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Characterization of presynaptic alpha-2 adrenoceptors in rat brain
COMPARISON OF SOME ALPHA-1 AND ALPHA-2 ADRENERGIC BLOCKERS IN EXPERIMENTAL MODELS OF PROSTATIC HYPERTROPHY M.K. TaP.fer, T.Yemane, J.l. Szekely and L.G. H~rsing, Jr. Institute for Drug Research, H-1325 Budapest, P.O.Box 82.
•CHARACTERIZATIC~ OF PRESYNAPTIC ALPHA-2 ADPd~NOCEPTORS IN RAT BRAIN G.Zsilla, J.P.Kiss, A.Mike, T.Zelles, A.Tdth +, A.Lajth + and E.S.Vizi Inst.of Experimental Medicine,Budapest,Hungary and N.S.Kline Inst. for Psychiatric Res. ,Orangeburg,N.Y+
Hungary It is well established that adrenergic hyperactivity is a contributing factor in urinary obstruction during benign prostatic hyperplasia (BPH). Therefore in the clinical practice adrenergic blockers are used for sympatomatic relief. Since both. alpha~ selective blockers (prazosin, terazosin, etc) and nonselective ones (as phentolamine, phenoxybenzamine) are used, the question arises whether alpha2 blockade in conjunction with alpha~ inhibition may be beneficial in the treatment of BPH. We compared the efficacies of several alpha~ and alpha2 blockers and some nonselective ones in isolated organ preparations (rat vas deferens and urinary bladder and rabbit prostate strip) and in vivo models of BPH. The urinary obstruction induced by phenylephrine infusion in acute experiments or chronic testosterone treatment was recorded by transvesical cystometry. In the acute model both alpha~ and a!pha2 inhibitors improved the voiding parameters. However, in the chronic model only the alpha1 blockers were effective in proportion with their potencies in vitro.
~ e release of norepinephrine (NE) in rat hippocampus was studied in vivo and in vitro. Results of microdialysis studies have shown that low doses of BRL 44408 an alpha-2AD antagonist had no effect on the basal release of NE, however in a dose Of 5 mg/kg i.p. significantly enhanched the extracellular level of NE. ARC 239 an alpha-2BC antagonist was ineffective. BRL 44408 increased the electrically induced release of 3H-NE in in vitro perfusion experiments while ARC 239 had no effect in a dose range of 10nM to luM. These results were in agreement with data of receptor b i ~ i n g studies. 3H-Yohimbine was displaced frc~a hippocampal membranes with high affinity by BRL 44408 (pKi = 8.26 + 0.15) whereas ARC 239 was less effective (pKi = 7.02 + 0.06). The pKi values of eight different alpha-2 adrenergic compounds showed a very good correlation in hippocampus and cortex suggesting the identity of the receptors. The pKi values in the spleen (alpha-2A) or in the kidney (alpha-2B) ccmparing with the hippocampus or cortex showed a poor correlation. Our data indicate that hippocampal NE release in the rat brain is regulated by alpha-2D adrenoceptors which are identical with the alpha-2 receptors located in the cortex.
ATP-EVOKED Ca z+ MOBILIZATION, lPs FORMATION AND PURINE RELEASE FROM RAT CULTURED ASTROCYTES Ballerini P.,Ciccarelli R.,Di Iorio P.,Giuliani P.,D'Alimonte I.,Renzetti A. and Caciagli F. Institute of Pharmacology~ University of Chieti, Chieti, Italy.
E F F E C T S OF ADENOSINE A G O N I S T S AND ANTAGONISTS ON T H E S L E E P - W A K I N G C Y C L E IN T H E RAT R.BertorellL N. Ferri, M. Adami and E. Ongini Research Laboratories, Schering-Plough, Comazzo, Milan, Italy.
ATP has been found to increase [Ca2+]i in a variety of excitable and nonexcitable cells and to act as a neurotransmitter and/or neuromodulator within the CNS. We previously reported that purines (P) are released at rest and uncle=:a field electrical stimulation from dissociated primary cultures of rat brain astrocytes. 2McSATP, a selective P~y receptor agenist and AMPPNP, a stable ATP analogue, increased P outflow from the glial cultures; this increase was counteracted by suramin, a P2 receptor antagonist. The effects of ATP on Ca 2+ mobilization, evaluated by the fluorescem probe fltra2/AM, and PI metabolism were then investigated, it increased both [Ca2~]i and PI hydrolysis. The responses to ATP were reduced by the P2 receptor antagonist reactive blue 2 (RB2). 2MeSATP was also able to cause a rise of [Ca¢+ll and of PI metabolism completely counteracted by RB2. c¢,[~methylene-ATP was ineffective whereas UTP, a P2~yagonist was able to induce 1Ps formation and Ca 2+ accumulation. Glial cell pretreatment with oATP, a P2z antagonist, only slightly reduced the intracellular ion increase evoked by 2MeSATP but significantly inhibited the ATP-induced rise of [CaZ+l~levels. The increase in [Ca2~]ievoked by ATP persisted, even thongh reduced, in a medium Ca2*free+EGTA, indicating a prevailing role of Ca2j from intracellular stores. Release of Ca 2' by ATP was strongly redhced by tbapsigargin and by acute treatment with PMA. Furthermore thapsigargin also increased P outflow evaluated in sister cultures, whereas 3,4,5,lrimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) decreased P oulflow induced by 2McSATP. These observations confirm that functional P2 receptors are present on astrocyte membranes and even though the nucleotide levels required to trigger Ca2~ mobilization, purinc release or IP breakdown are relatively high, p.M extracellular ATP concentrations can be achieved during several pathological events. The possibility that the ATP responses were mediated via P2 receptor subtypes other than P2y, as P2u and P~z is under investigation. Moreover our data strenghten a role for ATP as a medmtor of neuron-astrocytc slgnahng, the rise of [Ca ]l being a slgual for glial cells to release substances (e.g. purines) that feed back to modulate neuron excitability or regulatc synaptic transmission.
There is evidence that adenosine may participate in the regulation of sleep and wakefulness in the mammalian central nervous system. To understand whether the adenosine receptor subtypes A1 and A2~ are involved in sleep processes we carried out electroencephalographic (EEG) studies in the rat using agonists and antagonists for either receptors. EEG activity was recorded for 6 h after intraperitoneal administration of drugs, and the stages of wakefulness, rapid eye movements (REM) sleep and n o n - R E M sleep were classified thereafter. The dose-response effects of the A1 agonist 2-chloro-N 6cyclopentyladenosine (CCPA), the Aza agonist 2 - h e x y n y l - 5 ' - N ethylcarboxamidoadenosine (2HE-NECA) and the non-selective agonist 5'-N-ethylcarboxamidoadenosine (NECA) were examined. In addition, we have studied the effects of the selective A1 antagonist 8cyclopentyl-l,3-dipropylxanthine (DPCPX), and of the Aza adenosine antagonists 9-chloro-2-(2-furyl)-5,6-dihydro-[1,2,4]-triazolo [1,5-c] quinazolin-5-imine (CGS 15943) and 7-(2-phenylethyl)-5-amino-2(2-furyl)-pyrazolo [4,3-e]-l,2,4-triazolo [1,5-c] pyrimidine (SCH 58261). The results obtained with these antagonists were compared with those of caffeine (10 mg/ltg). CCPA and 2HE-NECA at all doses tested (0.003-0.03 and 0.03-0.3 mg/kg, respectively) did not significantly modify the sleep architecture over the 6 h recording time, while NECA, at 0.03 and 0.I mg/kg, increased the time spent in waking and decreased both n o a - R E M and REM sleep in the whole recording period. Like caffeine, both CGS 15943 (0.3-10 mg/kg) and SCH 58261 (0.3-10 mg/kg)i at the highest dose, produced sleep suppression, while DPCPX (0.3-10 mg/kg) did not affect sleep parameters. In conclusion, these studies suggest that both A1 and Aza adenosine receptors are involved in the regulation of mechanisms underlying sleep and wakefulness.
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194
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•CHARACTERIZATIC~ OF PRESYNAPTIC ALPHA-2 ADPd~NOCEPTORS IN RAT BRAIN G.Zsilla, J.P.Kiss, A.Mike, T.Zelles, A.Tdth +, A.Lajth + and E.S.Vizi Inst.of Experimental Medicine,Budapest,Hungary and N.S.Kline Inst. for Psychiatric Res. ,Orangeburg,N.Y+
Hungary It is well established that adrenergic hyperactivity is a contributing factor in urinary obstruction during benign prostatic hyperplasia (BPH). Therefore in the clinical practice adrenergic blockers are used for sympatomatic relief. Since both. alpha~ selective blockers (prazosin, terazosin, etc) and nonselective ones (as phentolamine, phenoxybenzamine) are used, the question arises whether alpha2 blockade in conjunction with alpha~ inhibition may be beneficial in the treatment of BPH. We compared the efficacies of several alpha~ and alpha2 blockers and some nonselective ones in isolated organ preparations (rat vas deferens and urinary bladder and rabbit prostate strip) and in vivo models of BPH. The urinary obstruction induced by phenylephrine infusion in acute experiments or chronic testosterone treatment was recorded by transvesical cystometry. In the acute model both alpha~ and a!pha2 inhibitors improved the voiding parameters. However, in the chronic model only the alpha1 blockers were effective in proportion with their potencies in vitro.
~ e release of norepinephrine (NE) in rat hippocampus was studied in vivo and in vitro. Results of microdialysis studies have shown that low doses of BRL 44408 an alpha-2AD antagonist had no effect on the basal release of NE, however in a dose Of 5 mg/kg i.p. significantly enhanched the extracellular level of NE. ARC 239 an alpha-2BC antagonist was ineffective. BRL 44408 increased the electrically induced release of 3H-NE in in vitro perfusion experiments while ARC 239 had no effect in a dose range of 10nM to luM. These results were in agreement with data of receptor b i ~ i n g studies. 3H-Yohimbine was displaced frc~a hippocampal membranes with high affinity by BRL 44408 (pKi = 8.26 + 0.15) whereas ARC 239 was less effective (pKi = 7.02 + 0.06). The pKi values of eight different alpha-2 adrenergic compounds showed a very good correlation in hippocampus and cortex suggesting the identity of the receptors. The pKi values in the spleen (alpha-2A) or in the kidney (alpha-2B) ccmparing with the hippocampus or cortex showed a poor correlation. Our data indicate that hippocampal NE release in the rat brain is regulated by alpha-2D adrenoceptors which are identical with the alpha-2 receptors located in the cortex.
ATP-EVOKED Ca z+ MOBILIZATION, lPs FORMATION AND PURINE RELEASE FROM RAT CULTURED ASTROCYTES Ballerini P.,Ciccarelli R.,Di Iorio P.,Giuliani P.,D'Alimonte I.,Renzetti A. and Caciagli F. Institute of Pharmacology~ University of Chieti, Chieti, Italy.
E F F E C T S OF ADENOSINE A G O N I S T S AND ANTAGONISTS ON T H E S L E E P - W A K I N G C Y C L E IN T H E RAT R.BertorellL N. Ferri, M. Adami and E. Ongini Research Laboratories, Schering-Plough, Comazzo, Milan, Italy.
ATP has been found to increase [Ca2+]i in a variety of excitable and nonexcitable cells and to act as a neurotransmitter and/or neuromodulator within the CNS. We previously reported that purines (P) are released at rest and uncle=:a field electrical stimulation from dissociated primary cultures of rat brain astrocytes. 2McSATP, a selective P~y receptor agenist and AMPPNP, a stable ATP analogue, increased P outflow from the glial cultures; this increase was counteracted by suramin, a P2 receptor antagonist. The effects of ATP on Ca 2+ mobilization, evaluated by the fluorescem probe fltra2/AM, and PI metabolism were then investigated, it increased both [Ca2~]i and PI hydrolysis. The responses to ATP were reduced by the P2 receptor antagonist reactive blue 2 (RB2). 2MeSATP was also able to cause a rise of [Ca¢+ll and of PI metabolism completely counteracted by RB2. c¢,[~methylene-ATP was ineffective whereas UTP, a P2~yagonist was able to induce 1Ps formation and Ca 2+ accumulation. Glial cell pretreatment with oATP, a P2z antagonist, only slightly reduced the intracellular ion increase evoked by 2MeSATP but significantly inhibited the ATP-induced rise of [CaZ+l~levels. The increase in [Ca2~]ievoked by ATP persisted, even thongh reduced, in a medium Ca2*free+EGTA, indicating a prevailing role of Ca2j from intracellular stores. Release of Ca 2' by ATP was strongly redhced by tbapsigargin and by acute treatment with PMA. Furthermore thapsigargin also increased P outflow evaluated in sister cultures, whereas 3,4,5,lrimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) decreased P oulflow induced by 2McSATP. These observations confirm that functional P2 receptors are present on astrocyte membranes and even though the nucleotide levels required to trigger Ca2~ mobilization, purinc release or IP breakdown are relatively high, p.M extracellular ATP concentrations can be achieved during several pathological events. The possibility that the ATP responses were mediated via P2 receptor subtypes other than P2y, as P2u and P~z is under investigation. Moreover our data strenghten a role for ATP as a medmtor of neuron-astrocytc slgnahng, the rise of [Ca ]l being a slgual for glial cells to release substances (e.g. purines) that feed back to modulate neuron excitability or regulatc synaptic transmission.
There is evidence that adenosine may participate in the regulation of sleep and wakefulness in the mammalian central nervous system. To understand whether the adenosine receptor subtypes A1 and A2~ are involved in sleep processes we carried out electroencephalographic (EEG) studies in the rat using agonists and antagonists for either receptors. EEG activity was recorded for 6 h after intraperitoneal administration of drugs, and the stages of wakefulness, rapid eye movements (REM) sleep and n o n - R E M sleep were classified thereafter. The dose-response effects of the A1 agonist 2-chloro-N 6cyclopentyladenosine (CCPA), the Aza agonist 2 - h e x y n y l - 5 ' - N ethylcarboxamidoadenosine (2HE-NECA) and the non-selective agonist 5'-N-ethylcarboxamidoadenosine (NECA) were examined. In addition, we have studied the effects of the selective A1 antagonist 8cyclopentyl-l,3-dipropylxanthine (DPCPX), and of the Aza adenosine antagonists 9-chloro-2-(2-furyl)-5,6-dihydro-[1,2,4]-triazolo [1,5-c] quinazolin-5-imine (CGS 15943) and 7-(2-phenylethyl)-5-amino-2(2-furyl)-pyrazolo [4,3-e]-l,2,4-triazolo [1,5-c] pyrimidine (SCH 58261). The results obtained with these antagonists were compared with those of caffeine (10 mg/ltg). CCPA and 2HE-NECA at all doses tested (0.003-0.03 and 0.03-0.3 mg/kg, respectively) did not significantly modify the sleep architecture over the 6 h recording time, while NECA, at 0.03 and 0.I mg/kg, increased the time spent in waking and decreased both n o a - R E M and REM sleep in the whole recording period. Like caffeine, both CGS 15943 (0.3-10 mg/kg) and SCH 58261 (0.3-10 mg/kg)i at the highest dose, produced sleep suppression, while DPCPX (0.3-10 mg/kg) did not affect sleep parameters. In conclusion, these studies suggest that both A1 and Aza adenosine receptors are involved in the regulation of mechanisms underlying sleep and wakefulness.
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194
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