Contractile actions of imidazoline alpha-adrenoceptor agonists and effects of noncompetitive alpha1-adrenoceptor antagonists in human vas deferens.

The contractile actions of imidazoline alpha-adrenoceptor agonists were investigated in human vas deferens longitudinal and circular muscle. The effects of phenoxybenzamine were studied in comparison to dibenamine and SZL-49 (4-amino-6,7-dimethoxy-2-quinazolinyl-4-(2-bicyclo[2,2,2]octa-2,5-dienylcarbonyl-2-piperazine), an alkylating prazosin analogue that discriminates between alpha(1H)- and alpha(1L)-adrenoceptor subtypes. The imidazoline alpha-adrenoceptor agonist, A-61603 (N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide), was a potent agonist (pD(2); longitudinal muscle 6.9, circular muscle 6.4) and cirazoline a partial agonist (pD(2); longitudinal muscle 6.1, circular muscle 5.1). Oxymetazoline was less effective, indanidine and clonidine were ineffective. SZL-49 produced a differential inhibition of contractions evoked by A-61603 in circular (alpha(1H)) compared to longitudinal (alpha(1L)) muscle and phenoxybenzamine had the opposite effect. Dibenamine inhibited the contractions comparably in both muscle types and analyses of its partial alkylation of receptors yielded identical estimates of equilibrium dissociation constant (pK(d)) for A-61603 in longitudinal (5.82) and circular (5.84) muscle. Receptor occupancy-response relationships revealed that whilst the muscle types are not different in receptor reserves for A-61603, contraction to the potent imidazoline is more efficiently coupled in longitudinal than in circular muscle. This underlies the markedly different responsiveness of the muscle types to cirazoline or oxymetazoline (alpha-adrenoceptor agonists with lower efficacies relative to A-61603). The differential inhibitory actions of phenoxybenzamine and SZL-49 are discussed.

Discrimination by SZL49 between contractions evoked by noradrenaline in longitudinal and circular muscle of human vas deferens.

The effects of irreversible alpha1-adrenoceptor antagonists, SZL-49 (an alkylating analogue of prazosin), dibenamine and benextramine on contractions to noradrenaline (NA) in longitudinal and circular muscle of human epididymal vas deferens were investigated. Competitive alpha1-adrenoceptor antagonists were also used to further characterize the alpha1-adrenoceptor subtype stimulated by NA in longitudinal and circular muscle. NA evoked concentration-dependent contractions of both muscle types (pD2; 5.4 and 5.2 respectively). The contraction of circular muscle was comparatively more sensitive than that of longitudinal muscle to pretreatment with SZL-49. In contrast, dibenamine or benextramine produced comparable effects in both muscle types. The relationship between receptor occupancy and contraction in either longitudinal or circular muscle was nonlinear, with half-maximal response requiring similar receptor occupancy (longitudinal muscle 14%, circular muscle 16%). Maximal response in both muscle types occurred with little or no receptor reserve (<10%). The competitive alpha1-adrenoceptor antagonists produced dextral shifts of the dose-response curves to NA in longitudinal and circular muscle. The inhibitory potencies, estimated from the apparent pKB values were significantly different in longitudinal and circular muscle respectively for either WB 4101 (pKB, 8.6 and 9.5) or RS-17053 (pKB, 7.1 and 9.0) but not for Rec 15/2739 (pKB, 9.2 and 9.8) or HV 723 (pKB, 8.3 and 8.4). In conclusion, the potency profile of the competitive alpha1-adrenoceptor antagonists and the lack of different receptor reserves for NA in the muscle types suggest that the discriminatory effects of SZL-49 is primarily due to a predominance of the alpha1L-adrenoceptor subtype in longitudinal muscle and alpha1A-subtype in circular muscle.

Mineralocorticoid receptor-mediated enhancement of neuronal excitability and synaptic plasticity in the dentate gyrus in vivo is dependent on the beta-adrenergic activity.

The dentate gyrus neurons in the hippocampus contain a high density of both mineralocorticoid and adrenergic receptors. By in vivo extracellular recording from adrenalectomized rats we investigated the possible relationships between the two systems with regard to neuronal excitability and activity-dependent synaptic plasticity. Pretreatment with aldosterone significantly enhanced both basal neuronal excitability and tetanically evoked synaptic plasticity in adrenalectomized, but not sham-operated rats. The enhancement was blocked by spironolactone, indicating a mineralocorticoid receptor-dependent effect. The adrenomedullary hormone epinephrine also significantly enhanced synaptic plasticity via activation of beta-adrenergic receptors. Beta-adrenergic antagonist propranolol, infused directly into the dentate gyrus granule cell layer, significantly reduced the effect of aldosterone on neuronal excitability and partly canceled the aldosterone-enhanced synaptic plasticity. No effect of propranolol was found after its amygdaloid infusion. The mineralocorticoid receptor antagonist spironolactone did not affect the epinephrine-induced effects. These results indicate that the pretreated adrenal steroids interact with the catecholaminergic system in the dentate gyrus of adrenalectomized rats and that the functional beta-adrenergic pathway is involved in the mechanism of mineralocorticoid-induced cellular effects in vivo.

Calculation of agonist efficacy, apparent affinity, and receptor population changes after administration of insurmountable antagonists: comparison of different analytical approaches.

The determination of the affinity and efficacy of an agonist in functional (as opposed to radioligand binding) experiments is necessary to explain its potency. Using modeled dose-response data both in their ideal form and with an added average deviation as well as previously published experimental data, a variety of analytical approaches were compared which differed in goodness-of-fit, ease of handling, and range of successful application. A nonlinear curve-fitting algorithm that analyzed several dose-response curves simultaneously and fitted them to an extended version of the operational model of Black and Leff (1983) (Proc R Soc Lond B 220:141-162) was demonstrated to be superior to the other approaches using the above criteria. However, judging from the limitations of the analytical approaches, claims of efficacy or affinity differences between agonists that are based on less than 10-fold numerical differences in the same behavioral paradigm should be viewed with skepticism. It was also found that simple inspection of dose-response curves obtained before and after administration of an insurmountable antagonist give estimates of fair accuracy under most circumstances.

Actions of galanin and some of its analogues on rat isolated gastric fundus.

The study was undertaken to characterize the effects of porcine galanin (pGal) and some of its analogues on rat gastric fundus muscle strips. pGal, galantide (M15) and pGal(1-14)-[Abu8]SCY-I evoked reproducible concentration-dependent contractions in concentrations of 1-300, 3-1,000 and 100-3,000 nM, respectively, with EC50 values of 13, 70 and 187 nM. Hill's coefficient for pGal is 1.03, indicating an interaction of one pGal molecule with one receptor, fulfilling criteria of classical receptor theory. For M15 and pGal(1-14)-[Abu8]SCY-I, Hill's coefficients are significantly different from 1, namely 0.73 and 1.56, so that one drug molecule may not interact with one receptor. The stimulatory effects of pGal were not modified by dibenamine 10 microM or glybenclamide 1 or 10 microM. Diltiazem 0.1, 1 and 10 microM, papaverine 0.1, 10 microM or dibutyryl cAMP (dib cAMP) 100 and 300 microM, blocked the contraction to pGal in a concentration-dependent manner, indicating an important role for the influx of extracellular calcium ions and regulation by cAMP the pGal-evoked contraction. Diltiazem, dibutyryl cAMP and papaverine were not competitive antagonists of pGal in the stomach smooth muscle.

Endothelium-dependent relaxation in response to acetylcholine in the human uterine artery.

The effect of acetylcholine on isolated human uterine artery rings was investigated. Acetylcholine induced concentration and endothelium-dependent relaxation (pD2 = 7.29 +/- 0.03) of the precontracted arterial segments. The dissociation constant (KA) for acetylcholine was 1.35 (0.92-1.77) mumol/l. The occupancy-response relationship was non-linear. Half-maximal response to acetylcholine was obtained with 5.25% receptor occupancy. Muscarinic receptor antagonists: atropine, pirenzepine, methoctramine, p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) and 4-diphenyl-acetoxy-N-methyl-piperidine (4-DAMP) competitively antagonized the response to acetylcholine. The constrained pA2 values were 9.32 +/- 0.03, 7.13 +/- 0.01, 6.26 +/- 0.01, 8.17 +/- 0.01 and 9.13 +/- 0.02, respectively. A selective muscarinic M2 receptor antagonist, gallamine, had no effect on acetylcholine-induced relaxation. It is concluded that in human uterine arteries acetylcholine induces endothelium-dependent relaxation and acts as a full agonist. We suggest that the muscarinic receptors involved in the acetylcholine-induced relaxation of the isolated human uterine artery are predominantly of the M3 subtype.

Interactions between responses mediated by activation of adenosine A2 receptors and alpha 1-adrenoceptors in the rabbit isolated aorta.

1. This paper describes aspects of the functional antagonism between the responses mediated by activated alpha 1-adrenoceptors and adenosine A2 receptors in the adventitia- and endothelium-denuded aorta of the rabbit. 2. Adenosine A2 receptor agonists relaxed aortic rings pre-contracted with phenylephrine. The relaxation response was agonist concentration-dependent and saturable. The respective contractile and relaxation responses were stable, reproducible, and reversible. 3. Increasing the phenylephrine concentration caused a progressive attenuation of the action of adenosine A2 receptor agonists, consisting of a decreased maximal response and a dextral shift of the adenosine agonist concentration-response curve. This functional antagonism could be completely reversed upon removal of adenosine by either the addition of adenosine deaminase or by wash-out of the adenosine agonist from the tissue. The relaxation response to the adenosine A2 receptor partial agonists, N6-cyclohexyladenosine and R-(-)-N6-(2-phenylisopropyl)adenosine, was abolished at higher phenylephrine concentrations (e.g. 30 EC50). 4. A 1000 fold increase in the adenosine concentration was required to shift the value of the EC50 of phenylephrine six fold, while a similar increase in the value of the EC50 of adenosine could be elicited by only a 32 fold increase in the phenylephrine concentration. A 30 fold increase in the phenylephrine concentration shifted the value of the EC50 of 5'-N-ethylcarboxamidoadenosine four fold. 5. Analysis of the functional antagonism between the responses mediated by these receptors using the Black & Leff (1983) operational model of agonism allowed for the estimation of the agonist dissociation constant, KA, and the apparent efficacy, tau, for both phenylephrine and adenosine A2 receptor agonists. Increasing the concentration of phenylephrine reduced the value of tau for adenosine agonists in a concentration-dependent and saturable manner. Similarly, increasing the concentration of adenosine reduced the value of tau for phenylephrine in a concentration-dependent and saturable manner. The phenylephrine KA value obtained by the method of functional antagonism (1.9 microM) was similar to that obtained by the receptor inactivation method (2.1 microM). 6. Partial occlusion of the alpha 1-adrenoceptor by the alkylating agent, dibenamine, demonstrated that the magnitude of the adenosine A2 receptor-mediated relaxation was inversely proportional to the number of functional alpha 1-adrenoceptors. 7. It is concluded that the magnitude of functional antagonism is proportional to the stimulus elicited through either receptor. We propose that this tissue preparation and pair of receptors is a good model to study quantitative aspects of functional antagonism between activated receptors.

In vitro effects of milbemycin oxime: mechanism of action against Angiostrongylus cantonensis and Dirofilaria immitis.

The neuropharmacological mechanisms underlying the action of milbemycin oxime on the motility of Angiostrongylus cantonensis and Dirofilaria immitis were examined in vitro. In A. cantonensis, milbemycin oxime caused inhibitory effects at low concentrations of > or = 10(-9) g/ml, and paralysis was elicited at 10(-8) - 10(-6) g/ml. The paralysis was antagonized by picrotoxin and bicuculline but not by dibenamine. In addition, stimulatory effects were observed when the antibiotic was used at higher concentrations of 3-5 x 10(-6) g/ml, and the action was antagonized by strychnine. Both effects were also observed in the preparation contracted by eserine or pyrantel. When D. immitis was treated with milbemycin oxime at concentrations of 10(-7) and 3-5 x 10(-6) g/ml, only slight inhibitory and stimulatory effects, respectively, were observed. These effects were partially antagonized by picrotoxin and strychnine, respectively. These results suggest that the inhibitory and stimulatory actions of milbemycin oxime are caused through gabergic and cholinergic mechanisms in A. cantonensis and D. immitis.

Mutual-effect amplification of contractile responses elicited by simultaneous activation of alpha-1 adrenergic and 5-hydroxytryptamine2 receptors in isolated rat aorta.

5-Hydroxytryptamine (5-HT), and the selective alpha-1 agonists phenylephrine (PE) and oxymetazoline (OXY), were used to study the effects of simultaneous coactivation of the 5-HT2 and alpha-1 adrenergic receptors, respectively, on the contractile responses of isolated rat aortic rings. Dissociation constants (KA) were determined for each of the agonists at their respective receptor subtypes. The KA values for PE and OXY at the alpha-1 receptor were 316 nM and 1.82 microM, respectively, while the KA for 5-HT at the 5-HT2 receptor was 478 nM. Concentration-response curves for each agonist were analyzed by the Black and Leff operational model of pharmacological agonism to determine efficacy (tau) and slope factor values. The estimated tau for PE (16.02) was much greater than the tau for either OXY (4.15) or 5-HT (2.95), which had similar efficacies. Using a previously described drug concentration paradigm, a mutual-effect amplification of the 5-HT-induced contractile response was observed with mixtures of 5-HT and PE, whereas mixtures of OXY and 5-HT elicited a mutual-effect amplification of the observed response to OXY alone. In both cases, the theoretical concentration-response curve constructed using the Poch and Holzmann method of equiactive substitution demonstrated that mutual-effect amplification was largely the result of simple additivity of agonist effects. In addition, estimates of tau and slope factor determined from the Black and Leff equation were substituted into the Leff model of mutual-effect amplification and used to accurately predict the location of the concentration-response curves elicited by mixtures of 5-HT and PE, as well as mixtures of OXY and 5-HT. This represents the first time a mathematical model has been used to accurately predict the outcome of coactivation of the alpha-1 and 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of 5-hydroxytryptamine receptors on isolated ovine uterine artery in late pregnancy.

5-Hydroxytryptamine (5-HT) and 2,5-dimethoxy-4-methyl-amphetamine (DOM) are potent agonists on isolated ovine uterine arteries in late pregnancy. Similar pA2 values (8.56 and 8.33, respectively) of ketanserin, tested against 5-HT and DOM, indicate that responses produced by both agonists are mediated by the 5-HT2 receptor. The contractions produced by 8-OH-DPAT and 2-methyl-5-HT were also blocked by ketanserin (10(-8) M) with the dissociation constants (KB) being 2.49 and 2.88 nM, respectively. This provides evidence that these agonists are activating 5-HT2 receptors in the ovine uterine artery. DOM was more potent than 5-HT, but had a similar efficacy to that of 5-HT. The greater affinity of DOM may explain its greater potency. The dissociation constants (KA) of 5-HT and DOM acting on 5-HT receptors were determined by analysis of concentration-response data before and after fractional inactivation of receptors with dibenamine. The mean KA values for 5-HT and DOM were 3.7 +/- 0.7 x 10(-7) and 1.8 +/- 0.3 x 10(-7) M, respectively. Assessment of receptor occupancy vs. functional response demonstrated little or no receptor reserve in this tissue. Several other 5-HT receptor agonists caused contractions but were much less potent than 5-HT. The order of potency of these agonists was determined to be DOM greater than 5-HT greater than or equal to alpha-methyl-5-HT much greater than 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) greater than 2-methyl-5-HT greater than 1-(3-chlorophenyl) piperazine (mCPP) greater than m-trifluoromethyl-phenylpiperazine (TFMPP).(ABSTRACT TRUNCATED AT 250 WORDS)

Peculiarities of neural regulation of the thyroid, adrenocortical and testicular functions in old age.

The age changes of neural control over the function of the thyroid, adrenal cortex and testicles were examined in adult (6 months) and old (28 months) male rats. In old age, there was a weakening of adrenergic control of thyroidogenesis, alpha-adrenergic and M-cholinergic control of glucocorticoid function of the adrenal cortex and reduction of adrenergic and M-cholinergic influences in the regulation of steroidogenic function of the testicles.

Do both adrenaline and noradrenaline stimulate cardiac alpha-adrenoceptors to induce positive inotropy of rat atria?

1. The positive inotropic responses of rat paced left atria to adrenaline and noradrenaline were recorded. Desmethylimipramine (DMI, 1 microM) and metanephrine (10 microM) were initially present throughout. 2. The positive chronotropic responses of spontaneously beating right atria to adrenaline were used as a reference. In these, pindolol, in increasing concentrations, caused progressive shift of the concentration-response curves to the right, which yielded a pA2 value (8.15) compatible with antagonism of beta-adrenoceptors. 3. The left atrial tension responses to adrenaline showed an initial progressive displacement by pindolol (up to 3 microM) which gave an unexpectedly low pA2 value (6.48). However, with further increases in pindolol concentration there was no additional shift of the curve. In the presence of pindolol (3 microM), prazosin (0.1 microM) displaced the curve to the right but the pA2 value derived from this shift (7.75) was less than expected for alpha 1-adrenoceptor antagonism. 4. When the experiments in the presence of pindolol (3 microM) were repeated in the absence of DMI, prazosin displaced the concentration-response curves for adrenaline-induced left atrial tension to a greater extent and the pA2 value (8.76) was now compatible with adrenaline stimulating typical alpha 1-adrenoceptors. 5. The concentration-response curves for noradrenaline-induced left atrial tension were also progressively displaced to the right by pindolol (0.1, 0.3 and 1.0 microM). These concentrations yielded a Schild plot of unity slope and a pA2 value of 7.94 +/- 0.04. This was not significantly different from the pA2 value of 8.02 +/- 0.07 determined for pindolol against isoprenaline in the left atria, which indicates a normal interaction of noradrenaline with beta-adrenoceptors in the absence and presence of low concentrations of pindolol. 6. A further increase in the concentration of pindolol to 3 microM failed to induce an additional shift of the noradrenaline curves, whether a 'before and after' antagonist or a 'naïve tissue' design was adopted. Similarly, the rightwards shift of the concentration-response curves by timolol reached a limit as the concentration was increased. In all cases the limit of shift occurred at a noradrenaline EC50 value of 5-10 microM. 7. At the limit of beta-adrenoceptor antagonism, prazosin and dibenamine did not displace the noradrenaline curves further. The residual inotropic response to noradrenaline therefore appeared to be mediated via neither alpha- nor beta-adrenoceptors. 8. DMI, in the absence of beta-blockade, produced the potentiation of adrenaline and noradrenaline expected of a neuronal uptake inhibitor. However, in the presence of pindolol, there was no potentiation of the right atrial rate response to adrenaline while its left atrial tension responses were antagonized. This suggested that DMI was acting as an alpha-adrenoceptor antagonist. It also explained the less-than-expected shift by prazosin of the adrenaline responses in the presence of both pindolol and DMI, the latter drug already exerting some alpha-blocking activity. In contrast, the left atrial tension responses to noradrenaline in the presence of pindolol (1 microM) were neither potentiated nor antagonized by DMI. 9. When the effects of prazosin upon left atrial tension responses to noradrenaline in the presence of pindolol (10 microM) were examined in the presence of a lower concentration of DMI (O.1 microM) or cocaine (1O microM), again there was no further shift of the curve. However, when the effect of prazosin) The Macmillan Press Ltd 1989 598 K.L. WILLIAMSON & K.J. BROADLEY was examined in the absence of DMI, but in the presence of pindolol (1 and 1O microM) or timolol (3 microM), there was a small shift of the curves by prazosin (0.1 microM). This yielded pA2 values of 7.19, 7.34 +/- 0.1 and 7.66 +/- 0.09, which were at least one order of magnitude less than literature values and that obtained with adrenaline (8.76 +/- 0.18), and are not consistent with noradrenaline stimulating an alpha 1-adrenoreceptor in the presence of beta-adrenoceptor blockade, the increase in left atrial tension by noradrenaline does not appear to be mediated by beta l- or typical alpha-adrenoceptors. This is in contrast to adrenaline which in these conditions stimulates typical alpha 1-adrenoceptors.

The effect of strontium on the drug-receptor interaction on cholinergic drugs.

The effect of replacing calcium with strontium in the perfusion fluid was qualitatively and quantitatively studied in the isolated longitudinal muscle of the guinea-pig ileum. In the presence of strontium hyoscine could be considered a competitive antagonist of acetylcholine for the acetylcholine receptor of the longitudinal muscle of the guinea-pig ileum; dibenamine still blocked this receptor in an irreversible way. The equilibrium constants for acetylcholine (KA) and hyoscine (KI) were obtained in the presence of calcium (KA = 3.16 +/- 0.63 microM; KI = 0.38 +/- 0.07 nM), and strontium (KA = 7.00 +/- 0.89 microM; KI = 0.93 +/- 0.16 nM). The results show a decrease in the affinity of both drugs for the muscarinic receptor in the presence of strontium.

Relationship between alpha-adrenoceptor occupancy and contractile response in rat vas deferens. Experimental and theoretical analysis.

The rat vas deferens has been considered to be the tissue of choice to study alpha-adrenergic drugs. However, some of these agonists have elicited complex responses in this organ. Therefore, detailed characterization of alpha-adrenoceptor-mediated responses of the rat vas deferens was the aim of this work. Experiments were designed to study the contractile response of this tissue to phenylethanolamine (noradrenaline) and to two imidazolines (oxymetazoline and naphazoline). These responses were related to receptor occupancy and to other parameters of drug action, i.e. dissociation constants, relative efficacies, ED50 and maximal responses. A theoretical model was used to check the experimental data. There was a non-linear relationship between response and receptor occupancy with all three agonists. The dissociation constants for noradrenaline, oxymetazoline and naphazoline were 11.06, 0.15 and 0.10 microM, respectively. The rat vas deferens had 75-80% spare receptors for noradrenaline. Oxymetazoline and naphazoline were shown to be partial agonists with low relative efficacies as compared to noradrenaline (0.0063 and 0.0056 respectively). The dose-response curves generated by the model for partial agonists were similar to the curves obtained experimentally in vitro.

Involvement of the sympathetic nervous system in the cardiovascular effects of ACTH-(1-24) during hemorrhagic shock in rats.

In urethane-anesthetized rats, removal of about 50% of the total blood volume over a period of 25-30 min caused hypovolemic shock, with extreme hypotension (MAP = 18-25 mm Hg and death of all animals within 22 +/- 5 min. The i.v. injection of ACTH-(1-24) in the dose range of 40-160 micrograms/kg induced a sustained, dose-dependent, and, at the highest dose used, an almost complete recovery of blood pressure, and 100% survival, at least for 2 h after treatment. The effect of ACTH-(1-24) was completely prevented by reserpine (5 mg/kg) and clonidine (0.1 mg/kg), significantly reduced by prazosin (0.1 mg/kg), dibenamine (15 mg/kg) and i.v. yohimbine (1 mg/kg) and unaffected by i.c.v. yohimbine (0.2 mg/kg) and i.v. practolol (15 mg/kg). These data suggest that the effect of ACTH-(1-24) in hypovolemic shock depends on the functional integrity of the sympathetic nervous system and is mediated through an activation of peripheral alpha-adrenoceptors.

Two apparently distinct muscarinic cholinoceptor mechanisms in guinea-pig taenia caecum.

Thirty-min treatment of guinea-pig taenia caecum with 300 nM propyl-benzilylcholine mustard (PrBCM) shifted the concentration-response curve for carbachol to the right with a reduction of the maximum contraction, but 90-min treatment did not result in further inhibition. Under these conditions, pilocarpine hardly contracted the preparations, and it competitively antagonized carbachol. Muscarinic agonists might interact with two types of receptor mechanisms and carbachol elicited a stimulus from both types, whereas pilocarpine did so predominantly from the PrBCM-sensitive one.

Motility and drug susceptibility of Angiostrongylus cantonensis developing from gamma-irradiated first-stage larvae.

The effects of neuropharmacological agents on the motility of irradiated and non-irradiated Angiostrongylus cantonensis adult females were studied. GABA induced complete paralysis in non-irradiated and 5,000 R-irradiated worms, but caused only slight paralysis on 10,000 R-irradiated worms. The paralytic effect of GABA was antagonised by picrotoxin. The reason for low susceptibility of heavily irradiated worms to GABA is not known. There was no difference in susceptibility of non-irradiated and irradiated worms to other neuropharmacological agents including eserine, phenylephrine and dibenamine.

Characterization of the alpha-1 adrenergic receptors in the thoracic aorta of control and aldosterone hypertensive rats: correlation of radioligand binding with potassium efflux and contraction.

Pharmacologic analysis of functional and radioligand binding studies were used to determine whether alterations in adrenergic receptors contribute to the catecholamine supersensitivity observed in the thoracic aorta of aldosterone hypertensive rats (AHR). Adrenergic function was evaluated using receptor-mediated contraction and rate of 42K efflux. The pA2 for phentolamine was the same in AHR (7.9 +/- 0.1 and 8.0 +/- 0.1) and control (7.9 +/- 0.2 and 8.1 +/- 0.1) rats whether measured by contraction or 42K efflux. The pA2 value for the selective alpha-1 antagonist prazosin (9.8 +/- 0.1 to 10.7 +/- 0.2) and the alpha-2 antagonist yohimbine (6.6 +/- 0.2 to 7.4 +/- 0.2) was similar in AHR and control groups using both norepinephrine and phenylephrine as agonists. The rank order of potency was prazosin greater than phentolamine greater than yohimbine in both groups. The KD for [3H]prazosin binding to AHR (26 +/- 3 pM) and control tissue (34 +/- 6 pM) agreed with the prazosin KB obtained by measurements of contraction and 42K efflux. The alpha-1 receptor density was also unaltered: 39 +/- 1 fmol/mg in AHR and 44 +/- 3 fmol/mg in control. Assessment of the NE dissociation constant (Ka) by method of fractional receptor inactivation indicated that the Ka was 4.8 X 10(-7) M in both AHR and control tissues. However, at the same receptor occupancy, the NE-induced increase in 42K efflux was elevated markedly in aorta from AHR. We conclude that alteration in adrenergic receptor density, affinity or type is not the cause of catecholamine supersensitivity in the aorta from AHR and that postreceptor events mediate this phenomenon.

The 5-hydroxytryptamine (5-HT2) receptor stimulates inositol phosphate formation in intact and broken WRK1 cells: determination of occupancy-response relationships for 5-HT agonists.

5-Hydroxytryptamine (5-HT) stimulates the accumulation of inositol-trisphosphate in WRK1 cells, a cell line originating from a rat mammary tumor. 5-HT acts via a single receptor type for which it has an affinity constant estimated to be 1.27 microM. A series of agonists known to act at 5-HT2 receptors are partial agonists in this system and have a rank order of relative intrinsic efficacies corresponding to that seen in other systems possessing 5-HT2 receptors. There is an essentially linear occupancy-response relationship for 5-HT and other agonists indicating the absence of a strong amplification mechanism between receptor activation and inositol phosphate formation. The selective blockade of the 5-HT response by nanomolar concentrations of 5-HT2 selective antagonists but not by drugs acting at other 5-HT receptor subtypes suggest that the receptor in WRK1 cells is of the 5-HT2 type. Additionally, we demonstrate that in WRK1 membranes 5-HT acts via the 5-HT2 receptor to elicit a GTP dependent increase in the production of inositol-bisphosphate and inositol-trisphosphate. These properties of the WRK1 cell line indicate that it is a useful model with which to study the nature of 5-HT receptor coupling to the putative second messenger(s), the inositol phosphates.