neurons undergo death by apoptosis. The mechanism by which high K⁺ promotes neuronal survival and conversely inhibits apoptosis) is unclear. Several pieces of evidence indicate that an increase in intracellular calcium (Ca²⁺) resulting from depolarization mediated-influx of extracellular Ca²⁺ is necessary. We examined the effect of thapsigargin on granule neuron cultures. Thapsigargin is an inhibitor of the endoplasmic reticular Ca²⁺ ATPase causing a depletion of Ca²⁺ from internal stores. this treatment would therefore be expected to raise intracellular cytosolic Ca²⁺ without membrane depolarization. We find that treatment of mature neurons with thapsigargin at doses 5 nM inhibits death resulting from the lowering of extracellular K⁺. The survival effect of thapsigargin was not affected by inhibitors of extracellular Ca²⁺ influx including nifedipine, verapamil, methoxyverapamil, Mg²⁺, and Ni²⁺, nor was it inhibited by the NMDA receptor antagonist, MK801. We have further examined whether thapsigargin could substitute for elevated K⁺ during the maturation of granule cells. Unexpectedly, treatment of younger (immature) neuronal cultures with the same dose of thapsigargin (5 nM) induced cell death. DNA fragmentation analysis suggested that death was due to apoptosis and not toxicity. As observed with the survival effect on mature neurons, the lethal effect of thapsigargin on immature granule cells was not prevented by inhibitors of Ca²⁺ influx.     

Release of dopamine from mesencephalic neurons in aggregate cultures: influence of target and non-target cells

Spontaneous release of [³H]dopamine (DA) was observed from reaggregates of dissociated cells from fetal rostral mesencephalic tegmentum (RMT) containing DA neurons cocultured with their axonal target cells from striatum (CS) or frontal cortex (FCx), or with non-target cells from occipital cortex (OCx), or tectum. Such release increased in response to 50 mM K⁺. Tetrodotoxin (TTX) suppressed the spontaneous release from RMT-CS and RMT-FCx reaggregates by 42%; from RMT-tectum reaggregates by 24%, and did not significantly inhibit the release from RMT-OCx cocultures. Since TTX blocks spontaneous neuronal activity, these results suggest that the presence of axonal target cells enhances the activity of the dopamine neurons. DA neurons within RMT-FCx reaggregates released significantly more [³H]DA in response to 50 mM K⁺ than in RMT-CS cocultures. This result is in accord with the findings in vivo that inhibitory feedback mechanisms on DA release, present in the striatum, are lacking in the frontal cortex.     

Acute and chronic losartan administration: effect on angiotensin II content and modulation of [3H]norepinephrine release from rat interscapular brown adipose tissue

Summary To determine if acute or chronic (21 days) losartan (10mg/kg, s.c.) regulates the renin-angiotensin system in interscapular brown adipose tissue, angiotensin II (AII) content and [³H]overflow from slices preloaded with [³H]norepinephrine were examined. Acute or chronic losartan administration had no effect on AII content. AII increased evoked [³H]overflow from slices from control rats. Losartan administration did not alter basal [³H]outflow or evoked [³H]overflow. Acute losartan administration inhibited AII-induced enhancement of evoked [³H]overflow. Tolerance developed to the inhibitory effect of losartan following chronic administration.Supported by a grant from the American Heart Association (Local Kentucky Affiliate) and by a gift from DuPont Merck Pharmaceuticals. Portions of this work have been presented in abstract form [The Pharmacologist 34(3): 157, 1992]     

In Vivo release by vagal stimulation of L-[H] glutamic acid in the nucleus tractus solitarius preloaded with L-[H] glutamine

In anesthetized and paralyzed rats, using a push-pull perfusion technique, we examined the effect of bilateral vagal stimulation on the release of L-[³H] glutamic acid (L-[³H] Glu) from the nucleus tractus solitarius (NTS), after preloading the tissue either with L-[³H] Glu or L-[³H] glutamine (L-[³H] Gln). Vagal stimulation sufficient to produce a maximum fall of arterial pressure (AP) evoked release of L-[³H] Glu from the NTS when the tissue was preloaded with either ³H-Glu or ³H-Gln, and of D-[³H] aspartic acid (D-[³H) Asp) when this stable Glu analogue was used to preload the tissue. Results demonstrate that the precursor L-Gln is a good marker of the releasable pool of L-Glu in vivo and are consistent with the hypothesis that L-[³H] Glu is a neurotransmitter in the NTS, mediating the vasodepressor response from cardiopulmonary mechanoreceptors.     

Effects of salt‐loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging

Salt‐loading (SL) impairs GABA_A inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down‐regulating the activity of K⁺/Cl^? co‐transporter2 (KCC2) and up‐regulating Na⁺/K⁺/Cl^? co‐transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl^?]_i in SON AVP neurones. The study combined virally‐mediated chloride imaging with ClopHensorN with a single‐cell western blot analysis. An adeno‐associated virus with ClopHensorN and a vasopressin promoter (AAV2‐0VP1‐ClopHensorN) was bilaterally injected in the SON of adult male Sprague‐Dawley rats that were either euhydrated (Eu) or salt‐loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl^?]_i in response to focal application of the GABA_A agonist muscimol (100 μmol L^‐1). SON AVP neurones from Eu rats showed muscimol‐induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β‐actin. Neurones that did not respond during chloride imaging did not show KCC2 and β‐actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L^‐1) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L^‐1) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol‐induced Cl^? influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA‐12) (50 μmol L^‐1) and protein kinase inhibitor (K252a) (100 nmol L^‐1) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt‐loading increases [Cl^?]_i in SON AVP neurones via a TrKB‐KCC2‐NKCC1‐dependent mechanism in rats.     

Evidence for NMDA receptor in the afferent synaptic transmission of the vestibular system

This study aimed to define the pharmacology and physiological role of the N-methyl-d-aspartate (NMDA) receptor in the synapse between the hair cells and primary afferent neurons in the vestibular system. The spontaneous and mechanically evoked spike discharges of vestibular nerve fibers were extracellularly recorded in isolated inner ear from the axolotl (Ambystoma tigrinum). Pressure ejection of NMDA (10⁻⁶ to 10⁻³ M) elicited a dose-dependent increase of the basal spike discharge from the vestibular nerve fibers. Extracellular magnesium antagonized the NMDA effect in a dose-dependent manner.d(-)-2-amino-5-phosphonovaleric acid (AP5, 10⁻⁵ to 10⁻³ M) and 7-chloro-kynurenic acid (7ClKyn, 10⁻⁶ to 10⁻³ M) inhibited the basal activity of the vestibular nerve fibers. 7ClKyn also diminished the responses elicited by the mechanical stimulation of the preparation. Glycine (10⁻⁹ to 10⁻⁶ M) applied by bath substitution enhanced the NMDA responses, and the glycine agonistd-serine partially reversed the 7ClKyn inhibitory action. These results suggest that NMDA receptors participate in the generation of the basal spike discharge of vestibular system primary afferent neurons, but its activation is not critical for the response to brief mechanical stimuli.     

Local and distal effects induced by unilateral striatal application of opiates in the absence or in the presence of naloxone on the release of dopamine in both caudate nuclei and substantiae nigrae of the cat

Halothane-anesthetized cats implanted with push-pull cannulae in both caudate nuclei (CN) and substantiae nigrae (SN) were used to study the effects of naloxone and various opiates when applied into the left CN on the release of newly synthetized tritiated dopamine (DA) from nerve terminals and dendrites of the two nigro-striatal dopaminergic pathways. In all cases, the drugs (naloxone, opiates alone or in the presence of naloxone) were applied for 30 min into the left CN. When applied alone, naloxone (10⁻⁶ M) induced a delayed reduction in tritiated DA release both in the ipsilateral and contralateral CN. These effects were seen after removal of the drug from the superfusion fluid. Complementary experiments made with tritiated naloxone (10⁻⁶ M) revealed that the contralateral effect on DA release was not due to a diffusion of the opiate antagonist from its application site. Locally,d-Ala₂, Met-enkephalinamide (d-Ala₂, Met-Enk, 10⁻⁶ M) and the potent δ agonist Tyr-d-Ser-Gly-Phe-Leu-Thr (DSThr, 5 × 10⁻⁸ M) induced a biphasic increase in tritiated DA release. The local changes in tritiated DA release evoked by morphine (10⁻⁶ M) and μ agonists such as Tyr-d-Ala-Gly-NH-C₆H₁₃ (10⁻⁸ M) and fentanyl (10⁻⁸ M) differed from those of δ agonists and furthermore differed from each other. For instance, morphine induced a delayed increase in tritiated DA release whereas a biphasic increase followed by a delayed inhibition occurred with fentanyl. Among all the opiates testedd-Ala₂-Met-Enk was the only one which elicited a distal effect, that is a reduction of tritiated DA release in the ipsilateral SN. Marked differences in these opiates' effects on tritiated DA release occurred both locally and in distal structures when opiates were applied simultaneously with naloxone (10⁻⁶ M). Locally, the changes induced by μ agonists were particularly altered since during morphine's application with naloxone a reduction of tritiated DA release occurred. In addition, the opiate antagonist prevented the second increase and the delayed inhibition of tritiated DA release evoked by fentanyl (10⁻⁸ M). Interestingly, the combined application of naloxone with eitherd-Ala₂,Met-Enk (10⁻⁶ M) DSThr (5 × 10⁻⁸ M) or morphine (10⁻⁶ M) resulted in the appearance of changes in tritiated DA release in contralateral structures. The most striking effect was seen withd-Ala₂,Met-Enk which enhanced tritiated DA release in the contralateral CN and SN. These results are discussed in the light of the involvement of several types of opiate receptors and of the polysynaptic pathways responsible for the distal changes in dopaminergic transmission.     

13C NMR spectroscopy study of cortical nerve cell cultures exposed to hypoxia

Primary cultures of cerebral cortical GABA-ergic neurons growing on top of a preformed layer of astrocytes (co-cultures) were incubated with [1-¹³C]glucose and exposed to a low oxygen atmosphere (2% O₂) for 17 hr. ¹³C, ¹H, and ¹³P nuclear magnetic resonance (NMR) spectroscopy was performed on perchloric acid (PCA) extracts of cells and of media collected from these cultures. In the control groups incorporation of ¹³C label into glutamine, citrate, and lactate could be demonstrated in both cell extracts and culture media. Labeled GABA and glutamate were only observed in cell extracts. During hypoxia high energy phosphates decreased but lactate production and glucose consumption increased. There was a decreased amount of citrate and glutamine in cell extracts and media of the hypoxic co-cultures. There was a change in distribution of the ¹³C label within the GABA molecule, with an increase of labeling in the C-2 position. This change in ¹³C distribution was not found in glutamine present in the media where it is a precursor for GABA in neurons. Instead a decrease in the corresponding C-4 position was observed. These results suggest that energy depletion during hypoxia leads to reduced export from the astrocytic tricarboxylic acid (TCA) cycle as demonstrated by a decreased amount of citrate and changed distribution of ¹³C in glutamine. The change in the distribution of label in GABA from cell extracts as compared to glutamine in the medium may indicate that neurons are synthesizing GABA using precursors supplied from their own TCA cycle and not from precursors supplied by astrocytes.     

Some metabolic effects of ammonia on astrocytes and neurons in primary cultures

Some metabolic effects on primary cultures of neurons or astrocytes were studied following acute or chronic exposure to pathophysiological concentrations (usually 3 mM) of ammonia. Three parameters were investigated: (1)¹⁴CO₂ production from¹⁴C-labeled substrates [glucose, pyruvate, branched-chain amino acids (leucine, valine, isoleucine), and glutamate]; (2) interconversion between glutamate and glutamine; and (3) incorporation of label from labeled branched-chain amino acids into proteins. Neither acute nor chronic exposure to ammonia had any effect on¹⁴CO₂ production from [U-¹⁴C]glucose in astrocytes and neurons, whereas under certain conditions¹⁴CO₂ production from [1-¹⁴C]pyruvate in astrocytes was inhibited by ammonia. Production of¹⁴CO₂ from [1-¹⁴C]branched-chain amino acids was inhibited by acute, but stimulated by chronic, exposure to ammonia (3 mM) in astrocytes, with less effect in neurons. Production of¹⁴CO₂ from [1-¹⁴C]glutamate in both astrocytes and neurons was inhibited by acute exposure to ammonia. In astrocytes, glutamate levels tended to decrease and glutamine levels tended to increase following acute exposure to ammonia; in neurons, both glutamine and glutamate levels decreased. Protein content (per culture dish) increased in astrocytes but not in neurons, after chronic exposure to ammonia, possibly as a result of enhanced protein synthesis and/or by inhibition of protein degradation.     

No enhancement by nitric oxide of glutamate release from P2 and P3 synaptosomes of rat hippocampus

Effects of nitric oxide on glutamate (Glu) release in long-term potentiation (LTP) were investigated by superfusion of conventional (P2) and large (P3) synaptosomes prepared from the rat hippocampus. Basal releasing rates of endogenous Glu from P2 and P3 fractions were 103.6 and 85.2 pmol/min/mg protein, respectively. Exposure to a depolarizing concentration of KCl (30 mM) evoked 3.58- and 4.52-fold increases in releasing rates of Glu from P2 and P3 fractions, respectively. Although the perfusion with sodium nitroprusside (NP, 10⁻³ M), a nitric oxide-releasing agent, failed to augment the K⁺-evoked releases of Glu from P2 and P3 synaptosomes, NP enhanced that from slices of the hippocampus by 39% without changing basal release. Similarly, 8-bromoguanosine3′ : 5′-cyclic monophosphate (10⁻⁴ M) increased the K⁺-evoked release of Glu from slices by 30%, but not from either synaptosomes. When synaptosomes were prepared from the hippocampus which was pretreated with two trains of electrical field stimulation (100 Hz, 0.1 ms, for 2 s), K⁺-evoked releases of Glu from P2 and P3 synaptosomes were increased by 15% and 23%, respectively. Although nitric oxide is postulated to function as a retrograde messenger to maintain LTP, present results suggest that nitric oxide may not directly act upon nerve terminals to enhance glutamate release, but that interventions of glias and short neurons may be involved in the presynaptic mechanism of LTP.     

Pharmacogenetic screening of carbamazepine-induced severe cutaneous allergic reactions

Recent studies associated the HLA-B^∗1502 allele with carbamazepine (CBZ)-induced Stevens–Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) in patients from China, Thailand and Malaysia. No association has been found in patients from Europe or Japan. Linkage summary reports from East and South-east Asia predict a highly significant odds ratio (OR) of 84.75 (95% confidence interval [CI] = 42.53–168.91; p = 8.96 × 10[–15]) with sensitivity and negative predictive values of 92% and 98%, respectively. The higher prevalence of HLA-B^∗1502 allele among certain Asian populations (10–15%) compared to Caucasians (1–2%) may explain a 10-fold to 25-fold higher incidence of CBZ-SJS/TEN in patients from Asia. Screening for HLA-B^∗1502 before using CBZ can prevent SJS/TEN in certain populations, but screening may be less beneficial in populations with low HLA-B^∗1502 allele frequency and in patients exposed to CBZ for more than 2 months. A retrospective study demonstrated that the costs of HLA-B^∗1502 screening were less than those of SJS treatment. This article reviews possible benefits and concerns of HLA-B^∗1502 screening in clinical practice.     

Neuropeptide Y increases the inhibitory effects of clonidine on potassium evoked3H-noradrenaline but not3H-5-hydroxytryptamine release from synaptosomes of the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat

Summary The release of³H-noradrenaline (³H-NA) and of³H-5-hydroxytryptamine (³H-5-HT) evoked by high-K⁺ (15 mM) was studied in synaptosomes isolated from the hypothalamus and the frontoparietal cortex of the male Sprague-Dawley rat using a superfusion apparatus. Based on concentration-response curves obtained by analyzing the full-time course of the inhibitory effects of clonidine on³H-NA and on³H-5-HT release neuropeptide Y (NPY) (1 nM) was shown to significantly increase the ability of clonidine to inhibit³H-NA release in synaptosomes isolated from the hypothalamus and from the frontoparietal cortex. NPY (1 nM) alone had no effect on K⁺-evoked³H-NA release from these regions. In contrast, NPY (1 nM) did not modulate the inhibitory effects of clonidine on³H-5-HT release in the above mentioned regions. These results indicate that NPY can increase the sensitivity of the 2-autoreceptors belonging to hypothalamic NA and/or to adrenaline nerve terminals and to cortical NA nerve terminals, while the 2-heteroreceptors inhibiting³H-HT release in the same brain regions appear not to be regulated by high affinity NPY receptors. Thus, 2-autoreceptors and 2-heteroreceptors appear to be differentially controlled by high affinity NPY receptors at least with regard to regulation of³H-NA and³H-5-HT release, respectively.     

Dynamics of testosterone,dihydrotestosterone and estradiol-17β uptake and metabolism in the brain of the male guinea pig

(1) In castrated male guinea pigs protracted infusion to steady state levels of ³H-testosterone (T) either alone or in combination with ¹⁴C-dihydrotestosterone (DHT) or ¹⁴C-estradiol-17β (E) was used to estimate uptake and retention of these steroids from the blood by the anterior pituitary (AP), posterior pituitary (PP), medial basal hypothalamus (MBH), cerebellum (CB) and cerebral cortex (CC). In addition, the amount of ³H-T converted by these various tissues to ³H-DHT and ³H-E was measured. (2) When ³H-T was infused alone, all tissues studied took up and retained the steroid in higher concentrations than blood. The relative order from highest to lowest concentration was as follows: AP>PP>MBH>CB>CC. When ³H-T was infused simultaneously with either ¹⁴C-DHT or ¹⁴C-E, the ¹⁴C-androgen interfered with ³H-T uptake in both the AP and MBH (not in the other tissues); whereas, the ¹⁴C-estrogen interfered with ³H-T uptake only in the AP. (3) ¹⁴C-DHT and ¹⁴C-E were sequestered by those tissues examined. Intertissue differences were not evident for ¹⁴C-DHT levels; in contrast, ¹⁴C-E was substantially more concentrated in the AP, PP and MBH. (4) Intratissue conversion of T to DHT and T to E were noted. For those tissues showing the greatest affinity for T (AP, PP and MBH), the percentage of DHT derived by in situ metabolic conversion was consistently greater (81–15%) than the percentage of E (12–0%) similarly derived. For those tissues showing a low affinity for T (CB and CC), the opposite was the case. (5) The brain metabolized (extracted) 28% of both incoming T and DHT and 35% of E. (6) Metabolic clearance rates of T, DHT and E were 54.2, 71.1 and 33.7 ml/min × kg^?1, respectively. (7) The results indicate extensive brain metabolism of the three hormones examined and also suggest that there is more than one population of androgen receptor. The localization of active T and E uptake and rapid metabolism of T to DHT in neuroendocrine areas reinforces the belief that hormonal actions depend upon these events in this species.     

Characterization of substance P and somatostatin receptors on adrenal chromaffin cells using structural analogues

Substance P (SP) and somatostatin (SRIF) are known to inhibit the nicotine-induced release of catecholamines (CAs) from isolated adrenal chromaffin cells in culture^22,24. In order to characterize the receptors mediating this action, we have tested several SP and SRIF analogues for their effects on release of [³H]l-norepinephrine ([³H]NE) from chromaffin cell cultures. SP-free acid and a series of 11 SP analogues, in which each amino acid of SP is replaced in turn byl-alanine, all inhibited the nicotine-induced release of [³H]NE from these cultures. The rank order of potency of the analogues for this action was similar to their reported order of potency in other SP-responsive tissues. The least potent were SP-free acid, [Ala⁷]-SP, [Ala¹⁰]-SP, [Ala⁸]-SP and [Ala¹¹]-SP, while the potencies of the Ala 1 to 6 analogues and [Ala⁹]-SP were closer to that of SP. [Leu⁷]- and [Leu^7,8]-SP had potencies similar to that of SP. SP itself had no effect on basal [³H]NE release. The results suggest that adrenal chromaffin cells possess a specific SP receptor mediating inhibition of agonist-induced CA release and that the binding site of this receptor shares similar structural requirements with the binding site of the SP receptor on other tissues.Several SRIF analogues, which have been previously shown to be more potent than native SRIF at selective SRIF receptors^{2,3,31, 35}, were compared to SRIF for effects on [³H]NE release from chromaffin cell cultures. These analogues were found to be active but less potent than SRIF in inhibiting nicotine-induced [³H]NE release from these cultures, suggesting that the site mediating this action differs in its structural requirements from the SRIF receptor found in some other tissues.     

Release of synaptosomal dopamine formed from tyrosine andl-dopa

The present report compares the synaptosomal release of [³H]dopamine, continuously forming from [³H]DOPA, with that of [¹⁴C]dopamine, forming from [¹⁴C]tyrosine as a marker of dopaminergic nerve endings. For the purpose of the comparison, synaptosomal (P₂) preparations from rat caudate nuclei were incubated withl-[³H]DOPA and [¹⁴C]tyrosine for 10 min and the particulates were rapidly separated from the medium postincubation. The separated fractions were analyzed for the level of double labelled (¹⁴C/³H) dopamine and the synaptosomal content of the labelled substrates. Of the total labelled dopamine formed, the fraction that was present in the medium, following the synaptosomal release, was determined. Tested were the release enhancing effects of various additions which included several known dopaminergic agents, serotonin and 5-hydroxytryptophan. The data show that the addition of dopamine to the incubation mixture to either 0.5 or 1.0 μM concentration markedly enhanced the release of labelled dopamine. Serotonin when added to 5.0 μM concentration also raised the medium content of labelled dopamine but it was ineffective at 1.0 μM. 5-Hydroxytryptophan, 1.0 or 5.0 μM, had no effect. For the comparison of the release enhancing effects of an addition of [¹⁴C]dopamine and [³H]dopamine, the corresponding release indices (release index = medium/total ratio of labelled dopamine in the presence of an addition divided by the same ratio in control (no addition) sample) were determined. The data indicate that the index of [¹⁴C]dopamine did not differ significantly from that of [³H]dopamine following the addition of either dopamine, serotonin or 5-hydroxytryptophan at any of the concentrations tested. A similar lack of difference between the index of [¹⁴C]dopamine and that of [³H]dopamine was observed following the addition of reserpine (1.8 μM), although a considerable enhancement of labelled dopamine release occurred. The addition of either amphetamine (9.1 μM) or amfonelic acid (9.1 μM) also enhanced labelled dopamine release but in their presence the index of [³H]dopamine was significantly higher than that of [¹⁴C]dopamine. Amfonelic acid preferentially raised the [³H]dopamine index at the lowest concentration of 0.91 μM that we have tested and also after only 5 min of incubation; coaddition of reserpine increased the [¹⁴C]dopamine release, thus abolishing the preferential effect of amfonelic acid on [³H]dopamine. When the incubation was performed without an addition (control sample), no difference (NS) was observed between the particulate to medium distribution of [¹⁴C]dopamine and that of [³H]dopamine after either 5, 10 or 15 min. The results suggest that (a) dopamine synaptosomally formed froml-DOPA may exist in a pool distinct from that dopamine arising from tyrosine hydroxylation and (b) the observed dopamine compartmentation may be due to some mechanism distinct from any possible participation of serotoninergic partivles. Also, the present results support the previously suggested existence of synaptosomal DOPA in pools.