G. Haeusler

Clonidine-induced inhibition of sympathetic nerve activity: No indication for a central presynaptic or an indirect sympathomimetic mode of action

SummaryThe effects of clonidine on blood pressure, heart rate, contractile state of the nictitating membranes, spontaneous sympathetic nerve activity and response of sympathetic nerves to hypothalamic stimulation were compared in normal anaesthetized cats and in anaesthetized cats pretreated with reserpine and α-methyl-p-tyrosine. The pretreatment lowered the noradrenaline content of various parts of the brain to less than 5 ng/g, i.e. to less than 1–3% of that of the controls. Under the conditions of this severe noradrenaline depletion, blood pressure and heart rate were low and spontaneous sympathetic nerve activity consisted of continous, high-amplitude discharges which contrasted with the low-amplitude bursts of activity—synchronous with the respiration—of the controls. In contrast to the controls, clonidine did not lower blood pressure and heart rate in the cats with noradrenaline depletion; however, the clonidine-induced contractions of the nictitating membranes were of similar magnitude and duration in both groups of animals. The efficacy of clonidine in reducing or abolishing spontaneous sympathetic nerve activity and in inhibiting the response of sympathetic nerves to hypothalamic stimulation was equal in controls and in cats with noradrenaline depletion, its potency being 3-fold higher in the former. The results indicate a direct stimulation of α-adrenoceptors by clonidine both in the periphery and in the central nervous system and make it unlikely that the central effect of clonidine on blood pressure is due to a release of noradrenaline from central adrenergic neurones. It is further concluded that clonidine activates an adrenergic mechanism in the central nervous system by stimulation of postsynaptic α-adrenoceptors. The inhibition of such a mechanism as a consequence of a diminished noradrenaline release due to stimulation of presynaptic α-adrenoceptors—as proposed from in vitro experiments—seems to be of no importance for the central effect of clonidine on sympathetic nerve activity and blood pressure.

Central adrenergic neurones and the initiation and development of experimental hypertension

Zerstörung zentraler adrenerger Neurone durch Injektion von 6-Hydroxydopamin in einen Seitenventrikel verursachte bei normotonen und genetisch hypertonen Ratten lediglich eine geringe und vorübergehende Blutdrucksenkung und beeinflusste die DOCA- und renale Hypertonie überhaupt nicht. Im Gegensatz dazu verhinderte 6-Hydroxydopamin die Entwicklung der DOCA- und renalen Hypertonie, wenn es 7–10 Tage vor deren Induktion in einen Seitenventrikel injiziert wurde. Bei genetisch hypertonen Ratten, bei denen eine allmähliche Blutdrucksteigerung schon bald nach der Geburt einsetzt, unterbrach intraventrikulär injiziertes 6-Hydroxydopamin die weitere Entwicklung der Hypertonie. Die Resultate weisen auf die Bedeutung zentraler adrenerger Neurone für die Pathogenese verschiedener experimenteller Hypertonieformen hin.

Evidence suggesting a transmitter or neuromodulatory role for substance P at the first synapse of the baroreceptor reflex

SummaryThere is evidence that the undecapeptide substance P is the transmitter released from pain fibres in the dorsal horn of the spinal cord. This, in turn, suggested to us the possibility of a similar role for substance P in another type of primary sensory structure, namely the baroreceptor neurones which terminate in the bulbar nucleus of the solitary tract (NTS). Substance P, injected into a lateral brain ventricle of urethane-anaesthetized rats, caused dose-dependent increases in blood pressure and heart rate. By contrast, local application of substance P to the region of the NTS, using small pieces of filter paper soaked in substance P-containing solution, resulted in hypotension and bradycardia. In cats anaesthetized with urethane, the same procedure also decreased blood pressure, heart rate and spontaneous sympathetic nervous activity. Release of substance P from nerve endings, through local application of capsaicin to the NTS, produced cardiovascular effects which were indistinguishable from those of substance P. A combined cannula-electrode with an uninsulated tip enabled identification of sites in the NTS of rats and cats, where electrical stimulation elicited decreases in blood pressure, heart rate and spontaneous sympathetic nervous activity. These sites were considered to contain the first synapse of the baroreceptor reflex. Subsequent microinjection of substance P through the cannula into these defined areas resulted in effects which were similar to those of the preceding electrical stimulation. The results suggest a transmitter or neuromodulatory role for substance P at the first synapse of the baroreceptor reflex in the NTS.

Activation of the central pathway of the baroreceptor reflex, a possible mechanism of the hypotensive action of clonidine

SummaryArterial blood pressure, heart rate and discharges in the preganglionic splanchnic and a postganglionic renal sympathetic nerve were recorded in cats anaesthetized with urethane. Electrical stimulation of the posterior hypothalamus or the fastigial nucleus of the cerebellum elicited an immediate increase in sympathetic nerve activity and a rise in blood pressure and heart rate. The stimulation-induced discharge pattern in the sympathetic nerves was characterized by a strong initial burst followed by a phase of inhibition and a final stabilization of the discharges at a level definitely lower than the initial burst. This pattern was reversibly converted into a constant high amplitude firing during a lowering of the blood pressure by bleeding the cats and irreversibly so after cutting the buffer nerves. These findings indicate that the inhibitory phase of the sympathetic discharge pattern during central stimulation is due to the rise in blood pressure and the ensuing baroreceptor-reflex activation.Clonidine (0.03 and 0.1 mg/kg i.v.) reduced the spontaneous sympathetic nerve activity and lowered blood pressure and heart rate. The sympathetic discharges evoked by central stimulation were partially inhibited by clonidine, an effect which could be overcome by raising the voltage used for central stimulation. Independent of the strength of this stimulation an inhibitory phase in the evoked discharge pattern was not observed after clonidine, even when the low blood pressure due to the drug action was raised by a noradrenaline infusion. After clonidine, the evoked discharge pattern closely resembled that after simultaneous stimulation of both sinus nerves and the hypothalamus or the fastigial nucleus, and it was not altered by additional stimulation of the sinus nerves. These observations have led to the hypothesis that clonidine causes a long-lasting activation within an as yet unidentified part of the central pathway of the depressor baroreceptor reflex. In view of the well-known α-adrenoceptor stimulating property of clonidine, and since the central effect of clonidine was antagonized by the α-adrenoceptor blocking agent piperoxan, it is likely that the central part of the baroreceptor-reflex pathway is modified by or contains adrenergic neurones.

On the mechanism of the adrenergic nerve blocking action of bretylium

SummaryBretylium and tetracaine when perfused at increasing concentrations inhibited and finally blocked discharges elicited by acetylcholine and KCl in adrenergic nerve endings of the isolated perfused heart and less regularly in the isolated perfused spleen of the cat.There was a direct correlation between the inhibition of the effects of sympathetic nerve stimulation and the inhibition of KCl-induced discharges by bretylium and tetracaine in the heart with regard to both intensity and time course. Acetylcholine-induced release of noradrenaline in the heart was somewhat more resistant to the action of bretylium than acetylcholine-induced antidromic discharges in cardiac adrenergic nerves.Bretylium and tetracaine inhibited and blocked discharges in sinus nerve afferents elicited by rises in the perfusion pressure and by injection of KCl.The concentration for complete block in the perfused carotid sinus preparation was 800–1,000 times higher for bretylium than for tetracaine, whereas bretylium was equally or more potent (depending on the time of interaction) than tetracaine in the isolated perfused heart. Moreover, the effect of bretylium in the heart and spleen developed slowly, increased continuously during the perfusion period and was very resistant to washing out. In contrast, tetracaine achieved its maximum effect on heart, spleen and carotid sinus preparations very rapidly and was readily washed out. The time course and reversibility of the effect of bretylium on baroreceptor afferents were not drastically different from those of tetracaine.All observations may be explained by the fact that bretylium is a very weak local anaesthetic causing, however, a marked selective local anaesthesia of adrenergic nerve terminals because of its high accumulation in these endings. The adrenergic nerve blocking effect of bretylium seems to be adequately explained by its stabilizing effect on the membrane of the nerve terminals.

Electrical events in cardiac adrenergic nerves and noradrenaline release from the heart induced by acetylcholine and KCl

SummaryThe isolated cat heart with intact sympathetic nerve supply was perfused with varying concentrations of acetylcholine, DMPP and KCl. The ensuing asynchronous discharge in cardiac sympathetic nerves was recorded and the noradrenaline liberated into the perfusate was measured.The infusion of acetylcholine for one minute at a relatively low concentration (5×10−5 to 8×10−5 g/ml) induced asynchronous firing over the entire period of infusion and an average liberation of 44 ng/min noradrenaline. Increasing the concentration of acetylcholine or adding atropine to the perfusion fluid greatly shortened the duration of the antidromic discharge but enhanced severalfold the amount of noradrenaline liberated.Within a narrow range of very low concentrations DMPP induced continuous firing. At higher concentrations antidromic discharges were restricted to the first few seconds of infusion. Muscarinic drugs such as pilocarpine and methacholine neither induced firing nor released noradrenaline. When added to acetylcholine, pilocarpine reduced the amplitude of acetylcholine-evoked firing and the amount of noradrenaline liberated.KCl in concentrations above 50 mM induced a very short-lasting firing and a considerable noradrenaline output which was concentration-dependent.It is concluded that the acetylcholine- and DMPP-induced action potentials by themselves contribute little to the noradrenaline-releasing activity of these drugs. As in the case of KCl, the sustained depolarization of the sympathetic nerve ending by acetylcholine and DMPP seems to enhance entry of Ca++ into the ending, an essential factor for the liberation of noradrenaline.ZusammenfassungAm isolierten, nach Langendorff perfundierten Katzenherzen wurden die auf Injektion oder Infusion von Acetylcholin, DMPP und KCl an den sympathischen Nervenendigungen asynchron auftretenden und antidrom fortgeleiteten Entladungen vom N. cardiacus medius oder inferior abgeleitet. Gleichzeitig wurde das Coronarperfusat gesammelt und sein Noradrenalingehalt fluorimetrisch gemessen.Eine 1minütige Infusion von Acetylcholin in verhältnismäßig niedriger Konzentration (5·10−5 bis 8·10−5 g/ml) verursachte asynchrone Entladungen, die praktisch unvermindert während der gesamten Infusionsdauer anhielten. Die Noradrenalinabgabe aus dem Herzen betrug dabei durchschnittlich 44 ng/min. Eine Steigerung der Acetylcholinkonzentration oder der Zusatz von Atropin zur Perfusionslösung verkürzte die Dauer der Entladungen auf die ersten Sekunden der Infusion, aber verstärkte die Freisetzung von Noradrenalin um das Mehrfache.Innerhalb eines sehr engen Konzentrationsbereiches führte auch die Infusion von DMPP zu anhaltenden antidromen Entladungen im kardialen Sympathicus. Jedoch bewirkte bereits eine geringe darüber hinausgehende Konzentrationserhöhung eine starke Verkürzung der Zeit, in der Entladungen registriert werden konnten.Unter der Infusion von muscarinartig wirkenden Substanzen wie Pilocarpin und Methacholin traten weder asynchrone Entladungen auf noch wurde Noradrenalin freigesetzt. Dem infundierten Acetylcholin zugesetztes Pilocarpin verringerte die Amplitude der Acetylcholin-bedingten Entladungen und reduzierte die Menge des abgegebenen Noradrenalins.In Konzentrationen über 50 mM verursachte KCl außerordentlich kurz dauernde Entladungen und eine während der gesamten Infusionsdauer anhaltende Noradrenalinausschüttung. Die Menge des abgegebenen Noradrenalins hing von der Konzentration des KCl ab.Aus der Gegenüberstellung des zeitlichen Verlaufs von elektrischer Aktivität und Noradrenalinfreisetzung sowie aus der Konzentrationsabhängigkeit beider Phänomene geht hervor, daß unter der Infusion von Acetylcholin, DMPP und KCl die Noradrenalinfreisetzung nur zu einem geringen Teil durch Auslösung von Aktionspotentialen erfolgt. Wesentlich mehr Noradrenalin wird während der permanenten Depolarisation der sympathischen Nervenendigungen abgegeben, deren Auftreten wahrscheinlich gemacht werden konnte. Dabei ist offenbar der während der Depolarisation erfolgende Ca++-Einstrom in die Nervenendigung entscheidend für die Einleitung der Noradrenalinausschüttung.

A comparison of the effects of chemical sympathectomy by 6‐hydroxydopamine in newborn and adult rats

1 The effects of chemical sympathectomy with 6‐hydroxydopamine (6‐OHDA) on the cardiovascular system of the rat were compared in, (a) 10‐week‐old rats treated during the first 14 days after birth with 150 μg/g subcutaneously, and (b) adult rats injected intravenously with 2 × 50 mg/kg on day 1 and 2 × 100 mg/kg on day 7 and the experiments performed on day 8. 2 Intravenous administration of 6‐OHDA to adult rats almost completely abolished the pressor responses to stimulation of the entire sympathetic outflow in the pithed rat, the contractions of the lower eyelid to stimulation of the cervical sympathetic trunk and the vasconstrictor responses produced by periarterial nerve stimulation of the isolated renal artery preparation. Pressor responses to physostigmine and to tyramine were markedly reduced or abolished in anaesthetized and pithed rat preparations, respectively. 3 In corresponding experiments, 10‐week‐old rats treated as newborns with 6‐OHDA showed a marked reduction in the stimulation‐induced pressor responses and contractions of the lower eyelid, but completely normal vasoconstrictor responses to periarterial nerve stimulation of the isolated perfused renal artery were obtained. The pressor responses to physostigmine were slightly reduced but the tyramine responses were unchanged. 4 Treatment with 6‐OHDA at birth caused an almost complete and long‐lasting noradrenaline depletion in the heart, spleen, salivary glands and ileum but only a partial depletion in the mesentery from 10‐week‐old rats. These low noradrenaline levels showed no recovery in rats up to an age of 4 months. The tyrosine hydroxylase activity in both the cervical and stellate ganglia from 10‐week‐old rats was markedly reduced by treatment with 6‐OHDA after birth. 5 Injections of 6‐OHDA after birth produce an almost complete and permanent sympathectomy of various adrenergically innervated organs in the rat. The vascular system represents a major exception, exhibiting a surprisingly high resistance to this type of chemical adrenergic denervation.

The Two Mechanisms of Action of Racemic Cardiotonic Emd 53998, Calcium Sensitization and Phosphodiesterase Inhibition, Reside in Different Enantiomers

The novel cardiotonic EMD 53 998 increases contractile force in vitro through both inhibition of phosphodiesterase III (PDE III) activity and increase in the responsiveness of the contractile proteins to calcium (“calcium sensitization”). Because EMD 53 998 is a racemate, the possibility arose that the two modes of action do not reside equally in the enantiomers. Therefore, the effects of the racemate and its two enantiomers [(+)EMD 57 033 and (-)EMD 57 439] were analyzed in guinea pig and rat cardiac tissue with respect to Ca2+ sensitization (Ca2+-induced force development in skinned cardiac myofibers and myofibrillar ATPase activity) and PDE III inhibition (isolated PDE isoenzymes and cyclic AMP level in isolated cardiac myocytes). In addition, the positive inotropic effects were compared in isometrically contracting papillary muscles. Enhancement of force of contraction (Fc) in submaximally activated skinned fibers showed a selectivity for the (+)enantiomer with EC50 = 1.7, 4.8, and <100 μM for EMD 57 033, EMD 53 998, and EMD 57 439, respectively. Ca2+ concentration for half-maximal activation was decreased by 0.5 log units, and Cmax was increased by 15% at 10 μM EMD 57 033. Similarly, myofibrillar ATPase activity was most potently enhanced by the (+)enantiomer, with EC50 values of 1.8, 2.5, and <30 μM for EMD 57 033, EMD 53 998, and EMD 57 439. respectively. PDE III activity was inhibited with greater potency by the (-)enantiomer, with IC50 values of 0.05, 0.06, and 1.94 μM for EMD 57 439, EMD 53 998, and EMD 57 033, respectively. The cyclic AMP content of isoprenaline-stimulated rat cardiac myocytes was increased by 50% at 13.6 and 0.71 μM for EMD 57 033 and EMD 57 439, respectively. In intact guinea pig papillary muscle, the positive inotropic effect of the (+)enantiomer was insensitive to isoprenaline pretreatment; in contrast, the (-)enantiomer showed only a weak positive inotropic action which was strongly enhanced in the presence of isoprenaline. We conclude that one of the two different mechanisms underlying the overall positive inotropic activity of EMD 53 998 can be assigned, almost exclusively, to one of the two enantiomers. Thus, the (-)enantiomer EMD 57 439 is a “pure” PDE III inhibitor with almost no Ca2+ sensitizing activity; the (+)enantiomer EMD 57 033 is a potent Ca2+ sensitizer with only a weak PDE III inhibitory activity as compared with the racemate. In contrast to other compounds with mixed activity, EMD 57 033 is unique in possessing both a high absolute potency at the level of the contractile elements and a favorable relation of Ca2+ sensitization to PDE inhibition.

Vascular reactivity to 5-hydroxytryptamine and hypertension in the rat

SummaryIsolated perfused mesenteric artery preparations of genetic (Japanese strain), renal and DOCA/saline hypertensive rats showed a marked increase in reactivity to the vasoconstrictor effect of 5-hydroxytryptamine (5-HT) as compared with normotensive controls. In the arteries of the hypertensive animals, the threshold vasoconstrictor doses of 5-HT were lower, the 5-HT dose-response curves were steeper and their maxima were increased by a factor of 2.5–4.2. These observation only partially fit into the concept that increased vascular reactivity in hypertension is due to an increased wall/lumen ratio of the arterial blood vessels. Pressor responses to 5-HT were also higher in isolated perfused hindquarter and in pithed preparations from renal and DOCA/saline hypertensive rats but not in those from genetic hypertensive rats. In isolated perfused renal artery preparations, the reactivity to 5-HT was equal for normotensive and hypertensive (genetic and DOCA/saline) animals, indicating that not all arteries of hypertensive rats share the increased reactivity to 5-HT. Pretreatment with reserpine slightly reduced the reactivity to 5-HT in arteries from both hypertensive and normotensive rats. Non-vascular smooth muscle of DOCA/saline hypertensive rats, for which isolated gastric strips were used as an example, responded to 5-HT in the same way as that of normotensive rats. Methysergide blocked the pressor responses to 5-HT but did not influence the blood pressure of the hypertensive animals, thus questioning a causal relationship between the increased vascular reactivity to 5-HT and the maintenance of high blood pressure in these three types of experimental hypertension.

Characterization of [3H]nifedipine binding sites in rabbit myocardium

A specific, high affinity (KD 1.8 nM) binding site for the calcium entry blocking drug [3H]nifedipine was identified in homogenates of rabbit myocardium. [3H]Nifedipine binding was rapid (t1/2 3 min) and reversible (t1/2 11 min). Calcium entry blockers with different chemical structures competed with [3H]nifedipine binding in the potency order: nifedipine much greater than D600 = verapamil greater than tiapamil greater than cinnarizine = prenylamine. Diltiazem and perhexiline did not significantly inhibit [3H]nifedipine binding. The potencies of these drugs to inhibit binding were similar to their abilities to depress contractions of the isolated rabbit papillary muscle. The stereoselectivity of D600 and verapamil ((-)-much greater than (+)-isomers) as inhibitors of papillary muscle contractions was not apparent in [3H]nifedipine competition experiments. The slopes of the concentration-inhibition curves for D600 and verapamil were significantly less than for nifedipine. It is concluded that [3H]nifedipine may be labelling part of the myocardial Ca2+ channel, and that verapamil-like substances and nifedipine differ in their mode of interaction with this binding site.