Further evidence for a cAMP dependent regulation of tyrosine-3-monoxygenase induction in adrenal medulla

Summary Unilateral adrenal denervation caused a gradual decrease of adenylate cyclase activity in rat adrenal medulla. The extent of the increase in adrenal medullary 3,5-cyclic adenosine monophosphate (cAMP) content elicited by injections of carbamylcholine declined gradually following adrenal denervation. Three or nine days after denervation carbamylcholine caused rise of cAMP and a delayed increase of tyrosine-3-mono-oxygenase (TH) activity of similar magnitude in intact and denervated adrenal medullae. However, after an interval of 15 days or longer following denervation the increase in TH activity elicited by carbamylcholine was greatly reduced. These results support previous proposals that cAMP is involved as a second messenger in the trans-synaptic induction of TH.     

Lack of correlation between changes in cyclic nucleotides and subsequent induction of tyrosine hydroxylase in rat adrenal medulla

Summary Pretreatment of rats with dexamethasone (2.5 mol/kg, a dose which blocks the release of ACTH from the pituitary gland) abolished the reserpine-mediated increase in cAMP and the increase in the cAMP/cGMP ratio in the adrenal medulla. In contrast, the reserpine-mediated induction of tyrosine hydroxylase (TH) remained unchanged. Hypophysectomy had a similar effect to dexamethasone treatment. Since changes in cAMP and changes in the cAMP/cGMP ratio are not indispensible prerequisites for the subsequent induction of TH, a causal relationship between the two phenomena seems to be excluded.     

Association between the increase of cAMP content and the trans-synaptic induction of tyrosine hydroxylase in rat adrenal medulla

Summary When dexamethasone 0.25 or 2.5 mole/kg i.p. was injected 2 h before reserpine (16 mol/kg i.p.) the time course of the increase in cAMP content of rat adrenal medulla was changed. Reserpine alone caused a monophasic increase lasting between 1–2 h; reserpine after dexamethasone caused a biphasic increase: the immediate response, lasting between 15 and 30 min, was followed by a secondary increase beginning 2–3 h after reserpine and lasting for several hours. The overall increase in cAMP content elicited by reserpine during the 8 h following injection remained unchanged or was even increased, depending on the dose of dexamethasone. Pretreatment with dexamethasone, which delayed the increase in cAMP, also delayed the activation and translocation of protein kinase and the induction of tyrosine hydroxylase caused by reserpine in adrenal medulla. The action of reserpine on the cAMP content of adrenal medulla required an intact innervation and did not appear to be related to increased secretion of ACTH from pituitary. In denervated adrenals reserpine failed to increase the cAMP content of the medulla but not that of the cortex.     

The relaxant action of osthole isolated from Angelica pubescens in guinea-pig trachea

The effect of osthole, isolated from Angelica pubescens, on the contraction of guinea-pig trachea was studied. Osthole (25–100 mol/l), theophylline (10–1000 mol/l) and higher concentrations of nifedipine (0.1–100 mol/l) suppressed the contraction response curves of tracheal smooth muscle caused by carbachol, prostaglandin F₂ (PGF₂), U46619 (thromboxane A₂ analogue) and leukotriene C₄ (LTC₄) in a concentration-dependent manner. The contraction caused by high K⁺ (120 mmol/1) and cumulative concentrations of CaCl₂ (0.03–3 mmol/1) was also inhibited concentration-dependently by osthole (25–100 mol/l), theophyl line(10–1000 mol/l) and lower concentrations of nifedipine (0.01–0.1 mol/l). The relaxant actions of osthole were not affected by propranolol (1 mol/l), glibenclamide (10 mol/l) or removal of tracheal epithelium. Osthole (100 mol/l) was still effective in causing tracheal relaxation in the presence of nifedipine (1 mol/l). In Ca²⁺-free- and EGTA (0.2 mmol/1)-containing medium, the relaxing effect of osthole was more potent than in normal Krebs solution. Osthole (25 and 50 mol/l) caused 2.9 and 6.5, or 3.0 and 5.6 fold, respectively, increase in potency of forskolin or sodium nitroprusside in causing tracheal relaxation but did not affect that by cromakalim. Osthole (50 mol/l) enhanced the increase in tissue cAMP and cGMP levels induced by forskolin and sodium nitroprusside, respectively, and in higher concentrations (100 and 250 mol/l), itself increased markedly tissue cAMP and cGMP contents. Osthole (10–250 mol/l) inhibited the activity of cAMP and cGMP phosphodiesterases in a concentration-dependent manner. It is concluded that osthole exerts a nonspecific relaxant effect on the trachealis by inhibiting the cAMP and cGMP phosphodiesterases. Correspondence to: C. M. Teng at the above address     

Activation of β2-adrenoceptors by isoprenaline and adrenaline enhances noradrenaline release in cortical kidney slices of young spontaneously hypertensive rats

Summary The aim of the present study was to investigate -adrenoceptor modulation of noradrenaline release from sympathetic nerves in superfused cortical kidney slices of 4-week-old spontaneously hypertensive rats (SHR) and age-matched controls (WKY). After preincubation with ³H-noradrenaline the kidney slices were electrically stimulated in superfusion chambers. The stimulation induced (S-I) outflow of radioactivity was mainly composed of unmetabolized 3H-noradrenaline in both strains and thus taken as an index of noradrenaline release. There was a frequency-dependent (1.25–20 Hz) increase in the S-1 outflow of radioactivity. At all stimulation frequencies tested S-I outflow of radioactivity was similar or even slightly lower in SHR than in WKY kidney slices in either the absence or presence of cocaine (10 mol/l). The non-selective -adrenoceptor agonists isoprenaline (0.l gmol/1) and adrenaline (0.01 and 0.1 mol/l) enhanced S-I outflow of radioactivity. The facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) were blocked by the selective ₂-adrenoceptor antagonist ICI 118551 (0.1 mol/l) but not by the selective ₁-adrenoceptor antagonist atenolol (0.3 mol/l). The cell-permeable CAMP analogue 8-bromo-cAMP (300 mol/l) enhanced S-1 outflow of radioactivity to a similar extent in both SHR and WKY kidney slices. A combination of 8-bromo-cAMP (300 mol/l) and adrenaline (0.1 mol/l) did not enhance S-1 outflow of radioactivity to a greater extent than 8-bromo cAMP (300 mol/l) alone in both strains. However, the facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) but not that of adrenaline (0.01 mol/l) were significantly greater in SHR than in WKY. The results suggest that stimulation of prejunctional ₂-adrenoceptors by adrenaline even in the absence of a-adrenoceptor blockade enhances noradrenaline release in kidney cortex of young SHR and WKY. This ₂-adrenoceptor mediated effect may possibly be dependent on cAMP formation. The greater facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) in SHR as compared to WKY are in accord with receptor binding studies which show a higher density of ₂-adrenoceptors in SHR than in WKY kidney cortex.Abbreviations SHR Spontaneously hypertensive rats - WKY WistarKyoto rats - cAMP 3-5-cyclic adenosine monophosphate - S-I stimulation induced Send offprint requests to: L. C. Rump     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Dopamine inhibits prostaglandin F2α-induced depolarization of rabbit jejunal arteries via activation of DA1-receptors

Summary In rabbit jejunal arteries, the membrane potential of single smooth muscle cells decreased on the application of noradrenaline 3 mol/1. LY 171555 1 mol/1 did not change, whereas SKF 38393 10 mol/1 reversed the effect of noradrenaline. When prostaglandin F₂ (PGF₂) was used to evoke depolarization in the presence of prazosin 0.1 mol/1, rauwolscine 1 mol/1 and propranolol 1 mol/1, both SKF 38393 10 mol/1 and dopamine 10 mol/1 repolarized the membrane. SCH 23390 1 mol/1 antagonized the effects of SKF 38393 10 mol/1 and dopamine 10 mol/1. Thus, the change in membrane potential is mediated by a DA₁-recep-tor.     

The sympathetic axons innervating the sinus node of the rabbit possess presynaptic opioid к- but not μ- or δ-receptors

Summary The postganglionic sympathic nerves of rabbit isolated hearts were stimulated with pulses delivered at 5 Hz and train durations of 1–5 s. Ethylketocyclazone 0.01–1 mol/l and fentanyl 1 and 10 mol/l but not morphine 1 and 10 mol/l, Met-enkephalin 1 and 4 mol/l or d-Ala², d-Leu⁵-enkephalin 0.5 and 5 mol/l diminished the stimulation-evoked increase in heart rate. The effect of ethylketocyclazocine 0.1 mol/l was antagonized by naloxone 1 and 10 mol/l. In contrast, the effect of fentanyl was not changed by naloxone 10 mol/l. Ethylketocyclazocine 0.03 and 1 mol/l did not reduce the tachycardia elicited by exogenous noradrenaline. The results suggest that, under in vitro conditions, only presynaptic opioid - but not - or -receptors inhibit the release of noradrenaline from the sympathetic neurones innervating the sinus node.     

Evidence for a vasoconstriction-mediating receptor for UTP,distinct from the P2 purinoceptor,in rabbit ear artery

Summary 1. The mechanism of uridine 5-triphosphate-(UTP-)induced vasoconstriction was studied in the rabbit ear artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure. 2. Noradrenaline, adenosine 5'-triphosphate (ATP) and UTP caused concentration-dependent vasoconstriction. ATP and UTP were approximately equipotent. 3. The vasoconstrictor effect of UTP 300 mol/l was enhanced by a mixture of atropine, diphenhydramine and methysergide (1 mol/l each) and not affected by indometacin 10 mol/l. 4. Prazosin (0.01 –1 mol/l) and phentolamine (1–10 mol/l) reduced the vasoconstrictor effect of UTP 300 mol/l by up to 34%. Prazosin 1 mol/l failed to diminish the vasoconstrictor effect of UTP 300 mol/l after the sympathetic nerves had been destroyed with 6-hydroxydopamine. 5. , -Methylene-ATP (10–50 ol/l) elicited transient vasoconstriction. Subsequently, vasoconstrictor responses to ATP 100 or 300 pmol/1 were reduced by 88%, whereas responses to UTP 100 gmol/1 were enhanced, responses to UTP 300 mol/l decreased by only 32% and responses to UTP 1000 gmol/1 reduced by 74%. After in vitro-denervation with 6-hydroxydopamine or in the presence of phentolamine 1 mol/l throughout, a, -methylene-ATP (10–50 mol/l) reduced the vasoconstrictor effect of UTP 300 mol/l by 44% and 43%, respectively. 6. We suggest that, in the rabbit ear artery, the non-adrenergic and , -methylene-ATP-resistant vasoconstrictor response to UTP is mediated by a separate receptor mechanism, distinct from the P₂ purinoceptor. Send offprint requests to K. Starke     

Evidence against a causal relationship between increase in c-AMP and induction of tyrosine hydroxylase in the rat adrenal medulla

Summary It was the purpose of the present experiments to establish whether the reserpine-mediated changes in adrenomedullary c-AMP result from - or -adrenergic activation of adenylate cyclase by catecholamines liberated from adrenal chromaffin cells and whether a causal relationship exists between these changes and subsequent induction of tyrosine hydroxylase (TH).The reserpine-mediated increase in c-AMP was blocked by neither - nor -adrenergic blocking agents. However, propranolol abolished the increase in c-AMP by a mechanism which is not related to the -blocking properties of this drug. Although propranolol abolished the reserpine-mediated increase in c-AMP it did not interfere with the subsequent TH induction. Moreover, administration of aminophylline together with reserpine produced a larger and more prolonged increase in c-AMP in denervated adrenals than reserpine alone in intact glands. Denervation prevented TH induction in spite of the more extensive increase in c-AMP in the adrenal medulla after the combined treatment. It is concluded that there is no causal relationship between the overall increase in c-AMP in the adrenal medulla and the subsequent TH induction. However, the possibility that a small pool of c-AMP which is overshadowed by the non-related overall changes may be involved in trans-synaptic TH induction cannot be excluded.     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

Inhibitory adenosine A1-receptors on rat locus coeruleus neurones

Summary Intracellular recordings were performed in ₁-pontine slice preparation of the rat brain containing the locus coeruleus (LC). Adenosine (100, 300 mol/l) and its structural analogues, namely (–)-N⁶-(R-phenyliso-propyl)-adenosine (R-PIA; 3 – 30 mol/l) and S-PIA (10, 30 mol/l), as well as 5-N-ethylcarboxamido-adenosine (NECA; 3–30 mol/l) inhibited the firing rate of spontaneous action potentials and produced hyperpolarization; their rank order of potency was RPIA - NECA > S-PIA > adenosine. When applied by superfusion, all agonists strongly desensitized the LC cells; the hyperpolarization never surmounted 6 mV. Upon pressure ejection of adenosine 10 mmol/l from ₁- micropipette positioned close to an LC neurone, the membrane potential was raised by 14 mV and the apparent input resistance decreased by 20%. When the membrane potential was hyperpolarized by current injection to ₁- similar extent as adenosine did, the fall in input resistance was only 7%. The adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) 30 mol/l decreased the frequency of action potentials alone; on simultaneous bath-application with adenosine 300 mol/l it potentiated the hyperpolarization caused by the purine derivative. 8-Cyclopentyl-1,3-dipropylxanthine (CPDPX) 0.1 mol/l had no effect on its own, but it antagonized both R-PIA 30 mol/l and NBTG 30 mol/l. A higher concentration of CPDPX (1 mol/l) facilitated the spontaneous firing. In conclusion, both exogenous and endogenous adenosine activates somatic and/or dendritic A₁-receptors of LC neurones leading to an enhancement of potassium conductance and thereby to ₁- decreased firing rate and ₁- hyperpolarization. Send offprint requests to P. Illes at the above address     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Identification of the neuroeffector transmitter in jejunal branches of the rabbit mesenteric artery

Summary Vasoconstriction or excitatory junction potentials (e.j.ps) evoked by nerve stimulation (15 field pulses at 2 Hz every 3 min) were recorded in rabbit isolated jejunal arteries. The resting diameter of the arteries and its decrease in response to stimulation was measured by a photoelectric method. Vasoconstriction was insensitive to prazosin 0.1 or 1 mol/l. Yohimbine 1 mol/l considerably enhanced, whereas ,-methylene ATP (,-meATP) 1 mol/l abolished the contractile response. In order to test the effect of exogenously applied transmitter candidates, noradrenaline (0.1–1 mol/l) and ATP (10–30 mol/l) were added in concentrations which evoked a vasoconstriction comparable to that induced by electrical stimulation. The action of noradrenaline was prevented by prazosin 0.1 mol/l, but was unaffected by both yohimbine 1 mol/l and ,-meATP 1 mol/l. ,-meATP 1 mol/l depressed the effect of ATP. The e.j.ps evoked by a train of 15 pulses showed facilitation up to the third response and thereafter depression; a partial summation was also observed. Prazosin 0.1 mol/l did not change the e j.p. amplitudes. By contrast, when yohimbine 0.1 or 1 mol/l was added to the prazosin-containing medium, both the late e j.ps in the train and the summation were enhanced in a concentration-dependent manner. ,-meATP 1 mol/l almost abolished the e.j.ps. In conclusion, in rabbit jejunal arteries, stimulation of postganglionic sympathetic nerves may release noradrenaline together with ATP which is probably the sole neuroeffector transmitter under our conditions. Transmitter release seems to be modulated by the activation of presynaptic ₂-adrenoceptors. Under the stimulation conditions of the present experiments the released transmitter does not activate postsynaptic ₁-adrenoceptors. Send offprint requests to P. Illes     

The involvement of central cholinergic system in the pressor effect of intracerebroventricularly injected U-46619, a thromboxane A2 analog, in conscious normotensive rats

The aim of this study was to determine the involvement of the central cholinergic system in the rise in blood pressure evoked by the thromboxane A2 (TxA2) analog, U-46619, given centrally. Intracerebroventricular (i.c.v.) injections of U-46619 (0.5, 1.0 and 2.0 g) caused dose- and time-related increases in blood pressure and decreased heart rate in awake rats. U-46619 (1 g; i.c.v.) also produced an approximately 65% increase in posterior hypothalamic extracellular acetylcholine and choline levels. Pretreatment with SQ-29548 (8 g; i.c.v.), selective TxA2 receptor antagonist, completely inhibited both the cardiovascular responses and the increase in acetylcholine and choline levels to subsequent injection of U-46619 (1 g; i.c.v.). Atropine (10 g; i.c.v.), nonselective muscarinic receptor antagonist, pretreatment did not affect the cardiovascular responses observed after U-46619 (1 g; i.c.v.). Pretreatment with the nonselective nicotinic receptor antagonist, mecamylamine (50 g; i.c.v.) attenuated the pressor effect of U-46619 (1 g; i.c.v.). Higher doses of mecamylamine (75 and 100 g; i.c.v.) pretreatments did not change the magnitude of the blockade of pressor response to U-46619; however, they abolished the bradycardic effect of U-46619 dose-dependently. Interestingly, pretreatment of rats with methyllycaconitine (10 g; i.c.v.) or -bungarotoxin (10 g; i.c.v.), selective antagonists of 7 subtype of nicotinic acetylcholine receptors (7nAChRs), partially abolished the pressor response to i.c.v. injection of U-46619 (1 g). Similar to the mecamylamine data, the use of higher doses of methyllycaconitine (25 and 50 g; i.c.v.) produced the same magnitude of blockade that was observed after the 10 g methyllycaconitine pretreatment, but it completely abolished the bradycardic effect of U-46619 (1 g; i.c.v.) at the dose of 25 g. The present results show that central administration of U-46619 produces pressor and bradycardic effect and increase in hypothalamic acetylcholine and choline levels by activating central TxA2 receptors. The activation of central nicotinic receptors, predominantly 7nAChRs, partially mediates the cardiovascular responses to i.c.v. injection of U-46619.     

Influence of papaverine derivatives on phosphodiesterase activity,cyclic 3′,5′-AMP levels and relaxing effect on rabbit ileum

Summary The correlations between the relaxing effect of papaverine derivatives, inhibition of low K_m-phosphodiesterase (cAMP-PDE=EC 3.1.4.17) activity and cyclic 3,5-AMP (cAMP) levels in isolated rabbit ileum were investigated. There was a strong correlation between the relaxing effect, inhibition of PDE activity and cAMP content for eupaverine, ethylpapaverine and papaverine. Eupaverine was the most effective relaxing agent (I₅₀=7.5 M) and the most potent inhibitor of PDE activity (K_i=0.6 M), followed by ethylpapaverine (I₅₀=10 M); K_i=0.8 M) and papaverine (I₅₀=20 M; K_i=2 M). In contrast, there was a strong relaxing effect (I₅₀=6 M) but only slight inhibition of PDE activity (K_i=350 M) by tetrahydropapaveroline (THP). The adenylate cyclase stimulating effect of THP which was shown by others is most likely the reason for comparatively higher cAMP levels, which were found to be elevated about seven times over basal levels of 0.35 nmoles/g wet weight, and effective relaxation. Relaxation could be induced by exogenously added cAMP (I₅₀=45 M) and dibutyryl-cAMP (I₅₀=450 M). Our results support the assumption that smooth muscle relaxation in rabbit ileum is mediated by cAMP. Some of these observations have been published in abstract form (Schulz and Berndt, 1972).     

SK&F 104078, a post-functionally selective α2-adrenoceptor antagonist in the human saphenous vein in vitro

Summary The present study investigated the effects of SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy]-3methyl-1H,2,3,4,-tetrahydro-3-benzazapine) at pre- and post functional ₂-adrenoceptors in the human isolated saphenous vein. Noradrenaline (0.001–100 mol/l) produced concentration-dependent contractions of the human saphenous vein which were competitively antagonised by the ₁-adrenoceptor antagonist prazosin (0.01–1.0 mol/l) and the ₂-adrenoceptor antagonist, rauwolscine (0.01–1.0 mol/l), indicating the presence of both post functional ₁- and ₂-adrenoceptors in this preparation. The selective ₂-adrenoceptor agonist, UK-14,304 (0.01–100 mol/l) also produced concentration-dependent contractions of the human saphenous vein which were antagonised by both rauwolscine (0.1 mol/l) and prazosin (0.1 mol/l). In the presence of angiotensin II (0.05 mol/l), which itself produced a transient contraction, rauwolscine (0.1 mol/l) produced a rightward shift of the UK-14,304 concentration-response curve while prazosin (0.1 mol/l) had no effect. SK&F 104078 (10.0 mol/l) under these conditions also produced a rightward shift of the concentration-response curve to UK-14,304, but was at least 100-fold less potent than rauwolscine. At pre functional ₂-adrenoceptors, exogenous noradrenaline (0.01 and 0.1 gmol/l) induced a concentration-dependent inhibition of stimulation-evoked [7-³H]-noradrenaline release from the human saphenous vein in vitro, which was antagonised by rauwolscine (0:1 mol/l) and tolazoline (10.0 mol/l) but not by SK&F 104078 (10.0 gmol/l).Rauwolscine (0.1 mol/l) produced a small increase in stimulation-evoked [7-³H]-noradrenaline release while both tolazoline and SK&F 104078 failed to produce any enhancement in release in the absence of exogenous agonist atconcentrationsupto10 gmol/l.Insummary, noradrenaline and UK-14,304 contracted the human isolated saphenous vein by an action at both postfunctional ₁- and ₂-adrenoceptors. These data demonstrate that SK&F 104078 discriminates between post- and pre-junctional ₂-adrenoceptors in the human isolated saphenous vein. Send offprint requests to M. V. Sennitt at the above address     

Noradrenaline release from rat sympathetic neurons evoked by P2-purinoceptor activation

The effects of ATP and analogues on the release of previously incorporated ³H-noradrenaline were studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Electrical field stimulation (40 mA at 3 Hz) of the neurons for 10 s markedly enhanced the outflow of tritium. ATP applied for 5 s to 2 min at concentrations of 0.01 to 1 mmol/l caused a time- and concentration-dependent overflow with half maximal effects at about 10 s and 100 mol/l, respectively. 2-Methylthio-ATP was equipotent to ATP in inducing ³H-overflow. ADP (100 mol/l), when applied for 2 min, also caused a small ³H-overflow, but , -methylene-ATP (100 mol/l), AMP (100 mol/l), R(–)N⁶-(2-phenylsiopropyl)-adenosine (R(–)-PIA; 10 mol/l) and 5-N-ethylcarboxamidoadenosine (NECA; 1 mol/l) did not. The ³H-overflow induced by 10 s applications of 100 mol/l ATP was abolished by suramin (100 mol/l) and reduced by about 70% by reactive blue 2 (3 mol/l). Electrically evoked overflow, in contrast, was slightly enhanced by suramin, but not modified by reactive blue 2. Xanthine amine congener (10 mol/l) and hexamethonium (10 mol/l) did not alter ATP-evoked release. Removal of extracellular Ca²⁺ from the medium reduced ATP- and electrically induced overflow by about 95%. Tetrodotoxin (1 mol/l) abolished electrically evoked ³H-overflow but inhibited ATP-induced overflow by only 70%. The ₂-adrenoceptor agonist UK 14,304 at a concentration of 1 mol/l diminished both electrically and ATP-evoked tritium overflow by approximately 70%. These results indicate that activation of P₂-purinoceptors stimulates noradrenaline release from rat sympathetic neurons. The release resembles electrically induced transmitter release, but additional mechanisms may contribute. Correspondence to: S. Boehm at the above address     

A comparison of the effects of TMB-8 and nifedipine on pressor responses to α1- and α2-adrenoceptor agonists in pithed rats

TMB-8 has been characterized as an inhibitor of the release of Ca⁺ from intracellular pools. We have studied the modification of the pressor responses to selective _l-adrenoceptor agonists (methoxamine and phenylephrine), and to selective ₂-adrenoceptor agonists (B-HT 920 and B-HT 933) in pithed rats, produced by TMB-8. We have compared this modification with that produced by the calcium antagonist nifedipine. Nifedipine (100 g/kg, 300 g/kg, and 1000 g/kg) inhibited in a dose-dependent manner the pressor responses to the ₁- and ₂-adrenoceptor agonists, the dose-response curves to the ₂-adrenoceptor agonists being shifted further to the right. TMB-8 at a dose of 3000 g/kg did not modify the pressor effects of the _l-adrenoceptor agonists, and neither did it reinforce the inhibition of such responses produced by nifedipine. By contrast, TMB-8 pretreatment (0.03 g/kg, 0.3 g/kg, 3 g/kg, 30 g/kg, 300 g/kg and 3000 g/kg) inhibited the responses to both ₂-adrenoceptor agonists, the inhibition being more pronounced with B-HT 920. A similar effect was obtained with 0.03 g/kg TMB-8 and 0.3 g/kg TMB-8, particularly in the case of B-HT 920. It was stronger with higher doses, but similar for all doses over 3 g/kg. The inhibition of the pressor responses mediated by the stimulation of ₂-adrenoceptors by TMB-8 was less in rats treated with the Ca²⁺ entry promoter BAY K 8644 (300 g/kg), and could also be reduced by the continuous infusion of CaCl₂ (0.25 g/min). These results suggest that in pithed rats TMB-8 may also behave as an inhibitor of the Ca⁺ influx into vascular smooth muscle.