Actions of alpha-adrenoceptor blocking agents on two types of intracellular calcium stores mobilized by noradrenaline in rat aorta

Summary In isolated rat aortic strips noradrenaline induces a biphasic contractile response in Ca-free medium, associated with two different intracellular calcium pools, one of which is common to caffeine. We analyzed the mechanisms involved in the depletion and repletion of both intracellular Ca pools sensitive to noradrenaline in different experimental procedures in presence of prazosin, phentolamine and yohimbine.At 37°C the -adrenergic blocking agents inhibited contractile responses to noradrenaline in Ca-free medium, with prazosin being highly selective. ₂-adrenoceptors probably do not participate in the release of Ca from internal stores, as no contractile response was observed after addition of clonidine in Ca-free medium. This indicates that noradrenaline-induced Ca-release from internal stores is mainly due to activation of ₁-adrenoceptors. At 25°C, these compounds failed to inhibit caffeine-induced contraction in Ca-free medium, but abolished the release of Ca from an intracellular store only sensitive to noradrenaline. This effect is attributale to a blockade of ₁-adrenoceptors and/or inhibition of receptor-mediated signal transduction. Correspondence to: M.P. D'Ocon at the above address     

Modulation of noradrenaline release by presynaptic alpha-2 and beta adrenoceptors in rat atria

Summary The effect of pretreatment with the beta-2-selective adrenoceptor agonist, (+-)-clenbuterol (0.3 mg/kg, twice daily, 14 days) on prejunctional alpha-2- and beta-adrenoceptors was studied in rat atria. When atria from non-pre-treated rats had been preincubated with (³H)-noradrenaline, (-)-isoprenaline (0.02 to 4.0 M) did not affect tritium overflow evoked by stimulation of the cardioaccelerant nerves, but a higher concentration (40 M) decreased it. Blockade of prejunctional inhibitory alpha-2-adrenoceptors by yohimbine (0.03, 0.3 and 0.8 M) enhanced the overflow of tritium. In the presence of yohimbine, isoprenaline (1.2 M) significantly increased stimulation-induced transmitter overflow, suggesting that in rat atria the facilitatory effect of isoprenaline mediated via prejunctional beta-adrenoceptors, is masked by the dominant influence of inhibitory alpha-2-adrenoceptors. (-)-Propranolol (0.1 M) prevented the isoprenaline-induced increase in atrial rate and the isoprenaline-induced enhancement of transmitter release in the presence of yohimbine (0.3 M), but did not modify by itself the stimulation-induced efflux of tritium, suggesting that neuronally released noradrenaline failed to activate facilitatory prejunctional beta-adrenoceptors. When atria from clenbuterol-pretreated rats had been preincubated with ³H-noradrenaline, the facilitatory effect of yohimbine 0.03 and 0.3 M was markedly enhanced and, in this case, isoprenaline (1.2 and 12.0 M) failed to cause its facilitatory effect in the presence of the alpha-2-adrenoceptor antagonist. Propranolol did not modify the facilitatory effect of yohimbine. No changes in the isoprenaline-induced increase in atrial rate were observed in clenbuterol-treated rats. In addition, the treatment reduced the positive chronotropic effect of nerve stimulation without affecting the response to exogenous noradrenaline, suggesting a reduction in the transmitter release induced by nerve stimulation in clenbuterol-treated rats. These results suggest that chronic treatment with clenbuterol desensitizes facilitatory prejunctional beta-adrenoceptors, without affecting the postsynaptic beta-adrenoceptors, thus implying that prejunctional beta-adrenoceptors possess properties of the beta-2-subtype. Send offprint requests to M. A. Enero at the above address     

Tyramine does not release noradrenaline from splenic nerve by exocytosis

Summary The release of noradrenaline by tyramine from bovine splenic nerves was found to be dose dependent. No dopamine--hydroxylase could be detected in perfusates from tyramine stimulated spleens in contrast to results obtained by electrical stimulation. It is concluded that the releasing action of tyramine differs fundamentally from that of electrical stimulation in that exocytosis is not involved.     

Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria     

Haemodynamic effects of adrenaline during treatment of hypertensive patients with propranolol and metoprolol

Summary A double blind cross-over trial of propranolol and metoprolol was carried out in eight hypertensive patients. At the end of each four-week period of medication, blood pressure and heart rate at rest were measured, and the haemodynamic effects of adrenaline infusion were studied. At rest, propranolol and metroprolol reduced the blood pressure and pulse rate to the same degree. Adrenaline infusion during propranolol medication caused a marked increase both in systolic and diastolic blood pressure, the blood flow in the forearm was unchanged, and the calculated vascular resistance showed a marked increase. Adrenaline infusion during metoprolol medication caused a less marked increase in systolic blood pressure and the diastolic pressure remained unchanged. Blood flow in the forearm increased and the vascular resistance in the forearm tended to decrease. Adrenaline infusion, therefore, caused different haemodynamic effects during non-selective -blockade with propranolol and during ₁-selective blockade with metoprolol. It seems probable that the adrenaline infusion test is comparable with adrenaline release during stress situations and the results may indicate that a ₁-selective blocker is to be preferred to a non-selective one as a therapeutic agent in the treatment of hypertension.     

Inhibition by interleukin-1\ of noradrenaline release in rat spleen: involvement of lymphocytes,NO and opioid receptors

Effects of indomethacin, N-nitro-L-arginine (NNA) and naloxone, and of pretreatment with cyclophosphamide (CY), on the interleukin (IL)-I\ induced inhibition of exocytotic noradrenaline release were investigated in the isolated, vascularly perfused spleen of the rat. Neurotransmitter release was evoked by perivascular electrical stimulation (4 Hz) and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection.Perfusion of the spleen with Tyrode's solution containing IL-1\ (100 pg/ml) for 90 min caused an inhibition of the stimulation-evoked noradrenaline overflow which persisted for at least 20 min after washout of the IL. The evoked overflow was reduced in the presence of NNA 30 mol/l, but remained unaffected by indomethacin 3 mol/l, naloxone 0.1 mol/l or treatment of the rats with CY (250 mg/kg). The opioid agonist etorphine 10 mol/1 inhibited the evoked overflow of noradrenaline and this effect was prevented by naloxone 0.1 mol/1. The inhibition of evoked overflow by IL-1\ was not affected by indomethacin but was reduced or even prevented in the presence of NNA or naloxone, or after lymphocyte depletion of spleens by CY.The results are compatible with the idea that in the rat spleen exocytotic noradrenaline release is accompanied by a concomitant secretion of a nitric oxide (NO)-like compound which, in turn, reinforces noradrenaline release, and that the release can be inhibited via prejunctional opioid receptors. The IL-1\ induced inhibition of evoked release appears to be a complex process which involves as one of many steps a decrease of the facilitatory NO-like compound and the release of endogenous opioids probably from spleen lymphocytes.     

Smoking patterns,nicotine intake at different times of day and changes in two cardiovascular variables while smoking cigarettes

Various smoking parameters were studied in 24 habitual smokers smoking cigarettes with known content of nicotine at fixed rates of puffing and times of day. Heart rate and skin temperature were recorded simultaneously. Doses of nicotine inhaled were estimated. Nicotine intake was seen to be closely related to puff length and rate. This occured, however, only when smoking in the afternoon. These findings are discussed in relation to differences in self-reported mood and activation changes at different times of day. Increases in heart rate were found to be dependent on the drug intake. Consistent decreases in skin temperature were observed during the smoking period, although not related to possible drug effects. The usefulness of this variable as an index of nicotine induced vasoconstriction is discussed.This work was supported by the Tobacco Research Council.     

Differences between full and partial α-adrenoceptor agonists in eliciting phasic and tonic types of responses in the longitudinal smooth muscle of the rat portal vein

Summary The aim of the present investigation was to study, taking into account both quantitative and qualitative differences, the influence of full and partial -adrenoceptor agonists on spontaneous myogenic activity in the rat portal vein.We found that the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, phenylephrine, St 587, Sgd 101/75, B-HT 920 and UK-14,304 could increase the amplitude of the phasic myogenic contractions in the rat portal vein with apparent differences in EC₅₀ and E_max values. In addition to an increase in phasic myogenic activity, the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, and phenylephrine were also able (in higher concentrations) to increase the basal tone of the rat portal vein preparation, again with apparent differences in EC₅₀ and E_max values. Changing the extracellular Ca²⁺ concentration from 0.9 mmol/l to 2.5 mmol/1 had no influence on the phasic character and the concentration range in which St 587 and UK-14,304 increased spontaneous myogenic activity, although changes in amplitude and frequency of the spontaneous myogenic contractions were less pronounced at a higher extracellular Ca²⁺ concentration (2.5 mmol/1). By the use of Schild analysis with the competitive a-adrenoceptor antagonists prazosin (pA₂ = 8.74) and 5-methyl-urapidil (pA₂ = 8.37), it was established that the contractile responses to St 587 were mediated by the same ₁-adrenoceptor subtype as the phasic and tonic type of contraction elicited by phenylephrine as described in a previous study. The concentration-response curve of UK-14,304 was significantly shifted to the right by low concentrations of prazosin (3 nmol/1–30 nmol/1), indicating stimulation of ₁-adrenoceptors by UK-14,304 in the rat portal vein. The -adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine irreversibly blocked the contractile responses to St 587. Based on the method of receptor alkylation with phenoxybenzamine an affinity constant was calculated for St 587 (pK_a = 5.91). Phenoxybenzamine was approximately 1000-fold more potent in inactivating ₁-adrenoceptors than chloroethylclonidine.In conclusion there appeared to be a divergence in the excitation-contraction coupling of ₁-adrenoceptors in the rat portal vein, which is reflected by two types of contraction (phasic versus tonic). The extent to which both the phasic and tonic types of contraction are stimulated by agonists depends on the affinity and intrinsic efficacy for each of the receptor-coupled effector pathways. Thus, partial and full agonism can only meaningfully be discussed if confined to one particular effector pathway. Send offprint requests to H. R. Schwietert at the above address     

Direct and indirect actions of dopamine on tracheal smooth muscle

Summary The effects of dopamine (DA) were studied on guinea-pig isolated tracheal chains. At a low concentration (10^–6M) DA occasionally produced a small contraction; this was followed by a dose-dependent relaxation 3×10^–6–3×10^–3 M).On a molar basis, DA was about 40 times less potent than noradrenaline (NA) in relaxing tracheal chains, and about 2700 times less potent than isoprenaline (ISO). The maximum degree of relaxation obtained with each drug was the same.Pretreatment of the guinea-pig with reserpine (5 mg/kg) resulted in a 3-fold shift of the DA curve to the right without concomitantly affecting the ISO doseresponse curve. Reserpine completely abolished the relaxant effects of tyramine, but a small contractile response remained.Desipramine (DMI), at a concentration of 10^–5 M, caused a 4-fold shift of the DA curve to th right. The same concentration of DMI resulted in a shift to the left of the NA dose-response curve by about 8-fold. Benztropine (10^–5 M) and haloperidol (10^–5 M and 3×10^–5 M) did not affect the DA dose-response curve.The DA-induced relaxation was inhibited by propranolol (10^–8–10^–6 M) in a dose-dependent manner. The higher concentrations of propranolol (10^–7 and 10^–6 M) unmasked the contractile effect of DA. In the presence of propranolol, phentolamine (10^–5 M) abolished the contractile effect of DA.It is concluded that DA has both direct and indirect actions on guinea-pig isolated tracheal smooth muscle. The relaxant effects of DA are predominantly due to a direct action on smooth muscle -adrenoceptors, with a component due to release of NA from adrenergic nerves. The contractile effects, which under normal conditions are masked by the relaxant effect of DA, are mediated by functional -adrenoceptors. There is no evidence for either specific dopaminergic nerves, uptake mechanisms or receptors in guinea-pig trachealis muscle.     

Interaction between narcotic antagonist (naloxone) and lysergic acid diethylamide (LSD) in the rat

LSD administration in rats elicited a diphasic reaction consisting of a brief excitable period (up to 8 min) followed by a prolonged catalepsy(8 min–1 h). While the cataleptic response was antagonized by a single injection of naloxone (given 30 min after LSD administration), pretreatment with naloxone shortened the excitable phase and potentiated the catalepsy.     

Influence of light and darkness on the turnover of noradrenaline in the rat heart

Summary Male Wistar rats (TNO W. 70) were kept under controlled conditions of light and darkness (12¬12 h). The cardiac turnover of noradrenaline was determined with two methods, either by the decay of the specific activity after i.v. injection of³H-(–)-noradrenaline or by inhibition of the tyrosine-hydroxylase withd,l--methyl-p-tyrosine. During the 12 h of darkness at night the turnover was significantly faster than during daytime (12h illumination). Reversal of the light schedule shifted the noradrenaline turnover by approximately 12 h. The experiments show that the cardiac turnover of noradrenaline can be influenced by environmental lighting. This may be of importance when effects of drugs are studied.Supported by a grant of the Deutsche Forschungsgemeinschaft.     

Cardiostimulatory effects of prenalterol,a beta-1 adrenoceptor partial agonist,in vivo and in vitro

Summary The cardiostimulatory effects of prenalterol, a beta-1-adrenoceptor partial agonist, were studied in vivo and in vitro and compared to those evoked by isoprenaline, a full agonist, and to those of other partial agonists.In the anaesthetized rat, prenalterol and terbutaline were found not to elevate the myocardial cyclic AMP content; this was in sharp contrast to isoprenaline. Both partial agonists did, however, produce significant effects on heart rate.In the anaesthetized cat, prenalterol exhibited chronotropic and inotropic intrinsic activities of 88 and 76% respectively in relation to isoprenaline. No statistically significant increase in myocardial cyclic AMP content could however be detected.Prenalterol did not stimulate adenylate cyclase significantly in the cat myocardial homogenate. This was also true of the beta-2-adrenoceptor selective partial agonist procaterol. In this preparation, isoprenaline, noradrenaline and adrenaline acted as full agonists. Furthermore, prenalterol produced a concentration-dependent inhibition of isoprenaline-activated adenylate cyclase.Our data indicate that maximal cardiac stimulation occurs at a low level of adenylate cyclase activation and low myocardial cyclic AMP concentration when provoked by a full beta-adrenoceptor agonist. The maximal physiological effects of a partial agonist such as prenalterol may consequently be achieved at a marginal activation of the adenylate cyclase.The present data may thus support the hypothesis of a large beta-adrenoceptor reserve for full agonists in the heart.     

Induction of immunotoxicity in mice by polyhalogenated biphenyls

Acute administration of Aroclor-1254 (500 mg/ kg) or 3,4,5,3,4,5-hexabromobiphenyl (HBB) (2–6 mg/ kg) IP, profoundly inhibited the plaque forming response to subsequent challenge with sheep erythrocytes in Ah locus positive (C57B1/6N or B6C3F1N) mice. These studies showed: 1) the immunotoxicity results paralleled enzyme induction results insofar as HBB was approximately 100 times more potent than Aroclor 1254; (2) neither Aroclor nor HBB treatment caused significant induction in the Ah locus negative DBA/2N mice; 3) when B6C3F1 mice were challenged with sheep red blood cells (SRBC) 6 or 16 weeks post Aroclor 1254 treatment, substantial recovery of a PFC response was observed; 4) when these compounds were administered to older (76-week-old) (B6C3F1 mice, severe depression of a PFC response was observed. In contrast to its profound depression of a PFC response, Aroclor-1254 (up to 1250 mg/kg) caused slight increases in lymphocyte proliferation induced by either T or B cell mitogens. A single 500 mg/kg dose of Aroclor-1254 also suppressed the ability of recipient B6C3F1 animals to reject a challenge with either the syngenic fibrosarcoma (PYB6) or the gram negative pathogen (Listeria monocytogenes).     

The functional pool of brain catecholamines: Its size and turnover rate

Behavioral data are reviewed that give evidence for an indiscriminate involvement of brain catecholamines (CA), especially dopamine (DA), in nervefunction, regardless of the time elapsed from their synthesis. Critical analysis of biochemical and pharmacological studies shows that a clear-cut distribution of brain catecholamines in two compartments [newly synthesized (NS) and main storage] is not at all established, and moreover that there is no adequate proof that the difference in turnover rates attributed to these two supposed pools is due to a preferential extraneuronal release of NS-CA during nerve function rather than to a preferential (nonfunctional) intraneuronal deamination of NS-CA, or at least of NS-DA.     

A simple method for sampling murine pulmonary and cardiac tissues for analysis of cyclic nucleotide levels

Summary A simple method for the rapid removal and freezing of mouse cardiac and pulmonary tissues is described. Samples thus obtained were judged to be suitable for valid estimation of in vivo levels of cyclic AMP and cyclic GMP based on the following findings: (a) the samples could be obtained and frozen in the very short time period of a few seconds; (b) no indication of adverse effects of the collection procedure was found upon examination of chemical indicators of energy metabolism; (c) the apparent rates of change of cyclic AMP and cyclic GMP levels during the first seconds after tissue isolation could produce small, but acceptable errors; and (d) dose-dependent elevations of pulmonary cAMP levels consistent with known effects in vitro were found after in vivo administration of isoproterenol.Abbreviations cAMP adenosine-3,5-cyclic monophosphate - cGMP guanosine-3,5-cyclic monophosphate     

Lysergic acid diethylamide antagonizes shaking induced in rats by five chemically different compounds

Thyrotropin-releasing hormone (TRH), sodium valproate, AG-3-5 (1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one), RX336-M (7,8-dihydro-5, 6-dimethylcyclohex-5-eno-1,2,8,14 codeinone), and Sgd 8473 (-[(4-chlorobenzylideneamino)-oxy]-isobutyric acid) each induced repetitive shaking of the body of rats after intraperitoneal injection. This action of the five diverse chemicals appears to be subserved by a common pharmacological component, because pretreatment with d-lysergic acid diethylamide (0.03–1.0 mg kg^-1, s.c.) attenuated the shaking behavior in a dose-related manner, and cross tolerance was found between RX 336-M and TRH, sodium valproate, and AG-3-5.     

Cardiac Ca current does not run down and is very sensitive to isoprenaline in the nystatin-method of whole cell recording

Summary The conventional l type Ca²⁺ channel current (I_Ca.L) was measured in single atrial and ventricular myocytes, with a new whole-cell recording technique using an ionophore, nystatin. The membrane of a cell-attached patch was gradually permeabilized by nystatin (100–200 gg/ml), added to the pipette solution, within 2–5 min after formation of a G-seal. The electrical activity of the cells was measured through the pipette. I_Ca.L, measured with the nystatin-whole cell recording technique, did not exhibit the so-called run-down phenomenon for up to 90 min. The response of I_Ca.L to isoprenaline was also well preserved during the measurement. The half maximal concentration for the isoprenaline-induced increase of I_Ca.L was 8.2 × 10^–9 M, which is a much smaller value than that reported previously. Thus, the nystatin-whole cell clamp recording is a useful technique to measure membrane currents of cardiac myocytes with preserving the physiological intracellular milieu. Send offprint requests to Y. Kurachi at the above address     

Effects of imidazolines on noradrenaline release in rat isolated kidney

The aim of the present study was to investigate whether or not activation of imidazoline receptors modulates noradrenaline release in the rat isolated kidney. Kidneys were pre-exposed to ³H-noradrenaline and the renal nerves were stimulated with 6 pulses at 100 Hz. The stimulation induced (S-I) outflow of radioactivity was taken as an index of endogenous noradrenaline release. The imidazoline derivatives clonidine (1–1000 nmol/l) and moxonidine (10–1000 nmol/l) inhibited S-I outflow of radioactivity with an EC₅₀ of 6.8 nmol/l and 62.5 nmol/l and a maximum of 88% and 97%, respectively. The concentration response curves for clonidine and moxonidine were shifted to the right by the selective ₂-adrenoceptor antagonist rauwolscine (0.1 mol/l) in a parallel manner with identical pK_B's of 8.52 and 8.46, respectively. Furthermore, the -adrenoceptor agonist (–)--methylnoradrenaline (0.1–30nmol/l), which has no affinity for imidazoline binding sites, inhibited S-I outflow of radioactivity with an EC₅₀ of 2.4 nmol/l and a maximum of 94%. Rauwolscine (0.1 mol/l) again shifted the concentration response curve for this -adrenoceptor agonist to the right with a pK_B of 8.40. Moreover, the selective ₂-adrenoceptor antagonist 2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline HCl(RX 821002,0.01 mol/l) shifted the concentration response curves for clonidine and moxonidine to the right with pK_B's of 9.46 and 9.18, respectively. The ₂-adrenoceptor antagonist 4-chloro-2-(2-imidazoline-2-ylamino)-isoindoline HCl (BDF 6143, 1–1000 nmol/l), which, in the presence of ₂-adrenoceptor blockade, has been shown to inhibit noradrenaline release through activation of imidazoline receptors, inhibited S-I outflow of radioactivity with an EC₅₀ of about 20.5 nmol/l (with 1000 nmol/l producing 60% inhibition). The inhibitory effects of BDF 6143 and clonidine were abolished after pretreatment of the kidneys with the irreversible ₂-adrenoceptor antagonist phenoxybenzamine (1 mol/l). However, RX 821002 did not alter and rauwolscine slightly antagonized the inhibitory effect of BDF 6143. It is concluded that the imidazoline derivatives clonidine and moxonidine as well as the phenylethylamine (–)--methylnoradrenaline inhibit noradrenaline release in rat isolated kidney exclusively through activation of prejunctional ₂-adrenoceptors. BDF 6143 inhibits noradrenaline release in rat isolated kidney through an ₂-adrenoceptor-independent, possibly an imidazoline receptor mechanism. Correspondence to: L. C. Rump at the above address     

Effect of phenothiazine neuroleptic drugs and tricyclic antidepressants on phosphodiesterase activity in rat cerebral cortex

The activity of phosphodiesterase (PDE) of rat cerebral cortex following the administration in vitro and in vivo of various concentrations of neuroleptic phenothiazine drugs and tricyclic antidepressive drugs has been investigated. It has been shown that PDE activity is inhibited by phenothiazine neuroleptic drugs (fluphenazine > trifluperazine > thioproperazine > chlorpromazine = thioridazine). Tricyclic antidepressants nortriptyline, chlorimipramine, protiptyline, imipramine and desipramine at a concentration of 10^–3 M caused 60–80% inhibition of PDE activity. It has also been found that the investigated phenothiazine compounds inhibit the high affinity PDE activity more than the PDE activity of low affinity to the substrate.The results obtained suggest that the mechanism of the neuroleptic action of phenothiazine drugs is partially connected with their influence on cyclic 3,5-AMP metabolism.Supported by Polish Academy of Sciences, 09.4.1.5.     

The reduction of excitability of the γ-loop by 1,3-dimethyl-5-aminoadamantane (DMAA)

Summary The -loop of pretibial flexor muscles was investigated before and after i.v. injection of 10 mg/kg DMAA in precollicular and prenigral decerebrate cats. DMMA reduces significantly the reflex discharge rate of Ia muscle spindle afferents, a result which might be important in regard of a hyperactive -motor system.Supported by the Deutsche Forschungsgemeinschaft, SFB 33.