Trendelenburg Au

Pharmacological characterization of presynaptic α2-autoreceptors in rat submaxillary gland and heart atrium

1 The pharmacological properties of presynaptic α2‐autoreceptors were studied in rat isolated submaxillary glands and atria. Tissue pieces were preincubated with [3H]‐noradrenaline, then superfused with medium containing desipramine, and stimulated electrically. In one series of experiments, pEC30 values of 12 α‐adrenoceptor antagonists were determined, i.e., negative logarithms of concentrations that increased the electrically evoked overflow of tritium by 30%. In another series, pKD values of 9 α‐adrenoceptor antagonists against the release‐inhibiting effect of 5‐bromo‐6‐(2‐imidazolin‐2‐ylamino)‐quinoxaline (UK 14304), and of 3 antagonists against the release‐inhibiting effect of methoxamine, were determined. 2 In submaxillary glands, the pEC30 values of the antagonists correlated well with their pKD values against UK 14304 (r = 0.93). The same was true for atria (r = 0.92). 3 In submaxillary glands, the pKD values of 3 antagonists against UK 14304 were very similar to their pKD values against methoxamine, with a maximal difference of 0.4. The same was true for atria where the maximal difference was 0.3. 4 The pEC30 values obtained in submaxillary glands correlated significantly with those obtained in atria (r = 0.81). The same was true for the pKD values (r = 0.79). However, the pEC30 and pKD values also indicated consistent differences between the two tissues. 5 It is concluded that the sites of action of the imidazoline UK 14304 (α2‐selective), the phenylethylamine noradrenaline, and the phenylethylamine methoxamine (α1‐selective) are exclusively α2‐adrenoceptors. There is no indication for presynaptic α1‐adrenoceptors or for an effect of UK 14304 mediated by presynaptic imidazoline receptors. The α2‐autoreceptor population in the submaxillary gland differs from that in the atrium. 6 Comparison with studies from the literature indicates that the submaxillary autoreceptors are closely similar to the α2D radioligand binding site found in the bovine pineal gland and probably the rat submaxillary gland. The atrial autoreceptors also conform best to this site, but the agreement is more limited; the atrial autoreceptors may represent a type related to, but distinct from, the α2D site, or a mixture of different types.

A re-investigation of questionable subclassifications of presynaptic α2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra

Abstract It has been suggested that at least the majority of mammalian presynaptic α2-autoreceptors belong to the genetic α2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the α2-autoreceptors in tissues in which previous assignments conflicted with this α2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as α2B or similar to, but not identical with, α2D, and in the human kidney, where they were classified as α2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be α2A, in agreement with the rule, but in contrast to indications that the α2A/D-adrenoceptor of the guinea pig possesses α2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the α2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined.EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known α2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were α2D and those in the human kidney α2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic α2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the α2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at α2A, α2B and α2C binding sites and α2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic α2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the α2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at α2B, α2C and α2D binding sites and α2D-autoreceptors.2D, those in the human kidney α2A, and those in the guinea-pig urethra equally α2D. All, therefore, conform to the rule that α2-autoreceptors belong at least predominantly to the genetic α2A/D subtype of the α2-adrenoceptor. The apparent paradox of an α2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic α2A/D-adrenoceptor otherwise has α2D pharmacological properties, is removed.

Modulation of 3H-noradrenaline release by presynaptic opioid, cannabinoid and bradykinin receptors and β-adrenoceptors in mouse tissues

Release‐modulating opioid and cannabinoid (CB) receptors, β‐adrenoceptors and bradykinin receptors at noradrenergic axons were studied in mouse tissues (occipito‐parietal cortex, heart atria, vas deferens and spleen) preincubated with 3H‐noradrenaline. Experiments using the OP1 receptor‐selective agonists DPDPE and DSLET, the OP2‐selective agonists U50488H and U69593, the OP3‐selective agonist DAMGO, the ORL1 receptor‐selective agonist nociceptin, and a number of selective antagonists showed that the noradrenergic axons innervating the occipito‐parietal cortex possess release‐inhibiting OP3 and ORL1 receptors, those innervating atria OP1, ORL1 and possibly OP3 receptors, and those innervating the vas deferens all four opioid receptor types. Experiments using the non‐selective CB agonists WIN 55,212‐2 and CP 55,940 and the CB1‐selective antagonist SR 141716A indicated that the noradrenergic axons of the vas deferens possess release‐inhibiting CB1 receptors. Presynaptic CB receptors were not found in the occipito‐parietal cortex, in atria or in the spleen. Experiments using the non‐selective β‐adrenoceptor agonist isoprenaline and the β2‐selective agonist salbutamol, as well as subtype‐selective antagonists, demonstrated the occurrence of release‐enhancing β2‐adrenoceptors at the sympathetic axons of atria and the spleen, but demonstrated their absence in the occipito‐parietal cortex and the vas deferens. Experiments with bradykinin and the B2‐selective antagonist Hoe 140 showed the operation of release‐enhancing B2 receptors at the sympathetic axons of atria, the vas deferens and the spleen, but showed their absence in the occipito‐parietal cortex. The experiments document a number of new presynaptic receptor locations. They confirm and extend the existence of marked tissue and species differences in presynaptic receptors at noradrenergic neurons.

α2‐Adrenoceptors in the enteric nervous system: a study in α2A‐adrenoceptor‐deficient mice

Mammals possess three types of α2‐adrenoceptor, α2A, α2B and α2C. Our aim was to determine the type of α2‐adrenoceptor involved in the control of gastrointestinal motility. In transmitter overflow experiments, myenteric plexus longitudinal muscle (MPLM) preparations of the ileum were preincubated with [3H]‐choline and then superfused. The α2‐adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium from preparations taken from wild type but not α2A‐adrenoceptor‐knockout mice. In a second series of overflow experiments, MPLM preparations were preincubated with [3H]‐noradrenaline and then superfused. Again medetomidine reduced the electrically evoked overflow of tritium from wild type but not α2A‐knockout preparations. In organ bath experiments, medetomidine reduced electrically evoked contractions of segments of the ileum from wild type but not α2A‐knockout mice. In each of these three series, phentolamine antagonized the effect of medetomidine in wild‐type preparations with greater potency than rauwolscine. In conscious mice, gastrointestinal transit was assessed by means of an intragastric charcoal bolus. In α2A‐knockout mice, the speed of gastrointestinal transit was doubled compared to wild‐type. Medetomidine, injected intraperitoneally, slowed gastrointestinal transit in wild type but not α2A‐knockout mice. We conclude that the cholinergic motor neurons of the enteric nervous system of mice possess α2‐heteroreceptors which mediate inhibition of acetylcholine release, of neurogenic contractions and of gastrointestinal transit. The noradrenergic axons innervating the intestine possess α2‐autoreceptors. Both hetero‐ and autoreceptors are exclusively α2A. It is the α2A‐adrenoceptor which in vivo mediates the inhibition of intestinal motility by the sympathetic nervous system.

Release-inhibiting α2-adrenoceptors at serotonergic axons in rat and rabbit brain cortex: evidence for pharmacological identity with α2-autoreceptors

The pharmacological properties of the presynaptic α2-adrenoceptors modulating the release of serotonin in rat and rabbit brain cortex (α2-heteroreceptors) were compared with the properties of presynaptic α2-autoreceptors in the same brain area. Brain cortex slices were preincubated with [3H]-serotonin or [3H]-noradrenaline and then superfused and stimulated by brief high-frequency pulse trains.The α2-adrenoceptor agonist bromoxidine reduced the electrically evoked overflow of tritium in experiments with both [3H]-noradrenaline and [3H]-serotonin and in brain slices from either species. The antagonists phentolamine, idazoxan, (+)-mianserin, rauwolscine, 5-chloro-4(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (ORG 20350), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), (−)-mianserin and corynanthine caused parallel shifts of the concentration-inhibition curves of bromoxidine to the right. Negative logarithms of antagonist dissociation constants pKd were calculated from the shifts. In the rat, the α2-autoreceptor pKd value of each single antagonist was similar to its α2-heteroreceptor pKd value, maximal difference 0.4, giving a close correlation, r = 0.97 (P<0.001). In the rabbit equally, the α2-autoreceptor pKd value of each single antagonist was similar to its α2-heteroreceptor pKd value, maximal difference 0.4, again yielding a close correlation, r = 0.96 (P < 0.001). However, antagonist pKd values at rat α2-autoreceptors differed from those at rabbit α2-autoreceptors, r = 0.70 (P > 0.05), and antagonist pKd values at rat α2-heteroreceptors differed from those at rabbit α2-heteroreceptors, r = 0.64 (P > 0.05). Comparison with radioligand binding experiments from the literature indicated that, in the rat, both auto- and heteroreceptors conformed best to the α2D subtype (r ⪖ 0.97, P < 0.01 for pKd correlation with binding sites in rat submaxillary gland) whereas, in the rabbit, they conformed best to the α2A subtype (r ⪖ 0.93, P < 0.01 for pKd correlation with binding sites in HT29 cells).It is concluded that, in both the rat and the rabbit, the α2-adrenoceptors modulating the release of serotonin are pharmacologically identical with the presynaptic α2-autoreceptors. However, rat α2-autoreceptors and -heteroreceptors differ pharmacologically from rabbit α2-autoreceptors and -heteroreceptors. Presynaptic α2-auto-as well as -heteroreceptors are α2D in the rat and α2A in the rabbit.

Enhancement of noradrenaline release by angiotensin II and bradykinin in mouse atria: evidence for cross-talk between Gq/11 protein- and Gi/o protein-coupled receptors

The interaction between α2‐autoreceptors and receptors for angiotensin (AT1) and bradykinin (B2) was studied in mouse isolated atria. The preparations were labelled with [3H]‐noradrenaline and then superfused with desipramine‐containing medium and stimulated electrically. Angiotensin II (10−11–10−7 M), angiotensin III (10−10–10−6 M) and bradykinin (10−11–10−7 M) enhanced the evoked overflow of tritium when preparations were stimulated with conditions that led to marked α2‐autoinhibition (120 pulses at 3 Hz), but not when stimulated with conditions that led to little α2‐autoinhibition (20 pulses at 50 Hz). Blockade of α‐adrenoceptors by phentolamine (1 or 10 μM) reduced or abolished the effect of angiotensin II and bradykinin on the overflow response to 120 pulses at 3 Hz. Addition of the δ‐opioid agonist [D‐Ser2]‐leucine enkephalin‐Thr (DSLET, 0.1 μM), or of neuropeptide Y (0.1 μM), together with phentolamine, restored the effect of angiotensin II and bradykinin. The β‐adrenoceptor agonist terbutaline (10−9–10−4 M) enhanced the evoked overflow of tritium irrespective of the degree of autoinhibition. The experiments show that (i) a marked prejunctional facilitatory effect of angiotensin and bradykinin in mouse isolated atria requires prejunctional α2‐autoinhibition; (ii) in the absence of α2‐autoinhibition, activation of other prejunctional Gi/o protein‐coupled reeptors, namely opioid and neuropeptide Y receptors, restores a marked effect of angiotensin II and bradykinin; and (iii) the facilitatory effect of terbutaline is not dependent upon the degree of α2‐autoinhibition. The findings indicate that the major part of the release‐enhancing effect elicited through prejunctional Gq/11 protein‐coupled receptors is due to disruption of an ongoing, α2‐autoreceptor‐triggered Gi/o protein mediated inhibition.

Prejunctional angiotensin receptors involved in the facilitation of noradrenaline release in mouse tissues

The effect of angiotensin II, angiotensin III, angiotensin IV and angiotensin‐(1–7) on the electrically induced release of noradrenaline was studied in preparations of mouse atria, spleen, hippocampus, occipito‐parietal cortex and hypothalamus preincubated with [3H]‐noradrenaline. The prejunctional angiotensin receptor type was investigated using the non‐selective receptor antagonist saralasin (AT1/AT2) and the AT1 and AT2 selective receptor antagonists losartan and PD 123319, respectively. In atrial and splenic preparations, angiotensin II (0.01 nM–0.1 μM) and angiotensin III (0.01 and 0.1 nM–1 μM) increased the stimulation‐induced overflow of tritium in a concentration‐dependent manner. Angiotensin IV, only at high concentrations (1 and 10 μM), enhanced tritium overflow in the atria, while angiotensin‐(1–7) (0.1 nM–10 μM) was without effect in both preparations. In preparations of hippocampus, occipito‐parietal cortex and hypothalamus, none of the angiotensin peptides altered the evoked overflow of tritium. In atrial and splenic preparations, saralasin (0.1 μM) and losartan (0.1 and 1 μM), but not PD 123319 (0.1 μM), shifted the concentration‐response curves of angiotensin II and angiotensin III to the right. In conclusion, in mouse atria and spleen, angiotensin II and angiotensin III facilitate the action potential induced release of noradrenaline via a prejunctional AT1 receptor. Only high concentrations of angiotensin IV are effective in the atria and angiotensin‐(1–7) is without effect in both preparations. In mouse brain areas, angiotensin II, angiotensin III, angiotensin IV and angiotensin‐(1–7) do not modulate the release of noradrenaline.

A search for presynaptic imidazoline receptors at rabbit and rat noradrenergic neurones in the absence of α2-autoinhibition

Presynaptic, release-inhibiting imidazoline receptors have hitherto been detected mainly under conditions of ongoing α2-autoinhibition. We tried to find them under α2-autoinhibition-free conditions, in the majority of experiments in the rabbit pulmonary artery but in a few experiments also in rabbit atria, rabbit brain cortex and rat brain cortex. Tissue segments were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Ten compounds, some thought to inhibit noradrenaline release through activation of presynaptic imidazoline receptors, some thought to act through α2-adrenoceptors, were tested. Rauwolscine and 6-chloro-N-methyl-2,3,4,5-tetrahydro-1-H-3-benzazepine (SKF 86466) were used as antagonists. In rabbit pulmonary artery segments stimulated by trains of 20 pulses/50 Hz (α2-autoinhibition-free conditions), all ten “agonists” [medetomidine, aganodine, 4-chloro-2-guanidyl-isoindoline (BDF 7579), 4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline (BDF 6143), 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304), α-methylnoradrenaline, clonidine, moxonidine, cirazoline and idazoxan] reduced the stimulation-evoked overflow of tritium, with potency decreasing in that order and with maximal inhibition by 59% (idazoxan) to 95% (moxonidine). Rauwolscine competitively antagonized the effects of all ten “agonists” with similar potency, the pKd-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pKd 7.6). Cirazoline was also studied in the other three tissues. In rabbit atrial segments stimulated by 20 pulses/50 Hz and rabbit brain cortex slices stimulated by 4 pulses/100 Hz (both autoinhibition-free), cirazoline reduced the evoked overflow of tritium with concentration-inhibition curves similar to the curve in the pulmonary artery. In the brain cortex, the pKd-value of rauwolscine against cirazoline was 7.7 (pulmonary artery: 7.6). In rat brain cortex slices stimulated by 3 pulses/100 Hz (autoinhibition-free), cirazoline failed to change the evoked overflow of tritium but antagonized the inhibitory effect of UK 14304, pKd of cirazoline against UK 14304 6.9. In rat brain cortex slices stimulated by trains of 36 pulses/3 Hz, finally (marked α2-autoinhibition), cirazoline increased the evoked overflow of tritium. In the rabbit pulmonary artery, rauwolscine and SKF 86466 acted with the same potency against typical presynaptic imidazoline receptor agonists (such as clonidine) and typical α2-adrenoceptor agonists (such as moxonidine). Hence, presynaptic imidazoline receptors were not detectable, in a tissue that is prototypical for presynaptic imidazoline receptors, in the absence of α2-autoinhibition, i.e. under conditions usually thought to be optimal for presynaptic receptor characterization. The pKd-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the α2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an α2(A)-autoreceptor agonist in rabbit tissues but as an α2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) α2A-adrenoceptors than the orthologous (rodent) α2D-adrenoceptors.

Noradrenaline release from cultured mouse postganglionic sympathetic neurons: autoreceptor-mediated modulation.

Abstract : The possible existence of α2‐autoreceptors, P2‐autoreceptors, and adenosine A1‐ or A2A‐receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective α2‐adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration‐inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six α‐adrenoceptor antagonists shifted the concentration‐inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3‐300 μM), adenosine (0.01‐100 μM), the selective A1‐receptor agonist cyclopentyladenosine (1‐1,000 nM), nor the selective A2A‐receptor agonist CGS‐21680 (1‐10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release‐inhibiting α2‐autoreceptors. As in intact tissues, the effectiveness of presynaptic α2‐adrenergic inhibition depends on the “strength” of the releasing stimulus. The pKD values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological α2D and hence the genetic α2A/D subtype of α2‐adrenoceptor. Neither P2‐autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.

Mouse postganglionic sympathetic neurons: primary culturing and noradrenaline release.

Abstract : Basic properties of noradrenaline release were studied in primary cultures of thoracolumbar postganglionic sympathetic neurons taken from 1‐3‐day‐old NMRI mice. After 7 days in vitro, the cultures were preincubated with [3H]noradrenaline and then superfused and stimulated electrically. Conventional trains of pulses (for example, 36 pulses at 3 Hz) as well as single pulses and brief high‐frequency trains (for example, four pulses at 100 Hz) elicited a well‐measurable overflow of tritium, which was abolished by 0.3 μM tetrodotoxin or omission of Ca2+, but not changed by 1 μM rauwolscine. In trains of one, two, four, six, eight, or 10 pulses at 3 Hz, the evoked overflow of tritium remained constant from pulse to pulse at 1.3 mM Ca2+, but declined slightly at 2.5 mM Ca2+. Tetraethylammonium at 10 mM selectively increased the overflow elicited by small pulse numbers and especially by a single pulse. In trains of 10 pulses delivered at 0.3, 1, 3, 10, 30, or 100 Hz, the evoked overflow of tritium increased from 0.3 to 30 Hz and then declined at 100 Hz. This relationship was particularly pronounced at low Ca2+ concentrations (for example, 0.3 mM) Tetraethylammonium at 10 mM selectively increased the overflow elicited by low frequencies of stimulation. It is concluded that primary cultures of mouse postganglionic sympathetic neurons can be used to investigate release of [3H]noradrenaline. The release is well measurable, even upon a single electrical pulse. It agrees with release in intact sympathetically innervated tissues in a number of fundamental properties, including the pulse number and frequency dependence. The preparation may be of special interest in conjunction with genetic manipulations in the donor animals.