Study of isolated apparent amniogenic limb deficiency in Hungary, 1975-1984

Rat brain cortex slices and synaptosomes preincubated with [3H]noradrenaline were used to investigate whether the NMDA-evoked noradrenaline release is modulated by agonists or antagonists at presynaptic alpha 2-adrenoceptors. In experiments on slices, noradrenaline and the preferential alpha-adrenoceptor agonists talipexole (former B-HT 920) and clonidine inhibited the NMDA-evoked tritium overflow whereas the selective alpha 1-adrenoceptor agonists cirazoline and methoxamine were ineffective. The alpha 2-adrenoceptor antagonists rauwolscine and idazoxan facilitated the NMDA-evoked tritium overflow whereas the preferential alpha 1-adrenoceptor antagonist prazosin was ineffective. The concentration-response curve of talipexole for its inhibitory effect on NMDA-evoked overflow was shifted to the right by idazoxan (apparent pA2 = 7.5). The EC50 of NMDA (97 mumol/l) for its stimulating effect on tritium overflow was not substantially changed by blockade of alpha 2-autoreceptors with 1 mumol/l rauwolscine (EC50 of NMDA in the presence of the alpha 2-adrenoceptor antagonist, 155 mumol/l), but the maximal overflow of tritium was increased 2.5 fold by this rauwolscine concentration. In experiments on synaptosomes, talipexole and noradrenaline inhibited the NMDA-evoked tritium overflow. The inhibitory effect of talipexole was abolished by idazoxan which, given alone, was ineffective, as was prazosin. Talipexole did also not produce an inhibition when tritium overflow was evoked by NMDA in the presence of omega-conotoxin GVIA 0.1 mumol/l; the latter, by itself, decreased the response to NMDA by about 55%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis in the male gonad

1. Previous work has shown that enalaprilat, an inhibitor of angiotensin-converting enzyme (ACE), potentiated the actions of alpha 1-adrenoceptor antagonists; it was hypothesized that angiotensin II (AngII) modulated the activity of alpha 1-adrenoceptors. This hypothesis was tested in Sprague-Dawley rat isolated perfused tail arteries using the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD123319. 2. Losartan had no alpha 1-adrenoceptor antagonist effects at concentrations below 1 mumol/L. Similarly, losartan (0.1 mumol/L) had no effect on the alpha 1-adrenoceptor antagonist action of doxazosin (1, 10 nmol/L) nor on the potentiation of doxazosin by enalaprilat (1 mumol/L). 3. PD123319 (0.1 mumol/L) had no alpha 1-adrenoceptor antagonist effect but altered the mode of action of the alpha 1-adrenoceptor antagonist doxazosin: PD123319 changed doxazosin from a competitive to a non-competitive antagonist, as evidenced by the reduced slope of the dose-response curve for the alpha 1-adrenoceptor agonist phenylephrine. 4. These results suggest that AngII can modulate alpha 1-adrenoceptor function in rat tail arteries via an indirect action at AT2 receptors. However, the present results do not rule out the involvement of bradykinin, endothelin or prostaglandin in the modulation of alpha 1-adrenoceptor function by angiotensin II.     

Effect of 8-sulfophenyl theophylline on endogenous noradrenaline release from sympathetic nerves of the rabbit ear artery

1. The release of endogenous noradrenaline (NA) and adenyl purine (ATP, ADP, AMP and adenosine) from the rabbit ear artery, evoked by electrical stimulation (ES; 16 Hz), was examined. 2. ES evoked a significant release of NA and purine; the ratio of the amount of total purine released to NA released was approximately 180 on a molar base. 3. ES-evoked purine release was significantly reduced by the denudation of the endothelium and abolished by the alpha 1-adrenoceptor antagonist, prazosin (1 mumol/L). 4. ES-evoked NA release was significantly reduced by a P1-purinoceptor antagonist, 8-sulfophenyl theophylline (8SPT). Purine release was slightly reduced by 8SPT. 5. These results suggest that endogenous NA released by ES results in the release of a large amount of purine, which may, in turn, increase the release of NA by acting on prejunctional purinoceptors on sympathetic nerve terminals.     

Nonexocytotic noradrenaline release and ventricular fibrillation in ischemic rat hearts

In myocardial ischemia, nonexocytotic noradrenaline release has been identified as underlying mechanism of ischemia-evoked noradrenaline release. Nonexocytotic noradrenaline release can be suppressed by inhibitors of the neuronal noradrenaline carrier (uptake), such as desipramine. Utilizing this pharmacological intervention the role of local noradrenaline release in the genesis of ischemia-induced ventricular arrhythmias was studied. Regional ischemia was induced in rat isolated perfused hearts by ligature of the left anterior descending coronary artery, and the venous effluent obtained during the first 2 min of reperfusion was used to measure the release of endogenous noradrenaline by high-performance liquid chromatography methods. Coronary occlusion caused ventricular fibrillation in a well reproducible manner with an incidence of 70 to 80% during a 30 min observation period. Blockage of uptake1 by desipramine decreased the occurrence of ischemia-induced ventricular fibrillation to 60% (0.01 mumol/l) or 20% (0.1 mumol/l), and ventricular fibrillation was completely suppressed by 1 mumol/l desipramine. Likewise, desipramine (0.01-1 mumol/l) concentration-dependently reduced endogenous noradrenaline release during 30 min of regional myocardial ischemia. Nisoxetine, a structurally unrelated inhibitor of uptake1, also suppressed ischemia-evoked ventricular fibrillation. In contrast to its antifibrillatory effect during regional myocardial ischemia, desipramine precipitated arrhythmias when ventricular fibrillation was induced by perfusing normoxic hearts with exogenous noradrenaline. Combination of desipramine (0.1 mumol/l) with exogenous noradrenaline (0.01 to 1 mumol/l) increased the incidence of ventricular fibrillation compared to noradrenaline perfusion alone. Under these conditions, uptake1-blockade is known to increase the extracellular concentration of the perfused noradrenaline. Finally, in the isolated, spontaneously beating papillary muscle of the left rat heart, desipramine (0.1 and 1.0 mumol/l) had no effect on the upstroke velocity of action potentials, the action potential duration and the effective refractory period. In conclusion, the findings demonstrate that nonexocytotic noradrenaline release is an important mediator of ischemia-induced ventricular fibrillation in isolated hearts of the rat. It is also documented that uptake1 inhibitors such as desipramine reveal their effects on ventricular fibrillation secondary to their action on transmembrane noradrenaline transport.     

Vascular actions of purines in the foetal circulation of the human placenta

1. The vasoactive effects of adenosine triphosphate (ATP), adenosine and other purines in the foetal circulation of the human placenta were examined. Single lobules of the placenta were bilaterally perfused in vitro with Krebs buffer (maternal and foetal sides 5 ml min-1 each, 95% O2:5% CO2, 37 degrees C). Changes in foetal vascular tone were assessed by recording perfusion pressure during constant infusion of each purine. To allow recording of the vasodilator effects, submaximal vasoconstriction was induced by concomitant infusion of prostaglandin F2 alpha (0.7-2.0 mumol l-1). 2. ATP (1.0-100 mumol l-1) usually caused concentration-dependent reductions in perfusion pressure. However, biphasic with initial transient increases, or only increases in pressure were sometimes observed. Falls in pressure caused by ATP were significantly reduced by addition to the perfusate of NG-nitro-L-arginine (L-NOARG) (100 mumol l-1) but not NG-nitro-D-arginine (D-NOARG) (100 mumol l-1). They were not influenced by addition of indomethacin (10 mumol l-1) or L-arginine (100 mumol l-1). 3. Adenosine (0.01-1.0 mmol l-1) consistently caused concentration-dependent reductions in perfusion pressure, this effect not being influenced by indomethacin. L-NOARG, but not D-NOARG, reduced the potency of adenosine approximately three fold. L-Arginine, but not D-arginine enhanced its potency by a similar amount. 4. 2-Methylthio-ATP, a selective P2 gamma agonist was approximately 50 times more potent than ATP as a vasodilator agent, always causing decreases in perfusion pressure. 5. Beta-gamma-Methylene ATP, a selective P20 agonist, was approximately 100 times more potent than ATP as a vasoconstrictor, but only caused transient increases in perfusion pressure.6. The rank order of vasodilator potencies of a selection of adenosine receptor agonists was, 2-chloroadenosine>5-(N-cyclopropyl)-carboxamidoadenosine, >5-N-ethylcarboxamidoadenosine, >2-chloro-N6-cyclopentyladenosine, >CGS-21680 > N6-cyclohexyladenosine = adenosine. Vasodilatation due to adenosine was inhibited by the PI-A2 receptor antagonist 3,7-dimethyl-l-propargylxanthine(DMPX).7. These results suggest that ATP may cause an endothelium-dependent vasodilatation in the foetal vessels of the human placenta via activation of a P2y receptor linked to the formation of nitric oxide(NO). Vasodilatation caused by ATP may mask an accompanying vasoconstrictor effect mediated, via a P2X receptor, in the villous vascular smooth muscle. Adenosine acting on P1-A2 receptors, which are also present in the foetal vasculature, may require synergistic interaction with NO to achieve a maximal vasodilator response.     

The interaction between enalaprilat and selected alpha-adrenoceptor antagonists in isolated rat tail arteries

1. Isolated perfused rat tail artery preparations were used to investigate the effects of the angiotensin converting enzyme inhibitor enalaprilat on the actions of a series of alpha-adrenoceptor antagonists. The agonist used was phenylephrine. 2. Enalaprilat (1 mumol/L) potentiated the competitive alpha 1-adrenoceptor antagonist actions of phentolamine (10-100 nmol/L) and yohimbine (0.3-3.0 mumol/L) as well as the non-competitive antagonist action of phenoxybenzamine (50-100 pmol/L). 3. The competitive alpha 1-adrenoceptor antagonist action of prazosin (1-10 nmol/L) was not affected by enalaprilat. 4. For the competitive alpha 1-adrenoceptor antagonists, including prazosin, there appeared to be an inverse relationship between antagonist potency and the extent of potentiation by enalaprilat. 5. The results support the hypothesis and angiotensin II modulates vascular smooth muscle alpha 1-adrenoceptor function.     

Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI

1. Long-term treatment with beta 2-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to beta-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with beta 2-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied. 2. The effect of beta 2-agonist incubation alone and after coincubation with dexamethasone on density and affinity of beta-adrenoceptors was investigated by radioligand binding experiments. 3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent beta-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73 +/- 4% in efficacy of isoprenaline to relax human bronchial smooth muscle. 4. After an incubation period of 60 min with 100 mumol l-1 terbutaline, a significant decline in its relaxing efficacy (81 +/- 8%) and potency (by a factor 5.5) occurred. 5. Incubation with 30 mumol l-1 isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4). 6. Coincubation of dexamethasone with isoprenaline (120 min; 30 mumol l-1) preserved the effect of isoprenaline on relaxation (129 +/- 15%). 7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 mumol l-1) resulted in a decrease in beta-adrenoceptor binding sites (Bmax) to 64 +/- 1.6% (P < 0.05), while the antagonist affinity (KD) for [3H]-CGP-12177 remained unchanged. 8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 mumol l-1) or isoprenaline (30 mumol l-1) plus dexamethasone (30 mumol l-1) for 120 min did not lead to a significant change of Bmax (160 +/- 22.1% vs 142.3 +/- 28.7%) or KD (5.0 nmol l-1 vs 3.5 nmol l-1) compared to the controls. 9. In conclusion, pretreatment of human bronchi with beta-adrenoceptor agonists leads to functional desensitization and, in lung tissue, to down-regulation of beta-adrenoceptors. This effect can be counteracted by additional administration of dexamethasone. Our model of desensitization has proved useful for the identification of mechanisms of beta-adrenoceptor desensitization and could be relevant for the evaluation of therapeutic strategies to counteract undesirable effects of long-term beta-adrenoceptor stimulation.     

Regionally specific changes in extracellular noradrenaline following chronic idazoxan as revealed by in vivo microdialysis

The selective alpha 2-adrenoceptor antagonist idazoxan was administered chronically (0.8 mg/kg per h) to rats for a period of 10 days via osmotic minipumps. On day 11, 24 h after removal of the pumps, the rats were anaesthetised and microdialysis probes were implanted into either the frontal cortex or hippocampus. Basal noradrenaline release in the frontal cortex was significantly elevated compared with the saline control group. Each animal was then challenged with idazoxan (10 mg/kg s.c.). Inhibition of presynaptic alpha 2-adrenoceptors resulted in a significant increase in noradrenaline release in the saline control group. However, animals treated chronically with idazoxan, showed a markedly attenuated response to the single dose idazoxan challenge in the frontal cortex. No significant change in either basal release or in response to idazoxan challenge was observed in the hippocampus in the chronic idazoxan-treated animals as compared with the chronic saline control group. Chronic idazoxan administration results in selective enhancement of noradrenaline release in the frontal cortex but not in the hippocampus. This would be consistent with a down-regulation of presynaptic alpha 2-adrenoceptors with the subsequent loss of presynaptic noradrenergic negative feedback inhibition.     

Effects of optical isomers of ephedrine and methylephedrine on the twitch response in the isolated rat vas deferens and the involvement of alpha 2-adrenoceptors

The effects of optical isomers of ephedrine (EPH) and methylephedrine (MEP) on the twitch response to electrical stimulation (1 msec, 1 Hz) in the isolated rat vas deferens were investigated to clarify the action on alpha 2-adrenoceptors. l-EPH (10(-7) 3 x 10(-5) M) and d-EPH (10(-6)-10(-4) M) markedly inhibited the twitch response in the presence of prazosin (10(-6) M). l-MEP also inhibited the twitch response at high concentrations (3 x 10(-5)-10(-3) M). The rank order of inhibitory potency was 1-EPH > d-EPH > l-MEP. Yohimbine (3 x 10(-7) M), a selective alpha 2-adrenoceptor antagonist, attenuated the twitch-inhibitory effects of EPH isomers and l-MEP. Furthermore, the twitch-inhibitory effects of EPH isomers and l-MEP were attenuated by reserpine treatment (8 mg/kg, s.c.). On the other hand, d-MEP showed the potentiation of twitch response, and competitively antagonized the twitch-inhibitory effect of clonidine (10(-9)-10(-6) M) with the pA2 value of 4.3 in the presence of prazosin. The results suggest that EPH isomers and l-MEP have stimulating activity for presynaptic alpha 2-adrenoceptors. In addition, the twitch-inhibitory effect of EPH isomers and l-MEP may be at least partly mediated through the release of noradrenaline. It is also suggested that d-MEP has competitive alpha 2-adrenoceptor antagonist activity.     

ORL1 receptor-mediated inhibition by nociceptin of noradrenaline release from perivascular sympathetic nerve endings of the rat tail artery

In the present study the effect of the opioid heptadecapeptide nociceptin, also termed orphanin FQ, an endogenous ligand for the orphan receptor named ORL1 (opioid receptor-like 1) receptor, was investigated on [3H]noradrenaline release induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery in the absence and presence of an alpha2-adrenoceptor antagonist, rauwolscine 3 microM. Nociceptin inhibited the electrically-evoked tritiated noradrenaline release in a concentration-dependent manner from rat tail arteries. This inhibitory effect of nociceptin was enhanced in the presence of the alpha2-adrenoceptor antagonist rauwolscine (maximum inhibition by 25% and 50% in the absence and presence of rauwolscine, respectively). At a supramaximal concentration (10 microM), the inhibitory action of DAGO, a selective micro-opioid receptor agonist, was less pronounced than that of nociceptin. The inhibitory effect of nociceptin was counteracted by naloxone benzoylhydrazone (3 microM) which by itself did not change the stimulation-evoked noradrenaline overflow. Naloxone (10 microM), a non-selective opioid receptor antagonist, did not affect the inhibitory effect of nociceptin whereas it abolished that of DAGO. In conclusion, these results suggest that nociceptin modulates noradrenergic neurotransmission by acting on prejunctional ORL1 receptors located on nerve terminals innervating the rat tail artery. They also demonstrate that prejunctional ORL1 receptors interact with prejunctional alpha2-adrenoceptors. The physiological significance of this phenomenon remains to be determined.     

Alterations in alpha 1-adrenergic receptor-mediated phosphatidylinositol turnover in hypoxic cardiac myocytes

The purpose of these studies was to examine the effects of hypoxia on alpha 1-adrenergic receptor (alpha 1AR) mediated phosphatidylinositol (PI) turnover in cultured neonatal rat cardiac myocytes. Cells were pre-labeled with [3H]-inositol and incubated for 1 h in either normoxia or hypoxia. Phenylephrine, an alpha 1AR agonist, was added at various time intervals (0-60 min) before termination of the incubation. There was a time-dependent release of radioactivity from the lipid fraction to the aqueous fraction with alpha 1AR stimulation. alpha 1AR-mediated PI turnover was biphasic in normoxic cells and monophasic in hypoxic cells. Using ion-exchange chromatography, radioactivity in the inositol trisphosphate (IP3) peak was increased with acute phenylephrine stimulation (5 min) in the normoxic cells, while inositol phosphate (IP) and inositol bisphosphate (IP2) were increased with chronic stimulation (60 min). After 5 min of alpha 1AR stimulation, hypoxia did not alter total aqueous radioactivity when compared to normoxia, but there was a significant increase in IP2. However, there was decreased PI turnover in chronically stimulated (30-60 min) hypoxic cells when compared to normoxic cells. Hypoxia had no effect on radioactivity in the IP3 fraction with either 0, 5, or 60 min of alpha 1AR stimulation, but there was a significant increase in [1,4,5]-IP3 in hypoxic cells with 30 s alpha 1AR stimulation. With hypoxia, there was no difference in radioactivity in the phosphatidylinositols with either 0 or 5 min stimulation when compared to normoxia. However, after 60 min of alpha 1AR stimulation, hypoxia resulted in increased PI and PIP, when compared to normoxic cells, but PIP2 radioactivity was unchanged. There was no effect of pertussis toxin on either the acute or chronic phase of PI turnover, negating involvement of Gi or G(o). These data suggest that alpha 1AR stimulation in neonatal rat cardiac myocytes is biphasic, and that hypoxia produces a slower monophasic response during extended alpha 1-agonist exposure as would be found with ischemia.     

Effects of alpha- and beta-adrenoceptor blockade on purine secretion induced by sympathetic nerve stimulation in the rat kidney

To characterize the effects of renal sympathetic nerve activation (RSNA) on renal purine secretion, 13 perfused rat kidneys were stimulated with periarterial electrodes at 7 Hz for 3 min, and purine secretion was determined by measuring with high-performance liquid chromatography purines in the renal venous perfusate 1 min before and during the last minute of RSNA. RSNA significantly increased renal perfusion pressure and significantly increased the secretion of adenosine and adenosine metabolites (inosine, hypoxanthine, and xanthine) by 2- to 5-fold. To investigate the participation of alpha- and beta-adrenoceptors in this response, four groups of perfused kidneys (n = 5/group) were pretreated with either vehicle, prazosin (alpha1-adrenoceptor antagonist; 0.03 microM), phentolamine (alpha1/2-adrenoceptor antagonist; 3 microM), or propranolol (beta1/2-adrenoceptor antagonist; 0.1 microM), and purine secretion was measured before and during RSNA at 1, 3, 5, 7, and 9 Hz. Prazosin, phentolamine, and propranolol abolished the RSNA-induced increase in the secretion of adenosine, inosine, hypoxanthine, and xanthine. In contrast, prazosin and phentolamine nearly abolished, whereas propranolol only slightly reduced, renal vascular responses to RSNA. Our results indicate that RSNA increases renal purine secretion via a mechanism that requires both alpha- and beta-adrenoceptors. It is well known that in the kidney adenosine activates renal afferent nerves, enhances renovascular responses to norepinephrine and angiotensin II, and increases sodium reabsorption; therefore, RSNA-induced adenosine production may contribute to the hypertensive effects of RSNA. Moreover, the antihypertensive effects of beta-adrenoceptor antagonists may in part be due to inhibition of RSNA-induced renal adenosine production.     

Interaction of the renin-angiotensin system, bradykinin and sympathetic nerves with cholinergic transmission in the rat isolated trachea

1. The present study was undertaken to investigate the interaction of the renin-angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release. 2. In both epithelium-intact and epithelium-denuded tracheal preparations, the alpha 2-adrenoceptor agonist UK14304 (0.1 and 1 microM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 microM) was antagonized by the selective alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Idazoxan (0.3 microM) alone had no effect on the S-I efflux. 3. Angiotensin II (0.1 and 1 microM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 microM), angiotensin II (1 microM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 microM), to block prejunctional alpha 2-adrenoceptors, angiotensin II (0.1 and 1 microM) did not alter the S-I efflux. When added alone, perindoprilat (10 microM) did not alter the S-I efflux. 4. In epithelium-denuded preparations, bradykinin (0.01-1 microM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 microM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 microM) and phosphoramidon (1 microM), respectively, bradykinin (1 microM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 microM) and phosphoramidon (1 microM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 microM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM), in either epithelium-intact or epithelium-denuded preparations. 5. In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with alpha 2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.     

Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell lineage

So far, no clear correlation has been found between the effects of dopamine D1 receptor agonists on motor behavior in primate models of Parkinson's disease and their ability to stimulate adenylate cyclase in rats, the benzazepine SKF 83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-]H- 3-benzazepine) being the most striking example. Since this discrepancy might be attributed to: (A) the different species used to study these effects or (B) the interaction of SKF 83959 with other catecholamine receptors, the aims of this study were: (1) to study the ability of SKF 83959 to stimulate adenylate cyclase in cultured human and monkey glial cells equipped with dopamine D1 receptors and (2) to evaluate the affinity for and the functional interaction of SKF 83959 with other catecholamine receptors. Binding studies revealed that SKF 83959 displayed the highest affinity for the dopamine D1 receptor (pKi=6.72) and the alpha2-adrenoceptor (pKi=6.41) and moderate affinity for the dopamine D2 receptor and the noradrenaline transporter. In monkey and human cells, SKF 83959 did not stimulate cyclic adenosine monophosphate (cAMP) formation to a significant extent, but antagonized very potently the dopamine-induced stimulation of cAMP formation in both cell types. The compound stimulated basal dopamine outflow and inhibited depolarization-induced acetylcholine release only at concentrations > 10 microM. Finally, SKF 83959 concentration dependently increased electrically evoked noradrenaline release, indicating that it had alpha2-adrenoceptor blocking activity and interfered with the noradrenaline transporter. In conclusion, SKF 83959 is a potent dopamine D1 receptor and alpha2-adrenoceptor antagonist. Thus, the anti-parkinsonian effects of SKF 83959 in primates are not mediated by striatal dopamine D1 receptors coupled to adenylate cyclase in a stimulatory way.     

Bradykinin B2-receptor-mediated stimulation of exocytotic noradrenaline release from cardiac sympathetic neurons

Myocardial ischemia, as well as angiotensin-converting-enzyme-inhibitors, increase cardiac concentrations of the non-apeptide bradykinin. Cardiac effects of bradykinin are potentially mediated by modulation of sympathoadrenergic neurotransmission. Accordingly, the present study was designed to examine the influence of bradykinin on exocytotic noradrenaline release from rat isolated perfused heart. Exocytotic noradrenaline release was induced by electrical field stimulation (1 min, 5 V, 6 Hz) twice to compare the effect of intervention (S2) with respective control stimulation (S1). The overflow of endogenous noradrenaline was determined by high pressure liquid chromatography and electrochemical detection. The results are expressed as the mean S2/S1 ratio+/-S.E.M. Bradykinin (1 micromol/l) evoked a significant increase in noradrenaline release (S2/S1: 1.60+/-0.12; P<0.01), which was even more pronounced after inhibition of neuronal reuptake of noradrenaline by desipramine (0.1 micromol/l: S2/S1: 1.83+/-0.15; P<0.01) excluding interference of bradykinin with the noradrenaline uptake1 carrier. The concentration-response curve for bradykinin (0.1 nmol/l to 10 micromol/l) revealed a maximum effect at 1 micromol/l and an EC50-value of 7.5 nmol/l. The effect of bradykinin was unaltered by the B1-receptor antagonist des-Arg9 (Leu8)-bradykinin (1 micromol/l; S2/S1: 1.69+/-0.17), whereas it was reduced significantly by the B2-receptor antagonist Hoe 140 (1 micromol/l; S2/S1: 1.14+/-0.11; P<0.05). Des-Arg9-bradykinin (1 micromol/l), a specific B1-agonist, had no effect on stimulation-induced noradrenaline release (S2/S1: 0.94+/-0.08). Utilizing pharmacological interventions, we attempted to characterize the intraneuronal signal transduction pathway mediating the effect of bradykinin on exocytosis. Neither inhibition of cyclooxygenase nor blockade of nitric oxide synthesis affected bradykinin-induced stimulation of noradrenaline release. Likewise, inhibition of protein kinase C by bisindolylmaleimide (1 micromol/l) or tyrosine kinase by genistein (10 micromol/l) had no effect on the promoting action of bradykinin. In contrast, inhibition of cytosolic phospholipase A2 activity by the specific inhibitor AACOCF3 (1 micromol/l) prevented bradykinin-induced increase in noradrenaline release (S2/S1: 1.09+/-0.15; P<0.01). In conclusion, bradykinin increases exocytotic release of endogenous noradrenaline from cardiac sympathetic neurons via activation of presynaptic B2-receptors. Intraneuronal coupling of B2-receptors to phospholipase A2 appears to mediate the facilitatory effect of bradykinin on noradrenaline release in rat heart.     

P2 purinoceptors modulating noradrenaline release from sympathetic neurons in culture

ATP (1 mM) inhibited, whereas 2-methylthio-ATP (30 microM), a P2Y-selective purinoceptor agonist, increased electrically evoked release of [3H]noradrenaline from chick sympathetic neurons. The P2X-selective purinoceptor agonist alpha,beta-methylene-ATP (30 microM) had no effect. The ATP-induced inhibition of release as well as the facilitation caused by 2-methylthio-ATP was not affected by the selective adenosine (P1) receptor antagonist 8-(p-sulfophenyl)-theophylline (8-PST; 100 microM), but completely prevented by the non-selective P2 antagonist suramin (300 microM). The present data reveal a dual regulation of noradrenaline release from sympathetic neurons. Facilitation seems to be mediated by a P2Y purinoceptor, whereas inhibition is caused by a P2 purinoceptor which needs further subtype characterization.     

Evidence for the presence of both P1 and P2 purinoceptors in the rat myometrium

1. Adenosine, adenosine triphosphate (ATP) and some stable analogues of adenosine inhibited field stimulation-induced contractions of the uterus from rats treated with oestradiol cypionate (20 micrograms/kg, s.c.) 1 day previously. Adenosine was twice as potent as ATP; both were potentiated by dipyridamole (10 mumol/L). 2. The order of agonist potency of adenosine and its analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) > N6-cyclohexyladenosine > or = R-phenylisopropyladenosine = S-phenylisopropyladenosine = 2-chloroadenosine > or = adenosine > or = ATP > > 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine. This order suggests the presence of P1 purinoceptors of the A2B subtype. 3. Responses to agonists were antagonized to differing extents by the P1 purinoceptor antagonist 8-phenyltheophylline (10 mumol/L). 4. In uterine preparations from rats pretreated for 2 days with oestrogen (20 micrograms/kg, s.c.) and for 1 day with progesterone (3 mg/animal, s.c.), the inhibitory potencies of adenosine and NECA were reduced, indicating hormonal regulation of uterine responsiveness to P1 purinoceptor agonists. 5. Stable analogues of ATP caused contractions of unstimulated myometrial preparations from oestrogen-treated animals, indicating activation of a P2 purinoceptor, possibly of the P2X subtype, because of the relative order of potency was alpha, beta-methylene ATP > beta, gamma-methylene ATP = ATP = 2-methylthio ATP.     

P2-receptor modulation of noradrenergic neurotransmission in rat kidney

1. ATP has previously been shown to act as a sympathetic cotransmitter in the rat kidney. The present study analyses the question of whether postganglionic sympathetic nerve endings in the kidney possess P2-receptors which modulate noradrenaline release. Rat kidneys were perfused with Krebs-Henseleit solution containing the noradrenaline uptake blockers cocaine and corticosterone and the alpha2-adrenoceptor antagonist rauwolscine. The renal nerves were electrically stimulated, in most experiments by 30 pulses applied at 1 Hz. The outflow of endogenous noradrenaline (or, in some experiments, of ATP and lactate dehydrogenase) as well as the perfusion pressure were measured simultaneously. 2. The P2-receptor agonist adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS, 3-30 microM) reduced the renal nerve stimulation (RNS)-induced outflow of noradrenaline (estimated EC50 =8 microM). The P2-receptor antagonist cibacron blue 3GA (30 microM) shifted the concentration-inhibition curve for ATPgammaS to the right (apparent pKB value 4.7). 3. Cibacron blue 3GA (3-30 microM) and its isomer reactive blue 2 (3-30 microM) significantly increased RNS-induced outflow of noradrenaline in the presence of the P1-receptor antagonist 8-(p-sulphophenyl)theophylline (8-SPT, 100 microM) by about 70% and 90%, respectively. The P2-receptor antagonist suramin (30-300 microM) only tended to enhance RNS-induced outflow of noradrenaline. When the nerves were stimulated by short pulse trains consisting of 6 pulses applied at 100 Hz (conditions under which autoinhibition is inoperative), reactive blue 2 did not affect the RNS-induced outflow of noradrenaline. 4. RNS (120 pulses applied at 4 Hz) induced the outflow of ATP but not of the cytoplasmatic enzyme lactate dehydrogenase. 5. ATPgammaS (3-30 microM) concentration-dependently reduced pressor responses to RNS at 1 Hz. Cibacron blue 3GA, reactive blue 2 as well as suramin also reduced pressor responses to RNS (maximally by 50 to 70%). 6. This study in rat isolated kidney, in which the release of endogenous noradrenaline was measured, demonstrates that renal sympathetic nerves possess prejunctional P2-receptors that mediate inhibition of transmitter release. These prejunctional P2-receptors are activated by endogenous ligands, most likely ATP, released upon nerve activity. Both, P2-receptor agonists and P2-receptor antagonists reduced pressor responses to RNS either by inhibiting transmitter release or by blocking postjunctional vasoconstrictor P2-receptors.     

Chronic administration of oxprenolol and metoprolol attenuate sympathetic cardiovascular responses only in non-adrenalectomized pithed rats

1. Oxprenolol and metoprolol (30 mg kg-1) were injected intraperitoneally (i.p.) for 1 day (acute treatment) and 6 weeks (chronic treatment) to Sprague-Dawley rats. 2. Increases of mean blood pressure and heart rate to noradrenaline (0.1-10 micrograms kg-1) and to electrical stimulation (0.5 msec, supramax V, 0.25-5 Hz) of the entire sympathetic outflow were measured in non-adrenalectomized (acute and chronic) and adrenalectomized (chronic) pithed rats. 3. Acute beta-adrenoceptor antagonist administration was without effect on mean blood pressure and heart rate increases to noradrenaline and electrical stimulation. 4. Chronic administration with oxprenolol significantly diminished the stimulation-induced increases of mean blood pressure and heart rate in non-adrenalectomized pithed rats. 5. Increases in heart rate, elicited by stimulation of the entire sympathetic outflow in non-adrenalectomized but not in adrenalectomized pithed rats, were decreased by metoprolol treatment. Both treatments were without effect on noradrenaline responses. 6. These results indicate that chronic beta-adrenoceptor antagonist treatment is associated with a reduction in the cardiovascular responses to sympathetic nerve-stimulation. However, this mechanism only operates when adrenomedullary adrenaline is present to facilitate the noradrenaline release through activation of presynaptic beta 2-adrenoceptors.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane- 1,3-dicarboxylic acid [(IS,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-alpha-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and I mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-(alpha-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methylD-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 microM) only the 50 microM infusion significantly decreased dialysate 5-HT. The GABA(A) receptor agonist, bicuculline (30 microM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG's group II/group II mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons.