Presence of opioid receptors in mesencephalic nucleus dorsalis raphe concerned in cardiovascular regulation in cats

Summary The effects of microinjecion of opioid receptor agonist and antagonist into mesencephalic nucleus dorsalis raphe, were studied on mean arterial pressure and heart rate to elucidate the nature and role of these opioid receptors in cardiovascular regulation. Microinjection of morphine (5 g and 10 g) into nucleus dorsalis raphe elicited both inhibitory and excitatory cardiovascular responses respectively, while microinjection of opioid receptor antagonist, naloxone (10 g) failed to produce any significant cardiovascular responses. However, local pretreatment with naloxone blocked both inhibitory and excitatory responses of graded doses of morphine. These opioid receptors seem to be localised in the neurons of the nucleus since microinjection of morphine into neural structures adjoining nucleus dorsalis raphe failed to induce any cardiovascular responses. Furthermore, the dose or morphine (2 g) which was ineffective when microinjected into nucleus dorsalis raphe, produced inhibitory cardiovascular responses after pretreatment with LM 5008, a 5-HT uptake blocker. Similarly, the excitatory cardiovascular responses of morphine microinjection were blocked by spinal cord transection (C₁) and p-CPA, guanethidine and piperoxan pretreatments, while bilateral cervical vagotomy failed to do so. Thus, it is likely that the inhibitory cardiovascular responses of morphine are mediated directly through stimulation of opioid receptors present in the neurons of nucleus dorsalis raphe while the excitatory responses to higher dose of morphine, appear to be due to a release of noradrenaline which in turn modulates the activity of neurons by acting on a adrenoceptors. Send offprint requests to K. K. Tangri at the above address     

Cardiovascular responses elicited by microinjection of monoamines into mesencephalic nucleus dorsalis raphe in cats

Summary The effect of monoaminergic agonists and antagonists microinjected into mesencephalic nucleus dorsalis raphe has been studied on blood pressure and heart rate to elucidate the nature and role of these monoaminergic receptors in cardiovascular regulation. Microinjection of monoamines, noradrenaline, phenylephrine and 5-hydroxytryptamine (5-HT) into nucleus dorsalis raphe elicited hypertension and tachycardia which could be blocked by local pretreatment with piperoxan (an -adrenoceptor blocker) and methysergide (a 5-HT receptor blocker) respectively. However, isoprenaline microinjections failed to evoke any response. Bilateral vagotomy did not prevent these cardiovascular responses evoked by monoamines microinjection, while cervical spinal cord (C₁) transection with bilateral vagotomy prevented these responses. These monoaminergic receptors seem to be localized in nucleus dorsalis raphe since microinjection of monoamines into neural structures adjoining nucleus dorsalis raphe, failed to induce any cardiovascular response. Monoaminergic receptors are present in nucleus dorsalis raphe which modulate cardiovascular activity by influencing sympathetic preganglionic neurons in the intermediolateral columns of the spinal cord.     

Regulation of adenylate cyclase activity in hamster adipocytes

Summary In ghosts of hamster adipocytes, the regulation of adenylate cyclase (ATP: pyrophosphate lyase, cyclizing; EC 4.6.1.1) activity by prostaglandins, -adrenergic agonists and nicotinic acid was studied. These three classes of antilipolytic agents caused adenylate cyclase inhibition without an apparent lag phase. Maximal inhibitions observed ranged between about 45% (by -adrenergic agonists) and 60% (by prostaglandins and nicotinic acid). The order of potency for the inhibitory prostaglandins (PG) was PGE₁ PGE₂>PGF₂PGI₂>PGD₂>6-keto PGF₁. The IC₅₀ values obtained were about 0.007, 0.06, 0.3 and 1 M for PGE₁, PGF₂, PGD₂ and 6-keto PGF₁, respectively. -Adrenergic agonists, studied in the presence of the -adrenergic blocking agent, propranolol (30 M), inhibited the fat cell enzyme with the order of potency (1)-adrenaline > (1)--methylnoradrenaline (1)-noradrenaline > clonidine tetryzoline > (1)-phenylephrine. The IC₅₀ values obtained for (1)-adrenaline and (1)-noradrenaline were about 3 and 10 M, respectively. The inhibitory effect of (1)-adrenaline was blocked by the -adrenergic antagonists with the potency order yohimbine phentolamine > prazosin. These findings suggest that an ₂ of receptors is involved in this catecholamine-induced inhibition. Nicotinic acid (10 M) reduced adenylate cyclase activity by about 60% with half-maximal effectiveness at about 0.6 M. The nicotinic acid derivatives, nicotinamide, -pyridylcarbinol and NAD (up to 100 M), had no effect on enzyme activity.Inhibition of the hamster adipocyte adenylate cyclase by the antilipolytic agents required the presence of both GTP, which reduced basal activity by about 80% at 10 M, and sodium ions, which specifically activated the GTP-affected from of the enzyme. Inhibition was also observed in the presence of ACTH, which in a GTP-dependent manner increased adenylate cyclase activity. Pretreatment of the enzyme preparation with NaF (10 mM) partially reduced the inhibitory effect, and preactivation with the stable GTP analogue, guanylyl 5-imidodiphosphate (100 M), abolished the adenylate cyclase inhibition by the antilipolytic agents.Abbreviations PG prostaglandin - GMP-P(NH)P guanylyl 5-imidodiphosphate Some of the data were presented in abstract form (Aktories et al., 1979a)     

The cardiovascular effects of intraventricularly administered histamine in the anaesthetised rat

Summary In urethane-anaesthetised rats intraventricular (i.c.v.) injections of histamine (0.1–10.0 g) elicited dose-related rises in both the resting blood pressure and heart rate. These cardiovascular effects of histamine were antagonised in a dose-dependent manner by i.c.v. pretreatments with the histamine H₁-receptor antagonists mepyramine (10, 50 and 100 g) and diphenylpyraline (100 and 200 g). Pretreatment with the histamine H₂-receptor antagonist metiamide (100 and 200 g i.c.v.) failed to modify either of the responses. A dose-related antagonism of the hypertensive response to histamine i.c.v. was elicited by phentolamine (100 and 200 g i.c.v.) but the positive chronotropic effect was not modified by this pretreatment. The cardiovascular responses to histamine i.c.v. were abolished by mecamylamine (5.0 mg/kg i.v.) and greatly reduced by 6-hydroxydopamine (3×250 g i.c.v.), but only the tachycardia was significantly modified by atropine (100 g i.c.v.) and propranolol (1 mg/kg i.v.). Propranolol (100 g i.c.v.), bilateral vagotomy, or acute bilateral adrenal demedullation failed to modify the cardiovascular responses to histamine i.c.v. The results suggest that histamine is able to modify the resting blood pressure and heart rate by independent central modes of action, which involve central adrenergic and cholinergic mechanisms.Preliminary findings of this study were presented at the Autumn meeting of the British Pharmacological Society (Finch and Hicks, 1975).     

Investigation into the positive inotropic effect of clondine in isolated hearts

Summary Clonidine in doses 2.5–40 g injected into the aortic cannula of isolated, perfused hearts of guinea pigs produced an increase in contraction amplitude. This positive inotropic effect was not antagonised by infusions of doberol (10 g/min), phentolamine (10 g/min) or pheniramine (50 g/min), it was antagonised by burimamide (30 g/min), a histamine H₂ receptor blocking agent. Clonidine showed some similarities to histamine such as parallel dose-response curves and during infusions of burimamide the curve of clonidine was shifted in a way which indicated competitive inhibition. It is suggested therefore that the action of clonidine is due to stimulation of histamine H₂ receptors.     

Cardiovascular effects of selective agonists and antagonists of histamine H3 receptors in the anaesthetized rat

The cardiovascular responses to a series of selective histamine H₃ receptor agonists, (R) -methylhistamine, imetit and immepip and selective antagonists, thioperamide, clobenpropit and clophenpropit, were studied in anaesthetized rats. At 0.003–1 mol/kg i.v. doses, H₃ agonists failed to produce any significant change in the basal blood pressure and heart rate. Larger doses of (R) -methylhistamine increased the blood pressure and heart rate and higher doses of imetit caused vasodepressor responses and reduced heart rate, whereas immepip proved virtually inactive. While (R) -methylhistamine-induced effects were not blocked by histamine H₁-, H₂- and H₃-receptor antagonists, they were however reduced by idazoxan and propranolol, which indicates that the mechanisms involved are adrenergic. The effects induced by imetit are not related to histamine H₃ receptors but are mediated by indirect (via 5HT₃ receptors) cholinergic mechanisms, since these effects were prevented by 1 mg/kg i.v. atropine and by 0.1 mg/kg i.v. ondansetron. Similarly, the H₃ antagonists per se failed to change basal cardiovascular function up to 10 mol/kg i.v. and only at 30 mol/kg i.v. were marked decreases observed in the blood pressure and heart rate with a significant reduction in the effects of noradrenaline. These data indicate that in anaesthetized rats, histamine H₃ receptor activation or blockade has no effect on basal cardiovascular function. The effects recorded after the administration of large doses of (R) -methylhistamine and imetit are clearly unrelated to histamine H₃ receptors and should be taken into account when using these compounds as H₃ ligands for in vivo experiments.     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

The subjective,behavioral and cognitive effects of subanesthetic concentrations of isoflurane and nitrous oxide in healthy volunteers

A prospective, crossover, double-blind trial was conducted in nine healthy volunteers in which the subjective, psychomotor and memory effects of isoflurane (0.0, 0.3 and 0.6%) and nitrous oxide (N₂O) (0, 20 and 40%) were examined. Dependent measures included visual analog scales and a standardized drug effects inventory (subjective effects), reaction time and eye-hand coordination (e.g., psychomotor performance), and immediate and delayed free recall (memory). There were some similarities in subjective effects between the two inhaled drugs (e.g., increased ratings of drunk and spaced out), but isoflurane had effects which N₂O did not have. Isoflurane but not N₂O increased visual analog scale ratings of confused, sedated, and carefree, and decreased ratings of in control of thoughts and in control of body. An odor was detected with isoflurane and it was disliked. Psychomotor performance was more grossly impaired during isoflurane inhalation than during N₂O inhalation. Psychomotor recovery from both agents was rapid and complete so that 5 min after the inhalation period had ceased, performance had returned to baseline levels. Both isoflurane and nitrous oxide impaired immediate and delayed free recall. The feasibility of using isoflurane in conscious sedation procedures is discussed.     

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     

Über die Wirkung von α-Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen

Zusammenfassung 1. Versuche über die Wirkung von -Methyl-Dopa auf den Brenzcatechinamingehalt von Meerschweinchenorganen haben ergeben, daß Noradrenalin im Herzen, sowie Dopamin und Noradrenalin im Gehirn durch äquimolare Mengen von -Methyl-Noradrenalin bzw. -Methyl-Dopamin ersetzt werden.2. Die Adrenalinverarmung der Nebennieren nach Vorbehandlung mit -Methyl-Dopa wird demgegenüber nur zu einem geringen Teil durch die methylierten Brenzcatechinamine -Methyl-Noradrenalin und -Methyl-Dopamin ausgeglichen. Ein weiteres Brenzcatechinamin konnte nicht nachgewiesen werden, so daß -Methyl-Adrenalin wahrscheinlich nicht gebildet wird.3. Im Meerschweinchenherzen gespeichertes -Methyl-Noradrenalin wird durch Reserpin in vivo schlechter freigesetzt als Noradrenalin.4. Das in isolierten Herzgranula (Sediment 100 000·g) gespeicherte -Methyl-Noradrenalin wird durch Segontin bei der Inkubation in vitro ebenfall schlechter freigesetzt als Noradrenalin aus normalen Herzgranula.5. Der Mechanismus der -Methyl-Dopa-Wirkung wird diskutiert.6. Es wird eine fluorimetrische Methode zur Bestimmung von -Methyl-Noradrenalin angegeben.

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Summary 1. The catecholamine content of guinea pig organs after administration of -methyldopa has been determined. The results show that the loss of noradrenaline from heart and that of noradrenaline and dopamine from brain is compensated by a stoichiometric uptake of -methylnoradrenaline and -methyldopamine respectively.2. On the contrary the loss of adrenaline from the suprarenal medulla induced by pretreatment with -methyldopa is not completely restored by the -methylated amines, -methyldopamine and -methylnoradrenaline. -methyladrenaline could not be detected even by paperchromatography.3. The release of the stored -methylnoradrenaline from guinea pig heart by reserpine is very small in comparison with that of noradrenaline.4. Furthermore only small amounts of the stored -methyl-noradrenaline are released from isolated granules of the guinea pig heart after incubation in vitro with segontin. Whereas the same amount of segontin depleted completely the noradrenaline content of the granules isolated from normal hearts.5. The mechanism of action of -methyldopa is discussed.6. A method for the fluorimetric determination of -methylnoradrenaline is described. It is at first separated from -methyldopamine, dopamine and histamine by column chromatography, condensed with o-phthaldialdehyd and the obtained fluorescense is measured by using an Aminco-Bowman spectrophotofluorimeter.

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Mit 6 Textabbildungen

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Herrn Dr. H. Blaschko zum 65. Geburtstag gewidmet.

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Über einen Teil der Ergebnisse wurde auf der Frühjahrstagung der Deutschen Pharmakologischen Ges. in Mainz, April 1964, berichtet. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 247, 297 (1964).

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Involvement of α-adrenoceptors in the excitatory effect of the A2 adenosine receptors agonist 5′-N-ethylcarboxamidoadenosine (NECA) on cardiac automaticity in the isolated right ventricle of the rat

The effects of the non-selective A₂ adenosine receptor agonist 5-N-ethyl-carboxamidoadenosine (NECA) were studied on ventricular automaticity induced by a local injury in the isolated right ventricle of the rat. In concentrations ranging from 0.1 to 100 nM, NECA significantly increased ventricular automaticity. This effect was not apparent when the nonselective -adrenoceptor blocker phenoxybenzamine was present at a concentration of 10 M, which antagonizes both ₁-and ₂-adrenoceptors, as well as when rats were pretreated with reserpine. In non-reserpinized rats, the excitatory effect of NECA was also abolished in the presence of the selective ₁-adrenoceptor antagonist prazosin, but not in the presence of the ₂-adrenoceptor antagonist idazoxan. In reserpinized rats, the excitatory effect of NECA was restored in the presence of the non specific -adrenoceptor agonist phenylephrine as well as in the presence of the selective ₁-adrenoceptor agonist amidephrine but not in the presence of the selective ₂-adrenoceptor agonist clonidine. These results suggest that the excitatory effect of NECA on ectopic ventricular automaticity is dependent on endogenous catecholamines and that -adrenoceptors of type 1 are, in some way, involved in this effect.     

The in vitro/in vivo comparative metabolism of 4-aminobiphenyl using isolated hepatocytes

The metabolism of 4-aminobiphenyl by isolated hepatocytes from various species was compared with urinary metabolite profiles in the same species. Radioactive compounds in concentrates of ether extracts from hepatocytes or urine following hydrolysis were analysed by TLC and reversed phase HPLC in conjunction with radioactivity monitoring and synthetic standards.The major metabolites from hepatocytes and in urine were 4-acetamidobiphenyl, 3-hydroxy-4-aminobiphenyl 4-hydroxy-4-aminobiphenyl and 4-hydroxy-4-acetamidobiphenyl. Oxidation of the amine nitrogen gave hydroxylamino, nitroso and nitro compounds. Minor metabolites were 2-hydroxy amine and amide, the hydroxamic acid and the oxamic acid. The urinary metabolite profiles correlated well with those from hepatocytes for each species.Abbreviations Used 4-ABP 4-aminobiphenyl - AA 4-acetamidobiphenyl - A3-OH 3-hydroxy-4-aminobiphenyl - A4-OH 4-hydroxy-4-aminobiphenyl - A2-OH 2-hydroxy-4-aminobiphenyl - AA4-OH 4-hydroxy-4-acetamidobiphenyl - AA3-OH 3-hydroxy-4-acetamidobiphenyl - AA2-OH 2-hydroxy-4-acetamidobiphenyl - AAN-OH N-hydroxy-4-acetamidobiphenyl - NBP 4-nitrobiphenyl - AN-OH 4-hydroxylaminobiphenyl - NOBP 4-nitrosobiphenyl Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions,G protein and adenylate cyclase

The effects of ₂-adrenoceptor agonists (dexmedetomidine, oxymetazoline), alone or in combination with various -adrenoceptor subtype-selective antagonists (CH-38083, idazoxan, WB4101, BRL44408, ARC-239, prazosin), on noradrenaline- and isoprenaline-induced lipolysis were investigated in human isolated abdominal subcutaneous fat cells. The rank order of potency of antagonists in preventing dexmedetomidine- and oxymetazoline-evoked suppression of isoprenaline-induced lipolysis was (pA₂-values): CH-38083 (7.69 and 7.48) idazoxan (7.5 and 7.41) > BRL 44408 (7.23 and 7.19) WB 4101 (7.13 and 7.12) > prazosin (5.18 and 5.17) > ARC-239 (4.72, 4.9). While CH-38083 and idazoxan, non-subtype selective ₂-adrenoceptor antagonists and BRL44408, a selective a_2A-adrenoceptor antagonist as well as WB4101 potentiated the lipolytic effect of noradrenaline, ARC-239, the selective _2B-adrenoceptor antagonist failed to affect it. In addition since the _2A-adrenoceptor selective agonist, oxymetazoline concentration dependently inhibited the lipolytic effect of isoprenaline, and WB4101 and BRL44408 (a_2A-adrenoceptor antagonists) antagonised the effect of oxymetazoline in a competitive manner, it is concluded that the a_2A-adrenoceptor subtype is involved in antilipolysis. In addition, functional evidence was obtained that there is an interaction between _2A- and -adrenoceptors located on the cell surface of adipocytes, through which locally released noradrenaline and/or circulating circulating adrenaline influence lipolysis.On leave from Institute of Medical Pharmacology, University of Pisa, Via Roma 55, I-5626 Pisa, Italy Correspondence to: E. S. Vizi at the above address     

The selective P2X purinoceptor agonist, β,γ-methylene-L-adenosine 5′-triphosphate,discriminates between smooth muscle and neuronal P2X purinoceptors

The effects of the putative selective P_2X purinoceptor agonist, ,-methylene-l-adenosine 5-triphosphate (me-l-ATP), were determined at rat neuronal and smooth muscle P_2X purinoceptors.Me-l-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 M. In contrast, the archetypal P_2X purinoceptor agonist, , methylene ATP (meATP;1–100 M), produced concentration-related depolarisation responses with a mean EC₅₀ value of 10.8 M. The depolarising effects of meATP were not attenuated by me-l-ATP (100 M). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 M), but not me-l.-ATP (1–300 M), evoked rapid ( < 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, me-d-ATP and meATP competed with high affinity for [³H]Lx meATP binding sites, with mean pIC₅₀ values of 7.7 and 8.3, respectively. However, me-l-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 M (mean pIC₅₀ value 4.1).In prostatic segments of the rat vas deferens, me-l-ATP (1–100 M) and meATP (0.3–100 M) each produced concentration-related contractile responses with mean EC₅₀ values of 17.1 and 3.6 M, respectively. Me-l-ATP (1–10 M) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [³H]meATP exhibited high affinity for me-l-ATP, meATP and me-d-ATP with mean PIC₅₀ values of 7.7, 8.4 and 7.3, respectively.These results indicate that me-l-ATP exhibits neither agonist nor antagonist properties at P_2X purinoceptors on rat vagal neurones and possesses only very low affinity for [³H]meATP binding sites in rat brain. In contrast, me-l-ATP is a potent, high affinity agonist at smooth muscle P_2X purinoceptors of the rat vas deferens. This selective agonist action of me-l-ATP suggests that P_2X purinoceptors in smooth muscle and neurones are different and represent distinct P_2X purinoceptor subtypes.     

Relaxation of hormonally stimulated smooth muscular tissues by the 8-bromo derivative of cyclic GMP

Summary Substances that cause contraction or relaxation of smooth muscle have been shown to increase intracellular levels of cyclic GMP. Because of the unclear role of cyclic GMP in the control of smooth muscle tone, cyclic GMP derivatives were exogenously applied to various smooth muscle preparations and their effects on tissue tone were studied.Whereas the basal tone of the rat ductus deferens was not affected by exogenous cyclic GMP or its dibutyryl or 8-bromo derivatives, the contractile responses of this tissue to noradrenaline and acetylcholine were depressed by preincubation with 10 M 8-bromo cyclic GMP (Br-cGMP). The 8-bromo derivatives of 2:3-cyclic GMP, 5-GMP and guanosine were without effects. Cyclic AMP levels were not changed by Br-cGMP. The frequency of oxytocin-stimulated rat uteri was also depressed by Br-cGMP (10 M). In helical strips of rat and rabbit aortae, Br-cGMP (1–100 M) caused a concentration-dependent, rapid decrease in noradrenaline-stimulated tissue tension. Br-2:3-cyclic GMP was ineffective. Noradrenaline-stimulated strips from hog spleen arteries were less sensitive to Br-cGMP than aortic tissue. In ductus deferentes and aortic strips stimulated by K⁺ at a depolarizing concentration, Br-cGMP caused less relaxation than under hormonal stimulation.These findings support the concept that cyclic GMP is involved in the control of smooth muscle tone and that hormone- and drug-induced elevations of the cyclic GMP level can reduce contractile responses to neurotransmitters and hormones.Abbreviations cGMP Guanosine 3:5-monophosphate, cyclic GMP - dibutyryl cGMP N², 2-O-dibutyryl guanosine 3:5-monophosphate - Br-cGMP 8-bromo guanosine 3:5-monophosphate - Br-2:3-cGMP 8-bromo guanosine 2:3-monophosphate - Br-GMP 8-bromo guanosine 5-monophosphate - Br-Guo 8-bromo guanosine, Br-guanosine - cAMP adenosine 3:5-monophosphate, cyclic AMP - dibutyryl cAMP N⁶, 2-O-dibutyryl adenosine 3:5-monophosphate - Br-cAMP 8-bromo adenosine 3:5-monophosphate This work was supported by grants from the Deutsche Forschungsgemeinschaft. Preliminary reports were presented (Schultz, 1977b; Schultz et al., 1978).     

β2-Adrenergic Receptor Genotype-Related Changes in cAMP Levels in Peripheral Blood Mononuclear Cells After Multiple-Dose Oral Procaterol

Purpose. To evaluate the ₂-adrenergic receptor (₂AR) genotype frequency in the Japanese population and the relationship between ₂AR genotype at amino acid position 16 (₂AR-16) and desensitization to ₂-agonist ex vivo. Methods. The ₂AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the ₂AR-16 genotype and the desensitization to ₂-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50g, Meptin®) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). Results. Allele frequency was Arg16:Gly16 = 46%:54%, Gln27:Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. Conclusions. The desensitization to ₂-agonist was associated more frequently with the mutation at ₂AR-16 (Gly16).     

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

Summary The effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 moles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 moles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 moles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 moles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 moles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 moles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 moles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 moles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 moles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 M ZK 62711 increased the level of cyclic AMP but 100 M histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 M ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 M cimetidine and enhanced the stimulatory effect of 100 M histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC₅₀=18 M) and guinea pig gastric mucosa (IC₅₀=1.5 M). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.     

Agonist-antagonist properties of fluorescent opioid probes in the guinea-pig myenteric plexus-longitudinal muscle preparation

Summary The behavioral consequences of -adrenoceptor subsensitivity were investigated by determining whether a physiological response that is mediated by -receptors, isoproterenol-induced drinking (IID), would be reduced by subacute antidepressant/ ₂-antagonist treatmentThe coadministration of typical (e.g., imipramine) or atypical (e.g., mianserin) antidepressants with yohimbine or piperoxan twice daily for four consecutive days reduced IID. Both the time course as well as the magnitude of -adrenoceptor subsensitivity could be behaviorally demonstrated. In addition, the reduction in IID observed after coadministration of imipramine with yohimbine was a centrally mediated effect since it was observed after systemic (subcutaneous) and central (intraventricular) administration of isoproterenol. These results provide evidence that IID is an appropriate behavioral model to demonstrate -adrenoceptor subsensitivity following subacute antidepressant/ ₂-antagonist treatment.Paper presented in part at FASEB, St. Louis, MO, USA (Abstract: Fedn Proc 43:941, 1984)     

Inhibitory dopamine receptors on sympathetic neurons innervating the cardiovascular system of the pithed rat

Summary Additional experimental evidence was obtained for an inhibitory function of prejunctional ₂-adrenoceptors and/or dopamine receptors located on noradrenergic neurons innervating the heart and resistance vessels of the pithed normotensive rat. Mixed ₂-adrenoceptor receptor agonists, differing in selectivity towards either receptor type, i.e. N,N-di-n-propyldopamine (DPDA), 2-N, N-di-n-propylamino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphthalene (DP-6,7-ADTN), B-HT 920 and B-HT 933 (azepexole) were used.In pithed normotensive rats, DPDA (30 and 100 g/kg/min) dose-dependently inhibited the electrical stimulation-induced increase in diastolic pressure, but did not significantly affect the stimulation-evoked increase in heart rate. The inhibition exerted by DPDA was blocked by haloperidol and sulpiride (0.3 mg/kg of each), but not by yohimbine (1 mg/kg), indicating the involvement of dopamine receptors. In this respect, sulpiride and haloperidol were found approximately equipotent.DP-6,7-ADTN (10 and 30 g/kg/min) impaired both tachycardic and vasoconstrictor responses in a dose-dependent manner. Sulpiride (0.3 mg/kg) only partially restored the DP-6,7-ADTN-depressed stimulation-evoked increase in diastolic pressure, whereas yohimbine (1 mg/kg) alone was without effect. The combination of both antagonists completely prevented the inhibition caused by DP-6,7-ADTN. On the other hand, yohimbine (1 mg/kg), but not sulpiride (0.3 mg/kg), selectively antagonized the DP-6,7-ADTN-induced inhibition of stimulation-evoked tachycardia.B-HT 920 (1, 3 and 10 g/kg/min) very effectively reduced the increase in diastolic pressure and heart rate caused by electrical stimulation. Inhibitory dopamine as well as ₂-adrenoceptors participated in the vascular effects of B-HT 920, whereas ₂-adrenoceptors were only involved in the cardioinhibitory response to this agonist.B-HT 933 (0.6 and 1 mg/kg/min) dose-dependently reduced the stimulation-evoked increase in arterial pressure through selective stimulation of inhibitory ₂-adrenoceptors, dopamine receptors not taking a part.The results confirm and extend the observations that in addition to ₂-adrenoceptors inhibitory dopamine receptors are located on the sympathetic neurons connected with the arterial vasculature of the pithed normotensive rat. The sympathetic nerves innervating the rat heart do not contain inhibitory dopamine receptors; their activity only can be modulated by ₂-adrenoceptor stimulation. In the pithed normotensive rat, activation of prejunctionally located ₂-adrenoceptors more effectively inhibits the sympathetic activity directed to the heart than that to the resistance vessels.     

Synthesis and Cytotoxic Evaluation of a Series of γ-Substituted γ-Aryloxymethyl-α-methylene-γ-butyrolactones Against Cancer Cells

Purpose. The main objective of this investigation was to explore thecytotoxic structure-activity relationships of -substituted -aryloxymethyl--methylene--butyrolactones against cancer cells. Methods. The target compounds were synthesized in two stepscommencing with aryl-OH which was treated with a bromomethyl ketonefollowed by the Reformatsky-type condensation. Results. Seven types of -methylene--butyrolactones were evaluatedin vitro against 60 human cancer cell lines derived from nine cancercell types. The average values of log G₅₀ indicated that for thearylportion, potencies of these -methylene--butyrolactones are in adecreasing order of quinolin-2(1H)-one (or 2-hydroxyquinoline, 21,–5.89) > quinoline (19, –5.79) > 2-methylquinoline (20, –5.69)> 8-hydroxyquinoline (17, –5.64) > 2-naphthalene (16, –5.59)> benzene (15, –4.90). The same order was obtained for both log TGIand log LC₅₀. However, for the -substituent, the potencies are in adecreasing order of biphenyl (16f–21f) > phenyl and4-substituted phenyl (16b-e–21b-e) > methyl (16a–21a). Conclusions. Unlike cardiovascular activities of -methylene--butyrolactones in which a -methyl substituent is necessary for vasorelaxingeffect while a phenyl or a halogen-substituted phenyl is prefer for theantiplatelet activities, a -biphenyl substituent proved to be the bestfor their cytotoxicities against various cancer cell lines tested.