The slope parameter of concentration-response curves used as a touchstone for the existence of spare receptors

The present work was stimulated by findings of a large reserve of presynaptic α₂-autoreceptors in rat neocortex by different investigators and our own group, using classical models of receptor agonism. The mathematical background of these classical models seems erroneous since the asymmetry that spare receptors introduce into concentration-response curves is not considered appropriately. This asymmetry leads to a steepening of curve fits based on the logistic function. Therefore, the slope parameter c of a logistically fitted concentration-response curve can be used as a touchstone for the existence of spare receptors. Spare receptors induce a c > 1. Concentration-response data of the α₂-autoreceptor-mediated inhibition of evoked [³H]-noradrenaline release in rat neocortex slices were re-analysed. The estimates of the slope parameter c of logistically fitted concentration-response curves obtained after treatment of rats with either vehicle or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to achieve an irreversible inactivation of α₂-autoreceptors, were not compatible with the existence of a large receptor reserve. A model for nonlinear regression analysis developed under the a priori assumption of spare receptors confirmed the absence of spare receptors. Received: 13 December 1996 / Accepted: 30 May 1997     

Presynaptic imidazoline receptors and α2-adrenoceptors in the human heart: discrimination by clonidine and moxonidine

The involvement of presynaptic ₂-autoreceptors and imidazoline receptors in the modulation of noradrenaline release was investigated in strips from human atrial appendages preincubated with [³H]noradrenaline and superfused with medium containing desipramine and corticosterone. Electrical impulses were applied transmurally at 2 Hz. The imidazoline derivatives moxonidine and clonidine reduced the evoked tritium overflow in a concentration-dependent manner. Moxonidine was 18-fold more potent than clonidine. The concentration-response curve for moxonidine, but not for clonidine was shifted to the right by the ₂-adrenoceptor antagonist rauwolscine. The apparent pA₂ value of rauwolscine against moxonidine was 8.41. An inhibitory effect was also observed for the imidazoline derivative BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), a mixed ₂-adrenoceptor antagonist/imidazoline receptor agonist; BDF 6143 was about 2-fold more potent than clonidine. Rauwolscine (1 M) did not substantially shift the concentration-response curve of BDF 6143.It is concluded that noradrenaline release in the human atrium is inhibited not only via presynaptic ₂-autoreceptors but also via presynaptic non-I₁, non-I₂ imidazoline receptors. The failure of rauwolscine to antagonize the inhibitory effect of clonidine suggests that clonidine preferentially stimulates the presynaptic imidazoline receptors; the ₂-adrenoceptor component of its action is probably suppressed by an inhibitory interaction between the two receptors. In contrast, moxonidine acts via presynaptic ₂-autoreceptors, leaving the presynaptic imidazoline receptor unaffected.     

Mathematical modelling and quantification of the autoinhibitory feedback control of noradrenaline release in brain slices

Summary Concentration-response curves, reflecting ₂-autoreceptor-mediated inhibition of [³H]-noradrenaline release by exogenous noradrenaline in rat cerebral cortex and rabbit hippocampus slices, were analysed in order to test the usefulness of a mathematical model describing the relation between the independent variable, exogenous noradrenaline, and the dependent variable, inhibition of release. This model was based on the assumption of direct proportionality between receptor occupation and response, implying that there is correspondence between the shape of a concentration-binding curve and a concentration-response curve. The experimental concentration-response curves were obtained by different approaches: noradrenaline release from brain slices prelabelled with [³H]-noradrenaline was elicited electrically either by pseudo-one-pulse (POP) stimulation or by stimulation with 36 pulses applied with a frequency of 3 Hz. POP stimulation avoids autoinhibition by released noradrenaline and, therefore, was a suitable touchstone for the applied mathematical model which evaluates by nonlinear regression analysis two primary parameters: the dissociation constant between noradrenaline and the ₂-adrenoceptor and the biophase concentration of noradrenaline which reflects the extent of autoinhibition and should be zero under POP conditions. In rat cerebral cortex tissue, the corresponding biophase concentration of endogenous noradrenaline was indeed estimated to be zero and the dissociation constant was K _d = 10^{–7.62±0.14} mol/l. With 3 Hz stimulation, the biophase concentration was 10^{–7.80±0.05} mol/l, which has to be interpreted with respect to a simultaneously estimated K _d of 10^{–7.63±0.12} mol/l. Since the K _d-values under POP or 3 Hz conditions were similar, the biophase concentration obviously had no influence on the estimate of the other primary parameter, K _d. With rabbit hippocampus, however, the main prerequisite of the mathematical model; i.e. direct proportionality between receptor occupation and response, was not established since the slope of the POP concentration-response curve (estimated as slope parameter c) did not correspond to that of a concentration-binding curve.In conclusion, mathematical modelling by nonlinear regression analysis of the autoinhibitory circuit of noradrenaline release allows the estimation of a parameter c of this feedback regulation which supports or rejects the assumption of direct proportionality between receptor occupation and functional response. When the given requirement of direct proportionality is shown to hold, this analysis allows the feedback circuit to be described quantitatively in terms of the affinity of noradrenaline for, and of the biophase concentration of noradrenaline at, the presynaptic ₂-adrenoceptors.Correspondence to T. J. Feuerstein     

PHARMACOLOGICAL INVESTIGATION OF α-ADRENORECEPTORS IN GUINEA-PIG SPLENIC CAPSULE

• 1 A study has been made of α-adrenoreceptors in the smooth muscle of the guinea-pig splenic capsule.
• 2 In this preparation, noradrenaline (NA), adrenaline (Adr) and α-methyl-NA were full agonists, while phenylephrine, dopamine and the catechol-imidazolidine (3,4-dihydroxyphenylamino)-2-imidazolidine, (DPI) were partial agonists.
• 3 The imidazolidines clonidine and oxymetazoline showed no agonist activity, but both inhibited the agonist activity of NA. pA₂ values calculated from Arunlakshana and Schild plots were 6.88 and 6.95 for clonidine and oxymetazoline respectively. Slopes of the Schild plots were close to unity, indicating competitive antagonism.
• 4 The α-adrenoreceptor antagonists phentolamine, prazosin and yohimbine also inhibited the agonist activity of NA with mean pA₂ values of 8.32, 9.22 and 6.90 respectively. Slopes of Schild plots were significantly less than 1, suggesting that the inhibition was not truly competitive in nature.
• 5 The irreversible α-adrenoreceptor antagonist phenoxybenzamine at low concentrations (10^–9M) shifted the log concentration-response curve to NA to the right and greatly reduced the maximum response.
• 6 It is suggested that the adrenoreceptors in the guinea-pig splenic capsule are probably of the α₁-type, and that this tissue possesses relatively few spare receptors.

     

Noradrenaline uptake inhibitors do not reduce the presynaptic action of clonidine on 3H-noradrenaline release in superfused synaptosomes

Summary The interaction between clonidine, as an agonist at the ₂-autoreceptors regulating noradrenaline release, and inhibitors of noradrenaline neuronal uptake was investigated in superfused synaptosomes, i.e. in conditions preventing accumulation of the released transmitter in the vicinity of presynaptic autoreceptors. Desipramine or cocaine did not decrease the inhibitory action of clonidine on the release of ³H-noradrenaline evoked by 15 mM KCl from rat cortex synaptosomes, even when the concentration ratio between uptake inhibitor and clonidine was very high. The present results do not support either the hypothesis of an interaction between imidazolines and noradrenaline uptake inhibitors at the level of ₂-autoreceptors, or that of a functional coupling between presynaptic ₂-autoreceptors and noradrenaline uptake mechanism.     

Antagonist inhibition curves and the measurement of dissociation constants

1. Experiments carried out on guinea-pig isolated ileum with carbachol as agonist and diphenyl- acetoxyethyl- dimethyl-ethyl- ammonium (DADMEA) bromide as antagonist gave results which fit the theoretical relation between fractional inhibition (Q) of the effects of an agonist ([A]) and the concentration of a competitive antagonist ([B]): this also involves the Hill coefficient (logistic slope factor, P) for the agonist concentration-response curve and the degree of agonist stimulation, [A]/[A]₅₀, where [A]₅₀ produces a half-maximum response.
2. Values of IC₅₀ and an exponent, P′, can be obtained by fitting Q to [B] using a logistic approximation to the relation. Both P′ and IC₅₀ should be greater with higher agonist stimulation but the increase in P′ may be masked by errors in extreme values of Q. Estimates of IC₅₀, however, invariably increased with higher agonist stimulation but with a steep concentration-response curve (P>1) and low agonist stimulation ([A]/[A]₅₀ <1), IC₅₀ can be less than K_D.
3. K_D was calculated from the results in three ways: (i) by a least-squares fit of Q to [B] using the values of P and [A]/[A]₅₀ calculated from the control concentration-response curve; (ii) from the value of IC₅₀ for each line and the values of P and [A]/[A]₅₀ and (iii) by using the agonist concentration-response curve to calculate the dose-ratio and estimate of K_D for each response in the presence of the antagonist. The methods gave similar results (nM: 11 experiments), 12.4±1.1 (i), 11.7±0.9 (ii), 14.8±1.6 (iii) but there are advantages in using methods (i) or (ii) rather than (iii).
4. The method by which K_D is calculated is less important than the experimental design: the plan used in this work, with alternative small and large responses from the tissue, is very suitable for estimating K_D with low concentrations of antagonists and small dose-ratios. Although it is not a sensitive test for competitive behaviour because only a small range of concentrations of antagonist is tested, the estimate of affinity should be free from complications involved in the use of higher concentrations of antagonist (and agonist) and the nature of the antagonism can always be checked by doing further experiments in the presence of a known competitive antagonist.

     

Characterization of the contractile responses to noradrenaline and adrenaline of aorta from normotensive and hypertensive rats

1. The effects of phenoxybenzamine treatment on the contractile responses of the aorta from WKY and SH rats to noradrenaline and adrenaline have been determined.2. There was no change in sensitivity of the aorta to noradrenaline or adrenaline with hypertension.3. Phenoxybenzamine treatment caused nonparallel rightward shifts of the concentration-response curves to the lower concentrations of noradrenaline and adrenaline.4. The K_A value for noradrenaline on the WKY rat aorta was 4.15 × 10⁻⁸ M and noradrenaline produced a 95% maximum response by occupying 95% of the available α₁-adrenoceptors. These parameters were significantly different on the SH rat aorta; thus the K_A value for noradrenaline was 76.40 × 10⁻⁸ M and noradrenaline produced a 95% maximum response by occupying 63% of the α₁-adrenoceptors.5. The K_A value for adrenaline on the WKY rat aorta was 5.03 × 10⁻⁸ M and adrenaline produced a 95% maximum response by occupying 95% of the available α₁-adrenoceptors. These adrenaline parameters were not significantly different on the SH rat aorta.6. In summary this study has demonstrated that although the sensitivities of the rat aorta to noradrenaline and adrenaline do not alter, there is a change in the K_A and receptor reserve for noradrenaline, but not adrenaline in hypertension.     

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 pK _d-values lying between 7.6 and 8.2. Similarly, SKF 86466 competitively antagonized the effects of clonidine and moxonidine with the same potency (pK _d 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 pK _d-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, pK _d 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 pK _d-values of rauwolscine and SKF 86466 indicate that all ten agonists activated the α_2A-autoreceptors of the pulmonary artery. Cirazoline behaved as an α_2(A)-autoreceptor agonist in rabbit tissues but as an α_2(D)-autoreceptor antagonist in rat tissues. Perhaps cirazoline generally possesses greater intrinsic activity at (human, rabbit) α_2A-adrenoceptors than the orthologous (rodent) α_2D-adrenoceptors. Received: 15 July 1998 / Accepted: 19 November 1998     

Pharmacological properties of two recombinant splice variants of the PACAP type I receptor,transfected and stably expressed in CHO cells

Two splice variants of the pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PACAP receptor and PACAP/HOP receptor isoform) were stably expressed in Chinese hamster ovary (CHO) cells that did not express constitutively receptors for this family. The PACAP./HOP receptor protein had a 28 amino acis extension in the C-terminal part of the third intracellular loop. The two cell lines studied, CHO 2–10 (PACAP receptor) and CHO 4–12 (PACAP/HOP receptor) expressed a receptor density of 4.6±0.3 and 2.6±0.2 pmol/mg protein, respectively, with corresponding K_d values of 14.2±2.0 and 8.2±1.0 nM for [Ac-His¹]PACAP-27 used as a tracer. Tracer binding was slightly decreased by GTP in both clones. The K_d values of PACAP-27, PACAP-38, vasoactive intestinal peptide (VIP), PACAP-27 fragments and analogues evaluated by binding competition curves, were higher in CHO 2–10 than in CHO 4–12, whereas the K_d for PACAP-38 fragments did not differ. The receptors were coupled to adenylate cyclase and the EC₅₀ values were lower than the K_d values in both cell lines, suggesting an amplification process due to the existence of spare receptors. Pretreatment of the CHO 4–12 cells with increasing concentrations of PACAP-27 for 24 h induced an increase in the K_act values and a decrease in the maximal stimulation; the same pretreatment of CHO 2–10 cells also induced an increase in the K_act values, but a marked increase in the adenylate cyclase activity in the absence of added peptide, suggesting that PACAP pretreatment had induced a permanent coupling of the receptor to the G_s site. Thus, the two splice variants differed in their capacity to recognize the ligand, and in their coupling to the G_s sites.     

Evidence for spare alpha 1-adrenoceptors for the accumulation of inositol phosphates in smooth muscle

The accumulation of inositol phosphates (IP) in smooth muscle from rat vas deferens and caudal artery was maximally increased 3- to 4-fold in response to exposure of the tissues to 100 microM noradrenaline. Clonidine (up to 3 mM) was a partial agonist. Pretreatment of the tissues with the irreversible alpha-adrenoceptor antagonist phenoxybenzamine (0.3-10 microM) shifted the noradrenaline concentration-response curve to the right before depressing the maximum. The maximum of the clonidine concentration-response curve was depressed without significant change in the EC50 by the same treatment. These data, which are most easily interpreted as demonstrating the presence of a receptor reserve for IP accumulation, are discussed.     

Postsynaptic α1-adrenoceptor mechanisms in rat vas deferens and ageing

Postsynaptic α₁-adrenoceptor mechanisms in vasa deferentia isolated from 3, 6, 18 and 40 week-old rats were studied by analysis of the concentration-response curve of noradrenaline and the Scatchard plot of specific binding of [³H]prazosin to microsomal fractions. The maximum tension developed by noradrenaline also increased with age from 3 to 18 weeks. The efficacy of noradrenaline and capacity of the maximum binding sites of [³H)prazosin increased with increasing age, while the dissociation constants of noradrenaline (K_A) and prazosin (K_d) were not changed with age. The increase of the maximum tension was proportional to the increase in efficacy. The increase of efficacy for noradrenaline in the vasa deferentia from rats of different ages is due to the increase in the total concentration of postsynaptic α₁-adrenoceptors.     

P2-Receptor-mediated inhibition of noradrenaline release in the rat pancreas

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1μM) and yohimbine (1μM), and stimulated electrically, in most experiments by 60 pulses/1Hz. The adenosine A₁-receptor agonist N⁶-cyclopentyl-adenosine (CPA; EC₅₀ 32nM), the non-subtype-selective adenosine receptor agonists adenosine (EC₅₀ 15μM) and 5’-N-ethylcarboxamidoadenosine (NECA; EC₅₀ 135nM), and the nucleotides ATP (EC₅₀ 13μM), adenosine-5’-O-(3-thiotriphosphate) (ATPγS; EC₅₀ 19μM) and adenosine-5’-O-(2-thiodiphosphate) (ADPβS; EC₅₀ 16μM) decreased the evoked overflow of tritium. The adenosine A_2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A₁-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10nM; pK_d 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30μM). Combined administration of a high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPβS were shifted to the right by both DPCPX (10nM; pK_d 8.7 and 8.9, respectively) and cibacron blue 3GA (30μM; pK_d 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10nM) and cibacron blue 3GA (30μM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM), ATP and ADPβS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30μM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A₁-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia. Received: 6 November 1997 / Accepted: 6 January 1998     

Mathematical analysis of the control of neurotransmitter release by presynaptic receptors as a supplement to experimental data

The quantitative analysis of receptor-mediated effects is based on experimental concentration-response curves in which an independent variable, the concentration of a receptor ligand, is linked with a dependent variable, the biological response. The steps that intervene between the ligand-receptor interaction and the subsequent biologic effect, i.e. modulation of transmitter release in our examples, are largely unknown. Nevertheless, the shape of a concentration-response curve may give some insights into the nature of the relation between receptor occupancy and ensuing response. The shape of the concentration-response curve can be evaluated by nonlinear regression analysis of the data points of the independent and dependent variable. If possible, the model applied should be mechanistically derived from a physical or chemical law, underlying the biological condition. For instance, the inherence of the Law of Mass Action allows to call the model mechanistic. The presence of spare receptors for an agonist must induce an alteration of the shape of the concentration-response curve as compared to a symmetric bimolecular concentration-binding curve. Evaluation methods which neglect the alteration of the geometrical form of concentration-response curves due to non-proportionality between receptor occupation and relative response do not seem appropriate to quantify spare receptors. The “general response function” may allow a mechanistic interpretation of the occupancy-response relationship. This function estimates roughly the number of “non-spare” receptors and of spare receptors on a functional unit that contribute to the response. Received: 13 July 1998 / Accepted: 18 February 1999     

Influence of cooling on the responsiveness of rat isolated anococcygeus muscle to noradrenaline

Summary The isolated anococcygeus muscle of the rat was used to study the effect of temperature on noradrenaline-induced contraction. The preparation was suspended in an organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of the bath temperature from 37°C to 20°C (cooling) produced an increase in tissue sensitivity to noradrenaline, as reflected in a 5.37-fold leftward shift in the concentration-response curve, and increased the maximum contractile response to this agonist (14.3%). Cooling had no effect on tissue sensitivity to a selective ₁-adrenoceptor agonist, methoxamine, but increased (12.4%) the maximum contraction to a similar extent to that to noradrenaline. 6-Hydroxydopamine pretreatment or nortriptyline (1 mol/l) induced a leftward shift of the noradrenaline concentration-response curve at 37°C, and profoundly inhibited the potentiating effect of cooling on tissue sensitivity to the catecholamine; the effect of cooling on the maximum response was unaffected. The affinity of noradrenaline or methoxamine for ₁-adrenoceptors at 37°C, determined from its dissociation constant (K _A), was not significantly different from that at 20°C. K _A values were determined by use of irreversible antagonism with phenoxybenzamine. On the other hand, diltiazem at concentration of 3 mol/1, which almost completely abolished the calcium ion-induced contraction of the potassium ion-depolarized muscle, caused only slight inhibition in the concentration-response curve for noradrenaline. The contractile responses to Ca²⁺ of the K⁺-depolarized muscle and of the tissue incubated in Ca²⁺-free (EGTA 0.1 mmol/1) Krebs solution containing diltiazem and noradrenaline were both depressed by cooling. However, the contractile response of the anococcygeus muscle caused by noradrenaline or methoxamine in Ca²⁺-free Krebs solution was augmented at 20°C. At 37°C, vanadate (1 mol/1) also augmented the methoxamine-induced contraction of the tissue incubated in Ca²⁺-free solution. This potentiating effect of vanadate was not observed at 20°C. From these observations, it is concluded that increased responsiveness of the rat anococcygeus muscle to noradrenaline with lowered temperature may involve the inhibition of neuronal uptake mechanism of the catecholamine and the inhibition of removal mechanism of Ca²⁺ which is released from intracellular stores associated with the postsynaptic ₁-adrenoceptors. Send offprint requests to T. Ishii at the above address     

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

It has been suggested that at least the majority of mammalian presynaptic α₂-autoreceptors belong to the genetic α_2A/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 α_2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as α_2B or similar to, but not identical with, α_2D, and in the human kidney, where they were classified as α_2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be α_2A, in agreement with the rule, but in contrast to indications that the α_2A/D-adrenoceptor of the guinea pig possesses α_2D pharmacological properties. Tissue pieces were preincubated with ³H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The K _d 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. EC ₅₀ 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 K _d values thus obtained with K _d values at known α₂ subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were α_2D and those in the human kidney α_2A. For example, the pK _d values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pK _d values at the prototypic α_2D 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 pK _d values at the α_2D-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 pK _d values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pK _d values at α_2A, α_2B and α_2C binding sites and α_2A-autoreceptors. On the other hand, the pK _d values of the antagonists in the human kidney were closely correlated with pK _d values at the prototypic α_2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pK _d values at the α_2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pK _d values at α_2B, α_2C and α_2D binding sites and α_2D-autoreceptors. 2D , those in the human kidney α_2A, and those in the guinea-pig urethra equally α_2D. All, therefore, conform to the rule that α₂-autoreceptors belong at least predominantly to the genetic α_2A/D subtype of the α₂-adrenoceptor. The apparent paradox of an α_2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic α_2A/D-adrenoceptor otherwise has α_2D pharmacological properties, is removed. Received: 30 May 1997 / Accepted: 1 September 1997     

Effect of Clonidine on Tyrosine Hydroxylase Activity in the Adrenal Medulla and Brain of Spontaneously Hypertensive Rats

Abstract: In the present study, we evaluated the effect of the α₂‐adrenoceptor agonist clonidine on tyrosine hydroxylase activity in adrenal medulla and brain of spontaneously hypertensive rats and Wistar Kyoto rats. Six‐week‐old animals were treated with clonidine (100 µg/kg body weight, daily, i.p.) for 4 weeks. Treatment with clonidine significantly reduced mean arterial blood pressure in spontaneously hypertensive rats to values similar to normotensive Wistar Kyoto rats. In the adrenal medulla of spontaneously hypertensive rats, clonidine treatment produced a significant increase in tyrosine hydroxylase activity with higher V_max values and no changes in K_M values. This effect was accompanied by a significant increase in tyrosine hydroxylase protein expression and of noradrenaline and adrenaline levels. In the brain of spontaneously hypertensive rats, treatment with clonidine produced a significant decrease in tyrosine hydroxylase activity with lower V_max values and no changes in K_M values accompanied by a significant decrease in tyrosine hydroxylase protein expression and of dopamine and noradrenaline levels. In Wistar Kyoto rats, clonidine treatment had no effect on tyrosine hydroxylase activity and protein expression or catecholamine levels in adrenal medulla or brain. Clonidine treatment significantly reduced noradrenaline and adrenaline plasma levels in spontaneously hypertensive rats and Wistar Kyoto rats. In conclusion, treatment with the α₂‐adrenoceptor agonist clonidine prevented the increase in mean arterial blood pressure in young spontaneously hypertensive rats. This effect was accompanied by opposite effects on tyrosine hydroxylase activity in spontaneously hypertensive rat adrenal medulla and brain: an increase in adrenal medulla and a decrease in brain, bringing sympathetic function to a similar profile found in normotensive Wistar Kyoto rats.     

The recovery of α-adrenoceptor function and binding sites after phenoxybenzamine

Summary The recovery of peripheral -adrenoceptor function and binding sites was studied in male New Zealand white rabbits after treatment with the irreversible adrenoceptor antagonist phenoxybenzamine. Phenoxybenzamine (5 mg/kg) was administered intravenously and the animals studied 30 min to 12 days later. Pressor dose response curves to intravenous phenylephrine, noradrenaline and guanabenz were constructed in vivo in conscious animals. The contractile response of abdominal aorta and renal artery to phenylephrine and noradrenaline was examined in vitro and the recovery of specific prazosin and clonidine binding to spleen membranes investigated in radioligand binding studies.The half life (t _1/2) for recovery of maximum pressor response in vivo ranged from 0.9±0.2 days for phenylephrine to 1.4±0.1 days for guanabenz. The t _1/2 for recovery of ED₅₀ was not significantly different to t _1/2 for recovery of maximum pressor response and ranged from 0.8±0.2 days for noradrenaline to 1.3±0.3 days for phenylephrine.Half life for recovery of maximum response and EC₅₀ in the isolated tissues was similar to that obtained in vivo for recovery of pressor responses and ranged from 0.4±0.1 days for the EC₅₀ of noradrenaline in the renal artery to 1.2±0.6 days for maximum response to phenylephrine in the abdominal aorta.The rate of recovery of specific clonidine binding did not differ significantly from the rate of recovery of pressor responses to the ₂-selective agonist guanabenz. t _1/2 for maximum number of specific clonidine binding sites, B _max was 1.6±0.9 days. However t _1/2 for recovery of specific prazosin binding was significantly longer than recovery of responses to phenylephrine and noradrenaline, t _1/2 for B _max was 3.6 ±0.1 day.     

Pilocarpine Treatments Differentially Affect α2-Adrenoceptors which Modulate the Firing Rate of Locus coeruleus Neurones and the Synthesis and Release of Noradrenaline in Rat Brain

Abstract: We have investigated the effect of treatments with the muscarinic acetylcholine receptor agonist, pilocarpine, on the sensitivity of central α₂-adrenoceptors that regulate the firing activity of rat locus coeruleus, the tyrosine hydroxylase activity in the rat cortex, hippocampus and hypothalamus, and the K⁺-evoked release of [³H]noradrenaline from rat cortical and hippocampal synaptosomes. Short-term (4 days), but not acute, treatment with pilocarpine caused a small but statistically significant increase in the inhibitory effect of the α₂-adrenoceptor agonist clonidine on the firing rate of locus coeruleus neurones, with a decrease in the ED₅₀ of 29% (P<0.001). However, no change in the effect of clonidine on the locus coeruleus was observed after longer pilocarpine (11 days) treatment. In the rat cerebral cortex, but not in hippocampus or hypothalamus, chronic (19 days) treatment with pilocarpine caused a decrease in the inhibitory effect of clonidine on tyrosine hydroxylase activity (55%, P<0.05), but did not change the stimulatory effect of the α₂-adrenoceptor antagonist idazoxan. Moreover, treatments (4, 11 and 19 days) with pilocarpine did not alter the inhibitory effect of clonidine [10^?8-10^?5 M] on the K⁺-evoked release of [³H]noradrenaline from rat cortical and hippocampal synaptosomes. These results indicate that administration of pilocarpine slightly potentiates some but not all the functional responses mediated by brain presynaptic α₂-adrenoceptors. In conclusion, these results do not support the hypothesis that chronic treatments with pilocarpine lead to a suitable model of α₂-adrenoceptor supersensitivity.     

Cisapride and a structural analogue,R 76186, are 5-hydroxytryptamine4 (5-HT4) receptor agonists on the guinea-pig colon ascendens

Summary The purpose of this study was to investigate whether the effects of cisapride and its close structural analogue R 76186 on the isolated guinea-pig colon ascendens, are mediated through 5-HT₄ receptors.Both cisapride and R 76186 induced contractions in a concentration-dependent fashion, giving monophasic concentration-response curves (cisapride: EC₅₀ = 1.1 × 10^–7 M, maximum effect = 40.3% of methacholine induced (3 × 10^–7 M) contractions; R 76186: EC_5o = 2.4 × 10^–8 M, maximum effect = 52.1%). Blockade of either 5-HT₂ or 5-HT₃ receptors did not affect the responses to cisapride. However, tropisetron (in 5-HT₄ receptor-blocking concentrations), and DAU 6285 and SDZ 205–557, two novel selective 5-HT₄ receptor antagonists, depressed the concentration-response curve to cisapride (to about 50%), and the curve to R 76186 was shifted to the right. The apparent pA₂ values were 6.6 (tropisetron), 6.3 (DAU 6285), and 7.5 (SDZ 205-557). However, none of these antagonisms was purely competitive as higher concentrations of these antagonists depressed the curve to R76186. Desensitization of the 5-HT₄ receptor with 5-methoxytryptamine (5-MeOT) inhibited the responses to cisapride, and abolished those to R 76186. The contractions to cisapride and R76186 were sensitive to mutual antagonism, depressing their concentration-response curves.Conclusions: Both cisapride and R 76186 mediate their contractile effects in the guinea-pig colon ascendens through agonism at the 5-HT₄ receptor, though cisapride also uses a non-5-HT mechanism. R 76 186 is a selective and potent 5-HT₄ receptor agonist. Correspondence to M. R. Briejer at the above address in Belgium     

Muscarinic inhibition of endogenous noradrenaline release from rabbit isolated trachea: receptor subtype and receptor reserve

The aim of the present study was to characterize putative muscarine receptors on sympathetic nerve terminals in the rabbit trachea. Release of endogenous noradrenaline from in vitro incubated rabbit tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) and quantified by high performance liquid chromatography with electrochemical detection.The muscarine receptor agonist oxotremorine inhibited the evoked release of noradrenaline completely at 1 ol/l (EC₅₀: 64 nmol/l). The concentration response curve was very steep (Hill coefficient of 2.3). Scopolamine shifted the concentration response curve of oxotremorine to the right (–log K_B 8.48) demonstrating specific, inhibitory muscarine receptors. Several subtype-preferring muscarine receptor antagonists also shifted the concentration response curve of oxotremorine to the right. The rank order of potency was (–log K_B or pA^* ₂): scopolamine (8.48) > AF DX 384 (7.88^*; slope of Schild plot 1.1) > (R)-trihexyphenidyl (7.87) > 4-DAMP (7.85) > AQ-RA 741 (7.77) methoctramine 6.18 > pirenzepine (6.0) >p-fluoro-hexahydrosiladifenidol (p-FHHSiD, 5.68). When these affinity constants were plotted against reported –log K_i values determined in binding studies on human cloned muscarine receptor subtypes (m₁-m₅), the best correlation was obtained for m₂. Indomethacin (3 mol/l), which on its own increased the evoked noradrenaline release by about 45%, affected neither the inhibitory effect of oxotremorine nor the antagonistic potency of methoctramine or p-FHHSiD. After preincubation for 48 min with 300 mol/l phenoxybenzamine, which has been shown to inactivate muscarine receptors irreversibly, the concentration response curve of oxotremorine was shifted 5.2 fold to the right and the maximal inhibition was reduced by 50%, whereas the slope remained steep (Hill coefficient 2.6). These experiments indicated that a fraction of about 22% of the muscarine receptors has to be occupied by oxotremorine to produce half-maximum inhibition of noradrenaline release; the dissociation constant of oxotremorine at the prejunctional muscarine receptors was 0.33 mol/l.In conclusion, the sympathetic nerve terminals in the rabbit trachea are endowed with inhibitory M₂-like muscarine receptors for which methoctramine displayed a low affinity. Since a large receptor reserve could be excluded, the steep concentration response curve of oxotremorine suggests that activation of muscarine receptors has to reach a threshold level before the onset of an inhibitory effect. Correspondence to: K. Racké at the above address