Allosteric properties of 5-HT2 receptors in tracheal smooth muscle

Summary 5-Hydroxytryptamine (5-HT)-induced contractions were investigated in isolated tracheal smooth muscle of guinea pig and calf. In guinea-pig tracheae, ketanserin reduced to 60% the maximum response to 5-HT, but also shifted the concentration-effect curve for 5-HT to higher 5-HT concentrations, as expected from its affinity for 5-HT₂ receptors [pK _B=–log K _B=9.6, K _B in mol/l]. The concentration effect-data for the depressant effect of ketanserin are closely associated with the curve for occupancy of 5-HT₂-receptors by ketanserin. In calf tracheae, ketanserin caused surmountable antagonism of the effects of 5-HT with a pK _B of 9.5. Methysergide reduced to 25% the maximum response to 5-HT in guinea-pig tracheae and to 20% in calf tracheae. The methysergide-depressed response to 5-HT was restored by ketanserin to 60% of maximum in guinea-pig tracheae, and to 100% in calf tracheae. The results support for tracheal smooth muscle a model of an allosteric regulation of 5-HT₂-receptors which was proposed for arterial smooth muscle by Kaumann and Frenken (this journal 328:295–300, 1985). The model requires that: 1) the 5-HT₂ receptor exists in two interconvertible states RR;2) 5-HT causes its effects through R; 3) methysergide, by acting on an allosteric site near or on the 5-HT₂ receptor, shifts the equilibrium into the inactive state R;4) ketanserin competes with 5-HT for the 5-HT₂ receptor and with methysergide for the allosteric site, thereby restoring the active state R of the 5-HT₂-receptor. All four requirements were experimentally verified in calf trachea. In guinea-pig trachea ketanscrin also appears to possess a partial ability to shift the 5-HT₂ receptor into the inactive R state. Thus, ketanserin is both a competitive antagonist at the 5-HT₂ receptor and also appears to be an allosteric modulator at the allosteric site.     

β 1- andβ 2-adrenoceptor binding and functional response in right and left atria of rat heart

Summary The properties of ₁- and ₂-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses to-adrenoceptor agonists were examined. [¹²⁵I](-)-pindolol (¹²⁵IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek ₁'s for association in right and left atria were 6.5×10⁹ l/mol-min and 2.3×10⁹ l/mol-min respectively, while thek _–1's for dissociation were 0.20 min^–1 and 0.17 min^–1. The kinetically determinedK _D's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumK _D's determined from Scatchard analysis of saturation isotherms of specific¹²⁵IPIN binding. Inhibition of¹²⁵IPIN binding by-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition by ₁- and ₂-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement by ₁-selective (practolol, atenolol and metoprolol) and ₂-selective (ICI 118,551) antagonists gave estimates of the proportion of ₁- and ₂-adrenoceptors present in rat atria. Right atria contained 67±4.2% ₂-adrenoceptors and 33±4.2% ₂-adrenoceptor, while left atria contained 67±2.8% ₁- and 33±2.8% ₂-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused by-adrenoceptor agonists were also measured. pA₂ values for non-subtype selective-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withK _D values determined for specific¹²⁵IPIN binding. pA₂ values for ₁- and ₂-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pK _D values of these drugs in binding to ₁-adrenoceptors, but not with the pK _D values in binding to ₂-adrenoceptors. Dose-response curves for stimulation of both rate and force by the ₂-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade of ₁-adrenoceptors with metoprolol than by selective blockade of ₂-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activating ₁-adrenoceptors. These results suggest that ₁- and ₂-adrenoceptors coexist in both left and right atria of rat heart in approximately a 21 ratio, however only ₁-adrenoceptors mediate the chronotropic and inotropic effects of-adrenoceptor agonists.Supported by a grant from the American Heart Association — Georgia Affiliate     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in guinea-pig atria and brain

The study was devised to classify, by means of antagonist and agonist affinities, the presynaptic ₂-autoreceptors in guinea-pig heart atria and brain cortex in terms of _2A, _2B, _2C and _2D. A set of antagonists and agonists was chosen that was able to discriminate between the four subtypes. Small pieces of the atria and slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.In one series of experiments (atria only), tissue pieces were stimulated by relatively long pulse trains (1 min) leading to marked ₂-autoinhibition. All 10 antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments (atria and brain slices), tissue pieces were stimulated by brief pulse trains (0.4 s or 40 ms) that led to little (atria) or no (brain slices) ₂-autoinhibition, and antagonist effects against the ₂-selective agonist 5-bromo-6(2-imidazolin-2-ylamino)-quinoxaline UK 14,304 were examined. All 10 (atria) or 8 (brain) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. In a third series of experiments (brain slices only), also with brief pulse trains (40 ms), pK_a values (negative logarithms of dissociation constants of agonist-₂-adrenoceptor complexes) were determined by comparison of concentration-inhibition curves of UK 14,304, guanoxabenz and oxymetazoline in normal tissue and in tissue in which a fraction of the receptors had been blocked by phenoxybenzamine. pEC_30% values in atria correlated with pK_d values (r = 0.942). pK_d values in atria correlated with pK_d values in the brain cortex (r = 0.970).It is concluded that the ₂-autoreceptors in atria and the brain cortex are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature - indicates that both autoreceptors are _2D. In atria, this identification is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d, and in the brain cortex it is supported by the agonist pK_a values. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2AD branch of the ₂-adrenoceptor tree.     

Differentiation of cardiac chronotropic and inotropic effects of β-adrenoceptor agonists

Summary The relative inotropic and chronotropic activity of -adrenoceptor agonists was studied in the noradrenaline-depleted, anaesthetized cat. Terbutaline, a selective ₂-adrenoceptor agonist, gave at a certain dose a more pronounced chronotropic than inotropic response, while a new ₁-selective adrenoceptor agonist (–)-H 80/62 produced the same degree of chronotropic and inotropic stimulation. The results indicate that there is some difference between the -adrenoceptors in the sinus node mediating chronotropic stimulation and -adrenoceptors in the ventricular myocardium mediating stimulation of the contractile force. It has been shown that there are both ₁- and ₂-adrenoceptors in the heart (Carlsson et al., 1972). In the light of this finding it is hypothetized that there are differences in the relative distribution of ₁- and ₂-adrenoceptors in the sinus node and in the myocardium. Although ₁ is the predominant type of -adrenoceptor in both regions, the ₁: ₂ concentration ratio seems to be higher in the myo-cardium, than in the sinus node.     

Subclassification of presynaptic α2-adrenoceptors: α2A-autoreceptors in rabbit atria and kidney

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors of rabbit atria and kidney in terms of _2A, _2B, _2C and _2D-A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with ³H-Noadrenaline and then superfused and stimulated electrically.In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to ₂-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to ₂-autoinhibition, and concentration-inhibition curves of the ₂-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pEC_30% values correlated with pK_d values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC_30% values in atria correlated with pEC_30% values in the kidney (r = 0.988) and pK_d values in atria correlated with pK_d values in kidney (r = 0.923).It is concluded that the ₂-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both autoreceptors are _2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.     

Modulation of L-type Ca current by denopamine, a nonparenteral partial β1 stimulant, in rabbit ventricular cells

The effects of denopamine, a nonparenteral partial agonist which is used clinically in Japan, on the L-type Ca²⁺ current (I _Ca) were examined in rabbit ventricular cells. Denopamine stimulated basal I _Ca with a maximum response of +33.2% and a concentration for half-maximal response (EC₅₀) of 0.039 M. The maximun response of I _Ca was only a quarter of that induced by isoprenaline (ISO), while 10 M denopamine elicited 70–75% of the maximum inotropic response in the papillary muscle preparations. The denopamine stimulation of I _Ca was abolished by selective ₁ antagonists (atenolol or bisoprolol). Pretreatment with forskolin or dialysis with cAMP also abolished the stimulation. Denopamine, in turn, inhibited ISO-stimulated I _Ca. This inhibition was not affected by pretreatment with pertussis toxin or prazosin. The presence of denopamine at various concentrations caused a rightward shift in the concentration/response curve for ISO stimulation of I _Ca. The Schild plot for this effect had a slope of 0.99 and K _p of 0.20 M. In the presence of guanosine-5-O-(3-thiotriphosphate) (GTPS) (0.5 mM) in the pipette, denopamine (10 M) stimulated the I _Ca to 86±5% of the maximum response induced by ISO. These findings indicate that denopamine modulates I _Ca exclusively through the ₁ adrenoceptor-adenylate cyclase pathway, that the stimulatory GTP-binding protein regulates the agonistic potency of denopamine, and that the signal from the ₁ adrenoceptors is amplified between I _Ca and the tension development, which would contribute to the spare capacity of adrenoceptors.     

Regional distribution of β1- and β2-adrenoceptors in the failing and nonfailing human heart

Summary Total -adrenoceptor density and ₁- and ₂-subtype distribution in right and left atria and in different ventricular regions from 14 failing and seven nonfailing human hearts have been compared. End-stage heart failure was due to idiopathic dilated cardiomyopathy (n=8) or ischaemic cardiomyopathy (n=6).In nonfailing hearts the total -adrenoceptor density was similar in the right and left atria and in all the ventricular regions studied (about 70 to 80 fmol/mg protein). The ₁:₂-adrenoceptor ratio in both nonfailing atria was similar (about 70:30%) and was significantly smaller than in the different regions of both ventricles (about 80:20%). The ₁-subtype density was similar in nonfailing atria and ventricles (about 55 fmol/mg protein). The ₂-subtype density was significantly higher in the right and left atrium (about 25 fmol/mg protein) than in both ventricles (about 15 fmol/mg protein).In patients with end-stage heart failure due to idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy the total -adrenoceptor density was reduced by 50–60% in all regions. On the other hand, the ₁- and ₂-subtype distribution differed with the cause of heart failure. In patients with idiopathic dilated cardiomyopathy, the ₁-adrenoceptor density was lower in all regions, but the ₂-adrenoceptor density was not significantly reduced. In patients with ischaemic cardiomyopathy both ₁- and ₂-adrenoceptors were reduced in all regions.It is concluded that downregulation of -adrenoceptors in patients with end-stage idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy occurs uniformly throughout the heart. The results support the hypothesis that changes in -adrenoceptor subtypes may be related to the cause of heart failure.     

SDZ ENS 163 is a selective M1 agonist and induces release of acetylcholine

Summary In the present study some pharmacological properties of the new muscarinic agonist SDZ ENS 163; (+)-(3S,cis)-3-ethyldihydro-4-[(1-methyl-1H-imidazol-5-yl)methyl-2(3H)-thiophenonedihydrogenphosphate] have been investigated. In the rat superior cervical ganglion, a model for M₁ muscarinic receptors, SDZ ENS 163 induced concentration-dependent depolarizations (pD₂ = 6.5 ± 0.3; efficacy = 128 ± 4.2% compared to carbachol). SDZ ENS 163 was a very weak partial agonist with respect to M₂ receptor-induced decrease in contractile force in rat left atria (efficacy = 14 ± 2.9%). In addition, SDZ ENS 163 competitively antagonized the effect of carbachol in rat left atria (pA₂ = 5.8 ± 0.2). In the guinea-pig ileum SDZ ENS 163 was a partial agonist with respect to force of contraction mediated by M₃ receptors (pD₂ = 5.3 ± 0.1; efficacy = 72 ± 4.2%). The oxotremorine-induced inhibition of the electrically stimulated release of acetylcholine (ACh) in rat hippocampal slices was reversed by SDZ ENS 163 (pA₂ = 5.5 ± 0.1). In addition after oral administration SDZ ENS 163 (3 – 10 mol/kg) reduced brain ACh levels, which is indicative of increased ACh turnover. Finally, increases in energy of the low frequency band (2–5 Hz) were observed in rat hippocampal EEG after intraperitoneal administration of SDZ ENS 163 (0.3 – 30 mol/kg). We conclude that SDZ ENS 163 is a selective M₁ agonist in vitro with an additional M₂ antagonistic effect. The in vivo effects of SDZ ENS 163 may result both from postsynaptic M₁ agonistic as well as M₂ receptor antagonistic activity. The unique pharmacological profile of SDZ ENS 163 may prove clinically favourable for treatment of cognitive deficits. Send offprint requests to H. W. G. M. Boddeke at the above address     

DPI 201-106, a novel cardioactive agent. Combination of cAMP-independent positive inotropic,negative chronotropic,action potential prolonging and coronary dilatory properties

Summary The in vitro cardiac effects of DPI 201-106, a novel piperazinyl-indole, were investigated.DPI 201-106 produced concentration-dependent positive inotropic effects in guinea-pig and rat left atria, kitten, rabbit and guinea-pig papillary muscles and Langendorff perfused hearts of rabbits between 10^–7 and 3×10^–6 mol/l. During isometric twitches, contraction and relaxation phases were prolonged in guinea-pig left atria and right ventricular papillary muscles from kitten and guinea-pigs. Spontaneous sinus rate was decreased in right atria of guinea-pigs and rats. Coronary flow increased in rabbit isolated hearts. Functional refractory period was increased in left atria from guinea-pigs and rats with EC₅₀ values of 1.7 and 0.24 mol/l respectively.In electrophysiological measurements, DPI 201-106 prolonged the action potential duration (APD₇₀) in guinea-pig papillary muscles up to 70% and in rabbit atria up to 120% at 3 mol/l. Other action potential characteristics were not changed in guinea-pig papillary muscles but V _max was decreased in rabbit left atria. The electrophysiological as well as the positive inotropic effects were stereoselective with the activity residing in the S-enantiomer.DPI 201-106 increased the Ca²⁺-sensitivity of skinned fibres from porcine trabecular septomarginalis with an EC₅₀ of 0.2 nmol/l. DPI 201-106 did not change cAMP levels in guinea-pig atria and rabbit papillary muscles. Slow action potentials were not induced by DPI 201-106 in partially depolarized guinea-pig papillary muscles. Phosphodiesterase activity of rat hearts was not inhibited by DPI 201-106 at pharmacologically relevant concentrations. The presence of propranolol did not influence the inotropic potency of DPI 201-106 in guinea-pig atria.In conclusion, DPI 201-106 represents a novel type of positive inotropic agents with a synergistic sarcolemmal and intracellular mechanism of action.     

Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC₅₀ values of 5.4±0.7 M and 6.2±0.4 M respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC₅₀ of 1.1±0.2 M. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation (_inact), and the integral current was inhibited with an IC₅₀ of 3.4±0.5 M. At 10 M AVE0118 _inact decreased from 9.3±0.6 ms (n=8, control) to 3.0±0.3 ms (n=8). The K_ACh current was investigated in isolated pig atrial myocytes by application of 10 M carbachol. At a clamp potential of –100 mV the I_KACh was half-maximally blocked by 4.5±1.6 M AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at –100 mV. Effects on the I_Kr current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 M. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I_Kr tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I_Ks, I_KATP (recorded in guinea pig ventricular myocytes), and L-type Ca²⁺ (recorded in pig atrial myocytes) were blocked by 10 M AVE0118 by 10±3% (n=6), 28±7% (n=4), and 22±13% (n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K⁺ channels I_Kur, I_to and I_KACH. This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.     

P2-purinoceptor antagonists: III. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to suramin

Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings.The compounds all shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 1 and 3672 M. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADPS to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values were between 10 and 786 M. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC_25% values between 170 and 590 M.The results indicate that P_2X-purinoceptor affinity, P_2Y-purinoceptor affinity and the ecto-nucleotidase effect all increase with the size of the molecule. BSt101 resembled NF023 in potency at all three sites, indicating that the possession of a second naphthalene-trisulphonate group is not a prerequisite for relatively high affinity. NF023 is interesting because it is P_2X- versus P_2Y-selective and, in addition, the compound with the highest P_2X- versus ecto-nucleotidase-selectivity presently available.     

5-Hydroxytryptamine receptors with a 5-HT6 receptor-like profile stimulating adenylyl cyclase activity in pig caudate membranes

This study deals with the characterization of 5-hydroxytryptamine (5-HT, serotonin) receptors positively linked to adenylyl cyclase in membranes from pig brain caudate. 5-HT and related agonists induced a concentration-dependent stimulation of adenylyl cyclase activity in pig caudate membranes, with the following rank order of potency (mean pEC₅₀ values): 5-HT (7.1) 5-methoxytryptamine (6.9) > 5-carboxamidotryptamine (5.6) > sumatriptan (<5). Maximal stimulation by 5-HT averaged 35 pmol cyclic AMP/min/mg protein over a basal activity of 159 pmol cyclic AMP/min/mg protein. 5-Methoxytryptamine and 5-carboxamidotryptamine had similar efficacies to that of 5-HT, whereas sumatriptan was about half efficacious. Other compounds known as agonists at some 5-HT receptors were weakly potent (mean pEC₅₀ values <5). They include the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT₄ receptor agonist, renzapride and the 5-HT₂ receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane) (DOI). In antagonist studies, methiothepin (0.1 and 1 mol/l) shifted the 5-HT curve to the right with no depression of the E_max, yielding pK_B values of 7.4–8.0. Clozapine (1 mol/l) also produced surmountable antagonism of 5-HT-induced effects (pK_B 6.9). Ketanserin (10 mol/l) weakly antagonized 5-HT (pK_B 5.0). The 5-HT₄ receptor antagonists, tropisetron (ICS 205–930) and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), each at 1 mol/l, did not significantly alter the concentration-response curve of 5-HT. The present receptor shares some characteristics of the recently cloned 5-HT₆ receptor (Monsma et al. (1993) Mol Pharmacol 43:320–327): similar pharmacological profile, location (striatum) and ability to stimulate adenylyl cyclase. It may thus represent the functional 5-HT₆ receptor in its natural environment. Correspondence to: P. Schoeffter at the above address     

Cross-talk between β<Subscript>1</Subscript>-adrenoceptors and ET<Subscript>A</Subscript> receptors in modulation of the slow component of delayed rectifier K<Superscript>+</Superscript> currents

Delayed rectifier K⁺ currents (I_K) play a critical role in determining cardiac action potential duration (APD). Modulation of I_K affects cardiac excitability critically. There are three components of cardiac delayed rectifier, and the slowly activating component (I_Ks) is influenced strongly by a variety of stimuli. Plasma levels of noradrenaline and endothelin are elevated in heart failure, and arrhythmias are promoted by such humoral abnormalities through modulation of ion channels. It has been reported that protein kinase A (PKA) and protein kinase C (PKC) modulate I_Ks from human minK in a complex manner. In the present study, we coexpressed human minK with the human ₁-adrenoceptor (h₁AR) and the endothelin receptor subtype A (hET_AR) in Xenopus oocytes and investigated the effects of receptor activation on the currents (I_Ks) flowing through the oocytes. ET-1 modulated I_Ks biphasically: a transient increase followed by a decrease. The PKC inhibitor chelerythrine completely inhibited the effects of ET-1. Intracellular EGTA abolished the transient increase by ET-1 and partially inhibited the subsequent decrease in the currents. When I_Ks was increased by 10^–6 M isoproterenol (ISO), ET-1 did not increase but rather decreased the current to an even greater extent than under control conditions. In addition, the effects of ISO on I_Ks were suppressed by ET_AR stimulation. These data indicate that I_Ks can be regulated by cross-talk between the ET_AR and ₁AR systems in addition to direct regulation by each receptor system.     

Interaction of enantiomers of hydroxy tolazoline with adrenoceptors

Summary Adrenoceptor-mediated effects of the enantiomers of hydroxytolazoline and tolazoline (i. e., desoxy derivative) have been investigated in vitro. The enantiomers and tolazoline were partial agonists of postjunctional ₁-adrenoceptors in rat aorta. The rank order of potencies of the compounds in this system was as follows: tolazoline > R(–)-hydroxytolazoline > S(+)-hydroxytolazoline. The efficacy of R(–)-hydroxytolazoline was higher than that of tolazoline, though its affinity for the receptor was less. The K _B values for prazosin against these agonists were nearly equal, which indicated that these imidazolines activate the same type of receptor in rat aorta. The S(+)-isomer, however, produced both a prazosin sensitive and resistant component of the response. The interactions of the derivatives with presynaptic ₂-adrenoceptors were studied in field-stimulated myenteric plexus-longitudinal muscle of guinea-pig ileum. These substances were blockers at presynaptic ₂-adrenoceptors. Based on K _B values, the order of affinity in this system was as follows: tolazoline > S(+)isomer R(–)-isomer. -Adrenoceptor mediated activity was quantitated in guinea-pig and rat atria. R(–)-hydroxytolazoline lacked chronotropic effects either in guinea pig or rat atria. At 3 × 10^–4 M the isomer did not antagonize the effect of isoproterenol in the atria. On the other hand, S(+)-hydroxytolazoline produced a variable chronotropic effect in guinea-pig atria, but failled to show any significant activity in rat atria. Thus, the -adrenoceptor mediated action appears to be insignificant. Steric aspects of -adrenoceptor mediated events are discussed.This investigation was, in part, supported by a grant from the United States Public Service, National Institutes of Health, GM 29358 Send offprint requests to P. N. Patil at the above address     

P2-purinoceptor antagonists: II. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to Evans blue and trypan blue

Effects of Evans blue and four derivatives as well as of trypan blue and four derivatives, mostly smaller fragments but two compounds with an additional ethylene bridge in the center of the molecule, were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.All compounds shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right, and most compounds increased the maximum of the curve. Each member of the Evans blue series was similar in potency to the corresponding member of the trypan blue series. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 0.8 and 385 M. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADPS to the right in a surmountable manner. In 2 of 3 cases where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values in the trypan blue group were between 5.2 and 324 M. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC_25% values between 13 and 158 (in 1 case >1000) M.The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P_2X purinoceptor affinity but greatly influences P_2Y affinity and ecto-nucleotidase blockade. Among active compounds, apparent purinoceptor affinity and ecto-nucleotidase blockade increase with the size of the molecules up to Evans blue and trypan blue themselves; introduction of a central ethylene bridge does not result in a further gain in potency. NH01, the desmethyl derivative of Evans blue, seems to be interesting because it is the compound with the highest P_2X- versus P_2Y-selectivity presently available.     

Presynaptic α2-autoreceptors in mouse heart atria: evidence for the α2D subtype

Presynaptic ₂-autoreceptors in mouse atria were characterized in terms of the _2A, _2B, _2C and _2D subtypes. Segments of the atria were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. The affinity of up to 16 antagonists for the autoreceptors was assessed as (1) pEC_30% values, i.e. concentrations that increased previously autoinhibited release of ³H-noradrenaline (120 pulses, 3 Hz) by 30%, and (2) pK_d values against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under conditions of no or little autoinhibition (2 trains of 20 pulses, 50 Hz, train interval 120 s).The pK_d values correlated well with the pEC_30% values (r = 0.98; P<0.001; slope of regression line 0.93), indicating that UK 14,304 and released noradrenaline modulated the release of noradrenaline through pharmacologically identical receptors. Comparison with antagonist affinities for (1) prototypic native ₂ radioligand binding sites, (2) radioligand binding sites in COS cells transfected with ₂ subtype genes, and (3) previously classified presynaptic ₂-autoreceptors — all taken from the literature — indicated that the mouse atrial autoreceptors corresponded to the _2D subtype. For example, the pK_d values at mouse atrial autoreceptors correlated closely with pK_d values at native _2D binding sites in the bovine pineal gland (r = 0.96; P<0.001); with pK_d values at _2D binding sites in COS cells transfected with the rat _2D gene (r= 0.85; P<0.01); and with pK_d values at guinea-pig cerebral and atrial and mouse cerebral _2D-autoreceptors (r=0.96–0.98; P<0.001). The antagonist pK_d values at mouse atrial autoreceptors correlated less with pK_d values at _2A, _2B, and _2C sites.It is concluded that the presynaptic ₂-autoreceptors in mouse atria are _2D This identification supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D pair of orthologous ₂-adrenoceptors.     

Cardiac postjunctional supersensitivity to β-agonists after chronic chemical sympathectomy with 6-hydroxydopamine

Summary The sensitivity to sympathomimetic amines of isolated atria removed from sham-injected and 6-hydroxydopamine-treated (6-OHDA) guinea-pigs was examined in the presence of an extraneuronal uptake blocker and an -adrenoceptor antagonist. Three weeks of pretreatment with 6-OHDA resulted in leftwards shifts of the dose-response curves for the positive chronotropic and inotropic responses of right and left atria to isoprenaline. The responses to the partial agonist salbutamol were also potentiated after 6-OHDA pretreatment, revealed as an increase in the maximum response relative to isoprenaline.The supersensitivity was post-synaptic in origin and independent of changes in disposition or metabolism, since it was observed with agonists immune to neuronal uptake and O-methylation, and in the presence of extraneuronal uptake inhibition by metanephrine. It was also specific for the -adrenoceptor, no supersensitivity to histamine being found. In the right atria, the supersensitivity was partially masked by an opposing depressant effect after 6-OHDA pretreatment which was observed with histamine.Dissociation constants (K _A) for the left atrial inotropic responses to orciprenaline were determined by use of the antagonist Ro 03-7894. Atria from 6-OHDA-pretreated animals were supersensitive to orciprenaline, but the K _A value did not differ from that after sham injection. It could therefore be concluded that the increase in sensitivity was not due to an increase in affinity for the -adrenoceptor.     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

β-Adrenoceptor regulation and functional responses in the guinea-pig following chronic administration of the long-acting β2-adrenoceptor agonist formoterol

Formoterol is a long acting ₂-adrenoceptor agonist designed for the alleviation of the symptoms of asthma. This study examined the effects of 14 day administration of formoterol (200 g/kg/day i.p.) on ₁- and ₂-adrenoceptors in guinea-pig cardiac and lung tissue. Quantitative autoradiography was used to measure changes in receptor density and organ bath studies determined alterations in functional response.Formoterol treatment produced marked reductions of between 43% and 77% in ₂-adrenoceptor density in all regions of the heart (atrioventricular node, bundle of His, right and left bundle branches, interventricular and interatrial septa, right and left atria, ventricles and apex) and lung (bronchial and vascular smooth muscle and parenchyma) (P < 0.01, n = 6). ₁-Adrenoceptor density remained unchanged in all cardiac and lung regions. In functional studies (–)-isoprenaline was 4 fold less potent at causing relaxation of carbachol (1 M) precontracted tracheal smooth muscle (pD₂: control 8.49 ± 0.03, formoterol 7.91 ± 0.10, P < 0.001, n = 4), but formoterol treatment did not change the ability of (–)-isoprenaline to elicit a maximum response. The pK_B values for ICI 118,551, 7.33 ± 0.08 in the control and 7.20 ± 0.01 in formoterol treated animals, were between those expected for ₁- and ₂-adrenoceptors suggesting involvement of both subtypes in the response. In spontaneously beating right atria and electrically paced left atria, tissues in which responses are largely mediated by ₁-adrenoceptors, there was no significant change in responses to (–)isoprenaline (right atria pD₂: control 8.45 ± 0.02; formoterol 8.42 ± 0.11; P = 0.77, n = 4) (left atria pD₂: control 8.25 ± 0.03; formoterol 8.47 ± 0.08; P = 0.09, n = 4). In the presence of CGP 20712A (100 nM) the pK_B values did not change with formoterol treatment (left atria: control 9.59 ± 0.12, formoterol 9.66 ± 0.12; P = 0.70, n = 4) (right atria: control 8.93 ± 0.11, formoterol 9.11 ± 0.07; P = 0.25, n = 4).The doses and route of administration of formoterol used in this study differs from those used clinically. However, this study demonstrates that chrome formoterol administration produces selective down-regulation of ₂-adrenoceptors in the lung and heart. The changes in the lung are accompanied by a shift to the right in the concentration-response curve to -agonist stimulation with no change in the maximum response.     

Application of a Combined “Effect Compartment/Indirect Response Model” to the Central Nervous System Effects of Tiagabine in the Rat

Pharmacological inhibition of GABA uptake transporters provides a mechanism for increasing GABAergic transmission, which may be useful in the treatment of various neurological disorders. The purpose of our investigations was to develop an integrated pharmacokinetic–pharmacodynamic (PK/PD) model for the characterization of the pharmacological effect of tiagabine, R-N-(4,4-di-(3-methylthien-2-yl)but-3-enyl)nipecotic acid, in individual rats in vivo. The tiagabine-induced increase in the amplitude of the EEG 11.5–30 Hz frequency band (), was used as pharmacodynamic endpoint. Chronically instrumented male Wistar rats were randomly allocated to four groups which received an infusion of 3, 10, or 30 mg kg ^–1 ml min ^-1 kg^–1, 1.50.1 L kg^–1 and 200.2 min.A time delay was observed between the occurrence of maximum plasma drug concentrations and maximal response. A physiological PK/PD model has been used to account for this time delay, in which a biophase was postulated to account for tiagabine available to the GABA uptake carriers in the synaptic cleft and the increase in EEG effect was considered an indirect response due to inhibition of GABA uptake carriers. The population values for the pharmacodynamic parameters characterizing the delay in pharmacological response relative to plasma concentrations were k_eo=0.030 min ^–1 and k_out=81 min^–1, respectively. Because of the large difference in these values the PK/PD model was simplified to the effect compartment model. Population estimates were E₀=155 6 V, E_max=100 5 V, EC₅₀=287 7 ng ml^–1, Hill factor=1.8 0.2 and k_eo=0.030 0.002 min ^–1. The results of this analysis show that for tiagabine the combined effect compartment-indirect response model can be simplified to the classical effect compartment model.